The major royal jelly proteins (MRJPs) as the alternative of fetal bovine serum (FBS) were used to culture Chinese hamster ovary cell line (CHO-K1). The effects of mixture media with different MRJPs/FBS (M/F) proportions on the proliferation of CHO-K1 cell were compared. The result of methyl thiazolyl tetrazolium (MTT) assay showed that the cells in the medium with M/F 50/50 possessed the highest relative proliferation activity. Compared to the CK (M/F 0/100), the relative cell proliferation activity in the medium with M/F 50/50 was increased by 25.1% (48 h) and 13.6% (72 h), respectively. Cell imager showed that the cell density and diameter in the medium with M/F 50/50 were significantly higher than those in the CK. The detection of cell cycle with flow cytometry showed that the cell proliferation index (PI) in the medium with M/F 50/50 was increased by 19.4% compared to that in the CK, indicating that MRJPs might promote the synthesis of DNA, proteins and enzymes. The Raman spectrum of cells in the medium with M/F 50/50 was similar with those in the CK and bovine serum albumin (BSA) medium, demonstrating that the complex serum maintains the cell type stable. In conclusion, the results suggest that MRJPs could partially replace FBS in the cultivation of CHO-K1 cell line and possess industry prospect.

A full-length sequence of phytase gene (phyA) from Aspergillus niger ZJUY was cloned and modified based on polymerase chain reaction (PCR)-directed mutagenesis, aiming at addressing phytase denaturalization and deactivation during the process of pelleting. The mutation types included codon optimization for key sites, the introduction of hydrogen bond (T295S, Q296R and V43N) and the deletion mutation of disulfide bond (Cys196-Cys446). The anchored-Flo1p (FS) and target-phyA were successfully inserted into the yeast expression vector pPICZαC by seamless cloning. Eight kinds of expression vector pPICZαC- FS/phyA were integrated into the Komagataella phaffii GS115 genome by homologous recombination using lithium chloride transformation method, respectively. Phytases were successfully displayed on the surface of K. phaffii GS115, verified by immunofluorescence staining and flow cytometry. The resulted recombinants A31, A61 and A84 all showed a high phytase activity of 7 000 U/g. In addition, phytase-displaying exhibited better thermostability and pH-stability than those of secretory phytase. Among them, A61 remained 18% phytase activity after water bath at 90 ℃ for 1 h, and still maintained more than 80% of phytase activity at a pH range of 1.6 to 4.0. This characteristic of strain A61 can overcome deactivation during the process of granulating at 60 to 90 ℃ and better meet the demand in animal feed and food processing.

It is important to investigate the mechanism of growth and development of edible fungi for genetic breeding and cultivation technique research. Based on the total expressed proteins of biological tissues and cells, a protein expression profile can be established by using proteomics technology, which can not only provide a material basis for the law of life, but also provide a theoretical basis and solution for biological genetic breeding.
In this study, in order to analyze the protein expression changes at different developmental phases and explore the law of growth and development of Pleurotus pulmonarius, we applied an integrated proteomic approach to comprehensively investigate the proteome change of P. pulmonarius at different developmental phases, including mycelium phase, primordium phase, coral-like phase, and fruit body phase, by using high-sensitive-mass spectrometer.
The results showed that, compared with the initial mycelium growth phase, a total of 885 differentially expressed proteins were isolated from the other three growth phases, with 376, 642 and 692 differentially expressed proteins from the primordium phase, coral-like phase and fruit body phase, respectively. The specifically expressed proteins were 11, 23, 136 and 181 respectively in the four different developmental phases. Gene ontology annotation showed that 26%, 20%, 15% and 11% of the differentially expressed proteins were related with carbohydrate synthesis, reproductive structure development, organ synthesis and protein metabolism. Moreover, the analysis of metabolic pathway showed that the differentially expressed proteins mainly involved in the pathway of tricarboxylic acid cycle and glycolysis/gluconeogenesis which may possibly play an important role in the growth and development of P. pulmonarius. The heat shock protein 70 (HSP70), belonging to a molecular chaperone, showed significant differences in mycelium phase, primordium phase, corallike phase, and fruit body phase, indicating that it is critical to regulate the growth of P. pulmonarius.
In conclusion, there are differentially expressed proteins and differential signaling pathways at different developmental phases of P. pulmonarius. These differentially expressed proteins play a very important role in the organic development, fruiting body morphogenesis and resistance to adverse environmental conditions. This study lays a theoretical basis for the research of breeding and cultivation techniques of P. pulmonarius.

Abscisic acid (ABA) is a vital hormone that regulates stomatal closure and responds to stress conditions in plants. Therefore, ABA signal pathway involves numerous physiological processes in plants, such as seed dormancy, germination, development and ripening. Among the families of ABA receptor genes, ABA-insensitive (ABI) is a major transcription factor that regulates ABA signal pathway. To date, little information is known about the ABI gene of buckwheat.
To characterize ABI gene sequence polymorphism and genetic relationship of genus Fagopyrum, the budlets of 30 germplasm resources from seven wild species including F. esculentum, F. tataricum, F. zuogongense, F. pilus, F. megaspartanium, F. cymosum and F. urophyllum were collected for DNA extraction. The ABI UniGene sequence of buckwheat was isolated from the previously performed transcriptome sequencing data (accession number: SRS1350375). Specific primers of ABI gene were designed based on the high conservative sequence for polymerase chain reaction (PCR) amplification, and the amplified fragments from 30 germplasm resources were processed for gene sequencing. Then the gene sequence analysis was performed on nucleotide
BLAST of NCBI database (http://blast.ncbi.nlm.nih.gov/Blast.cgi), and the genetic variance was analyzed by DNAsp5, and the analysis of ClustalW multiple sequence alignment, genetic distance and hierarchy were clustered by MEGA 5.05.
The results showed that 20.28% of polymorphic sites were found in ABI gene fragments among 30 germplasm resources of the seven wild species of genus Fagopyrum, whereas only 1.86% of polymorphic sites were found among 14 germplasm resources of F. tataricum and 1.81% of polymorphic sites were found among five germplasm resources of F. esculentum, indicating that ABI gene sequence is highly conserved in the intraspecies of genus Fagopyrum. Genetic distance analysis showed that both the values of interspecific genetic distance and interspecific net genetic distance between F. zuogongense and F. pilus were the largest, whereas those between F. cymosum and F. urophyllum were the least based on the ABI gene sequence analysis. Hierarchical cluster analysis indicated that the phylogenetic relationship of F. tataricum was close to F. megaspartanium, F. cymosum and F. urophyllum, then further grouped with F. pilus, while the F. esculentum was close to F. zuogongense based on the ABI gene sequence analysis. The F. tataricum and F. zuogongense collected from different regions were respectively clustered, suggesting that the mutation of ABI gene might be influenced by the growth environment during plant evolution.
Inconclusion, ABI gene sequence is highly conserved in the intraspecies of genus Fagopyrum; nevertheless, different growth environments caused distinctive mutations of ABI gene. Based on the gene sequence analysis, a close relationship was found between F. esculentum and F. zuogongense. In addition, close relationships were also found among F. tataricum, F. megaspartanium, F. cymosum and F.urophyllum.The above results provide a theoretical basis for ABI gene research and evolutionary relationship of genus Fagopyrum, but the exact evolution among different species of buckwheat still needs further research.

During development process of the Zhoushan archipelago, the original old-growth broadleaved forests on the islands have been severely destroyed due to intensive human activity. Neolitsea sericea (Lauraceae), distributed on a few islands of the Zhoushan archipelago, is facing the danger of extinction due to rapid degradation and destruction of original habitats. So far, plenty of studies on N. sericea have been focused on its population genetic diversity and genetic structure. Neolitsea sericea is well adaptable to the environment, thus can survive in the ravine on the islands. The strong tolerance of N.sericea to drought, wind, salt and barren soil also makes it optimal species for afforestation. However, few studies on drought tolerance of N. sericea have been reported yet.
Plant response to drought stress at a molecular level is a complex biological process, involving consideration of the stress effects and regulation events. Thus, it is of great importance to analyze the underlying mechanism systematically at the physiological level.
In this study, one-year-old seedlings of N. sericea were selected as test materials. To obtain a more complete physiological mechanism in responses to drought stress, N. sericea was exposed under the conditions of four different relative water contents (normal water supply, light drought, moderate drought and severe drought) in soil for 45 days. The relative water content in soil of normal water supply, light drought, moderate drought, and severe drought was controlled in 75%-80%, 55%-60%, 40%-45% and 30%-35% of field capacity, respectively. All plants were rehydrated after drought treatments by normal water supply for 10 d. The relative water content in soil was controlled by pot- weighing method through the experiment. The dynamic changes of the physiological and photosynthetic traits in leaves were measured every 15 days of stress treatments and at the end of rehydration. The traits measured in this study include net photosynthetic rate (Pn), stomatal conductance (Gs), water use efficiency (WUE), intercellular CO₂ concentration (Ci), transpiration rate (Tr), total content of chlorophyll (Chl), superoxide dismutase (SOD) and peroxidase (POD) activities, proline content, malondialdehyde (MDA) content, soluble sugar content and relative conductivity (Rc).
The results showed that the contents of Chl, Pn, Tr and Gs decreased constantly and significantly during severe drought, and the content of Ci decreased in 0-30 d but enhanced in 30-45 d during the severe drought, which indicated that the decrease of Pn was caused by stomatal limitation during the 0-30 d of severe drought, and by non-stomatal limitation during 30-45 d of severe drought. The continuous inhibition of Tr after rehydration from severe drought suggested the afunction of stomatal regulation. The drought stress increased the overall WUE of the leaves, although the WUE dropped significantly in 30-45 d of severe drought. The trend of MDA content, SOD and POD activities in N. sericea first increased and then decreased during the drought treatments, indicating the production and clearance of MDA had reached a dynamic balance. The significant increase of Rc indicated the elevation of cell membrane permeability. The overexpression of soluble sugar and proline in leaves during 30-45 d of drought treatment resulted in the reduction of Rc, which relieved the osmotic pressure in N. sericea. However, Rc staying at a high level after rehydration from severe drought indicated the damage of cell membrane.
In conclusion, N. sericea can conduct the instant stomatal regulation, eliminate active oxygen and regulate the osmotic pressure effectively, decrease the Tr and MDA content, increase the cell membrane permeability and enhance the WUE of leaves, to reduce the damage under the drought stress. However, sev

Sweet osmanthus (Osmanthus fragrans) is one of the top ten famous native horticultural plants in China. According to different flowering seasons, flower colors and inflorescence types, the cultivars are divided into four groups: O. fragrans Asiaticus Group, O. fragrans Albus Group, O. fragrans Luteus Group and O. fragrans Aurantiacus Group. Despite long-term cultivation of Osmanthus, little information was recorded on the formation of so many cultivars. O. fragrans Asiaticus Group was considered as the most primitive cultivars, and the ones with light color flowers formed earlier, then followed by deep color flowers. The cluster results based on various types of molecular markers were quite different from traditional classification system, indicating diversity of phenotypic traits might account for a tiny part of the whole genetic diversity of sweet osmanthus. However, at present, few studies can give evidence to further understand the evolution process of sweet osmanthus cultivars.
In this study, to further understand the evolution theory, a rooted phylogenetic tree for the cultivars of sweet osmanthus was constructed based on population structure analysis through sequence-related amplified polymorphism (SRAP) technology. Forty-five cultivars were used as plant materials; O. heterophyllus, O. fordii, O. cooperi “Yujie” and O. cooperi “Xuegui” were used as controls, and O. matsumuranus was used as an outgroup. Ten pairs of SRAP primers with high polymorphism were applied to amplify DNA of all samples, and the fragments were examined by capillary electrophoresis. POPGENE 1.32 software was applied to analyze genetic diversity and genetic differentiation. Structure 2.34 software was used to analyze population structure and divide cultivars into subgroups. Nei’s genetic distance among subgroups was calculated by NTSYSpc, then applied to construct a rooted phylogenetic tree by MEGA 6. The rate of hermaphrodite flower cultivars on each level of the phylogenetic tree was calculated to understand the sexual system evolution. Moreover, the genetic mechanism of flower color variation for sweet osmanthus was further speculated based on the result.
Results showed that the 10 pairs of SRAP primers produced 137 polymorphic bands among all the samples with an average of 13.7 bands per primer. Polymorphism information content ranged from 0.202 8 to 0.302 7, with an average of 0.250 7. Nei’s genetic diversity index ranged from 0.220 3 to 0.350 2, with an average of 0.283 5. The Shannon’s genetic diversity index ranged from 0.348 3 to 0.519 3, with an average of 0.436 4. There was significant population structure among sweet osmanthus cultivars, and 36 cultivars could be divided into seven subgroups with simple genetic background. Nine cultivars had complicated genetic background, which were identified as a mixed group. Gene differentiation coefficient (Gst) was 51.32% among subgroups, much higher than that of four cultivar groups. Moreover, less gene flow was observed among subgroups than that of four cultivar groups. These results indicated that the cultivars in the same subgroup had much closer genetic relationship than those in the same cultivar group. Using subgroups as the unit of evolution, a rooted phylogenetic tree was constructed. The sweet osmanthus cultivation had experienced about 10 stages (A-J level): subgroup 3 composed of major cultivars in O. fragrans Asiaticus Group and O. fragrans Albus Group formed first, and subgroup 5 composed of the male cultivars in O. fragrans Aurantiacus Group formed the latest, and the cultivars in O. fragrans Luteus Group formed in each stage after D level. With the evolution process, the rate of hermaphrodite flower cultivars dramatically reduced, proving that androdioecy sexual

Echinocandins are the first class of antifungals to target fungal cell wall. Two antifungals, including micafungin and anidulafungin, have been widely used in clinic. The synthesis of them includes an essential process: hydrolytic removal of fatty acyl side chains from FR901379 and echinocandin B (ECB) to generate cyclic hexapeptide nuclei. In this study, Actinoplanes utahensis SW1311 with acylase activity was isolated for both FR901379 and penicillin V. Then a recombinant Streptomyces coelicolor A3(2) harboring aculeacin A acylase (AAC) gene from A. utahensis SW1311 was constructed. The result showed that the AAC activity produced by the recombinant strain was 4.6-fold higher than that of A. utahensis SW1311. Additionally, the fermentation time of the recombinant strain was 30% shorter than that of A. utahensis SW1311. The results not only provide a new application of AAC for micafungin synthesis but also identify a new
suitable host for AAC gene.

Apis cerana cerana is a native honeybee species in China, which is widely distributed in all parts of the country, especially in the hills and mountains of the South China. Apis mellifera was introduced into China in the end of the 19th century. With interspecific competition and destruction of ecological environment, the number of Apis cerana cerana colonies in China declined sharply. In 2006, Apis cerana cerana was among the List of the Genetic Resources of Livestock and Poultry under Protection in China.
Located in the southwest of Zhejiang Province, China, Lishui City is a mountainous area and known as “the first ecological city in China” with the forest coverage rate of 80.8% and a variety of climate and vegetation. Lishui has a long history of raising Apis cerana cerana. In recent years, the number of Apis cerana cerana colonies in Lishui increased significantly. In order to understand the genetic diversity and taxonomic status of Apis cerana cerana in Lishui, the mitochondrial DNA tRNAleu-COⅡ sequences of 94 samples from each district and county of Lishui were determined and further analyzed in the study.
It was shown that the nucleotide polymorphism (Pi) was 0.003 52 and the haplotype diversity (Hd) was 0.772±0.036, which indicated that the genetic diversity of Apis cerana cerana in Lishui was high. Twenty haplotypes were found in 94 honeybee colonies. The main haplotype was found in 41.5% of the honeybee colonies and was identical to the main haplotype which was found in Apis cerana cerana in Fujian Province and also was identical to the sequence found in Guangzhou. Therefore, the results support Apis cerana cerana in Lishui belongs to the honeybee type of South China. However, the second most common haplotype in Lishui was different from that in Fujian Province. There were also two new haplotypes found in Lishui. The results indicated that Apis cerana cerana in Lishui had a unique genetic background. Furthermore, cluster analysis showed that the clusters of the haplotypes were closely associated with their geographical distribution, which indicated that different geographic Apis cerana cerana populations had been possibly differentiated by the geographical isolation in mountainous areas of Lishui. The above results are of great significance to the protection and reasonable use of the genetic resources of Apis cerana cerana in Lishui.

Chaetomium Kunze is mainly distributed in cellulose-containing substrates in the nature, including soil, plant residue, excrement of birds, omnivorous animals and rodents. Persistent organic compounds can be degraded by cellulase produced by Chaetomium Kunze, which also has antagonistic effects on certain microorganisms in the soil. However, littlehas been known about Chaetomium in Tibet. Therefore, a systematic research on resource distribution of Chaetomium in Tibet is necessary and urgent.
In this study, 355 samples of soil, plant residue and animal manure were collected from five regions of southeastern Tibet, in which plant residue and animal manure were separated by tissue separation, and soil samples were separated by dilution separation method. SPSS 13.5 software was used to analyze the diversity index of Chaetomium spp. from different regions, and 36 strains were preliminarily classified according to Arx system. The nucleotide sequence polymorphisms of rDNA-ITS and β-tubulin gene were analyzed by DnaSP version 5.0. The rDNA-ITS and β-tubulin gene sequences were used to analyze the diversity of Chaetomium species in southeastern Tibet.
The results showed that a total of 36 strains were successfully isolated from the 355 samples, and the average isolation rate was 10.14%. The diversity index data indicated that both the diversity index and species evenness index of Chaetomium spp. in Nyingchi was the highest, with the value of 1.178 5 and 0.605 6 respectively. But the richness index of Chaetomium was the highest in Chamdo, with the value of 0.861 4. Differences of dominant populations of Chaetomium were observed among the five regions: the dominant population in Gongbogyamda County, Medog County and Chamdo City, Zayü County, and Nyingchi County was C. globosum, C. funicola, C. bostrychodes, and C. convolutum, respectively. According to the morphological characteristics, the 36 strains were assigned to eight species. Moreover, rDNA-ITS sequences and β-tubulin gene were applied to conduct the diversity analysis, and the result showed that the variation sites, haplotype numbers, haplotype diversity, nucleotide diversity index and nucleotide difference of β-tubulin gene were significantly different from rDNA-ITS sequences, while β-tubulin gene showed greater base variability. The phylogenetic tree of rDNA-ITS and β-tubulin genes indicated that the 36 isolates were divided into seven groups. The two gene fragments can not only distinguish the species of Chaetomium with different morphologies (such as C. indicum, C. megalocarpum, C. globosum and C. funicola), but also those with very similar morphological characteristics (such as C. indicum and C. erectum) and some species hard to distinguish. However, several species such as C. convolutum, C. bostrychodes and C. nigricolor can’t be distinguished by rDNA-ITS and β-tubulin genes, which still need combining more gene fragments to differentiate.
In conclusion, the results confirm the rich resources of Chaetomium fungus in southeastern region of Tibet, providing data for abundant resources of Chaetomium in Tibet, and laying a foundation for the exploitation of the metabolites of Chaetomium.

SUPPRESSOR OF OVEREXPRESSION OF CO1 (SOC1) is a flowering integration factor that widely existed in flowering plants, which is necessary in multiple flowering induction pathways, including vernalization, photoperiod, gibberellin, and other endogenous or exogenous environmental signals. Therefore, research of the effect of SOC1 on plant growth and development can reveal the plant flowering mechanism and direct molecular breeding. Anthurium is a kind of high-grade potted flower in the worldwide. However, molecular biology research on this plant had just started. As a flowering integration factor, SOC1 in Anthurium may be a key to deeply understand the flower conversion process.
In this study, rapid amplification of cDNA ends (RACE) technology was used to obtain the full length of two SOC1 genes of Anthurium. Several biological softwares were used to analyze the biological information, including NCBI Blast, NCBI Conserved Domain Search, ProtParam, TMHMM Server version 2.0, SignalP 4.1 Server, iPSORT, PSIPRED, DNAman, and MEGA 6.0. Gene expression levels of the two SOC1 genes were detected by real-time reverse transcription?polymerase chain reaction (RT-PCR).
The main results were as follows: AaSOC1-1 and AaSOC1-2 were obtained from two independent SOC1 full coding sequence (CDS) regions, which shared 75.6% similarity at the nucleotide level. Their encoded proteins were composed of 216 and 213 amino acid residues, respectively. Both of the putative proteins had secondary structures such as helix, sheet, and turn, but no other special structures were found. Based on the online software analysis results, it was concluded that AaSOC1-1 and AaSOC1- 2 were located in the mitochondria but not in the nucleus. Phylogenetic tree analysis showed that the two SOC1 transcription factors from Anthurium were tightly clustered with each other, and also with other monocotyledons. Fluorescence quantitative RT?PCR analysis revealed that both genes from Anthurium were ubiquitously expressed in the vegetative and reproductive organs, but different in the expression patterns. Particularly, AaSOC1-1 was mainly expressed in the bract and tepal, whereas AaSOC1-2 was mainly expressed in the stem and pedicel.
In sum, this study reported the nucleotide and deduced amino acid composition of the two SOC1 transcription factors. Preliminary analysis of their characteristics, structure, and potential function were performed through bioinformatics software, combined with multiple sequence alignment and phylogenetic tree construction. Based on the results, the two genes found in Anthurium were identified as members of SOC1 gene family.Gene expression experiments confirmed the similarities and differences of their expression patterns, which were relatively conservative compared with other species. This study lays the foundation for understanding the functional properties of these two transcription factors, which also requires in?depth research to verify the above speculation and views.

With rapid development of industrialization and urbanization in China, large amounts of heavy metals have been directly or indirectly released into the soil environment through solid wastes, waste air, and waste water from industrial activities over the recent decades. In particular, cadmium (Cd) is one of the most toxic pollutants causing environmental problems. With excellent water solubility, Cd can be absorbed by plants easily and transferred to human body, eventually threatening the health of human beings. Lots of herb plants have tolerance for Cd and can accumulate to eliminate the Cd pollution in soils. However, with relative low biomass, the accumulation capacity of herb plants is limited. Little is known about the phytoremediation application of woody plants, which have large biomass and fast growth rate. Salix variegata has strong bioaccumulation on Cd and exceptionally high tolerance to Cd stress, especially planted along the sloping banks, thus it has the potential for phytoremediation of Cd polluted environments. However, the mechanism of tolerance and detoxification for Cd still needs further study. Metal binding ligands are effective for detoxification of heavy metals, mainly phytochelatins (PCs) and metallothioneins (MTs).
In this study, in order to explore the detoxification mechanism of S. variegata, and improve the potential enrichment and detoxification ability of S. variegata, hydroponic experiments were conducted under different Cd²⁺ concentrations, including 0 mg/L Cd²⁺ (CK), 2 mg/L Cd²⁺ (T₁), 10 mg/L Cd²⁺ (T₂), 20 mg/L Cd²⁺ (T₃) and 50 mg/L Cd²⁺ (T₄), and the Cd²⁺ and thiol-peptide contents in leaves and roots of S. variegata were determined.
The results showed that under the Cd stress, four kinds of thiol- peptides (PC2, PC3, PC4 and PC5) were detected in the leaves, and two kinds of thiol-peptides (PC2 and PC3) were detected in the roots. The total content of thiol-peptides in the leaves and roots of S. variegata increased with the Cd concentration and treatment time. The contents of glutathione (GSH) in the leaves and roots of S. variegata under different levels of Cd stress were significantly higher than that of the control treatment. Besides, the content of thiol-peptides in the leaves of S. variegata also increased significantly than the control. The increase of thiolpeptides was proportional related to the increase of Cd concentration. Meanwhile, the PC2 and PC3 contents of the T₄ group in the roots of S. variegata were significantly higher than the control. Significant positive correlations between thiol-peptide content and Cd accumulation were observed in the leaves and roots of S. variegata. Altogether, these results indicated that the thiolpeptide content increased with the Cd accumulation in the leaves and roots of S. variegata.
In conclusion, with intensified Cd stress, the chelating ability of phytochelatin to Cd increased significantly, and the tolerance and the detoxification ability of S. variegata to Cd stress were substantially improved. The PC2 content of each Cd concentration treatment in the leaves and roots of S. variegata was the highest among the thiol-peptides, especially in the roots of S. variegata. Therefore, the chelating ability of PC2 to Cd in S. variegata is the best among the thiol- peptides, especially the chelating ability of PC2 to Cd in the root.

Exiguobacterium sp. has been figured out to be the main spoilage bacteria on many kinds of food, and it has the potential to be the main spoiler. Exiguobacterium has already been isolated from extreme environments and they have evolved and acquired some resistance including thermotolerance, cold resistance, alkali resistance, and osmotolerance. The Exiguobacterium can normally metabolize under 4 ℃ storage temperature. Further, Exiguobacterium has strong proteolysis ability to damage nutrient and food, and it can form biofilm, not only increasing the resistance to detergents and preservatives, but also consuming nutrients to grow and reproduce. The present study aimed to establish a rapid identification method of Exiguobacterium acetylicum based on a double real-time polymerase chain reaction (RT-PCR). The study took the genes P401_RS0117025 and oxi_50582462, which were screened in the early experiment and were specific to Exiguobacterium sp. and E. acetylicum respectively, as detection targets. The specificity of the PCR assay was verified with the DNA of 12 E. acetylicum strains and 14 non-E. acetylicum strains; the effectiveness of the assay was evaluated by the foundation of standard curve, as well as its repeatability. The sensitivity of different DNA and cell concentrations was identified. The results demonstrated that the specific genes and primers of the early experiment were both well-specific. The specific evaluation experiment results showed that the probes T-291 and T-2B were also well-specific. The R2 values of standard curve were 0.994 and 0.999, respectively, indicating that the assay was credible, and the RT- PCR showed high repeatability. When DNA of different concentrations were added as templates, there was no obvious different in standard deviation of threshold (Ct), and the coefficient of variation of the same concentration was fluctuation during 0.51 to 1.12. Even though at 1.629×10^－4 ng/μL, its repeatability was well maintained, which indicated that the assay could showed high repeatability at low DNA concentration. The sensitivity-evaluation showed that the DNA detection limit of the assay was 1.629×10^－7 ng/μL, and the detection limit of pure bacteria colonies was 3.4 CFU/mL without enrichment culture. In addition, the high-sensitivity resulted in its outcoming of short- time cast, for it did not need the step of enrichment culture, which was different from other studies. Finally, the mathematical relationship between Ct and Nt colony-forming units (CFU) was developed by software Origin 9. It was C_t=－1.759× Nt +31.678, the R² value of which was 0.939. When applied to the fresh-cut leafy vegetables, the assay and the equation could detect rapidly and accurately the CFU on them. In sum, this study establish as an accurate, sensitive and efficient double RT- PCR assay to detect quantitatively food spoilage bacteria E. acetylicum. It lays a theoretical basis for food shelf- life prediction, and provides some references to the supply chain of fresh-cut vegetables.

Due to the rapid development of livestock breeding and poultry raising，non-point source pollution has become a significant threat to environmental management and aquaculture industry development，as well as to human health in the last few decades. Therefore，it is particularly urgent to establish a monitoring method that can be used as the efficient indicator of fecal pollution with high sensitivity and strong specificity. In animal guts，genes which are directly involved in bacterium-host interactions may display increased level of host-associated genetic variation，making them promising candidates for fecal source tracking. Specific markers targeting bacterium-host interaction genes for human，cattle and chicken were reported previously， however，swine-specific marker for fecal source tracking has not been found yet. We applied a genome fragment enrichment (GFE) method to enrich swine- specific metagenomic region that differ from those of other animal species. Briefly，a portion of swine fecal DNA was labeled with biotin and pre-hybridized with a composite fecal DNA pool of other animals including cow (n=20)，goat (n=20)，chicken (n=8)，duck (n=20) and goose (n=5) to block nonunique fragments. Then the pre-hybridized product and another portion of swine fecal DNA labeled with K9 primer were taken together to perform a competitive DNA hybridization. After streptavidin enrichment and long- linker PCR amplification by K9 primer，the products that were assumed as swine- specific fecal DNA were cloned into vectors and were sequenced. Dot blot hybridization with negative control fecal DNA (composite fecal DNA pool of other animals) was used to identify cloned GFE sequences which were not swine-specific. The cloned GFE sequences were assigned to bacterial class annotations based on the top BLASTX hit (the lowest E-value score) with the GenBank non-redundant database. The putative protein transcript of each sequence was analyzed based on the similarity of gene sequences by using BLASTX with GenBank non- redundant database， and their biochemical functions were therefrom predicted. Sequence analyses of five hundred randomly selected clones from the libraries obtained by three rounds of metagenomic GFE revealed that this subset contained a total of 384 non-redundant sequences and most sequences (87%) ranged from 400 bp to 600 bp in size. Dot blot hybridization using DNA composite of non- target animals as probes showed that only eight clones exhibited cross-reaction，indicating a very low false-positive rate of 2.8%. BLASTX searches identified homologous sequences in GenBank database for 315 non- redundant DNA inserts，with other 69 (17.9% of 384 swine fecal DNA sequences) inserts showed no homology with any previously reported genes. Based on top BLASTX hits，the sequences were putatively grouped into 20 bacterial classes including the predominant group of Bacteroidetes-like sequences (43.2%)，among which，120 sequences were similar to Bacteroidetes-Prevotella. Clostridia-like sequences were the second most abundant group (19.5%)，and Bacillilike sequences represented 8.6% of the clones. Moreover，three sequences exhibited identity to genes in Archaea. Biochemical function annotation revealed that 38.5% of the total analyzed sequences were predicted as genes with unknown functions. Among the fragments associated with characterized function genes (61.5%)，the sequences were most frequently assigned to putative proteins associated with metabolism (22% ，e.g.，carbohydrate metabolism and amino acid metabolism)，cellular processes (12.8%，e.g.，membrane transport and DNA repair/replication/recombination) and information storage and processing (7.6%). It is concluded that gene encod surface proteins，membrane associated proteins，secretary proteins and carbohydrate metabolism proteins of dominant bacterial classes could be regarded as putative targets for swine-specific microbial genetic markers.

Comparing to open-pollinated varieties, rapeseed hybrids had an obvious heterosis in seed yield and adaptation to environment. In the procedure of hybrid breeding, it is very important to select the male and female parents, which greatly affects the hybrid performance. For the traditional method of hybrid breeding, the selection of parent lines for combination is random, which lacks foreseeability of the hybrid performance. Application of marker-assisted selection (MAS) can enhance the efficiency and accuracy of the selection, and shorten the period of breeding program. Therefore, this study aimed to introduce MAS into the procedure of hybrid breeding in rapeseed, and to enhance the efficiency of breeding elite hybrids with good performance. In the present study, two male sterile lines and 26 cultivars (lines) were analyzed with 66 molecular markers. These makers were distributed on 19 chromosomes of Brassica napus and each chromosome contributed 2 to 4 markers. The two male sterile lines, MSL72A and Xiangyou 15A (XY15A), belong to a kind of genic male sterile (GMS) system named 9012A in rapeseed. The 26 cultivars (lines) included 19 semi-winter type cultivars (lines) from China, 4 winter type and 3 spring type cultivars (lines) from Germany. Fifty-two hybrids were derived by crossing the two male sterile lines with the 26 cultivars (lines). A dendrogram was established based on UPGMA (unweighted pair-group method with arithmetic means) cluster analysis. Nei72 distance between two male sterile lines and 26 cultivars (lines) were calculated, and the correlation between genetic distance and heterosis of hybrid performance was analyzed. The results showed that 254 polymorphic loci were derived from the 66 markers and each marker had a mean of 3.9 polymorphic loci. The mean genetic distance between XY15A and the 26 cultivars (lines) was 0.56, and the maximum and minimum genetic distances were 0.15 and 0.80, respectively. The mean genetic distance between MSL72A and the 26 cultivars (lines) was 0.58, and the maximum and minimum genetic distances were 0.28 and 0.84, respectively. The dendrogram and principal coordinate analysis of the two male sterile lines and the 26 cultivars (lines) showed that all the materials were divided into four primary groups, which were consistent with their geographical attribute. The four groups were the middle and lower reaches of Changjiang River, Huanghuai region, Europe spring type and Europe winter type. Correlation analysis indicated that the genetic distance based on molecular markers was significantly correlated with over male-parent heterosis of hybrid performance, and the correlation coefficients were 0.647** and 0.622** in hybrids derived from the two male sterile lines, XY15A and MSL72A， respectively. The seed yield of hybrids was also significantly correlated with the yield of male parents, and the correlation coefficients were 0.732** and 0.615**, respectively. All the results indicate that the hybrid performance depends on the performance of parent lines and the genetic distance between the two parent lines together. It will be highly efficient in hybrid breeding by combining male sterile lines with elite restorers which should have good performance and enough genetic distance with male sterile lines. Therefore, we consider that molecular markers could be used to select restorers for male sterile lines in hybrid breeding of rapeseed and enhance the breeding efficiency.

Cathepsin is a kind of protease that mainly exists in intracellular lysosome. Under the weak acid condition, cathepsin can be activated and acts as hydrolysate protein. Based on the different mechanisms of the protein hydrolysis, cathepsin is divided into four species, including aspartic acid protease, cysteine protease, serine protease and threonine protease. Cathepsin D and cathepsin E (CtpE) both belong to aspartic proteases, while the latter is fundamental basis for life activities of mammals. Moreover, CtpE is an important enzyme in participating physiological processes of aquatic animals, such as digestion, yolk formation and immune response. However, few researches were focused on immune function of CtpE gene in fish. In this study, a full- length cDNA of CtpE was cloned from medaka (Oryzias latipes), to identify the gene and protein sequences of CtpE in medaka, and to clarify the evolutionary relationship of O. latipes CtpE (OlCtpE) with other animals, providing a theoretical foundation for further research on the physiological function of CtpE in fish. The total RNA was extracted from medaka gut tissue, using Trizol kit according to the manual steps. The quality of total RNA was extracted by agarose gel electrophoresis. Reverse transcription- polymerase chain reaction (RT-PCR) and rapid amplification of cDNA ends (RACE) were used to clone the full-length cDNA of OlCtpE from gut tissue in medaka. According to GenBank, the CtpE gene and corresponding protein sequence of Fundulus heteroclitus, Poecilia formosa, Austrofundulus limnaeus and Larimichthys crocea were downloaded and then analyzed through the global Clustal X alignment. The conserved region of the medaka OlCtpE gene fragment was amplified using the degenerate primers PF1 and PR1. The 3' RACE specific primers PE2 and PE3 were designed according to the conserved sequence, which was amplified by degenerate primers PF1 and PR1 using DNAStar software. The 5' RACE specific primers PE4, PE5 and PE6 were designed according to the conserved sequence, which was also amplified by degenerate primers PF1 and PR1 using DNAStar software. The RT-PCR product sequence, the 3' RACE and 5' RACE product sequences were assembled by using DNAman software. Clustal X 1.81 and MEGA 4.0 softwares were used to analyze the amino acid homology. The basic physical and chemical properties of proteins were predicted by ExPASy- PROSITE and ExPASy- ProtParam. The signal peptide and the glycosylation sites were predicted by SignalP 4.1 and NetNGlyc 1.0, respectively. The tertiary structure of OlCtpE protein was predicted by homology modeling method using SWISS-MODEL software. The results showed that the full-length cDNA of the OlCtpE was 1 301 bp, containing 24 bp 5'-untranslated regions (UTR), 56 bp 3'-UTR and 1 221 bp open reading frame, presumably encoding 406 amino acids. The cloned cDNA sequence of OlCtpE gene has been submitted to the GenBank database (accession number: KP864679). The N-terminus of OlCtpE protein contained a signal peptide of 17 amino acids, and it was a secretory protein. The OlCtpE protein contained three N- linked glycosylation sites,“NPTI”(amino acids 26-29),“NFSV”(amino acids 95-98) and“NLTV”(amino acids 162-165). The sequence homology from medaka CtpE protein was 77% with those of F. heteroclitus and A. limnaeus, 74% with that of P. latipinna, and 71% with that of L. crocea. A total of 22 CtpE proteins in different fish species had the active sites of conserved aspartate protease, which were“VIFDTGSSDLWV”(amino acids 98-109) and“AIVDTGTSLIAG”(amino acids 284-295) by homology analysis. The amino acid sequence homology was 43.60% between the CtpE protein from medaka and the porcine pepsinogen, and the two tertiary structures were also very similar. The tertiary structural analysis showed that the substrate was fixed to the active site in the active center, and the hairpin of the active site could provide space for the combination of substrate and enzyme. The overall shape of CtpE protein from medaka and porcine pepsinogen showed ellipsoid, and formed a relatively independent space entity. In conclusion, OlCtpE might play a very important role in the immune processing and presentation of exogenous antigen. Cloning and function analysis of the OlCtpE provide essential evidence for further studies on immune gene function.

To investigate whether RIXI overexpressing transgenic plants influence the expression of other genes, we analyzed transcriptomic changes between R7 (RIXI overexpression transgenic line 7) plants and WT (wild-type) plants using deep RNA sequencing combined with digital gene expression profile analysis. The differentially expressed genes between the WT and R7 libraries were identified by DEG-Seq (differentially expressed genes from RNA-seq) software. The profiling analysis revealed that the overexpression of RIXI in rice resulted in numerous changes in gene expressions, including upregulation of 391 genes and downregulation of 905 genes. These differentially expressed genes were categorized into 30 groups with broad functions using gene ontology (GO) assignments. Among the 30 groups, five groups (singleorganism metabolic process, biological regulation, anion binding, small molecule binding and nucleotide binding） had more differentially expressed genes. Biological pathways affected by RIXI overexpression were mapped using the detected genes to reference canonical pathways in the Kyoto Encyclopedia of Genes and Genomes (KEGG). The differentially expressed genes were assigned to 98 KEGG pathways, and four enriched pathways were identified: metabolic, biosynthesis of secondary metabolites, plantpathogen interaction and plant hormone signal transduction. The measurement of agronomic traits of R7 showed that the overexpression of RIXI did not influence the growth and development of rice. Thus, we conclude that the xylanase inhibitor gene RIXI may play a role in activation of a complex signal transduction network in response to various biotic and abiotic stresses, but does not have a negative influence on growth and development of rice plants.

Microbial fuel cell (MFC) is an economic and effective way for wastewater treatment, which enables not only degradation of phenol but also conversion of biomass energy into electricity. Selection and breeding of electricigens from anode of a microbial fuel cell is the premise and foundation of MFC research; meanwhile, the problem of low energy efficiency can also be solved. Electronic delivery mechanisms of electricigens included biofilm mechanism and electron shuttle mechanism. Biofilm mechanism refers to the electricigens being attached to the electrode surface and then use cytochrome C or “nanowires” to transfer intracellular electrons to the electrode through the biofilm. Electronic shuttle mechanism concerns the use of a redox mediator to transfer electrons between the cell and the electrode. Currently, most Gram-negative bacteria with cell walls rich in cytochrome C, have been found to use cytochrome C to transfer electrons, such as Geobacter sulfurreducens, Aeromonas hydrophila and Rhodoferax ferrireducens, etc. In the process of electron transfer, the use of redox mediator for the electron transfer between the cell and the electrode is called electron shuttle mechanism. According to the source of redox mediator, it can be divided into exogenous redox mediator and endogenous redox mediator (cell autocrine). So far, little was known about the potential of Bacillus cereus to produce electricity. 
In this work, an efficient phenoldegrading electricigenic bacterium was separated and screened, and its MFC was built using the obtained strain, and the efficiencies of phenol degradation and electricity production were further investigated. Meanwhile, the anode carbon felt was analyzed by scanning electron microscopy, and the cyclic voltammetry curve of the obtained strain was measured during the four growth stages (7, 18, 31 and 52 h), to explore the potential related mechanism of electricity production.
Twentyone pure strains with potential ability of electricity production were isolated, of which WL013, WL024 and WL027 strains could produce electricity, and the WL027 was the favorite electricityproducing strain. Hence, the strain WL027 was selected as the dominant strain. Based on the combination results of 16S rDNA and physiological and biochemical characteristics, the strain WL027 was identified as Bacillus cereus. This strain WL027 was electrochemically active, and its electricity was mainly generated at the stable phase during the growth of the strain. When the strain WL027 was inoculated into the MFC, the maximum voltage increased by 179 mV compared with the start voltage, with Coulombic efficiency of 64.25% and phenol degradation rate of 68.62%. The intracellular and extracellular concentrations of riboflavin were 6.10×10^-3 and 1.32×10^-2 mg/L respectively during the process of electricity production. The voltage was increased by 18 mV when the riboflavin was added to the MFC at the stable phase. Therefore, it can be speculated that the strain WL027 could promote the electron transport through the secretion of riboflavin in microorganisms.
In conclusion, the MFC constructed by Bacillus cereus not only can degrade phenol efficiently, but also has obvious advantages in energy conversion efficiency. Besides, the strain WL027 can promote the electron transport through the secretion of riboflavin in microorganisms.

With the development of modern industries, drinking water in residential area and public building were supplied by water distribution systems. The drinking water distribution system is the final step from water plants to the users, and therefore is essential to assure drinking water safety for customers. Previous studies confirmed that many bacteria in the drinking water distribution systems existed as biofilm, which could induce corrosion and scaling of the pipe walls, reduce the water quality and water-carrying capacity of the pipelines. Moreover, as the pathogens growing in the biofilms transferred in the pipelines, the risks caused by these microorganisms would increase. Vibrio species is an estuarine bacterium widely distributed in the natural aquatic environment around the world. Among the discovered 76 kinds of Vibrio species, at least 12 kinds have pathogenic effect on human, and often result in severe diarrhea and dehydration. We found the Vibrio species in our previous survey of pathogenic bacteria in biofilm, indicating that the residents were at potential risk from Vibrio species. Therefore, the effect of pipe materials, pipe ages, and pipe diameters on amount of Vibrio species in biofilm of drinking water distribution system was necessary to investigate. 
In this study, 12 biofilm samples were collected from the drinking water distribution system in east China. Vibrio was isolated using thiosulfate-citrate-bile salts-sucrose (TCBS) agar medium and was identified according to their biochemical reaction characteristics using biochemical identification kit for Vibrionaceae GYZ9V. The amounts of total bacteria were determined by a spreadplating method according to the Ministry of Health Standard Examination Methods for Drinking Water (2006). Samples and their dilutions were spread on Petri dishes with nutrient agar medium. Analysis of variance (ANOVA) by a least significant difference test was used to compare Vibrio counts from different pipe materials, ages and diameters.
The results showed that Vibrio species could be detected in nodular cast iron, galvanized steel and stainless steel clad pipes, but not in plastic pipe. The Vibrio species amount on the biofilm attached to the nodular cast iron pipe (212±39.40 CFU/cm²) was significantly higher than those attached to the galvanized steel pipe (4.85±1.03 CFU/cm²) and stainless steel clad pipe (0.66±0.21 CFU/cm²). However, the rank of total bacteria amount was galvanized steel pipe > nodular cast iron pipe ≈ plastic pipe > stainless steel clad pipe. Pipe age had little influence on Vibrio amounts from galvanized steel and nodular cast iron pipes. Nodular cast iron pipe with a nominal diameter of 200 mm had the highest amount of Vibrio species (2.65×10³±204 CFU/cm²), followed by 150 mm (212±39.40 CFU/cm²) and 300 mm (44.20±6.88 CFU/cm²), and the tendency of total bacteria amount was similar with Vibrio species. 
In conclusion, contamination from Vibrio species with varying levels is observed in nodular cast iron, galvanized steel and stainless steel clad pipes, but not in plastic pipe. The different pipe materials, especially nodular cart iron pipe, have strong influence on Vibrio species amount in biofilm from drinking water distribution system; therefore, the potential risks of Vibrio species from nodular cast iron pipe should be taken into consideration while laying the pipelines.

China is the largest cotton producing country in the world. Zhejiang Province owned cotton growing area of about 17 264 hectares in 2013, was not major cotton producing area in China. Verticillium wilt, caused by Verticillium dahliae, is one of the most important disease of cotton and causes great economic losses in almost all cotton producing areas, but has not been found in fields in Zhejiang region. However, in 2014, the serious verticillium wilt disease of cotton was observed in the experimental field of Zhejiang University. The disease began to appear in the early June and occurred seriously in the early July. The disease caused the serious defoliation on the middle and lower parts of plants and the diseased plants were significantly dwarf. However, the disease became to remit gradually in the early August during high temperature in summer and dwarf phenomenon of the diseased plants was not obvious with new leaf formation. Since then, the diseased plants were still able to bloom and produce bolls. The symptom of the disease was similar to that caused by the defoliating pathotype strains of V. dahliae but in the later period, not identical to that. 
Based on the importance of verifying pathotype of pathogen and understanding the phenomenon of high temperature inhibiting disease for disease control, we detected the isolates causing the verticillium wilt disease of cotton by using the specific primers of defoliation and nondefoliation pathotypes of V. dahliae and characteristics of microsclerotia. Meanwhile, the effect of temperature on growth and development of pathogen was determined and the reason for the phenomenon of high temperature inhibiting disease was analyzed as well. 
Results of polymerase chain reaction (PCR) amplification using the specific primers showed that the isolates (VD-h₁, VD-h₂, VD-h₆, VD-h₃ and VD-h₅) obtained all belonged to the defoliating pathotype. The results of observation for characters of microsclerotia formation confirmed that the difference of microsclerotia length-to-width ratio between defoliating and nondefoliating pathotype isolates was remarkable. Temperature had a significant influence on Verticillium isolates. The optimal growth temperature of two isolates (VD-h₁ and VD-h₆) from Zhejiang was 22-28 ℃ and two (VD-101 and VD-086) from Xinjiang was 22-26 ℃, whereas at 32 ℃, fungal hyphal growth was all inhibited. At the same time, the numbers of produced conidial decreased and conidial germination was delayed. Combining biological properties of fungus and meteorological data, research results indirectly verified that the reason for disappearance of disease symptoms was that the growth and reproduction of pathogen were inhibited under the condition of high temperature in summer. 
In sum, this study provides important scientific data for studying the regulation of disease occurrence in future.

Peony (Paeonia suffruticosa) is one of the most famous traditional flowers with profound cultural connotation in China. With the fast development of gardenflower industry, the application of peony increased dramatically for the purpose of landscaping or medicinal uses. However, stress resistance of peony has been poor due to artificial domestication for thousands of years, which greatly restricted the development of peony. With the significant interest of adding new vitality into the flower industry by successfully introducing and cultivating peony in southern China, improving the tolerance of peony cultivars to stress has become crucial. The dehydration responsive element binding protein (DREB) gene is one of the most important transcription factors that was found to be related to adversity resistance. The objective of this study was to analyze the biological function of DREB gene from peony, named as PsDREB, with methods of yeast one-hybrid assay and transgenic technology. 
In this study, the ability of PsDREB protein binding with DRE and its transcriptional activation activity were tested by yeast onehybrid assay. Results showed that the PsDREB protein was able to combine specifically with the DRE, but unable to combine with the mutated DRE (mDRE), showing a strong transcriptional activity. Subsequently, the PsDREB gene was transferred into tobacco and was mediated by Agrobacterium to determine its biological function in response to adversity. Thereafter, the tolerance ability of the transgenic tobacco was detected under manipulated drought, low temperature, high salt, and abscisic acid (ABA) treatments, as compared to controls.
In conclusion, the PsDREB protein has strong binding and transcriptional activities. The overexpression of PsDREB can obviously enhance plant resistance to abiotic stresses, especially for drought and high salt treatments. These results lay the foundation for further research on the function mode of PsDREB transcription factor, and also supply theoretical and material evidence for excavating more possible stressresistant genes from peony so as to breed out highly stress-tolerant transgenic peony.

Within the genus Berberis L., B. amurensis Rupr. and B. anhweiensis Ahrendt are easily confused in the main taxonomic characters, and the demarcation between these two species usually depends on the site information of their living plants or specimens. 
In order to further reveal their interspecific relationship, the leaf morphological and micromorphological features of B. amurensis and B. anhweiensis were studied by means of field surveys and experimental observation, and their geographical distribution patterns were also mapped and analyzed based on specimen check and literature analysis. 
The results showed that, although the leaf shape of both B. amurensis and B. anhweiensis varied greatly, continuous variation patterns still existed in both species, and obvious similarities were found between the two species in features of leaf shape, dense spinescent teeth on the leaf margin, and the reticulate venation.
Light microscopy observation showed that, the outline of the periclinal walls of the leaf adaxial epidermal cells was approximately tetragonal to hexagonal in both B. amurensis and B. anhweiensis , but the anticlinal walls of the leaf adaxial epidermal cells were undulate or approximately straight in B. amurensis, and nearly straight or slightly undulate in B. anhweiensis. The outline of the periclinal walls of the leaf abaxial epidermal cells was irregularly polygonal in B. amurensis, and its anticlinal walls were undulate. The outline of the periclinal walls of the leaf abaxial epidermal cells was regularly polygonal in B. anhweiensis, and its anticlinal walls were nearly straight or slightly undulate. Both the two species were hypostomatic, and their stomata were all actinocytic and anomocytic.
Scanning electron microscope observation revealed that, the leaf adaxial epidermis of B. amurensis was nearly smooth, or was covered with dense granular wax ornamentation, and the outline of the periclinal walls of the epidermal cells was irregularly polygonal, and the anticlinal walls were undulate; the leaf abaxial epidermis was nearly smooth, or was covered with dense granular wax ornamentation and remarkable bifurcate ridges. The leaf adaxial epidermis of B. anhweiensis was covered with dense fine granular wax ornamentation, and the outline of the periclinal walls of the epidermal cells was irregularly polygonal, and the anticlinal walls were undulate or approximately straight; the leaf abaxial epidermis was covered with dense granular or short filiform wax ornamentation and notable bifurcate ridges. The outline of the periclinal walls and the anticlinal walls of the leaf abaxial epidermal cells were all obscure in both B. amurensis and B. anhweiensis, and the inner margin of the outer stomatal ledges was nearly smooth or partially notched.
There existed no obvious disjunctive geographical distribution between B. amurensis and B. anhweiensis in China, and the populations of B. amurensis might have extended its distribution from North China, via Qinling Mountains and Funiu Mountains, southward to Dabie Mountains, located across Central and East China, and spread closely to the distribution areas of B. anhweiensis in East China. 
Berberis anhweiensis was reported by AHRENDT in 1961, although AHRENDT considered that this species resembled B. chekiangensis Ahrendt; in fact, B. anhweiensis was much more similar to B. amurensis. Because of the considerable similarities between B. amurensis and B. anhweiensis in the morphological and micromorphological characteristics of leaves, together with the notable resemblances in other key taxonomic characters

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Potato virus Y (PVY) is one of the most important potato viruses causing serious economic loss in potato industry worldwide. Establishment of sensitive and specific virus detection technique is the key to prevent and control PVY. 　　In order to provide material and technical support for the diagnosis, scientific prevention and control of PVY in fields, four monoclonal antibodies (MAbs) against PVY were prepared with the hybridoma technique, and four serological assays were developed in this study. 　　PVY particles with 730 nm×11 nm were obtained from PVY^N-infected tobacco plants with a high yield purification method, and used as the immunogen. Four hybridoma cell lines (3B2, 3E4, 20B2 and 25C2) secreting sensitive and specific MAbs against PVY were prepared. Titers of the four MAbs in ascetic fluids secreted by prepared hybridoma cells ranged 10^-6 to 10^-7 by an indirect-ELISA. Western blot analyses indicated that three MAbs (3E4, 3B2 and 20B2) could specifically react with 30 ku protein, which was supposed to be the coat protein of PVY based on the size of the protein. The MAb 25C2 could react with an approximately 55 ku protein, which was supposed to be HC-Pro based on the molecular mass of the protein. Based on the prepared MAbs, four serological assays, ACP-ELISA, dot-ELISA, tissue blot-ELISA and immunocapture-reverse transcription-polymerase chain reaction (IC-RT-PCR) were developed for detecting PVY in plants. Specificity analyses of the four developed serological assays demonstrated that all assays could accurately detect virus in PVY-infected plant tissues, and have negative detection results with the leaf tissue crude extracts from PVS, PVA, PVX, PLRV-infected plant tissues or healthy potato and tobacco plant tissues. The sensitivity analyses indicated that the developed ACP-ELISA, dot-ELISA could specifically detect virus in PVY-infected plant tissue crude extracts diluted at 1∶81 920 and 1∶10 240 (g/mL), respectively. The developed IC-RT-PCR was the most sensitive, and could successfully detect virus in PVY-infected plant tissue crude extracts diluted at 1∶5 242 880 (g/mL).Thirty potato field samples from Yunnan Province were detected for the presence of PVY by the developed serological assays. The detection results of serological assays were the same as the result of RT-PCR, and 20 of the 30 potato field samples were PVY-positive. Sequencing and sequence comparative analysis of PCR products proved that the positive samples detected by the serological assays were really infected by PVY. 　　The anti-PVY MAbs and the developed serological detection assays will provide material and technology for detecting and quarantining PVY, breeding virus-free seed potatoes and establishing scientific prevention and control system of the disease.

Chinese kale (Brassica alboglabra Bailey) is an original Chinese vegetable belonging to the Brassicaceae family of cruciferae. It is commonly grown for its bolting stems and tender rosette leaves as the edible parts, which are tender and crisp. Chinese kale contains large amounts of health-promoting compounds, such as vitamin C, carotenoids, total phenolics, and glucosinolates. It is widely distributed in Guangdong, Fujian and Guangxi Provinces. 　　The typical yellow-flower Chinese kale cultivar “Fuzhou-Huanghua” was used as the plant material, the chromosome staining factors, including sampling time point, pretreatment and dissociation duration, were optimized. Karyotype characteristics were also investigated, and then the obtained results were compared with those from the reported white-flower Chinese kale so as to reveal the karyotype differences. 　　The seeds of “Fuzhou-Huanghua” were soaked for 2 hours, and then cultured in dark in petri dishes with moist filter paper at 25 ℃ for 3 days. The root tips were excised between 8:00—10:00 AM, with 30 min intervals to determine the optimum sampling time point; pretreatment in 0.002 mol/L 8-hydroxyquinoline were conducted for 1—10 h (1, 2.5, 5, 7.5 and 10 h, respectively), subsequently, the root tips were macerated in 1 mol/L hydrochloric acid at 60 ℃ for 1—16 min (1, 4, 8, 12 and 16 min， respectively). Thirty cells with clear metaphase chromosomes were selected, and then the numbers and karyotype analysis of chromosomes were done by the cytological standards. 　　The results showed that the percentage of the mitotic cells and metaphase cells reached maximum when sampled at 9:00 AM; the best contraction, shape and dispersion of chromosomes were found at 5-h pretreatment with 0.002 mol/L 8-hydroxyquinoline; the best cell dispersion, chromosome staining and cytoplasm transparency, as well as the highest contrast ratio were observed at 8-min dissociation. Yellow-flower Chinese kale has a total of 18 chromosomes. The chromosome types included large chromosomes (L), middle chromosomes 2 (M₂), middle chromosomes 1 (M₁), and the constitution of the relative length was 2L+2M₂+14M₁. The centromeric index ranged from 27.86% to 48.19%, and the arm ratio ranked from 1.08 to 2.59. There were four pairs (the first, forth, sixth and ninth chromosome) of submetacentric chromosomes (sm) and five pairs (number two, three, five, seven and eight chromosome) of metacentric chromosomes (m), while the two satellites (SAT) were observed at the sixth pair of chromosomes. The karyotype formula was 2n=2x=18=10m+8sm (2SAT). Karyotype asymmetry index (As.K) was 61.95%, and karyotype characteristics fell into type 2A according to STEBBINS classification criteria. Meanwhile, significant differences in karyotype formula, ratio of chromosome length (L/S), and range of chromosome relative length were observed when comparing the chromosome characteristics between yellow-flower and the reported white-flower Chinese kale. Specifically, compared with the above results, four m and five sm chromosomes were reported in six white-flower cultivars. Moreover, the ratio of chromosome length (L/S) in yellow-flower Chinese kale (1.48) was lower than that in white-flower Chinese kale cultivars (1.50—1.71), which is mainly due to the fact that the shortest chromosome relative length in yellow-flower Chinese kale (9.66%) was obviously higher than that in white-flower ones. However, no significant difference was observed in arm ratio range, As. K or satellite position between the two types. 　　In conclusion,the optimized chromosome preparation conditions and karyotype analysis of yellow-flower Chinese kale were revealed. These results will enrich the awareness of genetic composition of Chinese kale, and provide cytological evidence for the study on the taxonomic status and phylogenetic relationship of these taxa.

Alternaria brown spot (ABS), an emerging disease in China, is caused by Alternaria alternate pv. citri (A. alternata pathotype tangerine). The disease mainly affects tangerine and tangerine hybrid citrus, causing young leaf drop, fruit drop and dieback, and resulting in yield loss and affecting the commodity of fruits. ABS was first reported in Australia in 1962 and has become a serious problem in many citrus producing areas in South America, Florida, South Africa, Spain, Israel, Turkey and other countries in the Mediterranean area. ABS was first reported in China in 2010, affecting many important citrus varieties grown in Chongqing, Zhejiang, Yunnan, Hunan, Guangdong, Guangxi and Sichuan and causing significant yield losses. ABS can be controlled by fungicides; however, there is no existing fungicide that is currently registered for controlling ABS on citrus in China. 　　In order to identify effective fungicides for controlling ABS, an in vitro sensitivity assay was conducted. In total, 54 isolates of A. alternate pv. citri were single-spore collected from citrus-producing areas in Chongqing, Yunnan, Hunan, Zhejiang, Guangdong and Guangxi Provinces and were tested for sensitivity to fungicides, including boscalid, flutolanil, thifluzamide and fluazinam using a rapid Resazurin-based microtiterplate assay. Conidia were induced by growing fungal strains on V₈ medium at 25 ℃ with 16 h light/8 h dark cycle for 7 to 10 days. The concentration for 50% of maximal effect (EC₅0) of spore germination and mycelium was established for each isolate. All test isolates were sensitive to tested fungicides in varying degrees. In general, all isolates were highly sensitive to flutolanil, thifluzamide and fluazinam with an average EC₅0 of 0.010 3 μg/mL, 0.061 9 μg/mL and 0.010 0 μg/mL, respectively. When tested with boscalid, isolates had EC₅0 ranging from 0.238 3 μg/mL (C₁4 isolate collected from Chongqing) to 0.858 5 μg/mL (Z₁1 from Zhejiang) with an average of 0.467 7 μg/mL. It was observed that fungal strains collected from Guangdong and Zhejiang were less sensitive to boscalid than other test isolates and strains collected from Chongqing were more sensitive to thifluzamide than others. However, there was no correlation between the sensitivity to flutolanil and fluazinam and geographical origin of the isolates. 　　Overall, our studies revealed that boscalid, flutolanil, thifluzamide and fluazinam were effective for suppressing A. alternate pv. citri in vitro. It will be imperative to conduct field experiments for controlling ABS. The EC₅0values obtained from this study could be used as baselines for monitoring the development of fungicide-resistant subpopulation in future.

Connectivity map (CMap) technology was initially developed in Broad Institute of Massachusetts Institute of Technology and Harvard. It has been widely used to investigate mechanism of action of drug by academic research institutes and pharmaceutical companies globally. CMap technology, as one of the most important techniques in genomic era, provides a large public database of gene expression profiles and reveals how drugs affect gene expression under disease conditions. CMap database contained 1 309 FDA-approved small molecules and these small molecules were tested in five human cell lines to generate over 7 000 gene expression profiles. It also provided an interactive website and an online tool to conduct CMap queries against the chemical reference catalogues (http://www.broadinstitute.org/cmap). 　　Application of CMap technology in research on traditional Chinese medicines (TCMs) offers new avenues for understanding TCMs in terms of efficacy, dosage and safety. Since a prescribed TCM have complicated composition, it may exert therapeutic effects by a collective and interactive manner of the contained variety of compounds. Additionally, TCM often clashes with the knowledge of contemporary medicine, because its principles and concepts are difficult to be understood by scientists with background in contemporary medicine. For these reasons, using the usual “drug-target” measurement technology is difficult for comprehensive analysis of the mechanism of action of TCMs, resulting in less competitive of TCMs in international market. CMap technology is a gene expression signature-based approach and of a unique power for studying drugs with complex ingredients such as TCMs. By querying CMap database with a gene expression profile of TCM and comparing its similarity with CMap compounds, a list of ranked drugs can be obtained, which may provide clues for connections between the CMap drugs and TCM for sharing mechanism of action, chemical and physiological processes, and therapeutic potentials for particular diseases. In this article, we reviewed the advances in our understanding of application of the CMap technology for research on TCM, in respect to the mechanism of action, the drug repositioning and identification of active ingredients of TCM. In terms of the mechanism of TCMs, CMap technology is of potentials for predicting the efficacy and side effects of known TCMs and provides new ideas for further understanding of the biological pathways influenced under the treatment of TCMs. For application in drug repositioning, as CMap database achieves numerous existing drugs and their associated gene expression spectrums, it is possible to find new indications for TCMs by matching its gene expression profile to those in CMap database. Meanwhile, by comparing with the traditional thin-layer chromatography (TLC) and high performance liquid chromatography (HPLC), CMap technology can be very useful for identifying active ingredients of TCMs. 　　Up to date, CMap technology has been successfully used in several studies on TCMs. However, in respect to TCM research, there are limitations of the current CMap technology, in particular the limited number of CMap compounds available in the database. Thus, this genomic approach can be integrated with traditional chromatography-based fingerprinting method for better understanding of TCMs. Furthermore, it is important to establish CMap technology as the core platform for TCM research, including discovery of new TCMs and exploration of the new application of TCMs.

During the investigation of coastal plants in Ningbo, several species were discovered, which cannot be classified into any species previously reported in the flora of Zhejiang. Based on further comparison and information review, these species belonging to six genera in six families were identified as new records of Zhejiang Province, including Ophioglossum reticulatum Linn. (Ophioglossaceae), Gomphrena celosioides Mart. (Amaranthaceae), Nuphar sinensis Hand.Mazz. (Nymphaeaceae), Gratiola japonica Miq. (Scrophulariaceae), Gynostemma laxum (Wallich) Cogniaux (Cucurbitaceae) and Lycoris albiflora Koidz. (Amaryllidaceae). Gratiola Linn. is a new record genus of Zhejiang Province.
The discovery of these newly recorded plants enriched the content of flora of Zhejiang Province and provided fundamental materials for investigating their geographical distributions in China.

Nicotinamide adenine dinucleotide phosphate (NADPH)cytochrome P450 reductase (CPR) is an electron supplier for various cytochrome P450 monooxygenases. Most P450-mediated catalytic reactions in insects require involvement of CPR, such as detoxification of insecticides and plant secondary metabolites. So far, CPR genes have been identified and characterized from many model and non-model insect species. Since insect P450 system is one of the most important metabolic adaptive traits involved in the degradation of xenobiotics and regulation of endogenous substrates. CPR, as the indispensable electron donor of P450 system, has attracted increasing attention as a potential candidate to develop novel chemical inhibitor to manage target insect pests. Rice planthoppers, such as Nilaparvata lugens, Laodelphax striatellus and Sogatella furcifera, are considered to be the most serious pests of rice. Previously, some studies on L. striatellus and N. lugens found that silencing the CPR gene by RNAi technology could increase their sensitivity to insecticides, but little was known about S. furcifera. This work firstly reports the identification of CPR gene in S. furcifera and up-regulation of the CPR transcription by chemical insecticides. The research will facilitate further study on the function of CPR in S. furcifera.
In this study, a fulllength cDNA encoding CPR was cloned from S. furcifera. The phylogenetic relationships of SfCPR with other insect CPRs were estimated by neighbor-joining method, and its distribution in various tissues and different developmental stages were analyzed by realtime quantitative polymerase chain reaction (qPCR). Finally, after exposure of deltamethrin, buprofezin and imidacloprid at sublethal concentrations for 6, 12 and 24 h, the relative expression levels of SfCPR in the thirdinstar nymphs were investigated.
By searching the transcriptome data sets of S. furcifera, a cDNA fragment encoding putative CPR (named SfCPR) was identified. This cDNA fragment was then amplified by PCR and sequenced in order to confirm that the sequence was not chimeric. The SfCPR cDNA contained a complete open reading frame (ORF) of 2 034 bp nucleotides, encoding a protein of 677 amino acid residues. The theoretical isoelectric point (pI) and calculated molecular mass of SfCPR protein are 5.48 and 76.762 ku, respectively. The secondary structure of SfCPR protein showed the marked features of typical CPRs, such as N-terminal transmembrane region, FMN-, FAD- and NADPH-binding domains and conserved catalytic residues. In addition, a transmembrane anchor was predicted in the N-terminus of the protein, indicating that SfCPR is an endoplasmic reticulumlocated protein. Phylogenic analysis was used to gain insight into the phylogenetic relationships among CPR proteins from diverse insect species. We found that SfCPR and CPRs from other two planthoppers were clustered together. Realtime quantitative PCR showed that the expression of SfCPR was detectable in all developmental stages and the level in adults was the highest. The SfCPR transcripts were also expressed in all the tested tissues of the adults, and most were strongly expressed in the abdomen. The exposure at sublethal concentrations of deltamethrin, buprofezin and imidacloprid led to significantly elevated expression of SfCPR. The expression of SfCPR was activated soon (6 h) after treatment with buprofezin and imidacloprid, while the response of SfCPR expression to deltamethrin was relatively slow (12 h).
In conclusion, this work is the first report about the cDNA sequence information, secondary structure and transcription profiles of CPR gene in the S. furcifera. These findings provide foundation for further research on the physiological function of SfCPR.