L-phenylalanine, as an essential amino acid for human nutrition, is widely used in pharmaceutical and food industries. Using phenylalanine ammonia-lyase (PAL,EC 4.3.1.5) to produce L-phenylalanine is one of the major routes. However, most commercial enzymes are extracted from Rhodotorula glutinis, which is time-consuming and over-priced. Therefore, how to efficiently construct the genetic engineering strain to produce PAL is the hot topic. Pichia pastoris is popular in expressing heterologous proteins due to the advantages of low nutritional demands, excellent genetic stability and high-density fermentation. Inserting the heterologous gene into pPIC9K vector to achieve secreted expression in P. pastoris has been reported. However, unlike other vectors, pPIC9K has few desirable restriction enzyme cutting sites, which reduces vector construction efficiency when the classical method of digestion and then ligation is adopted. Under this condition, an efficient cloning strategy, independent of digestion and ligation, is required. Homologous recombination in vitro between pPIC9K and gene can settle this problem. Now, we intend to employ homologous recombination in vitro cloning method to insert the PAL gene into pPIC9K vector to obtain secreted expression in P. pastoris in order to lay the basis for industrial fermentation.
First, total RNA extracted from Astragalus membranaceus was used as template for isolating cDNA. Open reading frame (ORF) of PAL gene was amplified by PCR from cDNA with a pair of primers designed according to the sequence of PAL gene published in the GenBank. Then, ORF was cloned into vector pUCm-T. The transformant was selected to sequence for further analysis of the PAL gene sequence with the help of bioinformatics tools. After that, pPIC9K-PAL was constructed by homologous recombination in vitro. Similarly, the transformant was selected to sequence to investigate the base mutation caused by PCR. Linearized pPIC9K-PAL by SalⅠ was transformed into P. pastoris GS115 by electroporation. Positive strains were screened on MD and then YPD-G418 plates. The strain resistant to 2.0 mg/mL G418 was selected to express induced by 1% methanol every 24 hours. Supernatant was collected for expression analysis at 0, 12, 24, 48, 72, 96, 120 and 144 h. The recombined PAL protein was purified by Q sepharose fast flow chromatography. Finally, protein concentration was measured with Bradford method and enzyme activity was analysed by measuring the absorption of resultant trans-cinnamic acid at 290 nm.
After PAL was subcloned into pUCm-T, the result of sequencing indicated that about 2 200 bp ORF sequence of PAL gene was cloned from A. membranaceus. With the help of bioinformatics methods, it was predicted that PAL encoded a protein which was about 78 ku in molecular mass and 6.04 in pI, containing 718 amino acid residues. Amino acid sequence alignment revealed that the PAL shared 99% identity with PAL from A. membranaceus published in the NCBI. After homologous recombination in vitro between pPIC9K and PAL, the two evidences that about 900 bp fragment which was coincident with the expectation was obtained from the recombinant plasmid pPIC9K-PAL by EcoRⅠ double digestion and that furthering analysis of sequencing both suggested that PAL was successfully constructed into pPIC9K without any mutation compared with the first sequencing. After linearized pPIC9K-PAL was transformed into P. pastoris GS115, colony PCR indicated PAL gene was integrated into the yeast chromosome in contrast to the negative control. The strain resistant to 2.0 mg/mL G418 was selected to express. SDS-PAGE demonstrated that a sharp 78 ku protein which was equal to the predicted value was expressed in the supernatant in contrast to the parent pPIC9K. By Q sepharose fast flow chromatography, the protein was well purified. The concentration of purified protein was 0.08 mg/mL, accounting for 11.54% of total proteins and the speci

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Selenium (Se) was an important, biologically necessary trace element, which played an important biological roles in avian growth, reproduction, immune function and disease resistance. The biological significance of Se was attributed to its occurrence in selenoproteins in the form of selenocysteine (Sec) with a 21 amino acid genetic code. Se uses the stop codon UGA as the coding codon encoding Sec. Selenoproteins involved in many life processes such as antioxidant defense, cell signal transduction, metabolic pathway, development, immune function, and hormone regulates, and it was related to the occurrence and mechanism of many diseases. Selenoprotein T (SelT) was originally identified through silico studies, cloned and expressed in a mammalian cell line, confirmed as a selenoprotein. Previous studies have proved that SelT mainly occurred in Golgi apparatus and endoplasmic reticulum, with important biological function. SelT can act on the Ca2+ homeostasis, involved in neuroendocrine secretion. SelT has also been showed in alter cell adhesion. Up to now, 25 selenoprotein-encoding genes were identified in birds, however, only about half have been functionally characterized, most of which were involved in redox reactions. SelT was a Se-containing protein whose cellular function has not been characterized. Research on the structure and function of SelT was still in its infancy, but reports in mammals about SelT and the information about avian SelT were not clear. The purpose of this study is to explore the selenocysteine insertion sequence (SECIS) element, structures and functions of chicken SelT and the expression profiles of SelT in chicken tissues.
The chicken SelT sequence was used in the experiment. The SECIS elements of 12 vertebrates were analyzed by the SECISearch 2.19. The homologies of the molecule nucleotide and amino acid sequences on vertebrates were analyzed by DNAStar. The structure and functions of chicken SelT were predicted with the bioinformatics. The distribution of SelT in 30 tissues of 35-day-old chicken was analyzed by fluorescent quantitative real-time PCR (qRT-PCR).
The results showed that SECIS element of vertebrates belonged to the type Ⅱ SECIS element. Compared with chicken and other 11 vertebrates, the nucleotide sequence homologies of SelT were 48.0%85.1%. The homologies of amino acid sequences of chicken SelT with Xenopus laevis or Danio rerio were less than 90.0% and with other nine kinds of animals were among 90.6%94.9%. The chicken SelT was a transmembrane protein and had a signal peptide. It belonged to the RDx families and its enzyme classification was EC 2.5.1.18. SelT had binding sites with Ca2+. The results of fqRT-PCR showed that the SelT was widely expressed in chicken tissues, and the expression level in testis was the highest.
In summary, the SECIS element of vertebrate SelT belongs to the type Ⅱ and SelT is highly conserved in vertebrates. Avian SelT contains a CxxU motif in a thioredoxin-like fold, with glutathione S-transferase activity. SelT has binding sites with Ca2+, it was predicted that avian SelT could regulate the Ca2+ homeostasis. Avian SelT is widely expressed in chicken tissues, and the testis has the highest expression level, suggesting that the chicken SelT has a special function in the male reproductive organs. This study provides a basis for further experimental analysis of the structure-function of avian SelT.

The cotton bollworm, Helicoverpa armigera (Hübner), is a major pest of many agricultural crops around the world, and many of the classes of chemical insecticides are widely used for its control such as organophosphates (OPs), synthetic pyrethroids (SPs), and so on. Currently H.armigera has developed serious resistance to OPs and SPs all over the world. Carboxylesterases (CarEs) are a multi-gene family of enzymes that hydrolyze a diverse range of carboxylesters and are frequently implicated in the resistance of insects. Gene mutations of CarEs are a major mechanism of insects for the development of resistance to OPs in the OP-resistant Diptera pests like Musca domestica and Lucilia cuprina. It involves the substitution of a single amino acid within the active site of the esterase which convert it to an OP hydrolyase. However, no resembled mutation in nature was reported in the Lepidoptera pests like cotton bollworm, H. armigera to date. Hence, following our previous studies the aim of the current work was to elucidate the effects of specific point mutations of the carboxylesterases from H. armigera on the kinetic properties of the enzymes.
Two CarEs, 001F and 001G from H. armigera, were induced to mutate at positions 127 (A→D) or 238 (F→L) using a site-directed mutagenesis technique. They were then expressed with the baculovirus expression vector system (BEVS). The kinetic assays with α-naphthyl acetate (α-NA) and para-nitrophenyl acetate (p-NA) were carried out for all mutants using a spectrophotometer.
The results showed that the A127D mutations had dramatically reduced the hydrolytic activities of the CarEs toward the two substrates. The mutants all showed lower affinities to the substrates as the Km values were at least 1.6-fold higher than those of the wild type enzymes, and the kcat values (6.552.1 s-1) were decreased obviously, which were between 4- and 20-fold lower than those of the wild type enzymes. What’s more, the rate constants (kcat/Km values) of the A127D mutants (0.12 and 1.2 μmol-1·s-1·L) were also substantially declined, which were about between 20- and 90-fold lower than those of the wild type enzymes. By contrast, the F238L mutations had relatively less effects on the kinetic properties of the enzymes than the A127D mutations. The kcat values of these mutants were slightly lower than those of the wild type enzymes with the exception of the 001F F238L against α-NA, and the kcat/Km values were just between 1.5- and 7-fold lower than those of the wild type enzymes. For the double-mutation at both sites, however, they had remarkable effects on the hydrolytic activities toward the substrates. These mutants showed obviously poor affinities to the substrates as the Km values were between 4- and 30-fold higher compared to the the wild type CarEs. The kcat values of these mutants were between 10- and 15-fold lower than those of the wild type CarEs, and they nearly nullified all the hydrolytic activities shown by the very lower rate constants, which were only between 0.09 and 0.33 μmol-1·s-1·L, and between 70- to 370-fold lower than those of the wild type CarEs.
These results indicate that the mutation at positions 127 (A→D) and 238 (F→L) in CarEs has marked effects on their enzymatic activities, and it also strongly suggests that the positions A127 and F238 are two important active sites in enzymes involving in the catalytic function. Hence, this study can provide useful data for further understanding the functions of the CarEs in metabolic resistance of cotton bollworm, H. armigera in future.

Heterosis is a very important genetic phenomenon, and its application can significantly improve yields and enhance resistance in many crops. The use of this important tool, however, is limited in soybean production because: 1) The small size of soybean flowers makes it difficult and expensive to emasculate and hybridize； 2) There are only a limited number of male sterile lines available for hybrid seed production. Therefore, illuminating the mechanisms of male sterile in soybeans will benefit heterosis research of soybean.
Previous studies showed that ethylene biosynthesis in the soybean flower was closely related with male sterility. However, the mechanisms that ethylene induced male sterility of soybean remains unknown. Thus,exogenous ethylene treatment was carried out to study its fertility regulation role in soybean. Scanning electron microscopy (SEM) and palynology methods were used to study the effects of ethylene treatment, and real time quantitative PCR (RT-qPCR) was used to evaluate gene expression.
The results showed that flower numbers and pollen amount were significantly decreased, and abortion ratio of pollen was significantly promoted in the ethylene treatment compared with the control. The results of difference gene expression profile and RT-qPCR of the control and exogenous ethylene treatments indicated that the expression of eight genes which are related to cellular response to starvation, pollen growth and pollen wall assembly was changed significantly in the ethylene treatment compared with the control. Soluble sugar and starch content, activities of superoxide dismutase and peroxidase were all significantly lower while malondialdehyde content was significantly higher in flower tissue after ethephon treatment than the control.
In conclusion, it is confirmed that exogenous ethylene treatment will cause male sterility in soybeans, and the expression of eight genes responsible for pollen development has been significantly changed after ethylene treatment. Carbon starvation and proactive enzyme injury are involved in the process of male sterility formation. The study above provides a theoretical basis for future research on pollen sterility regulated by exogenous ethylene in soybean.

Water and fertilizer are very important for the growth and yield of Coffea arabica. C. arabica is an agricultural product with unique advantage in Yunnan Province, whose planting area and yield account for 90% of the whole country. However, its yield and quality are restricted because of seasonal drought and soil nutrient deficiency.
To investigate the optimal mode of water-saving and high-efficient utilization of water and fertilizer of C. arabica, the coupling effects of water and fertilization on growth and water use of C. arabica were studied by using four irrigation levels, i.e. sufficient irrigation (WS, soil moisture content was 75%85% of soil field capacity), high water (WH, soil moisture content was 65%75% of soil field capacity), middle water (WM, soil moisture content was 55%65% of soil field capacity) and low water (WL, soil moisture content was 45%55% of soil field capacity), and four fertilizer levels of NPK (total nitrogen was 10%, P2O5 was 30% and K2O was 20%), i.e. high fertilizer (FH, 4.5 g/kg dry soil), middle fertilizer (FM, 3.0 g/kg dry soil), low fertilizer (FL, 1.5 g/kg dry soil) and no fertilizer (FN, 0 g/kg dry soil)。 A completely design of pot experiments of C. arabica (Kadimu P796, a local variety) was conducted in intellectual control greenhouse from April to November in 2012, in Faculty of Modern Agricultural Engineering, Kunming University of Science & Technology in Kunming, Yunnan, China (102°79′E, 24°09′N). Irrigation was controlled by weighing method. Experimental soil bulk density was 1.2 g/cm3, field capacity (FC) of 24.3% (mass by mass). The temperature range of intellectual control greenhouse changed between 12 ℃ and 35 ℃ and air humidity range changed between 50% and 85%.
Water-soluble NPK compound fertilizer (total nitrogen was 10%, P2O5 was 30% and K2O was 20%) was applied twice on May 26th and August 26th in 2012. Morphological characteristics (basal diameter, plant height, leaf area, root shoot ratio, root mass ratio and specific leaf area) and biomass accumulation and distribution were measured on November 11th, and daily evapotranspiration was measured on October 14th in 2012, the day before irrigation in vigorous growth period of C. arabica seedling.
Results showed that the growth (plant height, basal diameter and leaf area) and biomass accumulation of C. arabica seedling increased with the increase of irrigation and fertilizer, but the effect of fertilizer was inferior to irrigation. Compared with low water (WL) treatment, irrigation could increase biomass accumulation by 59.03%369.77%, and root shoot ratio and root mass ratio increased with the increase of irrigation slightly. Moderate increase of water and fertilizer could greatly increase biomass accumulation and water use efficiency (WUE) under insufficient water and fertilizer, while marginal benefit significantly decreased while water and fertilizer supply increased under sufficient water and fertilizer. Daily evapotranspiration of C. arabica seedling decreased with the increase of fertilizer slightly, while increased with the increase of irrigation significantly. Compared with low water (WL) treatment, irrigation could increase water use efficiency by 7.39%128.96%.
In conclusion, faster growth and higher WUE could be obtained by high water and middle fertilizer (WHFM, soil moisture content was 65%75% of soil field capacity and fertilizer was 3.0 g/kg dry soil) simultaneously.

Competition between species was a fundamental ecological process, which can proceed by various mechanisms， and allelopathy of plants was strongly coupled with external biotic and abiotic stress factors. As one of the core contents of research to the modern agroforestry system, the allelopathy effects between different plants attracted more and more attention. Some studies were mainly concentrated in crops and weeds, the succession of forest and crops autotoxicity, but the allelopathic effect of Caragana intermedia on growth and distribution of the other plants around them, especially the crops, was still unknown.
Based on the above analysis and improved method, aqueous extracts of C. intermedia leaves and stems were used to test their allelopathic effects on seed germination and seedling growth of four crops (Zea mays, Triticum aestivum, Panicum miliaceum and Fagopyrum esculentum).
The results showed that: the seed germination index and germination rate of Z. mays, T. aestivum, P. miliaceum and F. esculentum were all decreased and their seed germination time were lengthened with the increasing of aqueous extracts of C. intermedia leaves and stems. But the effects of different concentrations of aqueous extracts on four test crop seeds were quite different. In the concentration of 12.5 mg/mL, the seed germination rates of three crops (Z. mays, T. aestivum, P. miliaceum) except F. esculentum had no significant differences (P ＞ 0.05), and the seed germination index of Z. mays decreased most (35.4%), and the mean germination day of T. aestivum seeds was significantly extended by 0.3 days, however, the Z. mays, P. miliaceum and F. esculentum seed germination showed no significant difference from the control (P ＞ 0.05).The aqueous extracts showed a “dose inhibition response” on the shoot growth of test crops, but for the root growth of Z. mays, the low concentrations of aqueous extracts showed no effect while high concentrations showed inhibitive effect. Compared with the shoot growth of F. esculentum, P. miliaceum and T. aestivum, the root growth showed higher sensitivity to aqueous extracts of C. intermedia. The comprehensive sensitivity of the allelopathic effect on the four test crops of aqueous extracts of C. intermedia leaves and stems was in the order of F. esculentum ＞ P. miliaceum ＞ T. aestivum ＞ Z. mays.

Mengding Ganlu produced in Mengshan Mountain of Sichuan Province has been one of the most famous teas and tribute teas in history. Like Mengding Ganlu, Mengshan Maofeng is highquality green tea produced in Mengshan Mountain area. The processing techniques of these two kinds of tea are typical of green tea processing. Historically, these two kinds of tea were produced in Mengquan, Houyan and Longxi villages of Mengshan Mountain, Mingshan County. With the increasing demands of Mengding Ganlu and Mengshan Maofeng, the teas similar to Mengding Ganlu and Mengshan Maofeng produced throughout Ya’an City area are sold as Mengding Ganlu and Mengshan Maofeng nowadays, which infringes the protection of origin place of Mengding Ganlu and Mengshan Maofeng. It is necessary to develop an instrumental technique to identify the authenticity of these two kinds of tea so as to protect consumer rights. Owing to the differences in tea cultivars, environmental conditions and processing methods, chemical compositions of tea show area specific characteristics, among which volatile aroma components are important indicators. The studies on geoaroma characteristics and brand protection of famous teas such as Westlake Longjing tea, Dongting Biluochun tea and Yunnan Puerh tea were reported. However, the study concerning Mengshan teas has not been available so far. The purpose of the present study was set to develop a method to discriminate the volatile aroma compounds of authentic Mengding Ganlu and Mengshan Maofeng produced in the three protected villages including Mengquan, Houyan and Longxi from those produced outside the three protected villages or nonprotected areas such as Hejiang.
Tea volatile aroma components were collected by simultaneous distillation and extraction (SDE) method using 30.00 g dry tea and 400 mL distilled water, with ethyl decanoate as internal standard，exacted at 45 ℃ for 1 h. The extracted volatile aroma components were identified by gas chromatographymass spectrometry (GCMS), based on the spectral library search (NIST147, NIST27 and WILEY7) and published references. The GCMS data of the relative contents of aroma components were expressed as the ratio of the peak area to internal standard (ethyl decanoate).
The tested results showed that there were 64 peaks detected, among which 63 were identified. The main volatile components of Mengding Ganlu and Mengshan Maofeng were alcohols and aldehydes, which accounted for 59.16%71.42% of the identified compounds．Furthermore, Mengding Ganlu and Mengshan Maofeng had high concentrations of phytol, geraniol, Llinalool, nonanal and nheptaldehyde, which could be used to discriminate from famous green teas produced outside the Mengshan Mountains such as Westlake Longjing tea and Dongting Biluochun tea. Mengding Ganlu and Mengshan Maofeng produced in the protected areas had higher concentrations of phytol and nonanal than those from the nonprotected areas. These were considered to be related to the unique natural climatic conditions of Mengshan Mountain and their special processing techniques. Phytol and nonanal had floral and fruity flavor. According to the quantitative evaluation of aroma components and tea aroma classification, the aroma types of Mengding Ganlu and Mengshan Maofeng produced in the central zone of the protected areas showed chestnut flavor, the teas produced in the edge areas showed honey and floral flavor, with fresh lily flower scent in the between.
It is concluded from this study that Mengding Ganlu and Mengshan Maofeng produced in the protected place of origin have higher concentrations of phytol and nonanal than those from nonprotected areas and they can be used to identify the authenticity of Mengding Ganlu and Mengshan Maofeng.

β-mannanases (endo-β-1,4-D-mannanases, EC 3.2.1.78), which exist widely in various organisms especially in microorganisms, can catalyze the cleavage of internal β-1,4-D-mannosidic linkages of mannan backbones. To date, almost all known β-mannanases have been classified into glycoside hydrolase (GH) families 5, 26 and 113 based on their amino acid sequence alignment and hydrophobic cluster analysis. Recently, they have attracted much attention due to their great potential applications in industrial processes, such as bioleaching pulps, depolymerizing anti-nutritional factors in feedstuffs, extracting oils from leguminous seeds, hydrolyzing mannan-based polymers in hydraulic fracturing of oil and gas wells, and producing mannooligosaccharides. However, most of commercial β-mannanases had some shortages in enzymatic properties, such as lower substrate affinity and poorer tolerance to extreme environments, which hindered the development of β-mannanases.
A GH family 5 β-mannanase (AuMan5A) from Aspergillus usamii YL-01-78 was used as the object of this study. The directed modification for its substrate affinity was subjected to the rational design and site-directed mutagenesis to gain a mutant enzyme AuMan5AY111F with higher affinity. Firstly, the three-dimensional (3-D) structure of a docked complex of Auman5A with mannobiose was predicted through homology modeling and molecular docking simulation. On the basis of this 3-D structure, 38 amino acid sites in proximity to mannobiose within 8  were located by using the PyMol software. Secondly, the multiple alignment of various β-mannanase sequences was performed, among which each sequence shared more than 50% identity with AuMan5A. According to the properties of amino acids at 17 non-conserved sites and their locations on the 3-D structure of AuMan5A, Tyr111, Phe206 and Tyr243 were selected to be substituted with the similar amino acids and/or high frequency ones in other β-mannanase sequences, respectively, forming a series of mutant enzymes. Lastly, binding free energies (ΔGbind) of various β-mannanases with mannobiose were calculated by using the molecular mechanics Poisson-Boltzmann surface area (MM-PBSA) method, respectively. The ΔGbind of AuMan5AY111F was -237.7 kJ/mol, lower than those of other enzymes. Based on the rational design, an AuMan5AY111F-encoding gene, Auman5AY111F, was constructed by mutating a Tyr111-encoding codon (TAC) of the Auman5A into a Phe111-encoding TTC by megaprimer PCR. Then, the Auman5AY111F and Auman5A were expressed in Pichia pastoris GS115, and kinetic parameters of the purified recombinant AuMan5AY111F and AuMan5A (reAuMan5AY111F and reAuMan5A) were determined, respectively. The results displayed that the Km value of reAuMan5AY111F, towards guar gum, dropped to 2.5 mg/mL from 3.9 mg/mL of reAuMan5A, indicating the substrate affinity of reAuMan5A increased correspondingly. While, the Vmax value of reAuMan5A kept almost unchanged after site-directed mutagenesis.
The directed modification of AuMan5A based on the rational design for enhancing its substrate affinity was firstly predicted by using various bioinformatics softwares, and then was confirmed by site-directed mutagenesis. This work provides a novel technology strategy for the directed modification of substrate affinities of β-mannanases and other enzymes.

Due to the high toxicity and low bioavailability, petroleum hydrophobic organic compounds (HOCs) have been found to be deposited in the natural environments and to present danger to human beings. A number of factors may affect the mass transfer of HOCs from nonaqueous phase liquids (NAPLs) into aqueous phase, thus affect their bioavailability and toxicity in aqueous phase.
This article reviewed the influence of the fungal species with ability to degrade petroleum HOCs and their physiological activities on the mass transfer of HOCs from NAPLs into aqueous phase, including the influence of fungi on the process of HOCs mass transfer, the metal toxicity changes and the biological effect resulting from mass transferring. This review firstly focused on the effect of fungi participation on the dynamics of mass transfer of the petroleum HOCs to aqueous phase, subsequently analyzed its influence on the metal toxicity and biological effects.
Compared to bacteria, fungi can use a variety of petroleum HOCs due to their hydrophobic mycelium networks, specific physiological biochemical and ecological advantages. In addition, fungi possess ecological capacity, dominating the living biomass in soil and being abundant in aqueous systems. These facts suggest that fungus may affect the mass transfer of petroleum HOCs to aqueous phase through its physiological activities. But biological factors affecting on this mass transfer and its mechanism of action are unknown. Fungi possess high ecological capacity participating in the bioremediation process of the polluted organic chemicals. Furthermore, the abilities of these fungi to form extended mycelial networks, the low specificity of their catabolic enzymes and their independence from using pollutants as growth substrate may make them better suited for bioremediation processes especially in the complicated pollution environments and which may affect the mass transfer of petroleum HOCs to aqueous phase.
These studies will be important for human beings to understand the effect of biological factors on the bioavailability and biological toxicity of petroleum HOCs and heavy metal pollution in ocean water. They also will be of great significance for us to understand the major environmental problems such as oil spills, and to further address the conversion rule of non-biomass HOCs to biomass in nature.

Antibiotics are medical drugs used to kill bacteria and treat infections. But in recent years, more and more antibiotics have been used in animal feed as growth promoters. Manure is used worldwide in agricultural practice as a fertilizer to enhance plant yields. Several studies revealed that manure also had a stimulating effect on the soil microbial biomass and community structure. However, in modern agriculture, the application of manure to field is very often combined with the spread of veterinary antibiotics. Tetracycline and penicillin are possible soil contaminants, since they are able to reach agricultural soils with manure of treated farm animals.
Zhejiang Province is a major tea producing region in China, whose tea exports amount to 100 000 tons and production value accounts for one-third of the country. Every year a large number of organic fertilizers are put into the tea garden and the spread of veterinary antibiotics increases with applying organic fertilizer. The organic fertilizer can increase the yield of tea tree, but the harm to soils caused by antibiotics can not be avoided in the process of using organic fertilizer. Microorganism is a promoter of soil nutrient transformation, which plays an important role in maintaining balance, health and high quality of soil ecosystem. Although it has been confirmed that organic fertilizer (especially manure) application could promote the growth of crops, the impact of antibiotics by applying organic fertilizer on soil microbial community is still unknown. Thus, the aim of this research is to investigate possible toxic effects of antibiotic exposure on the indigenous bacterial community of tea garden.
Topsoils (020 cm) were collected from Meijiawu Tea Village, Hangzhou, Zhejiang Province. Soils were spiked with a range of tetracycline or penicillin concentrations. The basic physicochemical properties of the tested soil were measured by routine analysis methods. The amounts of bacteria, fungi and actinomycetes of different treatments were calculated by plate count method. As we know, only 0.1%10% microbes can be cultivated by this method, therefore we also used the method of phospholipid fatty acid (PLFA) to analyze the PLFAs contents to make a quantitative description of microbial community. After cultivation for three weeks, compared with the control treatment (CK), the amounts of bacteria, fungi and actinomycetes in P5 treatment (100 mg/kg) decreased by 80%, 50% and 50%, respectively. Soil microorganisms showed resistance to antibiotics after incubation for four weeks. The results of phospholipid fatty acid method showed that soil microbial community structure changed obviously under different contents of antibiotics treatments. The ratio of G-/G+ increased after antibiotic treatments. Principal component analysis showed different treatments located in four different quadrants. The content of PLFA 20:0 was rich in T3, T4 and T5 treatments.
It is concluded that exogenous penicillin has certain inhibitory effect to bacteria, fungi and actinomycetes, and the inhibition increases with penicillin concentration. Tetracycline inhibits the growth of bacteria and actinomycetes in tea garden soil, but shows no obvious inhibitory effect against fungi. Inhibition of Gram-negative bacteria for both penicillin and tetracycline are stronger than Gram-positive bacteria. There is great difference in soil microbial community structure among different treatments. Microbes characterized by PLFA 20:0 may be the main groups of anti-tetracycline microbes.

Excessive fertilizer use has been recognized as a key factor in increasing agricultural and forestry greenhouse gases (GHG) emission. Previous studies found that proper fertilizer using control strategy can reduce agricultural CO2, N2O, CH4 emission, however, influence crop yields as well. Bamboo is an important economic plant in China, which has been intensively cultivated in recent years to satisfy increasing social demands of bamboo products. To maintain rapid growth of bamboo, the fertilizer using amount in bamboo forest is significantly higher than other human-planted forests such as Pinus massoniana and eucalyptus forest. Therefore, it is necessary to explore a sustainable fertilizer management approach which could achieve the optimum balance between low GHG emission and high production in bamboo forest.
The purpose of this study is to develop a sustainable fertilizer management approach by estimating the impact of different fertilization practices on GHG emission and biomass production of bamboo forest comprehensively. It is supposed to reduce current GHG emission from bamboo plantation without inhibiting bamboo production.
Integrated with the surveys/statistics data, the DNDC (denitrification-decomposition） biogeochemical model was applied to simulate seven typical fertilization scenarios of a Moso bamboo forest in Anji County, Zhejiang Province during 2000—2010. The fertilization scenarios were designed by different fertilizer types (commercial organic compound fertilizer, cow manure, ammonium bicarbonate) and application dosages (medium, low, high). Three indicators were adopted for the assessment: 1) the global warming potential (GWP) to assess the total CO2 and N2O emission from studied bamboo plantation; 2) the net primary production (NPP) to assess the bamboo production; 3) the GHG emission per bamboo biomass increments (Cflux/Cbiomass). All the indicator values were calculated from the DNDC modeling results.
According to the assessment results, manure had the strongest effect on reducing GHG emission by increasing the soil organic carbon (SOC), and the GWP under high amount of commercial organic fertilizer and manure application (scenario MH) decreased to -11.3 t/(hm2·a) while the value under none fertilization condition was 1.7 t/(hm2·a); the inorganic N fertilizer showed no obvious impact on CO2 emission but had strong effect on stimulating N2O emissions, and the high amount of inorganic N fertilizer (scenario ABH) led to a 12.2 times higher net GWP than the situation without fertilization, which was recognized as the most negative scenario for low-carbon fertilizer management. Under seven fertilization scenarios, the bamboo biomass increments showed no obvious difference compared with the situation without fertilization. However, the NPP under the M scenario (common amount of commercial organic fertilizer and manure) was slightly higher than other scenarios with the value of 4.71 t/(hm2·a), indicating that organic fertilizers and manure have stronger effect on improving bamboo production. Without inhibiting bamboo production, the high amount of manure application resulted in the lowest net GWP for per net primary production increment (-2.4 kg/kg), which had the most significant GHG reduction among all other scenarios.
As a consequence, organic fertilizer shows more potential on reducing CO2, N2O emission without decreasing bamboo production. From the GHG emission reduction perspective in sustainable fertilizer management for bamboo forest, advocating organic fertilizer application and reducing inorganic N fertilizer use can be a plausible approach towards the dual objectives of high bamboo production and GHG emission reduction.

Various plant proteins such as soybean meal and canola meal were considered promising protein sources as substitutes for fishmeal. However, they would enhance the presence of anti-nutritional factors like phytate. Phytate (myo-inositol-hexaphosphate phosphohydrolase) was the main storage form of phosphorus (P) in plant proteins and not available for fish due to the lack of intestinal phytase for efficient phytate hydrolysis during digestion. Therefore, most of the phytate-P was excreted into the water which contributed to eutrophication effects. Phytase could be added to plant-based diets to hydrolyze non-digestible phytate into available-P during passing through the fish gut and improve the P availability, which reduced P excretion and its loss into the environment in fish diets. Furthermore, phytase could also improve protein digestibility by hydrolyze the form of phytate-protein complexes that are resistant to proteolytic digestion. In the present, most researches on phytase were directed toward determining its effect on bioavailability of nutrients (P and protein) and the growth performance of fish. Based on the fact that nutrients (P and protein) were essential to maintain bone health and non-specific immune responses in fish, it was necessary to explore the effects of phytase on the bone health and non-specific immune responses in fish.
This experiment was conducted to evaluate the effects of different levels of liquid phytase on vertebral shapes and non-specific immune responses in channel catfish (Ictalurus punctatus). Six isonitrogenous (37.50% crude protein) and isocaloric (17.00 kJ/g gross energy) diets were formulated, a negative control （NC） diet: a basal diet was supplemented with 1% dicalcium phosphate (CaHPO4), a positive control （PC）diet: the NC diet was supplemented with another 1% dicalcium phosphate (CaHPO4), in addition, four phytase supplementation groups: the NC diets with graded levels (250, 500, 1 000, 2 000 U/kg) of phytase were assigned and fed channel catfish (5.78±0.07) g/individual with 5 replicates for 60 days after a foster period.
Results showed that the mass gain rate and feed conversion ratio were significantly improved by phytase supplementation compared with NC group (P ＜ 0.05). Especially in 1 000 U/kg group, the mass gain rate reached the most positive value by 117.42% compared with PC group (P ＜ 0.05). The protein retention efficiency was significantly improved by phytase supplementation compared with NC group (P ＜ 0.05) and no significant differences were observed between phytase addition group and PC group (P ＞ 0.05), and the P retention efficiency was increased significantly (P ＜ 0.05) with the level of phytase supplementation increasing. The curvature and scoliosis in the middle part of vertebrae were observed in NC group. In addition, the activity of lysozyme (LSZ) in liver was significantly higher than PC group when phytase was supplemented with 500 U/kg (P ＜ 0.05) and the activity of LSZ in head-kidney was highest in 2 000 U/kg group (P ＜ 0.05). A significant improvement (P ＜ 0.05) in PC group on the activity of superoxide dismutase (SOD) in liver was observed. For the activity of alkaline phosphatase (AKP) in liver, no significant differences were observed between 500 U/kg group and PC group (P ＞ 0.05).
In conclusion, the optimum level of phytase supplementation is 1 000 U/kg and phytase supplementation increas growth performance and protein retention efficiency, ameliorate vertebral malformations and improve non-specific immune related enzymes in channel catfish.

Wugu chicken is a marvel of traditional Chinese poultry breeds and is characterized by black skin and bones, and grayish-black meat. There are nearly 17 species of Wugu chicken, not including breeds with partially black skin and bones, recorded in Animal Genetic Resources in China published by the China National Commission of Animal Genetic Resources. Among these breeds, the Dongxiang blue-eggshell chicken was originally bred in Dongxiang County, Jiangxi Province, China. This breed is characterized by black feathers and most of the crest, beak, skin, muscle, bone, shins, and its toes are also black. The unique characteristic of the Dongxiang chicken is a blue eggshell. Mitochondrial DNA (mtDNA) analysis has become an effective method to explore genetic relation among populations because of its low molecular mass, simple structure, rapid evolutionary rate, maternal inheritance, and non-tissue specification, among other features. The aim of the present study was to analyze mtDNA control region polymorphisms and evolution in conservative groups of Dongxiang blue-eggshell chickens in comparison to other domestic Wugu breeds to better elucidate the genetic structure of Dongxiang chickens to further develop conservation and exploitation programs.
Blood samples were collected from 39 (19 cocks and 20 hens) eleventh generation Dongxiang blue-eggshell conservative chickens and DNA was extracted and then PCR-amplified and the products were sequenced. Nine other samples from domestic Wugu chicken breeds bred by our team or collected from the NCBI database were used for nucleotide sequence analysis. All breeds used in this study were recorded in Animal Genetic Resources in China and all collected data were analyzed using DNASTAR 5.0, DNASP 5.10, and MEGA 5.10 softwares.
The results showed that the 534 bp mtDNA control region sequence of the Dongxiang blue-eggshell chicken had a G+C content of 43.1% and included 21 variable sites, of which 71.4% (15/21) were transitional and 28.6% (7/21) were transversional. The nucleotide diversity was 0.008 29 ± 0.001 30. There were 73 haplotypes found in 248 samples from 10 Wugu chicken breeds. The Dongxiang blue-eggshell chicken had nine haplotypes with a diversity of 0.773 ± 0.048. Estimates of evolutionary divergence between breeds showed that the Dongxiang blue-eggshell chicken was genetically closest (0.009) to the Taihe silky chicken, which was also originally bred in Jiangxi Province. Yunxian Wugu chicken originally bred in Hubei Province had the farthest genetic distance (0.019, 0.020) from Yanjin Wugu and Tengchong snow chickens originally bred in Yunnan Province. A neighbor-joining phylogeny tree revealed six original clades among 10 domestic Wugu chicken breeds. Clade B included Gallus gallus spadiceus subspecies and had the largest number of samples (31.0%, 77/248), followed by clade A (27.8%, 69/248). The Dongxiang blue-eggshell chicken was closely related to three clades, of which clade A was the largest (64.1%, 25/39).
It is concluded that the Dongxiang blue-eggshell chicken is moderately genetically diverse and there is an apparent geographical association between domestic Wugu chicken breeds and their maternal origins, although each had individual characteristics.

In the southern China, the sugarcane is mostly planted on the slopes and hilly areas. Because of the influence caused by the typhoons and monsoons, the sugarcane always render in the state of lodging and bending. Lifting up the sugarcane is the first step of the mechanized harvesting of sugarcane. It is demanded that the harvest mechanic of sugarcane should be adapt to the planting patterns and growth condition, in order to lift up the sugarcane in different lodging status. Currently, the design and the research method on sugarcane lifter was traditional, which usually did static analysis only and did not take the dynamic characteristics into consideration.
The propped effect of the existing mechanism is poor and the efficiency is not really high, meanwhile, the sugarcane lifter has problems caused by the unreasonable structure design and operation parameters. In order to solve these problems, the virtual prototype technology will be used to do the virtual experiment on sugarcane lifter. At the same time, the technology will also analyze its key technology, save the cost of physical prototype research and development, and shorten the development cycle of the physical prototype as well.
The investigated subject of this paper was mainly the sugarcane lifter with the 60° installation angle and the equivalent pitch spiral. Aimed at the problems of the sugarcane lifting caused by the unreasonable structure design and the operation parameters, the three-dimensional model would be imported into the ADAMS software to do the dynamic simulation. The method of three levels and three factors comprehensive test was used, whose impact factors were sugarcane lifter speed, pitch and the forward speed. Regarding the lifting height as the evaluation index, it intended to find the optimum combination for the virtual experiment of the sugarcane lifter structure and the working parameters, meanwhile, the test data variance would be analyzed by the MATLAB software.
Through the analysis of the dynamic simulation of the sugarcane lifter, it was clear that when the time was 0 to 1.5 s, the sugarcane lifter would not get in touch with the sugarcane, and as a result, the centroid vertical displacement was not changed accordingly; but when the time was 1.5 to 4.5 s, the sugarcane would be lifted by the sugarcane lifter, and the centroid vertical displacement was correspondingly increased gradually. When the time was more than 4.5 s, the fluctuations of the centroid vertical displacement would come out because of the non-existence of the simulation of cutting part.
In the combination of the ADAMS virtual comprehensive test, the best operating and structure parameters for the angle of 60° installation sugarcane lifter was that: when the speed of the sugarcane lifter was up to 120 r/min, the pitch of thread was 360 mm, and the forward speed was 350 mm/s, the lifting height of the 60° side lodging sugarcane would reach the maximum 186.9 mm. In addition, the three factors have mutual interactions with each other, and this moment was the strongest time that the forward speed affected the height of the sugarcane lifter, all of which will eventually provide the theory basis for the improvement of sugarcane lifter.