Reducing methane emission from ruminants has important economic value and ecological significance. Anaerobic methane oxidation (AMO) is an important way to reduce methane emissions in different ecosystems, including wetlands, marine habitats and lakes. According to different electron acceptors, AMO can be divided into sulfate-dependent anaerobic methane oxidation (S-DAMO), nitrate/nitrite-dependent anaerobic methane oxidation (N-DAMO) and metal-dependent anaerobic methane oxidation (M-DAMO). Feeding nitrate and sulfate to ruminants also has methane-lowering effects; however, this process has always been thought to be the result of \mathrm{N}{\mathrm{O}}_{\mathrm{3}}^{－} or \mathrm{S}{\mathrm{O}}_{\mathrm{4}}^{\mathrm{2}－} competition for hydrogen as the electron acceptor. The thermodynamic reactions of N-DAMO and S-DAMO are superior to nitrate reduction reaction and sulfate reduction reaction, and if AMO can proceed in rumen, it will be of great significance to improve environment and feed utilization efficiency. Therefore, based on the description of the type of AMO, mechanisms of AMO and microorganisms involved, this paper compared the differences of nitrate and sulfate reducing methane production in the natural habitat and rumen, and found that AMO may occur in the rumen, which may be one of the reasons for the reduction of methane production in the rumen by nitrate and sulfate.

In order to further explore the mechanism of endogenous fatty acid synthesis and metabolism in goose granulosa cells, we used CRISPR-Cas9 technology to construct knockdown plasmids of a goose targeted stearoyl-coenzyme A desaturase (SCD) gene and package lentivirus. First, we designed the sequence of single-guide RNA (sgRNA) of goose SCD gene; second, synthesized in vitro and tested the lysis efficacy of sgRNA to target DNA site by endonuclease cleavage assays; finally, prepared the lentiviral plasmid of psgRNA-mCherry-T2A-Puro and pLenti-Cas9-T2A-EGFP using psPAX2 and pMD2.G as package plasmids. Results showed that the lentiviral plasmid was successfully constructed, and the strong double positive cell groups co-expressing the red fluorescent protein (mCherry) and enhanced green fluorescent protein (EGFP) were screened out when the lentiviral plasmid infected the Chinese hamster ovary (CHO) cells. The above results lay the foundation for infecting goose primary granulosa cells and targeting knockdown the SCD gene.

Transgelin-like protein (TLP) is a novel shell matrix protein identified previously from the myostracum layer of Mytilus coruscus shell. For exploring its function in the shell formation of mussel, the TLP was recombinantly expressed by Escherichia coli expression system based on the sequence analysis and codon optimization. The functions of recombinant TLP (rTLP) on calcite- and aragonite-type calcium carbonate crystals were then investigated, including morphology, polymorph, crystallization rate, and binding ability of calcium carbonate crystals. Sequence analysis showed that the TLP contained a calponin homology (CH) domain, and the spatial structure of TLP predicted by SWISS-MODEL presented a conformation formed predominately by α-helices. Functional analyses showed that the rTLP had significant effects on the morphological change of aragonite-type calcium carbonate crystal and polymorph change of calcite-type calcium carbonate crystal, suggesting the function of this protein in the transformation of calcite to aragonite. In addition, the rTLP showed inhibition of calcite-type calcium carbonate crystallization rate in vitro, and promotion of aragonite-type calcium carbonate crystallization rate under high protein concentration. Moreover, the rTLP presented binding abilities to the calcite-type calcium carbonate crystal rather than the aragonite-type calcium carbonate crystal. Considering the myostracum layer is composed of aragonite-type calcium carbonate crystal, we speculate that the TLP may play important roles in the shell biomineralization and the formation of myostracum layer.

In order to expand the germplasm resource bank of cotton after rape cultivation in the Yangtze River Basin, screen out excellent germplasm suitable for hybrid parents, and improve the efficiency of cotton breeding, 11 main agronomic traits of 72 upland cotton materials introduced were analyzed with the methods of variability, correlation analysis, principal component analysis and clustering analysis by statistical software SAS V8. The results showed that, the 72 Gossypium hirsutum germplasm resources had rich genetic diversity, and the variation coefficients of characters among 72 materials ranged from 1.15% to 30.31%. The variation coefficient of boll number per plant was the largest, was 30.31%. The correlation analysis showed that the growth period was significantly and positively correlated with plant type traits. The plant height was extremely significantly and positively correlated with the boll mass. The number of fruit branches per plant was extremely significantly and positively correlated with the boll number per plant, and significantly and negatively correlated with the boll mass. The lint percentage and boll mass were significantly and negatively correlated with some quality traits. The principal component analysis showed that the cumulative contribution rate of the first four principal components was 73.98%, which including most information of 11 traits. Clustering analysis showed that the 72 cotton materials were classified into six groups. Group Ⅰ belonged to high-quality materials, and group Ⅱ belonged to high-yield materials. Group Ⅴ belonged to high-yield and high-quality materials, which could be used in breeding. Two high-quality cotton materials and six high-yield cotton materials were screened out with the method of comprehensive evaluation, which would be used to cultivate new materials with combination of yield and quality traits.

In order to explore the varieties of ornamental crabapples with color leaves and a long viewing period in spring and the influence of pigment content on the leaf color, 10 ornamental crabapple varieties, Malus ‘Black Jade’, Malus ‘Pink Spires’, Malus ‘Profusion’, Malus ‘Prairifire’, Malus ‘Royal Raindrop’, Malus ‘Roudph’, Malus ‘Show Time’, Malus ‘Big Red’, Malus ‘Perfect Purple’ and Malus ‘Purple Prince’ from North America were selected as the experimental objects. Their leaf color parameters and pigment contents were tested at different developmental stages (prophase of leaf spreading L₁, early stage of leaf spreading L₂, late stage of leaf spreading L₃). The results showed that the change of lightness (L^*) in most varieties was relatively stable, while the fluctuations of redness (a^*) and yellowness (b^*) were large. The a^* values of ‘Black Jade’, ‘Pink Spires’, ‘Profusion’, ‘Roudph’,‘Big Red’, ‘Perfect Purple’, ‘Purple Prince’ varieties showed significant decreases (P＜0.05), and the a^* values of ‘Prairifire’, ‘Royal Raindrop’,‘Show Time’ varieties increased. The overall variation of b^* values was consistent and significantly increased (P＜0.05). Combining with the leaf color map, the a^* value can be used as the main indicator for the spring leaf enjoyment of the ornamental crabapple; and the b^* value is the auxiliary indicator for the leaf color change of the ornamental crabapple. The contents of chlorophyll and carotenoid increased and the anthocyanin content decreased with the developmental periods, while the content of chlorophyll was always higher than the contents of the other two pigments. Correlation analysis showed that, the anthocyanin contents of 10 ornamental crabapple varieties had significant or extremely significantly positive correlations with the a^* values. There was a significant positive correlation between carotenoid content and the b^* value of the leaves, and the correlation between chlorophyll content and leaf color parameters varied from variety to variety. In conclusion, among the 10 ornamental crabapple varieties, ‘Prairifire’, ‘Royal Raindrop’, and ‘Show Time’ have higher ornamental value, worthy of development and utilization as red leaf tree species in spring, as the leaves remain red for a long time (about 35 d) in the three developmental stages.

With the aim of exploring the regulating factors and involved mechanisms for the growth of Villosiclava virens mycelia, a strain (ZJ09) of this fungus was cultured on potato sucrose agar (PSA) plates under dark and lighting (with white light from fluorescents) conditions, respectively, and the growth states of the colonies were compared. A significant inhibitory effect was observed by lighting on the growth of V. virens mycelia, which suggested that lighting condition was a regulating factor for the mycelial growth of this fungus. The mycelial samples of the fungal strain ZJ09 from the dark and lighting groups were collected and their transcriptomes were sequenced, respectively. Six hundred and ninety-five differentially expressed genes (DEGs) caused by lighting were identified, including 359 up-regulated genes and 336 down-regulated ones. Six DEGs were selected out to check by real-time quantitative polymerase chain reaction (RT-qPCR) and the expression results were consistent with those by transcriptomic sequencing. During gene ontology (GO) enrichment analysis, 491 of the 695 DEGs were enriched to the terms of biological process, cellular component and molecular function, indicating that complex mechanisms may be involved in the inhibitory effect of lighting on mycelial growth. Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis results of the 695 DEGs indicated that influences on the structure and functions of ribosome, and amino acid metabolism were quite probably involved in the inhibitory effect of lighting on mycelial growth. As DEGs included a gene encoding acetyl-coenzyme A synthetase and the lighting condition significantly inhibited its expression, it was inferred that impairing the mycelial acetyl-coenzyme A synthesis ability was one of mechanisms of the inhibitory effect of lighting on mycelial growth. In contrast, as neither of the genes UvHOG1 and UvBI-1 was a DEG, the protein kinase HOG1 (high osmolarity glycerol 1) and Bax inhibitor-1 may both have nothing to do with the inhibitory effect of lighting on mycelial growth.

A membrane separation technology was used to make a grading preparation of theabrownines from Tibetan tea, and four theabrownine fractions (TBFs) with the molecular mass of 5-10, ＞10-50, ＞50-100, and ＞100 kDa were got, which were named as TBFs1, TBFs2, TBFs3 and TBFs4, respectively. The physicochemical properties, microstructure, and 1, 1-diphenyl-2-picrylhydrazyl (DPPH) scavenging activity of the four TBFs were comparatively studied. The results showed that the molecular mass of theabrownines was not less than 5 kDa, and the four TBFs with different ranges of molecular mass were all weakly acidic, which had different contents of phenolic hydroxyl, carboxyl group, and the complex compounds including proteins and polysaccharides. Spectroscopic analysis showed that the theabrownines were polyhydroxyphenols and were characterized by an absorption peak near 202 nm. The scanning electron microscope observation showed that the aggregation state of the TBFs was significantly different from each other. Within the range of 10-80 μg/mL, there was a positive linear correlation between the theabrownine concentration and its DPPH radical scavenging ability. In addition, the four TBFs showed significantly different antioxidant activities from each other, and the most active fraction was TBFs4 (＞100 kDa).

In order to understand the geochemical characteristics of soil cadmium (Cd) during weathering and soil formation of limestone, three types of limestone soils were sampled at the same time from western Zhejiang Province. The Cd enrichment in limestone hills and nearby valleys and the vertical distribution of Cd in the soil profiles were studied. The leaching characteristics of Cd during development of the soils were analyzed by mass balance method with reference to stable element zirconium (Zr), and the results were also compared with those of yellowish red soils developed from local shale. The results showed that the leaching and accumulation of Cd in the limestone soils occurred simultaneously. Because of the special soil-forming process of limestone, the enrichment of Zr and Cd in the soils was obvious, and the enrichment amounts of soil Cd in limestone hills and nearby valleys were obviously higher than that of shale-derived hilly soil in the same area. The vertical distribution of Cd in three kinds of limestone soils had obvious differences and the chemical forms of Cd in different limestone soils were also different. The high leaching characteristics of Cd in the soils during weathering of limestone and the surface erosion of the limestone soils with high Cd content may be the main reasons for the high Cd accumulation in the soils in the limestone distribution area and its vicinity.

One species of Leucopholiota was collected from Pangquangou National Nature Reserve in Jiaocheng County, Lüliang City of Shanxi Province during the investigation on species diversity of Tricholomataceae from 2017 to 2018, and it was identified by morphological and molecular methods. The morphological, growing habitat and ecological characteristics were recorded by fresh specimens in the field and the microscopical characters were examined and measured in detail under an optical microscope and a scanning electron microscope. Its main characters were as follows: the surface of pileus was dry with 3.0-5.5 cm in diameter, and covered with numerous, introvert and rusty-brown scales; the lamella was white, crowed, serrated on the edge, slightly depressed at the border with stipes, which was 2.5-5.1 cm long and 0.5-1.0 cm in diameter, and covered with rusty-brown scales as that on the surface of pileus from the bottom of stipes to the rings and with smooth stipes on the upper part of rings; spores were (4.11-5.95) μm×(3.19-4.09) μm, amyloid, hyaline, and oblong; basidia was (15.38-20.89) μm×(5.21-6.08) μm; cheilocystidia was (14.70-21.30) μm×(3.52-5.38) μm, nearly clavate or fusiform, hyaline, with a pointed bump at the top. The internal transcribed spacer (ITS) district of rDNA extracted from this species was specifically amplified and a fragment with 651 bp was acquired. The amplified sequence was similarity with Leucopholiota decorosa at a level of 99.2%. With combinations of morphology, molecular identification and phylogenetic analysis, the species collected on the rotten wood of Betula platyphylla Suk. was identified as L. decorosa, which is the first recorded species of Leucopholiota in China. It provides a basis for the research on the genus Leucopholiota in China.

In order to investigate the expression profile of β-defensin genes in the blood of Chinese alligator and the effect of stocking density on them, reverse transcription quantitative polymerase chain reaction (RT-qPCR) was used to detect the expression levels of β-defensin genes in blood samples from 41 seven-year-old Chinese alligators and study the defensin gene expression level differences between sexes and populations with different stocking densities. The results showed that only the AsBD5 and AsBD8 genes expressed in the blood of Chinese alligator, and there was a significantly positive correlation between their expression levels. There was no significant sex-biased difference of the defensin gene expression levels. However, their expression levels were significantly different between the two populations with different stocking densities. The defensin gene expression levels were significantly higher in the Chinese alligator population with higher stocking density, which indicating that the stocking density has some effect on the immune status of the captive Chinese alligator.

To understand the prevalence of porcine Pasteurella multocida in Tibet, a total of 60 samples (lungs, n=30; tonsil, n=30) were sterilely collected from the diseased and dead Tibetan swine in Tibet to analyze the capsular serotypes and virulence gene distribution of the porcine P. multocida. The results showed that one D-type porcine bacillus strain was obtained from the lung tissue, and the positive rate was 3.33% (1/30). However, there were not bacillus isolates from the tonsil. Moreover, the isolated strain carried 16 virulence genes and was closely related with P. multocida isolated from the poultry in Iran (GenBank No. AY225343). In addition, the strain was resistant to tetracycline, doxycycline, ampicillin and amoxicillin, but was sensitive to kanamycin, streptomycin, neomycin, spectinomycin, ciprofloxacin, norfloxacin and enrofloxacin. In conclusion, a capsular serotype D porcine P. multocida is isolated from the Tibetan swine, which provides references for the etiology and epidemiology investigation of porcine P. multocida in Tibet.

In order to realize the mechanization of the process of the orchard seedling leaving the nursery, the structure of seedling lifting and soil cleaning machine was designed, and the parametric design was carried out for key components such as seedling lifting device and soil cleaning device, and then finite element analysis and optimization were carried out for the seedling lifting shovel. The results showed that the maximum equivalent stress decreased from 5.026 MPa to 0.238 MPa, and the maximum equivalent strain decreased from 2.141 to 0.663. The overall deformation was far less than 5 mm, which met the design requirements. Finally, the reliability of the prototype was verified by field test. The results showed that the main root length of the seedlings was between 120 mm and 150 mm, and the injury rate of the seedlings was low, which met the requirements of agronomy. The size parameters of the machine are expected to provide a reference for the design and research of the seedling lifting and soil cleaning device.

In order to improve the uniformity of the flow when fertilizer apparatus is working, a kind of fertilizer apparatus owned circular arc gears was designed. Taking the circular arc gear discharging fertilizer apparatus as the research object, this study used discrete element method (DEM) simulation to analyze the influence of two key components including the arc radius of concave-groove of circular arc gear discharging fertilizer (r₁) and the minimal length between two mutual meshing arc gears (l). The results indicated that the arc radius of concave-groove of circular arc gear discharging fertilizer had significant effect on the coefficient of determination of discharging fertilizer amount in unit time, and the minimal length between two mutual meshing arc gears had significant effect on the coefficient of variation of the stability of fertilizer sowing amount. The optimum structural parameters were 8.54 mm as the arc radius of concave-groove and 5.22 mm as the minimal length between two mutual meshing arc gears. Upon this circumstance, the coefficient of variation was 0.28, and the coefficient of determination was 0.997 2. The optimum structure was selected to do the bench test. The results indicated that the coefficient of variation of the quality changes of discharging fertilizer was 0.27, and the coefficient of determination of fertilizer discharging amount in unit time was 0.998 0. The results of simulation experiment were basically consistent with the real result.

Aiming at the poor collection of the Hermetia illucens insect sand, combining with mechanical and physical properties of insect sand, the bidirectional spiral collecting device was designed, and the working principle and structural parameters of the device were analyzed and calculated. In order to determine the working performance of the bidirectional spiral collecting device for H. illucens insect sand and get the optimum parameters, the bidirectional spiral speed, discharging spiral speed, insect sand thickness were taken as the testing factors, and single lap discharge and homework time were taken as the evaluating indicators, with insect sand (moisture content of 43.6%) as experimental object. The single- and multi-factor orthogonal test analysis were carried out. The results showed that the factors affecting the single lap discharge were as follows: discharging spiral speed＞insect sand thickness＞bidirectional spiral speed. The factors affecting the homework time were as follows: insect sand thickness＞discharging spiral speed＞bidirectional spiral speed. The multi-factor orthogonal test had the same results with variance significance test, and discharging spiral speed and insect sand thickness had significant effects on single lap discharge (P＜0.05); for the homework time, insect sand thickness had significant effect (P＜0.05). The two evaluating indicators were analyzed based on the comprehensive scoring method, and the optimal operating parameters of the device were obtained with the bidirectional spiral speed of 78 r/min, discharging spiral speed of 68 r/min, insect sand thickness of 4 cm, mean single lap discharge of 142 g, and mean homework time of 48 s, which meet the requirements of H. illucens insect sand collection. This research provides a reference for the improvement and optimization of spiral collecting equipment for H. illucens insect sand.