Hemagglutinin (HA) protein is a kind of glycoprotein on the surface of Avian influenza virus, which is a main protective antigen inducing neutralizing antibody. Avian influenza virus H9N2 HA gene was successfully expressed in rice (Oryza sativa) endosperm. In order to establish a purification method for H9N2 Avian influenza virus HA protein expressing in rice, two methods were used in this study to purify HA protein. Method 1: Rice powder expressing HA protein was added into the extract in a ratio of 1∶5 mass to volume, stirred at room temperature, centrifuged and supernatant was taken. After filtration, crude HA protein was firstly captured by Q anion chromatography. Subsequently, HA protein was purified by HA protein antibody affinity chromatography, eluent was collected and the target protein was detected. Method 2: Crude HA protein solution was obtained in the same way as method 1. According to packing property of the Butyl hydrophobic, ammonium sulfate was added to HA protein solution, which was then stirred, centrifuged and filtered for a second stage of purification. The collected eluent was concentrated and changed, and purified by SP-cationic chromatography, and the eluent was collected to obtain the HA protein. The HA protein purified by the 2 methods was tested by SDS-PAGE and Western blot, and the HA protein was sent to the company for sequencing. The results showed that the purity of HA protein was about 90% by Q anion chromatography combined with HA affinity chromatography. The method of purification was relatively simple, but monoclonal antibodies need to be cultured and purified. This method was suitable for rapid and small-scale protein purification in laboratory. The purity of HA protein was about 90% by Q anion chromatography, Butyl hydrophobic chromatography and SP cationic chromatography. This method is suitable for establishing the purification process of HA protein and is used to purify HA protein on a large scale. The HA protein obtained by these two methods was sequenced and the result was consistent with the known amino acid sequence. In this study, two methods were established for the first time to obtain high purity recombinant H9N2 subtype HA protein from rice, it will provide basic data for the preparation of avian influenza H9N2 subunit vaccine in the future.

Superoxide dismutase (SOD) gene family plays an important role in the development and stress response in potato (Solanum tuberosum). However, there are few reports on the identification of SOD gene family members in potato and their gene expression change after tuber damage. In order to analyze the bioinformatics characteristics of potato SOD family members, investigate the expression difference of each family member in different organs of potato seedlings and response in damaged tubers, the softwares GSDS 2.0 and PLACE were used to analyze gene structure and cis-element of StSODs, and the softwares ProtParam, ProtComp 9.0, MEGA7.0, and MEME 4.11.1 were used to analyze physical and chemical properties, subcellular localization, phylogenetic relationship and conserved motifs. The expression level of the gene family numbers was determined in different organs and artificial wounded tuber by qRT-PCR. SOD activity and O₂^{• -} and H₂O₂ contents were analyzed at wounded sites of tubers with 10 mmol/L diethyldithiocarbamate (DDC) treatment by biochemical methods. The results showed that 8 StSODs were identified from the potato genome with different domains such as Cu/ZnSOD domain, Fe/MnSOD α-hairpin domain and C-terminal Fe/MnSOD domain. The amino acid lengths of these StSODs were 152~312 with isoelectric point 5.28~7.13. The predicted subcellular localizations were in the cytoplasm, chloroplast and mitochondria. The StSODs were distributed on 6 of 12 chromosomes unevenly, and divided into 3 subgroups Cu/ZnSOD, FeSOD, and MnSOD, phylogenetically based on their different domains. The promoters of StSODs contained a large number of cis-elements responding to lights, stresses and hormones. StSODs expressed in the roots, stems, leaves, buds and tubers of potato seedling, the expression levels of StCSD1 and StCSD2 in the tuber were significantly higher than that of other organs. The expression of StSODs could be induced at wounded sites of wounded tubers. The expression of StFSD1 and StFSD2 were significantly induced in the early stage, the increased expression of StCSD3 was observed both in early and late stages, while rest SOD genes were expressed in mid stage of the damage. DDC treatment significantly decreased SOD activity and H₂O₂ content, but increased O₂^{• -} content. The present study could provide a reference for further functional study of StSODs in wounded tubers.

Plants respond to the external environment by initiating a series of signaling pathways, which usually involve multiple protein kinases, such as CBL-interacting protein kinases (CIPKs). In this study, the sequence alignment method was used to identify the CIPK gene family within the whole genome of castor bean (Ricinus communis). The bioinformatics method was used to analyze the sequence structure, physical and chemical properties, chromosome positioning, cis-acting elements of promoters, conserved domains, phylogenetic relationships, and clustering methods of the encoded protein. RNA-seq and qRT-PCR were carried out to assess the expression of RcCIPKs. qRT-PCR was used to analyze the tissue-specific expression pattern of RcCIPKs and the expression level of CIPK affected by abscisic acid (ABA) hormone. The subcellular localization of RcCIPK16 were further analyzed in the epidermal cells of Nicotiana tabacum. Eighteen RcCIPKs family members were successfully identified. Gene analysis showed that RcCIPKs were divided into exon-rich and exon-poor types, which were located on 16 chromosome segments with uneven distribution. Most RcCIPKs contained MYB, MYC, ABRE and ERE elements, which indicated that these genes were responsive to plant hormone and stress. Protein analysis showed that majorities of RcCIPKs had the identical motif types and arrangement except RcCIPK3 and RcCIPK4. RcCIPKs were grouped into A, B, C, D and E categories based on phylogenetic analysis, which inferred that diverse types of CIPKs might perform various function. The results of RNA-Seq and qRT-PCR displayed that only RcCIPK1, RcCIPK12 and RcIPK16 were induced by cold stress. The tissue-specific analysis showed that RcCIPK1 and RcCIPK12 were mainly expressed in leaves and stems, and RcCIPK16 was mainly expressed in leaves; and the expression levels of the three genes were induced by ABA hormones. Subcellular localization analysis revealed that RcCIPK16 was mainly localized in the cytoplasm. This study firstly identified RcCIPKs gene family at the whole genome level and analyzed gene expression under cold stress, which will provide important candidate genes for further development of new cold tolerant castor germplasm.

Anthocyanins are important pigments for strawberry (Fragaria × ananassa) fruit coloring, and FAMYB10 is a key transcription factor for anthocyanin synthesis. To investigate the role of nucleotide polymorphisms of FaMYB10 alleles in anthocyanin accumulation in tobacco (Nicotiana benthamiana), in this study, 3 alleles of FaMYB10 were isolated from the fruit by homologous cloning. FaMYB10a (GenBank No. MW478285) and FaMYB10b (GenBank No. MG456859) were identified from the red strawberry variety 'Sweet Charlie', and FaMYB10c (GenBank No. MG456860) was identified in the fruit of the white variety 'Snow White'. Sequence analysis showed that the open reading frame of FaMYB10a and FaMYB10b was 702 bp, encoding 233 amino acids. There was a single nucleotide polymorphism at 94 bp from start codon ATG, where FaMYB10a was base A, and FaMYB10b was base C. The open reading frame of FaMYB10c was 710 bp, and there was a 8 base insertions at 491 bp starting from ATG, which led to premature termination and the encoding of 179 amino acids. The bases insertion in the open reading frame of FaMYB10c resulted in differences in physicochemical properties and protein structure from the other 2 alleles. When FaMYB10a and FaMYB10b were overexpressed, the tobacco plants showed anthocyanin accumulation and a red phenotype. However, when FaMYB10c was overexpressed, the tobacco plants showed no anthocyanin accumulation. The above results indicated that the nucleotide polymorphisms of FaMYB10a and FaMYB10b did not affect the anthocyanin accumulation, while the nucleotide polymorphisms of FaMYB10c led to functional changes, and ultimately tobacco leaves could not accumulate anthocyanins. The present study provides reference for the functional research of other important genes, and also provides a reference for the study of plant traits and evolution.

Peach (Prunus persica) is an important economic fruit tree in China, which plays an important role in the development of agricultural economy. Low temperature affects peach growth and development, which is one of the threats to peach production in early spring. In order to explore the low temperature adaptation and cold acclimation mechanism of peach trees in subtropical region, in this study, the changes of transcriptional expression profiles in leaves of peach germplasm 'mx14-1' with low chilling requirement were compared and analyzed, GO functional annotation and KEGG pathway functional enrichment analysis of differentially expressed genes showed that, under low temperature stress, differentially expressed genes in peach leaves were mainly related to stress response, and enriched in secondary metabolite synthesis pathway, involving flavonoids, terpenoids, diphenylheptanone, gingerol and phenylpropane synthesis, linoleic acid metabolism and other pathways. The genes related to flavonoid metabolism were analyzed by qRT-PCR, results showed that the expression of PAL (phenylalanine ammonialyase), C4H (cinnamic acid 4-hydroxylase), 4CL (4-coumarate:CoA ligase), CHS (chalcone synthase), CHI (chalcone isomerase) and F3H (flavanone 3-hydroxylase) gene, which encode key flavonoid biosynthetic enzymes, were significantly up-regulated in 4 ℃ 48 h treated leaves, the results were consistent with those of transcriptome sequencing. The determination of leaf metabolites showed that the contents of flavonols and derivatives related to flavonoid metabolism in peach leaves increased significantly under low temperature stress. 4 ℃ 48 h treatment group and control were treated with -2 ℃, the results showed that the total flavonoids and antioxidant capacity of peach leaves were significantly increased after 4 ℃ low temperature treatment, and the damage decreased at -2 ℃. This study provides a reference for further exploring the molecular mechanism of peach low temperature response and cold resistance.

Intramuscular fat is an important indicatior of beef quality, and preadipocyte differentiation is crucial for the formation of marbled beef. The purpose of this study was to explore the effect of propionic acid on the differentiation of intramuscular preadipocytes in bovine, and to analyze the signal mechanism of intramuscular preadipocyte differentiation from the cellular and molecular level. Bovine intramuscular preadipocytes were isolated by CollagenaseⅠunder sterile conditions, differentiation medium with different combinations of different doses of propionate (1, 3 and 6 mmol/L), 0 µg/mL insulin and 0.25 µmol/L dexamethasonethe were designed to induce the differentiation of bovine intramuscular preadipocytes for 6 d. The results of determination of intracytoplasmic lipid contents and qRT-PCR analysis showed that, propionate significantly enhanced the intracytoplasmic lipid contents of bovine intramuscular preadipocytes in a dose dependent manner (P<0.05), and 1, 3 and 6 mmol/L significantly up-regulated the expression of PPARγ and C/EBPα mRNA of bovine intramuscular preadipocytes (P<0.05). The combination of propionate, insulin, and dexamethasone resulted in the greatest intracytoplasmic lipid content, whereas the removal of dexamethasone, insulin or propionate reduced intracytoplasmic lipid content by 85%, 77% and 68% respectively compared with the combination of propionate, insulin, and dexamethasone (P<0.05). Furthermore, the expression level of PPARγ and C/EBPα mRNA were decreased significantly (P<0.05). These results indicated that propionate may stimulate the differentiation of bovine intramuscular preadipocytes by acting as the first messenger of hormones (such as insulin or dexamethasone), and acting as a signal molecule through signal transduction mechanism. This study provides basic data for elucidating the deposition mechanism of intramuscular fat in beef cattle and realizing the nutritional regulation mode to produce high quality beef.

Melatonin receptor 1A (MTNR1A) is an important factor affecting the seasonal estrus and litter size in sheep (Ovis aries). To study the correlation of MTNR1A gene with litter size in Chinese Merino (Xinjiang Junken type) and Prolific Suffolk sheep, In this work, PCR, sequencing and restriction fragment length polymorphism (RFLP) techniques were used to detect the SNPs and genotypes in exon 2 of the MTNR1A gene. Linkage disequilibrium analysis was used to construct haplotypes. The association of MTNR1A gene polymorphisms and haplotypes with litter size was analyzed by general linear model (GLM) in two sheep groups. The results showed that there were 3 SNPs (G735A, G753A and C845A), each with three genotypes, were detected in two sheep breeds. SNP1 and SNP2 were synonymous mutations, SNP3 was a missense mutation with a single A to C substitution at nucleotide position 845 resulting in an alanine replacing an aspartic acid. The genotype distribution and allele frequencies of three SNPs in two sheep groups were in Hardy-Weinberg equilibrium and moderate polymorphisms. The associated analysis showed that G735A and G753A loci were extremely significantly associated with average litter size in sheep (P<0.01), the litter size of GG genotype was significantly higher than those of AG and AA genotypes (P<0.01) in two loci. The C845A loci was significantly associated with litter size in sheep (P<0.05), the litter size of CC genotype was significantly higher than those of CC and CG genotypes (P<0.05). Four haplotypes were constructed for three SNPs of MTNR1A gene. The different haplotypes were extremely significantly correlated with litter size in sheep (P<0.01), and the H1 haplotype was significantly correlated with higher average litter size than H4 (P<0.01). The results showed that MTNR1A gene was closely related to litter size in Chinese Merino (Xinjiang Junken type) and Prolific Suffolk sheep. H1 was the main haplotype of high litter size of sheep, G735A, G753A and C845A loci could be used as useful genetic markers in marker assisted selection for breeding of fecundity sheep.

Leptin receptor (LEPR), a member of the class Ⅰ cytokine receptor superfamily, plays a key role in feed intake, energy balance and reproduction in mammals. Xupu goose (Anser cygnoides), an excellent local goose breed in Hunan Province, has excellent fatty liver performance and high nutritional value. To investigate the expression of LEPR at different tissues of Xupu goose, in this study, the coding sequence of LEPR was cloned by RT-PCR, and the putative protein spatial structure of LEPR was analyzed by biology software. Additionally, the expression of LEPR in Xupu goose different tissues and the correlation between LEPR expressions of adipose tissues and liver weight, ratio of liver to body weight, and blood biochemical indices were tested by quantitative Real-time PCR (qRT-PCR). The results showed that the coding sequence of LEPR was 3 468 bp, encoding 1 155 amino acids, which had high homology with LEPR gene of other poultry. phylogenetic analysis showed that Xupu goose had the closest genetic relationship with Zhedong white goose, followed by duck (Anas platyrhynchos) and chicken (Gallus gallus), and had a distant genetic relationship with mammals. LEPR gene was expressed in eight tissues of Xupu goose, including testis, spleen, sebum, liver, abdominal fat, heart, leg muscle, and chest muscle. The expression of LEPR gene in adipose tissue was negatively correlated with liver weight and aspartate aminotransferase (AST) content. This study provides a candidate gene for the molecular genetic study of liver type goose, and further provides a basis for studying the biological function of LEPR gene.

The genetic diversity of fish is closely correlated with its environmental adaptability and genetic evolution potential. In order to reveal the genetic background of different geographical populations of common carp (Cyprinus carpio) raised in paddy field, the genetic diversity and genetic structure of four populations of carp (Guangdong Liannan population (LN), Guangdong Ruyuan population (RY), Guangxi C. carpio var. Jinbian population (JB), Guangxi Sanjiang population (SJ)) were analyzed based on PCR amplification and sequence alignment of COⅠ(cytochrome oxidase subunit Ⅰ)-D-loop (displacement loop region)-ITS1 (internal transcribed spacer 1) sequences. The results showed that there were 71 polymorphic sites in the 1 842 bp COⅠ-D-loop-ITS1 sequences of four populations of carp, including 28 singleton variable sites and 43 parsimony informative sites, a total of 33 haplotypes were found in four populations of carp and 11 haplotypes were found in both JB and RY population. There were no shared haplotypes among the four popultions of carp. The haplotype diversity of LN population (Hd=0.824) and the nucleotide diversity of JB population (Pi=0.002 78) were the highest in the four populations. The haplotype diversity and nucleotide diversity of SJ population (Hd=0.662; Pi=0.000 56) were the lowest in the four populations. The results of genetic difference analysis showed that the genetic distance between SJ and JB populations was the smallest (0.004 6), while it was the largest between LN and RY populations (0.008 7). The genetic differentiation among these four populations were significant (FST>0.25, P<0.01). Molecular variance (AMOVA) analysis results indicated that a high proportion of the total genetic variance was attributable to variations among populations (70.65%). Individual phylogenetic tree constructed based on COⅠ-D-loop-ITS1 sequences could completely separate all the individuals of four populations, which had a better clustering effect than the individual phylogenetic tree constructed based on single gene (COⅠ, D-loop or ITS1). In the phylogenetic tree of four populations, SJ and JB populations firstly clustered into a small branch, and then clustered with RY population into a large branch, while LN population independently clustered into a branch. In conclusion, the haplotype diversity of the four populations of carp raised in paddy field were high, while the nucleotide diversity of them were quite low. The genetic differentiation among these four populations were significant, which indicated that the germplasm resources of these populations of carp should be protected and utilized as “evolutionarily significant units”. This study reveals the genetic structure and genetic relationship of four populations of common carp (Cyprinus carpio) raised in paddy field and enriches the basic information of the germplasm resources of carp in rice-fish system, which would lay a theoretical foundation for the preservation, development and utilization of the germplasm of common carp raised in paddy field.

Light plays an important role in regulating the physiological activities of fish, such as feeding, growth, stress, reproduction and endocrine. To enhance the health and welfare of farmed tilapia (Oreochromis niloticus), as well as explore the suitable light quality for the tilapia and reveal the underlying physiological mechanisms, tilapia (49.2±3.2) g, were exposed to different LED light quality (red, yellow, blue, green and white), and the body composition, growth performance, cortisol levels in plasma, activities of digestive enzymes in the stomach and immune-related enzymes in serum were measured in the current study. The results showed that different light quality affected the growth, stress and immunity status of tilapia. But the survival rate (SR) and condition factor (CF) of tilapia under different light quality had no significant difference. The feeding rate (FR) and weight gain rate (WGR) of the tilapia exposed to yellow or red light were higher, and feed conversion ratio (FCR) were significantly lower (P<0.05) than those in the other light qualities. The FR, WGR and specific growth rate (SGR) in the green light group and darkness group were the lowest. Although light qualities had no significant effect on ash and crude protein content of tilapia, they had certain effect on crude lipid and moisture content. The crude lipid content of tilapia in the yellow light group was not significantly different from the blue light group (P>0.05), but was significantly higher than the other 4 groups (P<0.05); the moisture content of tilapia in the yellow light group was not significantly different from the red and blue light groups (P>0.05) but significantly lower than the green, white and darkness groups (P<0.05). Activities of protease (PES) and α-amylase (α-AMS) in the stomach of tilapia of the red light group were significantly higher than those in the other light groups (P<0.05); The activity of lipase in the red light group was significantly lower compared with the white light group and the darkness group (P<0.05), but there was no significant difference among other groups. The cortisol concentration in the plasma of tilapia exposed to green light was significantly higher than that of the other groups (P<0.05), which indicated that the green light had a significant stress effect on tilapia. The alkaline phosphatase (AKP) activity in the plasma of tilapia in the green light group was significantly higher than that of the other light groups (P<0.05) but had no significant difference from the darkness group (P>0.05); Lysozyme (LZM) activity in the serum of tilapia exposed to yellow light was significantly higher than that in the other groups (P<0.05). The findings above indicated that yellow and red light decreased the FCR and promoted the growth of tilapia, but the green light induced a stress response in tilapia and had a negative effect on fish growth. Therefore, the red and yellow light were recommended in the industrial aquaculture of tilapia to improve fish welfare and productivity. The research provides a scientific reference for the light environment regulation of Oreochromis niloticus.

Environmental microbiota and intestinal microbiota play important roles in aquatic animals' health. This study investigated the changes of bacterial communities in recta and culture water of triangle sail mussel (Hyriopsis cumingii) fed by Monora phidiumcontortum and Cyclotella sp., respectively. The pH value, ammonia nitrogen (NH₄⁺-N), nitrite nitrogen (NO₂^--N), nitrate nitrogen (NO₃^--N) and total phosphorus (TP) were measured on days 0, 7, 14 and 28, and the 16S rRNA of bacterial communities in the recta and water of the mussels were analyzed through high-throughput sequencing. In total, 44 bacterial phyla and 1 171 genera were identified in 21 water samples and 21 rectal samples. At the phylum level, the composition of bacterial communities in the recta and water was similar, both containing Actinobacteria, Bacteroidetes, Firmicutes, Proteobacteria and Verrucomicrobia, but the differences in relative abundance were significant. The bacterial composition at the genus level was significantly different and only two dominant genera were the same, Polynucleobacter and Reyranella, which belong to the same phylum of Proteobacteria. The results of redundancy analysis (RDA) showed that pH and NO₂^--N had the greatest effects on the water and rectal microbiota, respectively. KEGG function predicted that the microbial functions in rectal and water microbiota had a clear difference, but both were mostly related to energy production and biosynthesis. Principal coordinates analysis (PCoA) and permutational multivariate analysis of variance (PERMANOVA) combined analyses showed that 2 microalgae feeding had no significant effect on the structure of rectal and water microbiota; the water microbiota varied significantly at different time points (P<0.05); the rectal microbiota was relatively stable. This study showed that the 2 microalgae had no significant effect on the structure and function of the rectal and water bacterical communities of H. cumingii, but the hydrochemical parameters had an important influence on the microbiota, the results provide a theoretical basis for further optimization of the intensive culture.

The fall webworm (Hyphantria cunea) is an important quarantine pest worldwide and a major invasive pest in China. Bacillus thuringiensis (Bt) is widely used insecticidal microorganisms, however the mechanism of Bt on H. cunea is still unclear. To analyze the binding characteristics of alkaline phosphatase (ALP) of H. cunea with Bt toxin and explore the insecticidal mechanism, the HcALP3 gene (GenBank No. MH796759) was cloned by PCR method, contained a 1 557 bp ORF which encoded 518 amino acids. The recombinant HcALP3 protein induced by isopropyl-β-D-thiogalactoside (IPTG) was expressed as a 56.65 kD recombinant protein in E. coli BL21. The transcription analysis of HcALP3 gene in different tissue and at different development stages were analyzed by qRT-PCR, the results showed that HcALP3 gene was expressed in every instar and was highly abundant in the fourth-instar larvae and midgut. Ligand blot assay showed that HcALP3 protein only bound to Bt cry1Ab35 toxin specifically, could not bind to Bt Cry1Ac and Cry9Ea10. The transcript levels of HcALP3 were reduced by 75.32% using RNAi technology for gene silencing of HcALP3. The dsRNA-HcALP3 injected larvae were reared on Bt Cry1Ab35, Cry1Ac and Cry9Ea10 protein, the corrected mortalities were 45.93%, 73.33% and 70%, respectively. The results revealed that the corrected mortalities induced by Bt Cry1Ab35 to dsRNA-HcALP3 injected larvae were significantly lower than those injected with dsRNA-egfp. In this study, the HcALP3 of H. cunea was identified and the function of HcALP3 was analysed, which provides basis for further study on the mechanism of Bt against H. cunea.

Agrilus zanthoxylumi is a significant trunk-boring pest of the Zanthoxylum bungeanum tree. In order to analyze its binding mode and ability with the host volatiles, the objective of this study is to clone the full-length cDNA sequence of the odor-binding protein gene (AzanOBP4), and analyze its binding mode and ability to host volatiles. Rapid amplification of cDNA ends (RACE) technique was used to clone the full-length cDNA of AzanOBP4 based on the transcriptome database of A. zanthoxylumi, and the nucleotide and deduced amino acid sequences of the gene were analyzed using different bioinformatics software. Moreover, the expression levels of AzanOBP4 in different tissues of adult were determined by qRT-PCR. I-TASSER software was used to develop the homologous model of AzanOBP4 and the model equality was evaluated using SAVES v6.0 and PROSA software. Using AutoDock to perform molecular docking analysis on the AzanOBP4 model and 14 host volatiles. The full-length cDNA of AzanOBP4 was 691 bp (GenBank No. MT318833), with the non-coding regions of 5' and 3' ends of 182 and 89 bp, respectively. ORF was 423 bp, encoding 140 amino acids. The encoded protein had 6 conserved cysteines belonging to the typical insect OBPs．Homologous sequence alignment analyses showed that AzanOBP4 had the highest amino acid sequence identity (83.57%) with AmalOBP3 from Agrilus mali, and the two sequence were clustered into one branch with 99% confidence in the phylogenetic tree. The AzanOBP4 gene was expressed in various tissues of both male and female adults, and the highest expression level was in female leg. Molecular docking showed that AzanOBP4 combined with 14 host volatiles mainly through hydrogen bonding, hydrophobic interaction and van der Waals force, and 4 cyclic host volatiles (α-caryophyllene, β-caryophyllene, α-cubebene and ç-muurolene) had a strong binding ability with AzanOBP4. Studies showed that AzanOBP4 was involved in the olfactory mechanism of identifying host plants, suggesting that it may play an important role during insect feeding and host locating and would lay a necessary foundation for revealing the molecular mechanism of olfaction in A. zanthoxylumi.

Aquatic plant Azolla is a mutualistic association among fern, Cyanobacterium and bacteria. At vegetative growth phase of Azolla, cyanobiont and bactbiont reside in the leaf cavity and symbiotically associate with the host plant. During the sexual reproduction of Azolla, cyanobiont was found to migrate into the sporocarp of Azolla. The presentt study aimed to explore the morphological and structural characteristics of the endo-bacteria during the sporocarp formation based on fluorescent microspcopic and electron microscopic observations using ultrathin sectioning, fluorescent in situ hybridazation and immno-gold labeling techniques. The results showed that the bacteria migrated into the developing sporocarp with cyanobiont and were positioned in the top area of pre-mature megasporocap. About 43% of the bacteria secreted numerous outer membrane vesicles. Some of them released DNA and fiberous polysaccharide-like substance, which became the main components of the matrix for the biofilm-like structure formed in the indusium of the megasporocarp. Around 25% bacterial cells were coated by the capsule, 17% of them accumulated poly-β-hydroxybutyrate granules in their cytoplasms, 9% of the bacteira appeared autophage-like or necrosis-like cell death. As megasporocarp matured completely, most of the bacteria were found to be inhabited in the biofilm-like matrix. These results illustrated that during the sporocarp formation of Azolla, the morphological and structural characteristics and cellular response of the endo-bactria might benefit them to keep the rhythm of the synchronous development with the host, then transfered to next symbiotic generation. Present study provides a new idea for artificial construction of crop-microbe symbiotic N₂-fixing system.

Screening lignin-degrading bacteria and studying its degradation properties could realize the efficient use of lignocellulosic, and provide high value-added raw materials for the chemical industry, and alleviate the contradiction of shortage of raw materials. In order to obtain more excellent and applicable bacteria for the biodegradation of lignin, a Bacillus sp. strain named MN-13 was screened from fresh manure of Bos taurus via heating enrichment, decolorization of aniline blue in plate cultivation combination with detection of enzyme activity. Strain MN-13 decolorized significantly aniline blue, oxidized Mn²⁺ and veratryl alcohol. According to morphology, physiological and biochemical characteristics, sequence analysis of 16S rDNA and gyrB gene, strain MN-13 was identified as Bacillus amyloliquefaciens (GenBank No. KP292553). Lignin degradation characteristics of strain MN-13 showed that this strain could efficiently degrade lignin in corn stalks with lignin-degradation rate of 44.8% and NDF-degradation rate of 29.0% after 24 d of inoculation. In the process of lignin-degradation mediated by strain MN-13, the ligninolytic peroxidase which was similar to lignin peroxidase (LiP) and manganese peroxidase (MnP) could be produced and the stage of highest enzyme activity was consistent with the period of the maximum lignin-degradation amplitude. Gas chromatography-mass spectrometry (GC-MS) analysis results of lignin degradation products showed that strain MN-13 could degrade lignin into H, G and S phenols by the fission of β-O-4, Cα-Cβ and Cβ-Cγ bonds and, oxidation of Cα and Cγ. Screening of B. amyloliquefaciens MN-13 provided a new ligninolytic microorganism which would enrich the bacteria resource capable of degrading lignin. Meanwhile, lignin degradation characteristics of B. amyloliquefaciens MN-13 would provide a reference for further application of Bacillus sp. strains in the lignin-degradation.

The growth differentiation factor 8 (GDF8) gene is a member of the transforming growth factor-β(TGF-β) superfamily. In addition to acting as a negative regulator of muscle growth, it also functions in various biological processes such as lipid metabolism and sugar metabolism. Recently, the functional role of GDF8 in reproductive organs has received increased interest. Studies have found that GDF8 and its receptors are expressed in uterus, ovaries, fallopian tubes, embryos and other tissues and play important physiological roles, including participating in follicular development and ovulation, regulating the synthesis of follicular steroid hormones, affecting the formation of extracellular matrix and the proliferation, differentiation and diffusion of granular cells, and is closely related to the outcome of pregnancy. This article will review the research progress of GDF8 in the reproductive system based on domestic and foreign literature, in order to provide a reference for subsequent research.

Pregnancy-associated glycoproteins (PAGs) immunoassay have been widely used for early pregnancy diagnosis in livestock. The use of an antibody as a probe for detection has some drawbacks of long preparation period and high cost. To enrich the diagnosis techniques of early pregnancy in dairy cows (Bos taurus) and reduce detection cost, a enzyme-linked aptamer assay (ELAA) was developed for visual detection of bovine pregnancy-associated glycoprotein 9 (bPAG9) based on sandwich-type format strategy. Several experimental conditions had been investigated, including the concentration of aptamer, horseradish peroxidase (HRP)-aptamer conjugates and blocking agent, incubation time, and coating buffer. Under the optimal conditions, the calibration curves for bPAG9 showed good linearities in the range of 0.5~100 ng/mL with correlation coefficients (R) better than 0.998. The detection limit of 0.22 ng/mL by the instrument, and the limit of visual detection was found to be 0.5 ng/mL by naked-eye observation. The recovery experiment was performed by spiking blank samples with 1, 10 and 100 ng of bPAG9. The average recoveries of bPAG9 were in the range of 93.2%~105.8%, with relative standard deviations of 3.51%~7.13%. Subsequently, the 60 serum samples from pregnant and nonpregnant cows at 30 d after insemination were analyzed by the proposed ELAA. The test results were confirmed with ultrasonography. The diagnostic accurate rate of ELAA was 91.7%. This suggested that the proposed method had good accuracy and reliability in real samples. The enzyme-linked aptamer assay was shown to provide a simple, economical, reliable rapid detection technology for pregnancy diagnosis in dairy cows.

Rhizoctonia solani, the pathogen of potato black scurf, has been a threatening factor to potato (Solanum tuberosum) production. R. solani in the soil mainly uses the sclerotium structure as the initial source of infection for latent dormancy, and its density is positively correlated with the incidence of potato black scurf disease. In order to quickly and accurately detect the density of R. solani and sclerotia in soil, TEF3/TEF7 specific primers were designed based on the conservative region of translation elongation factor (TEF) of R. solani anastomosis group 3 (AG3). A real-time quantitative PCR (qPCR) detection system for quantification of R. solani AG3 was developed using recombinant plasmid of 191-bp amplification product as the standard, and the sensitivity of qPCR (19.5 fg/µL) was 1 000 fold higher than that of common PCR. Common PCR and qPCR tests were carried out on suspected potato moles (n=18) and diseased soil (n=41) in 4 regions. The detection rate of potato moles was 100%, but common PCR (65.85%) failed to detect all DNA in the diseased soil, while the detection rate of the diseased soil by qPCR was 97.56%. In order to determine the pathogen quantity in the early detection of diseased soil in the field, the soil with different amount of sclerotium was artificially simulated, and qPCR detection was carried out by wet-sieving method. The curve relationship between cycle threshold (Ct) and sclerotium content in soil was established. The results showed that regression equation was y=-3.982x+12.687, and correlation coefficient R² was 0.986, showing good linear relationship, and the detection limit of artificially simulated pathogen quantity was 5×10^-6 g/g soil. The detection system developed in present study could detect sclerotium density of AG3 in soil rapidly and accurately. Detection of sclerotium content of R. solani in soil before planting could provide technical support for early warning of potato black scurf.

Sisal purple leafroll disease (SPLD) is a devastating disease that has occurred on sisal (Agave sisalana) in recent years. Previous studies by this research group have shown that the disease is highly associated with phytoplasmas. It is necessary to establish an efficient molecular detection technology for in-depth research on phytoplasma or disease monitoring and detection. To this end, this study intends to establish an efficient single-tube nested PCR detection technique for phytoplasma related to sisal purple leafroll disease. Firstly, the annealing temperature of inner and outer primers in the single tube nested PCR reaction system was studied by single-factor test. After the annealing temperature was determined, an orthogonal design was used to optimize key factors such as inner and outer primer concentration, dNTPs concentration, and Ex Taq enzyme dosage of single-tube nest PCR detection system. The results showed that when the annealing temperature of outer primer Sis-F1/R1 was determined to be 64 °C, and inner primer Sis-F2/R2 was 54 °C, the best reaction system (25 μL) was finally selected by orthogonal design test as follows: 10×Ex Taq Buffer 2.5 μL, 2.5 mmol/L dNTPs 5 μL, Ex Taq DNA Polymerase 1 μL, 15 μmol/L inner primers Sis-F2/R2 each 1 μL, 0.02 μmol/L outer primers Sis-F1/R1 each 1 μL, template DNA 1 μL, ddH₂O 11.5 μL. The above reaction system only amplified bands from the DNA template of the plants with purple leafroll disease, which had a high degree of specificity. The lowest detection limit of the detection system was a phytoplasma concentration of ≥ 1 fg/μL. The single tube nested PCR reaction system for the detection of phytoplasma related to sisal purple leafroll disease established in this research could provide technical support for follow-up researches on disease monitoring, correlation investigation, pathogenicity and functional verification.

Wheat crown rot is an important soil-borne disease occurring widely in most wheat (Triticum aestivum) growing areas in the world. Fusarium pseudograminearum is the dominant causal agent of wheat crown rot. In F. pseudograminearum strain FC136-2A, mycovirus Fusarium pseudograminearum megabirnavirus 1 (FpgMBV1) was found to cause the hypovirulence of FC136-2A on wheat. To explore the mechanism of hypovirulence caused by FpgMBV1, a pCAM-GFP-hyg construct was transformed into F. pseudograminearum strain FC136-2A by Agrobacterium tumefaciens-mediated genetic transformation technology. Molecular identification, fluorescent observation, biological characterization and pathogenicity test of the transformants were also conducted. Results showed that the GFP-labeled transformant FC136-2A-GFP had no biological differences compared to FC136-2A in growth rate and sporulation capacity. Besides, FpgMBV1 was still in the transformant FC136-2A-GFP and the pathogenicity of FC136-2A-GFP was low on wheat. The present study obtained GFP-labeled transformant FC136-2A-GFP of F. pseudograminearum, which could be used to observe the infection and pathogenic process of hypovirulence strain FC136-2A on wheat, and provide basic information for further study on the mechanism of hypovirulence effect of mycovirus FpgMBV1 on F. pseudograminearum.