Wheat (Triticum aestivum) is one of the important food crops in China. However, drought and other adverse conditions have seriously affected the quality and yield of wheat.Screening of stress-resistant genes and analyzing their mechanism of action can provide candidate genes and theoretical basis for wheat resistance molecular breeding. Xyloglucan endotransglucosylase/hydrolase (XET/XEH) is a class of enzymes with xyloglucan endoglycosyltransferase /hydrolase activity, which are collectively referred to as XTH, belonging to glycoside hydrolase 16 (GH 16) family. It can participate in the formation and remodeling of cell wall and plays an important role in plant growth and development and stress resistance. In this study, the cDNA sequence of the TaXTH-7A gene (GenBank No. MK395550) was isolated by PCR from wheat, and the coding region was 870 bp in length, which encoded 290 amino acid. The TaXTH-7A genome contained 3 exons and 2 introns. Protein sequence analysis indicated that TaXTH-7A contains several XET-specific domains and they were highly conserved across species. Phylogenetic tree analysis indicated that the XTH-7A protein evolved between monocotyledonous and dicotyledonous, and between C3 and C4 plants, and was closely related to Aegilops tauschii. The results of qRT-PCR showed that TaXTH-7A gene was expressed in roots, stems, leaves and spiket of wheat at heading stage, and it was dominant in roots. After drought stress, its expression increased and peaked at 4 hours. Subcellular localization results showed that TaXTH-7A was localized in the cytoplasm, cell membrane, and apoplast. Functional identification showed that over-expression of tTaXTH-7A enhanced drought resistance of Arabidopsis thaliana. This study is helpful to further study the function of TaXTH-7A gene in wheat, and also provides a theoretical basis for wheat resistance to abiotic stress.

Xyloglucan transglycosidase/hydrolase (XTH) is a key enzyme in the process of plant cell wall remodeling, and participates in the regulation of plant growth and development. To elucidate and explore the function of XTH in the process of stress response in maize (Zea mays), ZmXTH23 (GenBank No. LOC100191584) in XTH family was cloned from maize inbred line 'Chang7-2' and its biological function was studied. Bioinformatics analysis showed that ZmXTH23 contained a complete open reading frame of 897 bp encoded 298 amino acids and was a member of LamG superfamily. Besides, it performed xyloglucan endotransglycosidase (XET) activity and its amino acid sequence had the closest affinity with that of PmXTH25 (GenBank No. RLM56175.1) in Panicum miliaceum (similarity reached 90%). The expression pattern of ZmXTH23 was analyzed by qRT-PCR, and the results showed that the expression of ZmXTH23 was tissue-specific, it had the highest expression level in young stem (P＜0.01), and was induced by abscisic acid (ABA), NaCl and PEG6000. Prokaryotic expression analysis by SDS-PAGE and Western blot confirmed that ZmXTH23 protein could be expressed in Escherichia coli BL21 transferred with pET28a-ZmXTH23 recombinant plasmid. Besides, the growth of host bacteria pET28a-ZmXTH23 under different concentrations of salt and mannitol stress was better than that of host bacteria pET28a, which indicated that the ZmXTH23 protein enhanced the salt tolerance and drought resistance of pET28a-ZmXTH23 recombinant host strain. In conclusion, ZmXTH23 played an important role in response to abiotic stress and the results in this study provides theoretical basis for the creation of new maize germplasm resistant to stress.

Segregation distortion is an ubiquitous phenomenon in nature and an important driving force for biological evolution. With the development and progress of molecular marker technology, a large number of segregation distortion phenomena are reported in the genetic research of different crops and the proportion of marker segregation distortion and genetic effect vary according to species, population, marker type, etc., while there were segregation distortion regions (SDRs). In order to investigate the phenomenon of maize (Zea mays) segregation distortion and its causes, a F₈ population consisting of 198 recombinant inbred lines (RIL) was used in the study, which derived from a cross between a tropical maize inbred line B31-3 and a temperate maize inbred line HZ4. A total of 747 SNPs, covering 10 maize chromosomes, were detected with polymorphism between parent B31-3 and HZ4. Segregation distortion analysis was performed among the 747 SNPs for the RIL population. The results of chi-square test (χ² =96.92, >χ²_0.05=6.63)(P<0.05), showed that the distribution proportion of the female B31-3 and male HZ4 genotypes deviated from the theoretical ratio of 1∶1 in RIL population and the genetic contribution rate of female B31-3 to offspring was higher than that of male HZ4. The result indicated that 279 SNPs (37.3%) showed the genetic distortion (P<0.05). Of the total segregation distortion SNPs, 194 SNPs (69.5%) deviated toward female parent B31-3, while 85 SNPs (30.5%) deviated toward male parent HZ4. Totally, 10 SDRs were detected among 6 chromosomes. 3 SDRs were distorted to HZ4, while 7 SDRs were skewed to B31-3, which showed female parent gametophytes had an competitive advantage in forming a zygote in this study. There were 6~64 segregation distortion SNPs in SDRs and the span of SDRs were 3.69~78.11 cM. SDR5-1 and SDR5-3, detected in this study, were located in near regions where gametophyte genes related to segregation distortion had been reported. Additionally, in the study, the RIL population was affected by high temperature in the group building process. So, the occurrence of segregation distortion and the formation of SDRs might be related to gametic selection and high temperature environment in this study. This study provides basic data for understanding the law of segregation distortion of maize.

Breeding new herbicide-tolerant transgenic soybean (Glycine max) varieties is effective means to balance supply and demand of soybean in China. However, the safety evaluation of new varieties of transgenic crops should be carried out before they are commercialized, and genetic stability is one of the important evaluation factors. A new glyphosate-resistant gene AM79-EPSPS with national intellectual property rights has been cloned by Chinese researchers, but there is no report on its application in transgenic soybean. In this study, the progenies of A6 and A8, two transgenic AM79-EPSPS soybean lines, were taken as the research objects in order to analyze the genetic stability of T₁ and T₂ generation transformant materials based on DNA level, transcription level, translation level and tolerance level of the transformant to glyphosate, respectively. PCR was used to detect the major functional elements (including promoter, target gene and terminator) of the inserted fragments in two generation plants (T₁ and T₂) of A6 and A8. The main functional elements, the 35S promoter, AM79-EPSPS gene and nos terminator could amplify the target band, indicating that the main functional elements were inserted completely in different generations. Southern blot analysis was performed to detect the insertion copy number of the target gene AM79-EPSPS in T₁ and T₂ generation plants of A6 and A8 transformants. The result showed that the target genes at different generations were inserted through a single-copy insertion into the genomes of the 2 transformants, and the integration of every generation was consistent with each other. Transcription levels of AM79-EPSPS were detected by RT-PCR, and the result showed that AM79-EPSPS gene was expressed in different tissue of T₁ and T₂ generation plants in A6 and A8 transformants, indicating that the gene expression of AM79-EPSPS in A6 and A8 transformants remained stable between generations. The protein expression of AM79-EPSPS in T₁ and T₂ generation of A6 and A8 transformants was detected by Western blot and ELISA. The results of Western blot showed that in the T₁ and T₂ generation of A6 and A8, exogenous protein AM79-EPSPS could be labeled by EPSPS antibody, and the molecular weight of AM79 antibody-labeled protein was within the range of 47~50 kD, which was consistent with the predicted size of the protein. The results of ELISA analysis showed that the expression levels of AM79-EPSPS protein were different in the same tissues of A6 and A8 in different generations, but there was no significant difference. Therefore, the expression level of the target protein AM79-EPSPS was relatively stable between different generations. Verification of stable inheritance of transformant tolerance to glyphosate by spraying glyphosate. The results showed that the plants of different generations of transformants A6 and A8 could tolerate glyphosate at seedling stage, and there was no significant difference in tolerance between the 2 generations, but the negative control died. Therefore it could be concluded that the transformants demonstrate intergenerational stability in terms of the tolerance to glyphosate in herbicides. This study indicated that these 2 transformants were expected to become materials for future spreading and application,and that AM79-EPSPS gene could be used as a candidate gene for breeding commercial glyphosate-resistant soybean and other crops. Furthermore, this study also provides a methodological basis for the national genetically modified crops safety assessment, and promotes the development process of domestic genetically modified crops industry.

Flower development has vital significance for the reproduction of plant offspring, and the genes of MADS-box family play an important role in this process. In order to explore the function of the MADS-box gene transcription factor 6 (Trans6) in the flower development of sunflower (Helianthus annuus), HaTrans6 gene (GenBank No. XM_022141206.1) was cloned from sunflower and was analyzed systematically. The information analysis of gene sequence showed that HaTrans6 had typical MADS-box and K-box conserved domains, and HaTrans6 was a candidate member gene of MADS-box. Phylogenetic tree analysis showed that HaTrans6 gene had the closest homologous relationship with Agamous-like 13 gene (AGL13) and AGL6 gene of Arabidopsis thaliana. The expression pattern analysis indicated that HaTrans6 gene was highly expressed in flower and immature seed. The high and stable expression level was observed from floral organ primordial stage to 5 d after flowering, and reached its top level at floral organ primordial stage. In addition, it was shown that HaTrans6 gene was expressed in bract, crown, petal, stamen, pistil and ovary on 5 d before and the very day of flowering, and the highest expression abundance was found in the ovary, followed by bracts. The above results suggested that Hatrans6 gene might play an important role in regulating floral development and early fruit development of the sunflower. The present study provides basic data for further investigation about the function of Hatrans6 gene.

As a novel bioreactor, the salivary gland bioreactor specifically expresses various foreign proteins by using the regulatory region of salivary secretory proteins. The commonly used salivary secretory protein is parotid secretory protein (PSP).The aim of this study is to establish a method for site-specific integration of foreign genes into PSP gene using the CRISPR/Cas9 system and analyze the off-target effect of the selected gRNA on PSP gene. Firstly, sgRNA sequences were designed online according to PSP gene sequence and 5 sgRNA sequences were selected. Then, using sgRNA in vitro transcription kit and Cas9 in vitro digestion kit, the sgRNA sequence with high in vitro activity which targeting PSP gene were screened out. At the same time the co-expression vectors of pCas9-sgRNA for sgRNA and Cas9 and a targeting vector pMD19-5'arm-NeoR-3'arm carrying the expression structure of neomycin resistance gene (NeoR) were constructed. Finally, sgRNA and Cas9 co-expression vector and target vector were co-transfected into porcine kidney cells (PK-15 cells), screened with neomycin, and single cell clones were isolated. Positive cell clones which was knocked-in neomycin resistance gene (NeoR) were identified by PCR and sequencing, the off-target effects of every sgRNA were analyzd. The results of in vitro enzyme digestion showed that sgRNA1, sgRNA3, sgRNA4 and sgRNA5 had higher activity in vitro. Sequencing results indicated that the 4 sgRNAs were successfully targeted the forigen gene into the PSP gene of Sus scrofa. The knock-in efficiency of sgRNA1 and sgRNA5 was 22.7% (5/22) and 26.1% (6/23), with 1 potential off-target site had off-target effect; The efficiency of sgRNA3 and sgRNA4 was 50.0% (12/24) and 42.1% (8/19), with no off-target effect at the 5 potential off-target sites. In this study, the method for gene editing of pig PSP gene was successfully established, and screening for sgRNAs that efficiently guide foreign genes into the pig PSP gene provides basic data for further research on transgenic Sus scrofa.

Identification of functional genes and mutations that have large genetic effects on milk production traits can provide molecular information for marker-assisted selection in order to increase selection accuracy and accelerate genetic gain in dairy cattle (Bos Taurus). Nowadays RNA-seq is one of strategies for identification of key genes affecting complex traits in human (Homo sapiens), domestic animals and plants. In previous RNA-seq study on the transcriptomes of liver tissues from Chinese Holstein at different lactation stages (50 d before parturition , 15 d after parturition, 60 d after parturition), 40 candidate genes for milk production traits were identified. Of them, the fructose-bisphosphatase 2 gene (FBP2) was significantly differentially expressed among different periods. It encoded a gluconeogenesis regulatory enzyme which participated in glycogen synthesis from carbohydrate precursors and played an important role in gluconeogenesis. Thus, the purpose of this study was to further validate whether the FBP2 gene showed significant genetic effects on milk production traits in dairy cattle. By re-sequencing the entire exons, and 2 000 bp of 5' and 3' flanking regions of FBP2 gene using pooled DNA, 11 single nucleotide polymorphisms (SNPs) were detected, including 7 in 5' flanking region and 4 in 3' flanking region. These identified SNPs were genotyped with matrix-assisted laser adsorption time of flight mass spectrometry (MALDI-TOF-MS) technology and tested for association with 5 milk production traits in 1 099 Chinese Holstein from Beijing region. All 11 SNPs were found to be statistically significant for protein yield (P=0.0495~<0.0001). Also, 6 of them were significantly associated with protein percentage (P=0.0434~0.0004), 5 of them were significantly associated with milk yield (P=0.018~0.007) and 1 SNP was significantly associated with fat yield (P=0.0143). The additive and allele substitution effects of the most SNPs reached significance (P<0.05, P<0.01). On the other hand, with linkage disequilibrium (LD) analysis, the 11 SNPs were found to be highly linked (r²=0.48~0.99), and 2 blocks were inferred. Haplotype-based association analysis showed that the 6 haplotype combinations in block 1 were significantly associated with the 5 milk traits (P=0.0413~<0.0001), and the haplotype combinations in block 2 were significantly associated with milk yield, fat yield, protein percentage and protein yield (P=0.0413~<0.0001). H2 and H3 in block1 and H2 in block2 were advantegous haplotypes for higher milk yield and composition. This study first indicated that the FBP2 gene had significant genetic effects on milk yield and milk composition traits in dairy cattle, especially affecting milk protein, and could be useful to provide valuable gene information for molecular breeding programs of Chinese Holstein.

Long chain acyl-CoA synthetase 1 (ACSL1) plays a key role in the synthesis of triglycerides, phospholipids and cholesterol esters and the oxidation of fatty acids. The Bos grunniens milk has rich nutrition and higher milk fat. However, there are few studies on the genetic mechanism of milk quality traits. In this study, Gannan yak was used as the research object, and the polymorphic loci of the promoter region of yak ACSL1 gene were detected by DNA sequencing, and the association analysis were performed to evaluate the effects of different haplotypes combination and genotypes on milk quality traits. This study found that 3 SNPs in the promoter region of the ACSL1 gene in the Gannan yak population, which were g.2079A>T, g.2409G>A and g.1235_g1236delTG deletion. In the F1 region of the yak ACSL1 promoter, the animals with MN genotypes had significantly higher the milk protein rate than those with genotypes MN (P<0.05). The animals with BB genotypes had significantly higher total solid matter content and milk fat percentage than those with genotypes AA and AB (P<0.05). In the promoter F2 region, the animals with DD genotypes had significantly higher milk fat percentage than those with genotypes CC. 8 haplotypes and 5 diplotypes were constructed, in which the diplotype H4H4 had significantly higher (P<0.05) or extremely significantly higher milk fat content and total solid content than those with other doplotype (P<0.01). Therefore, the mutation in the promoter region of yak ACSL1 gene can be used as a potential genetic marker for the quality traits of Gannan yak milk which also provide theoretical basis for the molecular genetic research of yak milk quality traits.

Lysine-specific histone demethylase 1B (KDM1B) is required for de novo methylation of some imprinted genes in oocytes. The aim of this study was to clone the CDS sequence of the KDM1B gene of yak (Bos grunniens), and to analyze the expression characteristics of KDM1B in various tissues and the process of in vitro maturation of oocytes in yak. In this study, the CDS region of KDM1B was amplified from yak ovary cDNA by PCR.The physic-chemical properties, structure and conservation of KDM1B were predicted by bioinformatics. The expression of KDM1B in different tissues and in the process during oocyte in vitro maturation was detected by qRT-PCR. Result showed that the CDS of yak KDM1B gene was 1 737 bp (GenBank No. MK806390), encoding 578 amino acids. The molecular weight of protein encoded by KDM1B gene in yak was 64.90 kD. The theory isoelectric point was 7.20, and the grand average of hydroparthicity was -0.190, and the instability index was 41.73, predicted that KDM1B was a hydrophilic protein; The secondary structure of KDM1B consisted of α-helix, β-corner, and random coils. The amino acids sequence of KDM1B contains N-terminal double zinc finger (zf-CW), N-terminal SWIRM (Swi3p/Rsc8p/Moira, SWIRM) and C-terminal amine oxidase (AO) domain. The protein was predicted to be lacking signal peptides and cross membrane. Moreover, the conservation analysis showed that yak KDM1B gene had high homology comparison of mammals such as zebu (B. indicus) and cattle (B. taurus). The expression of KDM1B mRNA was found in various tissues of yak, and the relative expression was highest in small intestine. During in vitro maturation (GⅤ phase, MⅠphase, MⅡphase) of oocytes, the mRNA expression level of KDM1B was extremely significantly higher in MⅡ oocytes than that in GⅤ and MⅠ stages (P＜0.01), but no significant difference between GⅤ and MⅠ stages. These results would be beneficial for studying the role of KDM1B in the reproductive process in yak.

Mongolian sheep (Ovis aries) and Dorper sheep, as excellent sheep breeds in China and South Africa, have good performance in stress resistance and mutton quality, but that little is known about the regulation of their heat response. In this study, By Illumina HiSeq system, the skeletal muscles of Mongolian sheep and Dorper sheep were sampled and RNA-sequence (RNA-seq) under heat condition (＞30 ℃), and further detected 252 differentially expressed protein-coding genes (including 109 annotated genes) and 60 long non-coding RNA genes. Based on gene functional enrichment, pathway enrichment analysed and previous studied for differentially expressed genes, 19 heat-response-related protein-coding genes were screened out. Immediate early response 5 (IER5), arly growth response 1 (EGR1), sprouty related EVH1 domain containing 1 (SPRED1), testis expressed 15, meiosis and synapsis associated (TEX15), etc 15 genes were involved in heat-response of skeletal muscle in Mongolian sheep. Angiopoietin like 7 (ANGPTL7), circadian associated repressor of transcription (CIART), ATP binding cassette subfamily A member 12 (ABCA12), calcium and integrin binding family member 2 (CIB2) were involved in heat-response of skeletal muscle in Dorper sheep. The biological functions of above 19 protein-coding genes contained cellular heat response, heat shock protein binding, calcium ion binding, folding-competent state maintaining of denatured protein and DNA repair etc. Heat-response-related BCL2 associated athanogene 3 (BAG3), DnaJ heat shock protein family (Hsp40) member A4 (DNAJA4), heat shock protein family A (Hsp70) member 1 like (HSPA1L), EGR1, IER5, DNAJB1 and myosin light chain 10 (MYL10) were trans-regulated by lncRNA transcripts of long non-coding RNA genes XLOC_1944721, XLOC_3483583, XLOC_775492, etc. Above heat-response protein-coding genes and long non-coding RNA genes can make people comprehend heat regulation in sheep, while the genes also provide molecular theoretical basis for livestock breeding on targeting heat-resistant.

Major histocompatibility complex gene (MHC) can encode antigen-presenting molecule to initiate immune response of the body, which has great potential in animal disease-resistance breeding. In this study, the correlation between MHC-DQB2 gene and Coccidia infection in Shanbei white cashmere goats (Capra hircus) was analyzed to provide basic data for the mining and application of disease-resistant genes in goats. MHC-DQB2 gene exon 2 was analyzed by bioinformatics and population genetics in 104 Shanbei white cashmere goats, infection of Coccidia was detected and the correlation between DQB2 and coccidiosis was analysed. A total of 15 alleles and 27 genotypes were obtained. The results showed that DQB2 had high polymorphism and under positive selection pressure in the course of evolution, the frequency of each allele in the population had significant difference. In this study, the infection rate of Coccidia was 71.43%, and moderate intensity infection (1×105≥oocyst per gram (OPG) ≥1×104) was predominant. Correlation analysis revealed that DQB2*03 allele and 0311 genotype were negatively correlated with Coccidia infection (P＜0.05), indicating that DQB2*03 allele and 0311 genotype might probably be the susceptibility allele or genotype of coccidiosis. The results of this study will help to clarify the role of MHC molecular markers in the immunity of Shanbei white cashmere goats, provide experimental data support for the correlation between MHC genes and diseases, and promote the application in disease-resistance breeding.

Growth arrest-specific gene 6 (GAS6) is differentially expressed during the differentiation of chicken preadipocytes and may be involved in regulating the fat deposition of chicken (Gallus gallus domesticus). However, its tissue distribution and developmental expression pattern in chicken is yet unknown. To study the tissue distribution and developmental expression profiles of GAS6 gene, six 4-week old Jinmao Black chickens (3 males and 3 females) were used. qRT-PCR was used to detect the tissue distribution of chicken GAS6 genes in 8 tissues (heart, liver, spleen, lung, kidney, breast muscle, leg muscle and abdominal fat). The developmental expression pattern of GAS6 gene in chicken abdominal fat was detected at different growth stages (1-day, 4, 8, 12, 16-week old). The results showed that GAS6 gene was highly expressed in abdominal fat and moderately expressed in lung and kidney of male and female chickens. The expression pattern of GAS6 gene in the abdominal fat showed trends of “M” and “W” type at different developmental stages of male and female chickens. The expression peak appeared before 4-week old both in male and female chickens. This current study combined with high-throughput sequencing results showed that GAS6 gene might influence the early development of abdominal fat by regulating the proliferation and growth arrest of adipocytes in chicken. This study revealed the tissue expression profiles of GAS6 gene in Jinmao Black chickens and analyzed its developmental expression pattern in abdominal fat, which provides theoretical basis for the further study on the function and expression regulation mechanism of GAS6 gene in chicken fat deposition.

The large economic losses caused by leg disorders have raised concerns in the broiler (Gallus gallus) industry. Valgus-varus deformity (VVD) is an outward or inward angulation of the intertarsal joint tibiotarsal bone that results in deviation of the tarsometatarsus. The growth performance and the serum biochemical indicators of VVD broilers and normal broilers were measured at 18, 28 and 38 d. And the bone morphometry of VVD broilers and normal broilers was measured at 28 and 38 d. The results showed: 1) Body weight, shank length and girth of the VVD broilers were significantly or extremely significantly lower than those of normal birds at 18, 28 and 38 d (P＜0.05 or P＜0.01). 2) Compared with normal broilers, the contents of triglyceride (TG) (28 and 38 d) and high density lipoprotein cholesterol (HDL-C)(18 and 38 d) were significantly or extremely significantly lower in VVD groups (P＜0.05 or P＜0.01). The total cholesterol (T-CHO) content (18 d) and low density lipoprotein cholesterol (LDL-C)(18, 28 and 38 d) were significantly or extremely significantly higher in broilers with VVD than in normal birds (P＜0.05 or P＜0.01). 3) The serum Ca level was extremely significantly higher at 18 and 38 d (P＜0.01) in VVD groups.. The serum Ca, ALP (28 d) and the P (18 d) were significantly or extremely significantly lower at 28 d (P＜0.05 or P＜0.01) in VVD groups. Generally, VVD resulted in decreased growth performance and abnormal changes in blood lipid, serum calcium, serum phosphorus and serum alkaline phosphatase levels in broilers. The aim of this study was to explore the effects of VVD on growth performance, serum content and bone morphometry in broiler, to provides reference for study of VVD and leg health in broiler.

Different feeds will affect the metabolism and the intestinal structure and flora of grass carp (Ctenopharyngodon idellus), which further change its muscle quality and immunity. So, the purpose of this study was to explore the effects of three feeds on the metabolism and growth of grass carp, including grass feed (Pennisetum sinese), broad bean (Vicia faba) and artificial compound feed. Serum enzyme activities related to metabolism and intestinal histological were detected, and then the abundance of intestinal flora and changes in metabolic pathways were further analyzed by high-throughput sequencing technology. The results of three feeds showed that, in the grass carp fed with grass feed, there were the highest superoxide dismutase (SOD) activity, integral intestinal structure, the highest abundance of both probiotics and carbohydrate metabolic pathway. Broad bean feeding increased trypsin (TRY) activity, damaged intestinal tract, increased the abundance of pathogenic bacteria and disease transmission pathway. In the grass carp fed with artificial compound feed, the growth rate (74.52%), activity of alkaline phosphatase (ALP) and abundance of glycan biosynthesis and metabolism pathway were highest, and the intestinal tissue structure was relatively complete. These results demonstrated that, in the grass carp, Pennisetum sinese grass meal feed was helpful for maintaining intestinal health and enhancing metabolism, and broad bean led to the intestinal damage and inflammation, and artificial feed caused a certain degree of intestinal inflammation and immune suppression in spite of the highest nutritional value. This study would provide guidance for the selection and matching of different feeds and the research of new artificial feed in grass carp.

Endophytes, which can produce the same or similar metabolites as host plants, is an important resource for screening novel secondary metabolites. Thus, it has great significance to isolate and screen antagonistic endophytic bacterium from the wild Polygonatum cyrtonema and study the antibacterial activity of its metabolites. The endophytic bacterium HJ-2 (GenBank No. MK774703) isolated and screened from the rhizome of the wild P. cyrtonema was identified as Bacillus subtilis. In order to furtherly utilize the strain, different media were used for fermentation of the strain. The active substances were extracted from the bacterial cell of strain HJ-2 and its fermentation broth by methanol, ethanol, n-butanol, acetone and ethyl acetate, respectively. All the extracted products were used for physicochemical properties and antibacterial activity study. Our results showed that the antibacterial active substances produced by the strain were presented in the fermentation broth and had significant antibacterial effects against Escherichia coli, Staphylococcus aureus, and Pseudomonas aeruginosa. The fermentation products were further tested for acid and alkali resistance and heat resistance. The results showed that the products could resistance to high temperature of 65 ˚C, but had a poor performance of acid and alkali resistance. This study provides a theoretical basis for the exploration of endophytic bacteria resources of P. cyrtonema from Anhui province and the comprehensive development and utilization of strain HJ-2.

Mycoplasma synoviae (MS) is an important avian pathogenic Mycoplasmas that can lead to respiratory tract diseases, arthritis and synovitis in chickens (Gallus gallus) and turkeys (Meleagris gallopavo), which resulted in reduction in egg production and hatchability, poor egg quality, growth retardation, low feed conversion rate and carcass condemnation, thus caused serious economic losses to the poultry industry. Therefore, the study on detection method and good immune preparation for MS would provide good support for the rapid diagnosis and effective prevention of MS infection. Base on this, the primers were designed according to the sequence of the pdhA and pdhB gene of MS WVU1853 strain (GenBank No. CP011096.1) in GenBank, and the gene pdhA, pdhB and junction fragments AB of both in a single nucleotide overlap were amplified. After completing the point mutations of pdhA using over-lap PCR, the optimized pdhA and pdhB were respectively cloned into the pETDuet-1, the fragments AB was cloned into pET-28a(+), and the prokaryotic co-expression vectors pETDuet-AB and pET-AB were constructed. Then the pETDuet-AB and pET-AB were transformed into Escherichia coli BL21 (DE3), and the recombinant proteins were expressed with induction by isopropyl β-D-thiogalactoside (IPTG). Subsequently, the expression products were purified and its enzyme activity was analyzed. The results of SDS-PAGE have showed that the pdhA and pdhB of MS WVU1853 strain were co-expressed in E. coli BL21 (DE3), and both recombinant proteins were highly expressed in BL21 (DE3) transformed by pET-AB. However, the expression level of recombinant proteins pyruvate dehydrogenase E1 alpha subunit (PDHA) was significantly lower than that of pyruvate dehydrogenase E1 beta subunit (PDHB) in E.coli transformed by pETDuet-AB. The results of the enzyme activity analysis had showed that the purified expression products from BL21 (DE3) transformed by the pETDuet-AB or pET-AB had enzyme activity. This study provides a prospective material for further study of the biological functions of pyruvate dehydrogenase E1 alpha subunit and beta subunit from MS, also provides possible markers for the establishment of diagnostic methods and possible vaccine candidates for MS infection, as well as provides a new idea for the co-expression of the 2 genes.

The mastitis is one of the most common diseases mainly caused by pathogenic microorganisms for female mammals, especially in cattle and sheep, and also has long been focused highly by researchers in the field concerned. Under the background that mastitis cannot be effectively treated with conventional approaches because of its complexity in the pathogenesis, the rising of omics technologies including transcriptomics provide good technology platforms and research ideas for investigating pathogenic mechanism of pathogens and resistant mechanism of host during mastitis. In this paper, the recent progresses in research on the application of transcriptomics in cattle and sheep mastitis were reviewed. This review offers references for the in-depth study of molecular mechanisms of host defense against bacterial infections and diagnosis and prognosis of mastitis.

Arbuscular mycorrhizae (AM) fungi are the oldest and most extensive plant symbionts on the earth, which promotes good plant growth and is also a green environment-friendly pest control pathway. This paper is based on the research status of AM fungi control pests for many years. From the mechanism of AM fungi on plant pest control, it clarifies two ways in which AM fungi change the coping mechanism of symbiotic plants in the face of pest invasion: 1) Promote the plant to produce secondary metabolites to strengthen chemical defense and improve plant pests direct or indirect defense ability to enhance the insect resistance of plants; 2) Improve plant tolerance to pests by improving plant growth, plant nutrition, root activity, etc. This review provides a theoretical basis for biological control of plant pests and prospects for the development direction of AM fungi and the application prospects of insect pests.

Culture of hemocytes in vitro has been preliminarily studied in many aquatic crustaceans, but different researchers have not reached a broad consensus in the study of the key culture conditions, such as medium type, serum concentration and osmotic pressure. Culture of hemocytes in vitro is of great importance for further molecular study in Chinese mitten crab (Eriocheir sinensis). In order to investigate the optimal culture condition in vitro, such as medium, osmotic pressure and serum concentration, cultivation efficiencies of hemocytes in Chinese mitten crab from 5 different culture medium (1 × L-15, 2 × L-15, 3 × L-15, Sf-900 and TC-100 medium, respectively), osmotic pressures (345, 569, 803, 991 and 1215, mOsm/kg, respectively) and serum concentrations (0%, 5%, 10%, 15% and 20%, respectively) were analyzed. In addition, this study also detected the relative gene expression in the cultured hemocytes. The results showed that the TC-100 medium was the best culture medium according to morphological profiles and survival rate of cultured hemocytes. Furthermore, the best cultivation conditions were determined under the condition of osmotic pressure of 991 mOsm/kg and 0% bovine serum, which showed the best morphological profile and more than 50% of survival rates after 10 d cultivation. Expressions of the 3 house-keeping gene (ubiquitin-conjugating enzyme E2b (UBE), ubiquitin/ribosomal S27 fusion protein gene (S27) and β-actin) of Chinese mitten crab were steadily detected in hemocytes in continuously cultured for 10 consecutive days. The results have revealed that the medium and the key culture conditions could better cultivate hemocytes of Eriocheir sinensis, and the cultured cells could be used in gene function experiments. This study provides a new potential technical method for the study of gene function of Eriocheir sinensis.