Abstract Bacillus thuringiensis (Bt) has become one of the most widely uses and most successful microbial insecticides in the world because of its insecticidal effect, specificity to the target organism, harmlessness to humans and animals, non-polluting environment, no harm to the natural enemies of pests, and high efficiency. However, there is a serious problem that Bt and its ICPs sprayed on crops have a short survival period under sunlight, which seriously hinders the further utilization of Bt. Therefore, in order to enhance the insecticidal effect of Bt, it is a key measure to prolong the persistence time of Bt. Bacterial biofilm (BBF) can improve the UV resistance of bacteria and regulate the adaptability of bacteria to environmental stress. Based on a previous comparative genomic analysis of our lab, a putative biofilm gene 00940 from Bt XL6-was identified (GenBank No. CP013000.1). According to bioinformatics analysis, 00940 gene is likely to be a glutamine synthase, which is regulated by SigL, CcpA, DegU and LexA. In Bacillus subtilis, SigL is an enhancer located downstream of the B. subtilis glutamate dehydrogenase gene and transcribes the rocG gene encoding glutamate dehydrogenase;CcpA transcription factor is involved in the breakdown of metabolites; DegU controls the gene expression of flagella formation and biofilm formation; LexA protein is induced in the case of DNA damage and is transcription of the bacterial SOS DNA repair system inhibitor. It is speculated that these transcription factors also play a similar role in Bt. The 1 kb upstream sequence and 1 kb downstream sequence of 00940 gene were obtained using PCR, as well as the kanamycin resistant gene. The 00940 gene knockout box containing the above three sequences was inserted into the thermosensitive vector pMAD, and the resulting recombinant plasmid was transformed into Bt XL6-. A 00940 gene knockout mutant Bt XL6-Δ00940ΩKm was then obtained. Bt XL6- was used as a control for the phenotypic analysis of the knockout mutants. Growth curve measured data showed that the lack of 00940 gene did not affect the growth of strains.The swarming motility of Bt XL6-Δ00940ΩKm was significantly higher than that of Bt XL6-. Based on laser scanning confocal microscopy, the biofilm thickness of the mutant was higher than that of Bt XL6-. Herein, lack of the 00940 gene improve the biofilm formation of Bt XL6-. The results lay a good foundation for the elucidation of the 00940 gene function.

Abstract Termites (Isoptera) often eat wood rich in cellulose, and there are much bacteria producing cellulase in vivo. The aim of this study was to screen a strain of producing highly active cellulase from termites to amplify the cellulase gene of this strain and express it in Escherichia coli, which had a potential basis for the the construction of cellulase gene recombinant bacteria. Collecting termites samples from Foping County of Shaanxi province, this study screened 4 strains of producing cellulase from termites (Reticulitermes chinensis) in vivo by cellulase screening medium and measurements of cellulase activity; then optimized the fermentation conditions were conducted by single factor experiment on the strain which producing the highest active cellulase, and studied the enzymatic properties of cellulase. Finally, according to the data of NCBI primers which amplified the cellulase genes were designed and prokaryotic expression was completed. The results showed that the 4 cellulase producing strains screening from termites were Enterobacter asburiae, Bacillus cereus, Bacillus subtilis and Bacillus amyloliquefaciens, respectively. The cellulase activity of the Bacillus subtilis was the highest, and the optimum fermentation temperature and pH of the strain was 42 ℃ and 6.5. The optimum temperature and pH of enzymatic reaction was 70 ℃ and 4.5, under this condition, the cellulase activity reached 2.36 U/mL. Under the condition of 50 ℃ and pH 6.5, the thermal stability and pH stability of the cellulase produced by the strain were optimum. Using crude enzyme solution to ferment crude fiber, it was found that the degradation rate of crude enzyme solution to four kinds of feed, corn (Zea mays) stalk , wheat (Triticum aestivum) straw, alfalfa (Medicago polymorpha) hay and silage corn stalk was 12.21%, 1.30%, 3.24% and 17.42%, respectively. Gene cloning results showed that the Bacillus cereus strain contained two cellulase genes BaG and Cbs. Under prokaryotic expression of the cellulase gene, the proBaG didn't show any cellulase activity, while the proCbs showed the optimum conditions of 60 ℃, pH 5.0, cellulase activity reached 4.14 U/mL. Under the condition of 50 ℃ and pH 5.0, the thermal stability and pH stability of the cellulase were optimum. Finally, the fusion expression of BaG and Cbs gene showed a new protein of about 35 kD while BaG and Cbs gene expressed the protein with a molecular weight of 27.4 kD and 55.2 kD, respectively. In the optimum conditions of 70 ℃ and pH 4.5, the cellulase activity of the fusion product expression reached 4.57 U/mL. Under the condition of 50 ℃ and pH 4.5, the thermal stability and pH stability of the cellulase were optimum. This study indicated that the BaG gene without cellulase activity could interact with the Cbs gene and may produce a new protein with higher cellulase activity and smaller molecular weight. This study should have important theoretical value for the construction of recombinant bacteria which could degradate lignocellulose.

Abstract Japanese flounder (Paralichthys olivaceus) is one of the important commercial marine fish in China. The embryo cryopreservation of Japanese flounder have been successful, however, there has been no study on the gene expression profiling in its embryos under low temperature. In this study, embryos at somite stage, tail bud stage and heart-beating stage were treated at 0 ℃, respectively, while embryos was cultured at 16.5 ℃ as the control. The stability of 4 candidate genes were analyzed by using GeNorm and NormFinder software, including 18S rRNA, ACTB, GAPDH and EF1α. Results showed that the stability of 4 genes were 18S rRNA=EF1α＞ACTB＞GAPDH by using the GeNorm software; while, the stability of 4 genes were 18S rRNA＞ACTB＞EF1α＞GAPDH by using NormFinder software. Therefore, 18S rRNA was supposed as the most stable housekeeping gene in Japanese flounder embryos incubated in low temperature. In order to study the influence of low temperature on gene expression of Japanese flounder embryos, embryos at tail bud stage were treated at 0 ℃ as embryos at this stage were more adaptive to temperature variations. Embryos culturing at 16.5 ℃ were used as control group. The expression variations of 2 genes, including cold-inducible RNA-binding protein gene (CIRP) and heat shock protein gene (Hsp70) were measured and analyzed by using qRT-PCR. It was found that CIRP mRNA expression decreased within 30 min, increased and reached the peak in 120 min, and then decreased again in 180 min to the level that was no significant different from the control. Hsp70 mRNA expression dropped in the first 30 min of treatment, then increased and reached the peak in 120 min; however, it decreased during 120~180 min, which showed significant difference from the control group (P＜0.05). These results indicated that low temperature would trigger the change of gene expressions of CIRP and Hsp70. Meanwhile, the variation trend suggested that there could be a protection mechanism in cells against low temperature. This study is a good basis for the further exploration on the adaption to low temperature of Japanese flounder embryos.

Abstract Aspartic proteinases (APs) are a family of protease enzymes, which plays pivotal roles in plant growth and development. This paper aims to investigate the preliminary biological function of APs in Rosa hybrida. A aspartic protease gene (AP) was cloned from Rosa hybrida 'Samantha' and named RhASP1 (GenBank No.: MG384616) by using reverse transcription-PCR (RT-PCR) method. The RhASP1 transcription expression characteristics were also examined using quantitative real-time PCR (qRT-PCR). The biological function by overexpressing RhASP1 in Arabidopsis thaliana was also investigated. The results showed that the open reading frame (ORF) of RhASP1 was 1 287 bp, which encoded 428 amino acids and belonged to B type APs. Qualitative PCR analysis showed that RhASP1 transcript expression levels was induced by NaCl treatment. Moreover, 24 h ethylene treatment and 3 h dehydration treatment significantly upregulated RhASP1 expression, respectively (P＜0.05). RhASP1 was overexpressed into Arabidopsis thaliana wild type (WT) by agrobacterium-mediated method. Three independent RhASP1-overexpressing lines with different expression levels were obtained to explore its biological function. The morphological and physiological performance of RhASP1 overexpressing lines were also investigated under salt stress. The seeding rates, increment primary root length and later root number of RhASP1-overexpressing plants were significantly higher than that of the WT plants under salt stress conditions(P＜0.05). The reactive oxygen species (ROS) levels of WT and RhASP1-overexpressing plants were also examined by histochemical staining. Both the RhASP1 transgenic and controls plants accumulated much brown sediment and blue sediment, whereas the RhASP1 transgenic plants were much lower than in the WT plants. The RhASP1-transgenic plants accumulated significantly less O2· and H2O2 compared with WT plants (P＜0.05). These findings examined preliminary biological function of RhASP1, and will provide new gene resources and basic theory foundation for stress breeding in Rosa hybrida.

Abstract The premelanosome protein gene (PMEL) can directly initiate the formation of fibers in the melanin small body and promote the melanosome synthesis, which is one of the main candidate genes to regulate animal coat color. At present, there are relatively few researches on the differentially expressed genes of the coat color of the Vulpes lagopus. This research was designed to study the activity region and transcription factors of PMEL gene in Vulpes lagopus. The gene promoter sequence of Vulpes lagopus PMEL gene was obtained through transcriptome sequencing technology, and the method of bioinformatics was used to predict the core promoter region of PMEL gene and the transcription factor binding site. Using Vulpes lagopus DNA as template, promoter deletion fragments of different lengths of the gene were amplified and ligated to pGL3-Basic vector. The recombinant plasmid was transiently transfected into A375 and 293T cells, and the activity was verified by the dual-luciferase assays system. The results showed that 8 fragments with different lengths of promoter regions were amplified and cloned into the vector pGL3-Basic.The region -1 162/+8 of PMEL gene in Vulpes lagopus was detected by dual-luciferase assays system as the core promoter region. There were some positive regulatory elements in the region from -1 162/-655. The bioinformatics prediction analysis revealed that there were 5 transcription factor binding sites in the region. Using the overlap extension PCR technology successfully constructed 5 mutation vectors. Their activity was significantly decreased by dual luciferase assay. It was suggested that these 5 transcription factors were the positive regulatory elements in Vulpes lagopus PMEL gene transcription. In this study, the core region of PMEL gene promoter were identified that 1 162/+8. Sp1 (-966/-952), Sp1 (-912/-900), Sp1 (-750/-736), NF-1 (-712/-699) and NF-1 (-682/-673) binding sites were the positive regulatory elements of Vulpes lagopus PMEL gene.This study provides a scientific basis for exploring the gene expression regulation mechanism, and provides ideas for Vulpes lagopus fur quality breeding and creating new materials.

Abstract Porcine contagious pleuropneumonia (PCP) is a serious respiratory disease with great harm to pigs (Sus scrofa), caused by Actinobacillus pleuropneumoniae (APP) infection. It is one of the major epidemic diseases in the world pig industry. Existing vaccines can not give full protection against App infection, so it is necessary to screening new vaccines. In this study, immune protection of recombinant NLPI (rNLPI)vaccination against App challenged infection was detected. Based on APP nlpi sequences in GenBank, the primers were designed. APP nlpi sequence was obtained by PCR from APP genome. Sequence analysis and function predication was made by bioinformatics software. The results revealed that the length of this gene (GenBank No.: MH027649) was 900 bp and its predicat protein was a 299 amino acid resides of an puptative outer membrane lipoprotein. Homology analysis showed that it shared high sequence similarity between different serotypes of APP. The APP nlpi was inserted into pET32a (+). Recombinant protein rNLPI was produced in Escherichia coli BL21 after induced by isopropyl β-D-1-thiogalactopyranoside (IPTG) and then determined by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and Western blot. The 34 kD-protein was detected and showed good immunogenicity. The protective efficacy of rNLPI experiment was tested in 6-week-old specific-pathogen-free BALB/c mice (Mus musculus). The serum immunoglobulin G (IgG) level was assayed by enzyme-linked immune sorbent assay (ELISA). The results showed that 50 μg rNLPI+Frund's incompleted adjuvant, 30 μg rNLPI+ Frund's incompleted adjuvant and 10 μg rNLPI+Frund's incompleted adjuvant and 30 μg rNLPI respectively induced 41%, 22%, 10% and 20% survival rate. No mice survived the challenge in adjuvant group and control group. The rest living mice of experiment groups slowly returned to normal health status. From above, rNLPI induced a partial immune protection of mice against App challenged infection. The study accumulated data for exploring new vaccines of porcine contagious pleuropneumonia.

Abstract The patatin like phospholipase domain-containing 3 (PNPLA3), also known as fat cell nutrients or calcium-independent phospholipase A2ε (iPLA2ε), is a member of the PNPLAs family. PNPLA3 has the activity of hydrolyzing phospholipids and neutral lipids and ester acyltransferase activity, and is able to participate in fat accumulation and fat mobilization. The objective of the present study is to investigate the characteristics of expression and regulation of PNPPLA3 in liver of chicken (Gallus gallus) and to pave the way for further elucidating the biological function of PNPLA3 gene in lipid metabolism of liver in chicken. The amino acid sequences of PNPLA3 in chicken and 12 other different species were retrieved from the genomic data bank using the Basic Local Alignment Search Tool (BLAST) homology search. Bioinformatic methods were used for phylogenetic and syntenic analysis. The qRT-PCR technique was used to analyze the temporal and spatial expression of PNPLA3 gene in different tissues of chicken. The regulatory mechanism of PNPLA3 gene was analyzed through stimulating juvenile hens in vivo, and chicken embryo hepatocytes treated with 17β-estradiol and estrogen receptor antagonists MPP, ICI182780 (fulvestrant) and ICI146474 (tamoxifen) in vitro. The results showed that the amino acid sequence similarity of PNPLA3 among chicken and amphibian and fish species was relatively higher than that among chicken and mammalian species. The PNPLA3 gene was extensively expressed in various tissues tested, in particular, the expression levels were higher in pectoral muscle, abdominal fat and pancreas in 10-week-old chicken, higher in liver, pectoral muscle, abdominal fat in 30-week-old chicken (P＜0.01). The 17β-estradiol treatment significantly increased the expression level of PNPLA3 in chicken liver tissue and primary hepatocytes (P＜0.05). The MPP, a specific antagonist of estrogen receptor ER-α, could completely inhibit the effect of 17β-estradiol on PNPLA3 expression (P＜0.05). Therefore, it is concluded that the similarity of the amino acid sequence of PNPLA3 is higher between chickens and amphibian and fish, however, it is lower to mammalian. PNPLA3 was abundantly expressed in liver tissue, and its expression level was significantly increased after maturation. The expression of PNPLA3 was regulated by estrogen, and the regulative effect was predominantly mediated via ER-α in liver of chicken. These findings lay the foundation for further understanding the regulation mechanism of fat metabolism in chicken.

Abstract The traditional rice-fish coculture system is considered a sustainable form of agriculture that provides rice grain and fish for farmers in the world. Cyprinus carpio rubrofuscus, as a high quality local varieties paddy field carp, has been cultured in paddy fields of the north region of Guangdong province with a long history. In order to understand the effect of artificial culture and selective breeding on the genetic diversity and genetic structure of C. carpio rubrofuscus and to provide the study basis for preservation of germplasm resources and utilization, 16 microsatellite markers were selected and used to compare the genetic diversity of one generation of selectively bred population (F5) and 2 landraces (Ruyuan population and Lechang population) in C. carpio rubrofuscus in this study. The results showed that all selected primers were polymorphic and 119 alleles were detected from these 3 populations. The number of alleles detected on each locus varied from 3 to 10 with 7.44 alleles per primer pair on an average. In the 3 populations, the average expected heterozygosity (He) were 0.636 0, 0.698 9 and 0.775 1; the Shannon's diversity index (I) were 1.206 3, 1.402 0 and 1.612 2; and the average polymorphism information content (PIC) were 0.570 1, 0.645 8 and 0.720 7，respectively. It is suggested that the genetic diversity level of all the 3 populations were high (PIC＞0.5000), but the level of genetic diversity of the tow landraces was higher than that in F5 generation of selectively bred population. The genetic differentiation index (Fst) among all the locus was 0.102 8 with the value ranged from 0.044 0 to 0.246 8 indicated a moderate level of differentiation in the 3 populations. Pairwise Fst values also indicated that the 3 populations had moderate genetic differentiation with the highest value (0.182) between the selectively bred population (F5) and the Lechang population indicating a greater level of differentiation. While the value between the Ruyuan population and Lechang population was lowest (0.058), indicating a moderate level of differentiation. Among the 3 populations, the genetic distance was 0.205 6~0.622 4 and the genetic identify index was 0.536 6~0.814 2. The selectively bred population (F5) was the furthest with the Lechang population (0.6224) and the Ruyuan population was nearest with the Lechang population in terms of genetic distance (0.2056), while the genetic identity between the selectively bred population (F5) and Lechang population was lowest (0.5366) and the highest between the Ruyuan population and the Lechang population (0.8142). The NJ clustering tree based on the genetic distance by using the unweighted pair group method with arithmetic (UPGMA) demonstrated that the 2 landraces (Ruyuan population and Lechang population) clustered together firstly and then clustered with the selectively bred population (F5). This study suggested that the selective breeding work was efficient and the artificial selection has enlarged genetic differentiation between the breeding and landraces populations while decreased the genetic diversity within the breeding populations, but the high genetic diversity and genetic potential were maintained in the breeding population, indicating that there is great potential for future selections of C. carpio rubrofuscus through selective breeding. This study would provide valuable information for genetically breeding in C. carpio rubrofuscus in the future.

Abstract Streptococcus pneumoniae is one of the major human (Homo sapiens) pathogens and leads to approximately 1.6 million deaths annually in the world. Owing to the abuse of the antibiotics, S. pneumoniae generated antibiotic resistance, especially the multiresistance to many antibiotics. Thus it is urgent to develop new antibiotics and vaccines. Based on bioinformatics analysis，a gene SP_0010 (GenBank No.: A0A0H2UMY6-1) was identified from S. pneumoniae strain TIGR4 that encodes a putative β-lactamase. In this study, the potential β-lactamase gene SP_0010 was amplified by polymerase chain reaction and inserted into the pET28a(+) vector to obtain the recombinant expression plasmid, which was then transformed into Escherichia coli strain Rosetta (DE3) for expression. The sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) results indicated that the optimum induction temperature of the recombinant protein Sp_0010 was 16 ?C for 20 h, meanwhile, the target protein was eluted with 250 mmol/L imidazole from Ni-NTA and loaded onto a Superdex-200 column for further purification. The recombinant Sp_0010 existed as a monomer in solution and has high purity. Peptidoglycan binding assay revealed that the recombinant Sp_0010 showed a stronger binding affinity to teichoic acid-free peptidoglycan. All these findings revealed that protein Sp_0010 had an important role in peptidoglycan metabolism. Thus it would have great significance to elucidate the mechanism of pneumococcal antibiotics resistance.

Abstract Zinc finger proteins (ZFPs) are a class of transcriptional factors that play important roles in growth, development and stress responses in plants. Finding more genes which have key function in cold stress response can give support for cold tolerance breeding in Eucalyptus trees with molecular assisted selection. A zinc finger protein gene induced under treatment at low temperature was screened in Eucalyptus grandis. The gene was named EgrZFP7. EgrZFP7 protein structure and its function were analyzed in the research.This gene is a classical C2H2 type zinc finger protein gene. The protein sequence it encoding contains 2 zinc finger motifs, 1 L-Box motif and 1 EAR motif. EgrZFP7∷GFP vector was constructed and subcellular localization of EgrZFP7 was verified by bombardment of onion epidermis with a gene gun.The result showed that it localized in nuclear. The vector of 35S∷EgrZFP7 was also constructed and transformed into Arabidopsis thaliana. Two homozygoteslines of EgrZFP7-OX1 and EgrZFP7-OX2 were obtained. Under normal growth condition, the 2 EgrZFP7 over-expression lines showed more lateral roots and the length of lateral roots were also longer than that of the wide type. However, when EgrZFP7-OX1 and EgrZFP7-OX2 were treated at -8 ℃ for 3 d, then recovered for 3 d under normal temperature, the speed of growth recovery of over-expression lines was slower than that of the wild type. Finally, death percentage of total seedlings EgrZFP7-OX1 and EgrZFP7-OX2 lines were obviously higher than that of the wild type, implying that over-expression of EgrZFP7 could increase sensitivity to low temperature in seedlings. A protein, EgrERF4 (Eucalyptus grandis ethylene response factor 4), which can interact with EgrZFP7 was screened and verified from Yeast Two Hybrid Library. It could be concluded that EgrZFP7 possibly play a role in gene regulation in cold stress response through interact with EgrERF4 in Eucalyputs grandis. The present study gives more information to learn the mechanism of cold stress response in Eucalyptus.

Abstract Cucurbita ficifolia is one of characteristic germplasm crops that has strong resistance abilities to many disease pathogens stress, especially resistance to Fusarium oxysporum. Based on the degenerated primers being designed according to the conserved regions of NBS type genes in other plants, eight RGA sequences were cloned and sequenced from Cucurbita ficifolia genome by PCR amplification. Among them, the sequence of HQRGA2 had got NB-ARC structure domain and would be classified an individual category, yet the other seven sequences hadn’t got the NBS domains by multi alignment sequence contrast. Furthermore, the results of nucleotide homology analysis indicated that 98% and 97% of nucleotides of HQRGA2 were identity to the resistant genes SQRGA-13 and SQRGA-8 from Cucurbita moschata respectively. Furthermore, the amino acid sequence analysis results showed that the sequence of HQRGA2 belonged to the class of Non-TIR-NBS-LRR disease-resistance genes analogues, which contains the conserved regions of P-loop, Kinase-2, Kinase-3, and GLPL domain. We also obtained that 96.6% amino acids were homology to RGAs of Cucurbita moschata by comparison analysis of amino acid sequences. The results of qRT- PCR showed that the expression of HQRGA2 from C. ficifolia was influenced and induced by the fungi of Fusarium oxysporum, with the expression pattern of ‘up-down’, which indicated its relation to resistance gene in the aspect. The obtaining of NBS type of R-gene analogues provided us a clue for the further cloning of disease- resistance genes from Cucurbita ficifolia., Abstract Cucurbita ficifolia is one of characteristic germplasm crops that has strong resistance abilities to many disease pathogens stress, especially resistance to Fusarium oxysporum. Based on the degenerated primers being designed according to the conserved regions of NBS type genes in other plants, eight RGA sequences were cloned and sequenced from Cucurbita ficifolia genome by PCR amplification. Among them, the sequence of HQRGA2 had got NB-ARC structure domain and would be classified an individual category, yet the other seven sequences hadn’t got the NBS domains by multi alignment sequence contrast. Furthermore, the results of nucleotide homology analysis indicated that 98% and 97% of nucleotides of HQRGA2 were identity to the resistant genes SQRGA-13 and SQRGA-8 from Cucurbita moschata respectively. Furthermore, the amino acid sequence analysis results showed that the sequence of HQRGA2 belonged to the class of Non-TIR-NBS-LRR disease-resistance genes analogues, which contains the conserved regions of P-loop, Kinase-2, Kinase-3, and GLPL domain. We also obtained that 96.6% amino acids were homology to RGAs of Cucurbita moschata by comparison analysis of amino acid sequences. The results of qRT- PCR showed that the expression of HQRGA2 from C. ficifolia was influenced and induced by the fungi of Fusarium oxysporum, with the expression pattern of ‘up-down’, which indicated its relation to resistance gene in the aspect. The obtaining of NBS type of R-gene analogues provided us a clue for the further cloning of disease- resistance genes from Cucurbita ficifolia.

Abstract Astragalus sinicus is an important nectariferous plant and green manure crop. It plays an important role in promoting crop yield. Basing on the local farming system, selecting flowering varieties is an important breeding target of Astragalus sinicus. LEAFY (LFY) is a plant-specific flower meristem specific-gene. However, the study on LEAFY gene of Astragalus sinicus is not reported yet. In this study, in order to explore the function of LEAFY gene in Astragalus sinicus, the sequence of LEAFY in Astragalus sinicus was cloned by Illumina Hiseq method and rapid-amplification of cDNA ends (RACE) technology, and the bioinformatics analysis of LEAFY gene was performed by bioinformatics analysis software; The expression characteristics of the LEAFY gene in different tissues were analyzed by qRT-PCR; The function of LEAFY gene of Astragalus sinicus was identified after expression vector construction, infection and statistical analysis of phenotypic characteristics of T2 transgenic homozygous lines were performed. The results showed that the full-length cDNA of the LEAFY gene (GenBank No. MH352146) in Astragalus sinicus was 1 400 bp in length and contained the open reading frame of 1 191 bp; The encoded protein contains 396 amino acids, including 22 α-helices and 5 β-sheets, which has a molecular weight of 44.72 kD and a theoretical isoelectric point of 6.41; The similarity of the encoded amino acid sequences to the LEAFY proteins of Medicago truncatula, Medicago sativa, Cicer arietinum, and other species were all above 80%, which was highly homologous. The qRT-PCR analysis of LEAFY gene showed that the LEAFY gene was expressed in various organs, and the order from high to low of the expression levels were flower buds, flowers, leaves, leaf buds, roots, stems and pods. The results of the experiments with Arabidopsis thaliana over expressing LEAFY showed that the number of bolting days of transgenic Arabidopsis thaliana was 3 days earlier than that of wild type, and the time of transgenic A. thaliana flowering was 2 days earlier than that of wild type. The average number of flowers was 1.79 more than that of wild type. In summary, this study showed that the LEAFY gene had the high homology with the leguminous species, and was the highest expressed in the flower bud, and verified that the LEAFY gene might regulate the flowering mechanism. These results provide a scientific basis for the further development and utilization of A. thaliana by molecular technology, and are of great significance to agricultural production.

Abstract CD320 (cluster of differentiation 320) is the cobalamin transporter receptor, which belongs to the low-density lipoprotein receptor family. CD320 plays an important role in cobalamin transfer process and regulates many physiological processes in mammalian. This study was undertaken to obtain the sequence of goat CD320 gene and its expression patterns in different tissues and development stages in goat testicle. The cDNA of CD320 gene was cloned by RT-PCR, and was further bioinformatics analyzed. The qRT-PCR technology was utilized to detect the expression patterns of CD320 in different tissues (heart, liver, spleen, lung, kidney, skeletal muscle, ovary, testicle) in adult and expression profiles of various developmental stages. The results showed that the goat CD320 gene sequence was first obtained (GenBank No.:MG560828). The full-length cDNA sequence was 877 bp and the ORF was 768 bp; encoding a protein of 255 aa. The protein weight was 27.18 kD, had 2 LDLRA (low-density lipoprotein receptor type A) domains (residues 53~89 and residues 129~171) and a transmembrane region (residues 20~224). The results of the homologous sequence alignment showed that the protein of goat and other species in the core site of the LDLRA domain and the transmembrane region was highly conserved. The phylogenetic analysis showed that goat together with ruminants such as sheep (Ovis aries) and cattle (Bos taurus), belonged to the Bovidae of Ruminantia. The transcriptional expression displayed that CD320 expressed in all tissues (heart, liver, spleen, lung, kidney, skeletal muscle, ovary, testicle) and the expression of lung, testicle and muscle was significantly higher than other tissues (P<0.01). In the testicle, the expression of CD320 was lower in fetus and 2-months-old (suckling period), and increased rapidly in 6-month-old (sexual maturity), which was significantly higher than that in fetus and suckling period (P< 0.01), and then decreased. In the epididymis, overall, the expression of CD320 in the epididymis of fetal stage was lowest. The expression of sexual maturation was the highest in caput epididymidis and cauda epididymidis, and the difference was significant compared with other stages (P<0.01), and the expression decreased after body maturation. But the expression of suckling period was the highest in the corpora epididymidis, and there was a significant difference compared with other periods (P<0.01). These results suggested that CD320 may participate in the physiological process development of testicles, epididymis and spermatogonial differentiation in goat. This study provides the basic for further study on CD320 gene function in goat.

Abstract Goose hemorrhagic polyomavirus (GHPV) is a newly identified emerging infectious pathogen in ducks in recent years, China. In order to understand the genomic characterization of Cherry Valley duck (Anas platyrhynchos domestica) origin GHPV (designated as strain FJ201601), the whole genome of GHPV strain FJ201601 was amplified by PCR with five fragments by using specific primers. The obtained sequences were cloned and verified by Sanger sequence, and then the positive results were assembled. The results showed that the genome length of strain FJ201601 comprised of 5 254 nucleotides (GenBank accession number MG544856), with(G+C)% content at 48.01%, coding for 6 function genes with ORF-X (510 bp, 169 aa), VP2(981 bp, 326 aa), VP3(654 bp, 217 aa), VP1(1062 bp, 353 aa), Large T(1911 bp, 636 aa) and Small T(483 bp, 160 aa), respectively. Genomic nucleotide comparison demonstrated that GHPV strain FJ201601 shared nucleotide identity higher than 99.7% with other GHPV isolates, which were collected from different hosts and regions. The GHPV six functional genes were all showed high nucleotide and amino acids identity at ORF-X (99.4%~99.8% and 98.2%~99.4%), VP2 (99.7%~99.9% and 99.7%~100%), VP3 (99.7%~99.8% and 99.5%~100%), VP1 (99.5%~99.8% and 99.7%~100%), Large T (99.7%~100% and 99.8%~100%) and Small T (99.6%~100% and 99.4%~100%), respectively. Phylogenetic analysis indicated that GHPV isolates shared very closer evolution distance. However, there were subtle differences in genetic evolution different did not consistent with different hosts (different duck and goose species) and regions, the same as the Chinese Pekin ducks-origin GHPV and Cherry Valley ducks-origin GHPV. This study lays a good foundation for further epidemiological investigation and pathogenic mechanism study of the Goose hemorrhagic polyomavirus.

Abstract Myostatin (MSTN) is a negative regulator to muscle cells growth and differentiation. MSTN gene's mutation will lost its function, which will make animals have significantly more muscle mass. The purposes of this study are to find the best sgRNA which could edit sheep's (Ovis aries) MSTN gene efficiently and build EGFP and sgRNA co-expression vectors, with which CRISPR/Cas9 system could improve sheep MSTN gene's editing efficiency. First using Gibson Assembly method to incorporate the 2A+ enhanced green fluorescent protein (EGFP) into pX330, the study got the pX330-EGFP vector. Then 12 sgRNAs were designed and using Golden Gate method separately, inserted these single-guide RNA (sgRNA) oligonucleotides into pX330-EGFP plasmid, and 12 pX330-EGFP-sgRNA expression plasmids were got. The 12 pX330-EGFP-sgRNA vectors were transferred into sheep fibroblasts by electroporation. After 48 h, using SURVEROR analysis, it was be found that 3 groups (T2, T9, Q2) of cell's DNA were edited, which were groups of sgRNA. Then 100 green cells of each group were collected and extracted the DNA, after amplification of the MSTN gene by PCR, the productions were send for sequencing analysis. The results showed that the targeting efficiency of sgRNA-T2, sgRNA-T9 and sgRNA-Q2 were 40%, 40% and 60% respectively. In this study, we build EGFP and sgRNA co-expression vetors and selected the best sgRNA to sheep MSTN gene which was 60%. This protocol will be helpful to find more sgRNAs to different genes. These results provide a scientific basic for the production of MSTN gene editing sheep.

Abstract 5-hydroxytryptamine (5-HT) is essential to stimulate skeletal calcium reabsorption for milk synthesis by the mammary gland. Tryptophan hydroxylase (TPH1) is the rate-limiting enzyme in peripheral 5-hydroxytryphtamine (5-HT) synthesis and thus plays an essential role in 5-HT metabolism. Objective of this study is to generate stable TPH1 gene knockout goat (Capra hircus) mammary epithelial cell line by CRISPR/Cas9 system, which could be an important material for exploring functions of 5-HT and calcium circulatory metabolism in goat mammary glands. Firstly, single guide RNA (sgRNA) sequences were designed based on the sequence of TPH1 (GenBank No.: 102184739) and were inserted into pX458 and pX459 vectors, respectively. Then goat mammary epithelial cells (GMECs) were transfected with pCas-guide vectors and puromycin (1 μg/mL) was used for screening positive cells. Finally, Western blot and DNA sequencing were used for identifying if the TPH1 gene of the GMECs had been knocked out. In this study, 3 pairs of pCAS-sgRNA vectors targeting TPH1 gene exon 1 of dairy goat were designed and constructed by using CRISPR/CAS9 technique. The transfection efficiency reached 20%; Single cell line px459-sg2-SC5 was screened by puromycin and the editing efficiency was 32%; DNA sequencing result showed that there were 10 bp base deletion at sg2 targeting site. Protein expression of TPH1 was blocked. Taken together, goat mammary epithelial cell line with stable knockout of TPH1 gene was successfully obtained, which would provide important materials for the study of the regulation of mammary gland physiology by 5-HT.

Abstract Mycoplasma bovis (Mb) is an important pathogenic mycoplasma of cattle (Bos taurus). The biofilm formation of Mb can cause persistent infection in host cells and cause great economic loss to cattle industry. Therefore, the purpose of this study is to screen a dominant strain of Mb biofilm formation by identifying the biofilm formation ability of different Mb strains, and then optimize the cultural conditions on biofilm formation of Mb. First of all, the ability of biofilm formation of Mb PG45 strain, Hubei (HB) strain, Wuwei (WW) strain, Dingxi (DX) strain, and Lintao (LT) strain were identified by polystyrene microtiter plate assay, and then the strain with strong biofilm forming ability was used as the model strain to optimize the culture conditions of Mb biofilm formation. The results showed that the ability of biofilm formation was different among different strains of Mb, among which the biofilm formation ability of LT strain was the strongest, the next was HB strain. And the biofilm formation ability of PG45 strain, WW strain and DX strain was the weakest. The optimal cultural conditions for biofilm formation of Mb was that the Mb cultures (1.7E+08 CFU/mL) were inoculated at a dilution of 1∶10 into the Eaton medium containing 0.01 mg/mL fish sperm DNA, 20% serum of horse, 1% glucose and final pH 8.5, and then incubated at 37 ℃, with 5% CO2 for 72 h. Under the optimal cultural conditions, the biofilm formation ability of LT strain was sound. In this study, a dominant strain of Mb biofilm formation was obtained, and a stable and obvious biofilm was formed by optimizing the cultural conditions, which will provide excellent biomaterials for biofilm related study of Mb.

Abstract Long non-coding RNA (lncRNA) is defined as a class of long transcripts longer than 200 nucleotides which are not translated into protein. They were considered as dark matter of products in gene expression at the beginning. lncRNA plays an important role in diseases, cell cycle, stem cells differentiation and so on. The correlative molecular mechanisms are generally classified four types (signals, guides, decoys and scaffolds). Because of the huge quantities and complex mechanisms of long non-coding RNAs, this research field still needs to be explored. In this review, we combine researches in recent years and summarize the definition, classification, molecular mechanisms, biological functions and prospect, This article provides reference for the study of the mechanisms and functions of lncRNA.