Abstract Plant purple acid phosphatase (PAP) is crucial in response to low phosphorus stress. To further validate its function in phosphorus absorption, Pinus massoniana (PmPAP1) gene was genetically transformed into tobacco (Nicotiana tabacum) 'Xanthin' via Agrobacterium-mediated method, and 25 transgenic PmPAP1 lines were obtained through PCR detection in the current study. With semi-quantitative RT-PCR and qRT-PCR analysis, 13 transgenic PmPAP1 lines were highly over-expressed in transcriptional level of PmPAP1, among which 2 transgenic PmPAP1 lines (Line-13 and Line-10) were treated with different concentration of phosphorous for 45 d, and Semi-quantitative RT-PCR and qRT-PCR were used to detect the expression of PmPAP1 gene in transgenic plants under different phosphorous concentration. Meanwhile, the total phosphorus content, inorganic phosphorus content, phosphatase (APase) activity in roots and leaves, leaf protective enzyme activity, leaf content of malondialdehyde (MDA) as well as biomass indexes of wild type(WT) and transgenic PmPAP1 plants were measured. The results indicated that the expression of PmPAP1 were up-regulated in leaves and roots of the transgenic PmPAP1 tobacco as subjected to phosphorus deficiency in comparison with the normal condition (control), and the expression of its was bare in roots and leaves with the high phosphorus treatment. Under the condition of low phosphorus, the activities of acid phosphatase (APase) in roots and leaves of transgenic PmPAP1 tobacco were elevated 1.27 and 1.25 times respectively, compared to the WT plants, and the contents of total phosphorus and inorganic phosphorus increased by 28.32%, 45.48% in Line-10 and 36.45%, 62.31% in Line-13 respectively compared with the WT plants. Furthermore, the activities of peroxidase (POD) and superoxide dismutase (SOD) content analysis between transgenic PmPAP1 tobacco and WT plants revealed that the transgenic PmPAP1 tobacco were significantly elevated (P＜0.05) compared to those of the WT plants. The average activity of POD and SOD in transgenic PmPAP1 tobacco were 1.56 and 1.42 times of the WT plants respectively, while the results showed that the content of MDA in transgenic PmPAP1 tobacco leaves decreased significantly (P＜0.05). The MDA concent of transgenic PmPAP1 tobacco decreased 29.62% and 23.41% in line-10 and line-13 respectively compared with the WT plants. Effect of phosphorus on the growth of plants revealed that the total biomass and root biomass as well as the ratio of root to shoot of the transgenic significantly increased (P＜0.05) in transgenic PmPAP1 tobacco. Under the condition of no-phosphorus and low-phosphorus, the average root weight were 48.57% and 78.19% which were higher than that of the WT plants respectively, and the average root to shoot ratio increased by 60.28% and 53.02% compared with the WT plants respectively. Under the condition of normal phosphorus, there were no significant difference between the transgenic PmPAP1 tobacco and WT in phenotype of plants, while the transgenic PmPAP1 tobacco grew better than WT under phosphorus deficiency and demonstrated the high tolerance to low-phosphorus. There was no obvious difference in the enzyme activities, MDA concent, phosphorus concent and biomass of transgenic and WT plants under the condition of adequate phosphorus. Therefore, the over-expression of PmPAP1 might considerably enhance the tolerance to low-phosphorus stress in transgenic PmPAP1 tobacco, suggesting the involvement of this gene in phosphorus utility, particularly under low-phosphorus stress. These foundings provide basis theory for the better understanding for molecular mechanism of low-phosphorus tolerance in Pinus massoniana and creation of novel tobacco germplasm with low-phosphorus resistance.

Abstract In masson pine (Pinus massoniana) breeding programs, lack of co-dominant genetic markers highly constrained the development of molecular marker assisted breeding. In order to comprehensively elucidate the germplasm genetic information and develop more new molecular markers suitable for P. massoniana, currently, an EST-SSR marker system was established based on transcriptome data obtained by high-throughput sequencing technology of this tree, and their characteristics were analyzed. Consequently, the distribution patterns of the markers in the transcriptome sequences the SSR-PCR system for P. massoniana was established and examined with 27 individuals. Results indicated that a total of 70 896 unigenes were screened by MISA software from P. massoniana transcriptome, 3 329 SSR loci occurred in 3 074 unigenes. Around 2 750 unigenes contained a single SSR loci, and the occurrence frequency was 3.88%. Average of 223 unigenes contained 2 or more than 2 SSR loci, and the occurrence frequency was 0.31%. One hundred and one unigenes contained the mixed SSR loci, and the occurrence frequency was 0.14%. The frequency of these SSRs was 4.69%, and the mean distance was 20.94 kb. Among the SSR locis, mononucleotide, trinucleotide and dinucleotide were the major types, accounting for 40.16%, 32.83% and 20.94% of the total, respectively. A/T, AT/AT and AAG/CTT were the most frequent motifs in mononucleotide, dinucleotide and trinucleotide repeats, accounting for 97.68%, 71.45% and 21.70% of the total, respectively. Totally, 200 pairs of primers were randomly selected for amplification, and their amplification rate was 78.5%. A total of 137 SSR markers were scored from 27 accessions of P. massoniana amplified by 24 pairs of primers with superior polymorphism, and the polymorphism information content (PIC) was 0.703. Among selected pairs of primers, PmS33 belonged to low polymorphism loci, the polymorphism content was 0.166, PmS88 and primer PmS97 were moderate polymorphism loci, the polymorphism content were 0.346 and 0.263, respectively, and the others were highly polymorphic point. And the average observed number of alleles (Na), effective number of alleles (Ne), Nei's genetic diversity (I) and Shannon index of diversity (H) in the 27 P. massoniana gemplasms were 6, 3.9, 1.338 and 0.66, respectively. The tested germplasms could be completely distinguished by primers Pms164 and Pms184. The manual cultivar identification diagram (MCID) map of tested germplasms was constructed by EST-SSR markers. The coefficient of the 27 germplasms ranged from 0.32 to 0.92 with the threshold of 0.63. All the germplasms were grouped into 3 subclusters by the unweighted pair-group method with arithmetic means (UPGMA) the first major group included 3 red heartwood clones and some fast growing clones, the second major group included all the clones which were fast growing clones, and the third major group included 5 high fat clones. Therefore, a group of SSR primers with high polymorphic potential was designed based on the sequencing of the transcriptome of P. massoniana. The primers might be effectively used to classify 27 clones, the cluster analysis somewhat collaborated with that based on the wood characters as well as the origin of the germplasms, thus limited primers could provided a comparatively polymorphic genetic information. Conclusively, EST-SSR markers based on transcriptome information are feasible, which may provide a theoretical basis for the construction of genetic map, draw fingerprinting and calibration of target genes for P. massoniana germplasm resources.

Abstract　Low temperature is one of the major environmental factors that influence rice (Oryza sativa) growth, development and production. Rice seedlings are particularly sensitive to chilling in early spring. Therefore, improvement of chilling tolerance in rice may significantly increase rice quality and production. To identify genes participated in the regulation of plant chilling tolerance, the cold-resistant gene library was constructed through suppression subtractive hybridization (SSH) in a previous study. From the SSH library, a transcription factor TIFY1b (LOC_Os03g52450) with an conserved TIF[F/Y]XG domain, which might involve in the rice cold resistance, was isolated. To explore the function of TIFY1b and its homology gene TIFY1a (LOC_Os03g47970), the technology of “clustered regularly interspaced short palindromic repeats/CRISPR-associated protein 9 (CRISPR/Cas9)” was used to edit their genome. According to the characteristics of CRISPR/Cas9 editing system and the conservation of target genes (TIFY1a and TIFY1b), 2 sites of 20 nt guide RNA (gRNA) targeted to the exon of TIFY1a were designed and named A1 and A2; 2 sites of 20 nt gRNA targeted to the exon of TIFY1b were designed and named B1 and B2. A1, A2, B1 and B2 were all ligated and constructed individually to the CRISPR/Cas9 vector with single target site to create tify1a or tify1b single mutant; A1 and B1 were together ligated and constructed to another CRISPR/Cas9 vector which harbors 2 target sites to create tify1a & tify1b double mutant. Then, these recombinant plasmids were all transferred to a rice cultivar Nipponbare by Agrobacterium-mediated transformation method. T0 transgenic mutants were obtained and confirmed by hygromycin-resistance screening. Further sequencing for the genomic DNA of TIFY1a or TIFY1b gene locus in T0 transgenic lines showed 60% mutagenesis frequency in target A1 site, 87.5% mutagenesis frequency in target A2 site, 35% mutagenesis frequency in target B1 site, 75% mutagenesis frequency in target B2 site, and 62.5% mutant ratio of lines containing both target A1 and target B1. These results suggested that CRISPR/Cas9 systems can effectively induce site-specific mutations in T0 rice plants. Moreover, the results found various targets of mutant types presented in the T0 transgenic mutants, such as deletion of bases, insertion of bases, insertion behinds deletion of bases, and long fragment deletion of genomic DNA. The major mutation type in this study was 1 bp insertion and 1 bp deletion mutation. In addition, The results found that the positions of the mutation could occur in the upstream of the proto spacer adjacent motif (PAM) region ranging 4~7 bp, and the mutation occurred in the 4 bp upstream of the PAM region was more often. Consequently, 3 different mutation types mediated by the CRISPR/Cas9 system were formed in T0 transgenic lines, including homozygous mutation, biallelic mutation and heterozygous mutation. All these mutation types could descend stably into the next generation. Furthermore, protein analysis indicated that frameshift or premature of proteins which caused function loss of TIFY1a or TIFY1b gene happened in T0 transgenic mutants. Collectively, a series of different types of tify1 mutant lines were successfully obtained by using CRISPR/Cas9 technology in this study and could be used to investigate the role of TIFY1 genes in rice adaptation to chilling temperature. Our studies might reveal a novel pathway that controls cold adaptation in rice and will help to broaden the possibilities for genetically engineering cold-tolerant rice cultivars.

Abstract Stripe rust (Puccinia striiformis f.sp tritici) is a severe fungal disease in wheat (Triticum aestivum) that results in decreased yield and quality. It is important to explore a new way of improving stripe rust resistance with introducing exogenous resistance genes in wheat breeding. In this work, the Agrobacterium-mediated transformation method was adopted to introduce eucommia ulmoides chitinase1 (EuCHIT1) gene into wheat variety 'Guizi 3', and T1 trangenic plants were obtained according to the result of β-glucuronidase (GUS) histochemical staining and PCR identification.The chitinase activities in the transgenic wheat plants and wild-type were measured, and the resistance to stripe rust, and protective enzymes activities and relative expression level of pathogenesis-related protein genes were measured. The results indicated that chitinase activity in the transgenic plants, 3 328.63 U/g FW, was 42.21% which was higher than that in the wild-type. After inoculating with race CYR32 for 7 d, the catalase (CAT), super-oxide dismutase (SOD) and peroxidase (POD) activities were 211.91, 448.37 and 81.30 U/g FW in the transgenic plants and 159.95, 294.38 and 37.87 U/g FW in the wild-type plants, respectively. This showed that the CAT, SOD and POD activities in the transgenic wheats were 32.48%, 49.76%, and 114.68%, respectively, which were higher than those in the wild-type. The results also indicated that the malondialdehyde (MDA) content in the transgenic plants was 8.69 nmol/g FW which was 29.23% lower than that in the wild-type plants (12.28 nmol/g FW). After inoculating with the fungus, the transgenic plants delayed the occurrance of stripe rust disease for 9 d compared to the wild-type. The identification of the resistance to stripe rust on wheat leaves at 14 days after inoculating showed that the transgenic plants appeared high resistance, while the wild-type was moderately susceptibel. Additionally, the lengths of flag leaves with diseases in the transgenic wheats were shorter than those in the wild-type. The relative expression levels of pathogenesis-related protein1 gene (PR-1), pathogenesis-related protein2 gene (PR-2) and pathogenesis-related protein5 gene (PR-5) in the transgenic plants were respectively 1.14, 6.61 and 3.87 folds higher than those in the wild-type before inoculation of the pathogen. After inoculation with the fungus, the expression levels of PR-1, PR-2 and PR-5 in the transgenic plants were up-regulated and were 2.14, 3.41 and 7.55 folds higher than the wild-type, respectively. The results suggested that transgenic plants increased protective enzymes activities and relative expression level of pathogenesis-related protein genes, which thereby enhanced the disease resistance of transgenic plants. This study supplies the basis for the creation of transgenic stripe rust resistant wheat materials, and it also provides a theoretical basis on further investigations about the mechanism of EuCHIT1 gene.

Abstract Botrytis cinerea is a typical necrotrophic ascomycete fungus, known as grey mould. This grey mould can infect more than 200 plant species, leading to substantial losses in numerous crops including tomato (Lycopersicon esculentum), strawberry (Fragaria×ananassa), grape (Vitis vinifera), and so on. There is high genetic diversity in population of B. cinerea. In this paper, phenotypic diversity, pathogenicity differentiation, transposable element genotypes, phylogenetic group, genetic diversity of B. cinerea were reviewed.

Abstract CRISPR/Cas system in many bacteria and most archaea consists of clustered regularly interspaced short palindromic repeats (CRISPR) and associated proteins (Cas) and confers adaptive immunity resistance against exogenous invading plasmids and virus. Due to its site-specific, high-efficiency, easy-operation and low-cost, CRISPR/Cas9 has developed into the third generation of gene editing tool after Zinc-finger nuclease(ZFN) and transcription activator-like effector nuclease (TALEN) and gained tremendous popularity from researchers around the world. In this paper, the research progress on CRISPR/Cas system from the classification, structure and molecular mechanism was first reviewed. The diversified development of CRISPR/Cas system, profound exploration of new CRISPR system as well as different strategies for eliminating off-target effect, and improving specificity and extending targeting range was summarized, such as expanding protospacer adjacent motif (PAM) region, optimum design of sgRNA, processing construction of Cas9 and a series of work. The review pointed out its reformation and latest applications in establishment of animal models, gene therapy of human diseases, genome-wide screening and preparation of animal breeding materials , it also forecasts the research interests and application prospects of this promising technology. This article gives a detailed description of basic information of CRISPR/Cas9 which may provide references for researchers in related areas to do some futher research and application.

Abstract Porcine deltacoronavirus (PDCoV) is a new coronavirus that has been popular in China since the beginning of 2015. Development of a rapid and accurate detection method for the PDCoV is of great significance. A pair of primers were designed and synthesized according to the conserved region of the M gene sequence of PDCoV published in GenBank. The 260-bp fragment was successfully amplified and the recombinant plasmid of pMD18-T-PDCoV-M was constructed, and a SYBR GreenⅠreal-time RT-PCR was developed by optimization of the reacting conditions. The specificity, sensitivity and repeatability of the method were studied. The results demonstrated that the sensitivity of this assay was 54 copies/μL. In addition, the assay had good specificity for the PDCoV and had no cross-reaction with Transmissible gastroenteritis virus (TGEV), Pseudorabies virus (PRV), Porcine circovirus type 2 (PCV2), Porcine epidemic diarrhea virus (PEDV), and Porcine reproductive and respiratory syndrome virus (PRRSV). This SYBR Green Ⅰreal-time RT-PCR showed good repeatability, and the variations in intra- and inter-assays were both less than 1%. 198 clinical samples were tested for PDCoV by using this established method, and the positive rate was 20.71% (41/198). The established SYBR GreenⅠRT-PCR method for PDCoV provides a rapid and accurate diagnosis for PDCoV and provides a new technical means for the further study of the disease.

Abstract Mandarin fish (Siniperca chuatsi) is one of the most important cultured freshwater fish in China. In order to carry out pedigree analysis of selective mandarin fish, compare identification effects of double digest restriction-site associated DNA sequencing (ddRAD-seq) and microsatellites, and establish paternity identification in mandarin fish, 30 pairs of microsatellite primers were selected from GenBank, and 11 of them with good specificity and polymorphism were screened by polyacrylamide gel electrophoresis. Then, these 11 primers were used to amplify 4 full-sib families whose pedigree information was known. Capillary electrophoresis was used to carry out microsatellite genotyping. The result of cluster analysis showed these four full-sib families were divided into four groups using the 11 pairs of microsatellite primers. All 3 283 SNP markers were obtained by ddRAD-seq, and they were used to construct a clustering tree, including 155 samples from the same family and 12 samples from other families. Finally, the 12 samples were effectively distinguished. The above 11 pairs of microsatellite primers were used to detect the 12 samples from other families, eight samples from the same family and their parents, and clustering tree was constructed. The result showed that the 8 samples from the same family and their parents were clustered together, while the 12 samples were clustered in the other cluster. These results illustrated that these 11 pairs of microsatellite primers had good family identification ability. Then, parentage determination of 126 offsprings, which were bred by 7 male and 13 female breeding parents, were carried out by these microsatellite loci. Simulation analysis showed that the non-exclusion probability of each locus ranged from 0.276 to 0.895, and the highest calculate exclusion probability was 99.999%. Among these 126 offsprings, 105 offsprings had been successfully identified for their pedigree relationship by parentage analysis and the success rate was 83.3%. These results illustrated that these 11 pairs of microsatellite primers had high calculate exclusion probability and good identification effect in pedigree identification. The paternity identification method established in this study will be helpful for breeding of mandarin fish.

Abstract The increase of melatonin (Mel) can obstruct the development of reproductive organs and reduce the egg production in poultry. The arylalkylamine N-acetyltransferase (AANAT) is the major rate-limiting enzyme for melatonin synthesis from serotonin. In order to investigate the polymorphism of the AANAT gene on laying traits, SNPs in 5' regulatory region of the gene were detected with the direct sequencing and then their correlations with the laying traits were analyzed in Shouguang chickens (Gallus domesticus). The results showed that 10 low polymorphism loci and 11 moderate polymorphism loci were found in the 5? regulatory region, respectively. Three SNPs with moderate polymorphisms had significant effects on laying traits (P＜0.05). The locus of -803 could significantly advance the age at first egg and increase the egg production during 20~25 weeks of age. The loci of -561 and -522 increased the egg production during 26~45 weeks of age and during 46~57 weeks of age, respectively, so as to significantly increased the total egg production as well. In summary, the 5' regulatory region of AANAT gene have many SNPs, which were significantly associated with laying traits and thus are potential molecular markers to improve laying traits in Shouguang chickens.

Abstract Basic helix-loop-helix (bHLH) transcription factors play important roles in regulating plant growth-development and abiotic stress response. In order to enrich the research content of bHLH transcription factors and find new bHLH transcription factor in regulating secondary metabolite in Salvia miltiorrhiza, SmbHLH37 was cloned based on gDNA and cDNA. The gene sequence of SmbHLH37 was 1 506 bp with one intron of 27 bp, and 1 479 bp complete CDS (GenBank accession No. KP257470.1) encoding 492 amino acids. BlastP result showed that SmbHLH37 protein had high homology with bHLH3 in Sesamum indicum etc. Amino acid sequence analysis showed that SmbHLH37 contained a HLH domain and motif 7. Molecular evolutionary tree of SmbHLH37 protein and all the bHLH transcription factors in Arabidopsis thaliana indicated that SmbHLH37 protein was the closest relative to AtJAM3 (A. thaliana jasmonate-associated MYC2-like 3). Subcellular localization showed that SmbHLH37 protein was mainly localized in the nucleus. The expression pattern disclosed that the expression level of SmbHLH37 was the highest in leaves and the lowest in flowers. qRT-PCR analysis indicated that expression of SmbHLH37 could increase in a short time with 0.1 mmol/L abscisic acid (ABA) and wounding treatment. SmMYC2 and SmbHLH37 expression levels in SmMYC2 overexpressing transgenic S. miltiorrhiza lines OE-8 and OE-12 were detected, and the results showed SmMYC2 expression levels in OE-8 and OE-12 significantly or extremely significantly increased (P＜0.05 or P＜0.001). Meanwhile, the expression levels of SmbHLH37 in OE-8 and OE-12 were similar with SmMYC2 expression. The present data has laid a foundation for studying the function of SmbHLH37 in the future．

Abstract Metallothioneins, which are a kind of widely distributed and low molecular weight cysteine-rich proteins, were found to play an important role in heavy metal tolerance. In this study, a cDNA fragment corresponding to the type Ⅱ metallothionein (MT2) coding region (GenBank No. KY643823) was isolated from tartary buckwheat (Fagopyrum tataricum) leaf. FtMT2 encoded a deduced peptide of 78 aa residues with a calculated molecular mass of 7.87 kD and contained the cysteine-rich domains with the presence of C-C, C-X-C, and C-X-X-C (X was other amino acids other than cysteine) motifs at amino-terminus and C-X-C motifs at carboxy-terminus. Sequence and homology analysis showed that the FtMT2 protein sequence shared high homology with other plant type 2 MT-like proteins. In order to clarify the regulatory mechanism of FtMT2 in response to Cu2+ stress, 2.1 kb upstream sequence of FtMT2 was isolated by genome walking. The 2.1 kb fragment and its 5′ flanking deletions were tested for promoter activity using the dual luciferase reporter system and transient transformation in mesophyll protoplasts of F. tararicum. The promoter activities of all the fragments were up-regulated, and the full length promoter showed strongest inducibility in response to Cu2+ treatment. The role of FtMT2 protein in protecting against Cu2+ toxicity was demonstrated in pGEX4T-1-FtMT2-beared Escherichia coli, which could obviously increase the host E. coli cell's tolerance. Functional characterization of upstream region of FtMT2 provides the regulatory mechanism for establishing the method for copper contamination monitoring and offers an underlying basis for further revealing the molecular mechanisms of heavy metal resistance of Tartary buckwheat. Keywords Tartary buckwheat, Metallothioneins (MTs), Promoter, Heavy metal tolerance

Abstract Mink(Mustela vison) coat color, which is an important quality trait of mink, is also an extremely important factor affecting the price. Premelanosome protein gene (PMEL) is an important candidate gene that affects the variation of coat color and has been paid more and more attention in recent years. The aim of this study were to screen the mink coat color gene PMEL promoter active region and transcription factors binding sites, to provide a theoretical basis for elucidating the gene expression and regulation mechanism, and to provide ideas for color mink breeding and improvement. The specific primers were designed based on the domestic ferret (M. putorius furo) PMEL gene sequence (GenBank: NW_004569320.1), which was highly homologous to the mink. The fragment in a 5' flanking region was amplified and cloned into the vector pMD19 vector. The positive colonies were identified and sequenced. Six fragments with different lengths of promoter regions were amplified and cloned into the vector pMD19 vector. The positive colonies and vector pGL3-Basic were simultaneously digested with 2 restriction enzymes Kpn Ⅰ and Hind Ⅲ. The digested mixture were purified and ligated with T4 ligase to get the circular plasmid. The endo-free plamids were isolated after the positive colonies, which were identified by PCR, double enzyme digestion, sequencing. 293T and A375 cells were transiently transfected with lip2000 liposome. The dual-luciferase assay system was used to measure the luciferase activity. Transcription factor binding sites in core promoter region were predicted and verified. The length of the fragment in 5' flanking region of PMEL gene in mink was 1 401 bp. The predicted active region in the promoter, conserved motifs and multiple transcription factor binding sites were involved in the cloned fragment in the 5' flanking region. Six different lengths of fragments were obtained and ligated with luciferase reported vector. When the promoter 5' was truncated, luciferase transcriptional activity firstly increased and then decreased. The core promoter was involved in 5' flanking region of PMEL gene in mink and this region was from -671 bp to +87 bp. The activity of the promote decreased from -671 to -477, which indicated that there were some positive regulatory elements in the region from -671 to -477. The transcription factor binding site were predicted with bioinformatic methods and online program, which showed that there were many transcription factors binding sites in the strong active promoter. Sp1 (-516/-506), Sp1 (-505/-495) and Sp1 (-499/-489) binding sites were obtained based on the prediction of at least 2 softwares, and the 3 Sp1 binding sites were mutated, respectively. The results showed that -516/-506, -505/-495 and -499/-489 were positive transcription regulatory regions. The core promoter region from -671 bp to +87 bp was identified in mink PMEL gene, and 3 predicted Sp1 binding sites were positived regulatory regions and played critical role in regulating the activity of the promoter. The results provide important information for understanding the biological function of PMEL gene in mink and a new theoretical basis for further studying the molecular genetic mechanism of mink's coat color.

Abstract Reproductive performance is an important economic trait in sheep (Ovis aries). The study of its molecular mechanism is also extremely important. Reproductive performance of sheep is regulated by the hypothalamic-pituitary-gonad reproductive axis, and is also controlled by many growth factors. KITLG acting as ligand of KIT (KIT LIGAND, KL or KitL), as an important growth regulation factor, plays a key role in follicular development. The current study aimed to analyze the expression of KITLG gene and SNPs as well as their association with litter size in sheep, and to offer a reference data for the candidate gene research of reproduction trait in sheep. Therefore, Real time-PCR was conducted to investigate the expression patterns of KITLG gene in hypothalamus, pituitary, ovary, heart, muscle, lung, kidney, liver, spleen, duodenum, fat and rumen tissues of Hu sheep, and restriction fragment length polymorphism (RFLP) technologies were carried out to screen SNPs and analyze their association with litter size in sheep population of Hu sheep and Small tail Han sheep. The results revealed that KITLG gene expressed in heart, liver, spleen, lung, kidney, rumen, duodenum, muscle, fat, hypothalamus, pituitary, and the expression in ovary was high. According to PCR-RFLP technologies, two polymorphisms g.38473C＞T and g.47468C＞T2 were identified in intron 2 and 5 of KITLG gene respectively. In g.38473C＞T locus, two genotypes, CC and CT were found in both Hu sheep and Small tail Han sheep. Of them, CC was the dominant genotype, and C was the dominant allele. In addition, the homozygosity (Ho), heterozygosity (He), effective allele number (Ne), polymorphic information content (PIC) were 0.89, 0.11，1.31 and 0.11 for Hu sheep, and were 0.80, 0.20, 1.10 and 0.16 for Small tail Han sheep, respectively. The χ2 test indicated that Small tail Han sheep and Hu sheep populations were in Hardy-Weinberg equilibrium. Same as g.38473C＞T locus, two genotypes, CT and TT were identified in g.47468C＞T locus in both Hu sheep and Small tail Han sheep. Of which, TT was the dominant genotype, and T was the dominant allele. The Ho, He, Ne, PIC were 0.95, 0.05, 1.06, 0.05 for Hu sheep, and were 0.91, 0.09, 1.10 and 0.09 for Small tail Han sheep, respectively. The χ2 test indicated that Small tail Han sheep population and Hu sheep were all in Hardy-Weinberg equilibrium. The association analysis of polymorphisms in KITLG gene with litter size revealed that g.47468C＞T was significantly correlated with litter size in Hu sheep and Small tail Han sheep (P＜0.05). In summary, this study indicated that expression of KITLG gene in ovary was high. SNP (g.47468C>T) of KITLG gene was associated with litter size in Small tail Han sheep, and this gene could be used as a candidate gene for litter size selection at early stage of growth. This study provides a scientific basis for searching molecular markers associated with reproductive traits of the sheep, and setting breeding strategies, as well as improving the production performance of sheep. Compared with traditional breeding methods of sheep, the reaching results can also be used as a marker-assisted selection to greatly improve the breeding speed, and to achieve greater genetic progress in sheep.

Abstract The ubiquitin ligase E3 family plays an important role in the innate immunity of vertebrate, and many members of tripartite motif (TRIM) family have function as an E3 ligase in the ubiquitin modification based on the existence of the conserved RING finger domain. In order to evaluate the immune function of TRIM16 and TRIM25 genes from Nile tilapia (Oreochromis niloticus), the full-length cDNA sequences of TRIM16 and TRIM25 genes from were obtained by reverse transcription PCR and RACE. In addition, the gene structure and protein secondary structure were also analyzed. qRT-PCR was used for analyzing the expression of TRIM16 and TRIM25 genes in tissues and the process of embryonic development, and the response to Streptococcus agalactiae infection. Results showed that the TRIM16 gene (GenBank accession No. KY746714) was 2 314 bp in total length which contained an ORF of 1 677 bp encoding 558 amino acid residues. The 5'-UTR and 3'-UTR were 13 and 624 bp, respectively. TRIM25 gene (GenBank accession: No. KY968697) was 2 748 bp in total length which contained an ORF of 1 677 bp encoding 558 amino acid residues. The 5'-UTR and 3'-UTR were 21 and 1 050 bp, respectively. TRIM16 and TRIM25 genes were all without introns. The results of putative protein indicated that the TRIM16 and TRIM25 had conserved structures of the TRIM family, such as the RING finger domain and the B-box domain. The putative protein of TRIM16 had 29.0%~92.6% identities with other teleosts and mammals. The putative protein of TRIM25 had 20.0%~88.1% identities with other teleosts and mammals. The putative protein of TRIM16 and TRIM25 exhibited the highest identity with Maylandia zebra (92.6% and 88.1%, respectively). Tissue distribution analysis revealed that TRIM16 and TRIM25 genes expressed in all tested tissues with the highest expression level in blood and the lowest expression level in liver. The expression levels of TRIM16 and TRIM25 in the blood were 60.46 and 274.07 folds compared with that in the liver, respectively. Both TRIM16 and TRIM25 genes expressed during the embryonic development of Nile tilapia, which suggested that they played an important role in the immune process of Nile tilapia in the early stage of embryonic development. Upon stimulation with intraperitoneal injection with Streptococcus agalactiae, the expression level of TRIM16 and TRIM25 genes were up-regulated in all test tissues (intestine, spleen, gill, kidney and blood) at the same times. The highest expression levels of TRIM16 gene in intestinal, spleen, gill, kidney and blood were 1.30, 2.09, 1.61, 7.81 and 6.05 folds compared with 0 h (control group), respectively. The highest expression levels of TRIM25 gene in the intestine, spleen, gill, kidney and blood were 11.13, 1.22, 1.26, 61.41 and 77.80 folds compared with 0 h (control group), respectively. The results showed that TRIM16 and TRIM25 genes played an important role in the immunoreaction of Nile tilapia against S.agalactia infection. This study provides a theoretical basis for further understanding of the anti-infective immune mechanism of Nile tilapia and exploring new ways of disease prevention and control.

Abstract The canine blood typing reagents are lack of use in pet diagnosis and treatment in China. Our purpose is to develop canine DEA1.1 blood typing antibodies and provide materials for blood typing of pet dogs (Canis lupus familiaris) before blood transfusion. In this study, Beagle dog was taken as experimental animals, and antibody gene was derived from peripheral blood lymphocytes. Degenerate primers were designed for the variable region gene of canine antibody. Complementary DNA fragments of variable heavy (VH) and variable light (VL) chains of the antibodies were synthesized by reverse transcription RT-PCR, and were assembled into single-chain antibody variable fragment (scFv) by splice overlap extension PCR. Both scFv and the pCANTAB5E vector were double-digested with restriction endonuclease Sfi Ⅰ and Not Ⅰ, respectively, and they were connected with T4 ligase. Single chain antibody library was constructed. Dot enzyme-linked immunosorbent assay (Dot-ELISA) method was used to screen single-chain antibody for anti-DEA1.1 blood group. The positive clone of blood group antibody was identified, and the prokaryotic expression of recombinant scFv protein was constructed. Clinically samples tested validate specific antibodies with DEA1.1 blood can be used to pre-clinically test transfusion of dog DEA1.1. The results showed that the degenerate region of canine antibody variable region was available, and the canine scFv library was successfully constructed. The constructed immune library had an actual capacity of 7×105, being a diverse and large enough storage capacity. Finally, 3 strains of canine DEA1.1 blood group antigen with the single chain antibody were screened. Clone16 protein was identified as a soluble protein, being derived from prokaryotic expression protein. Clone16 single chain antibody was obtained by affinity purification, and further clinical samples showed strong binding characteristics. This study provides a theoretical basis for the preparation of canine DEA1.1 blood group identification reagents.

Abstract Litchi (Litchi chinensis Sonn.) is one of the most commercially important fruit in subtropical areas with good flavor and rich nutrients. However, pericarp browning and fruit decay during storage period with high temperature and high humidity had led to great economic loss. In order to reduce the abuse of artificial chemical fungicide, development of a new method for preservation of harvested litchi fruit had shown great importance. In this study, the fermentation broth of microbial community using Enteromorpha prolifera as subtract was used for postharvest treatment of litchi fruit. The effect of different concentrations of supernatant on visual quality and storability of harvested litchi fruit were investigated. The high-throughput sequencing technique of 16S rRNA V4 zone was used for analysis of structure and abundance of microbiota. Nuclear magnetic resonance (NMR) metabonomics technology was used for the determination of metabolites with fresh-keeping potential. Based on the sequencing of Hiseq2500 PE250 platform, the result indicated that Bacillaceae, Cyclobacteriaceae and Alteromonadaceae were main families in this microbial community, in which the Bacillus was the dominate genus with the the abundance of 32.31%. Furthermore, the abundance of Alteromonadaceae and Microbulbifer which could degrade the E. prolifera was 8.47% and 6.72%, respectively. The main preservation related substances in fermentation broth was determined as glycine betaine and trehalose, and the concentration was 0.001 5 and 0.007 9 mmol/L, respectively. Compared with the control group, the litchi fruit being dipped in the fermentation broth at different concentrations for 10 min could significantly maintain lower respiration rate of fruit, browning index, disease index and percentage of weight loss, so these after-treatment litchi fruit kept higher percentage of commercially acceptable fruit during ambient storage condition. This study provides a theoretical basis and technical method for postharvest bio-preservation of litchi fruit and other subtropical fruit.

Abstract Peanut (Arachis hypogaea) is an important oil and economic crop, but the breeding develops slowly for the narrow genetic resources and low genetic diversity. In order to search new breeding method and broaden the germplasm resources, new peanut germplasms were created by fast neutron irradiation and in vitro tissue. Dry seeds of peanut variety Luhua 11 were irradiated by fast neutron irradiation with dosage of 9.7 Gy. The explants of embryonic leaflets were separated from the irradiated seeds and inoculated on the media containing 10 mg/L 2,4-D for inducing somatic embryo formation. After 4 weeks culture, the subcultures were transferred to the media supplemented with 4 mg/L 6-benzylaminopurine (BAP) for inducing somatic embryo germination and plant regeneration. The regenerated plantlets were grafted and transplanted into field. Some mutants with characteristics of long stem and branch, early maturing, easy to sprout, variable pod shape and orange endopleura have been obtained. A new peanut variety Yuhua 5 with the characteristics of early maturity, lodging-resistant and suitable for mechanized harvesting was also obtained. Yuhua 5 had passed peanut variety regional multi-location trials in Liaoning province 2015, and the yield of which was 15.03% higher than control variety Baisha 17. The research showed that it is a new and effective way of creating new germplasms and breeding new varieties by irradiation and in vitro tissue in peanut. A new peanut variety Yuhua 5 has been cultivated by this way.