Transposon mediated insertional mutagenesis has great value in the discovery and interpretation of functional genes in aquaculture fish. In this paper, a preliminary study of insertion mutagenesis was carried out in 3 Cyprinidae fish mediated by Tgf2 transposon. On the basis of high efficient Tgf2 transposon, 4 donor plasmids pTgf2-β-actin-eGFP, pTgf2-EF1α-eGFP, pTgf2-MyoD-eGFP, pTgf2-Krt8-eGFP and the in vitro expression of mRNA helper plasmid gfTP in pCS2-gfTP were constructed. The donor plasmids and in vitro expressed gfTP transposase mRNA were co-injected into 1~2 cell stage fertilized eggs of grass carp (Ctenopharyngodon idellus), crucian carp (Carassius auratus) and blunt snout bream (Megalobrama amblycephala). By detection and screening of the eGFP gene integration rate, the insertional mutagenesis library mediated by Tgf2 transposon was initially established. In the early stage of embryos, the fluorescence ratio was very high. The fluorescence ratio in both blunt snout bream and crucian carp reached 90%, and even 95% in grass carp. According to the different donor plasmid, there were three types of fluorescence, such as in the muscle, skin and whole body. The integration ratio in larva fish was 50%~53%, and the integration ratio in the one year old fish was 33.0%~36.1%. In total, in grass carp, 162 positive mutants, and 45 individuals were got in them had obvious phenotypes. There were 60 positive individuals in crucian carp, including 14 obvious phenotypes. In blunt snout bream, there were 51 positive individuals, including 10 obvious phenotypes. These results suggest that insertional mutagenesis could be realized in grass carp, blunt snout bream and wild crucian carp by Tgf2 transposon inserted into fish genome. Our studies can accumulate some basis for the study of mining and functional mechanism of target character gene function.

Peroxidase (POD) is one of the major oxidases involving in the oxidation of phenolsand and plays important roles in browning of fruits and vegetables. To investigate the function of POD in Luffa (Luffa cylindrical), the full- length cDNA of POD was cloned from luffa fruit by using rapid-amplification of cDNA ends (RACE) and reverse transcription-PCR (RT-PCR) techniques. This gene was named LcPOD, and the GenBank accession was KM506755. The full length cDNA of LcPOD was 1 117 bp, and this cDNA contained a 993 bp open reading frame (ORF) that encoded 331 amino acids，with a predicted molecular weight of 36.32 kD and a hypothetical isoelectric point of 5.545. LcPOD shared over 80% identity with the homologous proteins from melon (Cucumis melo, XP_008449774.1), zucchini (Cucurbita pepo, ABF68751.1) and cucumber (C. sativus, NP_001295810.1), proving that it was highly conservative. Motif Scan analysis showed that LcPOD protein had the characteristic amino acid sequences of peroxidase proximal heme-ligand in the position of 81~191 and heme active sites in the position of 55-66 sites,respectively, which suggested that LcPOD protein was a typical ClassⅢ POD. Wolf Psort protection indicated that LcPOD protein was located in the cytoderm. qRT-PCR analysis revealed that LcPOD can be expressed in various luffa tissues (i.e., roots, stems, leaves, flowers and fruits), and the expression level of LcPOD was the highest in root, followed by leaves and flowers, and the expression level was the lowest in stems.The expression levels of LcPOD were different among eight luffa varieties，and the expression in L. cylindrica was higher than that in L. acutangula. During post-harvest storage at 4 ℃, LcPOD expression level gradually increased and reached maximum at 2 d, then was down-regulated in 3 d and 4 d. During fresh-cut placement at 25 ℃, LcPOD expression level gradually increased and reached maximum at 2 h, then was down-regulated in the remaining time points (i.e., 4 h, 6 h and 8 h). The expression of LcPOD gene, POD activity and total phenolics were highly related to the browning of luffa during fresh-cut and post-harvest storage. LcPOD may play a regulation role in luffa browning process. These results provide a scientific basis for further revealing the mechanism of luffa browning and the genetic improvement on luffa.

miRNA plays an important role in sheep (Ovis aries) skeletal muscle development. This study aimed to find out the differently expressed miRNA in Bashbay sheep skeletal muscle during different development stages, and to furtherly understand the relationship between miRNA expression and excellent meat traits of Bashbay sheep. In this study, the Solexa sequencing technology was applied to investigate the miRNA expressing profiles in Bashbay sheep skeletal muscle during fetal period (mixed sample of 40, 50, 60, 80, 100 and 120 d) and postnatal stage (mixed sample of newborn, 30, 60 and 90 d). By high-throughput sequencing and data analysis, 11.82 M and 11.51 M clean reads, and 0.31 M 0.28 M unique sequences were obtained. Among them, 20% and 17% were candidate miRNA sequences, respectively. Then blastn was performed between miRBase (V21.0) and unique sequences, and finally got 537 and 496 miRNAs which were conservative among species. There were 102 and 115 miRNAs of them were matched with ovine known miRNAs, respectively. The rest sequences were compared with EST database of sheep, and 486 and 510 new candidate miRNAs were obtained by Mfold and MiPred program. There were total 385 miRNAs which differently expressed between fetal and postnatal skeletal muscle of Bashbay sheep (expressing change were above 2 fold), including 203 up-regulated and 182 down-regulated miRNAs, respectively. Applying TargetScan, Pictar and miRanda, the candidate target genes of top 10 up- and down-regulated miRNAs were predicted, and the one which was predicted by at least two programs was chosen to be further analyzed. Total 3 852 candidate target genes were predicted, and most of them participated in many important signal pathways, just like mitogen-activated protein kinases (MAPK), wingless type MMTV (mouse mammary tumor virus) integration site family members (WNT) and adherens junction, etc. This study enriches the miRNA database of sheep skeletal muscle, and could help to further understand the regulation mechanism of miRNA in sheep skeletal muscle development.

Interleukin-6 (IL-6) is also called proinflammatory cytokine interleukin-6, plays a critical role in many chronic inflammatory and autoimmune diseases, especially inflammatory bowel disease. In this study, in order to investigate the regulation of IL-6 gene expression at the molecular level, Jinghai Yellow Chickens(Gallus gallus) were used as experimental materials, genomic DNA sequencing was used to detect single nucleotide polymorphisms (SNPs) of proinflammatory cytokine IL-6 gene from the upstream -2 200 bp to downstream 500 bp region, bioinformatics softwares were used to predict the core promoter region transcription factors and CpG islands of IL-6 gene, to analyze how the single nucleotide mutation of IL-6 gene changed the transcription factors and the CpG islands. The sequencing results showed that 28 SNPs sites were found in the gene, nineteen SNPs were located in the 5' regulatory region, two were located in exon (both of them are synonymous mutation), two were located in intron and 5 SNPs were located in the 3' region. Out of 28 mutation sites, there were 4 SNPs are not labeled in GenBank, three of them (G -357 A, C -447 G and A -663 G) were located in the 5' regulatory region and one in the 3' region (C3177T). Bioinformatics analysis showed that IL-6 gene had a very distinct core promoter recognition feature, there were 11 SNPs located in this core region, causing the change of transcription factor binding sites, the new discovered G-357A mutation may generate a new transcription factor binding site (Sp1) from the beginning of -360 bp, C-447G mutation can lead to one transcription factor change (HB become C/E BPalp), A-663G mutation may lead to the formation of 3 new transcription factor binding sites (AP-2alph and two Sp1), other detected SNPs could produce 5 new transcription factor binding sites (Sp1, ISGF-3, TEC1, IRF-1, ICS BP) and lead to 7 transcription factor binding sites (MEB-1, GLO, GATA-1, C/E BPal, Sp1, GR, Sp1) disappearing; meanwhile, the prediction results suggested that the SNP of C-939G may regulate the expression of IL-6 gene by affecting the number of the CpG island. C-939G mutation in the promoter region may lead the number of CpG island to change from 3 to 2, before the mutation, the CpG islands which were located in -1 010~-908 bp, -616~-509 bp and -370~-58 bp, respectively, after the mutation, the CpG islands were located in -615~-505 bp and -370~-58 bp, respectively. It can be speculated that SNPs in 5' regulatory region may influence the expression of IL-6 gene by changing the transcription factor or the methylation region. The results of this study have great significance for further exploring the function of IL-6 gene and the relationships between these SNPs and chicken intestinal inflammation resistance, and production performance.

Abstract：In this study, the primers was designed according to cDNA sequences of apolipoprotein gene from EST database of largemouth bass and the genomic fragments of Apo-A1, Apo-A4 and Apo-C1 were amplified, respectively. 4 single nucleotide mutations were identified by using directly sequencing. The A+489C mutation was located in Apo-A1 gene. The A+633T mutation was located in Apo-A4 gene. The two mutations (A+24G and A+75C) were located in Apo-C1 gene. The genotype of each SNP in 159 largemouth bass was analyzed with the method of SnaPshot. Chi-square test showed that four SNP loci were in Hardy-Weinberg equilibrium state in the largemouth bass population. A+633T, A+24G and A+75C had the same genotype frequency, which formed two haplotypes (A and B) and constituted with three genotypes (AA, AB, and BB). This suggested that the three SNPs might completely link. A general linear model was used to analyze the relationship between polymorphisms of apolipoprotein gene and major growth traits. The result indicated that A+489C site showed significant association with body weight and body high (P<0.05). The different genotypes of A+633T in Apo-A4, A+24G and A+75C sites in Apo-C1 genes showed significant association with body weight, body high and total length, respectively (P<0.05). Our results showed that 4 SNPs had genetic effects on the growth traits in largemouth bass, which could be important candidate molecular markers for largemouth bass molecular marker-assisted selection.

Columnar trait of apple (Malus × domestica Borkh.) is controlled by a single dominant Columnar (Co) gene, located in apple genome chromosome 10. Many putative genes are predicted in the Co region. Screening genes related to the columnar trait in apple provides theoretical basis for identification of the Co gene. Sixty-four putative genes in the Co region (18.51~19.1 Mb) were screened by SRA/EST (Sequence Read Archive/Expressed Sequence Tags) database. Samples of shoot tips, leaves and buds from columnar cultivar ‘Wijcik’ and non-columnar cultivar ‘McIntosh’ were used to extract total RNA, respectively, and the cDNA mixture of an equal volume of each sample after reverse transcription, was used as materials to conduct. Reverse transcription PCR (RT- PCR) was to amplify partial cDNA sequences of putative gene. Quantitative Real-time PCR (qRT-PCR) reactions were performed to analyze the differential expression of putative genes in apple shoot tips. 32 putative genes expressed in different apple tissues or organs were detected by searching SRA/EST database. 29 putative genes were obtained by RT-PCR amplification, and 15 genes were with significant differences in apical tips of the columnar apple by qRT-PCR analysis. Among them, 6 genes (No.26, No.41, No.33, No.38, No28 and No.32) were up-regulated significantly (P＜0.05), and 9 genes (No.30, No.9, No.37, No.6, No.44, No.10, No.27, No.11 and No.4) were down-regulated significantly in apical tips of the columnar cultivar(P＜0.05). In this study, 15 genes with significant differences in the apical tips of the columnar apple could be regarded as candidate genes of the Co. The candidate genes are identified to elucidate the function of critical genes and molecular mechanisms of the apple columnar mutation in the Co gene region, and serve as a directional genetic improvement for apple trees.

The selection of disease resistance related SNP markers is the basis and prerequisite for disease resistance breeding. Interferon-b promoter stimulator 1 (IPS-1) plays a pivotal role in the production of type I interferon (IFN) and pro-inflammatory cytokines. In order to obtain the SNPs associated with the disease resistance in nile tilapia (Oreochromis niloticus), in this paper we designed four pairs primers to amplify the IPS-1 gene (ENSONIG00000012278). Through sequencing by cloning or directly sequencing of PCR products, 36 SNPs were obtained from 39 individuals of 20 nile tilapia parents family. Seventeen sites (S1 and S21-S36) located in exon region, and 9 SNPs were nonsynonymous mutation, 4 SNPs were synonymous mutation. Nineteen sites located in intron region. By directly sequencing or SNaPshot method, 82 individuals from susceptible group and 84 individuals from resistance group of offspring were used to analyze polymorphisms and Genetic parameter. Popgen32 was used to calculated the polymorphisms and genetic parameter of the IPS-1 SNPs in susceptible group and resistance group. The results showed that the polymorphism information content (PIC) value of the IPS-1 SNPs ranged from 0.02 to 0.37, suggesting that all SNPs locus had low or moderate polymorphism. Significant departure from Hardy-Weinberg equilibrium was observed at 16 SNPs in resistance group and 5 SNPs in susceptible group (P＜0.001). The genotype frequency and allele frequency of the 36 SNPs in IPS-1 gene were analyzed by SPSS 20 and tested by χ2 test. And the correlation between the IPS-1 SNPs and the phenotype of Streptococcus agalactiae resistance was analyzed. The results showed that One SNP (S8(T-3756C)) was significantly associated with S. agalactiae susceptible trait (P＜0.05). In resistance group, the frequency of CC, CT, and TT genotype was 97.5%, 2.5% and 0%, respectively, and in susceptible group, the frequency was 75.7%, 12.2% and 12.2%, respectively. The linkage disequilibrium analysis and prediction of haplotypes showed that all the 36 IPS-1 SNPs formed 6 haplotype blocks and 22 haplotypes. Three of the haplotypes H2-3 (GTTG), H3-2 (CCDGTTGAGCGCCA) and H3-4 (TTDGTTGAGCGCCA) were significantly associated with S. agalactiae susceptible trait. And 3 of the haplotypes H3-5 (TCDGTTGAGCGCCA) were significantly associated with S. agalactiae resistance trait (P＜0.05). These results indicate that the SNP site S8 and the four haplotypes H2-3, H3-2, H3-4, H3-5 of IPS-1 gene can be candidate molecular marker for disease resistance breeding of nile tilapia. This study provided a data base for breeding resistant strains of Nile tilapia.

Molting is a vital biological phenomenon of crustaceans and is closely related to the growth and leg regeneration of Chinese mitten crab (Eriocheir sinensis). In order to detect the mechanism of molting and growth of Chinese mitten crab, the concentration of ecdysteroid hormone (EH), the expression of ecdysone receptor gene (EcR) and molt-inhibiting hormone (MIH) gene at different molting stages were investigated by ELISA and qRT-PCR, respectively. Meanwhile, the correlation analysis was also conducted between growth traits and the concentration of EH, expression of EcR or MIH genes. The results showed that the concentration of EH fluctuated regularly in one molt cycle, and molting would started when EH content reached about 12 U/mL. The concentration of EH was significantly positive correlation with expression of EcR(P＜0.01), but was significantly negative correlation with expression of MIH(P＜0.01). The concentration of EH and expression of EcR at premolt stage were significant higher than that at postmolt stage (P＜0.05), and they were the lowest at intermolt stage compared with that at other molting stages (P＜0.05). However, the expression of MIH showed completely opposite trend compared with the concentration of EH and expression of EcR, indicating the highest at intermolt stage compared with that at other molting stages (P＜0.05). There was a significantly negative correlation for the expression of EcR between intermolt and postmolt stage (P＜0.05), and significantly positive correlation for the expression of MIH between premolt and intermolt stage, as well as between premolt and postmolt stage (P＜0.05), while there was no significant correlation for the EcR and MIH genes at other molting stages (P＞0.05), which indicated that the expression of MIH at premolt stage had a remarkably effect on the expression of MIH at postmolt and intermolt stage; while the expression of EcR at intermolt stage had a significant effect on the expression of EcR at postmolt stage. The correlation analysis showed that the concentration of EH was remarkably positively correlated with body weight at both premolt and intermolt stages (P＜0.05), the expression of EcR at different molting stages was significantly positively correlated with body weight (P＜0.05), and it was also notably positively correlated with carapace length and carapace width (P＜0.05) at intermolt stage. However, the expression level of MIH was negatively correlated with body weight at postmolt stages (P＜0.05), while remarkably negatively correlated at premolt and intermolt stages (P＜0.05). and it was remarkably significantly negatively correlated with carapace length, carapace width at intermolt stage (P＜0.01). Briefly, the results indicated that the concentration of EH had a significant effect on body weight of Chinese mitten crab at premolt and intermolt stage. The expression of EcR and MIH at different molting stages had significant effect on body weight, and their expression at intermolt stage also had significant effect on morphological traits. This study gives new insight into molecular mechanism of molting and growth of Chinese mitten crab, and provides a theoretically molecular and physiological basis for weight increment differences after molting in this crab.

Abstract Stearyl-CoA desaturase (SCD) is a key enzyme in the biosynthesis ofmonounsaturated fatty acids in organisms. It has a wide range of physiological functions and plays an important role in the regulation of lipid dynamic equilibrium, fat generation and lipid oxidation. Transcriptomic analysis of livers of pre-laying (20 weeks old) and peak-laying (30 weeks old) hens showed that the expression level of SCD gene was significantly higher in the liver of laying hens than that in pre-laying hens. To further understand the regulation mechanism of expression and biological functions of the gene in chicken, real-time quantitative PCR (qPCR) was used to analyze the expression patterns of SCD in different tissues and various developmental stages. The regulation mechanism of SCD was exploried by a series of experiments in vivo and in vitro. The results showed that the SCD was extensively expressed in various tissues with relatively higher expression levels in liver, abdominal fat and kidney, and also relatively higher expression levels in peak-laying hens than that in young poults. The expression of the SCD gene was significantly regulated by estrogen. This study indicated that expression of the SCD gene is regulated by estrogen and might involve in liver lipid metabolism in chicken.

2,4-Dichlorophenoxyacetic acid (2,4-D) is a phenoxy herbicides that has been used to control broadleaf weeds in cereal and grass crops for over 70 years. When applied to dicotyledonous plants at effective doses, 2,4-D causes uncontrolled and disorganized plant growth that leads to death. It's necessary to improve 2,4-D resistance of sensitive plant. In soil, this herbicide is regarded as readily biodegradable and mostly depended on microbial degradation. A lot of degrading bacteria contain 2,4-D-catabolizing enzyme, which encodes an Fe (II)/α-ketoglutarate-dependent dioxygenase, could convert 2,4-D into 2,4-dichlorophenol as an initial step of 2,4-D mineralization. In our previous study, an effective 2,4-D-degrading strain, identified as Cupriavidus campinensis BJ71, was isolated from a wheat soil with a long-term history of 2,4-D use. And 2,4-D-degrading gene, named as cctfdA, was cloned from this strain BJ71. This gene's full length is 864 bp and encodes 287 amino acids. In this study, using cctfdA as model, man-made Nt-cctfdA with tobacco-preferred codon usage was designed and synthesized. An expression vector named pSH737-Nt-cctfdA, containing Nt-cctfdA gene, under control of a constitutive 35S promoter, was constructed. Two expression vector pSH737-Nt-cctfdA and pSH737 were introduced into Nicotiana tobacum through Agrobacterium tumefaciens-mediated gene delivery. 15 regenerated tobaccos resisted to 2,4-D contained pSH737-Nt-cctfdA and 10 genetic lines contained pSH737 were obtained. T1 generation plants were selected with kanamycin resistance and analyzed by PCR for presence of this gene. A series of three experiments were carried out to study 2,4-D resistance. Firstly, when leaves in vitro were soaked with 250 mg/L 2,4-D, pSH737-Nt-cctfdA transgenic tobacco leaves remained green and intact, whereas wild-type and pSH737 transgenic leaves were injured severely after seven days. Secondly, tobacco leaf discs were punched aseptically from fresh leaves and plated on shoot regeneration media containing different 2,4-D concentration and incubated for three weeks. Wild-type tobacco leaf discs generated two shoots on levels of 2,4-D up to 0.4 mg/L and pSH737 genetic plants generated one shoot on 0.6 mg/L concentration, whereas shoots rate of pSH737-Nt-cctfdA transgenetic tobacco were 58.97% on callus induction medium containing 8 mg/L, representing a 30-fold increase in tolerance to this herbicide compared with the control. Finally, when different concentrations of 2,4-D were sprayed on young plants, wild-type and pSH737 transgenic young plants were killed on 350 mg/L concentration. In contrast, transgenic plants expressing Nt-cctfdA exhibited no visible signs of 2,4-D damage, even when treated with up to 10 000 mg/L of the herbicide. It showed that 2,4-D resistant levels in transgenic pSH737-Nt-cctfdA tobacco was extremely exceeded 30 times than the control and was 10 times than the usual field application rate (P＜0.05). When plants were sprayed with 2,4-D, chlorophyll content was not changed basically in Nt-cctfdA transgenic plants, whereas it was obviously decreased in cotrol group (wild-type and pSH737 genetic plants, too). In conclusion, cctfdA gene from Cupriavidus campinensis BJ71 could obviously improve 2,4-D tolerance in transgenic tobacco. And the results could help us to illuminate 2,4-D resistance mode clearly and provide basic data for further studying 2,4-D resistance transgenic tobacco.

我国猕猴桃(Actinidia)种质资源极其丰富，可为猕猴桃遗传改良和资源利用提供广泛的背景材料，而从实生苗群中筛选变种是猕猴桃新品种培育途径之一。本研究以17份猕猴桃品种(系)(其中2份红阳变异系、1份金桃变异系)为材料，采用目标起始密码子多态性(start codon targeted polymorphism, SCoT)对其遗传多样性和变异进行分析。结果表明，基于优化后的适宜于猕猴桃SCoT-PCR反应体系，从64条引物中筛选出42条引物对17份猕猴桃材料扩增出487条清晰的条带，其中多态性条带为470条带，平均多态性比率为95.1%。17份猕猴桃材料之间的遗传距离范围在0.153~0.693，平均值为0.492。变异株桂乐1号、桂乐2号与对照红阳的遗传距离分别为0.276和0.299，金桃变异与金桃的遗传距离为0.539，3个变异株系均与对照有一定的亲缘关系与遗传距离。聚类分析表明，在遗传距离为0.460的水平上，可以将17份猕猴桃材料分为4组。桂乐1号、桂乐2号与对照红阳归为第Ⅲ组，金桃变异与对照金桃则未能聚为同一组。SCoT分子标记可以对猕猴桃性状变异进行初步鉴定，为进一步开展变异新品种的早期鉴定和保护提供技术参考，以及为猕猴桃种质资源遗传多样性分析及其分类提供理论支持。

CD9 and CD81 were both important candidate factors during sperm-egg fusion in mammals, but their tissue expression characteristics in vivo were still unknown. In order to elucidate their expression characteristics in sheep (Ovis aries), tissue expression of CD9 and CD81 genes in Small Tail Han sheep ewes (high fecundity) and Sunite sheep ewes (low fecundity) in the follicular phase was investigated using semi-quantitative reverse transcription polymerase chain reaction (sqRT-PCR) and quantitative real-time quantitative PCR (qRT-PCR) in the present study. The results showed that the expression of CD9 and CD81 genes was found in 14 tissues of two sheep breeds. The CD9 gene expression was the highest in oviduct tissue of two sheep breeds, and its expression in hypothalamus, pituitary and ovary tissues of Small Tail Han sheep was extremely higher than that of Sunite sheep (P＜0.01). The CD9 gene expression in ovary and hypothalamus tissues was significantly higher than that in pituitary and uterus in Small Tail Han sheep (P＜0.05), while its expression in hypothalamus and uterus tissues was significantly higher than that in ovary and pituitary in Sunite sheep (P＜0.05). The expression of CD81 gene in reproductive axis tissues in Small Tail Han sheep was extremely higher than that in Sunite sheep (P＜0.01). In Small Tail Han sheep, the CD81 gene expression was the highest in hypothalamus tissue (P＜0.01), and its expression in pituitary and oviduct tissues was extremely higher than that in ovary and uterus tissues (P＜0.01). While in Sunite sheep, the CD81 gene expression was the highest in ovary tissue (P＜0.01), and its expression in hypothalamus was extremely higher than that in pituitary, oviduct and uterus (P＜0.01). Our study preliminarily indicated that CD9 and CD81 genes maybe relate with difference of sheep prolificacy, which might provide a reference for revealing the molecular regulatory mechanism of sheep reproductive performance.

Largemouth bass (Micropterus salmoides) is a very important commercial farming fish with the annual production of more than 340 000 t in China. But it is a carnivorous teleost, and a large amount of forage fish and fish meal were used as food for largemouth bass every year. The residual forage fish and fish meal not only increase the culture costs, but also pollute waterbodies and farmlands seriously. In order to reduce culture costs and protect the natural environment, the selective breeding of the largemouth bass strain suitable for formulated feeds was carried out. It is a way to improve the effective conversion capability of largemouth bass for formulated feeds. In the previous study, the lipid levels of the formulated feeds have significant effects on the expression levels of 2 lipoprotein lipase genes (LPL type1 and LPL type2) in livers of largemouth bass. The report suggests the 1 genes potentially related to the adaptability of largemouth to formulated feed. In the current study, the primers of LPL genes were designed according to the known cDNA and the PCR amplifications were performed with largemouth bass genome DNAs as templates. After amplifying and sequencing, the genomic sequences of LPL type1 gene with the length of 6 170 bp were identified including 10 exons and 9 introns, and LPL type2 gene with the length of 4 419 bp were identified including 5 exons and 4 introns. By use of direct sequencing, eleven SNPs were detected in LPL type1 gene, and 6 SNPs were detected in LPL type2 gene. A total of 192 individuals from the cultured adult fish population fed on iced fresh fish and a total of 142 individuals from the cultured juvenile fish population fed on formulated feed were genotyped according to the SNP sites. The results showed that the 11 SNPs in LPL type1 were closely linked, and were named as T+156A. As well as the 6 SNPs in LPL type2 were closely linked, and named as C-224T. But the SNP sites were not closed linked between the 2 genes. The genotype frequency and gene frequency of the SNPs were similar between the 2 populations. And the calculations showed that the 2 populations were in Hardy-Weinberg equilibrium for the 2 SNPs. The general linear model was applied to analyze the relationship between SNPs and the full/empty traits, as well as the growth traits of largemouth bass. The results showed that there were no significant association between the LPL SNP sites and the growth traits of the adult fish fed on iced fish (P＞0.05). But in the juvenile fish population fed on formulated feed, the T+156A SNP was significantly related to the full or empty of the stomach and intestine. And the fish with AA genotype of T+156A SNP have significantly higher growth trait values in body weight and body length than those with TT genotype (P＜0.05). The results hinted that AA genotype in T+156A SNP has significant association with the adaptability of largemouth bass to the artificial diets. LPL type1 gene can be used as a candidate gene for the largemouth bass fed on formulated feeds, with high survival rate and fast growth rate. The current study not only provides the reliable research data to promote the breeding work of new strain of largemouth bass adaptable to formulated feed, but also provides the experiences and references for the bait domestication work of other carnivorous fish.

Signal transducer and activator of transcription 5a (STAT5a), considered as an important candidate gene for milk production traits, plays key roles in the process of development of regulating animal mammary gland. In the present study, seven tissues included heart, liver, spleen, lung, kidney, cerebellum and mammary gland of Tibetan and Small tail han sheep were detected by qRT-PCR to research the tissues distribution and the differences in expression between different sheep breeds and tissues. The P1 and P2 primer amplification regions of STAT5a in three sheep breeds (Tibetan sheep, Hu sheep and Duolang sheep) using PCR-SSCP and sequencing methods to analysis nucleotide sequence variation and population genetic characteristics. The results indicated that STAT5a gene was expressed in all seven tissues and showed significant differences in species and tissues. The expression of mammary gland was significantly higher than other six tissues in Tibetan and Small tail han sheep (P＜0.01). Between these two breeds, the expression of mammary gland in small han sheep was highly than Tibetan sheep(P＜0.05), but the expression of kidney, cerebellum, and liver were lower than Tibetan sheep (P＜0.05). Two SNPs (c.2150-154C＞T and c.2150-50T＞C) and corresponding alleles A and B were detected in P1 primer amplification regions of STAT5a in sheep. The allele B and genotype BB were the dominant allele and genotype and Tibetan population was moderately polymorphic (0.25＜PIC＜0.5), but other populations were low polymorphic (PIC＜0.25). The χ2 values of the three sheep populations were non-significant (P＞0.05), so those populations lay in the Hardy-Weinberg equilibrium. However, there was no polymorphism in the P2 primer amplification region. In conclusion, these results enrich the contents of the sheep genome and provide the basic information for the molecular mechanism of the formation of mammary gland.

Induced defense plays a key role in plant resistance against herbivores, and mediate interactions between phytophagous arthropods and plants via genes and signal pathways. The objective of this study is to understand the role of Ghppo1 gene in induced defense and cotton-cotton bollworm interaction in Bt cotton with Bt+CpTI (Bacillus thuringiensis+cowpea trypsin inhibitor) genes. Using the qRT-PCR, the expression changes of the Ghppo1 (gossypium hirsutum polyphenol oxidases 1 gene), jasmonic acid signaling pathway genes of cotton (Gossypium hirsutum) (i.e., gossypium hirsutum allene oxide synthase, GhAOS and gossypium hirsutum coronatine insensitive1, GhCOI1), which were damaged by Helicoverpa armigera and sprayed with Me-JA and Me-SA for 0, 6, 12, 18 and 24 h, respectively, were studied. The weight changes of Helicoverpa armigera and Spodoptera exigua which were fed on cotton plants that treated with first star H. armigera and Me-JA. Ghppo1 expression was up-regulated by infestations of first instar H. armigera larvae and applications of methyl jasmonate (Me-JA). In contrast, the third instar larvae and methyl salicylate (Me-SA) did not increase Ghppo1 expression levels in cotton plants. Additionally, cotton polyphenol oxidase activities were also up-regulated by first instar H. armigera larvae and Me-JA, but not by third instar larvae or Me-SA. The expression of GhAOS and GhCOI1 was induced by infestation of first instar H. armigera larvae. Infestations of H. armigera on cotton revealed that plants previously treated with first instar H. armigera larvae exhibit significantly increased resistance than third instar H. armigera and S. exigua. Ghppo1 expression was mediated by jasmonic acid, but not salicylic acid signaling pathway, and may be involved in this interaction among insect herbivores and cotton defense responses. This study shows that the first instar H. armigera larvae can induce resistance against H. armigera and S. exigua by regulating Ghppo1 expression levels.

Laccases are multi-copper containing glycoproteins. By catalyzation of various kinds of substrates through the oxidation-reduction reaction of copper ions in their conserved domains, Laccases involve in various physiological and biochemical processes, such as lignin synthesization, iron metabolism, flavonoid biosynthesis, wound healing, stress responses and et al. However, their functions in the post-harvest storage have not been reported. In order to study the function of the laccase genes in the kiwifruit storage process, two laccase genes (AdLAC3 and AdLAC7) were cloned from the Actinidia deliciosa cv. Miliang-1 using reverse transcription PCR(RT-PCR) and RACE-PCR methods. The full-length sequences of AdLAC3 were 2594 bp. It contained a 2205-bp open reading frame, encoding a 734 aa polypeptide and its accession number was MF405444. The full-length sequences of AdLAC7 were 2126 bp. It contained a 1695-bp open reading frame, encoding a 564 aa polypeptide and its accession number was MF405447. In addition, two alternative spliced variants (AdLAC3-variant1 and AdLAC3-variant2) of AdLAC3 were obtained. The accession number of AdLAC3-variant1 was MF405445, and the accession number of AdLAC3-variant2 was MF405446. Protein domain analysis showed that AdLAC3 and AdLAC7 both contained three typical copper binding domains (Cu-oxidase_3, Cu-oxidase and Cu-oxidase_2), but AdLAC3 and AdLAC7 were distributed to different evolutionary branching in the phylogenetic tree, which was due to the great difference in their sequences and implied that they may originate from different ancestral genes. Both AdLAC3 and AdLAC7 harbored five introns and six exons. qRT-PCR analysis in different tissues showed that AdLAC3 exhibited the highest expression levels in roots, followed by stems and leaves, but almost no expression level was detected in fruits. Similarly, AdLAC7 gene also showed the maximum expression in roots, followed by stems, while almost no expression was observed in the leaves and fruits. qRT-PCR analysis in the fruits treated with different storage conditions showed that AdLAC3 gene was significantly induced in the fruits either exposing to 25 ℃, 4 ℃or ABA, while the transcriptional level of AdLAC7 gene in fruits sharply increased only under ABA treatment. This suggested that AdLAC3 and AdLAC7 gene played distinct roles in the post-harvest storage of kiwifruit. This study provides new insights for the kiwifruit research in regard to post-harvest storages and quality regulating.

Bordetella bronchiseptica (Bb) is one of the pathogens of Swine infectious atrophic rhinitis (AR). Optimal conditions of Bb biofilm formation were developed, biofilm was well developed after 24 h, concentration of NaCl at 0.4%, pH 7.5, concentration of lactose at 0.5%. The protein expression profiles of whole Bb planktonic cells and biofilm-forming cells were investigated by comparative proteomics analysis, In this study, we compared the expression patterns of the whole Bb bacterial proteins originated from planktonic cells and biofilm-forming cells by a two-dimensional gel electrophoresis (2-DE). The results revealed that 15 protein spots were up-regulated and nine were down-regulated. Identified by mass spectrometry, the up-regulated protein spots mainly included elongation factor Tu (EF-Tu), superoxide dismutase (SOD) and molecular chaperone, involved in the stress, regulation and metabolism. The study is helpful to understand the Bb biofilm formation mechanism, and to prevent and control AR.

Based on the data from the International Agricultural Biotechnology Application Service Organization (ISAAA), the herbicide tolerance transgene events of 4 crops, including cotton (Gossypium hirsutum), soybean (Glycine max), canola (Brassica napus) and maize (Zea mays) were summarized. The aim is to provide important references for the development of herbicide tolerance transgenic crops in China. It was found that there were 328 herbicide tolerance transgene events by the end of 21 May 2017. These events are approved by the country concerned for direct consumption or as an additive or for cultivation. Herbicide tolerance transgene events of cotton, soybean, canola and maize were 39, 28, 32 and 201, respectively, accounted for 11.89%, 8.53%, 9.76% and 61.28% of the global herbicide tolerance transgenic events, respectively. Nineteen herbicide tolerance genes, which are derived from 16 kinds of organisms, involved in these herbicide tolerance events of these four crops. Seven out of 19 herbicide tolerance genes are derived from five plant genomes, including maize, arabidopsis (Arabidopsis thaliana), soybeans, tobacco (Nicotiana tabacum cv. Xanthi) and oats (Avena sativa), and the rest of 13 from microbial genomes. These 19 genes displayed tolerance to nine kinds of herbicides, which are glyphosate, glufosinate, imidazolinones, 2,4-dichlorophenoxy (2,4-D), isoxazolone, dicamba, sulfonylureas, mesotrione and bromoxynil. Singular herbicide tolerance events, multi herbicide tolerance events, stacked gene events (herbicide tolerance and other trait, such as insect resistance) were 25, 18 and 257, respectively, which accounted for 8.3%, 6% and 85.7% of the total herbicide tolerance events of four crops. These events were developed by eight companies, namely Syngenta, Monsanto Company, DuPont, Bayer CropScience, Dow AgroSciences LLC, BASF, Genective S.A. and the Stein Seed Farm Inc.(USA). Compared to the above international situation, the most of the herbicide tolerance events are glyphosate tolerance, mainly from microorganisms in China. Therefore several suggestions are put forward for developing herbicide tolerance events scientifically in China. Firstly, it is suggested to focus on the development of herbicide tolerance transgene events which should be from plants, because plants have abundant herbicide tolerance genes and herbicide metabolic genes. Secondly, it is recommended to develop glufosinate tolerance transgene events due to characteristics of glufosinate, such as broad spectrum, low toxicity and high activity, environmental compatibility, and excellent weed control effects. Thirdly, other herbicides tolerance transgene events, such as 2,4-D, mesotrione, bromoxynil and isoxazolones should be developed. The single herbicide tolerance crop would lead to the evolution of herbicide resistance weeds due to overreliance on a single herbicide or group of herbicides that share the same mechanism of action or of metabolism. Transgenic crops carried double herbicide tolerance and even more herbicide tolerance genes allow to rotations of herbicides with different modes of action and different metabolic pathways, which are more likely to delay the evolution of herbicide resistant weeds than using herbicide of the same mode of action. Finally, it is suggested to cultivate the event with stacked traits, which could improve the economic value and ecological benefit of the transgenic crops.

Bcl-2 associated athanogene (BAG) family proteins possess different biological functions, and widely participate in multiple biological processes such as tumor regulation, apoptosis and stress response. Currently, the researches of BAG family in plant mainly focus on Arabidopsis thaliana. In this review, we summarized the biological functions of BAG family in plant: (1) C terminal of BAG protein contains an evolutionarily conserved BAG domain, which can interact with Hsp70 related proteins. (2) Specific domains of N terminal such as ubiquitin-like domain and calmodulin binding domain, which promise BAG proteins to participate in different biological functions. (3) BAG proteins involve in many biological processes including plant response to stress such as temperature, salinity and pathogen infection, and programmed cell death. This article expounds reported researches of functions of BAG proteins in stress response of plant, which may be helpful in enhancing stress tolerance and then improving the study of molecular breeding in crops.