To investigate the genetic basis of heterosis in maize seedling traits, highly heterotic maize(Zea mays L.) hybrid Yuyu22 and RILs(recombinant inbred lines) were used as basic population to construct TTC(triple testcross) population, which included 312 testcross progenies by using TTC genetic mating design. Thirty QTLs were detected for the longest root length(LRL), shoot height(SH), primary root number(RN), root dry matter weight(RDW) and shoot dry matter weight(LDW) by using composite interval mapping. And some QTLs for different seedling traits were found to be located on the same chromosome regions, and a total of 4 regions were detected, which distributed on chromosome 2, 3 and 7, respectively. Further analysis indicated that 22 QTLs were detected by using Z1 and Z2 data, which were classified as overdominant(11), additive(5), partially dominant(5) and dominant(1). Furthermore, 8 genome regions of QTL× genetic background interactions and 16 markers pairs with epistatic effects were detected. Collectively, we propose that overdominance and epistasis are the main genetic basis of maize seedling traits and their heterosis.

In order to understand the internal relationship between the dynamic changes and characterization of microbial iron(Ⅲ) reduction in paddy soil, dynamic changes of community structure of Anaeromyxobacter were analyzed. Paddy soil samples were flooded 1 h, 1 d, 5 d, 10 d, 20 d, and 30 d respectively, and the dynamic changes of Anaeromyxobacter community structure were analyzed with PCR-RFLP technique. The 16S rRNA gene sequences obtained in this study have been submitted to NCBI under accession numbers JF430083~JF4301440. And reduction of Fe(Ⅲ) was determined with anaerobic slurry incubation method. The results showed that α diversity index among different flooding treatments illuminated distinct differences. The diversity was the highest in flooding 20 d treatment and the lowest in 10 d. In all samples, 11 RFLP-based dominant patterns(P1~P11) were found and divided into 3 groups based on 16S rRNA gene phylogenetic analysis. Group 1 and Group 2 included five dominant patterns respectively, one pattern(P1), was shared. And all the patterns in Group 1 and Group 2 were affiliated with Anaeromyxobacter capable of dissimilatory iron reduction. Group 3 was obviously different from the other two. In paddy soil treated with different flooding time, the dynamic succession of Anaeromyxobacter community structure occurred, and Anaeromyxobacter in Group 1 had close relationship with ferrihydrite reduction at flooding early phase and goethite reduction at the late stage. This research provides a theoretical basis to further clarify the environmental function of Anaeromyxobacter in soil.”

Flower development is a very important event in the development of higher plant, which can be divided into floral induction, floral initiation and floral organ specification. The genetic control of flower development is a very complex network of regulatory genes. In the past 2 to 3 decades, many genes or regulators of flower development have been identified and cloned in model plants, with a new class of regulatory factors- microRNAs(miRNAs) that were found at the beginning of this century. miRNAs are small approximately 21-nucleotide RNAs that function posttranscriptionally to regulate gene activity. miRNAs function by binding to complementary sites in target genes causing mRNA degradation and/or translational repression of the target. miRNAs function broadly to control many aspects of plant development. This review focuses on molecular mechanisms, the methodologies of miRNA study, and the roles of 7 miRNA families in flower development from the earliest stages(floral induction) to very late stages(floral organ cell type specification), and further research focuses are also discussed.

In order to clarify the genetic presentation of the Lycopene-β-cyclase(LYC-b) antisense gene in the progeny of transgenic tomato, the genetic stability of transgenic LYC-b antisense gene of T1 and T2 progenies of tomato(Lycopersicum esculentum) 2 varieties were studied, based on the previous getting transgenic LYC-b antisense gene plant, through detection of transgenicLYC-b antisense gene, measurement of fruit lycopene and soluble solid contents, and morphological and biological characteristics. The results showed that LYC-b antisense gene was exsisted and expressed in T2 plants, which was from positive T1 plant. This means target gene can be stably inherited from one generation to another. There were some differences between cultivars, generations, and plants of the same generation in the effect of LYC-b antisense gene on fruit lycopene content. The fruit lycopene content of T1 transgenetic plants increased by 24%~72% compare to the control while T1 transgenetic plants increased by 24.5%~105.3%. In addition, the flower number per infloresence was less thanthat of the negative control; the soluble solids content of transgenic tomato plants was different between generations and plants; one of T2 generation plants mutated from unlimited growth type to limited one. The results provides some basic data for extending the stable phenotype of transgenic tomato strains in production.

The purpose of this research is to explore the role of anthocyanidin synthesis pathway in pigmentation of brown cotton fibers. Two full-length cDNAs encoding flavonoid 3' -hydroxylase gene (F3'H) was cloned from XC-5 fibers of 16 days post anthesis (DPA) by rapid amplification of cDNA ends (RACE) and RT-PCR using specific primers based on the Gossypium hirsutum EST sequences(GenBank accession: DT545210, CO071403 and BG447485)from the blast results with the full-length cDNA of F3'H in Vitis vinifera that had already known and were named as GhF3'H-1 and GhF3'H-2 (GenBank accession: HM598123, HM598124). The length of the two cDNA sequences were 1 761 and 1 892 bp, respectively. They contained an identical opening reading frame (ORF) of 1 533 bp, encoding a protein of 510 amino acids. There were few differences between the two sequences except for the sequence length of the 3' untranslational region (UTR). The temporal and spatial expression patterns of chalcone synthase (CHS), flavonoid-3’，5'-hydroxylase as (F3'5'H) and flavonoid 3'-hydroxylase (F3'H) genes were examined by the semi-quantitative RT-PCR analysis with the specific primers. The expression analysis indicated that the expressed levels of CHS and F3'5'H gene in white and brown-fiber cotton cultivars during development were relatively high. But the F3'H gene was mainly express in the ovule of 1 day post anthesis, and were almost not expressed in the fibers and petals. All the results suggest that the production of pigments in brown cotton fibers is related to the biosynthesis pathway of anthocyanins, the principle of pigment formation in colored cotton has been disclosed, and this is of help to improve the colors of naturally colored cotton

Dihydroflavonol 4-reductase (DFR) is a key enzyme converting dihydroflavonols to leucoanthocyanidins in the biosynthesis of anthocyanins. EST sequencing was carried out using a cDNA library of peanut(Arachis hypogaea L.) immature seed and full length cDNA of peanut DFR was cloned. Sequence alignment showed that DFR was highly conserved among different plant species. Peanut cDNA microarray and semi-quantitative RT-PCR were used to analysis the expression of DFR in different tissues. Results indicated that the expression level was highest in gynophores and followed by flowers, while the expression in roots and leavies was low. DFR gene expression was also analysed in seeds from different cultivars, which suggested that it expressed higher in cultivars with dark-color seed coat, the expression was the highest in ZH9 seeds with black seed coat. We also analysed the expression level of DFR in a peanut mutation with purple leaves and stems, which demonstrated that DFR was highly expressed in the purple tissues. These results demonstrate that the expression level of DFR gene is positively correlation to the accumulation of anthocyanins, which indicates that DFR catalytic reaction is the key step in the biosynthesis of anthocyanins.

Angiopoietin-like protein 4(ANGPTL4) is a secretory protein associated with lipid metabolism. In order to study the function of ANGPTL4 gene in the regulation fat and energy metabolism, in this study, 789 blood samples were collected from 4 bovine populations Nanyang, Jiaxian Red, Luxi and Qinchuan cattles to extract genomic DNA, and SNPs of ANGPTL4 gene were detected by DNA sequencing. Then PCR-RFLP and association analysis between detected SNPs locus and growth traits were carried out. The results indicated that there were missense mutation(1422T>C) and synonymous mutation(4899C>T) in 3rd and 5th exon of ANGPTL4 gene (NC_007305.4), respectively. The association of polymorphisms revealed that the exon 3 and 5 of ANGPTL4 gene had significant correlation with average daily gain of 12-month-old Nanyang cattle(P<0.05); The 3rd exon of ANGPTL4 also had significant correlation with body weight of 12-month-old Nan Yang cattle(P<0.05). The results suggest that these loci may be considered as molecular marker in the marker-assisted selection program for growth traits in Nanyang cattle.

To study the genetic polymorphism and nomenclature of Mingguang small-ear pig(MG), Diannan small-ear pig(DN) and Bama xiang pig(BM), the polymorphic parameters were calculated using eighteen microsatellites (MS) in swine leukocyte antigen complex (SLA). The genetic parameters, including the observed heterozygosity(Ho), expected heterozygosity (He) and the polymorphism information content (PIC), were studied in three breed miniature pigs and Yunnan wild boar(YWB, Sus scrofa silvianus). The haplotypes, clustering and principal component (PC) analysis were further studied to elucidate the relationship in three breed miniature pigs. The results showed that the PIC of Yunnan wild boar was highest (0.694) versus Bama xiang pig was lowest(0.585), these breed miniature pigs were highly polymorphic. There were twelve and fourteen kinds of haplotypes in the regions of SLAⅠandⅡ, respectively. Four shared haplotypes were found among Mingguang small-ear pig, Diannan small-ear pig and Yunnan wild boar, only one haplotype was detected between Bama xiang pig and Mingguang small-ear pig. Comparison with Bama xiang pig, the relationship was close in Mingguang small-ear pig, Diannan small-ear pig and Yunnan wild boar by the clustering and PC analysis. These results suggest that Mingguang small-ear pig can belong to Diannan small-ear pig, because they are shared similar genetic background, this study gives us a molecular evidence for the synonym issue.

microRNAs(miRNAs) is a class of single-strand small RNAs that regulate gene expression by binding to complementary sequences in the 3' untranslated region(3'UTR) of target mRNAs. In a sense, identification of miRNAs and their targets in mammary gland tissue is essential to proclaim the function of miRNAs on gene expression and regulation. In the present study, small RNAs were extracted from porcine mammary gland tissue of Jinhua and Yorkshire breed respectively and used to construct a cDNA library. Then it was sequenced by Illumina Genome AnalyzerⅡx. Mappable sequences were obtained from raw sequences which filtered junk, short, simple, low copy and certain known types of RNA sequences. BLASTNs were performed between miRNABase Genome, EST database and mappable sequences. Different miRNAs detected between two pig breeds were conducted , and top ten abundance of miRNAs were selected for further bioinformatics analysis. The results showed that the reads of mappable sequence was 9 672 024 in the mammary gland tissue of Jinhua pig and 6 946 905 in Yorkshire pig, while the number of unique miRNA detected in Jinhua pig was 848 and 683 in Yorkshire pig. Moreover, 369 and 231potential novel unique miRNAs in Jinhua and Yorkshire pigs were discovered, respectively. Totally 288 miRNAs had significant difference between Jinhua and Yorkshire pigs(P<0.01), and 1 829 targets in accordance with top ten abundance of miRNAs were also revealed. These targets were primarily involved in many pathways such as antigen processing and presentation, typeⅠdiabetes mellitus, gap junction, allograft rejection and so on, suggest that these miRNAs have widely roles in physiological metabolism processing of mammary gland. Our study enriches the porcine miRNABase and provides a basis for researching the function and the regulated mechanism of miRNA in mammary gland tissue.

In general, gene expression changes when cotton is infested by phytophagous insects. Screening and identification of these regulatory genes induced by cotton bollworm (Helicoverpa armigera Hübner) infestation will help us understand the molecular mechanism of the host plants resistance to insect pests. In this paper, cotton (Gossypium spp.cv. Zhong12) was as test material. The cotton plants were divided into control groups and pest stress treatment groups (including 6, 12, 24 and 48 h, pest stress treatments, respectively). The total RNA were extracted from control groups and different pest stress treatments, and the Affymetrix cotton gene chip analysis were applied to explore gene expression profiles. It was found that a total of 4 109 genes (28%) in leaves were differentially expressed after induced 6 h, 1 917 of them was up-regulated and 2 192 of them was down-regulated, respectively. After 12 h, a total of 2 605 genes(18%) were differentially expressed, 1 326 of them was up-regulated and 1 279 of them was down-regulated, separately. After 24 h, a total of 3 213 genes(22%) were differentially expressed, 1 424 of them is up-regulated and 1 789 of them was down-regulated, respectively. After 48 h, a total of 2 763 genes(19%) were differentially expressed, 1 450 of them was up-regulated and 1 313 of them is down-regulated, separately. Bioinformatics analysis revealed that function of some differentially expressed genes involved roxidative stress response, defense response, signal transduction, transcriptional regulation, flavone and flavonol biosynthesis, terpenoids synthesis and metabolism, biosynthesis of alkaloids derived from many kinds of amino acids, etc. Furthermore, some potential genes which regulate specific volatiles emission in cotton were also identified. It was found that the gene expression of farnesyl pyrophosphate synthase was regulated by circadian rhythm. Meanwhile, (+)-delta-cadinenesynthase gene expression level increased when cotton plants was infested by H. armigera. Real-time quantitative PCR results indicated that farnesyl pyrophosphate synthase gene and chalcone--flavonone isomerase gene had the highest expression level when cotton infested 6 h by H. armigera. However, farnesyl pyrophosphate synthase gene and chalcone--flavonone isomerase gene had the lowest expression level when cotton infested 48 h by H. armigera. These findings proide base studying data for defense mechanism when cotton is infested by cotton bollworm, and help us further understand the molecular regulation of terpenoids synthesis under phytophagous insects stress.

In order to investigate technology of isolation and purification, and antimicrobial activity of pulp protein, in this paper, protein Mp-21.6 was isolated and purified from the pulp (Momordica charantia L.) by ammonium sulphate fractionation chromatographies on Sephadex G -100 and DEAE-52. And the protein was analyzed via SDS-PAGE and biochemical determination. The results showed that Mp-21.6 was homogeneous single band with molecular weight of 21.6 kD and pI 9.6. Mp-21.6 protein had intrachain disulfide bond by reductive two-dimensional electrophoresis. And Mp-21.6 was similar to RNMC_MOMCH[Ribonuclease MC(RNase MC, EC=3.1.27.1)] compared with amino acids by Aacomplement software. Antimicrobial activities of the protein were detected by antimicrobial testing. The results showed that Mp-21.6 protein from M. charantia had strong inhibition to testing Escherichia coli, Staphylococcus aureus, Salmonellab sp, Bacillus sabtilis,Saccharomyces cerevisiae and Aspergillus flavus respectively, whose minimal inhibitory concentration(MIC) was far below sodium benzoate value. In addition, the testing also showed that with grading separation and purification of Mp-21.6 protein from M. charantia, its total antioxidantive activity and clearance on ·OH free radical were rising. Therefore, protein Mp-21.6 has the significant antioxidant capacity and broad-spectrum antimicrobial activity. Biological active protein from M. charantia can exploit new type antimicrobial agents with development potential through grading purification by molecular weight interval fractionation way.

Nuclear receptor co-activator 1 gene (NCOA1) is one of the members of steroid hormone receptor which is essential for growth and differentiation of ovarian follicles. Single nucleotide polymorphisms (SNPs) were detected by PCR-SSCP, and two SNPs was found at exon 3 and 12. Rea1-time quantitative PCR was used for identification of expression changes of NCOA1 gene at exon 3 and 12. Results indicated that NCOA1 gene expression was increased along with growth as undulatory property and few before 14 weeks. NCOA1 gene expression was rapidly increased along with sex maturation, and high expression was found at 28 weeks then decreased. NCOA1 expression level of female line was easier than that of male line and significantly higher mRNA expression after 14 weeks (P<0.05). The advantage genotyps of AA and CC which significantly influenced the egg performance of individual expression, was significantly higher than that of the other genotype within the same site after 16 weeks (P<0.05). NCOA1 genes may play an important role in the process of sexual maturation and egg production performance.

Heat shock protein 90(HSP90) family is an evolutionarily conserved protein, to be involved in chaperone function, cell cycle control and antigen presentation. The cDNA sequences of heat shock protein 90 from Bursaphelenchus xylophilus, B. mucronatus and B. doui were cloned by RT-PCR and RACE methods, and named Bx-hsp90, Bm-hsp90, Bd-hsp90 (GenBank Accession No. GU013561, HMO34943 and HM347331) respectively. Theire full cDNA length were 2 255, 2 193 and 2 232 bp, the ORF were all 2 127 bp encoding 708 amino acid residues, 5'-untranslated region (UTR) was 33, 13 and 13 bp, and 3'-UTR was 95, 53 and 92 bp, respectively. And their full genomic length were 2 440, 2 388 and 2 407 bp, and all genomic sequences contained 3 introns. The amino acid sequence identities among Bx-hsp90, Bm-hsp90 and Bd-hsp90 was 98.78%, Evolutionary analyses showed that Bursaphelenchus xylophilus, B. mucronatus and B. doui had a close relationship with Caenorhabditis elegans, which might be indicative of their low evolution. Heat shock protein 90 from B. xylophilus, B. mucronatus and B. doui shared highly homologous, which made hsp90 to be a candidate gene for studying the biological evolution. The present study has a practical significance for understanding the invasive mechanism of B.xylophilus and lays a good foundation for understanding the environmental adaptabilities of these three plant parasitic nematodes.

Forkhead box transcription factor O1(FoxO1) plays a critical role in cell proliferation and differentiation in mammals. To interpret effects of the phosphorylation of FoxO1(p-FoxO1) on pig preadipocytes adipogenesis and it's underlying molecular mechanism, pig preadipocytes were cultured. After treated with wortmannin(WM), differentiation state of pig preadipocyte was detected by Oil Red O staining and the expression level of FoxO1 and adipocyte differentiation marker gene were analysed by qRT-PCR and Western blot. The results findings showed that as differentiation proceeded, FoxO1 mRNA, protein and p-FoxO1 protein were all increased. After the pig preadipocytes were treated with WM for 5 days, FoxO1 protein was notably increased(P<0.05), but p-FoxO1 protein was decreased significantly, meanwhile FoxO1 mRNA was not much changed. Oil red O staining showed that adipocytes differentiation was attenuated and adipocyte markers, such as PPARγ and C/EBPα, were remarkably lowered(P<0.05). Taken together, our data demonstrate WM decreases FoxO1 phosphorylation level and suppresses adipogenesis by reducing PPARγ and C/EBPα mRNA. Differentiation of pig preadipocytes is inhibited by dephosphorylation of FoxO1.

In order to enhance the industrial application of embryos produced by in vitro fertilization(IVF) with sex-sorted sperm, several main factors were investigated in the present study, including cumulus cell removal degree (intact, partial removal and complete removal), different bulls (A~C), volume of IVF drops (30~100 μL) and sperm concentrations(0.3×106~1.0×106/mL). The results showed that (1)The cleavage rate of oocytes with partial removal of cumulus cells (67±4.74)% was significantly higher than that of oocytes with intact cumulus cells (41±2.23)% or complete removal of cumulus cells (37.50±2.88)% (P<0.05); (2)The cleavage and blastocyst rates of oocytes fertilized with sex-sorted sperm of bull B [(61.25±5.78)%, (26.53±3.31)%] were significantly higher than those of bull A [(42.5±3.45)%, (17.65±3.65)%] and C [(30±2.62)%, (16.67±2.36)%] (P<0.05); (3)The cleavage and blastocysts rates of oocytes fertilized in 50~100 μL drops had no significant difference(60±3.54)%~(64.17±3.04)% and (20.41±4.33)%~(23.38±4.47)% (P>0.05), but significantly higher than those in 40 μL[(48.33±2.89)%, (13.79±2.02)%] or 30 μL [(33±2.74)%, (12.12±3.73)%](P<0.05);(4)The cleavage and blastocyst rates of oocytes fertilized with a sperm concentration of 0.5×106~1.0×106/mL showed no significant difference(62±4.12)%~(68.75±4.64)% and (22.58±2.17)%~(27.27±3.49)% (P>0.05), but were significantly higher than those with 0.3×106/mL[(38.75±3.58)%, (16.13±3.46)%] group(P<0.05). The results indicate that partial removal of cumulus cells and the selection of appropriate bulls combined with reasonable decrease of volume of drops and sperm concentration contribute greatly to increasing the efficiency of IVF with sex-sorted sperm, meanwhile decreasing the production cost of sexed embryos.

To elucidate the function of calsarcin-2(CS2) in the formation of skeletal muscle fiber and signaling pathway regulation, we used yeast two-hybrid approach to look for CS2 interacting proteins. The porcine skeletal library screen gave 180 positive interactions, in which 12 interacting proteins were obtained. Bioinformation analysis found that actin alpha 1(ACTA1), titin(TTN) and calmodulin 1 (CALM 1) were CS2 interaction partners. ACTA1 involved in the process of muscle contraction, TTN regulated the dynamics conduction of sarcomere and Z disc assembly, CALM 1 paticipated in calcium signal transduction. These results indicate that CS2 is implicated in the transduction of calcium signals and maintaining the structure of Z disc.

The present study established a porcine satellite cells in vitro model by means of the single fiber method to clarify the proliferation and myogenesis. Satellite cells can dissociate from the myofibers which isolated from porcine extensor digitorum longus (EDL) after digesting by collagenase Ⅰ. Then the cells were detected by their immunocytochemistry, growth curve, myogenesis induction and Western blot. The results showed that the cells adhered to the myofibers originally and then moved outside. More than 90 percent of the cells expressd the marker genes paired protein box(Pax7) and myogenic differentiation antigen(MyoD). After myogenesis induction, satellite cells began to convergence ,showed orientation growth, and finally formed multinucleated myotubule with expression of myogenin and myosin heavy chain(MyHC). Moreover, MyoD protein was highly expressed in proliferated stage, while the protein of myogenin and MyHC was detected only in the terminal differentiated stage.This study successfully cultured porcine satellite cells by means of the single fiber method and obtained high purity skeletal muscle satellite cells which can provide cell source for viable transplantation and cure related diseases.

Tail-interacting protein 47 gene(TIP47) is important for lipid droplet biogenesis and metabolism, to study the function and expression of goat TIP47 gene, we cloned TIP47 from Xinong Saanen goat(Capra hircus) mammary gland total RNA using RT-PCR and RACE PCR. The results showed that the cDNA sequence of goat TIP47 was 1 906 bp in length (GenBank accession No. HQ846827), including 82 bp of 5'UTR, 1 314 bp of CDS, and 510 bp of 3'UTR, encoding a protein of 437 amino acids with a molecular weight of 47.36 kD and pI 5.74. Nucleotides and amino acid(aa) sequences alignment showed that the nucleotides and aa of goat TIP47 had high score similarity with bovine (Bos taurus), human (Homo sapiens), mouse (Mus musculus) and pig (Sus scrofa) in GenBank. Primary protein structure analysis showed that TIP47 did not contain signal peptide sequence and transmembrane structure; the N-terminal of the protein was more hydrophobic than that of the C-terminal; the C-terminal of the protein was shaped like an L in tertiary structure. Goat TIP47 had the closest relationship with bovine, followed in pig and human, which was generated by phylogenetic tree analysis. TIP47 was expressed in all 12 tissues examined by Real-time fluorescence quantitative PCR, the result showed that the TIP47 mRNA expression in mammary gland was the highest and far higher than those of all other tissues examined, followed in spleen and liver; TIP47 expression in muscle was the lowest. The results suggest that TIP47 plays a potentially role in biogenesis and metabolism of lipid droplet in goat mammary gland, setting the base for function studies of this gene in lipid droplet activites.

In order to study DRB1 gene exon 3 polymorphism in Tibetan sheep, The number of alleles, single nucleotide polymorphisms(SNPs) sites, variation type, the genetic relationship and evolutionary significance of the alleles had been analyzed. Variation in the 500 Tibetan sheep (Ovis aries) DRB1 gene exon 3 was investigated by PCR-SSCP method, followed by cloning and DNA sequencing. The results showed that 8 alleles were identified, 15 nucleotide polymorphism sites were identified in 8 sheep DRB1 gene haplotypes and 7 novel DRB1 exon 3 alleles were found comparison with GenBank sequences. The phylogenetic tree results showed that the 8 haplotypes of Tibetan sheep DRB1 exon 3 could divide into two clusters. Allele B was the most common allele in three Tibetan sheep populations, and that allelic and genotype frequency were significantly deviated from Hardy-Weinberg balanced. The polymorphism was higher with PIC more than 0.5 at DRB1 gene exon 8 in three Tibetan sheep populations. The results suggest that Tibetan sheep DRB1 gene exon 3 has high level of sequence polymorphism，Tibetan sheep DRB1 gene is differentiated into two major category alleles from two mutant alleles at first.

The objective of this study was to investigate the protective effect and mechanism of angiotensin converting enzyme 2(ACE2) on renal oxidative stress injury induced by high glucose. The rats(Rattus norregicus) model of renal oxidative stress injury was established by intraperitoneal injection of streptozotocin(STZ) solution, and treated by subcutaneous injection of insulin once a day(1 mL (3.7×10-5 mol/L)). After 30 days, all the rats were decapitated and the serum and kidney tissue were collected. The contents of advanced glycation end products (AGEs), malondialdehyde(MDA), superoxide dismutase(SOD), angiotensinⅡ(AngⅡ), angiotensin1-7(Ang1-7) in serum and the mRNA levels of ACE2 and Mas receptor in kidneys were measured. Compared with control group, the contents of AGEs, MDA and AngII in serum of high glucose rats were significantly increased(P<0.01), and the levels of SOD and Ang1-7 in serum were significantly decreased(P<0.01). The mRNA expression of ACE2 in kidneys was obviously reduced(P<0.01), while the mRNA expression of Mas was obviously raised(P<0.01). After insulin treatment, the contents of AGEs and MDA in serum were obviously reduced(P<0.01); the activity of SOD in serum was significantly raised(P<0.01); the level of AngII in serum was significantly decreased(P<0.05) while the concentration of Ang1-7 in serum was significantly increased(P<0.05). The mRNA expression levels of ACE2 and Mas receptor in kidneys were significantly increased(P<0.01). Exparemental results suggest that ACE2 plays an important role on the renal oxidative stress injury induced by high glucose and the mechanism may be that the activation of the ACE2-Ang1-7-Mas axis reduces the effect of AngⅡ.

FMRFamide-like peptides (FLPs) play an important role in infection, feeding and reproduction of plant parasitic nematode. A novel FMRFamide-like neuropeptide gene was isolated from Ditylenchus destructor by EST analysis and RACE methods. The full length cDNA of Dd-flp-1 consisted of 891 bp, with a 681 bp ORF encoding a 227 amino acid protein and the predicted molecular weight was 25.36 kD. The protein included a signal peptide of 43 amino acids on the N- terminal and a predicted FMRF domain with five FLPs as NFLRFG on the C- terminal. It was a typical FLP-1-type protein, named Dd-flp-1(GenBank accession No: GU198750). Phylogenic analysis suggested that Dd-FLP-1 had a homology to free live nematodes, animal parasitism nematodes and other migratory plant-parasitic nematodes which might indicate association of the evolution of flp genes from plant parasitism nematodes and their habits. In situ hybridization analysis showed that the transcripts of Dd-flp-1 accumulated exclusively within the circumpharyngeal nerve ring, retrovesicular ganglion and ventral cord of D. destructor. The results suggested that the major functions of DD-FLP-1 were closed with feeding, moving and excreting in D.destructor. This study will provide the foundation and theoretical basis for research on the flp gene function and developing novel plant parasite control targets by RNAi

To find novel vip3 genes, the vip3-type genes in 72 Bt stains were identified by PCR method and high-resolution melting analysis(HRMA) system. The results indicated that three vip3 gene types, vip3Aa, vip3Af and vip3Ba were found in 18 positive strains. A full-length vip3 gene fragment, which obtained by PCR amplification with a pair of primers designed according to vip3-type gene sequences and DNA from T03B001(B. thuringiensis subsp. sumiyoshiensis) as template, was introduced into expression vector pEB and transformed into Escherichia coli Rosetta(DE3). At low temperatures, a peptide of 88 kD was expressed by IPTG induction. The encoded protein was composed of 789 amino acid rsidues. It shared a 96% sequence homology with Vip3Aa protein. This gene designated as vip3Aa39 by International Nomenclature Commitee of Bt endotoxin, GenBank accession No. was HMI17631. Vip3Aa11 protein was obtained in our previous study, 39 amino acid residues were found to be different between the Vip3Aa39 and Vip3Aa11 proteins. Insecticidal activities of soluble expressed products of vip3Aa39 and vip3Aa11 genes were tested against Agrotis ipsilon, Plutella xylostella, Helicoverpa armigera and Spodoptera exigua by feeding method. The bioassay results indicated that Vip3Aa39 had insecticidal activity against A.ipsilon with 50% lethal concentration(LC50) of 5.43 μg/mL compared to 73.62 μg/mL for Vip3Aa11; Vip3Aa39 showed insecticidal activity against P.xylostella with LC50 of 140.64 μg/mL while Vip3Aa11 protein had no activity against P.xylostella; Vip3Aa11 demonstrated insecticidal activity against H.armigera with LC50 of 35.18 μg/mL compared to 286.99 μg/mL for Vip3Aa39; Vip3Aa39 had insecticidal activity against S.exigua with LC50 of 2.02 μg/mL similar to 2.04 μg/mL for Vip3Aa11. The results indicated that insecticidal activity of Vip3Aa39 and Vip3Aa11 was different from each other against A.ipsilon, P.xylostella and H.armigera. Compared with the Vip3Aa11, Vip3Aa39 had high insecticidal activity against A.ipsilon and P.xylostella while showed low activity agaist H.armigera. Vip3Aa39 will provide more chocies to control agricultural pest, and it provide materials to study structure and insecticidal mechanism of the Vip3 protein.

A yeast one-hybrid library was constructed to screen the binding proteins of suspensor specific cis-element. In this study, five repeats of suspensor specific cis-element sequence(GAAAAGCGAA) were synthesized and ligated with reporter vector pHIS2.1 to construct the bait vector pHIS2.1-G564-5A. Total RNA was extracted from immature seeds of Arabidopsis thaliana, and the purified mRNA was used as template to synthesize ds cDNA with SMARTTMⅢ and CDSⅢ adapter. The purified ds cDNA, linearized pGADT7-Rec2 and pHIS2.1-G564-5A were simultaneously transformed into the yeast strain Y187 by LiAc-mediated method. The results showed that the co-transformation efficiency was 5.53×105 cfu/μg. After 3 times screening in SD/-Trp/-Leu/-His/ 100 mmol/L 3-AT medium, 276 positive clones were obtained and 142 of which were sequenced, whereafter the sequences were annotated by BLASTX against NCBI no-redundant protein database and 117 sequences were identified. Gene functional analysis showed that there were 14 proteins with DNA binding and 8 proteins with protein binding function, and this protein contained TATA binding protein 1(TBP1), Scaffold-associated region(SAR) DNA-binding protein, Ovule development protein, G-box binding factor 1(GBF1), and Histone H3. The results provide candidate genes for isolation and cloning transcription factors expressed specifically in suspensor.

In this study we construct a maker-free targeting vector of human leukemia inhibitory factor and verify its expression in goat mammary cells. First we use the recombination cassette LoxP sequence, goat β-casein 5′and 3′ homologous arms, positive and negative selection marker cassette to get targeting vector of human leukemia inhibitory factor. Transfection of the vector into goat mammary cells with LipofectamineTM 2000 conferred resistance to geneticin (G418) and resistance to ganciclovir (GANC) in the cells to get homologous recombination positive monoclonal, then we verify the hLIF expression by PCR, RT-PCR and Western blotting. The restriction and sequencing analysis resulst suggested that the targeting vector pLoxP-NT53L is constructed successfully, and after detected by PCR, RT-PCR and Western blotting, we find the target band, which demonstrate the targeting vector pLoxP-NT53L is integrated into the β-casein locus and the goat mammary cells can express protein specificity. Based on the study, it is very useful for produce marker-free darily goat of transfer hLIF gene.

Peste des petits ruminants(PPR) is an acute and highly contagious disease caused by Peste des petits ruminants virus (PPRV). For the detection of IgG against PPRV in sera, BALB/c mice(Mus musculus) were immunized with purified recombinant nucleoprotein of PPRV expressed in eukaryotic expression system. The hybridomas were screened by indirect ELISA using purified recombinant nucleoprotein of PPRV expressed in prokaryotic expression system as coating antigen. Eight monoclonal antibodies(mAbs) against PPRV N protein were filtered and the best was 1D5. Isotype of the 1D5 was IgG2b, and the titer of theascites induced by 1D5 was 1∶106. A competitive ELlSA(c-ELISA) for the detection of antibodies agaist PPRV was established using rpET-PPRN protein as coating antigen and mAb 1D5 as detecting antibody. The c-ELISA could differentiate rinderpest (RP) and PPR positive sera, and was sensitive and specific. According to the test results of 129 goat(Capre hircus) serum samples, the c-ELISA established in this study had a high coincidence rate of 96.9% compared with the c-ELISA kit supplied by CIRAD-EMVT. In this study a c-ELISA to detect IgG against PPRV in sera specifically is established, it’s significant for diagnosis and control of the disease and monitoring the effectiveness of vaccine.

The infection of Neotyphodium gansuense in drunken horse grass (Achnatherum inebrians) has caused great losses for livestock production in China. An effective molecular method for detection of N. gansuense is quite necessary to set up to replace the present identification method based on morphology and isolation culture. In this study, the N. gansuense isolated from the seed of drunken horse grass collected from Xinjiang, China was confirmed by aniline blue staining microscopy and used as the environmental samples. The reference strain collection used in this study included six species of Neotyphodium (N. gansuense, N. coenophialum, N. lolii, N. huerfanum, N. chisosum and N. aotearoae), and all of them had highly similar morphological patterns. A fragment of about 430 bp in size of the β-tubulin gene was first amplified and sequenced from each strain used. The universal Taqman probe and its primers for genus Neotyphodium, and specific Taqman probe and its primers for N. gansuense, were designed according to the sequence alignment results. The results showed the detection method based on the double-colored Real-time fluorescence PCR developed in this study was an effective, rapid and sensitive molecular method for detection of N. gansuense, which allowed us to detect the mycelia of N. gansuense from a single grass seed, and the detection duration only needed about seven hours. This method can be used to direct detect the infection rate of N. gansuense from the seeds of animal husbandry and lawn.