The aim of this study was to analyze the expression profile of the CD8B gene in different tissues of pigs (Sus scrofa), and to reveal the genetic effect of the CD8B gene polymorphism on blood immune traits. The expression level of the CD8B gene in seven tissues of Large White pigs was quantified by fluorescence quantitative polymerase chain reaction, and gene polymorphism was detected by direct sequencing and polymerase chain reaction-restriction fragment length polymorphism method in 382 Large White, 84 Landrace and 90 Songliao Black pigs, and effects of the CD8B gene mutation on peripheral blood T lymphocyte subsets and hematological traits were analyzed in the Large White samples. The results showed that the highest CD8B mRNA was expressed in the spleen, and a missense mutation (c.602 G>A) was found in exon 5 of the CD8B gene, and three genotypes (AA, AG and GG genotypes) were all detected in Large White, Landrace and Songliao Black populations. The CD8B gene polymorphism was significantly associated with blood CD4＋CD8－, CD4

Abstract The meiotic maturation of spermatocyte in mammal is controlled by the maturation promotion factor (maturation promoting factor, MPF), a complex of CDK1(cyclin-dependent kinase, CDK1) and CyclinB1 proteins. In the present study, we selected 0,2,6,12,24-month-old sheep testis to investigate the expression and cellular localization of CDK1 and CyclinB1 in the sheep testes on different months . CDK1 and CyclinB1 genes expression levels in different months testis were analyzed by qRT-PCR(fluorescence quantitative Realtime PCR,qRT-PCR) and CDK1 and CyclinB1 localization in testis were detected by immunohistochemistry. The results showed that with month increasing, the expression of CDK1 and CyclinB1 mRNA were firstly decreased and then increased. CDK1 and CyclinB1 mainly localized in the spermatogonia, primary spermatocytes and secondary spermatocytes. CDK1 protein always expressed at an extremely low level throughout all stage of development. However, its expression quantity was relatively stable. On the other side, the monthly expression difference of CyclinB1 protein in sheep testis of were remarkable, the expression level was always higer than that of CDK1 protein. These results indicated that the expression of CDK1 and CyclinB1 mRNA and protein in sheep testes at different months were significant. And it may play an important role in sheep testis development.

Cluster of differentiation antigen 14 (CD14) plays an important role in both the innate and adaptive immune responses. This study aimed at establishing the short hairpin RNA (shRNA) interference vectors with pig (Sus scrofa) CD14 gene silencing to package them into lentivirus for transfecting pig small intestinal epithelial cells (IPEC-J2) and conducting function analysis on cell level to provide fundamental basis for function and action mechanism of CD14 gene. Based on the whole coding sequence of pig CD14 gene (GenBank No. EF626695.1), 4 interfere sequences were constructed which encoded shRNAs against pig CD14 gene and cloned into lentivirus expression vectors of pGLV3-CD14-1, pGLV3-CD14-2, pGLV3-CD14-3, pGLV3-CD14-4 and negative control pGLV3-CD14-NC. IPEC-J2 was infected by lentivirus solution after packaging successfully and qRT-PCR was used to detect the interference efficiency of CD14 gene. Lentivirus with the highest interference efficiency continually infected cells through medicinal sieve to obtain pig small intestinal epithelial cell line with stable CD14 gene silencing. Escherichia coli adhesion test was conducted to detect the adhesion ability change of E. coli F18ab and F18ac to IPEC-J2. The results showed that 4 shRNA vectors were constructed successfully and the packaged lentivirus could reduce the mRNA expression level of pig CD14 gene, in which pGLV3-CD14-3 had the best interference efficiency of 94.6%. The adhesion results showed that the E. coli F18ab's adhesion ability to IPEC-J2 significantly enhanced after CD14 gene silencing and E. coli F18ac did not. This result showed that CD14 gene silencing enhanced the susceptibility to E. coli F18ab in small intestinal epithelial cells, which illustrated that CD14 gene may play an important regulation role in which small intestinal epithelial cells resist infection by E. coli F18ab. The establishment of pig small intestinal epithelial cell line with CD14 gene silencing stably mediated by lentivirus offered important material for mechanism research of CD14 and toll-like receptors/interleukin-1?receptor (TLRs/IL-1R) signal pathway, which played important roles in intestinal immune response and pathogen defense caused by pathogenic microorganisms. This study successfully screened out the efficient shRNA vector which could exclusively interfere porcine CD14 gene expression, which also provides experimental basis for further studying the CD14 gene function and mechanism for the resistance to gram-negative bacteria infection in porcine intestinal tract at the cellular level.

Arginine kinase (AK, L-arginine N-phosphotransferase, EC 2.7.3.3) is a phosphotransferase that plays a critical role in cellular energy metabolism in invertebrates such as insect, crustacean and some unicellular organisms. It catalyzes the reversible transfer of the phosphoric group of adenosine triphosphate (ATP) to arginine yielding adenosine diphosphate (ADP) and phosphoarginine. In order to better understand the role of AK gene and its regulatory mechanism during the cytochrome P450 (CYP6B6) expression of cotton bollworm(Helicoverpa armigera), the H. armigera AK (HarmAK) cDNA was cloned from midgut by RT-PCR on the basis of yeast one-hybrid results. The fragment digested by double enzymes was linked to a prokaryotic expression vector pET28a to construct the recombinant expression plasmid pET28a-HarmAK, and then converted into Escherichia coli BL21 competent cells. The fusion protein His-HarmAK was induced to express by isopropyl-β-D-thiogalactoside (IPTG), and purified by Ni2+ affinity chromatography. SDS-polyaerylamide gel electrophoresis (SDS-PAGE) and Western blot analysis were used to examine the fusion protein. The fusion protein activity was determined by the pH-spectrophotometric assay. qRT-PCR was used to test HarmAK expression level in both developmental stages of the cotton bollworm and different tissues of the 6th instar larvae. The results of sequencing and sequence analysis showed that the ORF of the HarmAK gene was 1 068 bp, encoding 355 amino acid residues and the predicted molecular weight and isoelectric point was 39.8 kD and 5.76, respectively. And the protein was deduced to have no signal peptide and transmembrane helices and abundant secondary structure. Besides, the HarmAK was monomer, which had 2 ligands including 1 L-arginine and 1 ADP. The recombinant pET28a-HarmAK expressed a soluble protein after IPTG induction. SDS-PAGE and Western blot analysis indicated that the fusion protein, purified using Ni2+ affinity chromatography, had the predicted size and higher purity. And the activity of the fusion protein was (5.5±0.85) μmol/(min·mg) protein by the pH-spectrophotometric assay, which showed that the fusion His-HarmAK was able to catalyze the phosphorylation of L-arginine. Expression profile results showed that HarmAK expressed in both different tissues and developmental stages. The HarmAK expression level was higher in midgut, head and integument than that of fat body. The amount of HarmAK was the highest in the 1st instar larvae and gut of 6th instar larvae, and then decreased to lower level from 2nd instar larvae to adult. Our results will provide very useful information for using HarmAK as a new molecular target to control H. armigera.

The cluster of differentiation 3 (CD3) molecules are expressed on the surface of T-cell and involved in T-cell activation by binding to T cell receptor complex non-covalently. In this paper, three CD3 subtypes (CD3γ/δ, CD3ε and CD3ζ) cDNA were obtained from Lateolabrax japonicus by RT-PCR and rapid amplification of cDNA ends (RACE). The full length cDNA sequence of CD3γ/δ was 1 231 bp in length, containing a 5'-UTR of 99 bp, 3'-UTR of 583 bp and ORF of 549 bp which encoded a putative protein of 182 amino acids. The CD3ε cDNA was 2 145 bp in length with 186 bp 5'-UTR, 1 434 bp 3'-UTR and 525 bp ORF coding a protein of 174 amino acids. The full length cDNA sequence of CD3ζ was 1 231 bp in length, including a 5'-UTR of 56 bp, 3'-UTR of 772 bp and ORF of 453 bp which encoded a deduced protein of 150 amino acids. The CD3γ/δ and CD3ε in Lateolabrax japonicus were similar in structure with an Ig-like extracellular domain, a transmembrane peptide and a cytoplasmic tail with one immunoreceptor tyrosine-based activation motif (ITAM). There were 4 cysteins in the extracellular domains of CD3γ/δ and CD3ε, the first 2 cysteins involved in Ig-fold stabilisation and the last two in CxxCxE motif important for dimerization. However, The CD3ζ molecule had a different structure with a short extracellular part of 5 amino acids, a transmembrane region and a long cytoplasmic tail with three ITAM motifs. Analysis of DNA and cDNA sequences showed that the ORF part of CD3γ/δ gene contained 6 exons and 5 introns while CD3ε gene contained 7 exons and 6 introns. qRT-PCR results showed that CD3γ/δ, CD3ε and CD3ζ were expressed in all tissues tested with higher levels in gill, spleen, headkidney and intestine and lower levels in fat, liver, eye and brain. The expression of CD3γ/δ, CD3ε and CD3ζ increased obviously in headkidney, intestine and spleen after intraperitoneal injection of Vibrio harveyi. The results indicated that CD3s were involved in immune response of T cells after bacterial infection. This obtained information will provide a theoretical basis for studying the molecular mechanism of fish CD3 in regulating fish immune response to pathogens.

Ustilago esculenta is the endophytic fungus of Zizania latifolia, and induces the plant stem swollen which may be related to its dimorphism. MAPK (mitogen activated protein kinase) signaling pathway has been known to play important regulatory role in the fungi dimorphism. In this study, the gene UeMkk1 (GenBank accession number: KR870332), encoding a mitogen activated protein kinase kinase (MAPKK), was cloned through analysis of the differentially protein profile and transcriptome sequencing data from yeast- and mycelium- form of U. esculenta. The full length of UeMkk1 was 2 204 bp, with a 2 043 bp open reading frame. Clustering analysis between UeMkk1 and MAPK signaling pathway proteins of yeast showed that UeMkk1 belonged to MAPKK protein. Blast results from NCBI and phylogenetic analysis results by MEGA 5.0 showed that UeMkk1 had higher homology to Mkk1 proteins in Ustilago, of which the highest homology was MKK1 of Ustilago hordei, reaching to the identify of 84%. Also the typical Serine/threonin dual specific protein kinase catalytic domain in UeMkk1 showed high conservation to other fungi. Meanwhile, UeMkk1 protein was successfully expressed in prokaryotic expression system, and purified from the lysate supernatant with good purity. The best expression system for UeMkk1 was that induced with 0.2 mmol/L isopropyl β-D-thiogalactoside (IPTG) at 37 ℃, and purified by Ni-NTA with 200 mmol/L imidazole elution. In addition, the morphology microscopic observation of U. esculenta cultured on basic medium with different carbon sources was carried out, showing that fungi hyphae formation was induced by sucrose, and the fungi maintained yeast-like growth type on the other culture medium (PDA, glucose or maltose medium). Meanwhile, real-time fluorescence quantitative PCR (qRT-PCR) analysis of UeMkk1 expression in the fungi after induced by different carbon sources was carried out, and the results showed that UeMkk1 was up-regulated at 4 d after cultured on maltose medium and at 6 d after cultured on PDA or glucose medium, showing a cyclical increased expression. Only on sucrose medium, UeMkk1 was earlier up-regulated (after 2 d, when the hyphae started formation) but fell down obviously when cultured after 8 d (when the growth of hyphae became slowly). The results showed that UeMkk1 was up-regulated during active differentiation and proliferation, indicating the involvement of UeMkk1 in cell wall integrity regulation of U. esculenta, which was similar to other fungi. All the work provideds research foundation for further studying the function of UeMkk1 and exploring its relationship with dimorphism of U. esculenta.

Bombyx mori Nucleopolyhedrovirus (BmNPV) 39K gene which encodes a phosphoprotein associated with viral gene expression, exists in genomes of all Lepidopteran Baculoviruses. The results of existing studies show that the 39K gene is related to the regulation of viral gene expression, but the specific regulation effects of BmNPV 39K gene on viral replication and transcription is still unknown. Red recombination technique and Bac-to-Bac system were respectively utilized to delete and recover 39K gene to construct a knockout bacmid (39K-ko-Bacmid) and 39K- rescued bacmid (39K-re-Bacmid). Then the constructed bacmids DNA were transfected into BmN cells. The results showed that both types of viruses could yield infectious virion in cells, indicating that 39K gene was not essential for the replication of BmNPV, however, the deletion of 39K gene could reduce virus titer significantly. In late transfection, the replication level of the viral genome was related to infectious budded virus (BV) which was generated by virus. Possibly, the reduced BV led to decreased replication capacity of deficient virus in late transfection. Furthermore, the resultes of qPCR showed that the deletion of 39K gene had no effect on virus early replication but would decrease the transcription level of viral late genes and caused extremely significant reduction of viral gene transcription level (P＜0.01) in different phases. In order to explore the role of 39K gene in the expression regulation of polyhedral promoter, we constructed a recombinant bacmid with BmNPV polyhedral promoter drived-firefly luciferase and Bombyx mori cytoplasm action A3 promoter drived-renilla luciferase. Quantitative detection of luciferase activity proved that 39K gene had significant regulation effect on polyhedral gene promoter, the deletion could enhance the activity of polyhedral gene promoter. In conclusion, 39K gene was not essential for the replication of BmNPV, but the deletion could reduce virus gene transcription level in different phases and enhance the activity of polyhedral promoter. We speculated that the 39K protein which may act as a repressor protein bound to polyhedron gene promoter, so that affected the polyhedron gene promoter combining with other transcription factors, and then resulted in decreasing of polyhedron gene promoter activity. This study will lay the foundation for further investigating the effect of BmNPV 39K gene on viral genome replication and transcription, also provides information on the efficient transcription mechanism of Bombyx mori baculovirus expression system.

Mycoplasma ovipneumoniae (MO) can lead to the atypical pneumonia for both sheep (Ovis aries) and goats (Capra hircus), which is wide- spreading and can cause serious harm. Based on the complete sequence of MO Y98 by our group, a potential hemolysin gene (hlyA) was obtained by using gene annotation and PCR method. In the present study, hlyA gene, which derived from MO Y98, was modified by using preference codons of Escherichia coli. And then prokaryotic expression vector (pET32a-hlyA) was constructed. pET32a-hlyA was transformed into E. coli BL21(DE3) polysS, and induced for the expression of HlyA. HlyA recombinant protein was visualized by SDS polyacrylamide gel electrophoresis (SDS-PAGE) and Western blot method, and purified by using Ni2+ metal chelate column. The results showed that the hly gene (GenBank No.KT598390) of MO Y98 strain contained 375 bp, encoding a polypeptide of 125 amino acid residues. After optimization, the codon adaptation index was 0.91, the modified hlyA is suitable for expression in E.coli. Recombinant fusion proteins were mainly existed in the form of inclusion body. Expressed HlyA protein was about 31.6 kD, consistenting with the expected size. The results of Western blot analysis showed that supernatant of purified protein could react with the primary antibody, indicating that HlyA proved an efficacious immunological reactivity. When purified recombinant HlyA protein concentration was diluted to 0.125 μg/mL, the ability of dissolving red blood cells had declined. This research contributes to the study of hymolysin pathogenicity in MO.

Pituitary adenylate cyclase-activating polypeptide (PACAP), a member of glucagon/secretes peptide family, is a pleiotropic hormone that enhances and stimulates the secretion of growth hormone, gonadotropin, prolactin and somatolactin. The single nucleotide polymorphism (SNP) in PACAP gene can affect its function or translation and transcription. Studying the association between the SNP and growth traits can provide candidate markers for marker-assisted selection. We detected the polymorphism of PACAP gene by DNA sequencing to explore the association between the polymorphism of PACAP and growth traits of largemouth bass (Micropterus salmoides). Five pairs of primers were designed to amplify the PACAP gene sequence, and a SNP A-2282C site was found in the 4th intron, and had 3 genotypes named as AA, AC and CC, respectively. The genotype and gene frequency of this SNP in 327 largemouth bass were further assayed with the method of Snapshot, the result showed that gene frequency of genotypes AA, AC and CC were 0.034, 0.645 and 0.321, respectively, and the frequency of A allele and C allele were 0.355 8 and 0.644 2. Chi-square test showed that the locus was in Hardy-Weinberg equilibrium in the largemouth bass population，the effective number(Ne) of the locus was 1.846 5, expected heterozygosity (Ho) and observed heterozygosity (He) were 0.540 9 and 0.644 2. The least squire method (LSM) was used to analyze the association of genotype with growth traits, and variance analysis indicated the mean values of AC genotype group was higher than that of AA and CC groups in body weight, body length, head length, total length, body depth and caudal peduncle length. And the body width and caudal peduncle length with AC genotype were significantly higher than that of AA (P＜0.05). The depth with AC genotype was extremely significantly higher than group with CC (P＜0.01), and those with AC genotype were significantly higher than those with AA genotype (P＜0.05). The locus can be a candidate molecular marker for largemouth bass breeding.

Strains producing cold-active amylase were isolated from the melt water of Yangbark glacier, East Pamirs, amongthem, the strain D5-2 with the highest amylase activity was identified as Pseudomonadaceae sp.by16S rDNA sequence analysis. The study on enzymatic properties showed that the optimum temperature and pH for catalytic activity was 25℃ and pH 6.5, respectively, but the thermal stability of the enzyme is relatively poor. The cold-adaptable amylase had high activities between pH 5.5 and 8.0. The amylase activity was activated by Mg2+ang Mn2+, and was inhibited by Fe2+, Na+and EDTA.The result indicates Pseudomonadaceae sp.D5-2 is a high-producing cold-active amylase strain which shows research and development potential, it deserves to be further researched.

Estrogen receptor (ESR) is a ligand-dependent transcription factor of nuclear receptor superfamily. Numerous studies showed that ESR genes areclosely related to animal reproduction activities. In order to analyze the correlation between the single nucleotide polymorphisms (SNPs) of ESR genes and egg-production trait of laying pigeon (Columba livia), the SNPs of partial ESR gene fragments were screened by the first-generation sequencing in 215 Taiping King pigeons(C. livia). Ten SNPs were detected in the threefragments, T129416C was located in intron 6 of ESRα gene, the other nine SNPs were detected in ESRβ. They were T365C, A388G, C389A and A429G in intron 1, G451A, G577Ain exon 2, T672C and A923C in intron 3, and T17167C in exon 5 of ESRβ gene. Among these ten sites, three of the mutations were located in exon, but only G577A made amino acid change from isoleucine (Ile) to valine (Val). These two amino acids are non-polar and hydrophobic, it is a common missense mutation that has little effect on protein polarity in vivo. All the ten sites were in balance based on the Hardy-Weinberg equilibrium test. The results of Linkage disequilibrium analysis among the ten sites showed that the sites S1 and S10 were not meaningful linkage disequilibrium relative to others, because there were greater physical distances between the two sits and the others. The sites between S2 and S4, S9 were strong linkage disequilibrium (D′≈1 and r2＞0.8) and site S2 could be completely represented by S4 or S9. The correlation analysis between SNPs of ESR genes and egg production showed that only site S8 was significantly correlated to egg production, genotype AB of this site was significantly higher than AA in egg production (P＜0.01), while BB genotype was higher than AA genotype and lower than AB genotype, but the differences were not significant (P＞0.05). The results indicate that the S8 site of ESRβ gene can be used as a molecular marker for egg-production traits in MAS breeding of laying pigeon.

Multiple endocrine neoplasia Ⅰ(MEN1) participates in regulatory role in the development of mammary gland and lactation behavior cycling. The objective of this study was to obtain full-length cDNA clone of bovine (Bos taurus) MEN1 gene (bMEN1), and analyze its expression levels of mRNA and target encoded protein menin in different cell lines. According to the deposited sequence of bMEN1 in GenBank, primers attached with EcoRⅠand HindⅢ restriction enzymes were designed and amplified by using qRT-PCR method. The digested PCR product was further ligated into eukaryotic vector pcDNA3.1-myshis-(A). Under in vitro transfection, the expected mRNA and target encoded protein menin were successively detected in homologous cell line bovine mammary epithelial cells (MAC-T) and heterologous cell lines Chinese hamster (Cricetidae) ovary cells (CHO) and mouse (Mus musculus) myoblast cells (C2C12), respectively by using qRT-PCR and Western blot. Double restriction enzymes digestion and sequencing results indicated that the eukaryotic expression vector of bMEN1 gene was successively cloned, named as pcDNA3.1-myc-his-bMEN1. Under the constructed transfection system, both mRNA and protein could expectedly express in all three different cell lines, with significantly highest expression levels after 24 h post transfection (P＜0.01) afterwards gradually decreased. Especially in CHO cells, bMEN1 mRNA expression level was 28 415-fold of negative control after 24 h post transfection, and 5.65-fold for encoded menin protein. This study can supply research tool and technical system for further functional or mechanism investigation of bovine MEN1 gene in mammary gland development and body metabolism.

Soybean (Glycine max) gibberellin-regulated MYB-related transcription factor binding protein 1 (GmGBP1) gene is a homologous gene of Sloan-Kettering retrovirus interacting protein (SKIP) gene . Proteins encoded by GmGBP1 not only participate in the regulation of a variety of abiotic stress signal transduction pathways in plants, but also play a role in the signal transduction of plant hormones, such as abscisic acid and gibberellin. In this study, soybean GmGBP1 gene was transformed into transgenic tobacco (Nicotiana tabacum) and overexpressed. After being treated at a high temperature of 48℃ for 48 h, leaf wilting and necrosis, leaf chlorosis and leaf etiolation were detected in the wild-type (WT) tobacco seedlings; while no obvious yellowing was found in the leaves of transgenic tobacco of overexpression GmGBP1 gene (GBP1ox) seedlings at a high temperature. After being treated at a high temperature of 55 ℃ for 12 h, tobacco seedlings were placed at room temperature for recovery growth for 15 d, and the survival rate of over-expression of GmGBP1 gene in tobacco seedlings was 24.14%. The survival rate of GBP1ox tobacco seedling was higher than the WT tobacco seedlings. The malondialdehyde (MDA) content of WT leaf tobacco seedlings was 2.07-fold more than that in GBP1ox after 48 ℃ treatment for 48 h. The leaves of tobacco seedlings were stained by using diaminobenzidine (DAB) after being treated at a high temperature, and the staining results demonstrated that the ratio of stained area of WT tobacco seedling leaves to total leaf area was 43.66%. The percentage of the total leaf area of DAB staining in WT seedling tobacco was significantly greater than GBP1ox tobacco seedlings (P＜0.05) as a result. High temperature treatment was performed in tobacco seedlings to analyze the changes in the relative expression levels of heat-resistance related genes in tobaccos. Zinc finger protein of Arabidopsis thaliana 12 (ZAT12) gene expression was significantly higher in GBP1ox leaf tobacco seedlings than that in WT (P＜0.05) for 3, 6, 7 and 8 h treatment at 55 ℃ high temperature. On the other hand, there was no significant difference in the relative expression levels of heat shock protein 40 (HSP40) gene in WT and GBP1ox the leaves of tobacco seedlings after being treated at a high temperature. The results showed that overexpression of GmGBP1 gene could improve the heat resistance of transgenic tobacco seedlings to some extent. The present study provides references for the cultivation of new heat-resistant transgenic crops, and lays a solid foundation on further investigations about the functions of soybean GmGBP1 gene.

Toll-like receptor 4 (TLR4) is a typeⅠtransmembrane protein and widely expressed in mammalian cell surface, which plays an important role in anti-infection immunity. To explore the expression of Toll-like receptor 4 (TLR4) in the Small Tail Han sheep (Ovis aries) mastitis tissue which caused by Escherichia coli. In the study, E.coli (O113 type) were used to infect Small Tail Han sheep breast and built the mastitis pathological model. The relative expression of TLR4 gene in the normal breast tissue and the breast tissue was infected with E.coli were detected by the methods of qRT-PCR. The expression and distribution of TLR4 protein in the normal breast tissue and the breast tissue infected with E.coli were examined by Western-blot and immunehistochemical SP method. The results showed that the TLR4 expressed in both normal breast tissue and the breast tissue infected with E.coli, but the TLR4 mRNA and protein expressed were highest in 48 h post-infection, and the expression of TLR4 mRNA and protein in 96 h post-infection had significantly lower than 48 h (P＜0.05), and the relative levels of TLR4 was lowest in normal control group (P＜0.05). The results of immunohistochemistry demonstrated that TLR4 expressed in the breast epithelium in both normal breast tissue and the breast tissue infected with E.coli. Compared with the control group, the positive expression was significantly enhanced after infected (P＜0.05). The results of this study showed that the expression of TLR4 was obviously increased after infected with E.coli in breast tissue of Small Tail Han sheep, which will establish the foundation the mechanism of TLR4 in sheep mastitis morbidity process during further study.

Dehydrins (DHNs), known as a group of late embryogenesis abundant proteins (LEA) in plants, are related to various environment stresses. In this study, CsDHN (GenBank accession No. FJ436978), a full length cDNA of DHN in tea plant(Camellia sinensis) was amplified via rapid-amplification of cDNA ends (RACE) technology. It was shown that the full-length cDNA was 960 bp, encoding 201 amino acids by sequence analysis. The deduced molecular weight of DHN protein was 21 kD, together with isoelectric point 8.3, which belonged to the Y3SK2 type of dehydrins. There were 2 exons and 1 intron in CsDHN. The bioinformatics prediction revealed that CsDHN located in the cytoplasm with no signal peptide or transmembrane domain. It was predicted to show strong hydrophilicity. The analysis of characteristic expression showed that CsDHN was up-regulated in tea plant exposed to low temperature, dehydration, abscisic acid(ABA) treatment and salt stress. The expression of CsDHN was increased not satisfactorily at 3 and 6 h under low temperature stress at 4 ℃, while the expression level was increased greatly (two times higher than control, P＜0.05) at 12 h. And the highest expression, 4.2-fold than that of control, was found at 18 h treatment, then decreased to its original level as control at 24 h. After the treatment of dehydration, the expression of CsDHN was increased dramatically. The expression level was 7.5-fold than that of control at 3 h and continuously increased to the highest level at 18 h, with the expression level was 93.4-fold (P＜0.05) than that of control. After that, the expression was decreased to some extent, not maintained a high level. In accordance to ABA treatment, the expression amount was markedly increased at 3 h, and reached to its greatest amount at 6 h (4.3-fold than that of control, P＜0.05), then began to decline. A little bit higher than its original level was found at 18 h to 24 h treatment. When treated with the solution of 300 mmol/L NaCl, the expression level of CsDHN was increased all the time till to 24 h, which was 2.5-fold and 17.8-fold than that of the original level at 3 h and 24 h, respectively. In the normal growth conditions, the expression of CsDHN existed in all organs in tea plant, which CsDHN was a constitutive expression gene. While, the expression patterns in all organs were obviously different. The highest expression was found in mature seeds (the expression was 11.2-fold than that of mature leaves); similar expression level were found in bud and leaves. But the expression levels in young stems, flowers and young roots were low with no significance, which were 0.24, 0.26, 0.32 -folds compared with leaves, respectively. This study showed that the CsDHN might participates with the defense against abiotic stress for tea plants and dehydration vitality protection during tea seeds maturation, which provides a certain theory foundation for understanding the molecular mechanism to tea stress resistance.

Doramectin is a novel macrolide antibiotics derived from avermectin and widely used as an anti-parasitic drug in animal husbandry. Doramectin can be produced by genetically modified Streptomyces avermitilis with altered avermectin synthesis pathways. Disruption of branched-chain alpha-keto acid dehydrogenase gene cluster (bkdFGH), which encodes the components of the branched-chain alpha-keto acid dehydrogenase(BCDH) complex multi-enzyme system, can direct S. avermitilis to produce effective doramectin (CHC-B1), along with other undesired analogues such as CHC-A1 and -A2, when cyclohexanecarboxylic acid (CHC) is added to the fermentation medium as a precursor. aveD, which encodes avermectin B 5-O-methyltransferase, is reported to be responsible for transforming avermectin "B" component to "A". Deletion of aveD may reduce the amount of CHC-A component in doramectin produced by bkdFGH-deleted S. avermitilis. Here we reported the construction of 2 mutant S. avermitilis strains capable of doramectin biosynthesis. S. avermitilis strain SAV939, an avermectin high-producing strain, was deleted for bkdFGH gene by homologous double crossover to generate mutant strain LZ-01. High performance liquid chromatography (HPLC) analysis demonstrated that LZ-01 could produce effective doramectin (CHC-B1) when CHC was added as a precursor in the fermentation process. Based on that, we further deleted avermectin D(aveD) in LZ-01 and obtained mutant strain LZ-02. Like LZ-01, LZ-02 could also produce CHC-B1 through fermentation, but without the ineffective component CHC-A. This work provides 2 novel doramectin producer mutants from an avermectin-overproducing industrial strain of S. avermitilis, which could potentially become industrial doramectin-producing strains after further improvement.

Spirulina is cultivated worldwide, used as a dietary supplement as well as a whole food, and is also available in tablet, flake and powder form. It is significant to improve the production capacity of Spirulina by optimization of production process. A biological reactor has a tank with an interior for growing the biological mass, and application of photobioreactor on high density culture of microalgae has become a hot spot in the domestic and foreign studies. The main economic spirulina is Spirulina platensis and S. maxima, however, S. platensis is the main Spirulina cultured in China. In this study, S. platensis was cultured by triangular flask and flat plate photobioreactor, and different culture conditions (concentration of glucose and antioxidants, and modes of ventilation and illumination) were designed to study the optimization of manufacture process. Results showed that adding glucose, Na2S2O3 and Na2SO3 to triangular flasks separately could significantly improve the algae biomass, and its optimal concentration was 3, 3 and 2 g/L, respectively. Under algae culture in flat plate photobioreactor, the biomass of algae was increased significantly with the increase of aeration intensity from 100 to 800 L/h, however, algae biomass improvement was more obvious under the treatment of gradually increasing aeration intensity from 100 L/h to 800 L/h. Algae biomass cultured by single-side light (4 500 lx) was obviously more than that cultured by double-side light (9 000 lx), and significantly less than that cultured by first single-side light and then double-side light at algae entering into logarithmic phase. The combination effect of Na2SO3 and glucose on algae biomass was better than that of Na2S2O3 and glucose. Further optimization of the above-mentioned conditions suggested that the algae biomass achieved the peak values under 4 g/L glucose and 3 g/L Na2SO3. Generally, adding glucose, Na2SO3, gradually increasing aeration intensity and illumination mode of the double side light source after the first one side light source increased algae production of S. platensis significantly. Under the condition of comprehensive optimization, the algae average growth rate and biomass production could be as high as 0.85 gDW/(L·d) and 10.02 gDW/L, respectively. Moreover, this photobioreactor had several advantages. That is small power consumption, small occupation area, relatively cheap cost, easy cleaning, simple structure, easy scale cultivation in room, etc. This culture technology can provide the theory basis and the technical method for scale cultivation of Spirulina.

Vascular endothelial growth factor (VEGF) is a multipotent factor implicated in vascular permeability, endothelial cell division, proliferation, migration, and angiogenesis, and also plays important and unique roles in reproductive system. The biological function of VEGF must be mediated through via binding to 2 high-affinity tyrosine kinase receptors, the fms-like tyrosine kinase (Flt-1) and fetal liver kinase-1/ kinase insert domain-containing receptor (Flk-1/KDR). A number of studies already found that, in the female mammalian reproductive process, VEGF and its receptors have been shown to be expressed in different tissues, including follicles, corpus luteal, embryo, placental and endometrium. VEGF-VEGFR systems act in an autocrine and paracrine manner and are thought to be involved in ovarian follicular development, oocytes maturation, luteal angiogenesis, endometrial change, embryo implantation, formation of placental blood vessel and embryo development. The mice carried out VEGF gene knockout and disrupted Flt-1 or KDR genes results in endothelial disorganization and abnormal vessel formation during early embryo development, even experience developmental defects and early embryonic lethality. Furthermore, the findings in recent years demonstrated that VEGF significantly promoted the oocytes maturation and embryo developmental competence in vitro. This article focused on the research progress on function and molecular aspects of VEGF and its receptors, as well as the relationship between VEGF and female mammalian genesial function. In the end, further research focuses were also discussed. Overall, this review may provide systematically theoretical references for VEGF and its receptor research.