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Construction of conjugal transfer system of Streptomyces cinnamonensis and effect of PCR-mediated nsdA gene disruption on its secondary metabolism 肉桂链霉菌偶联转移体系的构建及pcr介导的nsdA基因破坏对其次生代谢的影响
Pub Date : 2008-04-01 DOI: 10.1017/S1479236208002106
Chen Fen, Xiong Wei, M. Yong, Fan Yu-Qing, Liang Yunxiang, L. He-Ping, Xing Ren-Chang, Zheng Ying-hua
Intergeneric transfer of plasmid vectors pSET152 and pHL212 from donor Escherichia coli ET12567(pUZ8002) and S17-1 to Streptomyces cinnamonensis were demonstrated and optimized. The nsdA gene disruption structure was constructed by PCR-targeting system and then introduced into S. cinnamonensis BIB2005 through intergeneric conjugal transfer. After PCR analysis, the screened AprRKanS conjugant BIB309 was confirmed to be the nsdA mutant. Compared with the start strain, the yield of monensin of the nsdA mutant BIB309 increased 270 percent in the level of flask.
研究了供体大肠杆菌ET12567(pUZ8002)和S17-1质粒载体pSET152和pHL212向肉桂链霉菌的属间转移。利用pcr靶向系统构建nsdA基因断裂结构,并通过属间偶联转移将其导入S. cinnamonensis BIB2005中。经PCR分析,筛选到的AprRKanS偶联物BIB309确认为nsdA突变体。与起始菌株相比,nsdA突变体BIB309在瓶水平上的莫能菌素产量提高了270%。
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引用次数: 0
Screening, cloning and sequence analysis of gBTN1A1 gene in mammary gland of Xinong Saanen goat 西农萨嫩山羊乳腺gBTN1A1基因的筛选、克隆及序列分析
Pub Date : 2008-04-01 DOI: 10.1017/S1479236208001940
Zhang Lijuan, Wu Hui-juan, Luo Jun, L. Junxia, Z. Ning, Wang Hai-bing, Shan Cui-yan, Y. Gang
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引用次数: 3
Display of H5N1 Avian influenza virus haemagglutinin HA1 on Bacillus thuringiensis cell surface and its immunogenicity for mice H5N1禽流感病毒血凝素HA1在苏云金芽孢杆菌细胞表面的显示及其小鼠免疫原性
Pub Date : 2007-12-01 DOI: 10.1017/S1479236207001702
L. Mei, Lu Lin-Jing, Huang Jun-yan, Zhang Shu-huan, Bi Ding-ren, S. Ming
S-layer protein CTC surface display system of Bacillus thuringiensis (Bt) was used to test the possibility of displaying H5N1 Avian influenza virus haemagglutinin HA1 on B. thuringiensis cell surface. Two recombinant plasmids were constructed by replacing the central part below the surface anchor sequence slh of S-layer protein gene ctc with part ha1 gene (halp ). The two resulting plasmids were pCTC-HA1P (harboring fusion gene ctc-halp ) and pCSHA1P (harboring fusion gene csa-ctc-halp, csa represents csaAB operon which was very important to the anchoring of S-layer protein on the bacterial cell surface). Two recombinant B. thuringiensis strains were constructed by electro-transferring recombinant plasmids to B. thuringiensis plasmid-free derivative strain BMB171. The resulting strains were CH (harboring pCSHA1P) and BCCH (harboring pCTC-HA1P as well as the plasmid pMIL-CSA which carried csaAB operon). The vegetative cells of CH and BCCH were used as antigens of haemagglutination (HA) assay and haemagglutination inhibition (HI) assay. HA assay showed recombinant HA1 proteins were successfully displayed on the cell surface of CH and BCCH, respectively. HI assay showed recombinant HA1 proteins displayed on the cell surface were specific to standard positive HI (haemagglutination inhibition test) serum of subtype H5 AIV. After immunizing mice with vegetative cells of CH and BCCH respectively, CH and BCCH all elicited a humoral response to HA1 and exhibited immunogenicity as assayed by enzyme-linked immunosorbent assay (ELISA). ELISA also showed CH exhibited a higher immunogenicity than BCCH. The strategy developed in this study gives a possibility to generate heat-stable, oral, veterinary vaccine against AIV with B. thuringiensis S-layer protein CTC surface display system.
采用苏云金芽孢杆菌(Bacillus thuringiensis, Bt) s层蛋白CTC表面展示系统,对H5N1禽流感病毒血凝素HA1在苏云金芽孢杆菌(Bacillus thuringiensis, Bt)细胞表面展示的可能性进行了研究。用ha1部分基因(halp)取代s层蛋白基因ctc表面锚定序列slh以下的中心部分,构建了两个重组质粒。得到的两个质粒分别是pCTC-HA1P(包含融合基因ctc-halp)和pCSHA1P(包含融合基因csa-ctc-halp, csa代表对细菌细胞表面s层蛋白锚定非常重要的csaAB操纵子)。将重组质粒转移到苏云金芽孢杆菌无质粒衍生菌株BMB171上,构建了2株重组苏云金芽孢杆菌。得到的菌株为CH(携带pCSHA1P)和BCCH(携带pCTC-HA1P以及携带csaAB操纵子的质粒pMIL-CSA)。将CH和BCCH的营养细胞作为血凝(HA)试验和血凝抑制(HI)试验的抗原。HA实验显示重组HA1蛋白分别在CH和BCCH细胞表面成功表达。HI实验显示,细胞表面显示的重组HA1蛋白对H5亚型AIV标准阳性HI(血凝抑制试验)血清特异性。分别用CH和BCCH营养细胞免疫小鼠后,经酶联免疫吸附试验(ELISA)检测,CH和BCCH均对HA1产生体液应答,并表现出免疫原性。ELISA还显示CH比bch具有更高的免疫原性。本研究为利用苏云金芽孢杆菌s层蛋白CTC表面显示系统制备热稳定的口服兽用AIV疫苗提供了可能。
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引用次数: 0
Expression of H5N1 Avian influenza virus haemagglutinin in a baculovirus expression system H5N1禽流感病毒血凝素在杆状病毒表达系统中的表达
Pub Date : 2007-12-01 DOI: 10.1017/S1479236207001817
Dun Jifeng, Pu Juan, Z. Yingchun, L. Jinhua
The haemagglutinin (HA) gene of H5N1 Avian influenza virus (AIV) was amplified from the plasmid pGEM-HA and cloned into the baculovirus transfer plasmid pFastBacHT to construct the recombinant transfer vector pFastBacHT-HA. The pFastBacHT-HA was transformed into DH10Bac competent cells, transposed with a shuttle vector (Bacmid) and the transposition rBacmid-HA was constructed. The recombinant baculovirus was harvested from sf9 cells transfected with rBacmid-HA. The expressed HA protein was identified and analysed by SDS–PAGE, Western blot and haemadsorption assay. The 66 kDa protein could only react with chicken serum positive to H5 subtype AIV. The haemadsorption assay showed that the sf9 cells infected with rBacmid-HA baculovirus could absorb chicken red blood cells. These results indicated that the HA protein was successfully expressed in sf9 cells, with reaction specificity to H5 subtype antiserum.
将H5N1禽流感病毒(AIV)血凝素(HA)基因从质粒pGEM-HA中扩增到杆状病毒转移质粒pFastBacHT中,构建重组转移载体pFastBacHT-HA。将pFastBacHT-HA转化到DH10Bac的感受态细胞中,用穿梭载体(Bacmid)转座,构建转座rBacmid-HA。重组杆状病毒从转染rBacmid-HA的sf9细胞中获得。用SDS-PAGE、Western blot和血液吸附法对表达的HA蛋白进行鉴定和分析。66 kDa蛋白只与H5亚型AIV阳性的鸡血清发生反应。血液吸附实验表明,感染rBacmid-HA杆状病毒的sf9细胞能够吸附鸡红细胞。结果表明,HA蛋白在sf9细胞中成功表达,对H5亚型抗血清具有反应特异性。
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引用次数: 1
Cloning of ACO gene and inhibition of ethylene evolution in tomatoes with RNAi 用RNAi技术克隆ACO基因及抑制番茄乙烯进化
Pub Date : 2007-12-01 DOI: 10.1017/S1479236207001775
Cheng Yin-hua, Ouyang Bo, Li Hanxia, Ye Zhibiao
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引用次数: 0
QTL identification for seed setting rate of rice in various environments 不同环境下水稻结实率的QTL鉴定
Pub Date : 2007-12-01 DOI: 10.1017/S1479236207001970
C. Qingquan, Yu Si-Bin, Li Chunhai, Mou Tong-min
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引用次数: 1
Construction and evaluation of near-isogenic lines for major QTLs of basal root thickness and 1000-grain-weight in lowland and upland rice 陆稻基根粗和千粒重主要qtl近等基因系的构建与评价
Pub Date : 2007-12-01 DOI: 10.1017/S1479236207001787
L. Lifeng, Zhang Hongliang, Mu Ping, Qu Yan-ying, Li Zichao
The development of near-isogenic lines (NILs) is one of the important foundations for the establishment of molecular linkage map, the mapping of quantitative trait locus(QTL) and molecular marker-assisted selection (MAS). In the present study, NILs for two major QTLs of basal root thickness(BRT) and 1000-grain-weight(TGW) were obtained through the foreground selection for the target QTL, the background selection for the genome of the recurrent parents of 3 backcross populations( BC1F1, BC2F1 and BC3F1) and phenotypic selection for the population of BC3F2. There were 9 BRT QTL-NILs with BRT ranges of 1.07~1.16 mm, over the recurrent parent by 6.11%~15.18% and an average recovering ratio of genetic background (RRGB)of 97.22%; eleven NILs of TGW QTL with ranges of 21.25~26.25 g, over the recurrent parent by 7.05%~32.16% and 95.97% of RRGB.
近等基因系(NILs)的开发是建立分子连锁图谱、定位数量性状位点(QTL)和分子标记辅助选择(MAS)的重要基础之一。本研究通过对目标QTL的前景选择、对3个回交群体(BC1F1、BC2F1和BC3F1)的回交亲本基因组的背景选择和对BC3F2群体的表型选择,获得了基根厚度(BRT)和千粒重(TGW)两个主要QTL的NILs。共有9个BRT QTL-NILs, BRT范围为1.07~1.16 mm,比复发亲本高6.11%~15.18%,平均遗传背景恢复率(RRGB)为97.22%;11个TGW QTL的NILs值为21.25~26.25 g,分别比回亲本高7.05%~32.16%和95.97%。
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引用次数: 7
PCR–ELISA method for Mycoplasma contamination detection in veterinary vaccines 兽医疫苗支原体污染检测的PCR-ELISA方法
Pub Date : 2007-12-01 DOI: 10.1017/S1479236207001593
Z. Ying, Jiang Jun-bing, Liu Jin-ping, Jia Li-yan
A polymerase chain reaction–enzyme-linked immunosorbent assay (PCR-ELISA) method was set up to detect Mycoplasma in live vaccines and to establish the optimal experimental conditions. According to the 16S rRNA gene sequences published in GenBank, which include Mycoplasma gallisepticum of chicken and M. hyopneumoniae , M. hyosynoviae and M. flocculare of swine (submitted nos: Mg AY744942, Mhp AE017244, Mhs AY973563 and Mf X63377), PCR primers, labelled with digoxin (Dig), and probe, labelled with biotin, were designed using the software DNAstar and Primer Premier 5.0. The system and conditions of PCR–ELISA were optimized using purified genomic DNA extracted from Mycoplasma as a template. Samples of 30 batches of Newcastle disease live vaccines (I strain), 30 batches of Newcastle disease live vaccines (La Sota strain) and 30 batches of swine fever live vaccine were tested by PCR-ELISA. Results showed that the detection rate of Mycoplasma contamination from different vaccines was higher (36.5%) with this technique than with PCR (24.4%). The PCR–ELISA appeared to be a simple, fast and reliable method for qualitative and quantitative analysis of Mycoplasma . The practical use of PCR–ELISA as a kit to detect Mycoplasma contamination in live vaccine seems very promising.
建立了聚合酶链反应-酶联免疫吸附法(PCR-ELISA)检测活疫苗中支原体的方法,并确定了最佳实验条件。根据GenBank上已发表的鸡鸡感染支原体和猪肺炎支原体、水膜支原体和猪絮状支原体(提交号:Mg AY744942、Mhp AE017244、Mhs AY973563和Mf X63377)的16S rRNA基因序列,采用DNAstar软件和Primer Premier 5.0软件设计地高英标记的PCR引物和生物素标记的探针。以支原体基因组DNA为模板,对PCR-ELISA检测体系和条件进行了优化。采用PCR-ELISA法对30批次新城疫活疫苗(1株)、30批次新城疫活疫苗(La Sota株)和30批次猪瘟活疫苗进行检测。结果表明,该方法对不同疫苗支原体污染的检出率(36.5%)高于PCR法(24.4%)。PCR-ELISA方法简便、快速、可靠,可用于支原体的定性和定量分析。PCR-ELISA试剂盒在活疫苗支原体污染检测中的应用前景广阔。
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引用次数: 0
Genetic diversity analysis of diazotrophs in the rice rhizosphere 水稻根际重氮营养体遗传多样性分析
Pub Date : 2007-12-01 DOI: 10.1017/S1479236207001982
C. Bin, Zheng Si-ping, Zhou Li-juan, L. Zhimin, Song Yana, Zheng Wei-wen
The genetic diversity of dinitrogen-fixing bacteria associated with rice ( Oryza sativa ) was assessed by a polymerase chain reaction–restriction fragment length polymorphism (PCR–RFLP) approach on the nifH gene amplified directly from DNA extracted from washed rice roots and rhizospheric soil. Restriction digestion with the enzymes Mnl I and Hae III was performed to characterize 54 cloned nifH PCR products. RFLP profiles were clustered and analysed with the UPGMA program. Eight pairs of similar RFLP patterns (similarity>50%) and two pairs of homologous RFLP patterns (100% identity) were found from the washed roots and the rhizospheric soil, respectively. Three specific diazotrophic patterns were found from rhizospheric soil and rice roots. The analyses have revealed the presence of different nifH types, which appear to be significant components of the diazotrophic community in paddy fields, indicating that some of the diazotrophs may colonize the inside and the surface of the rice roots.
采用聚合酶链反应-限制性片段长度多态性(PCR-RFLP)方法对水稻(Oryza sativa)相关的二氮固定菌(Oryza sativa) nifH基因进行了遗传多样性分析。用Mnl I和Hae III酶切酶对54个克隆的nifH PCR产物进行了表征。采用UPGMA程序对RFLP谱进行聚类分析。在洗根和根际土壤中分别发现8对相似的RFLP模式(相似度为50%)和2对同源的RFLP模式(同源度为100%)。在根际土壤和水稻根系中发现了三种特殊的重氮营养模式。分析结果表明,水稻重氮营养菌群中存在不同类型的氮肥菌群,它们是水稻重氮营养菌群的重要组成部分,表明部分重氮营养菌可能在水稻根系内部和表面定植。
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引用次数: 3
Cloning and expression of Ma duck interleukin-18 mature protein gene and biological activity detection 麻鸭白介素-18成熟蛋白基因的克隆、表达及生物活性检测
Pub Date : 2007-12-01 DOI: 10.1017/S1479236207001842
Chen Hongying, Cui Bao-an, Xia PingAn, Liu Xinsheng, Yang Mingfan, Zheng Lanlan
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引用次数: 0
期刊
Chinese Journal of Agricultural Biotechnology
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