The cDNA fragments of interest were amplified using \%Sj\%λ ZipLox library as the templates by PCR and then cloned into a eukaryotic expression vector p\|CMV\|GH; A small number of DNA fragments inserted in the recom...The cDNA fragments of interest were amplified using \%Sj\%λ ZipLox library as the templates by PCR and then cloned into a eukaryotic expression vector p\|CMV\|GH; A small number of DNA fragments inserted in the recombinants was identified by restriction cleavage, EST sequencing and bioinformatical analysis; mice were injected intramuscularly with the expression library(L\|CMV\|SjR) or sublibraries(L\|CMV\|SjR 1,L\|CMV\|SjR 2 and L\|CMV\|SjR 3), immunized mice were challenged with \%Schistosoma japonicum\% cercariae on day 35, the levels of IgG antibodies in sera from the immunized mice were detected by ELISA. The results demonstrated that a partial cDNA expression library of \%S.j,\% with～10\+5 transformants, was constructed, most of the recombinants contained the insert DNA fragments of interest, and these fragments had the features of protein\|coding sequences for \%Schistosome.\% There were no significant differences for the levels of IgG antibodies in sera from all of the immunized groups. Mice immunized with L\|CMV\|SjR, L\|CMV\|SjR 1 and L\|CMV\|SjR 2 developed significant protective effect against \%Sj\% infection compared to control mice injected with the empty plasmid, the rate of worm reduction was about 30%.展开更多
目的构建亚洲带绦虫成虫全长cDNA质粒文库,为获取亚洲绦虫成虫的基因信息,建立基因表达谱,并为筛选疫苗基因和诊断抗原基因奠定基础。方法提取亚洲带绦虫成虫mRNA,构建pBluescript II SK全长cDNA质粒文库,测定扩增文库的滴度;用载体克...目的构建亚洲带绦虫成虫全长cDNA质粒文库,为获取亚洲绦虫成虫的基因信息,建立基因表达谱,并为筛选疫苗基因和诊断抗原基因奠定基础。方法提取亚洲带绦虫成虫mRNA,构建pBluescript II SK全长cDNA质粒文库,测定扩增文库的滴度;用载体克隆位点两端的引物进行PCR扩增,以检测所构建文库的质量。随机挑选质粒文库转化的阳性重组克隆,进行较大规模的5′端测序,归并unigene。结果测定文库的滴度为1011pfu/μl,插入片段的大小主要在1000bp以上。共测1495个克隆,获得有效EST序列1126个,归并为643条unigene。结论已成功获得一高质量的亚洲带绦虫成虫全长cDNA表达文库,并获得了较丰富的成虫表达基因数据。展开更多
利用SMART（switching mechanism at 5’end of the RNA transcript）技术构建了红肉猕猴桃品种‘红阳’（Actinidia chinesis cv‘Hongyang’）内果皮组织的全长cDNA文库，此文库的构建有助于克隆与次生代谢相关的基因，特别是红肉猕...利用SMART（switching mechanism at 5’end of the RNA transcript）技术构建了红肉猕猴桃品种‘红阳’（Actinidia chinesis cv‘Hongyang’）内果皮组织的全长cDNA文库，此文库的构建有助于克隆与次生代谢相关的基因，特别是红肉猕猴桃花青素特异合成代谢的基因。文库滴度为6．7×10^4cfu／mL，库容为2．72×10^8cfu／mL，文库重组率99．8％，插入片段多数分布在700～1000bp。随机挑选1014个克隆进行测序，测序成功963个，经过序列拼接去除低质量序列后获得632个unigenes，包括92个contigs和540个singletons，获得已知功能unigenes共441个。从所测克隆中得到一个花青素途径的结构基因AcF3H，其cDNA序列长1369bp（GenBank登录号：FJ542819），CDS区为1101bp，编码366个氨基酸的多肽。与拟南芥、葡萄及龙胆已知乃日氨基酸序列比对，发现该基因十分保守。通过RT—PCR技术对苍溪栽培的不同发育时期‘红阳’猕猴桃果实AcF3H基因的表达进行了分析。结果表明，果肉转色前AcF3H基因表达量较高，而转色初期表达量降低，此后随着果实着色加深表达量维持在较高水平。展开更多
【目的】研究防御基因表达模式,筛选抗枯萎病候选基因,进一步阐明抗病机制。【方法】以4号枯萎病生理小种诱导的‘农科1号’香蕉为试材,利用DSN(duplex-specific nuclease)均一化酶结合SMART(switching mechanism at 5′end of the RNA ...【目的】研究防御基因表达模式,筛选抗枯萎病候选基因,进一步阐明抗病机制。【方法】以4号枯萎病生理小种诱导的‘农科1号’香蕉为试材,利用DSN(duplex-specific nuclease)均一化酶结合SMART(switching mechanism at 5′end of the RNA transcript)技术构建香蕉根的均一化全长cDNA文库;并用多重半定量RT-PCR技术研究防御基因的表达模式。【结果】经检测,文库滴度为3.1×106 cfu.mL-1,扩增后库容为2.8×108 cfu.mL-1。随机挑选580个克隆进行测序,去除低质量序列后获得421个unigenes,片段分布在750—3 000 bp,文库重组率为100%,其中,未知功能基因占42%,全长cDNA序列为68%。文库筛选获得7个防御基因,进行不同时间的诱导表达分析,发现苯丙氨酸解氨酶(PAL)、过氧化氢酶(CAT)、β-1,3-葡聚糖酶(PR-2)和胶质乙酰酯酶(PAE)具有较低的本底表达水平;枯萎病菌浸染后,7个防御酶基因表达量增加,过氧化物酶(POX)、几丁质酶(PR-3)、PAE和PAL在第6天达到高峰;虽然7个防御基因在对照植株中的表达模式与诱导后的植株类似,但表达量均显著低于接菌后。【结论】所构建的均一化全长cDNA文库,片段重组性和完整性均达到分离筛选目的基因高质量文库的要求,且证明了7个防御基因不同程度地参与了抗枯萎病反应。展开更多
以小麦品种水源11和条锈菌CY31号小种为材料,用SMARTTMcDNA Library Construction Kit构建条锈菌诱导的小麦叶片cDNA文库。得到原始文库的滴度为6×106pfu/mL,扩增文库滴度9×109pfu/mL,重组率98%,插入片段在0.5～2.2kb。随机挑...以小麦品种水源11和条锈菌CY31号小种为材料,用SMARTTMcDNA Library Construction Kit构建条锈菌诱导的小麦叶片cDNA文库。得到原始文库的滴度为6×106pfu/mL,扩增文库滴度9×109pfu/mL,重组率98%,插入片段在0.5～2.2kb。随机挑取600个克隆,测序获得594条高质量ESTs,与GenBank序列进行BLASTx分析,已知功能ESTs与植物同源比例最高,占44%;其次为真菌,占32%;有18%ESTs在GenBank中没有匹配的同源产物。蛋白功能分析表明,PIG28编码蛋白、ABC转运子、金属硫因、泛素、质膜H+-ATPase和氨基酸透酶等可能参与了寄主与病原菌互作过程。展开更多
文摘The cDNA fragments of interest were amplified using \%Sj\%λ ZipLox library as the templates by PCR and then cloned into a eukaryotic expression vector p\|CMV\|GH; A small number of DNA fragments inserted in the recombinants was identified by restriction cleavage, EST sequencing and bioinformatical analysis; mice were injected intramuscularly with the expression library(L\|CMV\|SjR) or sublibraries(L\|CMV\|SjR 1,L\|CMV\|SjR 2 and L\|CMV\|SjR 3), immunized mice were challenged with \%Schistosoma japonicum\% cercariae on day 35, the levels of IgG antibodies in sera from the immunized mice were detected by ELISA. The results demonstrated that a partial cDNA expression library of \%S.j,\% with～10\+5 transformants, was constructed, most of the recombinants contained the insert DNA fragments of interest, and these fragments had the features of protein\|coding sequences for \%Schistosome.\% There were no significant differences for the levels of IgG antibodies in sera from all of the immunized groups. Mice immunized with L\|CMV\|SjR, L\|CMV\|SjR 1 and L\|CMV\|SjR 2 developed significant protective effect against \%Sj\% infection compared to control mice injected with the empty plasmid, the rate of worm reduction was about 30%.
文摘目的构建亚洲带绦虫成虫全长cDNA质粒文库,为获取亚洲绦虫成虫的基因信息,建立基因表达谱,并为筛选疫苗基因和诊断抗原基因奠定基础。方法提取亚洲带绦虫成虫mRNA,构建pBluescript II SK全长cDNA质粒文库,测定扩增文库的滴度;用载体克隆位点两端的引物进行PCR扩增,以检测所构建文库的质量。随机挑选质粒文库转化的阳性重组克隆,进行较大规模的5′端测序,归并unigene。结果测定文库的滴度为1011pfu/μl,插入片段的大小主要在1000bp以上。共测1495个克隆,获得有效EST序列1126个,归并为643条unigene。结论已成功获得一高质量的亚洲带绦虫成虫全长cDNA表达文库,并获得了较丰富的成虫表达基因数据。
文摘利用SMART（switching mechanism at 5’end of the RNA transcript）技术构建了红肉猕猴桃品种‘红阳’（Actinidia chinesis cv‘Hongyang’）内果皮组织的全长cDNA文库，此文库的构建有助于克隆与次生代谢相关的基因，特别是红肉猕猴桃花青素特异合成代谢的基因。文库滴度为6．7×10^4cfu／mL，库容为2．72×10^8cfu／mL，文库重组率99．8％，插入片段多数分布在700～1000bp。随机挑选1014个克隆进行测序，测序成功963个，经过序列拼接去除低质量序列后获得632个unigenes，包括92个contigs和540个singletons，获得已知功能unigenes共441个。从所测克隆中得到一个花青素途径的结构基因AcF3H，其cDNA序列长1369bp（GenBank登录号：FJ542819），CDS区为1101bp，编码366个氨基酸的多肽。与拟南芥、葡萄及龙胆已知乃日氨基酸序列比对，发现该基因十分保守。通过RT—PCR技术对苍溪栽培的不同发育时期‘红阳’猕猴桃果实AcF3H基因的表达进行了分析。结果表明，果肉转色前AcF3H基因表达量较高，而转色初期表达量降低，此后随着果实着色加深表达量维持在较高水平。
文摘【目的】研究防御基因表达模式,筛选抗枯萎病候选基因,进一步阐明抗病机制。【方法】以4号枯萎病生理小种诱导的‘农科1号’香蕉为试材,利用DSN(duplex-specific nuclease)均一化酶结合SMART(switching mechanism at 5′end of the RNA transcript)技术构建香蕉根的均一化全长cDNA文库;并用多重半定量RT-PCR技术研究防御基因的表达模式。【结果】经检测,文库滴度为3.1×106 cfu.mL-1,扩增后库容为2.8×108 cfu.mL-1。随机挑选580个克隆进行测序,去除低质量序列后获得421个unigenes,片段分布在750—3 000 bp,文库重组率为100%,其中,未知功能基因占42%,全长cDNA序列为68%。文库筛选获得7个防御基因,进行不同时间的诱导表达分析,发现苯丙氨酸解氨酶(PAL)、过氧化氢酶(CAT)、β-1,3-葡聚糖酶(PR-2)和胶质乙酰酯酶(PAE)具有较低的本底表达水平;枯萎病菌浸染后,7个防御酶基因表达量增加,过氧化物酶(POX)、几丁质酶(PR-3)、PAE和PAL在第6天达到高峰;虽然7个防御基因在对照植株中的表达模式与诱导后的植株类似,但表达量均显著低于接菌后。【结论】所构建的均一化全长cDNA文库,片段重组性和完整性均达到分离筛选目的基因高质量文库的要求,且证明了7个防御基因不同程度地参与了抗枯萎病反应。
文摘以小麦品种水源11和条锈菌CY31号小种为材料,用SMARTTMcDNA Library Construction Kit构建条锈菌诱导的小麦叶片cDNA文库。得到原始文库的滴度为6×106pfu/mL,扩增文库滴度9×109pfu/mL,重组率98%,插入片段在0.5～2.2kb。随机挑取600个克隆,测序获得594条高质量ESTs,与GenBank序列进行BLASTx分析,已知功能ESTs与植物同源比例最高,占44%;其次为真菌,占32%;有18%ESTs在GenBank中没有匹配的同源产物。蛋白功能分析表明,PIG28编码蛋白、ABC转运子、金属硫因、泛素、质膜H+-ATPase和氨基酸透酶等可能参与了寄主与病原菌互作过程。
基金The National Basic Research Program (973 program, No.2004CB117402)the National Natural Science Foundation of China(No.30671626)the Guangdong Provincial Science and technology Program (No.2006B36501006)
