【目的】深入探究麦根腐平脐蠕孢(Bipolaris sorokiniana)生长发育及致病力的分子作用机制,并鉴定BsTup1的互作蛋白。【方法】利用麦根腐平脐蠕孢(B.sorokiniana)孢子和不同时期的菌丝体为材料,构建酵母双杂交cDNA文库,以BsTup1基因为...【目的】深入探究麦根腐平脐蠕孢(Bipolaris sorokiniana)生长发育及致病力的分子作用机制,并鉴定BsTup1的互作蛋白。【方法】利用麦根腐平脐蠕孢(B.sorokiniana)孢子和不同时期的菌丝体为材料,构建酵母双杂交cDNA文库,以BsTup1基因为诱饵来筛选酵母双杂交文库,确定与BsTup1相互作用的蛋白。【结果】1)利用SMART(switching mechanism at 5′end of the RNA transcript)技术首次成功构建了麦根腐平脐蠕孢(B.sorokini-ana)分生孢子和菌丝体的混合cDNA文库。文库鉴定结果表明,构建的cDNA文库库容为4.8×10^(7) cfu·mL^(-1),文库插入片段重组率达100%且平均大小为1000 bp。2)构建了pGBKT7-BsTup1诱饵载体,无自激活活性。3)使用诱饵蛋白载体pGBKT7-BsTup1对麦根腐平脐蠕孢(B.sorokiniana)酵母双杂交cDNA文库进行筛选,经测序、序列比对和酵母回转验证,获得38个与BsTup1相互作用的候选蛋白。【结论】成功构建了麦根腐平脐蠕孢(B.sorokiniana)的cDNA文库,并鉴定出38个与BsTup1相互作用的候选蛋白。展开更多
Defensin is a kind of cationic, inducible antimicrobial peptide found in a large range of living organisms that contributes to host defense by disrupting the cytoplasmic membrane of microorganisms. With their broad an...Defensin is a kind of cationic, inducible antimicrobial peptide found in a large range of living organisms that contributes to host defense by disrupting the cytoplasmic membrane of microorganisms. With their broad antimicrobial spectrum and strong pharmaceutical effects, antimicrobial peptides, including defensins, represent a source of novel antibiotic agents. A novel full-length 430 base pairs cDNA of an insect defensin was cloned using polymerase chain reaction (PCR) from the cDNA library of houseflies (Musca domestica) that had been challenged by E. coli and Staphylococcus aureus. Sequence analysis revealed that the open reading frame of the cDNA encoded a 92-amino acid peptide, which contained an NH 2-terminal signal sequence (1-22) followed by a propeptide and the mature peptide (53-92). The sequence identity with other insect defensin is between 51% and 73%. The mature peptide, with a predicted molecular weight of 4\^0 kDa, and pI of 8\^69, has 1 negative charged amino acid and 4 positice ones. The putative housefly defensin is characterized by 6 invariant cysteine residues forming 3 disulfide bonds, Cys1-Cys4, Cys2-Cys5 and Cys3-Cys6. These results suggest that the novel full-length cDNA of the defensin gene, denominated Mdde, has been successfully cloned from houseflies.展开更多
目的获得我国广州地区粉尘螨 类变应原 (Der f ) c DNA片段 ,为构建 DNA疫苗或表达重组蛋白打下基础。方法挑取经选择鉴定的活粉尘螨 ,提取总 RNA,采用 RT- PCR的方法扩增 Der f 片断 ,进行克隆、测序和分析。结果获得长度为 6 32个碱...目的获得我国广州地区粉尘螨 类变应原 (Der f ) c DNA片段 ,为构建 DNA疫苗或表达重组蛋白打下基础。方法挑取经选择鉴定的活粉尘螨 ,提取总 RNA,采用 RT- PCR的方法扩增 Der f 片断 ,进行克隆、测序和分析。结果获得长度为 6 32个碱基对的核苷酸片断 ,序列分析结果和 Genebank上去除内含子后的基因序列 (em b| X6 5 196 .1| )同源性为99% ,其中有 6个碱基不同 ,推导的编码氨基酸序列同源性为 10 0 %。结论我们首次获得广州地区的 Der f 的 c DNA克隆 ,该克隆 c DNA序列与 Genebank上已公布的 Der f 序列高度同源。展开更多
文摘【目的】深入探究麦根腐平脐蠕孢(Bipolaris sorokiniana)生长发育及致病力的分子作用机制,并鉴定BsTup1的互作蛋白。【方法】利用麦根腐平脐蠕孢(B.sorokiniana)孢子和不同时期的菌丝体为材料,构建酵母双杂交cDNA文库,以BsTup1基因为诱饵来筛选酵母双杂交文库,确定与BsTup1相互作用的蛋白。【结果】1)利用SMART(switching mechanism at 5′end of the RNA transcript)技术首次成功构建了麦根腐平脐蠕孢(B.sorokini-ana)分生孢子和菌丝体的混合cDNA文库。文库鉴定结果表明,构建的cDNA文库库容为4.8×10^(7) cfu·mL^(-1),文库插入片段重组率达100%且平均大小为1000 bp。2)构建了pGBKT7-BsTup1诱饵载体,无自激活活性。3)使用诱饵蛋白载体pGBKT7-BsTup1对麦根腐平脐蠕孢(B.sorokiniana)酵母双杂交cDNA文库进行筛选,经测序、序列比对和酵母回转验证,获得38个与BsTup1相互作用的候选蛋白。【结论】成功构建了麦根腐平脐蠕孢(B.sorokiniana)的cDNA文库,并鉴定出38个与BsTup1相互作用的候选蛋白。
文摘Defensin is a kind of cationic, inducible antimicrobial peptide found in a large range of living organisms that contributes to host defense by disrupting the cytoplasmic membrane of microorganisms. With their broad antimicrobial spectrum and strong pharmaceutical effects, antimicrobial peptides, including defensins, represent a source of novel antibiotic agents. A novel full-length 430 base pairs cDNA of an insect defensin was cloned using polymerase chain reaction (PCR) from the cDNA library of houseflies (Musca domestica) that had been challenged by E. coli and Staphylococcus aureus. Sequence analysis revealed that the open reading frame of the cDNA encoded a 92-amino acid peptide, which contained an NH 2-terminal signal sequence (1-22) followed by a propeptide and the mature peptide (53-92). The sequence identity with other insect defensin is between 51% and 73%. The mature peptide, with a predicted molecular weight of 4\^0 kDa, and pI of 8\^69, has 1 negative charged amino acid and 4 positice ones. The putative housefly defensin is characterized by 6 invariant cysteine residues forming 3 disulfide bonds, Cys1-Cys4, Cys2-Cys5 and Cys3-Cys6. These results suggest that the novel full-length cDNA of the defensin gene, denominated Mdde, has been successfully cloned from houseflies.
文摘目的获得我国广州地区粉尘螨 类变应原 (Der f ) c DNA片段 ,为构建 DNA疫苗或表达重组蛋白打下基础。方法挑取经选择鉴定的活粉尘螨 ,提取总 RNA,采用 RT- PCR的方法扩增 Der f 片断 ,进行克隆、测序和分析。结果获得长度为 6 32个碱基对的核苷酸片断 ,序列分析结果和 Genebank上去除内含子后的基因序列 (em b| X6 5 196 .1| )同源性为99% ,其中有 6个碱基不同 ,推导的编码氨基酸序列同源性为 10 0 %。结论我们首次获得广州地区的 Der f 的 c DNA克隆 ,该克隆 c DNA序列与 Genebank上已公布的 Der f 序列高度同源。
