油菜细胞质雄性不育基因orf138、orf288的功能分析

Functional analysis of cytoplasmic male sterility genes orf138 and orf288 in Brassica

为确定油菜ogu CMS不育基因orf138编码蛋白的毒性与雄性不育的关系以及hau CMS不育基因orf288的核心功能区域,对2个不育基因进行不同长度的截短,进行原核表达和拟南芥、油菜遗传转化。不育基因orf138截短研究结果显示,IPTG诱导后,转入含有跨膜螺旋的pET32a^(1-138)和pET32a^(1-93)的大肠杆菌生长受到了明显的抑制,而转入去掉跨膜螺旋的pET32a^(45-138)的大肠杆菌生长不受影响,表明ORF138的毒性区域位于包含跨膜螺旋的N端;遗传转化结果证实PS∶Rfp138^(35-138)和PS∶Rfp138^(45-138)均不能引起拟南芥和油菜雄性不育,说明N端的毒性区域是ORF138引起植物雄性不育所必需的。不育基因orf 288不同长度截短的超表达载体拟南芥转化结果显示,PS∶Rfp288^(104-288)、PS∶Rfp288^(134-288)和PS∶Rfp288^(164-288)的转基因阳性苗出现雄性不育表型,PS∶Rfp288^(194-288)的转基因阳性苗则全部可育,初步证明orf 288的核心功能区域是orf 288^(164-288),ORF288的164-194 aa可能与hau细胞质雄性不育密切相关。

Two CMS genes were truncated in different lengths,expressed in prokaryotic cells and genetically transformed in Arabidopsis thaliana and Brassica napus.to determine the relationship between the toxicity of the protein encoded by the ogu CMS sterility gene orf138 and male sterility and the core functional region of the hau CMS sterility gene orf288.The results of truncated male sterile gene orf138 showed that the growth of E.coli expressing pET32a^(1-138) and pET32a^(1-93) containing transmembrane helix was significantly inhibited after the IPTG was added,while the growth of E.coli expressing pET32a^(45-138) without transmembrane helix was not affected.It is indicated that the toxic region of ORF138 is located at the N-end containing the transmembrane helix.Results of genetic transformation showed that PS:Rfp138^(35-138) and PS:Rfp138^(45-138) did not cause male sterility in Arabidopsis thaliana and B.napus,indicating that the complete N end is necessary for ORF138 to induce male sterility in plants.The results of genetic transformation of different truncated orf288 in Arabidopsis thaliana showed that positive seedlings transformed with PS:Rfp288^(104-288),PS:Rfp288^(134-288) and PS:Rfp288^(164-288) were male sterile,while all the positive seedlings transformed with PS:Rfp288^(194-288) were fertile.It is preliminarily proved that the core functional region of orf288 is orf288^(164-288),indicating that the 164th-194th aa of ORF288 may be closely related to the cytoplasmic male sterility in hau.