以谷蛋白GluA-2信号肽增强外源蛋白在转基因水稻胚乳中的表达与积累  

Enhancing Expression and Accumulation of Foreign Proteins by Using the Signal Peptide of Glutelin Glu A-2 in Endosperm of Transgenic Rice

作　　者：王红梅[1] 张昌泉[1] 李钱峰[1] 辛世文[2] 刘巧泉[1] 徐明良[1,3] 

机构地区：[1]扬州大学农学院/植物功能基因组学教育部重点实验室/江苏省作物遗传生理重点实验室/粮食作物现代产业技术协同创新中心,江苏扬州225009 [2]香港中文大学生物系 [3]中国农业大学农学与生物技术学院,北京100193

出　　处：《作物学报》2015年第4期524-530,共7页Acta Agronomica Sinica

基　　金：国家转基因生物新品种培育重大专项(2014ZX08001006-005;2014ZX08009-024B);江苏省杰出青年基金项目(BK2012010);江苏省高校优势学科建设工程和"青蓝工程"等项目资助

摘　　要：提高外源蛋白在特定目标组织器官中的表达量是转基因植物研究与开发的核心技术之一。谷蛋白是水稻种子中最主要的贮藏蛋白,其表达具有严格的时空特异性。为进一步研究谷蛋白信号肽序列在指导基因表达中的作用,本研究克隆了水稻谷蛋白Glu A-2基因的启动子及其信号肽编码序列,并与GUS报告基因编码区融合,构建了分别含有和不含有信号肽的表达载体p13GG和p13GSG;经农杆菌介导法分别转入同一水稻品种中,获得了20多个独立转化子,PCR证明外源基因都已整合进了水稻基因组中。Northern杂交结果表明,融合Glu A-2信号肽编码序列可显著提高GUS基因在水稻胚乳中的转录;利用GUS特异的抗体进行Western杂交分析,显示该信号肽序列可显著提高外源蛋白在转基因水稻胚乳中的积累,但是其所指导表达的GUS蛋白在水稻胚乳中并没有表现出相应的活性,其机制有待进一步深入解析。相关结果对于水稻品质改良基因工程研究以及以水稻种子作为生物反应器高效表达外源蛋白具有重要的指导作用。It is one of the key important techniques to enhance the expression of foreign proteins in target tissue/organ of trans- genic plants. Glutelin is the major component of storage proteins in rice seeds, and its expression was tightly temporal and tissue- specific, which is controlled by several mechanisms. To further reveal the function of the Glutelin signal peptide on expression of target gene, in present study, we isolated the promoter and signal peptide-coding sequences of the glutelin GluA-2 gene, and fused them transcriptionally to the GUS coding sequences. Beside, the construct without the GluA-2 signal peptide-coding sequences was also generated as a control. Both constructs with the GUS chimeric genes, named as pl3GSG and pl3GG~ were introduced into the same rice variety by Agrobacterium-mediated transformation. More than twenty independent transgenic lines were gener- ated for each construct, and the integration of the GUS chimeric gene was confirmed by PCR technique. The results from North- ern blot analysis showed that, after fusing the GluA-2 signal peptide coding sequences between the GluA-2 promoter and the GUS coding sequence, the transcription of GUS chimeric gene could be dramatically increased. Then, Western blot was carried out by using the GUS-specific antibody, and the results obviously revealed that the accumulation of foreign proteins was significantly enhanced in the endosperm of transgenic rice with the signal peptide. However, there was no or very low GUS activity in the endosperm of transgenic rice plants with the signal peptide. These results were very useful to improve the grain quality of rice via genetic engineering, especially produce foreign proteins in the seeds office as bioreactor.

关 键 词：转基因水稻 信号肽 谷蛋白 胚乳 基因表达 

分 类 号：S511.22[农业科学—作物学]