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

摘要

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 glutelinGluA-2 gene, and fused them transcriptionally to theGUS coding sequences. Beside, the construct without theGluA-2 signal peptide-coding sequences was also generated as a control. Both constructs with theGUS chimeric genes, named as p13GSG and p13GG, were introduced into the same rice variety byAgrobacterium-mediated transformation. More than twenty independent transgenic lines were gener-ated for each construct, and the integration of theGUS chimeric gene was confirmed by PCR technique. The results from North-ern blot analysis showed that, after fusing theGluA-2 signal peptide coding sequences between theGluA-2 promoter and theGUS coding sequence, the transcription ofGUS 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 of rice as bioreactor.%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GluA-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GluA-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。