种子特异启动子的克隆及植物表达载体构建

目的]启动子是植物基因工程中一个重要工具，对构建植物生物反应器有着重要意义。8S球蛋白是绿豆中含量最丰富的种子贮藏蛋白，因此，其调控序列可能提供了一个很好来源的种子特异性启动子。方法]通过基因组步移技术克隆了绿豆8s球蛋白a亚基基因8SGα的启动子区域，基因组步移使用的引物根据绿豆8SGα的cDNA序列设计。结果]通过3轮PCR的扩增，得到了472bp的上游序列片段，构建了植物双元表达载体pBI－8SGα-GUS。结论]研究结果可用于转化植物，并于转基因植物中分析启动表达的时空特异性及表达强度，并为植物种子生物反应器提供重要元件。

Cloning of Seed-specific Promoter and Construction of Plant Transformation Vector

Objective] Promoter is an important tool in plant genetic engineering, which is critical for developing an efficient plant bioreactor. 8S globulin is the most abundant seed storage protein in Vigna radiate, its regulator sequence may provide a good source of seed-specific promoter. Method]Vigna radiate 8S globulin α subunit promoter sequence was amplified by the method of Genome-walking in accordance with the Vigna radiate 8S globulin α subunit cDNA sequence. Result]The amplified sequence was cloned into pMD18-T vector, and then transformed into E. coli cells. Resultant plasmid was extracted and subjected to DNA sequencing. The cloned sequence was analyzed by various promoter prediction servers. The sequence was shown to contain a number of key components of seed-specific promoters; suggesting that we have successfully obtained the Vigna radiate 8SGα promoter. Conclusion]Subsequently, 8SGα was subcloned into plant binary vector pBI121 by replacing CaMV35S promoter upstream of GUS reporter gene with 8SGα promoter, thus providing a basis for investigation of 8SGα promoter function in planta.