【目的】克隆玉米Zea mays淀粉合成酶SSⅡa启动子,并分析其功能,为进一步研究和应用SSⅡa启动子奠定基础。【方法】通过NCBI上公布的玉米基因组序列,在网站MaizeGDB上BLAST查找到SSⅡa 5′侧翼序列,利用PCR方法从玉米B73中克隆SSⅡa启动子；通过PlantCare在线分析启动子顺式作用元件,用特异性引物分别克隆出长度为1407、867、633、483和365 bp的片段,与植物表达载体pCAMBIA3301连接,构建5种5′缺失体的植物表达载体,命名为P1、P2、P3、P4和P5。用农杆菌介导法转化拟南芥Arabidopsis thaliana,获得转基因拟南芥。【结果】以玉米B73基因组DNA为模板,用特异性引物SSⅡaF/SSⅡaR进行扩增,得到2526 bp序列；除草剂筛选的阳性拟南芥植株PCR验证均检测出gus基因；GUS组织化学分析表明,5种类型启动子构建的表达载体在成熟期叶片、果荚中均显蓝色；gus基因定量分析表明,成熟期5种转基因拟南芥叶片中, gus基因表达量P1最高,其他基本一致；种子中gus基因表达量P1和P2相近,且高于P3、P4和P5。【结论】成功克隆玉米SSⅡa启动子；构建的5种SSⅡa启动子缺失体表达载体在转基因拟南芥中均具有活性,长度为1407 bp(P1)和867 bp(P2)的启动子具有胚乳特异性。%Objective] To clone maize ( Zea mays) starch synthase SSⅡa promoter, analyze its function, and provide a basis for its future research and application. [Method] The SSⅡa 5′flanking sequence was found on Maize GDB by BLASTing the maize genome sequence published on NCBI, and the SSⅡa promoter was cloned from maize B73 using PCR. We analyzed the cis elements of the promoter using PlantCare. Fragments of 1 407, 867, 633, 483, and 365 bp were cloned with specific primers, and were inserted into the plant expression vector pCAMBIA3301, respectively. Five plant expression vectors with different 5′deletions of the SSⅡa promoter were constructed and named P1 , P2 , P3 , P4 and P5 . The transgenic Arabidopsis thaliana plants were obtained through Agrobacterium-mediated transformation.[Result] A DNA fragment of 2 526 bp was obtained by PCR amplification with maize B73 genome DNA as template and SSⅡaF/SSⅡaR as specific primers. The positive A. thaliana plants, which were screened by herbicide, had gus gene by PCR detection. The histochemical analysis of GUS showed that the expression vectors of five promoters were blue in leaves and pods at maturity. The quantitative analysis of gus gene showed that among five transgenic A. thaliana at maturity, the expression level of P1 in leaves was the highest and the others were basically the same, and the expression levels of P1 and P2 in seeds were similar, both being higher than those of P3, P4 and P5. [Conclusion] The maize SSⅡa promoter has been successfully cloned. The five constructed expression vectors with different 5′deletions of the SSⅡa promoter all have activities in transgenic A. thaliana, and the promoters with the length of 1 407 bp (P1) and 867 bp (P2) have endosperm specificity.
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