水稻种子特异表达启动子Ole18的克隆、序列分析及功能验证  

Cloning,sequence analysis and functional verification of seed specific expression promoter Ole18 gene from rice

作　　者：黄昕颖[1] 程祖锌[1] 肖长春[1] 黄志伟[1] 郑金贵 

机构地区：[1]福建农林大学农产品品质研究所,福州350002

出　　处：《南方农业学报》2015年第7期1147-1153,共7页Journal of Southern Agriculture

基　　金：国家科技支撑计划项目(2013BAD01B05);国家转基因重大专项项目(2009ZX08001-032B);福建农林大学青年教师科研基金项目(2013xjj06)

摘　　要：【目的】克隆水稻种子特异表达启动子Ole18,为实现外源基因在水稻胚、糊粉层特异表达奠定基础。【方法】采用PCR克隆扬稻6号Ole18启动子序列,并连接至p MD20-T载体上,经PCR验证后进行测序,并对测序结果进行生物信息学分析;用克隆获得的Ole18启动子取代p CAMBIA1301载体中GUS基因上游的Ca MV35S启动子构建重组表达载体,经农杆菌介导转化台粳9号,获得转基因植株,对其种子进行GUS染色。【结果】克隆获得大小为1261 bp的Ole18启动子序列,与japonica cultivar-group、IR36的18 k D Oleosin基因启动子同源性为99%。启动子预测软件NNPP推测该序列具有启动子的功能,含有种子特异表达启动子所必需的TATA-box、CAAT-box等顺式调控元件。GUS组织化学染色结果表明,该启动子序列能够驱动GUS基因在水稻种子胚及糊粉层中表达。【结论】克隆获得的Ole18启动子具有启动活性,且具有胚及糊粉层特异性表达功能。[Objective]The seed specific expression promoter Ole18 was cloned from rice in order to lay a foundation for the foreign target gene expressing specifically in rice embryo and aleurene layer. [Method]The sequence of promoter Ole18 was amplified by PCR from genomic DNA extracted from rice variety Yangdao 6 by homo-cloning, followed by linking into vector pMD20-T and sequencing to analyze its bioinformation. The CaMV35S promoter upstream of GUS gene in pCAMBIA1301 vector was replaced by cloned Ole18 promoter to construct recombinant expression vector pCAMBI- A1301-Ole18. The recombinant vector was transferred into rice variety Taijing 9 via Agrobacterium-mediated transforma- tion procedure. The expression of transgenic plants＇ seeds were detected by histochemical staining for GUS. [Result]A promoter sequence with 1261 bp was obtained from Ole18, which shared 99% nuleotide sequence homology with the 18 kD Oleosin promoter of japonica cuhivar-group and IR36. The function prediction and cis-element analysis indicated that the sequence had promoter function and contained cis-acting elements such as TATA-box, CAAT-box and so on, which was essential to seed-specific gene expression. Histochemical staining analysis of GUS showed that this promoter could drive the GUS gene expression only in seed embryo and aleurone layer of positive transgenic plants. [Conclusion]The cloned Ole18 promoter has promoter activation and can be specifically expressed in embryo and aleurone layerof seed.

关 键 词：Ole18启动子 基因克隆 序列分析 功能验证 

分 类 号：S511[农业科学—作物学]