大豆类受体蛋白激酶基因rlpk2启动子克隆及初步分析

Cloning and Preliminary Analysis of Promoter of Soybean Receptor-like Protein Kinase Gene rlpk2

用双链接头介导的基因组DNA步行技术获得了大豆中与衰老相关的类受体蛋白激酶基因rlpk25'侧翼1801bp启动子序列(Prlpk2,GenBank登录号:AY870314)。序列分析结果表明,这一启动子片段中有响应细胞分裂素、脱落酸、赤霉素和生长素等激素作用和响应干旱、寒冷及伤害等逆境胁迫的多个顺式作用元件。构建启动子Prlpk2与报告基因GUS融合基因的双元表达载体pRlpk2-GUS,并通过农杆菌介导法转化大豆幼苗和微型番茄Micro-Tom的子叶外植体,染色结果表明这1801bp的启动子Prlpk2可以有效地驱动GUS基因在大豆幼苗生长点和番茄愈伤组织中瞬时表达。

5＇-Flanking region of rlpk2 gene was cloned by using PCR-based genomic DNA walk- ing method （Fig. 1）, and designated Prlpk2 （GenBank Accession Number： AY870314）. Results of sequencing analysis showed that Prlpk2 contains several putative cis-elements that respond to CTK, ABA, GA or IAA, as well as elements that respond to different environmental stresses, such as dehydration, coldness, wounding and etc. An expression vector containing the Prlpk2-GUS fusion gene was constructed （Fig.2） and transferred into soybean seedlings and Micro-Tom cotyledon explants by Agrobacterium-mediated method. A strong transient expression of GUS was observed in both soybean plumule and Micro-Tom cotyledon callus （Fig.3）, thus confirming the promoter activity of Prlpk2.