甘蓝型油菜半胱氨酸蛋白酶基因BnCP51及其启动子的克隆与表达

Cloning and expression of cysteine proteinase Bn CP51 and its promoter from Brassica napus L.

为通过转基因技术建立新型授粉控制体系,克隆与雄性不育相关的基因及启动子,根据甘蓝型油菜全基因组信息设计特异引物,获得半胱氨酸蛋白酶家族基因Bn CP51的完整开放阅读框和上游启动子序列。Bn CP51基因全长1 032bp,在Gen Bank登录号KC898325,启动子长1 951bp。生物信息学分析表明,Bn CP51具有木瓜蛋白酶基因家族典型的保守结构域ERFNIN和GCNGG功能域。实时荧光定量PCR结果表明Bn CP51在花蕾中高量表达。在线启动子序列元件预测分析发现Bn CP51启动子序列中有多个花药特异表达元件。构建重组载体p Bn CP51::GUS,转化拟南芥,GUS染色结果表明在Bn CP51启动子的驱动下,报告基因GUS仅在中期花蕾和花药中特异表达,在其他组织中不表达。

To better understand the function of Brassica napus gene Bn CP51,specific primers were designed according to genomic information,Bn CP51 gene and its promoter were cloned from Brassica napus L. cv Zhongshuang 9. The complete open reading frame of Bn CP51（ KC898325 in Gen Bank） is 1 032 bp in length. The promoter of Bn CP51 is 1 951 bp in length. Bn CP51 was shown to be specifically expressed in Zhongshuang 9 buds by qRT- PCR. To investigate the expression pattern of Bn CP51 promoter,a recombinant vector was designated as p Bn CP51： ： GUS and transformed into Arabidopsis. GUS assay results showed that the promoter was specifically observed in buds and anthers at middle stage. No GUS expression was observed in roots,stem,leaves or siliques. It suggested that Bn CP51 played important role in bud and anther development in B. napus.