油菜种子特异表达启动子pNapB和pFAE1的结构与功能比较研究

Structural and function analysis of two seed-specific promoters:pNapB and pFAE1 from Brassica napus

启动子是基因在转录水平上的一种重要调控元件。本研究通过同源序列法从甘蓝型油菜品种中油821中分离到油菜基因NapB和FAE1的上游启动子序列pNapB和pFAE1,其中pNapB长1136bp,pFAE1长1420bp。两种启动子的核苷酸序列都含有种子特异表达启动子的顺式作用元件,包括RY重复、G-box和E-box等,但两种启动子顺式作用元件的序列分布不同。将pNapB和pFAE1两个启动子分别与报告基因GUS融合并通过农杆菌介导法导入烟草,得到大量转基因烟草植株。对T1代转基因烟草进行组织化学分析,比较了不同启动子的组织表达特异性。结果表明,两种启动子都只在种子中起作用,属于典型的种子特异性表达启动子,但是它们作用的时间和空间分布以及作用强度明显不同。pNapB启动子比pFAE1作用开始的时间早,持续时间长,在早期作用比pFAE1强,但pFAE1启动子在种子发育中期启动速度快,成熟种子的GUS染色深度在两种启动子间差别不大。

Promoter is an important regulatory element at the transcriptional level. The upstream regulatory regions of NapinB and FAE1 genes were separated from Brassica napus cv.Zhongyou 821 genomic DNA using homology-based method. Through analysis of 1 136bp pNapB and 1 420 bp pFAE1 promoter regions, such seed-specific cis-acting elements as the RY box, G-box, E-box were identified, while the distribution of the cis-acting elements was different in two promoters. Plant expression vector was constructed by ligating pNapB and pFAE1 with GUS gene and transferred into tobacco by Agrobacterium tumefaciens, respectively. A large number of transgenic tobacco plants were obtained. Histochemical analysis of T1 transgenic plants and comparion of the tissue specificity of two promoters showed that both pNapB and pFAE1 were seed-specific, though they showed temporal and spatial difference in GUS activity. In transgenic tobacco, pNapB started the GUS gene earlier and showed stronger GUS activity in the early stage of the embryo development while pFAE1 started the GUS gene at the middle stage of the embryo development showed stronger GUS activity at the later stage of the embryo development. The final color of the developed embryo which were histochemically stained showed a little difference between two promoters.