基于Tail-PCR分析AN2基因上游启动子活性

Analysis of AN2 Upstream Promoter Activity Based on Tail-PCR

摘要:

黑果枸杞(Lycium ruthenicum)富含花青素，AN2基因是调控黑果枸杞花青素合成代谢的主效基因。为解析AN2基因启动子的活性差异，采用Tail-PCR方法分别克隆了黑果枸杞和红果枸杞(L. barbarum) AN2基因起始密码子上游约1 686 bp (LrAN2p)和1 495 bp (LbAN2p)的序列。Plant CARE预测表明，LbAN2p和LrAN2p中分别有133和137个的顺式作用元件, 其中，参与光调控的顺式元件分别有11和15个；参与激素响应相关的顺式元件分别有13和16个。构建AN2启动子植物表达载体pKGWFS7:LbAN2p和pKGWFS7:LrAN2p，利用农杆菌介导的烟草遗传转化体系获得转基因烟草。GUS染色结果表明，LrAN2p能够驱动GUS在烟草中的表达，叶片呈现蓝色，具有较LbAN2p更强的启动活性，qRT-PCR结果表明，LrAN2p转基因烟草中GUS基因具有更高的转录水平，这可能会使AN2基因在黑果枸杞中具有更高的表达，激活黑果枸杞花青素合成代谢通路。这为解析枸杞果色形成及AN2基因的表达调控机制奠定了理论基础。

Abstract:

Lycium ruthenicum is rich in anthocyanin, and AN2 is the main gene regulating anthocyanin anmeta- bolism. In order to analyze the activity difference of AN2 gene promoter, the upstream sequence of AN2 gene start codon about 1 686 bp (LrAN2p) and 1 495 bp (LbAN2p) of L. barbarum were cloned by Tail-PCR method, respectively. Plant CARE predicted that there were 133 and 137 cis-acting elements in sequences of LbAN2p and LrAN2p, respectively, among which 11 and 15 cis-acting elements were involved in photoregulation, and 13 and 16 cis-elements involved in hormone response, respectively. The plant expression vectors pKGWFS7:LbAN2p and pKGWFS7:LrAN2p were constructed, and the transgenic tobacco was obtained by using Agrobacterium- mediated tobacco genetic transformation system. The results of GUS staining showed that LrAN2p could drive the expression of GUS in tobacco, and the leaves were blue, with stronger starting activity than LbAN2p. The results of qRT-PCR showed that GUS gene in LrAN2p transgenic tobacco had high transcription level, which might lead to high expression of AN2 gene in L. ruthenicum, and activating the anthocyanin anabolic pathway. Thus, these would provide a theoretical basis for understanding the mechanism of fruit color formation and AN2 gene expression.