桂花扩展蛋白基因OfEXPA2、OfEXPA4和OfEXLA1启动子克隆及活性分析

Cloning and activity analysis of promoters of expansin genes OfEXPA2, OfEXPA4 and OfEXLA1 from Osmanthus fragrans

为研究扩展蛋白基因在桂花花开放过程中的作用机制,利用染色体步移法,从桂花(Osmanthus fragrans)中克隆得到花开放相关基因OfEXPA2、OfEXPA4和OfEXLA1起始密码子上游1 108、808、945 bp的启动子序列。利用在线数据库Plantcare预测启动子中的顺式作用元件,发现这3个启动子序列中均存在基本元件TATA盒和CAAT盒,还含有脱落酸、生长素、水杨酸等激素诱导元件及响应干旱、高温等多个与植物非生物胁迫相关的元件,如MBS、HSE。分别将这3个启动子与β-葡萄糖苷酸酶(β-glucuronidase, GUS)报告基因融合进行瞬时表达,结果显示:OfEXPA2、OfEXPA4和OfEXLA1的启动子均能够驱动GUS基因在转基因烟草叶片中的表达。

Osmanthus fragrans is not only one of the top ten traditional famous flowers of our country,but also one of the famous greening and perfume plants in China.In order to study the flower opening mechanism of O.fragrans,expansin genes were identified and selected from the obtained transcriptome of O.fragrans.Three expansin genes,OfEXPA2,OfEXPA4 and OfEXLA1,were performed,which contributed to flower opening mechanism of O.fragrans.Promoters of the three genes were cloned by the method of Genome Walker.The lengths of 1 108,808 and 945 bp(OfEXPA2,OfEXPA4 and OfEXLA1)were obtained from O.fragrans,respectively.Online database'Plantcare'analysis revealed that the sequences of the three promoters contained basic cis-elements,such as TATA-box and CAAT-box.In addition,the promoter of OfEXPA2 contained hormone inducing elements ABRE,and the promoter of OfEXLA1 had AuxRR and TCA,indicating that the two genes would be regulated by corresponding hormones.Besides,there were many other elements involved in the plant abiotic stress.MBS,a cis-acting element in the promoter of OfEXPA2,was the MYB binding site,which responded to drought stress;there was a cis-acting element that responded to high temperature,HSE,in the promoter of OfEXLA1.In addition,the recombinant vectors were constructed and named OfEXPA2::GUS,OfEXPA4::GUS and OfEXLA1::GUS,respectively.The result indicated that the promoters of OfEXPA2,OfEXPA4 and OfEXLA1 could drive the GUS gene exclusively to express in leaves of transgenic tobacco.