黄毛草莓FnbHLH68转录因子基因和启动子的克隆及表达分析

Cloning and expression analysis of gene and promoter of transcription factor FnbHLH68 from Fragaria nilgerrensis

以黄毛草莓为试材,采用RT-PCR技术克隆了1个碱性螺旋—环—螺旋(bHLH)转录因子基因FnbHLH68的cDNA序列(GenBank登陆号:MN879282).序列分析显示,FnbHLH68基因开放阅读框长1161 bp,编码386个氨基酸,包含一个保守的碱性螺旋—环—螺旋(HLH)结构域.同源性分析表明,黄毛草莓FnbHLH68氨基酸序列与森林草莓FvbHLH68氨基酸序列的同源性为98.45%.进一步从黄毛草莓基因组DNA中克隆了FnbHLH68上游1849 bp的启动子序列pFnbHLH68(GenBank登录号:MW218450),预测含有多个响应逆境和激素诱导的顺式作用元件.成功构建了植物过表达载体pCAMBIA1300-HA-FnbHLH68和基因沉默载体pTRV2-FnbHLH68.实时荧光定量PCR检测显示:黄毛草莓叶片接种胶孢炭疽菌12 h后,FnbHLH68基因的表达量达到最高值,为0 h的10.6倍;黄毛草莓叶片用水杨酸处理后,FnbHLH68基因的表达量上升,12 h后达到最高值,为0 h的11.5倍.

We firstly isolated the cDNA sequence of a basic helix-loop-helix(bHLH)transcription factor FnbHLH68(GenBank accession number:MN879282)from Fragaria nilgerrensis Schltdl.ex Gay.by reverse transcription PCR.The full length of the FnbHLH68 gene was 1161 bp,encoding 386 amino acids,and consisted of a conserved helix-loop-helix(HLH)structural domain.Subsequent homology analysis showed that the encoding sequence of amino acid of FnbHLH68 had a high homology(98.45%)with Fragaria vesca Linn..The sequence of promoter FnbHLH68(GenBank accession number:MW218450),1849 bp upstream to the FnbHLH68 gene,was isolated from F.nilgerrensis.The FnbHLH68 gene was predicted to contain multiple cis-acting elements in response to stress and hormone.Then the overexpression vector pCAMBIA1300-HA-FnbHLH68 and gene silencing vector pTRV2-FnbHLH68 were further constructed.Real-time quantitative PCR assay demonstrated that after inoculated with Colletotrichum gloeosporioides(Penz.)Sacc for 12 h,the expression of the FnbHLH68 gene reached its height,over 10.6 times more than the expression level at 0 h.In the group treated with salicylic acid,the FnbHLH68 gene saw an upregulated expression,with the highest expression level being at 12 h,over 11.5-fold than the expression level at 0 h.