黄毛草莓FnMYB24转录因子基因和启动子的克隆及表达分析

Cloning and Expression Analysis of FnMYB24 Transcription Factor Gene and Promoter from Fragaria nilgerrensis Schltdl.

该研究以黄毛草莓(Fragaria nilgerrensis Schltdl.)为材料,采用RT-PCR技术克隆了黄毛草莓FnMYB24基因的cDNA和启动子序列。生物信息学分析表明,FnMYB24的cDNA序列长为1033 bp(GenBank登录号为MN879283),其开放阅读框(ORF)长为609 bp,编码202个氨基酸,含有1个保守的MYB_DNA-binding结构域。同源分析结果显示,黄毛草莓FnMYB24基因编码的氨基酸序列与森林草莓(Fragaria vesca)编码的氨基酸相似性较高;同时进一步克隆了该基因编码起始位点上游长度为718 bp启动子序列(GenBank登录号为MN879285),预测该序列包含激素响应元件、光调控元件等多个顺式作用元件。通过构建pFnMYB24∷GUS表达载体进行烟草瞬时转化,发现pFnMYB24启动子具有转录活性且能够驱动FnMYB24基因表达。实时荧光定量PCR结果显示:抗病品种黄毛草莓和易感病栽培品种‘妙香3号’的叶片接种胶孢炭疽菌(Colletotrichum gloeosporioides)后MYB24基因表达量均有上调,但‘妙香3号’的MYB24表达量始终低于黄毛草莓的表达量;SA处理后2个草莓品种的MYB24表达量均高于对照组,表明MYB24基因受水杨酸(SA)的诱导表达。研究表明,草莓MYB24基因可能参与调控抗炭疽病,为进一步研究MYB24基因在草莓抗炭疽病中的功能奠定了基础。

The cDNA and promoter sequence of FnMYB24 gene were cloned from Fragaria nilgerrensis Schltdl.by RT-PCR.Bioinformatics analysis showed that the cDNA sequence of FnMYB24 was 1033 bp(GenBank accession number MN879283)length,and its open reading frame(ORF)was 609 bp length,encoding 202 amino acids,including a conserved MYB_DNA-binding domain.Homogeneous analysis showed that the amino acid sequence encoded by FnMYB24 gene was 92.75%similar to that of encoded by Fragaria vesca.The promoter sequence of 718 bp upstream of the transcription initiation site(GenBank accession number MN879285)was further cloned,which was predicted to contain several cis-acting elements such as hormone response elements and optical regulation elements.By constructing pFnMYB24∷GUS expression vector and carrying out instantaneous tobacco transformation,it was found that the pFnMYB24 promoter had transcriptional activity and could drive the expression of FnMYB24 gene.Real-time quantitative PCR results showed that the expression of MYB24 gene in the leaves of resistant variety F.nilgerrensis Schltdl.and susceptible cultivar‘Miaoxiang No.3’was up-regulated after inoculation with Colletotrichum gloeosporioides,but the expression level of MYB24 in‘Miaoxiang No.3’was always lower than that in F.nilgerrensis Schltdl.This gene was also induced by SA since the MYB24 expression levels in two strawberry varieties after SA treatment were higher than those of controls.Our results suggest that MYB24 may involved in regulate resistance to strawberry anthracnose,this study laid foundation for further investigate the function of MYB24 confers resistance to strawberry anthracnose.