木薯转录因子基因MeHDZ14的克隆与分析

Cloning and Analysis of the Transcription Factor Gene MeHDZ14 in Cassava

HD-Zip家族基因在植物生长发育和逆境胁迫中起重要作用。为了研究MeHDZ14基因在非生物胁迫(尤其是干旱)应答中的作用,选用对干旱信号反应灵敏、相对耐旱的木薯品种"SC124"作为实验材料,利用RT-PCR克隆了MeHDZ14基因。生物信息学分析发现,MeHDZ14基因编码的蛋白具有典型的HD-Zip保守结构域。将该基因编码的蛋白与GFP融合,亚细胞MeHDZ14:GFP重组蛋白定位于细胞核。同时,酵母Y187中的转录自激活试验结果也表明,MeHDZ14蛋白具有明显转录自激活功能。推断MeHDZ14是一个典型的HD-Zip I转录因子。MeHDZ14启动子区具有多个ABA响应元件ABRE(ABA response element)。基因差异表达分析结果表明,MeHDZ14基因在叶片和根中的表达受干旱胁迫的诱导,并对外源ABA具有明显的响应。因此,认为MeHDZ14基因通过ABA依赖信号传导途径参与调控木薯干旱响应。此外,还发现MeHDZ14基因的编码区虽然存在数个SNP,但表现出高度保守性,且在不同木薯品种中的表达对干旱胁迫均有明显的响应,为进一步研究该基因的功能奠定了基础。

HD-Zip family genes play important roles in plant growth and stress response.To reveal the role of MeHDZ14gene inabiotic stresses(e.g.drought)in cassava,we cloned MeHDZ14gene by using RT-PCR from cassava cultivar SC124,which wasrelatively more resistant to drought stress.Bioinformatics methods were used to analyze its structural characteristics,andsemi-quantitative RT-PCR/qRT-PCR was used to explore its expression patterns in response to abiotic stresses in different planttissues and varieties.MeHDZ14has a726bp open reading frame,encoding241amino acids,and contains the typical HD and ZIPdomain.Blastp analysis showed that MeHDZ14has close genetic relationship with ATHB-7,which is a member of the family IHD-Zip gene.Yeast and subcellular localization test showed that the MeHDZ14gene is a transcription factor and specifically expressesin the nucleus.Genetic structural variation analysis revealed a total of four mis-sense mutations in eight tested varieties.However,amino acid mutations were not found between wild and cultivated cassavas.This indicates the MeHDZ14proteins arehighly conserved.Semi-quantitative RT PCR analysis revealed that MeHDZ14was specifically expressed in petioles,and inducedby drought stress in root and leaf,suggesting that MeHDZ14plays an important role in the early drought stage.Analysis byqRT-PCR showed that MeHDZ14gene had different expression levels in different cassava varieties,but the same mode underdrought stress and ABA treatment.These data indicate that MeHDZ14is a member of the ABA pathway responding to drought.Our results showed that MeHDZ14plays an important role in the molecular pathways of cassava drought resistance,underliningits potential in genetic improvement of cassava drought tolerance.