木薯MeAHL22基因的克隆及功能初步分析

Cloning and Functional Analysis of MeAHL22 Gene from Cassava

AT-hook是一类定位于细胞核的新型DNA结合蛋白基序,在细菌、真菌和动植物中普遍存在。AThook蛋白家族在植物生长发育、激素应答以及逆境胁迫中发挥着重要调节作用。本研究从木薯中克隆出MeAHL22基因,对其结构、亚细胞定位、启动子区域和表达模式进行分析。结果表明,MeAHL22基因的开放阅读框长度为873 bp,编码一个具有290个氨基酸的蛋白;结构分析发现该蛋白存在AT-hook基序和PPC/DUF296结构域,二级结构主要由α-螺旋(11.03%)、无规则卷曲(60%)、延伸链(21.72%)以及β-转角(7.24%)构成;生物信息学分析表明,该蛋白不存在跨膜结构域和信号肽,为可溶性的亲水蛋白,预测其定位在细胞核;利用农杆菌介导法在烟草中进行瞬时表达融合表达载体,结果表明MeAHL22蛋白的确定位于细胞核,与预测结果一致。MeAHL22基因启动子区域包含光响应、激素响应、非生物胁迫响应和生长进程相关的顺式作用元件。组织表达模式分析表明MeAHL22基因在木薯根、茎、叶中均表达,其中块根中的表达量明显高于其他组织。不同激素处理表明MeJA诱导MeAHL22在根茎叶中显著上调表达,ABA则抑制其表达,GA和IAA诱导在叶中上调表达。非生物胁迫处理表明MeAHL22对模拟干旱胁迫的诱导响应最为强烈,同时受到盐胁迫的诱导,但是对冷胁迫的敏感性较低。本研究初步确定了木薯MeAHL22基因在激素、非生物胁迫信号应答和生长发育进程中具有关键作用,为进一步研究MeAHL22基因的生物学功能提供了思路。

AT-hook is a class of novel DNA-binding protein motifs located in the nucleus,which are widely found in bacteria,fungi and animals and plants.AT-hook protein family plays an important regulatory role in plant growth and development,hormone response and stress.In this study,MeAHL22 was cloned from cassava and analyzed by bioinformatics and subcellular localization.The results showed that the length of open reading frame of MeAHL22 gene was 873 bp,encoding a protein with 290 amino acids.Structural analysis revealed the AT-hook motif and PPC/DUF296 domain of the protein.The secondary structure of the protein was mainly composed of alpha-helix(11.03%),random coil(60%),extended strand(21.72%)and beta-turn(7.24%).Bioinformatics analysis showed that the protein was a soluble hydrophilic protein without transmembrane domain and signal peptide,and it was predicted to be located in the nucleus.Agrobacterium-mediated transient expression of the fusion expression vector in tobacco indicated that MeAHL22 protein was located in the nucleus,which was consistent with the predicted results.The promoter region of MeAHL22 gene contained cis-acting elements related to light response,hormone response,abiotic stress response and growth process.The analysis of tissue expression pattern showed that MeAHL22 gene was expressed in roots,stems and leaves of cassava,and the expression level in storage root was significantly higher than that in other tissues.Different hormone treatments showed that MeJA induced the up-regulated expression of MeAHL22 in roots,stems and leaves,while ABA inhibited its expression,GA and IAA induced the up-regulated expression in leaves.The abiotic stress treatment showed that MeAHL22 had the strongest response to simulated drought stress and was also induced by salt stress,but its sensitivity to cold stress was low.The study preliminately confirmed that the MeAHL22 of cassava plays a key role in hormone,stress signal response and growth and development process,and provided a train of thought for further study of the biological function of MeAHL22 gene.