柑橘抗逆基因MYB96的克隆与表达分析

Cloning and Expression Analyses of Stress Resistant Gene MYB96 in Citrus

为了探讨柑橘MYB96基因在柑橘抗逆过程中的作用,以甜橙、柠檬、金柑、芦柑为试验材料,克隆得到4个柑橘MYB转录因子家族基因,分别命名为CsMYB96、ClMYB96、FmMYB96、CrMYB96,并对其生物信息学以及不同非生物胁迫处理下的表达模式进行分析。结果表明,CsMYB96、ClMYB96、FmMYB96和CrMYB96的开放阅读框长度分别为1032,1035,1035和1032 bp,其编码的蛋白分别由343,344,344,343个氨基酸组成,分子量分别约为38.16,38.27,38.22,38.13 ku,等电点分别为6.31,6.35,6.35,6.31,均为不稳定亲水性蛋白;亚细胞定位预测结果均定位于细胞核。这4个基因编码的蛋白二级结构相似,主要由α螺旋和无规卷曲构成;具有高度保守的R2和R3结构域;在进化关系上,与克莱门氏小柑橘CcMYB96亲缘关系最近。CsMYB96基因的启动子中含有脱落酸响应元件(ABRE)、低温响应元件(LTR)、厌氧响应元件(ARE)、参与干旱诱导的MYB结合位点(MBS)等非生物胁迫响应相关顺式作用元件。qRT-PCR结果分析表明,甜橙CsMYB96能被低温和干旱胁迫诱导表达;柠檬ClMYB96和金柑FmMYB96在高盐胁迫下被诱导表达;而芦柑CrMYB96的表达量在低温、干旱和高盐胁迫下都有不同程度的下调表达。

In order to explore the role of citrus MYB96 gene in the process of citrus stress resistance,four MYB transcription factor genes were cloned from sweet orange,lemon,kumquat and ponkan,and named CsMYB96,ClMYB96,FmMYB96 and CrMYB96,respectively.Their bioinformatics and expression patterns under different abiotic stress treatments were analyzed.The results showed that the open reading frame of CsMYB96,ClMYB96,FmMYB96 and CrMYB96 were 1032,1035,1035 and 1032 bp,respectively.The citrus MYB96 proteins were composed of 343,344,344 and 343 amino acids,respectively,with a molecular weight of about 38.16,38.27,38.22 and 38.13 ku and an isoelectric points of 6.31,6.35,6.35 and 6.31,these proteins were all unstable hydrophilic proteins and the prediction results of subcellular localization were all located in the nucleus.The secondary structures of four MYB96 proteins were similar,mainly composed ofα-helix and random curls.And all of four MYB96 proteins had a highly conserved R2 and R3 domains.In terms of evolutionary relationship,the proteins encoded by these four genes are most closely related to Citrus clementine CcMYB96 protein.The promoter of CsMYB96 gene contained abscisic acid response element(ABRE),low temperature response element(LTR),anaerobic response element(ARE),MYB binding site(MBS)involved in drought induction and other abiotic stress response related cis acting elements.Real-time quantitative PCR analysis showed that CsMYB96 could be induced by low temperature and drought stress,while ClMYB96 and FmMYB96 were induced under high salt stress.The expression of CrMYB96 was down-regulated in different degrees under low temperature,drought and high salt stress.