陆地棉小GTP结合蛋白基因GhROP6的克隆及表达初步分析

Molecular Cloning and Expression Preliminary Analysis of a Small GTP-Binding Protein Gene GhROP6 in Gossypium hirsutum L.

为探究ROP基因在棉花抵御逆境胁迫中的生物学功能,利用同源克隆的方法获得一个陆地棉GhROP6基因,通过生物信息学方法分析其理化性质、结构及进化关系,利用实时荧光定量PCR(qRTPCR)技术探究GhROP6基因的组织表达特异性及不同逆境胁迫和外源激素处理下的表达模式,构建GhROP6基因的VIGS载体并转化棉花,利用qRT-PCR技术检测其沉默效率。结果显示,GhROP6基因开放阅读框(ORF)为597 bp,编码一个含198个氨基酸的I类ROP蛋白;多重序列比对结果显示,GhROP6符合ROP蛋白结构特征,且与其他物种ROP蛋白高度同源;进化树分析结果显示GhROP6蛋白与拟南芥AtROP6蛋白同源性最高;GhROP6基因在棉花根、茎、真叶及子叶中均有表达,且在真叶中表达量最高;GhROP6基因对干旱、高盐、低温、高温等胁迫和外源脱落酸(ABA)、生长素(IAA)处理均有不同程度的响应,可能在棉花抗逆反应中扮演着重要角色。GhROP6在棉花的叶片和根部均得到有效沉默,表明已获得GhROP6基因沉默植株。本研究为进一步了解GhROP6基因的分子生物学功能奠定了基础。

In order to explore the biological function of ROP gene in cotton resistance to stress,homologous approach was used to obtain the GhROP6,and its physicochemical properties,structure and evolutionary relationship were analyzed by bioinformatic methods.Real-time quantitative polymerase chain reaction(qRTPCR)technology was used to detect the tissue expression specificity of GhROP6 gene and the expression pattern under different stress and exogenous hormone treatments.The VIGS vector of GhROP6 gene was constructed and transformed into cotton,and its silencing efficiency was detected by qRT-PCR.The results showed that the open reading frame(ORF)of GhROP6 gene was 597 bp in length,encoding a class I ROP protein containing 198 amino acid.Multiple sequence alignment showed that GhROP6 was consistent with ROP protein structure and highly homologous to ROP proteins of other species;Phylogenetic tree analysis showed that GhROP6 protein had the highest homology with Arabidopsis AtROP6 protein;GhROP6 gene was expressed in roots,stems,true leaves and cotyledons of cotton,and the expression level was the highest in true leaves;GhROP6 gene has different responses to drought,high salt,low temperature,high temperature stress and exogenous abscisic acid(ABA),auxin(IAA)treatments,and may play an important role in cotton stress response.GhROP6 was effectively silenced in leaves and roots of cotton,indicating that GhROP6 gene silencing plants were obtained.The present study provides a foundation and deepens our understanding about molecular and biological functions of GhROP6 genes.