蓝莓GEF家族基因鉴定与功能分析

Functional characterization of guanylate exchange factorgene family in blueberry(Vaccinium spp.)

鸟苷酸交换因子(guanylate exchange factors,GEFs)是广泛存在于动植物中的一类信号传导因子,通过三磷酸鸟苷(GTP)替换分子开关上的二磷酸鸟苷(GDP),调节分子开关的活性和功能。为研究蓝莓GEF蛋白在调控根毛和表皮毛发育过程中的功能,本研究利用生物信息学技术从蓝莓基因组中鉴定出32个GEF基因,系统进化分析将其划分为3个亚组。氨基酸序列比对和保守结构域分析表明蓝莓GEF均含有PRONE(plant-specific ROP nucleotide exchanger)结构域,属于PRONE超家族。进一步以蓝莓‘Emerald’为实验材料,克隆了VcGEF3序列,该基因开放阅读框长度1401 bp,编码467个氨基酸,分子质量为52.86kDa,理论等电点为5.40,预测VcGEF3蛋白为不稳定的亲水性蛋白。RT-PCR分析表明,VcGEF3在茎和花中表达,不在根、叶、果实中表达。在拟南芥中超量表达VcGEF3后,根毛长度被显著抑制,表皮毛呈不同程度弯曲生长,说明蓝莓VcGEF3参与根毛和表皮毛发育调控。

Guanylate exchange factors(GEFs)are a kind of signal transduction factor widely existing in animals and plants.It regulates the activity and function of molecular switches by replacing guanosine diphosphate(GDP)on molecular switches with guanosine triphosphate(GTP).In order to reveal the function of blueberry GEFs in the development of root and epidermal hair,32 GEF genes were identified,which were divided into three groups by phylogenetic relationship analysis.Amino acid sequence alignment and conserved domain analysis showed that VcGEFs contained a PRONE(plant-specific ROP nucleoside exchanger)domain,indicating that VcGEFs belong to PRONE superfamily.The VcGEF3 sequence was cloned from blueberry‘Emerald’.The open reading frame length of the gene was 1401 bp and encoded 467 amino acids.The molecular weight is 52.86 kDa and the theoretical isoelectric point is 5.40.It is predicted that VcGEF3 is an unstable hydrophilic protein.RT-PCR analysis showed that VcGEF3 expresses specifically in stems and flowers,but not in roots,leaves and fruits.Overexpression of VcGEF3 significantly shortened the length of root hair and curved the trichome in transgenic Arabidopsis thaliana.These results indicated that VcGEF3 was involved in the regulation of root and epidermal hair development.