葡萄VvRGL基因应答GA调控无核果实发育的研究

VvRGL Genes Regulate Seedless Fruit Development by Responding to GA in Grapevine

DELLA蛋白是GA信号传导途径的核心作用元件,属于GRAS核转录调节因子家族。该研究以‘魏可’葡萄品种为试材,通过基因克隆、启动子分析、染色体定位、基因蛋白结构及系统进化分析,鉴定VvRGL1和VvRGL2精确序列,预测其潜在功能,并采用qRT-PCR技术检测VvRGL基因应答GA在果皮、果肉及种子(区)的时空表达特征。结果表明:(1)VvRGL1及VvRGL2的染色体定位为Chr14和Chr7,开放阅读框(ORF)为2 007bp和1 815bp,编码氨基酸数量为668和604个;二者均含有GRAS保守结构域,但却不具备DELLA结构域,属于GRAS转录因子大家族成员,而不属于DELLA亚家族;基因结构分析表明,VvRGL1的DNA序列有1个内含子,2个外显子,而VvRGL2的DNA序列无内含子,有1个外显子,基因结构高度保守;进化分析表明,VvRGL1与拟南芥和柑桔的亲缘关系较近,而VvRGL2与草莓和杨树的亲缘关系较近。(2)2个基因的启动子均含有响应赤霉素和胚乳发育相关的作用元件,表明它们可能参与响应GA信号传导和种子胚乳发育过程。(3)qRT-PCR结果显示,外源GA处理均不同程度降低了2个基因在葡萄果皮和种子区的表达,但却上调了其在幼果期果肉中的表达,表明在果皮与种子(区)中,GA可能通过抑制葡萄VvRGL1/RGL2基因的表达参与调控葡萄无核果实的发育。本研究结果为进一步阐明VvRGL在赤霉素信号传导及葡萄无核果实发育机理中的作用机制提供重要依据。

DELLA protein is a core component of GA signaling pathway,and belongs to the GRAS nuclear transcription regulator family.In the present study,we used the grapevine cv.‘Wink’as the test material to identify the precise sequences and predict the potential functions of VvRGL1 and VvRGL2 through gene cloning,promoter analysis,chromosomal location,gene protein structure and phylogenetic analysis.And the qRT-PCR method was used to detect the temporal and spatial expression of VvRGLgenes in the grape berry peel,berry flesh and seed(or seed area)induced by exogenous GA.Result showed that:(1)the chromosomal localization of VvRGL1/RGL2 were Chr14 and Chr7,the open reading frame(ORF)were 2007 bp and 1 815 bp,and the number of amino acid were 668 and 604,respectively.Both of VvRGL1 andVvRGL2 contained a GRAS conserved domain,but did not have a DELLA domain,and were members of the GRAS transcription factor gene family,but not the DELLA subfamily.Gene structure analysis result showed that their gene structures were highly conserved with the DNA sequence of VvRGL1 was only one intron and two exons,while the DNA sequence of VvRGL2 had no intron and only one exon.Phylogenetic analysis showed that VvRGL1 was closely related to Arabidopsis and citrus,while VvRGL2 was closely related to strawberry and poplar.(2)The promoters of two genes both contained elements that were responsible for GA and endosperm development,suggesting that they might be involved in response to GA signaling and endosperm development.(3)The results of qRT-PCR showed that exogenous GA treatment reduced the expression of two genes in grape peel and seed area,but up-regulated their expression in the flesh of young fruit,suggesting that in the peel and seed areas,GA might participate in the regulation of grape seedless fruit development by inhibiting the expression of VvRGL1/RGL2.This study provides an important basis for further explore the function of VvRGLin GA signaling pathway and in seedless fruit development of grapevine.