葡萄细胞分裂素响应调节因子VlRRA1的克隆、表达及启动子活性分析

Cloning,Expression and Promoter Activity Analysis of VlRRA1 Gene in Grape

在‘巨峰’葡萄中克隆获得细胞分裂素响应调节因子VlRRA1及其启动子,分析了VlRRA1的表达特征,并对其启动子进行活性分析。结果显示,VlRRA1的cDNA全长为666 bp,编码221个氨基酸;保守结构域预测结果显示该基因仅含有1个磷酸接受结构域(REC),属于A型RR基因;系统进化关系表明VlRRA1与其他物种中的A型RR亲缘关系较近。酵母自激活试验说明VlRRA1不具有转录激活活性,符合A型RR基因的典型特征。qRT-PCR结果表明VlRRA1具有组织表达特异性,主要在茎、叶以及幼果中表达;外源细胞分裂素氯吡苯脲(CPPU)可以促进VlRRA1的表达,而细胞分裂素生物合成抑制剂洛伐他汀(lovastatin,LOV)则抑制VlRRA1的转录。VlRRA1启动子上有多种与非生物胁迫和激素响应相关的元件。GUS组织染色的结果表明,VlRRA1启动子具有活性,可以响应多种激素信号,包括与坐果相关的细胞分裂素、生长素和赤霉素。以上结果表明VlRRA1在细胞分裂素介导的葡萄坐果与幼果发育中具有重要作用。

The cytokinin response regulator VlRRA1 and its promoter were cloned from‘Kyoho’grape.The expression characteristics of VlRRA1 and the activity of VlRRA1 promoter were analyzed.The results showed that the full-length cDNA of VlRRA1 was 666 bp and encoded 221 amino acids.Conservative domain analysis showed that the gene contained only one phosphoacceptor receiver domain(REC),which belongs to type-A RR genes and can only accept phosphate groups;phylogenetic relationships showed that VlRRA1 was closely related to type-A RR in other species.The yeast self-activation assay illustrates that VlRRA1 has no transcriptional activation activity in agreement with other A-type RR genes and does not have a function similar to that of the B-type RR transcription factor.The results of qRT-PCR showed that VlRRA1 had tissue-specific expression and was mainly expressed in stems,leaves and young fruit,slightly lower expression in inflorescence and ripe fruit,suggesting that VlRRA1 may play an important role in the process of fruit set;exogenous cytokinin forchlorfenuron(CPPU)can promote VlRRA1 expression,while the cytokinin biosynthesis inhibitor lovastatin(LOV)inhibits VlRRA1 transcription.Many elements related to abiotic stress and hormone response were found in VlRRA1 promoter.The results of GUS tissue staining showed that VlRRA1 promoter had promoter activity and could respond to a variety of hormone signals,including three plant hormones related to fruit setting,cytokinin,auxin and gibberellin.These results suggest that VlRRA1 plays an important role in grape fruit setting and young fruit development mediated by cytokinin.