甘蓝自交不亲和相关转录因子BosiP_(A1)蛋白基因的克隆与表达分析

Molecular Cloning and Expression Analysis of Transcription Factor BosiP_(A1) in Brasscia oleracea

通过自交不亲和甘蓝‘A1’自花授粉1 h和未授粉柱头蛋白质表达谱的对比,鉴定出1个受自花授粉诱导上调表达的蛋白,通过PCR技术获取了其编码序列,命名为BosiPA1蛋白,其基因全长为3 730 bp,具有1个1 092 bp的完整编码框(KF314579)。BosiPA1由6个外显子、5个内含子组成,编码363个氨基酸。序列分析表明BosiPA1蛋白具有典型的basic helix-loop-helix(bHLH)功能域,在SCR、SRK、Exo70A1基因的ATG上游启动子序列中存在可以被bHLH功能区识别并结合的E-box(CANNTG)序列。在分子进化上甘蓝BosiPA1与AtbHLH128距离最近,与AtHEC1/2/3和TgGBOF-1处在同一个分支。RT-PCR检测表明甘蓝BosiPA1基因在花瓣、萼片、花粉、柱头和叶片中均有表达;荧光定量PCR分析表明BosiPA1基因在自花授粉10 min、30 min、1 h的柱头中呈逐渐上升趋势。上述结果说明BosiPA1与bHLH的进化分支较早,可能是一个参与甘蓝多器官发育的自交不亲和相关的转录因子。

Through the contrast of the stigma protein expression profiling between after self-pollination and none-pollination of Brasscia oleracea L. SI line A1,an up-regulated protein was identified,which was named BosiPA1 protein. The full-length DNA of this gene was 3 730 bp,and the ORF（open reading frame）was 1 092 bp. The gene contained six exons and five introns,encoded 363 amino acids,the amino acids sequence analysis showed that BosiPA1 contained a typical basic helix-loop-helix（bHLH）domains,which can recognize and bound the E-box（CANNTG）sequence. There are the E-box sequence in the promotersequences upstream of the ATG of SCR,SRK,Exo70A1. Phylogenetic tree analysis showed that Brassica oleracea L. BosiPA1 was most close to AtbHLH128,and grouped into one clade with AtHEC1/2/3 and TgGBOF-1. RT-PCR analysis indicated that BosiPA1 was expressed in petal,sepal,pollen,stigma and leaf,the further real-time fluorescence quantitative PCR analysis showed that the relative expression level of BosiPA1 in the stigma increased continually after 10 min,30 min,1 h self-pollination. All the results indicated that BosiPA1 may be a transcription factor related with SI and involved in multi-organs development in Brasscia oleracea L.