橡胶树树皮HbbHLH基因三端序列的克隆及表达分析

Cloning and Expression Analysis of 3′ End Region of HbbHLH Gene in Bark from Hevea Brasiliensis

[目的]为今后深入了解茉莉酸诱导橡胶树乳管分化的分子机理奠定基础。[方法]根据bHLH转录因子基因保守区设计引物,以橡胶树树皮RNA反转录第一链cDNA为模板,获得橡胶树树皮组织bHLH基因的EST序列,以此序列设计巢式引物,应用3′RACE技术对其三端序列进行克隆,并利用半定量RT-PCR分析该基因在茉莉酸处理后4 h、8 h、1 d、2 d、3 d的表达情况。[结果]克隆共得到1 084 bp的HbbHLH基因3端序列,序列分析发现该基因属于MYC型HLH结构域。表达分析结果显示,茉莉酸处理后,bHLH基因在8 h内表达逐渐增强,1 d、2 d时表达稳定最高,3 d时表达水平开始减弱。[结论]茉莉酸对HbbHLH基因的表达具有调控作用。

[ Objective ] The aim was to lay the foundation for further understanding the molecular mechanism of the jasmonate acid inducing laticifer differentiation in Hevea brasiliensis. [ Method] The EST sequence of HbbHLH gene in bark from H. brasiliensis was obtained from the first strand cDNA by reverse transcript mRNA from the bark of H. brasiliensis by using the primers designed according to the conservative region of bHLH transcription factor gene and the 3＇ end region of this gene was cloned by rapid amplification cDNA ends （RACE） technique using nested primers designed according to the obtained sequence. Further more, the expressions of the HbbHLH gene treated with jasmonate acid after 4 h, 8 h, 1 d, 2 d and 3 d were analyzed by semi-quantitative RT-PCR. [ Result] A 1 084 bp cDNA fragment of the 3＇ end region from HbbHLH gene was obtained by clone and the sequence analysis showed that the gene belonged to the HLH domain from MYC type. The expression analysis result showed that after the treatment with jasmonic acid, the expression level of HbbHLH gene was increased gradually within 8 hours, and kept stable at a high level after treatment with 1 d and 2 d, but decreased at 3 d. [ Conclusion] The jasmonic acid had the regulatory effect on the expression of HbbHLH gene.