美容杜鹃RcHsfB3基因的克隆及表达分析

Cloning and Expression analysis of RcHsfB3 from Rhododendron calophytum

摘要:

通过RT-PCR和RACE方法从美容杜鹃（Rhododendron calophytum）中克隆到一个热激转录因子（Heat shock factors, Hsfs），命名为RcHsfB3（GenBank登录号KU145694）。RcHsfB3基因全长1130bp，完整的开放读码框（ORF）长771bp，预测可编码257个氨基酸，分子量为28.8KD，等电点为5.38。系统进化树分析发现，该基因与其他几种植物的HsfB3基因属于同一分支，氨基酸同源序列比对结果表明RcHsfB3与葡萄的VvHsfB3的同源性最高，达67.29%。原生质体亚细胞定位表明，RcHsfB3编码蛋白定位在细胞核上。荧光定量PCR分析显示，美容杜鹃分别受到热激、低温和盐害胁迫处理后，RcHsfB3的相对表达量总体呈现先上升，后下降，且“上升-下降”交替的表达趋势。初步研究表明RcHsfB3基因受高温、低温和高盐胁迫的诱导表达。

Abstract:

A Heat shock factor (Hsf) from Rhododendron calophytum was cloned using RT-PCR and rapid amplification of cDNA ends (RACE) techniques and it was named RcHsfB3 (accession no. KU145694). The full length was 1130bp, including an open reading frame of 774bp and encoding a protein of 257 amino acids. Its molecular weight and theoretical isoelectric point were 28.8 kDa and 5.38 respectively. Phylogenetic analysis showed that this gene was classified as the same branch with HsfB3 of other plants, indicating that it belongs to the HsfB3 gene family. Multiple sequence alignment analysis indicated that the homology reaches 67.29% with VvHsfB3, which is highest. The result of subcellular localization in protoplasts exhibited that RcHsfB3 is located in the cell nucleus. The expression of RcHsfB3 were examined using real-time polymerase chain reaction. Under heat、cold and salt stress respectively, RcHsfB3 expression level showed an alternate trend of ‘rising and falling’, remarkably up-regulated.Preliminary studies indicate that RcHsfB3 will be induced by high temperature, low temperature and high salt stress.