GmHSFA1基因克隆及其过量表达提高转基因大豆的耐热性

Cloning of GmHSFA1 Gene and its Overexpression Leading to Enhancement of Heat Tolerance in Transgenic Soybean

热激转录因子在调节植物对逆境胁迫应答和热激蛋白基因表达方面起重要作用。采用生物信息学和比较基因组学方法结合RACE技术从大豆基因组中克隆到一个新的热激转录因子基因GmHsfA1,其cDNA全长1 781 bp,包含1个1 533 bp的开放阅读框,编码含有510个氨基酸残基的蛋白质(GenBank登录号为AY458843)。与其他转录因子的分子结构相似,GmHSFA1也含有4个典型的结构功能域-DNA结合域、寡聚域、核定位信号和C端激活域。BLAST分析表明,GmHSFA1与其同源性最高的番茄热激转录因子LpHSFA1之间的氨基酸序列相似性为52．46％。RT-PCR、Northern和遗传转化结果显示:1)GmHsfA1在大豆的不同组织中呈现组成型表达模式;2)常温下转基因大豆植株的GmHsfA1表达水平明显高于非转基因对照;3)GmHsfA1的过量表达激活了转基因大豆植株中热激蛋白基因GmHsp22在非诱导条件下的转录,并加强了高温胁迫下另2个热激蛋白基因GmHsp23和GmHsp70的表达;4)转GmHsfA1大豆植株的耐热温度(达52℃)明显高于非转基因植株。上述结果说明,GmHsfA1的过量表达激活或促进其下游3个热激蛋白基因的转录或表达,明显提高了转基因大豆植株的耐热能力。

Heat shock transcription factors （HSFs） are important in regulating heat stress response by mediating expression of heat shock protein （HSP） genes in various plant species. In the present study, a novel GmHSFA1 with an ORF of 1 533 bp （full-length cDNA sequence of 1 781 bp） was cloned from soybean genome via comparative genomic approach and RACE （rapid amplification of cDNA ends）. This gene encodes 510 amino acids consisting of a protein of 56.2 kDa （GenBank accession number： AY458843）. Similar to other HSFs, GmHSFA 1 has the basic modular structure including DBD, OD, NLS, and CTAD. BLAST analysis revealed the identity of 52.46% between amino acid sequences between GmHSFA1 and LpHSFA1 that has the highest similarity to GmHSFA1 in all HSFAls in various plant species. The results from RT-PCR, Northern blotting, and transformation showed： 1） GmHsfA1 exhibited the constitutive expression patterns in different tissues of soybean; 2） The expression level of GmHsfA1 in transgenic plants was notably higher than that in non-transgenic plants; 3） Overexpression of GmHsfA 1 activated transcription of GmHSP22 in transgenic plants under normal conditions and enhanced obviously expressions of GmHSP23 and GmHSP70 in transgenic plants under heat stress conditions; 4） Heat tolerant temperature （as high as 52℃） of transgenic plants was remarkably higher than that of non-transgenic plants. These results preliminarily proved that the overexpression of GmHsfA1 possibly led to the notable enhancement of heat-tolerant level of transgenic plants by mediating the activation of transcription or improvement of expression of some GmHSPs in the GmHsfA 1＇s downstream in transgenic plants, suggesting GmHSFA1 is a novel and functional heat shock transcription factor of soybean.