杨树热激转录因子HsfA1d的克隆、表达及单核苷酸多态性分析

Isolation, Expression and Single Nucleotide Polymorphisms Analysis of Heat Shock Transcription Factor HsfA1d in Populus

利用RT-PCR方法,首次由小叶杨受热激胁迫后构建的cDNA文库中扩增获得一热激转录因子HsfA1d cDNA克隆,测序结果表明克隆的PsHsfA1d cDNA片段总长为2036bp,基因内部含有完整的开放阅读框架,大小为1449bp,可编码长度为482个氨基酸残基的蛋白质,所推导的蛋白质氨基酸序列在HSF DBD结构功能域与拟南芥AtHsfA1d和水稻OsHsfA1蛋白的相似性分别为86.3%和87.4%。组织特异性Realtime-PCR结果显示,PsHsfA1d主要在杨树叶片和根部组织中高丰度表达。非生物胁迫、激素及糖诱导表达表明,PsHsfA1d不仅受高温、干旱与盐诱导表达,还受到激素中GA_3与ABA,及葡萄糖与蔗糖信号的上调表达。组合利用MEGA4.0和DnaSP4.50.7软件对小叶杨36株基因型个体的PsHsfA1d序列进行比对和分析,共检测到207个单核苷酸多态性(SNP)位点,SNP频率为1/16bp,多样性指数π为0.00772。在编码区域,共检测到69个SNP位点,其中37个为同义突变,31个为错义突变,1个为无义突变;非同义突变与同义突变的比率0.132<1,推测在小叶杨物种演化过程中,纯化选择是该基因内同义SNP位点主要的进化驱动力。

A cDNA clone encoding HsfA1d was isolated from a cDNA library prepared under heat shock stress in Populus simonii by the RT-PCR method. The cDNA of PsHsfA1d is 2 036 bp in length with an open reading frame （ORF, 1 449 bp in length） which is capable of encoding a protein of 482 amino acids. The deduced protein sequence of the PsHsfA1d shares 86.3%, and 87.4% identity with functional domain of HSF DBD of Arabidopsis thaliana AtHsfA1d, and Oryza sativa OsHsfA1, respectively. Tissue differential expression with realtime-PCR indicated that PsHsfA1d was expressed predominantly in the leaves and roots. We analyzed the expression patterns of PsHsfA1d under abiotic stress conditions and phytohormone treatment, and the result revealed that expression of PsHsfA1d was induced not only by heat-shock, drought and high-salt stress, but also up regulated by GA3, and ABA, and glucose and sucrose signals. The genomic sequences of PsHsfA1d in 36 individuals were aligned, compared and analyzed using the software MEGA4.0 and DnaSP4.50.7. A total of 207 single nucleotide polymorphisms （SNPs） were detected and the frequency and diversity of SNPs were 1/16 bp and 0.007 72, respectively. In total, 69 SNPs were detected in the coding regions of PsHsfA1d, of which 37, 31 and 1 were silent, missense, and nonsense mutations, respectively; the nonsynonymous nucleotide substitutions （πnonsyn） was markedly lower than πsyn, with the πnonsyn/πsyn ratio 0.1321, suggesting that diversity at the synonymous sites of exon regions would have resulted from strong purifying selection.