毛白杨PtDREB2A基因的克隆、表达及单核苷酸多态性分析

Isolation, Expression and Single Nucleotide Polymorphisms Analysis of PtDREB2A in Populus tomentosa

利用基因特异的PCR引物由毛白杨受干旱胁迫后构建的cDNA文库中扩增获得一PtDREB2A cDNA克隆,并进行测序和序列分析,结果表明:PtDREB2A cDNA片段总长为946 bp,基因内部含有完整的开放阅读框架,大小为864 bp,可编码长度为287个氨基酸残基的蛋白质,所推导的蛋白质氨基酸序列在AP2/ERF结构功能域与拟南芥AtDREB2A和水稻OsDREB2A蛋白的同源性分别为80.3%和83.3%。组织特异性Realtime-PCR结果显示,PtDREB2A在杨树根、茎、叶片和顶端分生组织中均有表达,但其表达模式不同:PtDREB2A在叶片表达丰度最高,在树干皮层及根部组织表达丰度中等,而在顶端分生组织及主干韧皮部、形成层与木质部表达丰度最低。非生物胁迫及激素诱导差异表达显示,PtDREB2A不仅受高温、低温、干旱与盐诱导表达,还受到激素IAA,NAA及GA3的上调表达,但与ABA的诱导无关。组合利用MEGA4.0和DnaSP4.50.4软件对毛白杨45株基因型个体的PtDREB2A序列进行比对和分析,共检测到49个单核苷酸多态性(SNP)位点,SNP频率为1/18 bp,多样性指数π为0.010 48。在外显子区域,共检测到46个SNP位点,其中19个为同义突变,27个为错义突变。

A cDNA clone encoding DREB2A was isolated from a cDNA library prepared under drought stress by the gene-specific PCR amplification from Populus tomentosa.The cDNA was 946 bp in length with an open reading frame （ORF,864 bp in length） which is capable of encoding a protein of 287 amino acids.The deduced protein sequence of the PtDREB2A shares 80.3%,and 83.3% identity with functional domain of AP2/ERF of Arabidopsis thaliana AtDREB2A,and Oryza sativa OsDREB2A,respectively.Tissue differential expression detected with realtime-PCR indicated that PtDREB2A transcripts had their mRNA products in roots,stems,leaves and apical shoot meristems with varied levels.The PtDREB2A transcripts were the most abundant mRNA products in leaves,with medium expression in stem bark and roots,but a weak expression was detected in apical shoot meristems,and in the phloem,cambium,and xylem of stem.We analyzed the expression patterns of PtDREB2A under abiotic stress conditions and phytohormone treatment,and revealed that expression of PtDREB2A was induced by heat-shock,cold,drought and high-salt stress,but also by IAA,NAA,and GA3,not by ABA.The genomic sequences of PtDREB2A in 45 individuals were aligned,compared and analyzed using the software MEGA4.0 and DnaSP4.50.4.A total of 49 single nucleotide polymorphisms （SNPs） were detected and the frequency and diversity of SNPs were 1/18 bp and 0.010 48,respectively.In total,46 SNPs were detected in the coding regions of PtDREB2A,of which 19 and 27 were silent and missense mutations,respectively.The results,therefore,would provide the important foundation for gene-assisted improvement of tolerance to abiotic stresses in P.tomentosa.