湖北海棠MhPR1a基因的克隆与表达特性分析

以水杨酸诱导的湖北海棠 Malus hupehensis (Pamp.) Rehd.]全长cDNA文库和基因组DNA为模板,克隆其PR1a基因(MhPR1a)的全编码区序列,并对该序列进行生物信息学分析；在此基础上利用荧光定量RT-PCR技术对湖北海棠根、茎和叶中该基因的表达特性及经过10μmol·L-1ABA、4℃低温处理及苹果蚜虫(Aphis citricola van der Goot)侵染后叶中该基因的表达特性进行了测定.结果表明:克隆获得的MhPR1a基因全长518 bp,最大开放阅读框为492 bp,编码162个氨基酸残基；编码的蛋白质为酸性蛋白,其相对分子质量为16 960,等电点pI 5.46；其基因组DNA序列与cDNA序列完全一致,说明MhPR1a基因内部没有内含子.湖北海棠MhPR1a基因与苹果(M.domestic Borkh.)和沙梨Pyrus pyrifolia( Burm.f.)Nakai] PR1基因的cDNA序列及其编码的氨基酸序列同源性均较高,其中cDNA序列的同源性均为97％,氨基酸序列的同源性分别为95％和97％；系统树也显示MhPR1a基因编码的氨基酸序列与苹果和沙梨的亲缘关系最近,聚为一类.MhPR1a基因编码的氨基酸序列具有SCP保守结构域,含有1个信号肽和6个保守的半胱氨酸残基.在湖北海棠的叶、茎和根中MhPR1a基因均能表达,在根中的表达量最高.10 μmol·L-1ABA和4℃低温处理48 h后均可诱导MhPR1a基因的表达,且相对表达量明显高于对照(处理0h)；苹果蚜虫也可诱导MhPR1a基因的表达,说明MhPR1a基因在湖北海棠抵抗植食昆虫和低温胁迫的过程中可能发挥着重要作用.

Cloning and expression characteristics analysis of MhPR1a gene in Malus hupehensis

Taking full-length cDNA library and genomic DNA of Malus hupehensis(Pamp.) Rehd.treated by salicylic acid(SA) as the templates,the whole coding region sequence of PR1a gene of M.hupehensis(MhPR1a) was cloned and its bioinformatics was analyzed.On the basis,the expression characteristics of MhPR1a gene in root,stem and leaf of M.hupehensis and that in leaf after treated by 10 μmol·L-1ABA,4 ℃ low temperature and infected by Aphis citricola van der Goot were determined by fluorescence quantitative RT-PCR technology.The results show that the full length of MhPR1a gene is 518 bp with the biggest open reading frame of 492 bp,it encodes 162 amino acid residues,its encoding protein is acidic protein with a relative molecular weight of 16 960 and isoelectric point of pI 5.46.Its genomic DNA sequence is full identical with its cDNA sequence,indicating that there is no intron in MhPR1a gene.The homology of cDNA sequence of MhPR1a gene of M.hupehensis and PR1 gene of M.domestic Borkh.and Pyrus pyrifolia(Burm.f.) Nakai and that of amino acid sequence encoded by those genes all are higher.In which,both homology of cDNA sequence reach 97%,while that of amino acid sequence reaches 95% and 97%,respectively.Also,phylogenetic tree shows that amino acid sequence encoded by MhPR1a gene has a closest genetic relationship with that of M.domestic and P.pyrifolia, which are clustered together.The amino acid sequence encoded by MhPR1a gene contains a conserved SCP domain,including one signal peptide and six conserved cysteine residues.MhPR1a gene can express in leaf,stem and root of M.hupehensis with the highest expression in root.The expression of MhPR1a gene can be induced after treated by 10 μmol·L-1 ABA and 4 ℃ low temperature for 48 h,and the relative expression is obviously higher than that of the control(treated for 0 h).And the expression of MhPR1a gene can also be induced by Aphis citricola.It is suggested that MhPR1a gene may play an important role in resistance of M.hupehensis to phytophagous insects and low temperature stresses.