从基因组DNA 及经水杨酸处理的全长cDNA 文库中分别克隆获得湖北海棠[Malus hupehensis (Pamp.)Rehd.]类甜蛋白基因MhPR5 的全编码区序列,并对其进行生物信息学分析;利用实时荧光定量RT-PCR 技术分析了该基因在湖北海棠各组织中的表达特性以及经非生物胁迫(200 mmol·L^-1 NaCl 和4 ℃低温)和生物胁迫[苹果轮纹病病原菌(Physalospora piricola Nose)和苹果蚜虫(Aphis citricola van der Goot)]后MhPR5 基因的表达特性。结果表明:克隆获得的MhPR5 基因全编码区的cDNA 序列长度为741 bp,编码246 个氨基酸残基;该基因的基因组DNA 序列长度为1 106 bp,包含1 个内含子;该基因编码的蛋白质相对分子质量为25 520,等电点为pI 4. 72。MhPR5 基因的核苷酸序列与苹果(M. domestic Borkh.)、梨[Pyrus pyrifolia (Burm. f.) Nakai]和桃[Prunus persica(Linn.) Batsch.]PR5 基因核苷酸序列的同源性分别为98%、95%和82%,该基因编码的氨基酸序列与苹果、梨和桃PR5 基因编码的氨基酸序列的同源性分别为97%、94%和52%。MhPR5 基因编码的蛋白质含有16 个保守的半胱氨酸残基和5 个保守的带负电荷的氨基酸残基,在N 端还含有1 个由25 个氨基酸残基组成的信号肽。MhPR5 基因在湖北海棠组培苗的根、茎和叶中均能表达,在根中的相对表达量最高、叶中最低;生物和非生物胁迫均可诱导MhPR5 基因的表达,说明MhPR5 基因在湖北海棠抵御生物和非生物胁迫过程中可能具有非常重要的作用。

The whole coding region sequence of thaumatin-like protein gene MhPR5 of Malus hupehensis   (Pamp.)  Rehd. was cloned and obtained from genomic DNA and full-length cDNA library treated by salicylic acid (SA), and its bioinformatics was analyzed. The expression characteristics of MhPR5 gene in different organs of M. hupehensis and that under abiotic stress (200 mmol·L^-1 NaCl and 4 ℃ low temperature) and biotic stress ( Physalospora piricola Nose and Aphis citricola van der Goot) were analyzed by real-time fluorescence quantitative RT-PCR technology. The results show that the cDNA sequence length of the whole coding region of MhPR5 gene is 741 bp, it encodes 246 amino acid residues, and its genomic DNA sequence length is 1 106 bp with a intron. The relative molecular weight of the protein encoded by MhPR5 gene is 25 520 and the isoelectric point is pI 4. 72. The homology of nucleotide sequence of MhPR5 gene with that of PR5 gene from M. domestic Borkh., Pyrus pyrifolia (Burm. f.) Nakai and Prunus persica (Linn.) Batsch. is 98%, 95% and 82%, respectively, and the homology of amino acid sequence encoded by MhPR5 gene and that encoded by PR5 gene of the above three species is 97%, 94% and 52%, respectively. The protein encoded by MhPR5 gene contains 16 conserved cysteine residues, 5 conserved amino acid residues with negative charge and a signal peptide composed by 25 amino acid residues in its N-terminal. MhPR5 gene can express in root, stem and leaf of tissue culture seedlings of M. hupehensis with the highest relative expression in root and the lowest in leaf. Expression of MhPR5 gene can be induced by biotic and abiotic stresses, meaning that MhPR5 gene might play an important role in resistance of M. hupehensis to biotic and abiotic stresses.