‘黄花’梨及其芽变‘绿黄花’梨HHT基因克隆与表达分析

Cloning and Expression of HHTGene in‘Huanghua'Pear and Its Bud Mutant‘Lühuanghua'Pear(Pyrus pyrifolia Nakai)

该试验以砂梨品种‘黄花’梨（果皮褐色）及其芽变‘绿黄花’梨（果皮绿色）盛花后第8周的果皮为试材,利用常规PCR和巢式PCR技术克隆了ω-羟基棕榈酸O-阿魏酰转移酶（ω-hydroxypalmitate O-feruloyl transferase,HHT）基因cDNA的全长,命名为PpyHHT（登录号为KX131155）。序列分析结果表明,该基因开放阅读框（ORF）为1 335bp,编码444个氨基酸。生物信息学分析显示,推定的PpyHHT蛋白质相对分子质量为49.91kD,等电点是4.75,与白梨相似性高达98%,亲缘关系最近。实时荧光定量PCR（qRT-PCR）表达分析显示,2种梨果皮中PpyHHT基因在盛花后6～9周的4个转色关键期表达量不断变化,在‘黄花’梨果皮中的表达量明显高于‘绿黄花’梨。推测PpyHHT基因可能参与砂梨果实褐色/绿色性状的形成。

The study used fruit peel of sand pear（Pyrus pyrifolia Nakai）cultivars‘Huanghua＇pear（russet fruit）and its bud mutant‘Lühuanghua＇pear（green fruit）at 8weeks after full bloom（WAFB）as experiment materials.the cDNA full-length of HHTgene,which was named PpyHHT（GenBank accession No.KX131155）,was cloned by conventional and nest PCR techniques.Sequence analysis showed that the full-length of the PpyHHTortholog consisted of a 1 335 bp open reading frame（ORF）encoding apolypeptide containing 444 amino acid residues.The molecular weight of deduced amino acids was 49.91 kD,with an isoelectric point（pI）of 4.75,which had the highest similarity（98%）and closest relationship with that in Pyrus bretschneideri.Real-time fluorescence quantitative PCR（qRT-PCR）analysis demonstrated that the expression of PpyHHTgene in‘Huanghua＇and‘Lühuanghua＇pear peel were changing constantly during the key period（6-9weeks）,and significantly higher in‘Huanghua＇pear peel than that in‘Lühuanghua＇.PpyHHTgene involved in the formation of sand pear russet/green traits,its expression level differences may play a role in the formation of sand pear skin color.