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.