‘黄花’梨及其芽变‘绿黄花’梨HHT基因克隆与表达分析

摘要

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, H HT)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1335 bp,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49.91 kD,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 (rus-set fruit)and its bud mutant ‘Lühuanghua’pear (green fruit)at 8 weeks after full bloom (WAFB)as ex-periment materials.the cDNA full-length of HHT gene,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 PpyHHT ortholog consisted of a 1 335 bp open reading frame (ORF)encoding a poly-peptide 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 demon-strated that the expression of PpyHHT gene in ‘Huanghua’and ‘Lühuanghua’pear peel were changing constantly during the key period (6-9 weeks),and significantly higher in‘Huanghua’pear peel than that in ‘Lühuanghua’.PpyHHT gene involved in the formation of sand pear russet/green traits,its expres-sion level differences may play a role in the formation of sand pear skin color.