梨贝壳杉烯酸氧化酶基因PcKAO1的克隆与表达分析

Cloning and Expression Analysis of ent-Kaurenoic Acid Oxidase Gene (PcKAO1)in Pear

以梨矮化砧木‘中矮1号’新梢叶片为试材,根据苹果基因组中相关序列信息设计特异引物,通过RT-PCR的方法克隆了梨赤霉素合成过程中关键酶——贝壳杉烯酸氧化酶(KAO)基因的编码框全长序列。该序列全长1568bp,开放阅读框(ORF)编码503个氨基酸,相对分子量为57.59kD,等电点为9.47,氨基酸序列与其它物种已知的KAO序列具有53.14%～98.61%的相似性,且具有细胞色素P450家族蛋白典型的功能结构域和跨膜结构域,将该基因命名为PcKAO1,GenBank登录号为KC153027.1。用同样的方法克隆了对照梨品种‘早酥’(乔化)和‘锦香’(半矮化)的PcKAO1基因,通过比对分析发现3个品种的PcKAO1基因间仅有个别碱基差异,其编码的蛋白序列完全一致。RT-PCR半定量分析表明:PcKAO1基因在‘中矮1号’各个检测的器官组织中均有表达,其中以种子和子房中的表达量最高;在3个品种的新梢叶片中,除新梢生长初期(5月10日)之外,其他时期矮化砧木‘中矮1号’均低于乔化和半矮化的对照品种‘早酥’和‘锦香’,而在这些时期‘中矮1号’新梢生长逐渐减缓乃至停长。

Ent-kaurenoic acid oxidase （KAO） is a critical enzyme in the pathway of gibberellins biosynthesis. In this research, specific primers were designed based on sequence information of apple genome and a coding sequence of ent-kaurenoic acid oxidase gene was isolated from new shoot leaves of pear dwarfing inter-stock ＇Zhongai 1＇ by RT-PCR method. The sequence was 1 568 bp in length with an open reading frame （ORF） encoding a protein of 503 amino acids. Molecular weight and isoelectric point of the protein was 57.59 kD and 9.47, respectively. Amino acids homology analysis indicated that the sequence shared 53.14% - 98.61% similarity with other reported KAOs, and it had the typical functionalregions of cytochrome P450 superfamily proteins, so named the gene as PcKAO1, GenBank accession number was KC153027.1. The other two PcKA01 genes from control pear varieties ＇Zaosu＇ （tall） and ＇Jinxiang＇ （semi-dwarf） also were isolated by the same method. Multiple sequence alignment showed that the PcKAO1 sequence only had few nucleotide bases difference among three varieties, but their encoding amino acids sequences were completely consistent. Half quantitative RT-PCR results showed that PcKA01 gene expressed in every tested organs of ＇Zhongai 1＇, and in the seeds and ovary it had the highest expression. In all the growth periods except the initial growth period （ 10th May） the PcKA01 gene expression of new shoot leaves in ＇Zhongai 1＇ was lower than in control varieties tall ＇Zaosu＇ and semi-dwarf ＇Jinxiang＇ . In these periods the new shoot growth speed of ＇Zhongai 1＇ was gradually slowed and stopped growing.