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园艺学报 ›› 2013, Vol. 40 ›› Issue (5): 849-.

• 果树 • 上一篇    下一篇

梨贝壳杉烯酸氧化酶基因PcKAO1 的克隆与表达分析

欧春青,姜淑苓,王 斐,王志刚,马 力,李连文   

  1. 中国农业科学院果树研究所,农业部园艺作物种质资源利用重点实验室,辽宁兴城 125100
  • 出版日期:2013-05-25 发布日期:2013-05-25

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

OU Chun-Qing , JIANG  Shu-Ling, WANG   Fei, WANG  Zhi-Gang, MA   Li, LI  Lian-Wen   

  1. Research Institute of Pomology,Chinese Academy of Agricultural Sciences,Key Laboratory of Horticultural Crops
    Germplasm Resourses Utilization,Ministry of Agriculture,Xingcheng,Liaoning 125100,China
  • Online:2013-05-25 Published:2013-05-25

摘要: 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1 568 bp,开放阅读框(ORF)编码503 个氨基酸,相对分子量为57.59 kD,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。

关键词: 梨, 贝壳杉烯酸氧化酶基因(KAO), 基因表达, 矮化

Abstract: 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 functional
regions of cytochrome P450 superfamily proteins,so named the gene as PcKAO1,GenBank accession
number was KC153027.1. The other two PcKAO1 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
PcKAO1 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 PcKAO1 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.

Key words: pear, ent-kaurenoic acid oxidase gene(KAO), gene expression, dwarf

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