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作物学报 ›› 2017, Vol. 43 ›› Issue (05): 640-647.doi: 10.3724/SP.J.1006.2017.00640

• 作物遗传育种·种质资源·分子遗传学 • 上一篇    下一篇

甘蓝型油菜烷羟化酶基因MAH1的克隆与表达分析

徐熠**,彭阳**,李帅,赵秋棱,张双娟,李加纳,倪郁*   

  1. 西南大学农学与生物科技学院,重庆 400715
  • 收稿日期:2016-11-04 修回日期:2017-03-01 出版日期:2017-05-12 网络出版日期:2017-03-08
  • 通讯作者: 倪郁, E-mail: nmniyu@126.com
  • 基金资助:

    本研究由重庆市基础与前沿研究计划项目(cstc2016jcyjA0170)和中央高校基本科研业务费专项资金(XDJK2014B037)资助。

Cloning and Expression Analysis of Alkane Hydroxylase Gene MAH1 from Brassica napus

XU Yi**,PENG Yang**,LI Shuai,ZHAO Qiu-Ling,ZHANG Shuang-Juan,LI Jia-Na,NI Yu*   

  1. College of Agronomy and Biotechnology, Southwest University, Chongqing 400715, China
  • Received:2016-11-04 Revised:2017-03-01 Published:2017-05-12 Published online:2017-03-08
  • Contact: Ni Yu, E-mail: nmniyu@126.com
  • Supported by:

    This study was supported by the Chongqing Basic and Advanced Research Project (cstc2016jcyjA0170), and the Fundamental Research Funds for the Central Universities (XDJK2014B037).

摘要:

在植物蜡质合成途径中,中链烷烃羟化酶(mid-chain alkane hydroxylase, MAH) 催化烷烃羟基化形成二级醇,进一步氧化为酮。本研究以拟南芥P450依赖性酶CYP96A15/MAH1基因为探针,采用电子克隆与RT-PCR技术,获得2个甘蓝型油菜MAH1的全长编码区序列,分别命名为BnMAH1-1和BnMAH1-2 (GenBank登录号分别为KT795344和KT795345)。二者ORF长度均为1491 bp,无内含子,核苷酸与氨基酸序列分别有92.4%与90.9%的一致性。根据编码区预测的BnMAH1-1和BnMAH1-2前体蛋白均为包含496个氨基酸残基的多肽链,具有典型的P450蛋白家族保守结构P415xR417x、K螺旋(E359xxR362)、C末端的血红素结合域(F436xxGxRxCxG445) 以及氧结合带保守区域(A/G)G309x(D/E)T312(T/S)。NCBI BlastN、氨基酸序列多重比对与系统学分析表明, 两者与拟南芥MAH1/CYP96A15同源性最高。实时荧光定量PCR表明,BnMAH1-1与BnMAH1-2主要在甘蓝型油菜茎、叶、花、及角果中表达,其中在叶片中的表达量最高,在根系中的表达量很低,这与角质层蜡质主要沉积在植株地上部分相一致。BnMAH1-1和BnMAH1-2在无蜡粉材料茎、叶片中几乎不表达,表明蜡质的减少与MAH1的转录下调有关。BnMAH1-1与BnMAH1-2受SA、MeJA、ACC、ABA、NaCl及干旱胁迫诱导表达,其中BnMAH1-1可能在水分胁迫响应中起主要作用。

关键词: 甘蓝型油菜, 角质层蜡质, 烷羟化酶

Abstract:

In wax biosynthesis, the mid-chain alkane hydroxylase (MAH) catalyzes the hydroxylation reaction from alkanes to secondary alcohols and further to corresponding ketones. In this study, using Arabidopsis P450-dependent enzyme CYP96A15 / MAH1 gene as a probe, the full-length coding sequences of two Brassica napus MAH1 genes, BnMAH 1-1 (GenBank Accession: KT795344) and BnMAH1-2 (GenBank Accession: KT795345), were isolated by in silico cloning and RT-PCR. The ORF lengths of BnMAH1-1 and BnMAH1-2 were 1491 bp, and no intron was included. BnMAH1-1 and BnMAH1-2 shared 92.4% and 90.9% of sequence identity at nucleotide and amino acid level, respectively. The predicted BnMAH1-1 and BnMAH1-2 protein contained 496 amino acid residues, with typical P450 protein family conserved domains P415xR417x, K helix (E359xxR362), C-terminal hemopexin-like domain (F436xxGxRxCxG445) and oxygen binding zone (A/G)G309x(D/E)T312(T/S). NCBI BlastN, multiple alignment of amino acid sequence, and phylogenetic analysis showed that they were most homologous to A. thaliana CYP96A15/MAH1. Real-time quantitative PCR showed that BnMAH1-1 and BnMAH1-2 were mainly expressed in stem, leaf, flower, and silique. The highest expression level was found in leaf and the lowest was in root, which was consistent with the wax deposition on aerial part of plant. The expression of BnMAH1-1 and BnMAH1-2 was barely detected in stem and leaf of wax deficient cultivar, suggesting that the reduced wax deposition is due to the down-regulation of MAH1 transcription. The expression of BnMAH1-1 and BnMAH1-2 was significantly induced by exogenous application of SA, MeJA, ACC, and ABA and exposure to NaCl and drought, among which BnMAH1-1 may play a major role in response to water stress.

Key words: Brassica nupas, Cuticular wax, Alkane hydroxylase

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