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Acta Agron Sin ›› 2017, Vol. 43 ›› Issue (05): 640-647.doi: 10.3724/SP.J.1006.2017.00640

• CROP GENETICS & BREEDING·GERMPLASM RESOURCES·MOLECULAR GENETICS • Previous Articles     Next Articles

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 Online:2017-05-12 Published:2017-03-08
  • Contact: Ni Yu, E-mail: nmniyu@126.com E-mail:714979637@qq.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).

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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