甘蓝型油菜烷羟化酶基因MAH1的克隆与表达分析

发布时间：2018-05-04 20:29

  本文选题：甘蓝型油菜 + 角质层蜡质　； 参考：《作物学报》2017年05期

【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(Gen Bank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P415x R417x、K螺旋(E359xx R362)、C末端的血红素结合域(F436xx Gx Rx Cx G445)以及氧结合带保守区域(A/G)G309x(D/E)T312(T/S)。NCBI Blast N、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、Me JA、ACC、ABA、Na Cl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。
[Abstract]:In the waxy synthesis pathway of plant, mid-chain alkane hydroxylase- (MAH) catalyzes the hydroxylation of alkanes to form secondary alcohols, which are further oxidized to ketones. In this study, two full-length coding regions of Arabidopsis thaliana P450 dependent enzyme CYP96A15/MAH1 were obtained by means of electronic cloning and RT-PCR techniques. They were named BnMAH1-1 and BnMAH1-2(Gen Bank accession numbers KT795344 and KT79534545, respectively. The length of ORF was 1491 BP, with no intron. The nucleotide and amino acid sequence were 92.4% and 90.9% respectively. The predicted BnMAH1-1 and BnMAH1-2 precursor proteins based on the coding region are polypeptide chains containing 496 amino acid residues. The conserved structure of P450 protein family, P415x R417xK helix E359xx R362C terminal heme binding domain F436xx Gx Rx Cx G445) and the conserved region of oxygen binding band, A / G ~ (309xN) D312x / T / S.NCBI Blast Ns, amino acid sequence multiple weight alignment and phylogenetic analysis showed that, They share the highest homology with Arabidopsis MAH1/CYP96A15. Real-time fluorescence quantitative PCR showed that BnMAH1-1 and BnMAH1-2 were mainly expressed in stems, leaves, flowers, and pods of Brassica napus. The results indicated that the decrease of wax was related to the down-regulation of MAH1 transcription. BnMAH1-1 and BnMAH1-2 were induced by SAMe JACC-ABANa NaCl and drought stress. BnMAH1-1 may play a major role in water stress response.
【作者单位】： 西南大学农学与生物科技学院;
【基金】：重庆市基础与前沿研究计划项目(cstc2016jcyjA0170) 中央高校基本科研业务费专项资金(XDJK2014B037)资助~~
【分类号】：S565.4
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本文编号：1844542