甘蔗ABA生物合成关键酶SoNCED基因克隆及表达分析

摘要

[目的]克隆甘蔗体内9-顺式环氧类胡萝卜素双加氧酶(9-cis-epoxycarotenoid dioxygenase,NCED)基因,分析其基本生物学信息及在逆境胁迫下的表达分析,为进一步探讨SoNCED基因在甘蔗脱落酸(ABA)生物合成途径中的作用提供理论依据.[方法]以甘蔗品种桂糖21号为材料,待蔗苗长至5～6片叶时分别进行干旱、低温、高盐和氧化胁迫处理,利用反转录PCR(RT-PCR)和cDNA未端快速扩增(RACE-PCR)克隆SoNCED基因,应用生物信息学方法对获得的氨基酸序列进行分析,构建原核表达载体并进行目的蛋白的诱导表达,通过实时荧光定量PCR(qRT-PCR)分析SoNCED基因在受到不同逆境胁迫时的表达情况.[结果]克隆获得的SoNCED基因(GenBank登录号JQ314108),cDNA全长为2521 bp,包含1个1827 bp的开放阅读框(ORF),编码608个氨基酸.多重序列比对及系统发育进化树分析结果表明,SoNCED基因编码的氨基酸序列与其他植物有一定的相似性,尤其与禾本科C4植物高粱和玉米的同源性最高,均达96％.成功构建SoNCED基因的原核表达载体pET-SoNCED,且通过IPTG诱导时可得到约66.0 kD的蛋白,确定该蛋白为SoNCE基因的表达蛋白.qRT-PCR结果分析表明,SoNCED基因在干旱、低温、高盐和氧化胁迫下均能诱导表达,其中氧化胁迫下其表达量在6h时达峰值,其他胁迫方式下其表达量在12h时达峰值.[结论]成功克隆获得甘蔗SoNCED基因,并从不同逆境下的表达情况推测该基因在甘蔗体内参与了干旱、低温、高盐和氧化等非生物胁迫的调控过程.%[Objective]The research cloned 9-cis-epoxycarotenoid dioxygenase gene (SoNCED) from sugarcane,analyzed its biological information and expression under stress,in order to provide theoretical basis for further exploring the role of SoNCED gene in sugarcane ABA biosynthesis.[Method]Taking sugarcane variety Guitang 21 as materials,when sugarcane seedlings grew 5-6 leaves,drought,low temperature,high salt and oxidation stresses were applied to them.SoNCED gene was cloned using reverse transcriptional PCR (RT-PCR) and rapid amplification of cDNA end (RACE-PCR).Bioinformatics was used to analyze amino acid sequence.Prokaryotic expression vector was established and induced expression of target protein was conducted.Real-time fluorescence quantitative PCR (qRT-PCR)method was adopted to study the expressions of SoNCED geneunderdifferent stresses.[Result]The cDNA of cloned SoNCED(GenBank accession number JQ314108) in sugarcane was 2521 bp in length with an open reading frame(ORF) of 1827 bp,encoding 608 amino acids.Multiple sequence alignment and phylogenetic tree analysis showed that amino acid sequence of SoNCED gene coding was similar to that of other plants.It showed the highest homology with Sorghum bicolor and Zea may,which reached 96％.Prokaryotic expression vector of gene pET-SoNCED was established.And 66.0 kD protein was obtained under IPTG induction,which was determined as expression protein of SoNCED gene.Results of qRT-PCR analysis showed that SoNCED gene was able to express under drought,low temperature,high salt,and oxidation stresses.Under oxidative stress,the expression level reached the peak in 6 h;and the expressions reached the peak in 12 h under other stresses.[Conclusion]Gene SoNCED was cloned from sugarcane.Different stresses are employed to predict the role of SoNCED gene in regulation of abiotic stresses such as drought,high salt,low temperature and oxidation.