Brassinosteroids (BRs) as an important phytohormone,play essential roles in plant growth,development and responses to adversity stresses.The key enzyme encoded by CYP90B1 is involved in the BRs biosynthesis.However,the characteristic of CYP90B1 gene response to stress has not been reported in maize.In this study,we cloned ZmCYP90B1 gene from Zea mays,GenBank accession number KY242373,via RT-PCR in combination of RACE technique.The full-length sequence of this gene is 2058 bp and the the complete open reading frame is 1518 bp,encoding 506 amino acid peptides.The predicted protein has the molecular weight of 57.66 kD and isoelectric point of 9.54,containing one transmembrane domain and one conserved domain of p450.Multiple sequences alignment analysis indicated the predicted protein shared high similarities with CYP90B1 from other plant species.The phylogenetic tree revealed a notable difference in the evolution of CYP90B1 between dicotyledons and monocotyledons.The qRT-PCR result showed that the expression of ZmCYP90B1 was induced by drought,high salt,low temperature,ABA,Spodoptera exigua attack,and methyl jasmonate (MeJA),suggesting that ZmCYP90B1 was involved in various abiotic stresses,insect resistance and response to MeJA.Overexpressing ZmCYP90B1 in tobacco seedlings could enhance drought resistance with less water loss rate and higher SPAD value in the transgenic lines.In addition,the activities of SOD,CAT,and POD and the content of proline increased significantly in all transgenic tobacco lines than in the wild type;both MDA and ABA contents were obviously lower in the transgenic lines than in the wild type.Through expression analysis of the down-stream stress-responsive genes,we demonstrated that drought tolerance enhancement by overexpressing ZmCYP90B1 might be involved in ABA-independent pathway and related to transcriptional regulation of antioxidant-related genes.%油菜素甾醇作为一类重要植物激素,在植物生长发育和抵御逆境胁迫等过程中发挥重要作用.CYP90B1基因编码酶是其合成途径中关键限速酶.本研究针对迄今有关玉米CYP90B1基因应答逆境胁迫特征尚未见报道现状,采用RT-PCR结合RACE技术,从玉米中克隆了油菜素甾醇生物合成关键基因ZmCYP90B1 (GenBank登录号KY242373).ZmCYP90B1全长2058 bp,开放阅读框为1518 bp,编码506个氨基酸.ZmCYP90B1蛋白预测分子量为57.66 kD,等电点为9.54,含有1个跨膜结构域及1个p450保守结构域.序列比对表明,ZmCYP90B1与其他物种CYP90B1蛋白高度相似,但在单双子叶植物中进化上具有明显差异.实时荧光定量PCR结果表明,非生物胁迫(干旱、高盐、低温和脱落酸)、虫害(甜菜夜蛾取食)和茉莉酸甲酯均诱导ZmCYP90B1表达,表明该基因响应多种非生物胁迫,参与植物对虫害和茉莉酸甲酯的响应.进一步构建过量表达Zm CYP90B1基因的烟草株系并检测表明该烟草株系抗旱性增强,叶片失水率下降,SPAD值增大.此外,干旱胁迫下转基因烟草超氧化物歧化酶(SOD)、过氧化氢酶(CAT)和过氧化物酶(POD)的活性以及游离脯氨酸(Pro)的积累量均显著高于野生型,丙二醛(MDA)和脱落酸(ABA)含量明显低于野生型.通过检测下游胁迫响应基因表达,表明ZmCYP90B1提高植物抗旱性可能不依赖ABA途径,而与其对抗氧化相关途径相关基因的转录调控有关.
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