苎麻ACC氧化酶基因(BnA CO2)的克隆、时空表达及原核表达分析  被引量:1

Cloning,Spatio-temporal Expression and Prokaryotic Expression Analysis of ACC Oxidase Gene(Bn ACO2)from Ramie

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作  者:罗金凤 彭文仙 肖旭 薛丽君[1] 邢虎成[1,2] Luo Jinfeng;Peng Wenxian;Xiao Xu;Xue Lijun;Xing Hucheng(Institute of Ramie,Hunan Agricultural University,Changsha,410128;Hunan Provincial Key Laboratory of Corp Germplasm Innovation and Utilization,Hunan Agricultural University,Changsha,410128)

机构地区:[1]湖南农业大学苎麻研究所,长沙410128 [2]湖南农业大学湖南省种质资源创新与资源利用重点实验室,长沙410128

出  处:《分子植物育种》2022年第8期2545-2553,共9页Molecular Plant Breeding

基  金:湖南省教育厅科学研究重点项目(18A092);作物种质创新与资源利用重点实验室科学基金开放项目(16KFXM04);国家自然科学基金项目(31101098)共同资助。

摘  要:ACC氧化酶(ACC oxidase,ACO)是乙烯合成途径中的一个关键酶。本研究通过RT-PCR结合RACE技术克隆得到一个ACO基因,命名为Bn ACO2,该基因cDNA序列全长为1377 bp,开放阅读框为957 bp,编码318个氨基酸多肽,预测其分子量和等电点(pI)分别为36.145 kD和5.60。生物信息学分析表明,BnACO2编码蛋白没有信号肽和跨膜结构域,亚细胞定位于细胞质。与川桑等物种ACC氧化酶基因核苷酸序列同源性在83%以上,氨基酸序列同源性在87%以上。通过系统发育树发现该基因和山黄麻、大麻ACO基因亲缘关系最近。实时荧光定量PCR分析表明,苎麻Bn ACO2基因在苎麻各部位均有表达,特别是在雌花花芽中表达显著。最后成功构建原核表达载体p QE-Bn ACO2,诱导表达出的目的蛋白分子量大小约为36.15 kD,与预测的蛋白大小一致。这为进一步研究BnACO2基因的功能提供了科学依据。ACC oxidase(ACO)is a key enzyme in ethylene synthesis pathway.In this study,An ACO gene was cloned by RT-PCR combined with RACE,named Bn ACO2.Total length of the gene cDNA sequence was 1377 bp,the open reading frame of 957 bp,coded 318 amino acid polypeptide,predict its molecular weight and isoelectric point(pI)36.145 kD and 5.60,respectively.Bioinformatics analysis showed that BnACO2 encoded protein had no signal peptide and transmembrane domain structure,and was subcellular localized in the cytoplasm.The similarity of nucleotide and amino acid sequences of ACC oxidase gene in ramie and other plants likes Morus notabilis were more than 83%and 87%respectively.Phylogenetic analysis showed that the ACO gene was closely related to jute and hemp.Real-time fluorescence quantitative PCR analysis showed that BnACO2 gene was expressed in all parts of ramie,especially in female flower buds.The prokaryotic expression vector pQE-BnACO2 was successfully constructed by double enzyme digestion.At 37℃,1.0 mmol/L IPTG induced PQe-BnACO2 to express the target protein,and the molecular weight of the induced protein was about 36.15 kD,which was consistent with the predicted protein size.This provides a scientific basis for further research on the function of BnACO2 gene.

关 键 词:苎麻 ACC氧化酶 克隆 表达分析 

分 类 号:S563.1[农业科学—作物学]

 

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