大豆GmVE2基因克隆及其在大豆籽粒维生素E动态积累中的表达分析

GmVE2 Gene Clone and Expression in the Dynamic Accumulation of Vitamin E in Soybean

大豆GmVE2基因是一种NAD(P)H脱氢酶,主要存在于高等植物叶绿体类囊体膜上,可参与氧化应激反应以及叶绿体PSⅠ电子循环传递。为了探究其功能,通过RT-PCR方法从东农50中克隆获得GmVE2基因的CDS全长序列。利用ExPASy-ProtParam等在线软件对GmVE2蛋白序列进行理化性质分析,预测结果表明,GmVE2基因编码区共编码167个氨基酸,分子式为C904H1326N208O246S10,分子质量为19.364 3 ku,总原子数为2 694,理论等电点(pI)为4.78,脂肪指数为87.01;蛋白正负电荷残基数分别为11和23;GmVE2蛋白的不稳定系数和GRAVY值分别为25.02和0.043,说明该蛋白是稳定的疏水性蛋白;蛋白存在跨膜结构、信号肽和磷酸化位点。将线性植物表达载体与目的片段进行连接,成功构建pCambia3300-GmVE2植物表达载体。通过qRT-PCR技术和高效液相色谱(HPLC)分别对动态籽粒及组织基因表达量和维生素E含量进行测定,结果表明,该基因表达量与生育期籽粒维生素E含量呈负相关(-0.951*),且维生素E含量在绿色组织中显著高于其他组织。本研究对大豆基因GmVE2与维生素E含量的调控关系进行初步研究,为高大豆维生素E含量的大豆新品种选育提供理论和实践基础,为后期研究GmVE2基因功能提供科学依据。

Soybean GmVE2 gene is a NAD(P)H dehydrogenase,which is mainly present in thylakoid membranes of chloroplasts of higher plants and can participate in oxidative stress reactions and cyclic PSⅠelectron circulation.In order to explore its function,the full-length CDS sequence of GmVE2 gene was cloned from Dongnong 50 by RT-PCR.Use ExPASy-ProtParam and other online software to analyze the physical and chemical properties of the GmVE2 protein sequence.The prediction results showed that the coding region of the GmVE2 gene encoded a total of 167 amino acids,the molecular formula was C 904 H 1326 N 208 O 246 S 10,the molecular weight was 19.3643 ku,the total number of atoms was 2694,the theoretical isoelectric point(pI)was 4.78,the fat index was 87.01;the residue bases of positive and negative charges were 11 and 23,respectively.The instability coefficient and GRAVY value of GmVE2 protein were 25.02 and 0.043,respectively,indicating that the protein was a stable hydrophobic protein;the protein had a transmembrane structure,a signal peptide,and a phosphorylation site.The linear plant expression vector was ligated with the target fragment and the pCambia3300-GmVE2 plant expression vector was successfully constructed.The qRT-PCR technology and high performance liquid chromatography(HPLC)were used to determine the gene expression and vitamin E content in dynamic grain and tissue,respectively.The results showed that the gene expression was negatively correlated with the vitamin E content in the growing period grains(-0.951*),and the vitamin E content was significant higher in green tissues than in other tissues.This study conducted a preliminary study on the regulatory relationship between soybean gene GmVE2 and vitamin E content,providing a theoretical and practical basis for the selection of new soybean varieties with high soybean vitamin E content,and providing a scientific basis for later research on the function of GmVE2 gene.