甘蓝型油菜cyMDH基因的克隆及干旱胁迫下的表达与光合特性的关系分析

为了解甘蓝型油菜苹果酸脱氢酶(cyMDH)的生理特性、结构,同时探究cyMDH基因对干旱胁迫的响应规律。本研究以抗旱性强的甘蓝型油菜‘94005’为研究材料,在初花期采用盆栽控水试验,设置了4个干旱处理:轻度干旱(LD)、中度干旱(MD)、重度干旱(SD)和极度干旱(ED),以正常浇水作为对照(CK),测定叶片光合指标、cyMDH酶活性,通过实时荧光定量PCR (RT-qPCR)分析cyMDH基因的表达量,采用3'RACE方法克隆cyMDH基因全长序列,并对测序结果进行生物信息学分析,复水后生长至成熟期测定产量。结果表明,cyMDH基因序列全长1 440 bp,996 bp的开放阅读框(ORF)位于207~1 205区域,编码332个氨基酸(GenBank登录号MH316130);Softberry亚细胞定位分析表明该蛋白是亲水细胞质蛋白,无跨膜区域;cyMDH蛋白分子量为35.69 KD,等电点为6.11;蛋白的二级结构预测结果表明,cyMDH蛋白由α-螺旋(44.88%)、无规则卷曲(34.04%)、延伸链(17.17%)以及β-转角(3.92%)组成;甘蓝型油菜‘94005’的cyMDH与拟南芥的cyMDH同源性最高达到93%。随着干旱胁迫加剧MDH基因表达量无显著变化。干旱胁迫下cyMDH酶活性与甘蓝型油菜‘94005’叶片净光合速率、气孔导度、蒸腾速率、胞间CO2浓度、产量成负相关,与水分利用效率成正相关。本研究获得了甘蓝型油菜cyMDH基因,并对其理化性质、结构等进行了预测与分析,同时对干旱胁迫下cyMDH酶活性与油菜叶片光合特性进行相关性分析,为进一步研究油菜抗旱分子机制提供科学依据,同时为油菜抗旱品种的选育提供理论支持。

Cloning of cyMDH Gene and Analysis of the Relationship with Photosynthetic Characteristics under Drought Stress in B.napus

In order to explore the physiological characteristics,structure and the response to drought stress of malic acid dehydrogenase(MDH)gene in Brassica napus L.,’94005’as experimental materials which the high drought-tolerant variety were treated with drought stress(mild drought,moderate drought,severe drought and extreme drought)during the flowering stage,with normal irrigation as the control.Measuring photosynthetic parameters and MDH enzyme activity of the leaves,then analyzing the expression of MDH through real-time fluorescence quantitative PCR(RT-qPCR),using RACE method to get the full-length clone of MDH gene,and sequencing results were analyzed with bioinformatics.Rehydration to grown to maturity to get the yield.The results showed that the full length of MDH gene sequence was 1440 bp,and the open reading frame(ORF)of 996 bp was located in 207~1205 bp region,encoding a protein of 332 amino acids with a predicted molecular weight of 35.69 kD and isoelectric point of 6.11.And secondary structure prediction showed MDH protein consists ofα-helix,random coil,extended strand andβ-turn,proportion of four kinds of secondary structural elements was 44.88%,34.04%,17.17%and 3.92%respect ively,MDH of rapeseed had a consistency over 93%with Arabidopsis MDH.There was no significant change of MDH gene expression value drought stress.Correlation analysis between photosynthetic index,yield and cyMDH activity showed that cyMDH activity was negatively correlated with Pn,Gs,Tr,Ci and yield,and positively correlated with WUE.The cyMDH gene of Brassica napus L.was obtained in this study,its physicochemical properties,structure was predicted and the relationship between cyMDH activity and photosynthetic characteristics was analyzed.It will provide theoretical basis for further study on molecular mechanism of drought resistance in rapeseed,and also provide theoretical support for the breeding of drought-resistant varieties in rapeseed.