红花非生物胁迫相关CtDHN1基因的克隆及功能研究  

Cloning and functional analysis of CtDHN1 gene related to abiotic stress in Carthamus tinctorius

作　　者：董程 杨景松 刘紫嫣 李海龙 初卓 文秋菊 解子洋 刘树明[2] 官丽莉[1] DONG Cheng;YANG Jing-song;LIU Zi-yan;LI Hai-long;CHU Zhuo;WEN Qiu-ju;XIE Zi-yang;LIU Shu-ming;GUAN Li-li(College of Life Sciences,Jilin Agricultural University,Changchun 130118,China;College of Animal Science and Technology,Jilin Agricultural University,Changchun 130118,China)

机构地区：[1]吉林农业大学生命科学学院,吉林长春130118 [2]吉林农业大学动物科学技术学院,吉林长春130118

出　　处：《中国油料作物学报》2020年第1期85-90,共6页Chinese Journal of Oil Crop Sciences

基　　金：吉林省教育厅“十三五”科学技术研究项目(JJKH20190916KJ);国家自然科学基金(31201237);吉林省大学生创新创业项目(2018红花干旱响应基因DHN的克隆及功能鉴定)。

摘　　要：脱水素(Dehydrins)属于LEA (Late embrygenesis abundant protein)蛋白家族的第二亚家族成员,是一类高度无序的响应逆境胁迫的蛋白。本研究通过对前期红花干旱胁迫转录组数据的挖掘,利用RT-PCR(reverse transcription-PCR)方法从油料作物红花中首次克隆得到一条全长为957bp响应干旱胁迫脱水素基因序列,命名为CtDHN1,其编码318个氨基酸,预测分子量为31.1kDa,等电点为6.36。生物信息学分析表明CtDHN1蛋白属于YnSKn型脱水素,原核表达分析表明CtDHN1蛋白为可溶性蛋白,在IPTG浓度为0.4mmol/L,OD600值为0.8,诱导时间为4h,诱导温度为37℃时表达量最高。大肠杆菌耐逆性分析表明CtDHN1基因可耐受1.5mol/L山梨醇和1.3mol/L氯化钠的干旱和高盐环境。体外酶活测定表明CtDHN1蛋白在500mmol/L山梨醇和300mmol/L氯化钠环境下可保护LDH酶活性。本研究初步鉴定红花脱水素蛋白的功能,为研究CtDHN1基因在红花抵抗非生物胁迫中的分子机制提供理论基础。Dehydrates was a member of the group 2 late embryogenesis abundant protein family,which were high disorder proteins and responded to adversity stress.Based on the transcriptome data of safflower under drought stress,CtDHN1,a dehydrin gene respondimg to drought stress was identified and cloned by using RT-PCR tech niques,which encoded 318 amino acids with the molecular weight of 31.1 kDa and the pI value of 6.36.Bioinformat ics analysis indicated that CtDHN1 protein belonged to YnSKn-type dehydrin.Prokaryotic expression analysis re vealed that CtDHN1 protein was a soluble protein.The protein had the highest expression level when the concentra tion of IPTG was 0.4 mmol/L,the OD600 value was 0.8,the induction time was 4 h,and the induction temperature was 37℃.The E.coli stress tolerance analysis demonstrated that CtDHN1 gene tolerated a drought and a high salt environment of 1.5 mol/L sorbitol and 1.3 mol/L sodium chloride.In vitro enzyme activity assays indicated that Ct DHN1 protein protected LDH enzyme activity in 500 mmol/L and sorbitol 300 mmol/L sodium chloride.This re search provided a theoretical basis for the molecular mechanism of CtDHN1 gene in safflower resistance to abiotic stress.

关 键 词：脱水素 非生物胁迫 原核表达 LDH活性 

分 类 号：Q943.2[生物学—植物学] S567.219[农业科学—中草药栽培]