StSRK2E-like基因的序列分析及表达载体构建

Sequence Analysis and Expression Vector Construction of St SRK2E-like Gene

SnRK2作为脱落酸(abscisic acid,ABA)信号转导途径的关键因子,对应答非生物胁迫防御机制的形成具有重要作用。为验证马铃薯SnRK2s的功能,本试验以‘青薯9号’为材料,利用RT-PCR对SnRK2家族中的StSRK2E-like基因进行克隆。序列分析显示,该基因CDS序列为1089 bp,编码蛋白全长为362 aa,相对分子质量为41.06 kD,脂溶指数为89.67,亲水指数为-0.321,为亲水性蛋白;进化分析显示该基因与番茄中同源基因的亲缘关系最近,相似性高达98.44%;该基因上游2000 bp启动子序列中除了含有TATA-box和CAAT-box核心元件,还含有干旱相关元件(MBS)及激素响应元件(TCA-element,ABRE,TGA-element),推测该基因参与干旱和激素胁迫应答。用RT-qPCR定量检测不同干旱胁迫时间下马铃薯根、茎和叶中StSRK2E-like基因表达水平。结果显示:StSRK2E-like基因在胁迫处理下的表达量显著低于对照,该结果表示该基因对干旱胁迫敏感;且随胁迫时间增加,对照组茎和叶中StSRK2E-like基因的表达量下调显著,胁迫组叶中下调显著(P<0.05),推测该基因在马铃薯抵御干旱胁迫过程中扮演着重要角色。StSRK2E-like基因的具体功能需要进一步研究。本试验成功构建pJAM1502-StSRK2E-like表达载体,为后续进行StSRK2E-like基因的功能验证提供试验基础。

Sn RK2,as a key factor of abscisic acid(ABA)signal transduction pathway,plays crucial role in the formation of defense mechanism in response to abiotic stress.In order to verify the function of potato SnRK2 s in potato,this experiment used’Qingshu9’as the material and used RT-PCR to cloned the StSRK2 E-like gene in the SnRK2 family.Sequence analysis showed that the CDS sequence of StSRK2 E-like gene was 1089 bp,the entire length of the encoded protein was 362 aa,the relative molecular mass was 41.06 kD,the fat solubility index was89.67,and the hydrophilic index was-0.321,which was a hydrophilic protein.Evolutionary analysis showed that StSRK2 E-like gene had the closest genetic relationship with the homologous gene in tomato,the similarity up to98.44%.In addition to the core elements of TATA-box and CAAT-box,the 2000 bp promoter sequence upstream of StSRK2 E-like gene also contains drought-related elements(MBS)and hormone response elements(TCA-element,ABRE,TGA-element),speculating that this gene was involved in response to drought and hormonal stress.RT-qPCR was used to quantitatively detect the expression level of StSRK2 E-like gene in potato roots,stems and leaves under different drought stress times.The results showed that the expression of StSRK2 E-like gene under stress treatment was significantly lower than that of the control,which indicated that StSRK2 E-like gene was sensitive to drought stress.With the increase of stress time,the expression of StSRK2 E-like gene was significantly down-regulated in stem and leaves of the control group,and significantly down-regulated in leaves of the stress group(P<0.05),which speculated that StSRK2 E-like gene played a role in the process of potato resisting drought stress.The specific function of StSRK2 E-like gene needs further study.The pJAM1502-StSRK2 E-like expression vector was successfully constructed in this study,which provided experimental basis for the subsequent functional verification of StSRK2 E-like gene.