虎杖PcMYB1启动子的克隆及其活性分析

Cloning and Activity Analysis of PcMYB1 Promoter from Polygonum cuspidatum

获得药用植物虎杖(Polygonum cuspidatum)PcMYB1启动子,并分析其活性,为进一步研究虎杖转录因子PcMYB1的功能奠定基础。通过hiTAIL-PCR方法,从虎杖叶片基因组DNA中克隆PcMYB1启动子并进行生物信息学分析。分别构建由全长启动子或5'端缺失启动子驱动GUS的重组表达载体P1∷GUS、P2∷GUS、P3∷GUS和P4∷GUS。将重组表达载体转入发根农杆菌中进行虎杖毛状根转化试验,以转化的阳性毛状根为材料,通过GUS组织化学染色分析启动子的活性。成功获得2 884 bp的PcMYB1启动子序列(GenBank登录号MT811057)。该启动子具有真核生物启动子必需的核心元件TATA-box和CAAT-box,还含有茉莉酸甲酯和低温响应元件以及光响应、厌氧应答等相关的顺式调控元件。重组表达载体经PCR鉴定,证实已构建成功。转化的虎杖毛状根染色之后显蓝色,但同对照相比颜色较浅,说明全长启动子或5'端缺失启动子都具有驱动GUS的活性,但启动活性都比CaMV35S启动子的启动活性弱。PcMYB1的启动子被成功克隆,其具有驱动下游GUS表达的活性。

This work aims to obtain PcMYB1 promoter from Polygonum cuspidatum and analyze its activity,and thus to lay a basis for further investigating the functions of promoter PcMYB1 promoter.hiTAIL-PCR was used to clone the PcMYB1 promoter from the leave genome DNA of P.cuspidatum,and bioinformatics analysis of it was conducted.The full-length promoter P1(+1 to-2884)and a series of progressive 5’truncated promoters P2(+1 to-2259),P3(+1 to-1807)and P4(+1 to-1362)were fused with GUS gene,and the recombinant expression vector P1∷GUS,P2∷GUS,P3∷GUS,P4∷GUS were constructed,respectively.Each recombinant expression vector was transferred into Agrobacterium rhizogenes to transform the hairy root of P.cuspidatum,and the transformed positive hairy root was used as the material.GUS histochemical staining was performed to analyze the activity of the promoter.The 2884 bp promoter sequence(GenBank accession number:MT811057)of the promoter was obtained.The promoter contained several core fragments(TATA-box and CAAT-box)essential for eukaryotic promoters,and also cis-elements that were responsive to MeJA and low-temperature and cis-acting regulatory elements that were associated with light and anaerobic responses.The successful constructions of the recombinant vectors were confirmed via PCR.The transgenic hairy roots of P.cuspidatum can be stained to have blue,but the color was light compared to the control,that demonstrated that both the full-length promoter and the other 5’truncated promoters drove the GUS gene expression,but the driving ability was lower than that of CaMV35S promoter.In sum,PcMYB1 promoter is successfully cloned and it shows the activity of driving the expression of downstream GUS.