茶树CsbHLH基因启动子的克隆与活性

Isolation and functional analysis of the CsbHLH promoter from Camellia sinensis

为探明茶树bHLH(CsbHLH)基因启动子在基因表达过程的调控作用,以"1005"茶树新品系芽为材料,采用染色体步移技术,分离克隆CsbHLH的上游5′端启动子序列,相关生物信息学分析分别运用在线软件Neural Network PromoterPrediction与Plantcare,克隆的启动子同向置换pCAMBIA1301载体上的CaMv35S启动子来构建重组表达质粒,然后分别进行注射烟草叶片瞬时表达实验和转化拟南芥功能验证.分离克隆得到CsbHLH基因启动子片段长度为810 bp,命名为probHLH,该启动子除含有与高转录水平相关的顺式作用元件5′UTR Py-rich stretch,CAAT-Box与TATA-Box核心启动子元件外,也包含多个激素应答元件、逆境响应元件和功能特异或者未知功能的作用元件;probHLH具有启动子驱动活性,转基因拟南芥实验结果显示probHLH启动子驱动的下游GUS基因在整个拟南芥植株都可以表达,而且能响应脱落酸ABA、茉莉酸甲酯MeJA、赤霉素GA3、NaCl、PEG-6000和光等非生物胁迫.本研究表明CsbHLH基因启动子为诱导型启动子特性,可能具有多重因子的诱导活性,CsbHLH基因可能参与ABA、MeJA和GA3多个激素信号转导和非生物胁迫应答过程.

Our previous study reported that CsbHLH gene is a key basic-helix-loop-helix(bHLH)transcriptional factor associated with epigallocatechin-3-gallate(EGCG)biosynthesis.However,the upstream regulatory sequence of CsbHLH has not been reported yet.To understand the function of CsbHLH promoter in tea plant(Camellia sinensis),this research aimed to isolate the 5’-flanking sequence of CsbHLH,and thereafter study its function.The 5’-flanking upstream region of CsbHHLH was isolated from the shoots of the tea cultivar‘1005’through genome-walking method.Then,sequence analysis was carried out using online bioinformatics resources,such as Neural Network Promoter Prediction and PlantCARE to reveal its basic core promoter,transcription start site,and cis-regulatory elements.The CsbHLH promoter sequence was cloned into the expression vector pCAMBIA1301 to replace CaMV35 S promoter and produce a new recombinant expression vector and then tested in infected tobacco leaves and transgenic Arabidopsis thaliana.A sequence of 810 bp,located in the 5’-flanking upstream of CsbHLH gene,was finally cloned and designated as ProbHLH.Sequence analysis showed that some of the 5’-UTR Py-rich stretch motifs correlated with transcription,and some of the typical cis-acting elements,such as the CAAT-Box and TATA-Box elements,number of cis-regulatory elements responding to exogenous hormones,stresses,and other cis-elements with specific or unknown functions were contained within probHLH.The expression of GUS gene can be driven by the promoter,probHLH.The experimental results from transgenic A.thaliana showed that probHLH promoter can drive the expression of GUS gene in A.thaliana plants and can execute the responses to abiotic stresses(light,NaCl or PEG-6000)and hormonal treatments(abscisic acid(ABA),methyl jasmonate(MeJA),or gibberellic acid(GA3)).ProbHLH is an inducible promoter with multiple inducible activities.CsbHLH may be involved in a variety of hormonal signal transductions including ABA,MeJA,and GA3 and abiotic stress responses in tea plants.