摘要：
[Objective] As an important component in plant cell wall,expansins are not only involved in most of the physiological process in plants,such as seed germination,vegetative organ development,fruit ripening and organ abscission but also involved in the process of stress resistance in plant.In this study,the promoter sequence of a expansin gene CpEXP1 was isolated from Chimonanthus praecox ‘ Qingkousuxin'The promoter activity study laid a theoretical foundation for exploring the molecular regulation mechanism of CpEXP1 gene during development in C.praecox.[Method] The promoter of the CpEXP1 gene was cloned by hiTAIL-PCR method.The cis-elements were analyzed by online bioinformatics tools.The promoter-reporter vector was constructed and introduced into tobacco by Agrobacterium-mediated method for transient-expression.Then the plant expression vector was transferred into Arabidopsis thaliana for further stable expression study.[Result] The regulative sequence(2485 bp)of the expansin gene CpEXP1 promoter(GenBank Accession:MG452931) was cloned from genomic DNA of C.praecox.Bioinformatics analysis revealed that the promoter sequence contained basic cis-elements,such as TATA-box and CAAT-box and many elements involved in the plant tissue-specific expression and abiotic stress.The result of transient-expression showed that CpEXP1 gene promoter could drive the GUS report gene expressed in leaf dish of tobacco.Analysis of T3 transgenic plants with histochemical staining and GUS gene expression with the quantitative real-time PCR showed that strong GUS activity appeared in seed germination stage and almost no expression in cotyledon and tender leaves.There is a weak expression of GUS gene in root and flower in transgenic Arabidopsis.With the maturation and senescence of Arabidopsis leaf and flower,the strong GUS activity can be detected at the basal of petiole and carpopodium.GUS gene expression level of transgenic plants have increased with different treatments,including high temperature(42°C),low temperature(4°C) and salicylic acid(SA).[Conclusion]Based on the time-space specificity of CpEXP1 gene promoter activity in various organs during different development stages in transgenic Arabidopsis,we speculated that the CpEXP1 gene promoter would be closely related with seed germination,fruit expanding growth and leaf abscission.On the other side,GUS gene expression was induced by different abiotic stress treatments such as high temperature,low temperature and salicylic acid,which indicated that the CpEXP1 gene promoter may also play a role in abiotic stress resistance.%[目的]细胞扩展蛋白(EXP)作为植物细胞壁的重要组成部分,参与种子萌发、营养器官发育、果实成熟、器官脱落、植物抗逆等植物生长发育过程中的多个环节.通过研究蜡梅细胞扩展蛋白基因CpEXP1启动子活性功能,为研究CpEXP1基因在蜡梅生长发育过程中的功能提供理论依据.[方法]以蜡梅‘磬口素心’为材料,通过hi-TAIL PCR法从蜡梅基因组DNA中克隆CpEXP1基因上游调控序列,利用生物信息学软件,分析CpEXP1基因启动子序列中潜在的顺式调控元件.构建该基因与GUS报告基因融合的植物表达载体,利用农杆菌介导的叶盘法在烟草叶片中进行启动子活性的瞬时表达研究,并进一步利用花序侵染法在拟南芥中进行稳定表达,利用GUS组织化学染色和GUS报告基因的实时荧光定量PCR检测,分析CpEXP1基因启动子的活性.[结果]获得了长度为2485 bp的蜡梅细胞扩展蛋白基因CpEXP1上游调控序列(GenBank Accession:MG452931),序列分析表明该启动子中除含有核心元件TATA-box和CAAT-box外,还包含多个与植物非生物胁迫及组织特异表达相关的顺式作用元件.在烟草中的瞬时表达分析表明该启动子具备驱动报告基因GUS表达的功能.进一步对转基因拟南芥植株的GUS组织化学染色和GUS基因表达分析结果显示,CpEXP1基因启动子在转基因拟南芥种子萌发初期活性较强,在子叶以及幼苗真叶中未检测到GUS活性;在花和根中活性较弱;在成熟叶片中可以检测到GUS活性,特别是在衰老叶片叶柄脱落区表达强烈.此外,该启动子在幼果中具有较强活性,随后启动活性逐渐下降,成熟果荚中仅在果柄脱落处能够检测到GUS活性.同时,转基因拟南芥植株中的CpEXP1基因启动子活性受高温(42°C)、低温(4°C)和水杨酸(SA)诱导,特别是对低温胁迫响应强烈,在4°C低温处理后,转基因拟南芥叶片中GUS基因的表达量是处理前的的8.7倍.[结论]CpEXP1基因启动子在转基因拟南芥不同发育阶段、不同器官中的活性具有明显差异,推测该启动子可能在种子发芽、叶片脱落以及果实脱落中发挥作用,同时CpEXP1基因启动子活性可被不同非生物胁迫诱导,可能参与植物抵御非生物胁迫的调控途径.
