关键词: 蜡梅, CpEXP1, 启动子, GUS基因, 非生物胁迫

Abstract: [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(2 485 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℃), low temperature(4℃)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.

Key words: Chimonanthus praecox, CpEXP1 gene, promoter, GUS gene, abiotic stress