Cloning And Activity Analysis Of ACS Promoter In Vitis Vinifera

Stress can seriously affect the plant growth and development, which will lead to lesscrop yield and worse quality. Ethylene as an important endogenous signal molecule, plays avery important role in the process of stress response in plant. ACC synthase is a keyrate-limiting enzyme that can catalyze S-adenosylmethionine into1-aminocyclopropane-1-carboxylic acid in the ethylene biosynthesis pathway. The production and activity of ACCsynthase in plant tissue often determines the ethylene production, therefore, the research onmolecular mechanism of ACC synthase involved in gene expression regulation is necessary.In this study, the promoter of the VvACS5gene and VvACS7gene were cloned from the grape,then the cis-acting elements of the two gene promoters were analyzed, and the influence ofcis-acting elements on the promoter activity were also determined, the specific results are asfollows:1. The promoter of VvACS5and VvACS7gene were cloned, and the length of sequencewas1019bp and1103bp individually. The cis-element of two gene promoter was analyed byusing online database PLACE and PlantCARE, various cis-acting elements involved in plantresponse stress were found, such as ABRE, ERE, TGACG-motif, CGTCA-motif, P-box,TATC-box, MBS, TC-rich et al.2.5’ deletion of VvACS5and VvACS7gene promoters were cloned with rechnstrictionenzyme site specific primers. Seven unequal length of5’ deletion fragments were respectivelyfused with pBI-221-LUC to construct seven expression vectors.3. By PEG-Ca²⁺mediated method, seven recombinant expression vectors weretransfected into wild-type Arabidopsis protoplast, then the expression level of LUC reportgene were detected. The results indicated that VvACS5and VvACS7gene promoters couldboth drive LUC reporter gene expression, and two active regions （-1015to-717,-717to-269）of the VvACS5gene promoter was found; an active region （-364to-139） and two repressiveregions （-848to-648,-648to-364） of theVvACS7gene promoter were also found.In summary, the promoter of two VvACS gene were cloned, seven expression vectorswith unequal length of5’ deletion fragments were constructed.The expression level of reportgene was tested by using transient expression system in order to determin the promotersactivity. The above experimental results laid the theoretical foundation of the expressionregulation of VvACS genes, and provided theoretical guidance for the research on expressionregulation of VvACS gene further.