Cloning And Expression Analysis Of Grape1-aminocyclopropane-1-carboxylate Synthase Genes

ACS (1-aminocyclopropane-1-carboxylate synthase) is one of the key enzymes inethylene biosynthesis and pathway. The ACS genes of Vitis vinifera were studied in thisresearch. The results were as follows:By comparing the methods of CTAB, improved SDS and Trizol which are commonlyused for extracting RNAs, we found that the RNAs of grape could be extracted by both theCTAB and the improved SDS methods. High-quality-RNAs without protein contaminationand degradation were obtained by the CTAB method, while a high output of grape RNAscould be extracted with the problem of degradation and contaminations of proteins and DNA.The grape RNA couldn’t been successfully extracted by the Trizol method which might beinfluenced by the guanidine salt in the extraction buffer.Three VvACS genes (VvACS3, VvACS4and VvACS5) were cloned successfully by PCRamplification after the reverse transcription of the total grape RNA extracted by the CTABmethod. Deduced Amino acid sequences of the3genes were analyzed. The results showedthat all of the three proteins had the AAT-like conservative domain, and have somephosphorylation loci, which implied that the VvACS protein could be phosphorylated afterthe translation. This property might be related to the regulation of ACS activity. Six VvACSgenes have been cloned by our lab. A phylogenetic tree was constructed after alignment of theamino acid sequences deduced from the ACS genes of tomatoes, arabidopsis, maize andgrapes. Results showed that these ACS have different levels of similarities and homologiesand clustered into different groups. The phylogenetic tree could provide references for furtherqualitative research of VvACS genes.After treatments of ethylene, abscisic acid and wounding, relative quantitative expressionlevels of VvACS5gene were measured by real-time PCR. It was found that both of theexogenous ethylene and abscisic acid could first promote and then inhibit the expression ofVvACS5. Treatment of wounding could increase the expression of VvACS5. All of the resultsindicated that VvACS5could respond to the stress stimuli. This finding preliminarilydemonstrated the involvement of ACS in the stress regulation of plant.For further analysis of the VvACS5function, a binary expression vector was constructedand transformed into Arabidopsis thaliana by agrobacterium-mediated floral dip method.Seven Arabidopsis plants of VvACS5transgenic pure line were obtained by PCR verifying and antibiotic screening. The transgenic plants of A. thaliana showed the characteristics oflarge leaf blades, strong seedlings, which might be the results of the VvACS5gene overexpression.The studies on the cloning, sequence analysis and functional analysis of the ACS gene inV. Vinifera provided an important theoretical reference for elucidating the mechanism of theinvolvement of VvACS genes in the regulation of resistance and function.