The Cloning And Genetic Transformation Of Grape ACC Oxidase Genes

As a major economic crop, grapes account for almost twenty five percent of the fruityield in the world. Grapes have a high value of nutrition and can be made into raisins andwine.But grapes are easily affected by those factors, such as, plant diseases and insect pests,mechanical injury, physiological disorders and fruit maturity, which will inevitably causeeconomic loss. As well known, ethylene is a gaseous phytohormone. It plays a significant rolein the regulation of plant growth and development as well as the responses to diverse stresses.1-aminocyclopropane-1-carboxylic acid oxidase is the last rate-limiting enzyme in ethylenebiosynthesis, converting ACC to ethylene. It influences the rate of ethylene biosynthesis. So,it is necessary to intensively study ACC oxidase in characteristics and functions.In this study, two ACO genes were cloned from grape by using reverse transcription PCR,which were named VvACO1and VvACO2. VvACO1comprised an open reading frame of1020bp, encoding a protein of339amino acids. The length of VvACO2open reading frame is957bp, encoding318amino acids. The amino acid sequences of both ACO genes have theconserved domain of ACC oxidase. Multiple sequences alignment results showed that theamino acid sequences of VvACO1and VvACO2shared a high similarity with those of otherhigher plants. In order to further analyze the function of ACO gene, VvACO2gene was theninserted into vector pCAMBIA Super-1300with forward orientation, constructing a senseexpression vector VvACO2/p1300. Micro-Tom and Arabidopsis thaliana Columbia weretransformed with the sense expression vector VvACO2/p1300by using Agrobacteriummediated leaf disc transformation and floral dip methods. The transgenic plants were screenedby PCR analysis and antibiotic screening. Thirty five transgenic tomato plants and T1seeds oftransgenic Arabidopsis thaliana plants have been obtained. Transgenic tomato plants floweredearlier and fruit ripened faster than wild-type plants.