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Expressing Characteristics Waco Genes In Grapevine Vegetative Tissues And Over-Expression Of VVACO Genes In Tomato

Posted on:2014-01-24Degree:MasterType:Thesis
Country:ChinaCandidate:T CaiFull Text:PDF
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Ethylene is an important plant endogenous hormone which controls many processes of plant life. ACO gene is the key gene in the biosynthesis of ethylene which regulates the synthesis of ACC oxidase.In this study, expression characteristics in various tissues and organs and in response to ethylene induction were investgated in grapevine (Vitis vinifera).Three grapevine genes (VvACO) were over-expressed in tomato (Solanum lycopersicum) by Agrobacterium mediated method. Transgenic tomatoes that over-expressedof the each of the VvACO genes were analyzed for their growth physiological indexs, ethylene release rates, and ACO enzyme activies., based on these results, functions of VvACO genes were prelimaryly predicted. The main results are as follows:1. Expresion characteritics of three VvACO genes in different tissues and organs and in response to ethylene treatments were analyzed using real-time RT-PCR methods. The results indicated that expression levels of VvACO1were the highest in young stem, and the lowest in old petioles. The highest expression of VvACO2gene was observed in peduncles and berries, and lowest in young stem. The highest expression of VvACO3gene was observed in young stem, and the lowest in shoot tips. The expression levels of VvACO1and VvACO2genes generally increased after72h of ethelyene treatment, and the increase of expression level of VvACO2gene was relatively small, whereas the that of expression levels of VvACO3gene was large, with a25-fold change at8h after ethylene treatments.2. The optimal tomato genetic transformation system was established using cotyledons as explants by Agrobacterium mediated method. Three VvACO genes were transferred into tomato, and the results showed that the maximum transformation efficiency was obtained when Agrobacterium cell density was OD6000.5with15minutes of infection. After explants were precultured, the optimum regeneration rate was observed at medium supplementaled with2mg/L Zeatin (ZT) and200mg/L Timentin. Strong shoots were then transferred to subculture medium with1mg/L ZT, and the root medium was supplemented with0.5mg/L IBA and100mg/L Timentin.3. RT-PCR analysis indicated that three, four, and six transgenic tomato lines with over-expressing VvACO1, VvACO2, VvACO3gene were obtained, respectively. The ACO enzyme activities and ethylene release rates were higher in the transgenic plant than those in non-tansgenic tomato leaves. The highest ACO enzyme activities and ethylene release rates in the tomato transgenic lines was observed with over-expressing VvACO1gene. The transgenic tomato plants which overexpressed both VvACO1and3were normal in morphological growth. The lowest ACO enzyme activities and ethylene release rates were observed in the tomato transgenic lines with over-expressing VvACO2gene. The VvACO1overexpressing plants showed dwarf phenotypes at the the young growth stage, and were normal morphologically at the maturity stage, but did not set fruit.
Keywords/Search Tags:Grapevine, Tomato, ACO genes, Genetic transformation, Ethylene, ACC oxidase enzyme activity, Ethylene release rate
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