Cloning Of Fruit Bending Related Gene Cs14-3-3in Cucumis Sativusl. And Genetic Transformation To Arabidopsis Thaliana

Cucumber (Cucumis sativus L.) is an important vegetable varieties for protected cultivation inChina.Fruit shape is one of the most important appearance qualities in cucumber, fruit bentingseriously affect the quality of cucumber appearance and significantly decreased its marketability,which caused great economic losses to farmers.Therefore, exploring the curved cucumber fruitsmechanism to reduce the bending fruit ratio is essential in breeding and production in cucumber.14-3-3protein is a highly conserved regulatory protein,which is ubiquitous ineukaryotes.through interaction with target proteins,it participates in a variety of regulatoryprocesses including metabolism in vivo, signal transduction, cell division, photoperiod, biologicalstress and abiotic stress regulation. The laboratory early results show that14-3-3protein is adifferentially expressed protein in the abdomen and the ridge of fruit benting varieties‘ChangchunMici’.the expression levels of this protein in the bending fruit abdomen is significantly higher thanthat in the bending fruit ridge. the nucleotide sequence encoding this protein located on the samechromosome with the molecular markers that tightly linked cucumber fruit bending, which impliesthat the14-3-3protein gene may play an important role in the process of cucumber fruit bending.In this study,we separated and cloned the14-3-3protein gene from the peel of the cucumber（Cucumis sativus L.）bending variety‘Changchunmici’ by RT-PCR technology. At the same time,by using Real-time fluorescence quantitative PCR （RT-qPCR）, we studied the expression ofmRNA in straight fruit and the abdomen and the ridge of the bending fruit in the periods of2、4、6、8、10、12days after flowering. We constructed the over expression vector and RNA interferencevector, through the flral dipping method, the gene has be transformed to Columbia wildArabidopsis.We hope the test can lay a foundation for revealing the molecular mechanism ofcucumber fruit bending. The main results obtained in this trial are as follows:(1) we cloned the14-3-3protein gene from the peel of the cucumber（Cucumis sativus L.）bending variety‘Changchunmici’, named Cs14-3-3. The full length cDNA of Cs14-3-3is792bp,encoding a protein of263amino acids.The protein contains the conserved domain of the14-3-3protein, belonging to the14-3-3superfamily.Gene sequence homology comparison found that thehomology of cucumber with grapes up to86%, and homology with rubber, tapioca, rhubarb, tobacco, arabidopsis thaliana up to80%above.Mapping of the amino acids found that, cucumberand rubber trees, cassava, longan, Arabidopsis share the higher similarity, up to90%above.Molecular phylogenetic tree showed that the14-3-3protein in cucumber,grapes, rubber, tapiocaand European beech are in the same evolutionary branch.14-3-3protein encoded by Cs14-3-3is aunstable hydrophilic protein in vitro. It does not have a transmembrane structure.The proteincontains114-3-3protein signal sequence I site and114-3-3protein signal sequence II site,lots ofphosphorylation sites、myristoylation sites and glycosylation sites. subcellular localization resultsshowed that the protein was localized in the nucleus and the cytoplasm。(2) we studied the expression of mRNA in straight fruit and the abdomen and the ridge of thebending fruit in the periods of2、4、6、8、10、12days after flowering.The result showed that ineach period, the expression level of Cs14-3-3from high to low order is bending fruit abdomen、straight fruit、bending fruit ridge,and in each part, the expression level of Cs14-3-3in2days afterflowering obviously higher than that in the other periods. In conclusion,it was preliminarilyrevealed that Cs14-3-3gene is related to fruit bending in cucumber,and plays an important role inthe period of early fruit development after flowering.(3) Cs14-3-3gene over-expression vector and RNA interference vector were constructed andnamed pBI121-M14-3-3and pBI121-F14-3-3respectively. Then the plant expression vector weresuccessfully transformed into Agrobacterium by Freeze-thaw method.(4) The plant expression vector was transformed into Arabidopsis, and the PBI121-M14-3-3transformed resistant plants was8, the PCR-positive plants was6. PBI121-F14-3-3transformedresistant plants was8, the PCR-positive plants was6. The T1generation seeds of transgenicplants were harvested.