Clone And Functional Analysis Of Bitter Gene Bi In Cucumber

Cucumber is one of important vegetables in the world, which plays an extremely important role in vegetable consumption in the while globe. However, the fruit of Cucumber, especially the cucumber in protected field, often results in bitterness due to genetic factor and the unreasonable management and technology of cultivation. Cucurbitacins C in cucumber results in production of bitter in friut, seriously affecting its quality. Bi gene controls the bitterness of cucumber vegetative and the bitterness in friut is controled by Bt gene. What’s more, the genetic segregation is comprehensive when bi and Bt genes are in the same genetic background. We will research the inheritance of bitter gene of cucumber and the key genes in the process of the synthesis of Cucurbitacins C. It is extremely significant to definite the molecular mechanism of the synthesis of bitterness so as to guide breeding workers to correctly select parents and to perform the molecular marker breeding.Since the sequencing of cucumber genome is complished, Bi gene is located at35Kb physical interval in sixth chromosome by density heredity mapping and high-resolution drawing. In this study, the condinate gene of Bi, Csa680, was found by comparative genomics. we researched the function of Csa680using the eucaryon expression of yeast、 the transient expression of tobacco and genetic transformation of cucumber.The main research results are as following:(1) The cloning of Bi gene:According to comparative genomics, four cyclases:Csa846、 Csa507、Csa680、Csa845were found. Phylogenetic analysis of four cyclases with the cyclases of Arabidopsis and Cucurbitapepo showed that the homology between Csa680and cucurbitadienol synthetase of Cucurbitapepo is up to90%.(2)The expression system of yeast:The constructed expression vector pYES2-Csa680was transformed to yeast. After the induction of yeast contained Csa680, the metabolites were extracted to detect cucurbitadienol by GC-MS. The result indicated that Csa680gene could cyclize2,3-oxidesqualene to generate cucurbitadienol.(3)The transient expression of tobacco:The constructed expression vector pCAMBIA1300-Csa680was transformed into Agrobacterium tumefaciens. The Agrobacterium tumefaciens containing Csa680was then introducted into tobacco. After transient expression, the functional protein Csa680made2,3-oxidesqualene cyclized to generate cucurbitadienol.(4) The genetic transformation of Cucumber with Csa680:The constructed expression vector pCAMBIA1300-Csa6850was transformted into Agrobacterium tumefaciens. Csa680was successfully introducted into the genome of non-bitter cucumber. Compared with non-bitter cucumber, the expression level of Csa680in transgenic cucumber is obviously higher than the control. What’s more, the transgenic cucumber produced Cucurbitacins C by LC-MS detection. The result indicated that Csa680played an extremely important role in the synthesis of Cucurbitacins C.