Screening And Identification Of Transcription Factors Related To Bitter Substances Biosynthesis Regulation In Cucumber (Cucumis Sativus L.)

Cucumis sativus L. is a kind of important vegetable crop, which is cultivated in worldwide area in the world. The accumulation of cucurbitacin C in fruits will result in bitterness in cucumber and seriously affect the quality of cucumber. In order to solve the problem of cucumber bitterness in fundamental, we must make clear the mechanism of bitterness formation in cucumber, only in this way we can lay a solid scientific basis to improved cucumber quality with the molecular breeding tools.In eukaryotes, regulation in transcription level is the main way of gene expression regulation. In this paper, Screening and identification of transcription factors related to bitter substances biosynthesis regulation were performed with cucumber cDNA library through yeast one-hybrid technology. The main results are as follows:The cucumber bitterness biosynthetic gene cluster promoter was cloned from the North China cucumber9930and introduced to the yeast one-hybrid reporter vector pHIS2. The pHIS2-BPr reporter vector was transformed into yeast Y187, with the optimal3-AT concentration (15mM) that can eliminate the basal expression of reporter gene in yeast cells. This concentration was used as3-AT working concentration of a follow-up screening cucucmber cDNA library.Using yeast one-hybrid technology, in the study cucumber cDNA library was screened several times and16transcription factors were detected. Among them,4transcription factors belong to bHLH family、4bZIP family、4AP2/ERF family、2MYB family and2zinc finger protein, respectively. Candidate transcription factors were identified by restoring verification and peer-to-peer activation verification with P450oxidase, showing that the4transcription factors can activate the expression of cucumber bitter substances biosynthetic gene cluster. They are Csa3G270(bHLHX)、 Csa6G020(bZIP)、Csa4G970(bZIP) and Csa5G220(bHLH) respectively.Reporter vector containing the luciferase and effector vector connected candidate transcription factors were constructed. The transcription factor transient expression analysis is directly converted to the leaves of tobacco plants using Agrobacterium infiltration method. The results proved that Csa3G270(bHLH), Csa4G970(ZIP) and Csa5G220(bHLH) could activate the expression of cucumber bitter substances biosynthetic gene cluster.Applying prokaryotic expression and purification system, prokaryotic expression vector pET32a-Csa5G220was constructed to induce and purify the fusion protein of Csa5G220gene expression. Csa5G220gene coded a same-size protein (251amino acid residues) and its encoding protein had a calculated molecular weight of33kDa with an isoelectric point of7.54. The binding capacity of the purified protein with the cis-acting element was further validated using electrophoretic mobility shift assay in vitro.