黄瓜是一类重要的蔬菜作物，在世界上被广泛栽培。果实中积累葫芦素C会导致黄瓜苦味，严重影响黄瓜品质。从根本上真正解决黄瓜苦味问题，必须弄清楚黄瓜苦味形成的机制，才能为后续利用分子育种工具改良黄瓜品质打下坚实的科学基础。 真核生物中，转录水平的调控是基因表达调控的主要方式。本研究使用黄瓜cDNA文库，通过酵母单杂交技术筛选与苦味物质生物合成调控相关的转录因子。主要结果如下： 从华北型黄瓜9930中克隆苦味生物合成基因簇的启动子序列并构建到酵母单杂交报告载体pHIS2上。pHIS2-BPr报告载体转化酵母Y187，获得能够消除报道基因在酵母细胞中的本底表达的最适3-AT浓度（15mM），此浓度作为后续筛选黄瓜cDNA文库的3-AT工作浓度。 使用酵母单杂交技术，对黄瓜cDNA文库进行多次筛选，共鉴定出16个转录因子，其中有4个bHLH家族、4个bZIP家族、4个AP2/ERF家族、2个MYB家族和2个锌指蛋白。候选转录因子通过恢复验证及与p450氧化酶进行一对一的激活验证，筛选出的4个转录因子可以激活黄瓜苦味物质生物合成基因簇的表达，分别是Csa3G270（bHLH）、Csa6G020（bZIP）、Csa4G970（bZIP）和Csa5G220（bHLH）。 构建含有荧光素酶的报告载体和连有候选转录因子的效应载体，利用农杆菌渗透法直接转化烟草植株上的叶片，进行转录因子的瞬时表达分析。结果证明Csa3G270（bHLH）、Csa4G970（bZIP）和Csa5G220（bHLH）可以激活黄瓜苦味物质生物合成基因簇的表达。 通过原核表达纯化系统，构建原核表达载体pET32a-Csa5G220，诱导纯化Csa5G220基因表达的融合蛋白。Csa5G220基因编码251个氨基酸的蛋白，相对分子量为33KDa，理论等电点为7.54。体外用凝胶阻滞试验进一步验证纯化蛋白与其顺式作用元件的结合能力。

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 cucumber 9930 and introduced to the yeast one-hybrid reporter vector pHIS2. The pHIS2-BPr reporter vector was transformed into yeast Y187, with the optimal 3-AT concentration (15mM) that can eliminate the basal expression of reporter gene in yeast cells. This concentration was used as 3-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 and 16 transcription factors were detected. Among them, 4 transcription factors belong to bHLH family、4 bZIP family、4 AP2/ERF family、2 MYB family and 2 zinc finger protein, respectively. Candidate transcription factors were identified by restoring verification and peer-to-peer activation verification with P450 oxidase, showing that the 4 transcription factors can activate the expression of cucumber bitter substances biosynthetic gene cluster. They are Csa3G270（bHLH）、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-Csa5G220 was constructed to induce and purify the fusion protein of Csa5G220 gene expression. Csa5G220 gene coded a same-size protein (251 amino acid residues) and its encoding protein had a calculated molecular weight of 33 kDa with an isoelectric point of 7.54. The binding capacity of the purified protein with the cis-acting element was further validated using electrophoretic mobility shift assay in vitro.