葡萄VvCCD1b基因克隆及与其启动子互作的转录因子筛选

Cloning of grape VvCCD1b gene and screening of transcription factors interacting with its promoter

  • 摘要: 【目的】筛选葡萄类胡萝卜素裂解双加氧酶(CCD)基因VvCCD1b启动子互作的转录因子,挖掘参与调控葡萄类胡萝卜素代谢的潜在因子,为深入探究葡萄类胡萝卜素的代谢调控网络提供理论参考。【方法】克隆VvCCD1b基因的cDNA及其启动子序列,利用生物信息学软件对VvCCD1b基因的染色体位置、启动子顺式作用元件及其编码蛋白的理化性质、保守结构域等进行预测分析。基于转录组数据分析不同葡萄品种及不同处理下葡萄CCD家族成员表达模式。通过酵母单杂交技术筛选与VvCCD1b基因启动子互作的转录因子,并进行点对点验证。【结果】通过PCR克隆获得VvCCD1b基因序列全长1641 bp,位于13号染色体上,编码546个氨基酸残基,为亲水性蛋白,二级结构由α-螺旋(16.67%)、β-折叠(5.49%)、延伸链(24.54%)和无规则卷曲(53.30%)构成,具有RPE65保守结构域。VvCCD1b基因启动子含有光响应元件(G-box、3-AF1 binding site、GATA-motif、TCT-motif)、水杨酸(SA)响应元件(SARE)、脱落酸(ABA)响应元件(ABRE)及AP2/ERF、WRKY、MADS-box转录因子结合元件。VvCCD1b基因表达水平随果实发育逐渐升高且受光照与逆境胁迫的影响。以VvCCD1b基因启动子为诱饵,初步筛选出VvRAP2-4、VvWRKY4、VvBHLH137转录因子及葡萄液泡加工酶、葡萄糖苷酶等18个功能蛋白,其中VvRAP2-4、VvWRKY4转录因子与VvCCD1b基因启动子间存在互作。【结论】VvCCD1b为亲水性蛋白,基因表达水平随果实发育逐渐升高,且受到干旱、高温、水涝胁迫和光照的影响。通过酵母单杂交技术筛选出与VvCCD1b启动子互作的候选转录因子。

     

    Abstract: 【Objective】To screen transcription factors that interacted with the promoter of the grape carotenoid cleavage dixoygenase(CCD)gene VvCCD1b,and explore the potential factors that involved in the regulation of carotenoid metabolism of grape,which provided theoretical reference for further analysis in the metobolic regulation network of grape carotenoid.【Method】The cDNA and promoter sequences of the VvCCD1b gene were cloned. Bioinformatics softwares were utilized to predict and analyze the chromosomal location of the VvCCD1b gene,the cis-acting elements of the promoter,as well as the physicochemical properties and conserved domains of its encoded protein. Based on the transcriptome data,the expression patterns of grape CCD family members in different grape varieties and under different treatments were analyzed. Additionally,yeast one-hybrid technique was employed to screen for transcription factors that interated with the VvCCD1b gene promoter,and the point-to-point validation was performed.【Result】The 1641 bp full-length sequence of the VvCCD1b gene was obtained through PCR cloning,located on chromosome 13,encoding 546 amino acid residues. It was a hydrophilic protein with a secondary structure composed of α-helix(16.67%),β-fold(5.49%),extended chain(24.54%)and random coil(53.30%),and contained the RPE65 conserved domain. The VvCCD1b gene promoter contained transcription factors binding elements of light-responsive elements(G-box,3-AF1 binding site,GATA-motif, TCT-motif),salicylic acid(SA)responsive element SARE,and abscisic acid(ABA)responsive elements(ABRE)and AP2/ERF,WRKY,MADS-box. The expression level of the VvCCD1b gene gradually increased with fruit development and was affected by light and adversity stress. In addition,using the VvCCD1b gene promoter as bait,the transcription factors VvRAP2-4,VvWRKY4,VvbHLH137 and 18 functional proteins including grape vacuolar processing enzymes and glucosidases were preliminarily screened,among which the transcription factors VvRAP2-4 and VvWRKY4 interacted with the VvCCD1b gene promoter.【Conclusion】VvCCD1b is a hydrophilic protein,the gene expression level gradually increases with the fruit development,and is affected by drought,high temperature,waterlogging stress and light. Candidate transcription factors interacting with the VvCCD1b promoter are screened by yeast one-hybrid technology

     

/

返回文章
返回