调控辣椒素生物合成的MYB家族转录因子筛选和功能研究

Capsaicin and its analogues, collectively called capsaicinoids are the pungent principle of peppers, which strictly biosynthesis in Capsicumis, an important constituent of fruit quality. The capsaicin demand gap is as high as 80% above in the world. To date, the enzyme genes involved incapsaicin biosynthesis have been elucidated, which showedtheirexpression levelswereconsistent with capsaicinoids contents. But the transcriptional regulation mechanism of capsaicin biosyntheticgenes is still largelyunknown. In our previous study, ranscriptome sequencing and qRT-PCR revealed that the expression levels of seven MYB TFs were significantly up-regulated or specifically expressed in placenta, and phylogenetic analysis showed that these transcription factors (TFs) share high homology with the MYB TFs involved in secondary metabolism from other plant species, suggesting that these TFs may play important roles in transcriptional regulation of capsaicinoids biosynthesis . In this study, the regulation mechanism of MYB TFs on capsaicin biosynthesis will be investigated. Firstly, the expression patterns of these MYB TFs will be analyzedinvarieties with differentpungency levels. Secondly, the roles of sevenTFs involved in capsaicin biosynthesis will be investigated in chili pepper by virus-induced gene silencing (VIGS), CRISPR-Cas9 and gene overexpression in chili pepper. Thirdly, yeast one-hybrid(Y1H) and chromatin immunoprecipitation(ChIP) are used to analyze the MYB TFs binding topromoters of MYB binding site (MBS) of capsaicin biosynthesis genes.In addition, yeast tow-hybrid (Y2H) and bimolecular fluorescence complementation (BiFC) were used to find key proteins interacted with MYB TFs proteins.The results from this work will help to uncover the regulation mechanisms of these MYB TFs on capsaicin biosynthesis, and consequently provide a basis for the generation of different pepper cultivars with expected fruit pungency levels by genetic engineering and breeding.

辣椒素是只在辣椒属植物中合成的辣味物质，是辣椒果实品质重要性状，需求缺口高达80%。目前，辣椒素生物合成途径关键基因基本阐明，且其在胎座中的表达量和辣味显著正相关，但是转录调控机制尚不清楚。我们的前期研究已经发现7个在胎座中显著上调或特异表达的MYB转录因子，该转录因子和其它植物中参与次生代谢的MYB转录因子具有较高的同源性，暗示这些转录因子可能参与辣椒素生物合成的转录调控。本项目拟深入研究7个MYB转录因子在辣椒素生物合成中的功能。分析MYB在不同辣度材料中的表达模式；分析VIGS沉默、CRISPR-Cas9基因敲除和超表达MYB转录因子对辣椒素合成的影响；用酵母单杂交和ChIP法分析MYB在辣椒素合成酶关键基因启动子上的结合位点；用酵母双杂交和BiFC法找到与转录因子互作蛋白。研究结果将阐明MYB转录因子调控辣椒素生物合成的分子机制，为辣椒果实辣椒素含量分子改良和调控奠定基础。

1、分析了辣椒素的含量. 发现辣椒素含量存在基因型、发育阶段和器官的差异,中国辣椒的辣椒素含量高于其他种，胎座中的辣椒素含量高于其他器官。. 2 、研究了控制辣椒素类物质含量的主效位点. 采用740（C. chinense）自交系做母本，CA1（C. annuum）自交系做父本构建了中间杂交群体。采用高通量测序技术运用简化基因组策略分析了150个F2单株的SNP，并构建了一个包含9038个标记的高密度的遗传图谱，证明Cap1是本研究群体中控制辣椒素类物质含量的主效位点。. 3、 Cap1调控辣椒素生物合成的研究.为了进一步确定Cap1位点候选基因，明确其调控辣椒素类物质生物合成的分子机制，本研究采用精细定位策略将Cap1位点限定到510 kb的范围，发现该位点包含6个基因编码产物为MYB转录因子。在这6个基因中只有1个基因Cap1在胎座中特异表达，并且其表达模式和辣椒素合成关键基因即辣椒素类物质积累特征高度一致。采用VIGS、酵母单杂交、双荧光素酶报告系统、染色质免疫共沉淀等技术证实了Cap1可以直接调控辣椒素的生物合成。. 4、 Cap1调控辣椒素合成分子机制研究. 分析Cap1编码区发现SNP较少出现在蛋白的DNA结合区域和激活区域。分析5个栽培驯化种Cap1基因启动子发现存在大量的SNP和InDel，启动子活性分析表明起始密码子上游1102~1292是C. chinense活性显著高于其他4个种的关键区域。进一步分析发现该区域存在1个W-box元件，值得注意的是该元件C. chinense正常，然而在C. annuum、C. baccatum、C. frutescens和C. pubescens存在缺失或者SNP突变。进一步分析表明该W-box可以被1个胎座特异表达的WRKY9转录因子识别和结合，使得C. chinense种的Cap1基因转录水平明显高于其他4个基因型，进而导致极辣C. chinense种的形成。. 5、茉莉酸诱导的R2R3-MYB 转录因子调控辣椒素的生物合成. 茉莉酸甲酯可以诱导3个转录因子CaMYB108、CaMYC2a CaMYC2b的表达，进而诱导主要结构基因的表达，导致辣椒素和二氢辣椒素合成的增加。