SmMYB6和SmbHLH1互作调控茄子花青素生物合成的机理研究

Eggplant (Solanum melongena L.), especially for purple eggplant, is known to be a novel anthocyanin-rich vegetable crop. It has been reported that the major anthocyanins in eggplant peels are delphinidin 3- (p- coumaroylrutinoside) -5 – glucoside (nasunin) and delphinidin 3-rutinoside (tulipanin, D3R), which has a strong antioxidant activity and ability of scavenging superoxide anino. However, little is known about the regulation of anthocyanin biosynthesis in eggplant. Our preliminary studies found that transcription factors (including SmMYB6 and SmbHLH1) and anthocyanin structural genes (including SmDFR and SmF3GT) are the key regulators in regulation of anthocyanin biosynthesis in eggplant. On this basis, EMSA and CHIP-PCR assays are to be used to verify whether SmMYB6 and SmbHLH1 directly binds to the promoters of SmDFR and SmF3GT respectively. Meanwhile, transcriptional activation and functional verification assays, and SmMYB6 specifically interacts with SmbHLH1 using both yeast two-hybrid and BiFC assays will be performed. Based on the verification of interaction of SmMYB6 and SmbHLH1, furthermore, a series of transgenic analyses in eggplant by Agrobacterium-mediated methods will be used to determine the expression of these TFs and structural genes, anthocyanin content and component, as well as antioxidant activity. Collectively, our findings will provide new insights into the mechanism at molecular level by which SmMYB6 interacts with SmbHLH1 to regulate the anthocyanin biosynthesis in eggplant, and have an important guiding significance in improving and creating the varieties of high anthocyanin content of eggplant in the future.

茄子，尤其是紫色茄子，是为数不多富含花青素的蔬菜作物。研究表明紫色茄皮中的主要花青素是飞燕草素3-（p-香豆酰鼠李糖苷）-5-葡萄糖苷和飞燕草素3-鼠李糖苷，具有较强抗氧化活性和清除过氧化物阴离子能力，但其花青素的生物合成途径是如何被调控的？至今尚未见报道。本课题组前期研究发现，SmMYB6和SmbHLH1、SmDFR和SmF3GT分别是调控茄子花青素合成的关键转录因子和结构基因。在此基础上，通过检测SmMYB6、SmbHLH1能否直接结合到SmDFR和SmF3GT的启动子上；再进行转录激活和功能鉴定及两者互作效应分析；在探明两者存在互作基础上，利用农杆菌介导法获得转基因茄子株系，系统研究其茄皮和果肉中结构基因表达、花青素含量和组分及抗氧化活性变化，进一步明确SmMYB6和SmbHLH1互作调控茄子花青素生物合成的机理，为茄子遗传改良和创造高花青素含量的茄子新种质奠定理论基础。

基于2014年和2019年公布的茄子基因组，利用生物信息学分析、转录组学分析、品种和组织表达分析，确定SmMYB6 和 SmbHLH1可能在茄子花青素生物合成中起关键调控作用。酵母双杂、双荧光素酶活性实验和遗传转化实验等方法被用来对其功能和分子机制进行研究。酵母双杂交和双分子荧光互补试验发现SmbHLH1并不能与SmMYB6互作。双荧光素酶活性试验发现SmMYB6可激活的SmDFR的转录活性，而SmbHLH1不仅显著降低SmDFR和SmANS的启动子转录活性，且能降低SmMYB6所激活的SmDFR启动子转录活性，这说明SmbHLH1可不依赖于SmMYB6直接调控花青素的合成。故分别对SmMYB6 和 SmbHLH1在茄子花青素生物合成中的调控作用进行了研究。.利用农杆菌介导法分别将SmMYB6和SmbHLH1过表达载体成功导入茄子中并获得多棵可稳定遗传的转基因茄子株系。在SmMYB6过表达茄子株系中，根、茎、叶片、茄皮、果肉和种子均有大量花青素积累且呈现紫色，花青素合成结构基因显著上调表达；通过对WT和两个SmMYB6过表达株系茄皮和果肉转录组数据分析发现，共有显著上调的基因112条，显著下调的基因60条。KEGG通路富集分析发现，显著上调的基因主要富集在次生代谢途径（主要在类黄酮合成途径）、植物的信号转导途径和环境适应途径中。代谢组学分析表明，在转基因株系茄皮和果肉中共检测到8种共有的差异代谢物，我们推测这应该是导致茄子显紫色的主要原因。.而SmbHLH1过表达茄子株系中，茎由紫色变为绿色。茎中花青素含量显著降低，花青素合成结构基因显著下调。转录组学分析发现，SmbHLH1还可使茎中碳代谢途径中的基因上调表达。WT和两个过表达SmbHLH1茄子株系叶片和茎中的叶绿素含量均增加，但仅茎中的净光合速率显著提高。而且茎、功能叶和新叶中的葡萄糖、果糖、蔗糖和淀粉含量均显著增加。