Fa-bHLH对草莓果实花青苷合成的转录调控机制研究

Anthocyanin is an important factor of fruit quality formation. It not only affects the appearance and flavor of fruit, but also functions as a healthy nutritional element for consumers. Increasing fruit anthocyanin content can meliorate fruit appearance and enhance its health function. Anthocyanin content is of significant difference among strawberry varieties. Therefore, strawberry serves a good material to study the metabolic regulation of anthocyanin. This project plans to construct the RNAi and over-expression vectors and make genetic transformation of strawberry with varieties of colors. Then assay the different expression profilings between the transgenic and control plants using gene chips. The interacted proteins will be screened using the yeast two-hybrid system. The interacted genes will be cloned and agrobacterium-mediated transformation of arabidopsis thaliana will be performed using the over-expression vector. The Fa-bHLH promoter will be amplified and the transformation of arabidopsis thaliana will be performed using an over-expression vector. The transgenic plants will be treated with different light times and temperatures. The genes combined with the promoter will be screened using the yeast one-hybrid system and the gene function will be confirmed. The expression profiles of Fa-bHLH and related genes will be assayed using the real time quantitative PCR and situ hybridization. And the anthocyanin content of transgenic plants will be analyzed using HPLC method. This project not only clarify the strawberry fruit anthocyanin accumulation mechanism, but also lay the foundation for exploring the regulation of other Rosaceae fruit trees' anthocyanin content measures and the breeding of anthocyanin-rich fruit varieties.

花青苷是形成果实品质的重要因素,提高果实花青苷含量既可改进果实色泽又能增强果实保健功能。草莓果实花青苷含量在品种间存在显著性差异,是研究花青苷代谢调控的良好试材。本项目拟在获得草莓Fa-bHLH基础上,通过RNAi和超表达转化不同果实颜色的草莓，利用基因芯片分析转基因植株与对照植株的表达谱差异；应用酵母双杂交筛选与Fa-bHLH互作的蛋白,分离互作基因、构建超表达载体，然后转化拟南芥进行功能分析；分离Fa-bHLH启动子，利用花序浸染法转化拟南芥，通过不同光温处理检测该启动子对不同环境因子的响应情况；利用酵母单杂交寻找与Fa-bHLH启动子序列结合的调控基因，回转验证后预测基因功能；利用荧光定量PCR和原位杂交分析Fa-bHLH及其调控基因在草莓中的表达模式。本项目不仅对阐明草莓果实花青苷积累机制具有重要意义，也为探索调控其他蔷薇科果树果实花青苷含量的措施和培育富含花青苷的品种奠定基础。

花青苷是形成果实品质的重要因素，提高果实花青苷含量既可改进果实色泽又能增强果实保健功能。本项目测定了草莓发育过程中的花青苷组分与含量变化，确定了栽培草莓花青苷的主要成分是Pg3G（天竺葵素-3-O葡萄糖苷）、Pg3MG（天竺葵素-丙二酰葡萄糖苷）和Cy3G（矢车菊素-3-O葡萄糖苷）；分离了草莓中MYB10和MYB1的序列，并进行了基因表达分析，分离了MYB10基因的启动子序列，预测了其功能；研究了不同光质下Fa-bHLH基因及其调控的下游基因在草莓果实中的空间表达模式，结果表明，这些结构基因的表达明显受到光质的影响。此外，光质对转录因子基因的表达也有影响；利用酵母杂交初步筛选到了与FabHLH互作的FaEPSP基因，分别构建了正义、反义和RNAi表达载体，进行了草莓遗传转化研究；用FabHLH和FaEPSP基因的RNAi表达载体进行了草莓果实的瞬时表达验证，结果表明能够影响草莓花青苷的合成代谢。本项目初步揭示了FabHLH和FaEPSP基因在草莓花青苷合成代谢中的作用，不仅对阐明草莓果实花青苷积累机制具有重要意义，也为探索调控其他蔷薇科果树果实花青苷含量的措施和培育富含花青苷的品种奠定基础。