DkMYB14上游调控基因筛选及其调控网络解析

中国甜柿自然脱涩性状受显性单基因控制，因而在完全甜柿遗传改良中具有重大应用潜力。前期研究表明DkMYB14通过抑制可溶性单宁（产生涩感的主要原因）的合成，并促进其向不溶性单宁转化的双重作用降低可溶性单宁含量，从而促进中国甜柿自然脱涩。由于其编码序列在甜、涩柿上无差异，因此，中国甜柿自然脱涩控制基因很可能通过控制DkMYB14的特异表达，从而控制这一自然脱涩过程，但具体细节尚不完全清楚。该项目计划以DkMYB14基因为基础，通过分离其启动子序列，利用酵母单杂交文库筛选上游候选调控因子；通过互作验证和转录活性分析，并在不同脱涩类型、不同组织器官和不同发育时期进行表达模式分析，从而筛选关键调控因子。进一步通过瞬时转化和稳定转化技术鉴定候选基因功能，结合DkMYB14上下游调控关系解析其分子调控网络。以期为中国甜柿自然脱涩的分子机理的解析乃至主栽涩柿的“甜柿化”遗传改造研究提供科学依据和功能基因。

The genetic trait of natural deastringency in pollination constant non-astringent (PCNA) of Chinese origin (C-PCNA) is controlled by a single dominant locus (CPCNA) against non-PCNA. Thus, using the C-PCNA for PCNA breeding is an expected strategy to overcome the inbreeding depression. The soluble proanthocyanidin (PA) is the main factor for astringency sensation. Our recent research work has characterized an R2R3-MYB transcription factor, DkMYB14, which regulates PA accumulation in C-PCNA fruit flesh, is a bifunctional transcription factor that acts as a repressor in PA biosynthesis but becomes an activator when involved in acetaldehyde biosynthesis. Interestingly, both functions contribute to the elimination of astringency by decreasing PA biosynthesis and promoting PA insolubilization, resulting in a non-astringency trait in C-PCNA. However, the regulator gene of DkMYB14 is still unknown. To identify the regulators, we plan to use a yeast one-hybrid (Y1H) system to screen the candidate regulators upstream of DkMYB14. The capability of candidate regulators binds to the DkMYB14 promoter will be verified using the electrophoretic mobility-shift assay shift assay (EMSA) and Y1H analysis, and dual-luciferase (LUC) and GUS assays will be conducted to analyze the transcription activity. Then the candidate will be selected again according to their expression pattern in different de-astringency types of persimmons at different development stages and different tissues. The genes that show fruit-specific expression and are correlated with tannin accumulation in C-PCNA will be further characterized by transient overexpression and silencing in persimmon leaves and fruit discs. Finally, the key candidate genes will be confirmed by stable transformation analysis. Based on all of the above experiments the key regulator of the natural deastringency of C-PCNA will be isolated. The research results will aid to clarify the mechanism of natural deastringency in C-PCNA persimmon and provide functional genes for genetic improvement of PCNA breeding in the future.