与中国甜柿可溶性单宁自然凝固相关的转录因子及其调控网络研究

Persimmon cultivars can be divided into two types, pollination constant non-astringent (PCNA) and non-PCNA. The former includes Chinese PCNA persimmon (C-PCNA) and Japanese PCNA persimmon (J-PCNA), the difference is in their genetic characteristic of PCNA deastringency trait that C-PCNA is controlled by a single locus and dominant against J-PCNA, suggested that C-PCNA could be a candidate for breeding new PCNA type. However, the mechanism of natural de-astringency of C-PCNA persimmon has not been understood clearly. Our previous study showed that ADH1 and PDC2 have an important role involved in soluble tannin coagulation. The first step of this research is to isolate their promoter sequences respectively, using the Y1H (Yeast one-hybrid) system to select the candidate interaction transcription factors (TFs) and cloning the full length of TFs cDNA. Secondly, using Y1H, EMSA and dual luciferase assays to verify they’re truly interaction. Furthermore, their structure and function could be analyzed through bioinformatics tools. Expression of the TFs will be analyzed using different de-astringency types (C-PCNA, J-PCNA and non-PCNA) at different development stages (2.5, 10, 20, 25 and 27.5 weeks after bloom), different fruit tissues (calyx, peel, flesh, core and seed), and combined with the result of soluble and insoluble tannin content analysis, the candidates TFs would be confirmed. In addition, subcellular localization and over expression through Agrobacterium-mediated genetic transformation will be carried out to confirm the function of these candidate TFs. And speculated the potential structural and regulatory genes that involved in tannin biosynthesis which regulated by the key TFs selected. Finally, based on the results of genes expression analysis, and through Agrobacterium-mediated genetic transformation and the natural de-astringency regulatory network in fruits from C-PCNA can be explored. This research will provide gene resources and technical basis for further elucidating the mechanism of the natural loss of astringency and breeding new cultivar with independent intellectual property rights in C-PCNA persimmon.

中国甜柿自然脱涩性状受显性单基因位点控制，因而在甜柿遗传改良中具有重大应用潜力；已知其自然脱涩可能涉及可溶性单宁的自然凝固过程，但其机理尚不完全清楚。该项目以功能明确的两个与自然凝固相关的结构基因DkPDH1和DkPDC2为基础，首先分离其启动子序列，通过酵母单杂交文库筛选其候选转录因子cDNA片段并克隆其全长；进而通过DNA/蛋白质互作和不同脱涩类型（中国甜柿、日本甜柿和非完全甜柿）、不同组织器官（萼片、果皮、果肉、果心和种子）和不同发育时期（花后2.5、10、20、25和27.5周）的表达模式分析，以及亚细胞定位和遗传转化进一步筛选其关键转录因子；然后通过靶基因预测和互作基因阳性鉴定初步明确受其调控的与单宁代谢有关的结构和调控基因；此外，通过对初选靶基因的表达模式分析和遗传转化确认其调控网络。以期为中国甜柿可溶性单宁自然凝固的分子机理乃至主栽涩柿的“甜柿化”遗传改造研究提供科学依据。

中国甜柿自然脱涩性状受显性单基因控制，因而在甜柿遗传改良中具有重大应用潜力；已知其自然脱涩可能涉及可溶性单宁的自然凝固过程，但其机理尚不清楚。通过对中国甜柿自然脱涩关键时期的转录组和蛋白组数据分析鉴定到7个候选MYB转录因子（DkMYB14-20）。进一步的序列和表达分析结果显示，DkMYB14具C2抑制基序，且在中国甜柿自然脱涩关键时期（花后15-20周）特异上调表达，与中国甜柿可溶性及不溶性单宁累积模式紧密相关；亚细胞定位分析结果表明，DkMYB14定位于细胞核；通过活体柿叶瞬时超表达和干涉表达、拟南芥稳定遗传转化以及离体柿果圆片转化试验证实DkMYB14通过负调控单宁的生物合成，同时正调控乙醛的生物合成而促进可溶性单宁的不溶化的双重作用减少可溶性单宁的累积；利用酵母单杂交和双荧光素酶及GUS活性分析实验鉴定了其双重调控作用的靶基因，即负调控单宁生物合成关键基因DkF3’5’H和DkANR，同时正调控乙醛生物合成关键基因DkADH1和DkPDC2；进一步的序列突变分析证实其C端ERA-like基序和R2结构域的第39位甘氨酸（Gly）是其双重调控作用的分子基础。研究结果为中国甜柿自然脱涩机理解析及完全甜柿遗传改良研究提供了新的科学依据。在国外专业期刊上在投1篇，培养博士1名，硕士3名；组织2次大型学术会议。

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