miRNAs调控柿单宁合成代谢机理研究

The process of de-astringency in Chinese PCNA (CPCNA) and Japanese PCNA is greatly different and the tannin metabolism is not well understanding in both CPCNA and JPCNA. Fruits of CPCNA, JPCNA and artificial de-astringency CPCNA will be sampled at the precise period of de-astringency occurrence using Printing method, proanthocyanidins concentration with taste judging. A high-throughput miRNAs sequence profiling will be performed by Solexa technology. After bioinformatics analysis, the differential miRNAs will be screened under different materials and treatments. The relationship of differential miRNAs and the targeted genes or DkMYB, DKMYC and DkbZIP5 will be termed by GO. The targeted genes will be validated by 5' RACE, meanwhile the expression pattern of recommended miRNAs and targeted genes and structural genes with transcription factors at different organs and development stages should be detected for finding key miRNAs in de-astringency. The key miRNAs' function in persimmon proanthocyanidins metabolism will be verified by VIGS and over-expression in Arabidopsis. Finally, the down-regulated genes of key miRNAs will be detected via gene chips. Based on above, we hope to elucidate the molecular mechanism of miRNAs concerning the persimmon tannin synthesis and metabolism. The results will provide scientific evidence for regulation of persimmon tannin metabolism and creation of new germplasm with high tannin concentration, also establish a platform for PCNA genetic modification and molecular breeding.

中国甜柿与日本甜柿自然脱涩过程明显不同，调控中、日甜柿单宁代谢的分子机理尚不明晰。本项目计划以中、日甜柿为主要试材，首先通过印迹法及单宁含量测定分别确定中国甜柿和日本甜柿自然脱涩以及人工脱涩关键时期，进而利用Solexa测序技术进行中国甜柿和日本甜柿RNA文库测序，比较和筛选中国甜柿和日本甜柿脱涩过程miRNAs差异序列；分析miRNAs可能靶定单宁代谢相关的靶标基因以及同DkMyb、DkMYC和DkbZIP5等转录因子关系并通过5'RACE验证；对不同发育时期果实和不同器官的miRNAs及潜在作用靶基因进行表达分析，找出其中影响单宁合成代谢的转录因子的关键miRNAs；通过VIGS系统和超表达验证所寻找到的miRNAs功能，并分析其调控下游基因，从而探讨中国甜柿与日本甜柿单宁生物合成代谢过程中miRNAs作用机制。本项目可为阐明柿单宁代谢的分子机理和遗传改良奠定基础。

中国甜柿与日本甜柿自然脱涩过程明显不同，调控中、日甜柿单宁代谢的分子机理尚不明晰。本研究通过高通量测序挖掘中国甜柿、日本甜柿自然脱涩过程中miRNAs及其靶标基因，筛选参与柿单宁生物合成代谢相关转录因子和结构基因并探讨其作用分子机理。本研究以中国甜柿‘鄂柿1号’、日本甜柿‘阳丰’、非完全甜柿‘磨盘柿’为主要试材，首先开展了不同脱涩类型果实发育过程中单宁含量动态变化测定，获得了中国甜柿和日本甜柿脱涩关键时期；构建了sRNA文库，利用Illumina测序技术筛选获得33个候选miRNA；通过qRT-PCR筛选获得3个参与中国甜柿单宁累积和自然脱涩相关的miRNA（miRNA858，miRNA143c和miRNA397）。利用3种类型柿单宁含量年动态变化、基因克隆与序列比对及进化树分析、亚细胞定位、RLM-5’RACE、qRT-PCR分析、柿叶片和果实圆片瞬时转化证实了miRNA通过抑制靶基因（DkMYB15，DkMYB2-like，DkALDH10和DkLAC2）活性，参与了柿原花青素累积和自然脱涩过程。本研究结果为解析中国甜柿和日本甜柿果实单宁累积过程和自然脱涩过程差异的分子机理提供了参考。在国内外专业学术期刊发表学术论文4篇，参与培养博士1名，硕士3名，参与组织召开国内学术会议4次。