中国原产完全甜柿自然脱涩关键基因的分离及其功能解析

PCNA (Pollination-constant non-astringent) persimmon (Diospyros kaki Thunb.) originated in China (termed CPCNA), which trait of de-astringent naturally controlled by a single gene, are important and valuable for PCNA genetic improvement. But the key gene which controlled loss astringent naturally are not vague. In this study, we plan to ‘Luotian-tianshi' (CPCNA), ‘Huashi No. 1’ (non-PCNA) and their hybrid F1 population (which were in fruiting stage) as materials. Then, the high density linkage map was constructed based on SNP marker which were developed by RAD-seq. Subsequently, the SNP marker which linked tightly to the traits of losing astringent naturally will be screened and the flanking sequences of the marker will be isolated from the Fosmid library of ‘Luotian-tianshi’ by PCR. Through compared with transcriptome of ‘Luotian-tiansh’ and micro RNA library of 'Eshi 1', the genes which are different in expression rate were found, and the full-length gene related to de-astringency naturally of CPCNA would be isolated by PCR. Subsequently, the expression of the genes may be analyzed in persimmon using different astringent types (CPCNA and non-PCNA) at different development stages, different fruit parts. Combined with tannin content and cell size analysis, the gene that directly controlled the trait of loss astringent naturally could be isolated. At last, over expression and suppression expression vectors could be constructed and transient transformation of persimmon leaf for further function confirmation. Expected results from this work will help clarify the key reason of loss astringent naturally in CPCNA, and consequently provide gene resources for breeding new CPCNA cultivars with independent intellectual property rights.

中国原产完全甜柿（CPCNA）的自然脱涩性状受单个基因控制，故在柿品种遗传改良中具有重大的应用价值，但控制其自然脱涩的关键基因尚不明确。本研究计划以‘罗田甜柿’（CPCNA）、‘华柿1号’（non-PCNA）及其二者杂交F1群体（已进入结果期）为试材，利用简化基因组测序技术构建基于SNP标记的高密度连锁图谱，并从中筛选出与自然脱涩性状紧密连锁的SNP标记，然后从‘罗田甜柿’Fosmid文库中分离标记的侧翼序列；将该序列与已有的两个转录组文库和两个microRNA文库比对，搜寻表达量存在差异的基因片段，获取与CPCNA自然脱涩相关基因；通过分析其在不同脱涩类型、不同发育阶段和不同部位的表达模式并结合单宁含量和细胞大小分析，筛选控制其自然脱涩的关键基因；最后通过超表达及反义表达载体瞬时转化柿叶片验证其功能；最终为明晰CPCNA自然脱涩机理提供科学依据，为培育自主知识产权新品种提供基因资源。