中国甜柿自然脱涩基因DkPK调控因子鉴定及其功能解析

The astringency of Chinese pollination-constant non-astringent (C-PCNA) persimmon (Diospyros kaki Thunb.) can be naturally removed on the tree. This process is controlled by a single locus and is dominant against other types of persimmons; therefore, the regulators of natural de-astringency constitutes an important gene resource for PCNA genetic improvement. Pyruvate kinase (PK; EC 2.7.1.40) is a key regulatory enzyme of acetaldehyde metabolism. The accumulation of acetaldehyde in C-PCNA persimmon fruit is thought to be directly involved in the coagulation of soluble PAs, resulting in a natural loss of astringency. In our previous study, 14 DkPK family members (DkPK1-14) were isolated, and DkPK1, DkPK7, DkPK8 were involved in the natural de-astringency of C-PCNA persimmon fruit. However, the regulation factors of DkPKs has not been understood clearly. In this work, the transcriptional regulatory factors interacted with DkPK1, DkPK7, DkPK8 will be isolated using yeast one-hybrid screens firstly. Then, their interaction with DkPKs would be further verified via the yeast one-hybrid assay and dual-luciferase reporter system. Meanwhile, candidate regulators associated with natural de-astringency in C-PCNA will be screened by analysis of qRT-PCR, PA content, and acetaldehyde concentration during fruit development in different types of persimmon. Furthermore, the function of candidate regulators will be confirmed by subcellular localization and genetic transformation analysis for further understanding the regulation mechanism of DkPKs involved in C-PCNA astringency removal. Our study will be helpful for understanding the mechanism of astringency removal naturally and for the breeding of PCNA cultivars in the future.

中国甜柿（C-PCNA）的自然脱涩性状受显性单基因位点控制，因而脱涩调控因子在甜柿遗传改良中具有重大的应用价值。已知中国甜柿自然脱涩可能涉及乙醛与可溶性单宁的结合（“凝固效应”），丙酮酸激酶（PK）是乙醛代谢的关键酶。项目组前期研究发现DkPK1、DkPK7、DkPK8参与中国甜柿的自然脱涩，但其调控因子及作用机理有待深入探究。本项目首先以上述3个功能明确的DkPK家族成员为诱饵，利用中国甜柿‘罗田甜柿’酵母cDNA文库筛选与其互作的转录因子并克隆全长；进而通过酵母单杂交和双荧光素酶活性检测进行互作验证，并结合不同脱涩类型、不同发育时期的表达分析、单宁及乙醛含量变化，筛选参与中国甜柿自然脱涩的调控因子；然后利用结构域预测、亚细胞定位及柿遗传转化确认其功能，从而明晰DkPK参与中国甜柿自然脱涩的作用机理。研究结果为中国甜柿自然脱涩机理的解析和完全甜柿的遗传改良提供科学依据。

柿（Diospyros kaki Thunb.; 2n = 6x = 90）原产中国，是柿属植物中最具经济价值的栽培种，也是我国特色传统果种。中国甜柿（C-PCNA）可以在树上自然脱涩，具有重要的育种及推广价值，但中国甜柿自然脱涩的机理尚不清楚。项目组以前期获得的中国甜柿脱涩关键基因DkPK1为诱饵，通过酵母文库筛选获得两个WRKY转录因子（DkWRKY3和DkWRKY15）。二者在中国甜柿发育时期的表达量均与可溶性单宁含量变化呈正相关。多序列比对及亚细胞定位均表明DkWRKY3和DkWRKY15为核蛋白。双荧光素酶及酵母单杂交试验表明DkWRKY3和DkWRKY15均可激活DkPK1的表达，且两蛋白表现出显著的叠加激活效应。此外，组织特异性表达分析表明DkWRKY3和DkWRKY15在柿种子中显著上调表达。在柿叶片中分别瞬时转化DkWRKY3和DkWRKY15，可显著降低柿叶片中可溶性单宁的含量，同时提高乙醛代谢相关的基因DkPK、DkADH、DkPDC的表达。因此我们认为DkWRKY3和DkWRKY15通过调控乙醛代谢相关基因，进而参与中国甜柿自然脱涩进程。.通过比较转录组对9种不同柿种进行亲缘关系分析，结果表明六倍体栽培柿（DK）与二倍体油柿（DO）、四倍体德阳柿（DD）亲缘关系最近，由此推测：油柿和德阳柿自然杂交后形成三倍体，而后又自然加倍形成六倍体的柿种，进而通过人为筛选驯化形成目前的栽培柿。单拷贝同源基因Ka/Ks分析显示，栽培柿中糖类代谢、醛类代谢相关基因的表达量显著高于其他近缘种，此类基因可能与柿的进化脱涩有密切关系。通过对不同地区（西南、西北、华南、华东、华北）228份栽培柿进行SSR分子标记分析、268份柿属种质进行SCoT和IRAP分析，结果显示不同地区的栽培柿相互交流较为频繁；西南地区柿遗传多样性较高，与其他四个地区遗传距离较远；鉴定同物异名或者同名异物分别13和16个；基于IRAP、SCoT分子标记种间分析，结果显示栽培柿与二倍体油柿、四倍体德阳柿亲缘关系最近，与比较转录组分析结果相一致，进一步表明栽培柿可能由油柿和德阳柿杂交形成。.