ALDH2基因家族与中国甜柿自然脱涩间的关系研究

Persimmon cultivars are classified into two types, pollination constant non-astringent(PCNA) and non-PCNA. The former includes Chinese PCNA persimmon (CPCNA) and Japanese PCNA persimmon (JPCNA), the latter comprises pollination variant non-astringent(PVNA), pollination variant astringent (PVA) and pollination constant astringent persimmon(PCA). The trait of loss astringency naturally in CPCNA persimmon controlled by single dominant locus, which is great breeding benefits for PCNA genetic improvement. However, the mechanism of natural de-astringency of CPCNA persimmon has not been understood clearly. Three kinds of persimmon cultivar, 'Luotian-tianshi'(CPCNA), 'Yohou' (JPCNA)and 'Mopanshi'(non-PCNA), will be used as examined materials in this project. Firstly, a screening will been performed to get all aldehyde dehydrogenase 2 (ALDH2) gene family mermbers basing a result of bioinformationa analysis on transcriptome database of the 'Luotian-tianshi'. Then, by RACE and Tail-PCR, full length cDNA sequences of candidate genes can be obtained. Furthermore, their structure and function could be analyzed through bioinformatics tools. Expression of the genes will be analyzed using different de-astringency types (CPCNA, JPCNA and non-PCNA) at different development stages (2, 10, 20 and 25 weeks after bloom), different fruit parts (calyx, peel, flesh, core and seed), and fruits befor or after ethanol treatment, and combined with the result of proanthocyanidin composition and content analysis, the candidates of cDNA clones of ALDH2 would be confirmed. In addition, subcellular localization and over expression and interference expression through Agrobacterium-mediated genetic transformation will be conducted to confirm the function of these candidate genes. Finally, based on the results of ALDH2, ADH (alcohol dehydrogenase) and PDC (pyruvate decarboxylase) genes expression analysis, and ALDH2, ADH and PDC enzymes activity assay, as well as acetaldehyde content changes before or after ethanol treatment in fruits, the relationship of between ALDH2 gene family and the natural de-astringency in fruits from CPCNA can be explored. This research will provide gene resources and technical basis for further elucidating the mechanism of natural loss of astringency and breeding new cultivar with independent intellectual property rights in CPCNA persimmon.

中国甜柿（CPCNA）自然脱涩受显性单基因位点控制，因而在完全甜柿遗传改良中具有重大的应用潜力，但其脱涩机理尚不完全清楚。该项目以'罗田甜柿'转录组数据库为基础，首先通过生物信息学技术进行数据库序列筛选和分析获得醛脱氢酶2（ALDH2）基因家族成员cDNA片段；进而通过RACE和Tail-PCR获得其全长并预测其结构和功能；然后根据其在不同脱涩类型（中国甜柿、日本甜柿和非完全甜柿）、不同发育阶段（花后2、10和20-25周）、不同部位（萼片,果皮,果肉,果心和种子）以及乙醇处理前后表达模式与原花色素成分和含量变化筛选候选基因家族序列。此外通过亚细胞定位及遗传转化确认其功能。最后基于乙醇处理前后目标基因家族表达模式及ALDH2酶活变化，以及与乙醛、乙醇脱氢酶（ADH）和丙酮酸脱羧酶（PDC）的相关性分析阐述其与自然脱涩间的关系。从而为中国甜柿自然脱涩机理以及甜柿遗传改良研究提供科学依据。

中国甜柿自然脱涩性状受显性单基因控制，因而在完全甜柿遗传改良中具有重大的应用价值，但其自然脱涩关键基因尚未明确。已知中国甜柿自然脱涩过程与乙醛介导的“凝固效应”（即可溶性单宁向不溶性单宁的转化过程）有关，但乙醛代谢下游的关键基因ALDH2（醛脱氢酶2基因家族）在其中的功能特性尚不清楚。本研究以中国甜柿（‘鄂柿1号’和‘罗田甜柿’），日本甜柿（‘阳丰’）和非完全甜柿（‘磨盘柿’）为试材，首先基于转录组数据库的序列信息，通过RACE技术分离获得ALDH2基因家族两个成员—DkALDH2a和DkALDH2b的cDNA序列全长。DkALDH2a和DkALDH2b在中国甜柿叶片瞬时超表达后，发现二者均可通过减少可溶性单宁的消耗进而抑制脱涩。DkALDH2a主要作用于种子器官，其表达量在果实发育过程中逐渐降低，以致大量乙醛从种子扩散到果肉中，进而通过凝固效应促进脱涩；DkALDH2b主要是直接在果肉中大量表达，其消耗的乙醛不足以阻止中国甜柿自然脱涩。DkALDH2a和DkALDH2b的表达量与中国甜柿自然脱涩呈负相关关系。以DkALDH2b启动子构建pALDH2b-AbAi诱饵载体并转化Y1HGold酵母菌株，应用酵母单杂交技术从酵母文库中筛选到1个与ALDH2b互作的BBR/BPC1转录因子。结合转录组测序技术和瞬时转化体系，筛选ALDH2及单宁代谢关键基因LAC1和ANR在中国甜柿叶片中瞬时超表达后的特异表达基因发现，ERF家族的转录因子基因可能参与中国甜柿自然脱涩的调控过程。该结果为中国甜柿自然脱涩分子机理的解析及其遗传改良提供了参考。在国外专业期刊上发表学术论文4篇，培养博士2名，硕士3名，组织召开学术会议2次。