脱落酸尿苷二磷酸葡糖基转移酶在柿子果实发育与成熟中的作用及功能分析

China's persimmon production accounts for two-thirds of the world's total persimmon production, however its market share is very low. The main reason is that the astringent persimmons once ripe fruit rapidly softening. Therefore molecular mechanism of softening urgent need to be clear. Recent studies have shown that ABA plays an important role in the regulation of persimmon ripening and senescence. However, the physiology and molecular mechanism of ABA regulating these process still is unclear. ABA specific Uridine Diphosphate Glucosyltransferases (ABA-UGT) can catalyze the reaction of ABA and sugar producing ABA-GE, and it plays an important role in the ABA accumulation. It means that ABA-UGT may play the key role in the development and ripening in persimmon fruits. Therefore, in this study, we are going to clone UGT genes from persimmon fruits, study the spatio-temporal expression and response character of these genes, find the relationship among them and measure a series of values in ripening physiology and biochemistry. At the same time, we will detect the activity of these UGT proteins which can catalyze the reaction of ABA and sugar forming ABA-GE, in order to confirm the ABA-UGT. On this basis, the function of UGT genes will be verified in which UGT-OE and UGT-RNAi will be transformed into the tomato and then the expression pattern of UGT genes and ABA levels in the transgenic fruits will be analyzed, especially the role of ABA-UGT in fruits development and ripening. The transcriptome changes of these UGT genes will be analyzed as well, and we will seek a regulatory network of these genes. To sum up, the role and molecular mechanism of ABA-UGT in persimmon fruits in development and ripening will be explored and their regulatory network will be preliminary revealed.

我国柿子产量占世界总产的三分之二，但由于果实成熟后极易软化造成市场占有率很低，因此柿果成熟软化的分子机制亟待解明。近年的研究表明，脱落酸在柿果成熟衰老调控中起着重要的作用，但ABA调控这些过程的分子机理尚不清楚。ABA尿苷二磷酸葡萄糖基转移酶（ABA-UGT）催化ABA与糖结合生成ABA葡萄糖酯的反应，对ABA的代谢起重要作用，这意味着该酶可能参与柿果成熟衰老过程的调控。为此，本文拟从柿果克隆UGTs基因，研究基因的表达特性及其和柿果发育与成熟的关系。同时检测UGTs蛋白活性，筛选出ABA-UGT。在此基础上，以与柿果发育和成熟调控机理相似、已建立转基因体系的番茄为试材，分析和验证该基因的功能，即将DkUGT-OE及DkUGT-RNAi转化番茄，分析转基因果实中UGT基因对脱落酸水平的调控，特别是分析UGT在果实发育和成熟中的作用等。综上，初步阐明UGT在柿果成熟过程中的作用和分子机理。

果实早期发育和养分积累是果实成熟的物质基础和能量来源，关于早期柿果发育，以及未成熟柿果向成熟柿果转变过程的研究较少。尿苷二磷酸葡萄糖基转移酶（UGT）介导脱落酸可逆糖基化途径的调节从而调控植株内源ABA水平。本研究通过对35 d和170 d两套柿果转录组数据的筛选，一系列胁迫应答特性分析以及体外蛋白酶活性试验确定了柿果中能特异性催化ABA糖基化的UGT基因DkUGT3。并通过异源表达在两个番茄品种中验证了目的基因的功能，结果发现：DkUGT3在果实早期发育过程中表达量较高，过表达DkUGT3的转基因植株叶片黄化，外源ABA处理和干旱处理都可以部分恢复转基因叶片的黄化表型，说明DkUGT3-OE转基因植株的叶片黄化是由于内源ABA水平降低造成的，这种ABA缺陷表型可以被外源ABA处理或者干旱胁迫诱导的内源ABA水平的增加部分恢复。转基因果实绿果期颜色较浅，果实糖类等营养物质以及类胡萝卜素、番茄红素等色素含量降低。转基因叶片和果实中大量叶绿体发育、叶绿素生物合成和光合作用相关基因的表达均被显著下调。总之，我们的研究结果表明过表达DkUGT3影响了果实发育，初步阐明了尿苷二磷酸葡萄糖基转移酶在果实发育过程中的作用和分子机理。