水稻黄酮醇糖基转移酶基因OsFUGT1调控种子活力的分子机制研究

Flavonols are able to regulate the plant development and growth by inhibiting auxin polar transport. A glycosylated flavonol, Kaempferol 3-O-rhamnoside-7-O-rhamnoside, was recently proven to be the active compound, which can block the auxin polar transport, but its target proteins remains unclear. We postulated that it acts by targeting to auxin influx carrier. In our previous study, by using a set of recombined inbreed lines derived from crossing rice ZS97 with MH63, a flavonol transferase gene (named as OsFUGT1) was identified by mapping based cloning, which largely affects rice seed vigor and my involved in the synthesis of Kaempferol 3-O-rhamnoside-7-O-rhamnoside in rice seeds. Based on our previous studies, we plan to continue investigating the detailed properties and functions of OsFUGT1 to see if it relates to the production of Kaempferol 3-O-rhamnoside-7-O-rhamnoside by performing in vitro enzymatic reaction and producing transgenic plant. By co-expression analysis with OsFUGT1 gene, we will find other glycosyltransferase genes that attributed to Kaempferol 3-O-rhamnoside-7-O-rhamnoside, aiming to reconstruct the whole pathway. We will also try to find the target proteins of Kaempferol 3-O-rhamnoside-7-O-rhamnoside by testing its interaction with auxin influx carriers expressed in yeasts. In this study, we will functionally characterize OsFUGT1 gene and clarify the molecular mechanism of flavonol glycoside on seed vigor. We expect to elucidate the genetic basis that controls rice seed vigor, which will be beneficial to the rice breeding in production of rice cultivar with higher seed vigor.

黄酮醇能够调节生长素极性运输，影响植物生长发育。山奈酚二鼠李糖苷被证实为真正的活性成分，但是它的靶蛋白是什么尚不清楚。我们推测它很可能是通过抑制生长素输入载体起作用。我们前期已经利用水稻ZS97和MH63的120个重组自交系材料，通过QTL定位和图位克隆，鉴定了一个对水稻种子活力有显著影响的黄酮醇糖基转移酶基因OsFUGT1，它可能参与了山奈酚二鼠李糖苷合成。拟在此基础上，通过体外生化活性分析和体内转基因实验，确认OsFUGT1参与山奈酚二鼠李糖苷合成的调控；通过鉴定与OsFUGT1共表达的糖基转移酶,分析它们的功能，阐明水稻种子山奈酚二鼠李糖苷的合成途径；通过酵母异源表达生长素输入载体蛋白，确认山奈酚二鼠李糖苷能够结合生长素输入载体，并抑制生长素运输，这些工作将深入解析黄酮醇糖苷影响生长素运输的分子机制，进一步阐明水稻种子活力调控的遗传和代谢基础，为水稻育种提供理论依据和参考。

黄酮醇能够调节生长素极性运输，影响植物生长发育。我们利用水稻ZS97和MH63的120个重组自交系材料，通过QTL定位和图位克隆，鉴定了一个对水稻种子活力有显著影响的黄酮醇糖基转移酶基因OsFUGT1。经过序列比对分析，发现OsFUGT1属于UGT84家族。 通过构建CRISPR-Cas9载体，转化水稻ZH11，获得基因敲除突变的水稻植株。经过代谢组分析，发现突变体缺失了一个代谢物[M-H]-643.24，其分子量大小与杨梅素二葡萄糖苷一致。基因敲除水稻幼根生长整体变慢，成熟期植株变矮，但是对有效穗数没有显著影响。构建原核表达载体，纯化细菌重组蛋白，通过体外酶活分析发现，OsFUGT1对多种类黄酮成分有活性，但是对杨梅素没有活性，推测可能OsFUGT1的底物不是杨梅素苷元，而是杨梅素单糖。这些结果说明水稻中黄酮醇抑制生长素运输的机制可能与双子叶植物拟南芥不同。这些工作为深入解析黄酮醇糖苷影响生长素运输的分子机制，进一步阐明水稻种子活力调控的遗传和代谢基础，为水稻育种提供理论依据和参考。项目资助发表论文2篇，待发表1篇。培养硕士研究生1名，博士生2名，其中1名已经取得硕士学位，2名在读。项目投入经费63.00万元，支出44.6744万元，各项支出与预算基本相符。剩余经费18.325万元，剩余经费计划用于本项目后续研究支出。