OsCESA4、7、9蛋白P-CR区域在水稻茎秆纤维素合成中的功能研究

It has been defined that cellulose biosynthesis of secondary cell walls affects plant growth, agronomic traits (lodging resistance) and biomass saccharification in rice. As we have recently found that cellulose synthase (CESA) complex integrity and activity could largely be affected by P-CR region site-mutation of OsCESA9, this project will focus to select a large number of mutants by gene-editing of 15 P-CR sites of OsCESA4, 7, 9 using CRISPR/Cas9 technology. Biochemical approaches will be applied to detect CESA complex compositions and enzymatic activities in the mutants, whereas physical and chemical analyses will be performed to determine their cell wall compositions (cellulose, hemicelluloses, lignin), lignocellulose crystallinity (CrI) and degree of polymerization (DP) of crystalline cellulose. Furthermore, the cell wall structures of mutants will be observed using various microscopies, and their major agronomic traits will be evaluated including lodging index. Finally, biomass enzymatic digestibility of mature stem tissues will be examined in the mutants under various physical and chemical pretreatments. Hence, this study will generate several desire mutants that could not only have normal growth and grain yields similar to wild type, but also exhibit the improved lodging resistance and enhanced biomass saccharification, which provides insights into genetics and breeding in rice and beyond.

次生细胞壁纤维素的合成对水稻生长发育、主要农艺性状、抗倒伏能力和茎秆纤维素糖化效率有重要影响。申请人前期研究发现，水稻次生壁纤维素合酶OsCESA9的P-CR区域与纤维素合酶复合体的形成和活性密切相关。本项目以日本晴水稻为材料，用CRISPR/Cas9技术分别对OsCESA4、7、9的P-CR区域中15个靶点进行基因编辑，获得大量的突变体材料。用生物化学方法鉴定纤维素合酶复合体中OsCESA4、7、9的组成和催化活性，用物理化学方法测定细胞壁组成（纤维素、半纤维素和木质素的含量）、纤维素的聚合度和结晶度，用细胞生物学方法观察细胞壁结构，综合关联分析主要农艺性状和抗倒伏能力，用生物工程方法分析茎秆纤维素糖化效率。本研究项目旨在阐明OsCESA4、7、9的P-CR区域在纤维素合成中的功能，获得生长正常、农艺性状优良、抗倒伏能力和茎秆纤维素糖化效率高的材料，为水稻遗传改良提供理论基础和可行方法。

次生细胞壁纤维素的合成对水稻生长发育、主要农艺性状、抗倒伏能力和茎秆纤维素糖化效率有重要影响。申请人前期研究发现，水稻次生壁纤维素合酶OsCESA9的P-CR区域与纤维素合酶复合体的形成和活性密切相关。本项目以日本晴水稻为材料，用CRISPR/Cas9技术分别对OsCESA4、7、9的P-CR区域中15个靶点进行基因编辑，获得大量的突变体材料。OsCESA4, 7, 9不同位点的点突变对株高、茎秆折断力、拉断力和倒伏指数具有不同的影响。大部分突变体株高下降和折断力无显著变化，引起植株抗倒伏能力增强。P-CR突变后的不完整蛋白质，虽然不具有合成纤维素的能力，但仍能参与合酶复合体组装，与正常OsCESA9蛋白竞争与另外两种亚基结合形成复合体，从而干扰纤维素的正常合成。通过编辑水稻次生细胞壁纤维素合酶基因的P-CR区，改变秸秆纤维素结构特征，可以显著提高水稻秸秆纤维素的酶解效率。突变体细胞壁中纤维素含量下降，半纤维素含量增加，木质素和果胶含量基本不变。纤维素的结晶度和聚合度下降，引起酶解效率显著升高。本研究项目探索了OsCESA4、7、9的P-CR区域在纤维素合成中的功能，获得了生长正常、农艺性状优良、抗倒伏能力和茎秆纤维素糖化效率高的材料，为水稻茎杆细胞壁遗传改良提供了理论基础和可行方法。