SMK11表观遗传调控玉米籽粒发育的分子机制

At present, the genetic regulation mechanism of maize grain development is mainly limited from the perspective of omics and molecular genetics. The epigenetic regulation mechanism of maize seed development needs further study. In the previous work, we found a new small kernel mutant (tentatively named as smk11), and cloned the candidate SMK11 gene by map-based cloning. Further analysis showed that the sequences near the splice site of the third intron has decreased DNA methylation level which lead to the intron retention. This project intends to further analyze the expression characteristics of SMK11 gene and its subcellular localization; It will be studied whether the complexes of the respiration chain was affected in mutant kernels, and the yeast two hybrid will be performed to identify the proteins interacting with SMK11. The methyl-CpG-binding domain (MBD) proteins will be identified, and the RNA cleavage factor which bind MBD proteins will be investigated by immunoprecipitation mass spectrometry. The results of this project will illustrate the molecular mechanism of SMK11 epigenetic regulation of maize kernel development, expand the understanding of the epigenetic regulation mechanism of the important agronomic traits of maize seed, and provide important theoretical support for the genetic improvement of maize.

目前，玉米籽粒发育的遗传调控机制局限于组学及分子遗传学解析，其表观遗传调控机制亟待深入研究。本项目组前期发现一个新的玉米小粒突变体smk11，图位克隆到候选基因SMK11。该突变体DNA甲基化水平在SMK11基因启动子区升高而在基因第三个内含子5’剪接位点附近降低，从而导致SMK11基因表达量下调和内含子滞留，影响籽粒发育。本项目拟在上述工作基础上，明确SMK11基因的组织表达特性及亚细胞定位；分析smk11突变体中呼吸链复合物变化及籽粒代谢组变化；鉴定SMK11的互作蛋白、揭示与SMK11第三个内含子5’剪接位点甲基化DNA结合的MBD(methyl-CpG-binding domain)蛋白及与MBD蛋白互作的RNA剪接因子。本项目研究结果将系统解析SMK11表观遗传调控玉米籽粒发育的分子机制，拓展对玉米籽粒形成这一重要农艺性状表观遗传调控机制的新认识，为玉米遗传改良奠定新的理论基础。

线粒体为籽粒发育提供能量，在玉米籽粒发育的遗传调控中具有重要作用，线粒体呼吸链功能复合物的组装需要复杂而有效的调节机制。与哺乳动物和酵母相比，植物细胞色素c氧化酶（复合物IV）所涉及的亚基组成和组装因子研究较少。在这项研究中，我们以玉米小粒突变体smk11为研究材料，图位克隆了玉米Smk11基因。Smk11编码线粒体定位的三肽重复序列（TPR）蛋白，Smk11的功能缺失严重影响线粒体复合物IV的组装和活性，导致玉米生长和种子发育延迟。蛋白质相互作用研究表明，SMK11可能与四种假定的复合物IV组装因子相互作用，即内膜肽酶1A（ZmIMP1A）、MYB结构域蛋白3R3（ZmMYB3R-3）、细胞色素c氧化酶23（ZmCOX23）和线粒体铁氧还蛋白1（ZmMFDX1），其中ZmMFDX可能与亚基ZmCOX6a和ZmCOX-X1相互作用；ZmMYB3R-3也可能与ZmCOX6a相互作用。SMK11的突变扰乱了这些亚基的正常组装，导致复合物IV的失活。本研究的结果表明，SMK11作为辅助的组装因子介导复合物IV亚基的正常组装，研究结果将有助于阐明植物线粒体复合物IV的组装在玉米籽粒发育调控中的重要作用。