纤维素合酶ZmCesA5在玉米籽粒发育中的功能研究

Maize kernel is mainly composed of embryo and endosperm，where endosperm accounted for about 85% of the entire kernel weight, is the main source of food for human and livestock. Zmcesa5 is a spontaneous mutant that shows smaller kernel than that of wild-type. We have cloned the gene by using the fine mapping strategy through large BC1F1 population derived from Zmcesa5 and B73, as well as candidate gene analysis. A 196 bp in second exon of ZmCesA5 causes the mutation of ZmCesA5. ZmCesA5 is a cellulose synthase gene, and is mainly expressed in kernel.This project will study on the molecular mechanism of the ZmCesA5 gene during the kernel development from the following points: 1) Check if the phenotype of Zmcesa5 mutant can be recovered by functional complementary test. 2) Determine the expression patterns of ZmCesA5 gene in the specific cell types of kernel by transformation of the constructions of ZmCesA5 promoter with GUS and GFP fusion vector. 3) Analysis the differentially expressed genes between wild-type and mutant-type kernel of ZmCesA5, and investigate the biological function of ZmCesA5 in kernel development. 4) Identify the interacting protein of ZmCesA5 by CoIP technology, and explore the functional network of ZmCesA5. This study will facilitate the understanding of the role of cellulose synthase in kernel development.

玉米籽粒主要由胚和胚乳组成，其中胚乳占整个籽粒重量的85%左右，是人类和家畜的主要食物来源。ZmCesA5是一个玉米自然诱发突变体，表现为籽粒扁小。课题组利用ZmCesA5突变体与B73杂交构建作图群体，通过精细定位和候选基因分析，成功克隆了该基因。ZmCesA5基因第二个外显子上196 bp的插入导致了该突变。ZmCesA5是一个编码纤维素合酶的基因，主要在籽粒中表达。本项目将在现有基础上开展以下研究：1)利用转基因互补实验，检测Zmcesa5突变表型能否恢复；2)构建ZmCesA5与GUS或GFP融合表达载体，利用转基因技术明确ZmCesA5在籽粒特定细胞中的表达情况；3)通过RNA-seq分析突变体与野生型籽粒转录本差异，探讨ZmCesA5在籽粒发育中的功能；4)利用免疫共沉淀技术寻找ZmCesA5的互作蛋白，探索其可能的互作网络。本研究将有助于阐明纤维素合酶在玉米籽粒发育中的功能。

纤维素合酶在植物细胞壁的形成中起着重要作用。但目前关于纤维素合酶功能机制的研究主要集中在拟南芥中，在玉米中的报道还十分有限，特别是其在籽粒发育中的功能还不清楚。前期，我们发现了一个玉米籽粒扁小的自然诱发突变体。本项目通过图位克隆的方法克隆了该突变基因ZmCesA5，其编码纤维素合酶。细胞学分析表明ZmCesA5的突变主要影响胚乳的发育，其硬质胚乳和粉质胚乳都明显发育异常。基因的时空表达模式分析显示ZmCesA5基因主要在胚乳中表达，其蛋白定位于细胞质。与其它纤维素合酶基因家族基因相比，ZmCesA5是胚乳中最主要的纤维素合酶基因。功能分析表明ZmCesA5突变可以影响类固醇生物合成、碳水化合物代谢、DNA复制等相关基因的表达，特别是营养物质存储相关基因的表达在突变体中显著下调。通过绘制籽粒发育早期高时间分辨率转录图谱并构建基因表达调控网络，挖掘到了一个ZmCesA5基因的上游调控转录因子ZmEreb126。另外通过CrY2H-seq (Cre reporter-mediated yeast two hybrid coupled with next-generation sequencing) 技术挖掘到了4个与ZmCesA5互作的蛋白。研究结果表明ZmCesA5基因的正常表达对胚乳的发育和营养物质积累至关重要，并初步明确了ZmCesA5基因的作用机制。本项目的研究为不仅有助于对胚乳发育调控机制的理解，同时也为玉米产量和品质的改良提供了重要的基因资源。