小G蛋白基因ZmArf2在玉米籽粒发育中的功能研究

Small GTP-binding proteins act in and regulate almost all life reactions in cells of plant, but the cloning and functional study for related genes were rarely reported in maize. In our previous research, a gene for small G protein, ZmArf2, was cloned from four suppression subtractive hybridization libraries, which were constructed using two maize inbreds with large and small grain at two key endosperm development stages. Preliminary studies in several aspects has showed that this gene might be related with kernel development and grain weight, including sequence comparison, expression analysis, in silico mapping and comparative mapping, gene mapping and sub-cell mapping. Further researches in this study are as follows: (1) constructing the prokaryotic expression vector of ZmArf2, expressing and purifying the fusion protein to test the binding ability of ZmArf2 protein with GTP in vitro; (2) constructing the antisense, interference and overexpression vectors of ZmArf2, genetically transforming maize using gene-gun bombardment and agrobacterium-mediated methods, screening transgenic plants and analyzing the effect of the target gene with different regulation levels on kernel cell development, grain weight, ion concentration and sucrose synthase activity, etc; (3) construcing tissue-specific expression vector of ZmArf2 to detect its expression site using GUS gene; (4) screening and verifying the interactive protein of ZmArf2 protein using yeast two-hybrid and bimolecular fluorescence complementation methods. These results will reveal the role of ZmArf2 in the the development of kernel and grain weight, and thus provide a new gene for molecular breeding, lay good foundation for developing functional markers and elite germplasm resources, and reveal its mechanism in activity.

小G蛋白几乎参与和调节植物所有细胞生命活动，但玉米相关基因的克隆及功能研究较少。课题组前期研究利用大粒和小粒玉米自交系构建了两个胚乳发育关键时期4个抑制差减杂交文库，从中克隆到小G蛋白基因ZmArf2，并通过序列和表达分析以及序列定位、基因定位、亚细胞定位初步证明其与籽粒发育和粒重有关。本项目拟开展研究内容：①构建ZmArf2的原核表达载体，体外检测其与GTP结合能力；②构建ZmArf2的反义、干扰和超表达载体遗传转化玉米，筛选转基因植株，研究不同程度调控目的基因表达对籽粒细胞发育、粒重、离子浓度和蔗糖合成酶活性等的影响；③构建组织特异性表达载体，检测目的基因的表达部位；④利用酵母双杂交和双分子荧光互补筛选并验证与其互作的蛋白分子。研究结果将揭示ZmArf2在玉米籽粒发育中的功能和对粒重的影响，为分子育种提供新基因，为发掘功能性标记和优异新种质、揭示其作用机制奠定基础。

本项目基于课题组前期研究已经从构建的胚乳发育关键时期抑制差减杂交（SSH）文库中克隆到的小G蛋白基因ZmArf2，通过亚细胞定位、体外GTP结合活性检测、拟南芥遗传转化、互作蛋白筛选及启动子克隆分析得到结果如下：①亚细胞定位将ZmArf2基因定位在细胞质和细胞质膜上；②构建原核表达载体ZmArf2-6 X his融合蛋白，体外诱导纯化后进行GTP结合活性实验检测到ZmArf2蛋白具有GTP结合活性；③构建ZmArf2基因的超表达载体并成功转入拟南芥，对3个T3代转基因纯合株系进行表型观察，不同转基因株系的平均叶面积、株高、千粒重、籽粒面积、粒长和粒宽分别比野生型显著增加，叶片表皮细胞和成熟胚子叶细胞的细胞大小分别显著增大，而细胞数目没有明显变化；④高通量测序分析获得ZmArf2转基因拟南芥中745个差异表达基因，其中上调表达基因224个，下调表达基因521个。GO功能注释分析差异基因主要参与的细胞成分包括叶绿体、叶绿体部分、类囊体以及光合系统，分子功能主要包括氧化还原酶活性，电子载体活性和四吡咯结合，涉及的生物过程主要在代谢前体和能量传递、防御响应和氧化还原过程；⑤以不同发育时期玉米籽粒为材料构建了酵母双杂交cDNA文库，ZmArf2蛋白作为诱饵蛋白筛选互作蛋白，初步筛选到7个有功能注释、1个没有功能注释的互作蛋白，回转验证发现共3个阳性互作蛋白；⑥分段扩增ZmArf2基因并测序，获得ZmArf2基因启动子序列，并对其顺式表达元件进行分析发现该序列中除了含有多个启动子核心元件，另外还存在多种功能性及与非生物胁迫相关的顺式作用元件。上述结果初步明确了ZmArf2基因在籽粒发育和粒重形成中的作用，这不仅有助于揭示籽粒发育的分子遗传机理，为改良粒重进而提高产量提供理论依据，同时也为通过基因工程途径提高粒重提供新基因，为进一步发掘功能性标记和优异新种质、揭示其作用机制奠定基础。