葡萄胚珠特异表达基因VvABCG2跨膜域变异对种子发育的影响

Seedlessness is a form of ovule development in grapes, where pollination and fertilization occurs normally but seeds fail to develop completely because of embryo and/or endosperm abortion. The ATP-binding cassette transporter (ABC) is a crucial kind of transmembrane transporter, and ABCG subfamily transporters play an important role in plant physiological processes. From the ovule specific VvABCG2 gene which is obtained in the differentially expressed genes analysis at an early stage of seed development, we found that of the VvABCG2 gene sequence in a typical seedlessness variety, Thompson seedless, is 78bp fragment less than the gene in a seed variety, and the membrane spaning domain in these two VvABCG2 genes contain different transmembrane helices. In this project, we will transfer VvABCG2 genes which contain different transmembrane helices to ABCG2 gene knockout Arabidopsis mutations to perform the gene complementary function test. Then, we'll observe and compare the phenotype differences among VvABCG2 overexpressed Arabidopsis mutants, completely knockout mutants of homologous VvABCG2 gene, the revertants and wild-type. In addition, we'll conduct subcellular localization experiment to determine the executive functions of dimer types VvABCG2 and their corresponding proteins; to analyze the effect on interactive proteins for transmembrane helical quantity deficiency; to analyze the correlation between transmembrane helix number and seed development among different degree of abortive seedless grapes and seed grapes. At last, we'll make a comprehensive analysis of the relationship between VvABCG2 and seed development in grape, and provide a foundation for revealing the mechanism of seedlessness formation.

无核葡萄是由正常授粉受精的胚珠在发育过程中败育解体而形成的。ATP结合盒转运蛋白（ABC转运蛋白）是一类重要的跨膜运输蛋白，G亚家族参与植物重要的生理过程。在前期差异表达基因分析中获得的胚珠特异性VvABCG2基因，发现典型无核品种无核白等VvABCG2基因缺失78bp片段，有核与无核品种VvABCG2跨膜结构域中跨膜螺旋数量不同。本项目将不同数量跨膜螺旋的VvABCG2基因转入拟南芥完全敲除ABCG2基因突变体中进行功能互补，对比超表达拟南芥突变体、同源基因完全敲除突变体、回复突变体和野生型的表现型差异；进行亚细胞定位，通过蛋白相互作用确定执行功能时VvABCG2的二聚体类型和相应蛋白，分析跨膜螺旋数量缺失对相互作用的蛋白的影响；分析有核、不同败育程度无核葡萄和有核葡萄的跨膜螺旋数量与种子发育相关性，综合确定葡萄VvABCG2基因与种子发育的关系，为揭示无核葡萄形成机理提供基础。

葡萄无核是一个优良的经济性状，研究其种子发育有重要理论和应用价值。ATP结合盒转运蛋白是一类跨膜运输蛋白，G亚家族的葡萄VvABCG20（原名VvABCG2）基因在有核和无核品种种子发育中有明显差异表达。项目开展了基因及启动子序列的多态性分析，在有核和无核品种中VvABCG20基因序列有9个SNP位点，其中1个引起氨基酸变异，相似性达到99.28%；但启动子序列差异较大，有1个41bp 的InDel标记，8个品种同时有序列a及序列b，7个品种仅有缺失的序列b，23条序列共有66个SNP位点，启动子序列表现较高的保守性。sqRT-PCR表明VvABCG20基因在花蕾、花、胚珠中高表达，qPCR分析胚珠发育不同时期表达存在明显差异，随着种子发育而表达增强，有核品种的表达量明显高于无核品种，并对几种外源激素有明显响应。亚细胞定位表明VvABCG20定位于质膜。分析悬浮细胞中添加脂类物质前体后基因的表达变化，初步说明VvABCG20可能运输香豆酸、丙三醇和琥珀酸3种底物。利用VIGS技术沉默直系同源基因后番茄种子数量明显减少，间接验证VvABCG20基因对葡萄胚珠发育具有重要调控作用。VvABCG20基因启动子驱动GUS在拟南芥各器官表达，5’端缺失分析初步确定一些激素结合的正负调控元件。在葡萄基因组中鉴定到30个ABCG基因半分子成员并克隆，生物信息学分析其序列特征、内含子/外显子结构、染色体位置、同线性和进化关系。分析了30个基因在不同组织的表达，检测到一些组织特异表达基因；分析13个基因在有核和无核品种种子发育过程的表达变化规律，确定了与胚珠败育相关的基因。克隆了VvABCG14及其可能互作蛋白的6个基因，通过酵母双杂交和BiFC确定VviABCG14和VviABCG7之间的互作，过表达VvABCG14的拟南芥植株生长加快，生育期延迟。