葡萄ATG基因家族的表达分析和VvATG7互作蛋白的筛选

发布时间：2018-07-24 10:59

【摘要】：假单性结实型无核葡萄的胚珠经过授粉受精,但却在发育中途败育解体,在这一过程中伴随着细胞程序性死亡(PCD)。细胞自噬是蛋白降解的一个重要途径,在真核生物体内非常保守,参与了植物生长发育、胁迫应答、衰老以及细胞死亡等诸多过程。迄今为止,自噬基因已经在多种植物中展开了研究,但多数集中于衰老和逆境响应等方面,关于种子形成过程中作用的研究很少。因此本试验结合无核葡萄胚珠发育特性,展开对自噬相关基因(Au Topha Gy-related gene,ATG)的研究。通过生物信息学方法鉴定葡萄中的ATG基因,利用RT-PCR技术探究ATG基因在葡萄不同组织和胚珠发育过程中的表达模式。主要取得以下研究成果:1.通过生物信息学方法,在葡萄基因组中鉴定出33个ATG基因,其蛋白序列长度94到2003个氨基酸不等。不同物种的进化分析表明葡萄ATG基因与双子叶植物拟南芥和烟草亲缘关系更近,与单子叶植物水稻亲缘关系较远。葡萄ATG基因的聚类分析及基因结构分析显示具有相同结构的基因亲缘关系更近,表明该基因家族在葡萄基因进化过程中比较保守。染色体定位发现Vv ATG基因分布在葡萄中的18条染色体。同线性分析表明,部分重复在Vv ATG基因家族的扩增中发挥了作用,共有17对ATG基因位于葡萄和拟南芥的线性相关区域,表明它们可能具有共同的祖先和相似的功能,因此我们根据已知的拟南芥自噬相关基因的功能去预测葡萄基因组中的自噬基因的功能。2.通过半定量PCR对Vv ATG基因进行组织表达模式分析,结果显示这些基因在黑比诺葡萄根、茎、叶、花、花蕾、果皮、卷须和胚珠中都有表达,只是表达水平略有不同,表明自噬在植物发育过程中具有广泛的作用。Vv ATG基因在有核品种黑比诺和无核品种无核白的胚珠发育过程中表达存在差异,实时荧光定量PCR结果显示,大部分Vv ATG基因在无核白中的表达水平较高,可能暗示着在无核白中自噬相关活动比较活跃,因此推测自噬可能与无核白胚珠败育相关。其中,ATG7是一个类泛素活化酶,在自噬体的形成过程中不可或缺。Vv ATG7基因在无核白中的表达量明显高于黑比诺中,在花后20-30天,无核白处于表达峰值,而在有核品种黑比诺中,该基因表达呈下调趋势。3.本试验预测发现葡萄基因组中有10个蛋白能够与Vv ATG7相互作用。因此我们从黑比诺葡萄中克隆了Vv ATG7、Vv ATG10和Vv ATG12三个基因,测序结果显示它们分别编码了711、230和94个氨基酸。利用酵母双杂交技术证明Vv ATG7蛋白分别与Vv ATG10和Vv ATG12蛋白存在互作。
[Abstract]:The ovule of pseudo-parthenocarpy seedless grape was pollinated and fertilized, but disintegrated in the middle of development, and accompanied by programmed cell death (PCD). Autophagy is an important pathway of protein degradation, which is very conserved in eukaryotes and participates in many processes such as plant growth and development, stress response, senescence and cell death. Up to now, autophagy genes have been studied in a variety of plants, but most of them focus on senescence and stress response, and few studies have been done on the role of seed formation in the process of seed formation. Therefore, the study of autophagy related gene (Au Topha Gy-related gene was carried out based on the characteristics of ovule development in seedless grape. The ATG gene in grape was identified by bioinformatics and the expression pattern of ATG gene in different tissues and ovules was investigated by RT-PCR technique. The main achievements are as follows: 1: 1. By bioinformatics, 33 ATG genes were identified in the grape genome with protein sequences ranging from 94 to 2003 amino acids. The phylogenetic analysis of different species showed that the ATG gene of grape was more closely related to Arabidopsis thaliana and tobacco than to monocotyledonous rice. Cluster analysis and gene structure analysis of grape ATG gene showed that the genetic relationship of the same structure gene was closer, which indicated that the gene family was conservative in the evolution of grape gene. VV ATG gene was found to be located on 18 chromosomes in grape. The same linear analysis showed that partial duplication played a role in the amplification of VV ATG gene family. A total of 17 pairs of ATG genes were located in the linear correlation region between grape and Arabidopsis, indicating that they may have common ancestors and similar functions. Therefore, we predict the function of autophagy gene in grape genome based on the function of Arabidopsis thaliana autophagy related gene. The expression patterns of VV ATG genes in Pinot Noir grape roots, stems, leaves, buds, pericarp, tendrils and ovules were analyzed by semi-quantitative PCR. The results indicated that autophagy had a wide function in plant development. Vv ATG gene was expressed differently in the ovule development of Pinot Noir, a nucleated variety, and denuclearized variety, and the results of real-time fluorescence quantitative PCR showed that there was a significant difference in the expression of Vv ATG gene in the ovule of Pinot Noir. The high expression level of VV ATG gene in denuclearized white may suggest that autophagy is more active in denuclearized white, so it is speculated that autophagy may be related to ovule abortion of denuclearized ovules. ATG7 is a Ubiquitin activator-like enzyme, and the expression of .Vv ATG7 gene in denuclearization is significantly higher than that in Pinot Noir during autophagy formation. The expression peak in denuclearized Pinot Noir is 20 to 30 days after anthesis, while in Pinot Noir, a nucleated variety, ATG7 is expressed at a peak level at 20 to 30 days after anthesis. The expression of the gene was down-regulated. This study predicted that 10 proteins in the grape genome could interact with VV ATG7. Therefore, we cloned Vv ATG7 VV ATG10 and VV ATG12 from Pinot Noir grape. The sequencing results showed that they encoded 711230 and 94 amino acids, respectively. The interaction of VV ATG7 protein with VV ATG10 and VV ATG12 proteins was proved by yeast two hybrid technique.
【学位授予单位】：西北农林科技大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：S663.1

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