葡萄VvGCN5基因的鉴定及互作蛋白筛选

Identification of grape VvGCN5 gene and screening of interacting proteins

[目的]本文旨在揭示组蛋白乙酰转移酶GCN5(general control non-repressible 5)在葡萄生长发育过程中的调控机制,并筛选出与VvGCN5互作的蛋白,以期为进一步构建VvGCN5在葡萄生长发育中的调控网络提供理论基础。[方法]利用生物信息学技术对VvGCN5的蛋白二级结构、基因结构、功能域、启动子的顺式作用元件以及抗逆的表达量和时空表达进行分析,并在烟草中进行亚细胞定位,研究VvGCN5表达位置。通过酵母双杂交技术筛选与VvGCN5互作的蛋白,并通过双分子荧光互补(BIFC)技术进行验证。[结果]VvGCN5的二级结构由α-螺旋、延伸链、β-折叠以及无规则卷曲构成,含有10段内含子序列和11段CDS序列,具有Acetyltransf_1和Bromodomain结构域,以及ARE、GT1-motif、TGACG-motif、CAT-box、CGTCA-motif等顺式作用元件。通过查询GEO数据库发现VvGCN5可能受到光照和灰霉病的影响。VvGCN5主要表达于葡萄的成熟茎、萌芽初期、心皮、种子中。亚细胞定位发现VvGCN5位于细胞核和细胞膜上。此外,以pGBKT7-VvGCN5作为诱饵蛋白,通过酵母双杂交技术和双分子荧光互补技术发现8个阳性蛋白与VvGCN5互作。[结论]本研究鉴定并分析了VvGCN5的基本信息,并得到了8个与VvGCN5互作的蛋白。

[Objectives]The purpose of this paper was to reveal the regulatory mechanism of general control non-repressible 5(GCN5)in the growth and development of grape,and to screen proteins that interacted with GCN5,in order to provide a theoretical basis for the further construction of the regulatory network of VvGCN5 in grape growth and development.[Methods]Using bioinformatics technology,the protein secondary structure,gene structure,functional domains,cis-acting elements of the promoter,as well as space-time expression and expression level of stress resistance of VvGCN5 were analyzed,and subcellular localization in tobacco was conducted to study the expression position of VvGCN5.Proteins interacting with VvGCN5 were screened by yeast two-hybrid technology and verified by bimolecular fluorescence complementation(BIFC)technology.[Results]The secondary structure of VvGCN5 consisted ofα-helix,extended chain,β-sheet and random coil,contained 10 intron sequences and 11 CDS sequences,and had Acetyltransf_1 and Bromodomain domains,and ARE,GT1-motif,TGACG-motif,CAT-box,CGTCA-motif and other cis-acting elements.By querying the GEO database,it was found that VvGCN5 might be affected by light and downy mildew.VvGCN5 was mainly expressed in mature stems,early germination stages,carpels and seeds of grapes.Subcellular localization found that VvGCN5 was located on the nucleus and cell membrane.In addition,using pGBKT7-VvGCN5 as a bait protein,8 positive proteins were found to interact with VvGCN5 through yeast two-hybrid technology and BIFC.[Conclusions]In this study,the basic information of VvGCN5 was identified,and 8 proteins interacting with VvGCN5 were obtained.