摘要
甘蔗杆状病毒(sugarcane bacilliform virus,SCBV)是广泛分布于世界甘蔗种植区的严重危害甘蔗种植的植物病毒之一。研究SCBV编码的病毒蛋白功能对于了解该病毒致病机制及抗病育种具有重要意义。本实验室前期从云南勐海的一株Badila甘蔗中分离获得了SCBV的全长基因组序列,并开展了其开放阅读框2(open reading frame 2, ORF2)编码的未知蛋白P2的功能研究。通过原核表达SCBV P2蛋白并经体外核酸亲和结合试验分析,发现SCBV编码的P2蛋白能以序列非特异性方式与同源或异源核酸亲和结合。进一步对P2蛋白中预测的coiled-coil like domain及C-端保守的脯氨酸、赖氨酸富集区域进行缺失及点突变分析,以及凝胶阻滞试验发现,随着C-端逐步截短,P2突变体蛋白与核酸结合的活性也逐渐减弱,其C-端保守的含脯氨酸、赖氨酸的21个氨基酸(amino acids,aa)对P2蛋白与核酸结合是必需的。同时,在P2蛋白与核酸的相互作用中,coiled-coil like domain被证明是不可或缺的。进一步酵母双杂交实验证明P2蛋白能与自身互作,且P2 C-端保守的21个氨基酸及coiled-coil like domain的缺失或替换直接影响了P2蛋白的自身互作。综上所述,本研究发现SCBV编码的P2蛋白是一个序列非特异性的核酸结合蛋白,且coiled-coil like domain和C-端保守的富含脯氨酸、赖氨酸区域通过影响其自身互作而影响其核酸结合活性。研究结果为进一步深入研究P2蛋白功能奠定了基础。
Sugarcane bacilliform virus(SCBV) is one of the severe plant virus that seriously threaten sugarcane production and is widely distributed in the sugarcane cultivation areas worldwide. It is of great significance to study the function of SCBV-encoded viral proteins for understanding the pathogenic mechanism of virus and viral resistance breeding. The complete genome of SCBV was obtained from cultivar Badila from Yunnan, China, and the research was focused on the function of the unknown protein P2 encoded by open reading frame 2(ORF2). P2 was expressed in Escherichia coli and purified and was found to be able to bind to cognate and heterogenous DNAs fragments in a sequence-nonspecific manner, when verified by affinity binding assay in vitro. Further, a series of mutants of truncation or point substitution of the predicted coiled-coil like domain or carboxyl terminal conserved proline and lysine rich region were also constructed and expressed. Electrophoretic mobility shift assay(EMSA) shows that the interaction between P2 and nucleic acids was progressively impaired as the C-terminus was gradually truncated, and the C-terminal conserved proline and lysine rich 21 amino acids was proved to be essential for P2-nucleic acids binding. While the coiled-coil like domain was proved to be indispensable for P2-nucleic acids interaction. Further yeast two-hybrid assays demonstrates that P2 can interact with itself, with reduced interacting strength other as the P2 C-terminus is progressively truncated, or deletion and replacement of the coiled-coil like region directly impacts P2 self-interaction. In summary, in this study we proved that the SCBV-encoded P2 protein is a sequence non-specific nucleic acid binding protein, and the coiled-coil like domain and C-terminus containing conserved prolines and lysine can influence its nucleic acid binding activity by regulating self-interactions, which lays the basis for further studies of P2 function.
作者
娄义念
梁凯莉
刘靖颖
王致远
许雄彪
LOU Yinian;LIANG Kaili;LIU Jingying;WANG Zhiyuan;XU Xiongbiao(Guangxi Key Laboratory for Sugarcane Biology,Guangxi University,Nanning,530005;State Key Laboratory for Conservation and Utilization of Subtropical Agro-bioresources,Nanning,530004)
出处
《基因组学与应用生物学》
CAS
CSCD
北大核心
2023年第12期1353-1363,共11页
Genomics and Applied Biology
基金
广西自然科学基金项目(2023GXNSFBA026058)
广西大学农学院科研发展基金(EE101705)
2022年广西研究生教育创新计划项目(YCSW2022065)共同资助。
