利用PLDMV/Twin-Strep侵染性克隆纯化HC-Pro病毒蛋白  

Purification of the Potyviral Helper Component-proteinase Using Agroinfection-compatible Twin-Strep-tagged Infectious cDNA Clone of Papaya Leaf Distortion Virus

作　　者：杨秀坤 沈文涛[2] 庹德财[2] 王赫 言普[2] 黎小瑛[2] 朱国鹏[1] 周鹏[1,2] YANG Xiukun;SHEN Wentao;TUO Decai;WANG He;YAN Pu;LI Xiaoying;ZHU Guopeng;ZHOU Peng(College of Horticulture,Hainan University/Key Laboratory of Quality Control of Tropical Horticultural Crops of Hainan Prov-ince,Haikou,Hainan 570228,China;Institute of Tropical Bioscience and Biotechnology,Chinese Academy of Tropical Agricul-tural Science,Haikou,Hainan 571101,China)

机构地区：[1]海南大学园艺学院/海南省热带园艺作物品质调控重点实验室,海南海口570228 [2]中国热带农业科学院热带生物技术研究所,海南海口571101

出　　处：《热带作物学报》2022年第4期684-692,共9页Chinese Journal of Tropical Crops

基　　金：海南省自然科学基金高层次人才项目(No.320RC717);国家自然科学基金项目(No.32072390)。

摘　　要：番木瓜畸形花叶病毒(Papaya leaf distortion mosaic virus,PLDMV)是一种新的潜在威胁番木瓜种植业的病毒,其辅助成分蛋白酶(helper component-proteinase,HC-Pro)是PLDMV编码参与病毒复制、运动、寄主植物症状表现的多功能蛋白,因此纯化获得具有功能活性的HC-Pro蛋白,研究其多功能性具有重要意义。本研究利用In-Fusion拼接策略和E.coli Cell-Free快速构建植物病毒侵染性克隆法,一步快速地将28个氨基酸组成的蛋白标签Twin-Strep插入到PLDMV HC-Pro氨基端,成功获得了基于农杆菌的携带Twin-Strep标签的PLDMV侵染性克隆pPLDMV-Strep。通过农杆菌渗透注射接种番木瓜植株,从感染PLDMV-Strep的番木瓜叶片总蛋白中,利用Twin-Strep蛋白标签亲和层析分离纯化获得PLDMVHC-Pro蛋白,并通过LC/ESI-MS/MS(liquid chromatography electrospray ionization tandem mass spectrometric)对其进行蛋白N端和C端序列测序,证明了纯化获得完整的PLDMVHC-Pro蛋白。研究结果为后续解析PLDMV HC-Pro在病毒侵染过程中的功能及与寄主蛋白相互作用奠定了基础。Papaya leaf distortion mosaic virus(PLDMV, genus Potyvirus) is an emerging threat to papaya production.The helper component proteinase(HC-Pro) encoded by potyvirus is a multifunctional protein involved in different steps of the viral cycle including aphid-vector transmission, virus replication and movement, cleavage of the polyprotein and suppression of RNA silencing. To enable the study of HC-Pro functions, high quantities of protein are required. Here we describe the construction of a PLDMV infectious clone that produces HC-Pro protein with a Twin-Strep-tag fused at the N-terminus, and the development of an efficient method to purify large amounts of PLDMV HC-Pro protein for the virus-infected papaya plants. To construct an agroinfection-compatible Twin-Strep-tagged PLDMV infectious cDNA clone designated pPLDMV-Strep, the previously constructed pPLDMV-WT plasmid that contained the full-length cDNA of the wild-type PLDMV genome was used as the template to amplify two DNA fragments containing all PLDMV viral sequences, the backbone of binary vector pGreenII-35S and Twin-Strep-tag by PCR with specific two primer pairs. Then,both PCR amplified fragments were assembled to produce the pPLDMV-Strep vector using one-step In-Fusion Cloning.Based on the Escherichia coli Cell-Free method, the pPLDMV-Strep were directly transformed into Agrobacterium tumefaciens to prevent potential problems such as plasmid instability during propagation in E. coli. Sequencing of the PCR products from A. tumefaciens transformants confirmed that the full-length viral sequences in pPLDMV-Strep was identical to that of the wild-type PLDMV isolate and the Twin-Strep-tag was introduced correctly into N-terminus of the PLDMV HC-Pro. The agroinoculated papaya seedlings with A. tumefaciens transformant harboring the plasmid pPLDMV-Strep developed systemic mosaic symptoms on leaves at 20 days post inoculation(dpi) which were similar to those caused by wild-type PLDMV. The results suggested that the PLDMV-Strep was infectious and insertion of Twin-S

关 键 词：番木瓜畸形花叶病毒 HC-PRO 马铃薯Y病毒 蛋白纯化 

分 类 号：S436.67[农业科学—农业昆虫与害虫防治]