http://www.ahs.ac.cn/images/0513-353X/images/top-banner1.jpg|#|苹果
http://www.ahs.ac.cn/images/0513-353X/images/top-banner2.jpg|#|甘蓝
http://www.ahs.ac.cn/images/0513-353X/images/top-banner3.jpg|#|菊花
http://www.ahs.ac.cn/images/0513-353X/images/top-banner4.jpg|#|灵芝
http://www.ahs.ac.cn/images/0513-353X/images/top-banner5.jpg|#|桃
http://www.ahs.ac.cn/images/0513-353X/images/top-banner6.jpg|#|黄瓜
http://www.ahs.ac.cn/images/0513-353X/images/top-banner7.jpg|#|蝴蝶兰
http://www.ahs.ac.cn/images/0513-353X/images/top-banner8.jpg|#|樱桃
http://www.ahs.ac.cn/images/0513-353X/images/top-banner9.jpg|#|观赏荷花
http://www.ahs.ac.cn/images/0513-353X/images/top-banner10.jpg|#|菊花
http://www.ahs.ac.cn/images/0513-353X/images/top-banner11.jpg|#|月季
http://www.ahs.ac.cn/images/0513-353X/images/top-banner12.jpg|#|菊花

园艺学报 ›› 2016, Vol. 43 ›› Issue (1): 15-24.doi: 10.16420/j.issn.0513-353x.2015-0574

• 果树 • 上一篇    下一篇

响应葡萄根瘤蚜侵染的扩展蛋白基因筛选及其多克隆抗体的制备

季兴龙1,韩 宁2,翟 衡1,杜远鹏1,*   

  1. 1山东农业大学园艺科学与工程学院,作物生物学国家重点实验室,山东泰安 271018;2齐鲁工业大学生物工程学院,济南 250353
  • 出版日期:2016-01-25 发布日期:2016-01-25
  • 基金资助:

    国家自然科学基金项目(31201609);山东省中青年科学家基金项目(BS2012NY007);山东省自然科学基金项目
    (ZR2013CQ022);高等学校博士学科点专项科研基金项目(20113702120007)

Identification of VvEXP Gene from Grape Roots Infected by Phylloxera and Preparation of Polyclonal Antibody to Grape Expansin

JI Xing-long1,HAN Ning2,ZHAI Heng1,and DU Yuan-peng1,*   

  1. 1College of Horticulture Science and Engineering,Shandong Agricultural University,State Key Lab of Crop Biology,Tai’an,Shandong 271018,China;2College of Bioengineering,Qilu Technology of University,Ji’nan 250353,China
  • Online:2016-01-25 Published:2016-01-25

摘要:

以对葡萄根瘤蚜敏感的‘克瑞森无核’葡萄(Vitis vinifera‘Crimson Seedless’)组培苗和抗性砧木‘1103P’组培苗为试材,通过荧光定量PCR技术从扩展蛋白基因家族中筛选到经根瘤蚜侵染后表达上调的扩展蛋白基因家族成员VvEXPA1。采用RT-PCR方法克隆到VvEXPA1基因片段,连接至原核表达载体pET-32a中,转化大肠杆菌DE3菌株,测序确定构建的重组载体pET32a-VvEXPA1开放阅读框正确,并经IPTG诱导表达了融合蛋白。该蛋白以包涵体的形式存在,纯化后免疫新西兰兔,制备扩展蛋白多克隆抗体,通过Western-blot和ELISA检测抗体的特异性及效价。结果显示制备的抗体具有较高的特异性,ELISA显示效价为1︰51 200。

关键词: 葡萄, 根瘤蚜, 扩展蛋白, VvEXPA1, 多克隆抗体制备

Abstract:

Based on qRT-PCR method,an expansin(VvEXPA1)was acquired and investigated from infected Crimson Seedless grape and 1103P grape rootstock in present study. VvEXPA1 was cloned into prokaryotic expression vector pET32a after identification by enzymatic digestion and sequencing,and then the recombinant plasmid with VvEXPA1 was transformed into E. coli DE3. The results showed that the protein was highly expressed in E. coli and the molecular weight of the recombinant protein was 41 kD. The specific antibody was prepared by immunizing rabbit with the purified protein,and then detected by Western-blotting and ELISA. Finally,the high specific antibody was obtained and the titers of anti-VvEXPA1 antibody were 1︰51 200.

Key words: grape, phylloxera, expansin, VvEXPA1, poly-antibody preparation

中图分类号: