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致病疫霉坏死基因PcF/SCR.l的克隆及功能分析
  • 摘要

    PcF(Phytophthora cactorum-fragaria)蛋白是一类只存在于疫霉属(Phytophthora)中的重要致病因子.致病疫霉(Phytophthora infestans)基因组学研究预测出了20个PcF/SCR(small cysteine-rich)-like基因,但其对寄主植物的致坏死功能尚未被证实.本研究选取其中编码蛋白被预测具有4个二硫键的基因PcF/SCR.l(GenBank:XM_002902885),对其进行基因克隆和致坏死功能分析.利用RT-PCR获得336 nt的全长cDNA,将其连入表达载体pBI121后,转化农杆菌(Agrobacterium tumefaciens)LBA4404,注射接种本生烟(Nicotiana benthamiana),观察叶片症状.在注射接种后5d时叶片开始出现黄化,7d时叶片出现严重皱缩和坏死症状,初步表明该基因具有致坏死功能.序列比对分析表明,PcF/SCR.l基因编码的蛋白具有与PcF蛋白相类似的3个二硫键(C89和C99,C68和C100,C73和C104)和1个仅存在于PcF/SCR.l的潜在二硫键(C88和C110),推测其在维持蛋白结构功能方面发挥重要作用.为深入了解PcF/SCR.l基因的致坏死功能,利用试剂盒定点突变该基因编码蛋白的第99、100、104和110位上的半胱氨酸,构建缺失二硫键突变体的重组质粒,并在本生烟中进行瞬时表达.仅突变110位半胱氨酸,或者同时突变99位和110位两个位点的半胱氨酸时,PcF/SCR.l基因的致坏死功能并没有受到影响;当半胱氨酸的突变个数增加到3个(C99/100/110A)或者4个(C99/100/104/110A)时,接种烟草叶片没有出现坏死症状,表明二硫键在该基因的致坏死功能中具有重要作用.为进一步了解PcF/SCR.l基因在致病疫霉侵染马铃薯(Solanum tuberosum)过程中的表达模式,利用荧光定量RT-PCR分析其在不同侵染时期表达量的变化,结果显示,该基因在整个侵染过程中均有不同程度的上调,而且在接种48 h时表达量达到最高值,说明该基因可作为毒素因子在病菌侵染马铃薯叶片的整个过程中都起作用,而且主要作用可能是在侵染后期(48 h)通过分泌大量的毒素蛋白来破坏寄主的防御系统进而促进自身的定殖.对PcF/SCR.l基因致坏死功能的研究,有助于深入了解该基因在致病过程中的作用及机制,为PcF/SCR-like家族其他基因的功能研究提供参考.

  • 作者

    赵冬梅  徐进  杨志辉  朱杰华  朱丽丹  ZHAO Dong-Mei  XU Jin  YANG Zhi-Hui  ZHU Jie-Hua  ZHU Li-Dan 

  • 作者单位

    河北农业大学植物保护学院,保定,071000

  • 刊期

    2014年6期 ISTIC PKU

  • 关键词

    PcF(Phytophthora cactorum-fragaria)/SCR.l(small cysteine-rich)-like基因  致病疫霉  致坏死功能  PcF(Phytophthora cactorum-fragaria)/SCR.l(small cysteine-rich)-like gene  Phytophthora infestans  Necrosis-inducing function 

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