• 中文核心期刊
  • CSCD来源期刊
  • 中国科技核心期刊
  • CA、CABI、ZR收录期刊

木薯MeABF1基因的克隆及功能

Cloning and Function of MeABF1 in Cassava

  • 摘要:
    目的 克隆木薯(Manihot esculenta Crantz)转录因子ABF1并诱导其原核表达和分析抗病功能,为进一步研究ABF1抗病功能提供参考依据。
    方法 从木薯‘华南124’(‘South China124’, ‘SC124’)中扩增MeABF1基因编码区序列(coding sequence, CDS),并构建MeABF1-pET32a融合表达载体,转化至DE3感受态细胞中进行蛋白诱导,通过SDS-PAGE以及Western blotting确定MeABF1蛋白的表达情况,使用MeABF1蛋白喷施木薯叶片后测定木薯叶片活性氧(reactive oxygen species, ROS)爆发、病程相关(pathogenesis-related, PR)基因表达及对地毯草黄单胞菌木薯萎蔫致病变种(Xanthomonas axonopodis pv. manihotis, Xam)的抗病性测定。
    结果 MeABF1基因CDS序列长度为1 491 bp。在37 ℃、1 mmol·L−1异丙基-β-D-硫代半乳糖苷(isopropyl β-D-thiogalactoside, IPTG)条件下诱导并取样,IPTG诱导1 h后即能检测到MeABF1-Myc融合蛋白,其条带强度随诱导时间延长而递增。ABF1蛋白能够诱导木薯叶片ROS爆发且最高爆发值是对照组的3倍,蛋白处理后PR基因的表达量提高,MePR1MePR2 表达水平分别是对照组的3.5倍和1.75倍,蛋白连续2 d喷施后侵染Xam测定木薯叶片6 dpi(days post inoculation)细菌数、病斑面积以及木薯叶片表型与对照组相比均表现出抗病特征。
    结论 在37 ℃下,1 mmol·L−1 IPTG能诱导MeABF1-pET32a融合蛋白正确表达,MeABF1蛋白可通过诱导ROS爆发和PR基因的表达,抑制细菌性枯萎病菌在木薯中的侵染。

     

    Abstract:
    Objective The gene of transcription factor ABF1 from Manihot esculenta Crantz (MeABF1) was cloned, and its prokaryotic expression and disease resistance-related function analyzed.
    Method Coding sequence (CDS) of MeABF1 was amplified from the M. esculenta cultivar, 'South China 124' ('SC124'). MeABF1-pET32a fusion expression vector was constructed and transformed into DE3 competent cells for protein induction. Expression of the gene was determined by SDS-PAGE and western blotting. Cassava leaves were sprayed with a solution of purified MeABF1 protein and tested for burst of reactive oxygen species (ROS), expression of pathogenesis-related gene (PR), and disease resistance to Xanthomonas axonopodis pv. manihotis (Xam).
    Result The CDS sequence of MeABF1 was 1491 bp long. The MeABF1-Myc fusion protein with increasing band intensity over time was detected after 1 mmol·L−1 isopropyl β-D-thiogalactoside (IPTG) induction at 37 ℃ for 1 h. The peak ROS burst in the treated leaves was 3-fold of control, and the MePR1 and MePR2 expressions upregulated 3.5-fold and 1.75-fold, respectively. A 2 d treatment significantly enhanced the disease resistance to Xam of the cassava leaves. The bacterial counts and lesion area on the leaves 6 d post inoculation (dpi) were significantly reduced with improved phenotypic outcomes over control.
    Conclusion MeABF1-pET32a was successfully expressed in cassava leaves with the induction by 1 mmol·L−1 IPTG at 37 °C. The foliar MeABF1 application significantly enhanced the resistance of the leaves to Xam infection with burst of ROS and heightened PR expression.

     

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