• 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 1,491bp long. The MeABF1-Myc fusion protein with increasing band intensity over time was detected after 1mmol·L1 isopropyl β-D-thiogalactoside (IPTG) induction at 37℃ for 1h. 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 2d treatment significantly enhanced the disease resistance to Xam of the cassava leaves. The bacterial counts and lesion area on the leaves 6d 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 1mmol·L1 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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