对地中海粉螟（Ephestia kuehniella Zeller，鳞翅目：螟蛾科）具有高毒性的苏云金芽孢杆菌MEB4菌株的分子特征分析

Molecular characterisation of Bacillus thuringiensis strain MEB4 highly toxic to the Mediterranean flour moth Ephestia kuehniella Zeller (Lepidoptera: Pyralidae).

作者信息

Abdelmalek Nouha, Sellami Sameh, Ben Kridis Asma, Tounsi Slim, Rouis Souad

机构信息

Laboratory of Plant Protection and Improvement (Biopesticides Team), Centre of Biotechnology, University of Sfax, Sfax, Tunisia.

出版信息

Pest Manag Sci. 2016 May;72(5):913-21. doi: 10.1002/ps.4066. Epub 2015 Jul 24.

DOI:10.1002/ps.4066

PMID:26103535

Abstract

BACKGROUND

Cry2 proteins play an essential role in current Bacillus thuringiensis (Bt) applications and in the prevention of insect resistance to Cry1A toxins. This paper reports on the screening and characterisation of novel Bt strains harbouring effective cry2A-type genes and higher insecticidal activity to Ephestia kuehniella.

RESULTS

A total of 29 native Bt strains were screened to search for the potent strain against E. kuehniella. The plasmid pattern of the selected strains showed interesting variability. PCR-RFLP analysis of two amplified regions showed high sequence identity within the selected cry2A-type genes. SDS-PAGE and western blot analysis revealed the presence of Cry2Aa toxin only in the MEB4 and BLB240 strains. The activation of Cry2Aa protoxins by larval midgut juice, trypsin or chymotrypsin enzymes revealed significant differences in terms of proteolysis profiles. Interestingly, a 49 kDa band was detected in the proteolysis pattern of BLB240, suggesting the presence of a chymotrypsin cleavage site that might have affected its insecticidal activity. Further, bioassays demonstrated that MEB4 (103.08 ± 36 µg g(-1)) was more active than BLB240 (153.77 ± 45.65 µg g(-1)) against E. kuehniella.

CONCLUSION

Based on its potent insecticidal activity, the MEB4 strain could be considered to be an effective alternative agent for the control of E. kuehniella.

摘要

背景

Cry2蛋白在当前苏云金芽孢杆菌（Bt）的应用以及预防昆虫对Cry1A毒素产生抗性方面发挥着重要作用。本文报道了携带有效cry2A类基因且对地中海粉螟具有更高杀虫活性的新型Bt菌株的筛选与特性研究。

结果

共筛选了29株本地Bt菌株以寻找对地中海粉螟有效的菌株。所选菌株的质粒图谱显示出有趣的变异性。对两个扩增区域的PCR-RFLP分析表明，所选cry2A类基因内具有高度的序列同一性。SDS-PAGE和western blot分析表明，仅在MEB4和BLB240菌株中存在Cry2Aa毒素。幼虫中肠液、胰蛋白酶或糜蛋白酶对Cry2Aa原毒素的激活在蛋白水解谱方面显示出显著差异。有趣的是，在BLB240的蛋白水解图谱中检测到一条49 kDa的条带，这表明存在一个可能影响其杀虫活性的糜蛋白酶切割位点。此外，生物测定表明，MEB4（103.08±36 μg g(-1)）对地中海粉螟的活性高于BLB240（153.77±45.65 μg g(-1)）。