Juárez-Hernández E O, Casados-Vázquez L E, del Rincón-Castro M C, Salcedo-Hernández R, Bideshi D K, Barboza-Corona J E
Life Science Division, Graduate Program in Biosciences Irapuato, University of Guanajuato Campus Irapuato-Salamanca, Irapuato, Guanajuato, México.
Food Department, University of Guanajuato Campus Irapuato-Salamanca, Irapuato, Guanajuato, México.
J Appl Microbiol. 2015 Dec;119(6):1692-9. doi: 10.1111/jam.12962.
The objective of this study was to produce stable inclusions of chitinase ChiA74Δsp in Bacillus thuringiensis subsp. israelensis (Bti) and to assay its insecticidal activity against Aedes aegypti larvae.
Bti was transformed with chiA74Δsp regulated by its own promoter or by the strong chimeric cytAp/STAB-SD promoter system to generate two recombinant Bti strains. These recombinants produced their native parasporal bodies composed of Cry4Aa, Cry4Ba, Cry11Aa and Cyt1Aa and ChiA74Δsp inclusions, and showed a approx. threefold increase in both endochitinase activity and viable spore count when compared with the parental strain. Both recombinants were approximately twofold more toxic (LC50s 8·02, 9·6 ng ml(-1) ) than parental Bti (19·8 ng ml(-1) ) against 4(th) instars of A. aegypti larvae.
ChiA74Δsp inclusions, together with the insecticidal crystals and spores of Bti increased the toxicity against A. aegypti larvae by at least twofold.
We report for the first time the engineering of Bti to produce spore-parasporal body-ChiA74∆sp inclusions in the same sporangium, which are released together following autolysis. Our work lays a foundation for engineering Bti to produce more efficacious combinations of Cry4Aa, Cry4Ba, Cry11Aa, Cyt1Aa and chitinase inclusions.
本研究的目的是在苏云金芽孢杆菌以色列亚种（Bti）中产生几丁质酶ChiA74Δsp的稳定包涵体，并测定其对埃及伊蚊幼虫的杀虫活性。
用由其自身启动子或强嵌合cytAp/STAB-SD启动子系统调控的chiA74Δsp转化Bti，以产生两种重组Bti菌株。这些重组体产生了由Cry4Aa、Cry4Ba、Cry11Aa和Cyt1Aa以及ChiA74Δsp包涵体组成的天然伴孢晶体，与亲本菌株相比，内切几丁质酶活性和活孢子数均增加了约三倍。两种重组体对埃及伊蚊4龄幼虫的毒性（LC50分别为8.02、9.6 ng/ml）比亲本Bti（19.8 ng/ml）高约两倍。
ChiA74Δsp包涵体与Bti的杀虫晶体和孢子一起，使对埃及伊蚊幼虫的毒性至少提高了两倍。
我们首次报道了对Bti进行工程改造，使其在同一个孢子囊中产生孢子-伴孢晶体-ChiA74∆sp包涵体，自溶后它们一起释放。我们的工作为改造Bti以产生更有效的Cry4Aa、Cry4Ba、Cry11Aa、Cyt1Aa和几丁质酶包涵体组合奠定了基础。
