Environmental Biotechnology Department, Centre for Genetic Engineering and Biotechnology (CIGB), Havana 10600, Cuba.
Environ Microbiol. 2010 Nov;12(11):2894-903. doi: 10.1111/j.1462-2920.2010.02259.x.
SfT6 has been identified in a subtracted cDNA library of Spodoptera frugiperda larval midgut transcripts as a serine-protease gene downregulated within 24 h of intoxication with Bacillus thuringiensis Cry1Ca1 protein. In the present study, the specific role of SfT6 during Cry1Ca1 intoxication was investigated by RT-PCR and in vivo RNA interference. Quantitative real-time RT-PCR analysis showed SfT6 mRNA levels in the midgut tissue were significantly reduced after injecting or feeding 4th-instar larvae with specific long-size dsRNA. Gut juice-mediated in vitro protoxin activation and susceptibility for Cry1Ca1 were investigated in Sft6-knockdown larvae and compared with control treated with nonspecific dsRNA. Our results demonstrate SfT6 plays a determinant role in Cry1Ca1 toxicity against S. frugiperda since a decreased expression caused a reduced protoxin activation by larval gut juice and reduced susceptibility of insects to toxin in bioassays. We propose SfT6 downregulation occurring at the early stages of Cry1Ca1 intoxication is part of a complex and multifaceted defensive mechanism triggered in the insect gut to withstand B. thuringiensis pathogenesis.
SfT6 在被苏云金芽孢杆菌 Cry1Ca1 蛋白中毒 24 小时内下调的 Spodoptera frugiperda 幼虫中肠转录本的消减 cDNA 文库中被鉴定为丝氨酸蛋白酶基因。在本研究中，通过 RT-PCR 和体内 RNA 干扰研究了 SfT6 在 Cry1Ca1 中毒过程中的特定作用。定量实时 RT-PCR 分析显示，在注射或喂食 4 龄幼虫特异性长 dsRNA 后，中肠组织中的 SfT6 mRNA 水平显著降低。在 SfT6 敲低幼虫中研究了肠液介导的原毒素激活和对 Cry1Ca1 的敏感性，并与用非特异性 dsRNA 处理的对照进行了比较。我们的结果表明 SfT6 在 Cry1Ca1 对 S. frugiperda 的毒性中起决定作用，因为表达水平降低导致幼虫肠液中原毒素的激活减少，以及昆虫在生物测定中对毒素的敏感性降低。我们提出，Cry1Ca1 中毒早期发生的 SfT6 下调是昆虫肠道中触发的复杂和多方面防御机制的一部分，以抵御苏云金芽孢杆菌的发病机制。
