Reduced Expression of a Novel Midgut Trypsin Gene Involved in Protoxin Activation Correlates with Cry1Ac Resistance in a Laboratory-Selected Strain of Plutella xylostella (L.)
Reduced Expression of a Novel Midgut Trypsin Gene Involved in Protoxin Activation Correlates with Cry1Ac Resistance in a Laboratory-Selected Strain of Plutella xylostella (L.)
复制标题
在实验室选定的小菜蛾 (L.) 菌株中，参与原毒素激活的新型中肠胰蛋白酶基因的表达减少与 Cry1Ac 抗性相关
DOI：
10.3390/toxins12020076
复制
发表时间：
2020-02-01
期刊：
影响因子：
4.2
通讯作者：
Zhang, Youjun
中科院分区：
文献类型：
--
作者：
Gong, Lijun;Kang, Shi;Zhang, Youjun
苏云金芽孢杆菌（Bacillus thuringiensis，Bt）产生多种杀虫蛋白以杀灭害虫。然而，对Bt毒素的抗性的进化阻碍了该技术的可持续利用。在此之前，我们在实验室选择的抗Cry 1Ac小菜蛾菌株SZ-R的中肠转录组和RNA-Seq数据中发现了胰蛋白酶样丝氨酸蛋白酶基因PxTryp_SPc1的下调。我们在这里表明，减少PxTryp_SPc1的表达显着降低酪蛋白和胰蛋白酶的活性影响Cry 1Ac原毒素激活，从而赋予更高的抗性Cry 1Ac原毒素比激活毒素在SZ-R菌株。本研究克隆了PxTryp_SPc1基因的全长cDNA序列，发现该基因在各龄期幼虫中主要在中肠组织中表达。随后，我们证实了PxTryp_SPc1基因在SZ-R幼虫中肠中显著降低，并且当用高剂量的Cry 1Ac原毒素选择时进一步降低。此外，PxTryp_SPc1基因的下调与SZ-R菌株中对Cry 1Ac的抗性遗传相关。RNAi沉默PxTryp_SPc1基因表达降低了敏感品系DBM 1Ac-S对Cry 1Ac原毒素的敏感性，支持PxTryp_SPc1基因的低表达与小菜蛾对Cry 1Ac的抗性有关。这些发现有助于了解中肠蛋白酶在昆虫对Bt毒素抗性机制中的作用。
Bacillus thuringiensis (Bt) produce diverse insecticidal proteins to kill insect pests. Nevertheless, evolution of resistance to Bt toxins hampers the sustainable use of this technology. Previously, we identified down-regulation of a trypsin-like serine protease gene PxTryp_SPc1 in the midgut transcriptome and RNA-Seq data of a laboratory-selected Cry1Ac-resistant Plutella xylostella strain, SZ-R. We show here that reduced PxTryp_SPc1 expression significantly reduced caseinolytic and trypsin protease activities affecting Cry1Ac protoxin activation, thereby conferring higher resistance to Cry1Ac protoxin than activated toxin in SZ-R strain. Herein, the full-length cDNA sequence of PxTryp_SPc1 gene was cloned, and we found that it was mainly expressed in midgut tissue in all larval instars. Subsequently, we confirmed that the PxTryp_SPc1 gene was significantly decreased in SZ-R larval midgut and was further reduced when selected with high dose of Cry1Ac protoxin. Moreover, down-regulation of the PxTryp_SPc1 gene was genetically linked to resistance to Cry1Ac in the SZ-R strain. Finally, RNAi-mediated silencing of PxTryp_SPc1 gene expression decreased larval susceptibility to Cry1Ac protoxin in the susceptible DBM1Ac-S strain, supporting that low expression of PxTryp_SPc1 gene is involved in Cry1Ac resistance in P. xylostella. These findings contribute to understanding the role of midgut proteases in the mechanisms underlying insect resistance to Bt toxins.