碱性磷酸酶和氨肽酶在埃及伊蚊的Cry11Aa抗性品系中发生了改变。
Alkaline phosphatases and aminopeptidases are altered in a Cry11Aa resistant strain of Aedes aegypti.
作者信息
Lee Su-Bum, Aimanova Karlygash G, Gill Sarjeet S
机构信息
Environmental Toxicology Graduate Program, University of California, Riverside, CA 92521, USA; Department of Cell Biology and Neuroscience, University of California, Riverside, CA 92521, USA.
Department of Cell Biology and Neuroscience, University of California, Riverside, CA 92521, USA.
出版信息
Insect Biochem Mol Biol. 2014 Nov;54:112-21. doi: 10.1016/j.ibmb.2014.09.004. Epub 2014 Sep 19.
Bacillus thuringiensis subsp. israelensis (Bti) is widely used for the biological control of mosquito populations. However, the mechanism of Bti toxins is still not fully understood. To further elucidate the mechanism of Bti toxins, we developed an Aedes aegypti resistant strain that shows high-level resistance to Cry11Aa toxin. After 27 selections with Cry11Aa toxin, the larvae showed a 124-fold resistance ratio for Cry11Aa (strain G30). G30 larvae showed cross-resistance to Cry4Aa (66-fold resistance), less to Cry4Ba (13-fold), but not to Cry11Ba (2-fold). Midguts from these resistant larvae did not show detectable difference in the processing of the Cry11Aa toxin compared to that in susceptible larvae (WT). Brush border membrane vesicles (BBMV) from resistant larvae bound slightly less Cry11Aa compared to WT BBMV. To identify potential proteins associated with Cry11A resistance, not only transcript changes in the larval midgut were analyzed using Illumina sequencing and qPCR, but alterations of previously identified receptor proteins were investigated using immunoblots. The transcripts of 375 genes were significantly increased and those of 208 genes were down regulated in the resistant larvae midgut compared to the WT. None of the transcripts for previously identified receptors of Cry11Aa (Aedes cadherin, ALP1, APN1, and APN2) were altered in these analyses. The genes for the identified functional receptors in resistant larvae midgut did not contain any mutation in their sequences nor was there any change in their transcript expression levels compared to WT. However, ALP proteins were expressed at reduced levels (∼ 40%) in the resistant strain BBMV. APN proteins and their activity were also slightly reduced in resistance strain. The transcript levels of ALPs (AAEL013330 and AAEL015070) and APNs (AAEL008158, AAEL008162) were significantly reduced. These results strongly suggest that ALPs and APNs could be associated with Cry11Aa resistance in Ae. aegypti.
苏云金芽孢杆菌以色列亚种（Bti）被广泛用于蚊虫种群的生物防治。然而，Bti毒素的作用机制仍未完全明确。为了进一步阐明Bti毒素的作用机制，我们培育了一种对埃及伊蚊具有高抗性的品系，该品系对Cry11Aa毒素表现出高水平抗性。在用Cry11Aa毒素进行27次筛选后，幼虫对Cry11Aa的抗性比达到了124倍（品系G30）。G30幼虫对Cry4Aa表现出交叉抗性（66倍抗性），对Cry4Ba的抗性较低（13倍），但对Cry11Ba没有抗性（2倍）。与敏感幼虫（WT）相比，这些抗性幼虫的中肠在Cry11Aa毒素的加工过程中未显示出可检测到的差异。与WT的刷状缘膜囊泡（BBMV）相比，抗性幼虫的BBMV与Cry11Aa的结合略少。为了鉴定与Cry11A抗性相关的潜在蛋白质，不仅使用Illumina测序和qPCR分析了幼虫中肠的转录本变化，还使用免疫印迹研究了先前鉴定的受体蛋白的改变。与WT相比，抗性幼虫中肠中有375个基因的转录本显著增加，208个基因的转录本下调。在这些分析中，先前鉴定的Cry11Aa受体（埃及伊蚊钙黏蛋白、碱性磷酸酶1、氨肽酶1和氨肽酶2）的转录本均未发生改变。抗性幼虫中肠中已鉴定的功能性受体基因在序列上没有任何突变，与WT相比，其转录本表达水平也没有任何变化。然而，碱性磷酸酶蛋白在抗性品系BBMV中的表达水平降低（约40%）。氨肽酶蛋白及其活性在抗性品系中也略有降低。碱性磷酸酶（AAEL013330和AAEL015070）和氨肽酶（AAEL008158、AAEL008162）的转录水平显著降低。这些结果有力地表明，碱性磷酸酶和氨肽酶可能与埃及伊蚊对Cry11Aa的抗性有关。
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