Laboratório de Bioquímica Estrutural de Proteínas, Departamento de Bioquímica, Centro de Ciências Matemáticas e da Natureza, Instituto de Química, Universidade Federal do Rio de Janeiro, Rio de Janeiro CEP, Brazil.
Laboratório de Bioquímica de Insetos e Parasitos, Instituto de Biofísica Carlos Chagas Filho, Universidade Federal do Rio de Janeiro, Rio de Janeiro CEP, Brazil.
Arch Insect Biochem Physiol. 2019 Sep;102(1):e21591. doi: 10.1002/arch.21591. Epub 2019 Jul 1.
In Brazil, the use of transgenic plants expressing the insect-toxic Bacillus thuringiensis endotoxin has been successfully used as pest control management since 2013 in transgenic soybean lineages against pest caterpillars such as Helicoverpa armigera. These toxins, endogenously expressed by the plants or sprayed over the crops, are ingested by the insect and bind to receptors in the midgut of these animals, resulting in disruption of digestion and lower insect survival rates. Here, we identified and characterized a membrane-associated alkaline phosphatase (ALP) in the midgut of Anticarsia gemmatalis, the main soybean defoliator pest in Brazil, and data suggested that it binds to Cry1Ac toxin in vitro. Our data showed a peak of ALP activity in homogenate samples of the midgut dissected from the 4th and 5th instars larvae. The brush border membrane vesicles obtained from the midgut of these larvae were used to purify a 60 kDa ALP, as detected by in-gel activity and in vitro biochemical characterization using pharmacological inhibitors and mass spectrometry. When Cry1Ac toxin was supplied to the diet, it was efficient in decreasing larval weight gain and survival. Indeed, in vitro incubation of Cry1Ac toxin with the purified ALP resulted in a 43% decrease in ALP specific activity and enzyme-linked immunosorbent assay showed that ALP interacts with Cry1Ac toxin in vitro, thus suggesting that ALP could function as a Cry toxin ligand. This is a first report characterizing an ALP in A. gemmatalis.
在巴西，自 2013 年以来，转 Bt 基因抗鳞翅目害虫（如棉铃虫 Helicoverpa armigera）的大豆品系已成功将表达杀虫毒素的转基因植物用于害虫防治管理。这些毒素由植物内源表达或喷洒在作物上，被昆虫摄入并与这些动物中肠的受体结合，导致消化中断和昆虫存活率降低。在这里，我们鉴定并表征了巴西主要大豆食叶害虫 A. gemmatalis 中肠的膜结合碱性磷酸酶（ALP），并表明其在体外与 Cry1Ac 毒素结合。我们的数据显示，在第 4 和第 5 龄幼虫中肠匀浆样本中，ALP 活性峰值最高。从中肠获得的刷状缘膜囊泡用于纯化 60 kDa 的 ALP，通过凝胶内活性和使用药理抑制剂和质谱的体外生化特性进行检测。当 Cry1Ac 毒素供应到饮食中时，它能有效降低幼虫体重增加和存活率。事实上，Cry1Ac 毒素与纯化的 ALP 在体外孵育导致 ALP 比活降低了 43%，酶联免疫吸附试验表明 ALP 在体外与 Cry1Ac 毒素相互作用，因此表明 ALP 可以作为 Cry 毒素的配体。这是首次在 A. gemmatalis 中鉴定 ALP。
