Role of two arginine residues in domain II, loop 2 of Cry1Ab and Cry1Ac Bacillus thuringiensis delta-endotoxin in toxicity and binding to Manduca sexta and Lymantria dispar aminopeptidase N.
Role of two arginine residues in domain II, loop 2 of Cry1Ab and Cry1Ac Bacillus thuringiensis delta-endotoxin in toxicity and binding to Manduca sexta and Lymantria dispar aminopeptidase N.
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Cry1Ab 和 Cry1Ac 苏云金芽孢杆菌 δ-内毒素的结构域 II、环 2 中的两个精氨酸残基在毒性以及与 Manduca sexta 和 Lymantria dispar 氨肽酶 N 结合中的作用。
DOI：
10.1046/j.1365-2958.2000.02109.x
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发表时间：
2000
影响因子：
3.6
通讯作者：
Dean,DH
中科院分区：
文献类型：
--
作者：
Lee,MK;Rajamohan,F;Jenkins,JL;Curtiss,AS;Dean,DH
通过定点突变将Cry1Ab和Cry1Ac的两个精氨酸残基（368 - 369）突变为丙氨酸、谷氨酸和赖氨酸。研究了突变体毒素对烟草天蛾（Manduca sexta）和毒蛾（Lymantria sativa）幼虫的抑制活性。Cry1Ac突变体毒素（c）RR-AA和（c）RR-EE和Cry1Ab突变体毒素（B）RR-AA和（B）RR-EE显示出对两种昆虫的毒性大大降低。相比之下，保守改变的（c）RR-KK和（B）RR-KK突变体不会改变对任一昆虫的毒性。使用由mL制备的刷状缘膜囊泡（BBMV）的结合测定。中肠证实，与它们各自的野生型毒素相比，（c）RR ‐ AA、（c）RR ‐ EE、（B）RR ‐ AA和（B）RR ‐ EE以较低的亲和力结合。汤姆. sextaBBMV，（c）RR-AA和（c）RR-EE显示BBMV结合大大降低。然而，（B）RR ‐ AA和（B）RR ‐ EE没有改变BBMV竞争模式，尽管它们的毒性降低。用从mL中纯化的氨肽酶N（APN）进行进一步的结合试验。M.使用表面等离子体共振（BIAcore）对SextaBBMV进行了分析。使用这种技术观察到突变体毒素的毒性和APN结合之间的直接相关性。BBMV和APN与Cry1Ab toM结合数据之间的不一致性。六表明不同的Cry 1Ab毒素结合机制的可能性或M中另一种受体的重要性。六。
Two arginine residues (368–369) of Cry1Ab and Cry1Ac were mutated to alanine, glutamic acid and lysine by site‐directed mutagenesis. Insecticidal activities of the mutant toxins onManduca sextaandLymantria disparlarvae were examined. Cry1Ac mutant toxins (c)RR‐AA and (c)RR‐EE and Cry1Ab mutant toxins (b)RR‐AA and (b)RR‐EE showed great reductions in toxicity against both insects. In contrast, conservatively changed (c)RR‐KK and (b)RR‐KK mutants did not alter toxicity to either insect. Binding assays with brush border membrane vesicles (BBMVs) prepared fromL. disparmidguts demonstrated that (c)RR‐AA, (c)RR‐EE, (b)RR‐AA and (b)RR‐EE bound with lower affinities compared with their respective wild‐type toxins. ToM. sextaBBMVs, (c)RR‐AA and (c)RR‐EE showed great reductions in BBMV binding. However, (b)RR‐AA and (b)RR‐EE did not alter BBMV competition patterns, despite their reduced toxicity. Further binding assays were performed with aminopeptidase N (APN) purified fromL. disparandM. sextaBBMVs using surface plasmon resonance (BIAcore). Direct correlation between toxicity and APN binding was observed for the mutant toxins using this technique. The inconsistency between BBMV and APN binding data with Cry1Ab toM. sextasuggests the possibility of a different Cry1Ab toxin‐binding mechanism or the importance of another receptor inM. sexta.