Characterization of a novel Cry9Bb delta-endotoxin from Bacillus thuringiensis.
Characterization of a novel Cry9Bb delta-endotoxin from Bacillus thuringiensis.
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DOI：
10.1016/j.jip.2008.03.012
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发表时间：
2008-07
影响因子：
3.4
通讯作者：
J. O. Silva-Werneck;D. Ellar
中科院分区：
文献类型：
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作者：
J. O. Silva-Werneck;D. Ellar
选择巴西苏云金芽孢杆菌日本血清型菌株S725，以测定其对绒豆毛虫（Anticarsia gemmatalis）的毒性。该菌株产生球形晶体，其含有约130 kDa的主要蛋白质，其在胰蛋白酶活化时产生50至70 kDa的片段。该蛋白对Cry 9类δ-内毒素具有高度的同一性和免疫亲和性。克隆的cry 9类似基因序列包含一个3492 bp的开放阅读框，编码1163个氨基酸，预测分子量为131.4kDa。推导的氨基酸序列与Cry 9 Ba、Cry 9 Ea、Cry 9Da和Cry 9 Ca蛋白的同源性分别为73%、64%、63%和59%。新的δ-内毒素被Bt毒素命名委员会指定为一个新的亚类Cry 9 Bb。Cry 9 Bb蛋白在无晶体Bt菌株中表达，并表现出对烟草天蛾Manduca sexta和绒豆毛虫A.芽状体研究了氨基酸残基变化A84 P的生物学效应。Cry 9 Bb晶体对M.突变体Cry 9 Bb晶体对六龄幼虫的LC_（50）为6.84μg/cm ~ 2，对六龄幼虫的LC_（50）为0.78μg/cm ~ 2。PCR筛选结果表明，Bt菌株S725中除cry 9 Bb基因外，还含有cry 1 I和vip 3基因。利用RT-PCR对cry 1 I基因进行转录分析，结果表明cry 1 I基因在孢子形成的T2和T5期转录。
The Brazilian Bacillus thuringiensis serovar japonensis strain S725 was selected for its toxicity to the velvetbean caterpillar, Anticarsia gemmatalis. This strain produces spherical crystals harbouring a major protein of about 130kDa which yields fragments of between 50 and 70kDa upon trypsin activation. The protein showed a high level of identity and immunoafinity to the Cry9 class of δ-endotoxins. The cloned cry9-like gene sequence contains a 3492bp ORF, which encodes a polypeptide of 1163 amino acids, with a predicted molecular mass of 131.4kDa. The deduced amino acid sequence is unique and shows 73% identity to Cry9Ba, 64% identity to Cry9Ea, 63% identity to Cry9Da, and 59% identity to Cry9Ca proteins. The novel δ-endotoxin was assigned to a new subclass, Cry9Bb, by the Bt Toxin Nomenclature Committee. The Cry9Bb protein was expressed in an acrystalliferous Bt strain, and exhibited activity against the tobacco hornworm, Manduca sexta, and the velvetbean caterpillar, A. gemmatalis. The biological effect of an amino acid residue change, A84P, was investigated. The LC50for the Cry9Bb crystals against M. sexta neonate larvae was 6.84μg/cm2, while the LC50for the mutant’s Cry9Bb crystals was 0.78μg/cm2. PCR screening revealed that in addition to cry9Bb, Bt strain S725 also contains cry1I and vip3 genes. Transcription analysis, using RT-PCR, showed that the cry1I gene was transcribed at T2and T5stages of sporulation.