[Identification and cloning of vip3A genes from isolates of Bacillus thuringiensis and their bioactivity analysis].
OBJECTIVE
To survey the distribution of vip3A-type genes from isolates of Bacillus thuringiensis in China and to clone novel vip3A genes encoding Vip3A proteins with high insecticidal activity against Lepidopteran insect larvae.


METHODS
We applied PCR-RFLP method to identify vip3A-type genes from 171 isolates and cloned novel vip3Aa genes.


RESULTS
The vip3A-type genes appeared in 63 of 117 B. thuringiensis isolates. We cloned 2 novel vip3Aa genes from isolates of TF9 and Bt16. Then, we subcloned vip3Aa26 and vip3Aa27 into vector pQE30 and transformed into Escherichi coli M15, respectively. The results of SDS-PAGE and Western blot analyses showed that an 88 kDa peptide was expressed in E. coli M15 with 1 mmol/L of IPTG induction at 37 degrees C, respectively. The International Nomenclature Committee of Bt nominated these two genes as the novel vip genes of vip3Aa26 and vip3Aa27, respectively. The bioassay results indicated that the Vip3Aa27 proteins were highly toxic to Trichoplusia ni, Spodoptera exigua and Helicoverpa armigera larvae and the LC50 values were 0.1251 microg/mL, 0.238 microg/mL and 9.238 microg/mL, respectively. However, the Vip3Aa26 protein only possessed toxicity to T. ni larvae.


CONCLUSIONS
The novel Vip3Aa27 protein had higher activity to Lepidopteran insect larvae compared with that for Vip3Aa26 protein. The results demonstrated that some amino acid changes had remarkable effect on the insecticidal activity.
Shen
Jianru
J
College of Plant Protection, Biological Control Center of Plant Diseases and Plant Pests of Hebei Province, Agricultural University of Hebei, Baoding 071001, China. shenjianru1981@yahoo.com.cn
Hou
Mingyu
M
Guo
Wei
W
D000818:Animals; D001413:Bacillus thuringiensis / Q000737:chemistry*; D001426:Bacterial Proteins / Q000302:isolation & purification / Q000494:pharmacology*; D002681:China; D004591:Electrophoresis, Polyacrylamide Gel; D005822:Genetic Vectors; D006460:Hemolysin Proteins / Q000494:pharmacology; D007306:Insecticides / Q000494:pharmacology*; D007814:Larva / Q000187:drug effects* / Q000382:microbiology; D007915:Lepidoptera / Q000187:drug effects*; D010572:Pest Control, Biological
D001426:Bacterial Proteins; D006460:Hemolysin Proteins; D007306:Insecticides; C447139:Vip3A protein, Bacillus thuringiensis