Isolation Of Bacillus Thuringiensis From Dicranopteris Dichotoma And Analysis Of Novel Cyt1Aa7 Gene

With heat treatment and sodium acetate-antibiotics culture medium,1 novel Bacillus thuringiensis strain named BRC-HQY1 had been isolated from 118 phylloplane samples of Dicranopteris dichotoma.Physiological and biochemical reaction were used to the novel strain for understanding its biological characteristics.Its morphology characteristics was then examined for the presence of parasporal crystals and spores by biological microscope after 72 h incubation at 30℃.The vegetative cell was rhabdoid in a single or twin short chains.They were found that sporogonium contained transparent elliptic spore and roundness or irregular parasporal crystal.In vegetative period,the surface morphology of this novel strain was also observed by atomic force microscopy(AFM).It was found that the vegetative cell with rounded ends had full and smooth surface and was peritrichous flagellated.In order to learn more about the physiological and biochemical characteristics of this novel isolate,sugar fermentation(sucrose,mannose,glucose),amylolysis,voges-proskauer,urea hydrolysis and esculin hydrolysis were performed,respectively.It was found that D-glucose was positive,but mannose and sucrose were negative.Meanwhile,amylolysis,urease,esculin,arginine decarboxylase and gelatin were positive,while Voges-Proskauer test was negative.cry genotypes of BRC-HQY1 were identified by PCR-RFLP,with 10 pairs of universal primers and relevant restriction enzymes previously reported.The results showed that BRC-HQY1 harbored cry4/10 and cry11.However,no other cry gene fragments were detected.PCR technique was also showed that cytl and cyt2 genes were detected in this novel isolate with two pairs of specific primers.SDS-PAGE analysis showed that the binds of 130 kDa,75 kDa and 27 kDa were detected,which were applied to B.thuringiensis subsp.israelensis.Bioassays showed that this novel isolate had high toxicity against Culex quinquefasciatus and Aedes aegypti.It was more toxic for A.aegypti to the former.In order to further study this novel gene which might prolong high-level resistance in the mosquito management,cyr1gene of BRC-HQY1 was successfully cloned and sequenced.Homology analysis showed that it was up to 99%similarity with the reported genes,which was a kind of widely insecticidal spectrum and high mosquitocidal toxin gene.There were about 3-4 nucleotides different from the reported cytlAa genes,and 2 encoded amino acids were different from other CytlAa proteins.The full sequence was obtained as an accession number of KF152888,which was assigned as cytlAa7 by the Bt Nomenclature Committee.Bioinformatics analysis of cytlAa7 gene was found that there was an open reading frame of 750 bp,encoding a protein of 249 residues with a calculated molecular weight of 27.3841 kDa and a predicted pI value of 4.77.Cyt1Aa7 protein had 1 domains,Bac_thur_toxin(Lys18 to Leu249),consisting of 232 amino acide.It was also a hybrid protein,which contained 35.34%of helix,22.49%of E-sheet,8.03%of β-turn,34.14%of random coil,respectively.The three-dimensional protein structure of Cytl Aa7 protein was also modeled by Homology-modeling Server.