Mining Of Insecticidal Gene Resources, Functional Verification And Evolutionary Study Of Bacillus Thuringiensis

The substance of this study is to mining, identification and cloning insecticidal strains and insecticidal gene resources from Bacillus thuringiensis which come from different ecological regions and to finding the candidate genes which have production potential application in insecticidal genetic resources for insect-resistant crops breeding. Around these goals, this study was carried out following experiments:1) Create a new method of screening insecticidal gene resources, established a high-throughput, efficient, economical, comprehensive and accurate new system used for identification insecticidal genes from Bacillus thuringiensis, and obtain some new modal insecticidal genes from some known strains through this system. Novel modal insecticidal genes were examined by expression validation of crystal morphology and insecticidal activities;2) Collect soil samples from two regions XiChang and PanZhiHua which have special ecological features compared with the other regions in SiChuan Basion and isolated Bacillus thuringiensis trains from these soil samples. Identification and obtain different Bacillus thuringiensis producted different types of parasporal crystals and new modal insecticidal genes;3) Analysed the genome phylogenetic of the strains containing different types of insecticidal genes.Bacillus thuringiensis (Bt) is an environment friendly green microbial insecticides which is widely used to control agriculture pests, forest pests, fruit trees and stored product insects. However, the target insect adapt to the use of single insecticidal and genes. New gene mining strategy may overcome the resistance problem caused by large-scale applications of the cry1type insecticidal genes and the problem of poor insecticidal genes resource, because new gene mining strategy may offer candidate gene resources of insect-resistant crops germplasm and candidate problem of resource-poor insecticidal genes for breeding. Based on this, the main results of this study are as follows:1. The construction of new system and method for mining novel insecticidal gene resources. Twenty-two strains were screened and finally five strains were selected out as candidate strains by screening the crystal types and the plasmid bands. The plasmids from each strain were extracted and mixed as one DNA sample and was constructed a paired-End DNA library with an insert sequence of460bp for sequencing (Illumina HiSeq2000). Finally we obtained57,153,336paired-end Reads. The short Illumina reads were de novo assembled by using Velvet1.1.05and2,286Contigs with an average length of5,347were obtained. GLIMMER3.02was used with the model parameter trained on assembled contig sequences to predict all of the open reading frams(ORFs) and12,696ORFs with an average length of756bp were obtained. Combined with the gene annotation in database PFAM, GO, NR, KEGG, Swiss-Prot, TrEMBL and the gene sets of cry, cyt and vip and the BLASTX (NCBI). Finally17fragments showed some amino acids identities to insecticidal genes were obtained. There were13cry-like genes, three cyt-like genes and one vip-like gene were selected out. The primers were designed and used for amplification full length genes. Finally,17full length genes were obtained. The study results showed that our study constructed a high-throughput, economical, rapid, comprehensive, accurate system for identification toxin genes in Bacillus thuringiensis.2. Full length gene cloning, expression and function verification. Using the PCR technology cry1Ac, cry2Aa, cry54Aal, cry30Fal, cry54-like, cry30Fal, cry52Bal, cytlDa2, cyt1Dal, cyt2Aa3, vip3Aa29and one binary gene were obtained. Three genes: cry59Bal, cry56Aa3, cry70Aal and one cry39ORF2were novel genes. Gene cry59Bal, cry56Aa3and cry70Aal were chosen for expression in a shuttle expression plasmid pSTK and constructed engineering strains by introducing the recombinant plasmids into the crystal-negative mutant strain HD73-by electropotation. The positive recombinant strains were named as HD56Aa3, HD59Bal and HD70Aal. The SEM analysis and SDS-PAGE showed the recombinant strains HD56Aa3, HD59Bal produced spherical parasporal crystals. The insecticidal activities assay against the modal Leipidoptera insects (Spodoptera exihgue) and the modal Diptera insects(Culex quinquefasciatus) indicate Cry56Aa3and Cry59Bal had insecticidal activities against S. exigua, while the Cry70Aal had no insecticidal activities. The three protein had no insecticidal activities C. quinquefasciatus. The LCso of Cry56Aa3toxic to S. exigua was15.69μg/mL,95%confidence limit=12.89-19.01pg/mL; the LCso of Cry59Bal toxic to S. exigua was26.2μg/mL,95%confidence limit=16.2-75.3μg/mL.3. Collecting the soil samples and analysis of the resources from Xichang and Panzhihua. 729soil samples were collected from Xichang and Panzhihua,695Bt-like strains were isolated using sodium acetate antibiotic medium. All of the695strains were observed by microscope and15strains had the abilities to produce parasporal crystals. The rate of Bt which produced parasporal crystals was2.15%. Nine strains were isolated from Lushan, and produced spheres crystals and bipyramidal crystals; four strains were isolated from Panzhihua and can produced spheres crystals and bipyramidal crystals; two strains were isolated from the mountains surround by Xichang downtown and produced spheres crystals. There were no Bt strains isolate from Luoji Mountain. The37pair of primers collected in our lab were used for preliminary identification the toxin genes of the15strains.17genotype including cry3, cry7, cry9, cry15, cryl8, cry20, cry26, cry28, cry30, cry34, cry36/49, cry52, cry59, cytl and vip1were identified. Gene type cyt1, cry56and cry9were the more type in these five strains, while gene type cry3, cry7, cry18, cry34and cry52were identified from one strain. The SDS-PAGE analysis and the primer analysis indicate the strains in Xichang and Panzhihua had a big different compared with the strains in other regions of Sichuan basin. This study may had the ability to enriching the resources of Bt strains and toxin genes.4. The analysis of genome phylogenetic:24strains including11strains stored in our lab,8strains isolated from Xichang and Panzhihua and5strains collected from NCBI. The sequences information of local strains were obtained by sequencing, the other sequences information was collected from NCBI. The phylogenetic analysis of the strains from Panxi area and the strains from other ecological zone showed the evolution position was different from the other strains and had a special position.