Discovery And Activity Analysis Of Novel Insecticidal Genes From Bacillus Thuringiensis

Bacillus thuringiensis(Bt)has become the most widely used microbial pesticides in the world as its advantages that specific insecticidal activity against many agricultural pests,safety for nontarget organisms and environmental friendliness.Its insecticidal activity is attributed largely to insecticidal proteins produced by itself.Most insecticidal proteins are encoded by cry genes and vip genes,and cry genes have been applied in the transgenic crops for over twenty years.Therefore,Bt insecticidal gene discovery has a great significance for pest control.Currently,high-throughput sequencing techniques are the principle means to discover insecticidal genes.But the higher price of sequencing and duplication in Bt resource will cause a great waste of money.So there is an urgent need to establish a method of analyzing and comparing large numbers of isolated strains,and further removing duplicate strains to obtain the strains used for insecticidal gene discovery.When finished sequencing of large numbers of Bt strains,subsequently it usually take more time on the genome sequencing data assembly and insecticidal genes annotation of strains.So it is necessary to improve the efficiency of insecticidal gene discovery.Now discovery of Bt insecticidal genes still has a great demond due to certain pests less sensitive to the existing Bt insecticidal proteins,lack of Bt insecticidal proteins against some new target pests,and insect resistance to Cry proteins expressed in transgenic crops,thus it is very important to discover novel Bt insecticidal proteins with high insecticidal acticivity and no cross-resistance with those Cry proteins in transgenic crops.For the above needs,this thesis is focused on the research of Bt strain isolation technique,insecticidal gene discovery method,and screening and identification of novel Bt genes with insecticidal activity.The main results are as follows:1.Study on Bt strain isolation techniqueThis study compared and analyzed a large number of isolated strains on the basis of Bt insecticidal gene fingerprint using PCR-RFLP technique.Firstly,strain genome extraction method was improved.Magnetic bead method of DNA extraction can achieve rapid and high-throughput extraction of strain genomic DNA.Then,the primer system was improved to detect more insecticidal genes by adding some pairs of primers.The PCR-RFLP pattern analysis system was improved using nucleic acid fragment analyzer on the principle of capillary electrophoresis system which can analyze and compare the PCR-RFLP patterns of many strains,and the pattern information of strains can be output in a digital form.Furthermore,some bioinformatics methods were applied to calculate the similarity values among PCR-RFLP patterns,which could effectively remove the duplicates from a large number of isolated strains.Finally,the remaining strains will be used for further insecticidal gene discovery.2.Study on discovery of insecticidal genes from Bt mixed genome sequencing dataThe simulated Bt mixed genome data was respectively assembled by single-strain genomic assembly software(IDBA and Velvet)and metagenomic assembly software(IDBA-UD and Meta Velvet).Assembly results showed that the IDBA-UD assembly software was the best of four assembly softwares,with the longest N50 and average sequence length.Furthermore,the coding genes were predicted by the Meta Gene Mark software that the longest N50 was obtained from the IDBA-UD assembly results.Insecticidal gene prediction results showed that the largest numbers of insecticidal genes were discovered from the IDBA-UD coding gene pool,which reached 78 percent of the total insecticidal genes from 14 Bt strains.3.Screening of novel Bt genes with insecticidal activityIn this study,we analyzed the genomes of 78 Bt strains using the second-generation sequencing technique,and obtained a large number of novel genes that were similar to the existing insecticidal genes.Partial novel genes(96 novel genes)were screened by insecticidal activity assays,including 31 novel genes ranking the first level,48 novel genes ranking the second level,16 novel genes ranking the third level,and 1 novel gene ranking the fourth level.These novel genes were cloned and expressed in Escherichia.coli.The results showed that 92 novel insecticidal genes could be expressed in E.coli Rosseta(DE3).Finally,the insecticidal activity of these expressed proteins were analyzed,and the results showed a novel insecticidal gene cry9C-like with insecticidal activity against Plutella xylostella and Ostrinia furnacalis was screened from the 92 novel genes,and another 91 novel genes showed no insecticidal activity against several common lepidopteran pests and coleopteran Colaphellus bowringi Baly.4.Characterization of a novel insecticidal protein Cry9Cb1 from BtFurther studies were carried out on the cry9C-like novel insecticidal gene obtained in the above study.Firstly,this gene sequence was submitted to NCBI Gen Bank and the accession number was MK005301,and named cry9Cb1 by Bacillus thuringiensis delta-endotoxin nomenclature committee.SDS-PAGE showed the expressed cry9Cb1 in the acrystalliferous mutant Bt strain HD73-to have a molecular mass of approximately 130 k Da,and this protein was found to be spherical crystal protein by atomic force microscope.Bioassay results showed high insecticidal activity of Cry9Cb1 against some lepidopteran pests including Plutella xylostella with LC50 value of 0.38 ?g/m L,Ostrinia furnacalis with LC50 value of 1.28 ?g/m L,and Chilo suppressalis with LC50 value of 0.50 ?g/m L.In addition,Cry9Cb1 also exhibited medium activity against Agrotis ipsilon and Mythimna separata larvae with LC50 values of 130.22 and 244.77 ?g/m L,respectively.Cry9Cb1 showed no cross-resistance with Cry1 Fa in Ostrinia furnacalis.Its minimal active fragments against Plutella xylostella and Ostrinia furnacalis were identified to be 72T–657V and 68D–655A,respectively;food safety assessment showed no sequence homology with any known allergic protein,and rapid degradation and inactivation by both heat and gastrointestinal environment.In a word,this thesis studied on discovery of Bt insecticidal genes.Improvement of discovery methods will play an important role in improving the efficiency of Bt insecticidal gene discovery.Screening and identification of novel insecticidal genes with high insecticidal activity will lay a good foundation for the development of Bt pesticides and Bt transgenic crops.