Identification And Cloning Of Novel Cry Genes From Bacillus Thuringiensis Using Redundant Exclusion PCR

Bacillus thuringiensis(Bt), one of the most effective microbial pesticides for pest control, had high specificity to agricultural pests, which showed tremendous application value and broad application prospect. However, Bt strains could only be identified one by one with the traditional methods, which were slow speed and inefficient. To solve this problem, a method named redundant exclusion PCR(RE-PCR) were designed. In RE-PCR, the Bt pooled genomic DNA was used as the template, and a pair of generic amplification primers and special primers were added in a same reaction system. The special primers designed to specifically bind to redundant, unwanted genes that were a subset of those copied by the amplification primers.A pooled genemic DNA prepared from 2000 Bt strains was established and used to clone cry8 and cry9 novel genes. With the aim of optimizing the cloning of novel genes from a genomic pool containing many previously identified, homologous, genes we designed a redundant exclusion PCR technique. The generic amplification primers were used to construct a gene library to identify the cry8 and cry9 genes of Bt pooled genomic DNA. Result showed that more than 95% was known genes, of which cry9 Ea and cry9 Da genes had a higher proportion, and the proportion of novel cry8-like(accession: KU143733)gene was 4.5%. The specific primers of cry9Ea/b and cry9 Da were designed(RE9Ea/b_F and RE9Da_F) and used in RE-PCR to block amplification of the full length redundant gene. A new gene library was constructed and quite different from the former library. In new gene library, two of these profiles(cry9Ea and cry8-like) were the same as previously identified while the other three corresponded to cry8 Fa, cry8Ab-like and cry8 Ea. The cry9 Ea profile was detected and its frequency had dropped to 7% left. The cry9 Da and cry9 Eb profiles had successfully been excluded. In contrast the frequency of the cry8-like profile rose to 42.5%. The method was proved very efficient at increasing the number of rare genes in the resulting libraryNew cry8-like gene was highly similar to Cry8 Ab sharing 79%. The truncated cry8-like gene was fused to cry8 Ea C-terminal coding region and then sub-cloned into p STK and introduced into HD73- for expression and the engineered Bt strain named HD8. The Hycry8 protein expressed 130 k Da protein in this host and accumulated as spherical crystals. When the protein was treated with chymotrypsin, a 60 k Da protein was obtained as expected.The insecticidal activity of hycry8 was tested against Colaphellus bowringi and the insecticidal activity of spore-crystal mix from the recombinant Bt strain was tested against larvae of 15-day-old Anomala corpulenta, 5-day-old Holotrichia parallela, and 5-day-old Holotrichia oblita. Strain HD8 was shown to be toxic to A. corpulenta larvae.In conclusion, the RE-PCR method we established was an efficient way to mine novel genes from a pooled DNA sample from a very large culture collection. The method could improve the efficiency of new gene identification and cloning effectively, and will play an important role in discovery and identification for pesticidal genes of Bacillus thuringiensis.