The Cloning,Expression And Function Analysis Of Nine Novel Insecticidal Genes From Bacillus Thuringiensis

The Cry toxins, which are encoded by the cry genes of Bacillus thuringiensis(Bt), were shown to be toxic to major insects, nematodes, and even cancer cells, but not to humans and animals. Thus, Bt become the most successful used biopesticide. However, the widespread and persistent application of Bt based products has created many problems, such as the relatively narrow insecticidal spectrum, potentially pest-resistant insect strains and insufficient of insecticidal genes resource. Mining and cloning of more novel insecticidal genes is urgent to overcome the above problems. Here, we mainly focused on the following aspects:1. Cloning nine novel insecticidal genes by the high-thoughout platform. Using a fast, efficient platform which was based on high-thoughout, we selected nine novel insecticidal genes which amino acid sequence with less then 45% identity differ known insecticidal genes and completed its original strains identifing and cloning, including 3 cry genes and 6 vip genes. Only three genes have been named by Bacillus thuringiensis nomenclature committee. Cry73Aal is in the primary rank and Cry29Bal, Cry21Eal are in the secondary rank, respectively.2. The expression of Cry toxins. In the current study, the Cry toxins proteins are devided into two parts. Firstly, three cry genes cry29Ba1, cry21Eal and cry73Aal can express crystal proteins. Supprisingly, we could get predicted proteins, only when cry73Aa1 coexpressed with the downstream gene orf2. This indicates that orf2 may play the role as a novel help protein.Secondly, eight cry genes, which have been constructed in the Bt recombination stains in our group(cry2385, cry67Aa1, cry479, cry 1719, cry2986, cry21Cal and cry21Dal), can observate the predicted crystal proteins by using SDS-PAGE and microscope observation.3. Determination of activity of Cry toxins. We selected ten proteins (Cry29Ba1, Cry21Ea1, Cry73Aa, Cry2385, Cry67Aa1, Cry479, Cry 1719, Cry2986,Cry21Cal and Cry21Da1)for insect bioassay. Among them, we chose Cry2986, Cry21Eal, Cry73Aal, Cry21Da1 and Cry21Cal to test against Caenorhabditis elegans and discovering they displayed obvious insect growth-inhibitory activities. We selected Cry2986, Cry21Eal, Cry73Aa1 and Cry29Ba1 to test against Helicoverpa armigera and Plutella xylostella and finding they had no activity (lethal or inhibitory). Except for Cry2385, all of ten proteins showed activity against Culex pipiens fatigans at various degrees. Cry2986 had a high degree of identify (83%) with Cry8Ab which have high activity against Lepidoptera. Supprisingly, instead of being toxic to Lepidoptera, Cry2986 have high activity against Caenorhabditis elegans, which will broaden the insecticidal spectrum of Cry8.The novel insecticidal genes that cloned in this study greatly enriched the types of insecticidal protein genes. The can be used as topical pesticides to protect crops, and the proteins will be expressed in transgenic plants to confer inherent pest resistance.