Clone, Expression And Function Of Bm-lethal Genes In Silkworm, Bombyx Mori

More than 60% of pests are Lepidoptera insects. By studying and making use of lethal genes of lepidopterous insects, we are able to spread lethal genes in pest population by creating and releasing in the environment certain quantity of male pests of recessive or conditional dominant lethal genes to restrain the number of pests and control insect pests in the long run, which bring forth great significance for biological insects control.The silkworm is modal insect of Lepidoptera, and also the only modal lepidopterous living creature whose genome has been sequenced, which makes the research of functional genes of lepidopterous insects much more convenient. Our researches are based on the silkworm database such as dbWGS, dbEST, dbProtein and so forth. With the published lethal gene sequences of Drosophila melanogaster and Caenorhabditis elegans as original data, we successfully cloned homologous lethal genes in silkworm for the first time and analyzed their configuration and function by means of bioinformatics methods and biological experiments, such as RT-PCR, molecular clone, prokaryotic expression, RNAi and so forth. The main results are as followed:1 The insilico cloning of silkworm homologous lethal geneWith the published 146 lethal gene sequences of Drosophila and Caenorhabditis elegans as original data, we cloned 7 homologous lethal genes from EST database of the silkworm. The 7 genes were named Bm-lethal (2) (GeneBank accession number: EF157831), Bm-lethal (3) s1921, Bm-lethal (3) neo18, Bm-lethal (3) 07882, Bm-lethal (3) 02640, Bm-female lethal and Bm-let-2 (GeneBank accession number: EU183409) respectively. Our researches lay out the most homologous lethal genes of lepidopterous insects among the current reports so far.2 cDNA clone and sequence analysis of silkworm homologous lethal geneWe cloned and sequenced the six homologous gene: Bm-lethal (2), Bm-lethal (3) s1921, Bm-lethal (3) neo18, Bm-lethal (3) 07882, Bm-lethal (3) 02640 and Bm-let-2 from the transcription products of fat bodies in fifth instar larvae of the silkworm C108. Using RACE technology, we successfully got the full-length mRNA sequences of Bm-lethal (2) and Bm-let-2.Bioinformatics analyses display that the amino acids of Bm-lethal(2) contain a DAO (D-amino acid oxidase) conserved domain, including a Glyceraldehyde-3-phosphate dehydrogenase conserved domain, and two Ca2+ binding domains and that it's a kind of FAD dependent oxidoreductase. The amino acids of Bm-lethal (3) s1921 contain six EZ-HEAT repeats, having more than 50 percent of homology with deoxyhypusine hydroxylase of other species, so we assumed it to be a deoxyhypusine hydroxylase. There are two transcript variants of Bm-lethal (3) neo18 in silkworm, encoding the same protein, which have 40 to 60 percent of homology with C-terminal amino acids of NADH dehydrogenase (ubiquinone) 1 beta subcomplex 5. Thus it may take part in oxidation-reduction reaction as a subuit of ubiquinone subcomplex. The amino acids of Bm-lethal (3) 07882 contain Nop14 (nucleolar protein 14) conserved domain, which have 40 to 50 percent of homology with the Nop14 of Homo sapiens and Mus musculus. Therefore, we classified it as Nop14 gene. The amino acids of Bm-lethal(3) 02640 contain a porphobilinogen deaminase conserved domain which is also the binding domain of dipyromethane cofactor, having 60 to 70 percent of homology with N-terminal amino acids of dipyromethane, so we presumed it as dipyromethane gene. The protein of Bm-let-2 has a high homology with the type IV collagenα1(IV) chain of Anopheles gambiae, Apis mellifera and Homo sapiens, the C-terminal amino acids of which contain two overlap type IV collagen conserved domains and 9 collagen conserved domains, indicating it to be type IV collagen gene.Using dbWGS of silkworm, we spliced Bm-lethal (2) and Bm-let-2 full-length genic sequences by means of biological technology and RT-PCR. Through phylogenetic analysis using MAGE3 we found they are closely phylogenetic with insects as Apis mellifera and Drosophila, whereas far away from mammals such as Homo sapiens and Bos Taurus, which is consistent with traditional theory.3 Prokaryotic expression and space-time expression analysis of Bm-lethal(2) gene, and the function studies by RNAi.The prokaryotic expression experiment proves that Bm-lethal (2) is able to be induced to express abundantly in prokaryotic cells. The space-time expression experiments show that Bm-lethal (2) gene expresses slightly in blood, no tissue specificity of expression is found in gonad, fat body and midgut. The expression amount of Bm-lethal (2) gene increases gradually in the period of fifth instar larvae but decreases dramatically in pupa period. The expression amount peaks in the eggs of virgin moth and decreases later on, and the gene hardly expresses in the eggs oviposited 72 h later, even activation treatment by HCl was imposed on the eggs.RNAi research makes out that, after injection of Bm-lethal (2) dsRNA, the expression amount of Bm-lethal (2) decreases remarkably, however, the vitality of the silkworms doesn't change significantly. 9 days after the injection, the silkworms treated by Bm-lethal (2) dsRNA weigh much heavier than those of negative and positive controls. The experimental results above indicate that there may be alternatives to replace Bm-lethal (2) gene and carry out its function after it was repressed.