Study On The Lethal-related Genes In The Silkworm, Bombyx Mori And Cotton Bollworm, Helicoverpa Armigera

Lepidoptera insects are the second biggest order in Insecta and have closely related with human in production and life. Much of Lepidoptera insects are agricultural pests. Silkworm is the model insect of Lepidoptera, which was determined by international non-vertebrate association, and is the only Lepidoptera biology that completed the whole genome sequencing. So it provided a convenient technology platform for functional genomics research.Since 2000, release of insects carrying a dominant lethal technology (RIDL) has been caught extensive attention. It's principle is raised the male that carried conditional lethal genes, and then released into the wild.They will mate with the native population, then all the progeny of such matings will die , so it achieved the purpose of pest control. This technology has a species-specific and environmental characteristic. It has great significance to control agricultural pests. Currently, RIDL technology has been successfully tested in Drosophila melanggaster. In Lepidoptera pests, such as Helicoverpa armigera, also has yielded some results.In this study, we closely around the key word "lethal", relied on the protein sequence which encoded by Drosophila melanggaster lethal genes(P-element induced mutations), Osiris gene family that in the Triplo-lethal (triploid and haploid lethal) locus and thermo-sensitive lethal mutation Pros (Pros261 and Prosβ21) genes respectively, made use of bioinformatics, RT-PCR, RACE, expression and RNA interference technologies, cloned the relevant genes of Bombyx mori and down structural analysis and functional study. The major results are as follows:1. Cloning and expression analysus of the lethal-related genes (P-element induced lethal mutation) from Bombyx moril (3)neo18 gene of fruit fly is the lethal mutation of NADH dehydrogenase gene by inserted P element, so its'encoded protein has the function of NADH dehydrogenase. By using the methods of bioinformatics, RT-PCR and RACE technology, a full-length cDNA was isolated from silkworm, named as Bm-l(3)neo18. We had analyzed the structure and expression of Bm-l(3)neo18. It is 868 bp nucleotide long, contains an ORF (573 bp), 25 bp nucleotide sequence in 5'UTR and 251 bp nucleotide sequence in 3'UTR. It encodes 190 amino acids, with their predicted mass 22.7 kD and isoelectric point 9.60. Bm-l(3)neo18 contains 3 exons and 2 introns. Gene localization indicated that Bm-l(3)neo18 mapped within from 120.3knt to 120.4knt region of nscaf2930 in No. 3 chromosome. The deduced amino acid showed that a NADH dehydrogenase SGDH subunit domain between 1 to 185 amino-acid residue and a transmembrane region between 61-83 amino-acid residue. It has more than 50% identity to other insects such as Aedes aegypti. The NADH dehydrogenase conservative regions were much similar with each other. Molecular evolution by Neighbor Joining method indicated that Bm-l(3)neo18 had homologous with other NADH dehydrogenase of insects. Bm-l(3)neo18 was expressed lowly at egg stage, while expressed highly at larva, pupa and moth stages in our tested samples.lethal (1) G0334 gene of fruit fly is the lethal mutation of pyruvate dehydrogenase (PDH) gene by inserted P element, so its'encoded protein has the function of PDH. By using the methods of bioinformatics, RACE and RT-PCR technology, a full-length cDNA was isolated from silkworm, named as Bm-l (1). We had analyzed the structure and expression of Bm-l (1). It is 1630 bp nucleotide long, contains an ORF (1200 bp), 186 bp nucleotide sequence in 5'UTR and 207 bp nucleotide sequence in 3'UTR. It encodes 399 amino acids, with their predicted mass 43.93 KD and isoelectric point 8.07. Bm-l (1) contains 8 exons and 7 introns. The deduced amino acid showed that a E1-dh domain between 69 to 365 amino-acid residue and it is dehydrogenases (all of which use thiamine pyrophosphate as a cofactor) specificity. Secondary structure prognosticate results revealed:αhelix is 28.8%,βstrand is about 12.0%.It has more than 63% identity to other insects such as Tribolium castaneum. The conservative regions were much similar with each other. Bm-l(1) was expressed at egg stage, larva, pupa and no tissue and stage specific in our tested samples.Deoxyhypusine snyhtase (DHS) and deoxyhypusine hydroxylase (DOHH) are the two enzymes that catalyze the synthesis of hypusine within eukaryotic initiation factor 5A (eIF5A). Synthesis of hypusine is essential for the function of eIF5A in eukaryotic cell proliferation and survival. lethal(3) s1921 gene of fruit fly is the lethal mutation of DOHH gene by inserted P element, so its'encoded protein has the function of DOHH. Here we described the cloning and expression of two full length cDNAs, encoding respectively DHS-like protein and DOHH-like protein from Bombyx mori by using the methods of bioinformatics, RT-PCR and RACE technology, named as BmDHS and BmDOHH. Sequencing results indicate that they are 1311 bp and 1874 bp in length including complete open reading frame (ORF) 1116 bp and 915 bp, which encode 371 amino acids (molecular weight is about 41.11 kD and isoelectric point is 5.84) and 304 amino acids (molecular weight is about 34.30 kD and isoelectric point is 4.86), respectively. BmDHS contains only one exon, and BmDOHH contains 4 exons and 3 introns. The deduced amino acid sequence of BmDHS contains a deoxyhypusine synthase domain from 47 to 361 amino acid residues, and the deduced amino acid sequence of BmDOHH contains six E-Z type HEAT repeat domains (23-52, 54-83, 87-116, 177-206, 208-237, 241-270). Compared to DHS and DOHH amino acid sequences from other species, such as Homo sapiens, Drosophila melanogaster, both silkworm DHS protein and DOHH protein have more than 55% identity. The conservative regions are very similar with each other. The phylogenetic tree analysis indicated that not only DHS but also DOHH from different species has genus-specific features. The expressions of BmDHS and BmDOHH are no tissue and stage specific in our tested samples.By using mulberry antifreeze protein gene'dsRNA and autoclaved distilled water as control, based on microinjection RNA interference technology, we have preliminarily got BmDHS gene silence phenotype by injecting 4th larval instar. Compared with control group, in the silent silkworm, the average level of BmDHS mRNA was reduced almost 25%; the average level of BmeIF5A mRNA was reduced about 4.8%; the average larval weight increased by 42.9% 10 days after injection; the average cocoons weighet increased by 35.4% at the fifth day after mounting. It preliminarily proofed that BmDHS gene plays an important role in silkworm's growth and development.2. Cloning and expression analysis of the Osiris-related genes from Bombyx moriTriplo-lethal Locus (Tpl) was surveyed as the only one that both triplo-lethal and haplo-lethal gene locus in the Drosophila genome. There was an Osiris gene family named gene Osiris1 through Osiris20 in the Tpl region. Through BLASTTP and PSI-BLAST analysis, there are only three other members of this family located outside of this locus. We called them Osiris21 to Osiris23. This gene family has the Insecta specificity. Through bioinformatics, RT-PCR and RACE methods, six silkworm genes belonging to Osiris gene family have been cloned and named as BmOsiris7,BmOsiris9,BmOsiris18,BmOsiris19,BmOsiris20 and BmOsiris21, respectively. Sequence analysis indicated that BmOsiris gene's ORF are between 728 bp and 882 bp in length. The molecular size of deduced amino acid which seemed quite similar with Drosophila Osiris protein and are 26 kD-31 kD range. The analysis of isoelectric point indicated that BmOsiris21's isoelectric point is 9.0, others are at between 6.41 and 8.66. SMART conservative region prediction on line found that this protein family belong to transmembrane protein and have specific conservative regions for the Osiris family, called DUF1676. All above dated supported the speculation that the cloned genes belonged to Osiris gene family. Phylogenetic tree analysis demonstrated paralogs of Bombyx mori and Drosophila melanogaster are much closer to the infraspecific orthologs. Expression pattern analysis indicated that each gene in our tested samples has no tissue and space-time speciality, but expression amount had significant differences. chromosome location showed that BmOsiris7,BmOsiris9,BmOsiris18,BmOsiris19 and BmOsiris20 located on the Chr.26, however, BmOsiris21 individually located on Chr.4. This result is very similar with Osiris genes from Drosophila melanogaster.3. Cloning and sequence alignment investigation of Pros-related genes from Bombyx moriBoth Pros261 and Prosβ21 in Drosophila belong to dominant mutation of temperature-sensitive lethality, and they are caused by mononucleotide mutation of proteasomeβ6 subunit and proteasomeβ2 subunit, respectively. Pros-related genes in Dazao breed of Bombyx mori have been registered in NCBI, but have not yet reported in written. In this article we named them as DzPros26 and DzProsβ2, respectively. This experiment designed primers by amino acid sequence of DzPros26 and DzProsβ2 registered, and cloned Pros-related genes in temperature-sensitive lethal system of Sch breed, named them as SchPros26 and SchProsβ2. Sequence alignment of ORF indicates the sequence of SchProsβ2 is is in perfect accordance with DzProsβ2. Sequence alignment of SchPros26 ORF and DzPros26 ORF showed that SchPros26 has mutated at the site of 223 bp, 556 bp, 621 bp, 624 bp, 658 bp and 659 bp. The results of the online software analysis displays that the base mutation brings the changes of primary structure, secondary structure and tertiary structure of deduced amino-acid residue sequence. Predicting results of proteinic functional site shows that Sch breed runs short of signal site of proteasomeβsubunit, because the seventy-fifth residue of SchPros26 protein, Glycine(G), has changed into Serine(S). However, DzPros26 protein in normal Dazao breed has the signal site of proteasomeβsubunit. It displays that SchPros26 may be relating with temperature-sensitive lethality of Sch breed.4. Cloning and sequence analysis of Pros-related genes from Helicoverpa armigera Homologous gene of cotton bollworm Pros261 has been cloned by using bioinformatics, RT-PCR, RACE and so on, named it as Haproβ1. Proteasomeβsubunits have the same conserved region and homologous with one another, so taking Drosophila Melanogaster Prosβ21 as question sequence, we find two EST sequences in cotton bollworm EST database. But they can not being contig with each other. Two homologous genes of cotton bollworm Prosβ21'have been cloned by the way of RT-PCR and RACE, called them Haproβ5 and Haproβ7, respectively. Protein encoded by Haproβ1 is 25.54kD, pI is 5.96, and amino-acid residues from 22 to 232 is the conserved region of proteasomeβ1 subunit; Protein encoded by Haproβ5 is 30.87kD, pI is 6.53, and amino-acid residues from 74 to 261 is the conserved region of proteasomeβ5 subunit; Protein encoded by Haproβ7 is 30.07kD, pI is 8.04, and amino-acid residues from 40 to 229 is the conserved region of proteasomeβ7 subunit. Alignment analysis indicates that Haproβ1,Haproβ5 and Haproβ7 are conserved in different species. They have the similar system evolution relationship that consistent with species evolution. The results of gene expression profiling analysis showed that Haproβ1,Haproβ5 and Haproβ7 are pretty similar, and high expression in body wall, midgut, gonad, but low expression in head, which is possibly because proteinic metabolic activity of head is lower than other detected tissues.