Identification Of Chromodomain-containing Genes And Function Of MSL-3 Homologs And Bm-MOF In Bombyx Mori

The silkworm, Bombyx mori, an important economic insect, can secrete silk to form cocoon. There are all 20,000,000 sericulturists now in our country and it is an characteristic industry in advantage preponderance in international market. But because of the low economy character, the quality of most of the raw silk is poor in our country compared to developed countries such as Japan, which decreases the economic profit of sericulturists and our country. The foremost character in silk manufacture, output and quality of silk, can be easily affected by environment, epigenetic factor. Chromodomain, a recognizer of epigenetic codon, participates in the heredity of epigenetic codon. The role of chromodomain in epigenetic and in chromatin conformation could redound to the study of quantitive heredity and it also has great significance in reinforcing the world station of our silk industry, improving the competition and increasing the income of farmer. Based on the silkworm genome draft and EST(expressed sequence tag), we analyzed the chromodomain-containing(CD gene) proteins of silkworm and studied on the cloning and function of MSL-3 homologs and MOF genes in them. The main results present as follows.1. Identification of homologs of CD genes in silkworm genomeHomology analysis between the silkworm genome and all the known genes of yeast, Drosophila and human CD genes shown that 11 homologs were found in silkworm and all of them expressed in silkworm by EST proof and RT-PCR. Homologs of CD genes were reported firstly in silkworm genome.Analysis of the CD genes between silkworm and Drosophila indicated that almost all the CD genes of Drosophila has homologs in silkworm. There are 3 CHD family genes, one less than in Drosophila, but obvious decreased to in mammal such as human. The expression of the 3 CHD family genes has obvious difference by RT-PCR. BmCHD1 has no expression in all the tested stages, BmCHD3 has visible expression except in dispaused eggs, faint expression in it, and the expression of BmCHD6 is high in 1st day of 2nd instar larva and pupa,low in the other stages.The amount of SUV39, HP1 and PC family genes between silkworm and Drosophila is equal. The expression detected by RT-PCR indicate that there are distinct difference among them. BmHP1αexpresses in all the tested periods, BmHP1γis notably difference, high in moth, pupa is second and relative low in 1st day of 2nd instar larva and dispaused eggs, while BmHP1βhas no expression in pupa and moth, although it expresses in the other two stages. Similar to BmCHD6, BmSu(var)39 is expressed in 1st day of 2nd instar larva and pupa, no expression in the other two stages. The expression of BmPC is decreasing in pupa, moth, 1st day of 2nd instar larva and even no in dispaused eggs.The msl3 and mof gene of Drosophila function in dosage compensation. It is no evidence to make shown that there are dosage compensation in silkworm now. The being of msl3 and mof in silkworm is interesting. The report that MOF and MSL3 in mammals compose a transcriptional complex and not involved in dosage compensation indicates that their function in silkworm maybe the same with that in mammals. 2. The cloning and prokaryotic expression of MSL-3 homologs in silkwormThe MSL-3 homologs in silkworm Bm-msl3 and Bm-mrg15 were cloned by RT-PCR and the accession number of them in GenBank are DQ887343 and DQ888173. The cDNA length of them are 2356 bp and 1113 bp, respectively, with an ORF of 1665 bp and 1020 bp coding 554 and 339 residues, which contain a N-ter CD and an MRG domain, no signal peptide. Multiple sequence alignment of homologs indicate that the two structure domains of coded proteins are conserved, especially the MRG domain, while the CD of some genes are lack. The Bm-msl3 has high similarity with drosophila homologs, 61% identity between them. While the similarity of MRG15 gene between silkworm and human is high, 47% identity between them. The results of BLASTN search of the two genes in silkworm genome shown that they both have only one copy in genome. The comparison between cDNA and genome indicates that 13 exons and 12 introns are in Bm-msl3 and all of the boundaries of exon/intron is GT-AG except one. Similarly, gene Bm-mrg15 has 6 exons and 5 introns and the boundaries of them are all GT-AG. RT-PCR experiment indicated that the two genes were expressed in all tested tissues and stages, which seem to imply its key role in silkworm development.Prokaryotic expression of the two genes was carried out through sub-cloning into pGEX-4t-2 vector. SDS-PAGE electrophoresis of the product after inducement indicated that pGEX-4t-2-BmMSL3 had no expression except in 18℃, low incubation temperature and the specific band is about 70 KDa and the solute composition has GST enzyme activity, which is postulated the cut short expression of recombinant Bm-msl3. pGEX-4t-2-BmMRG15 is successfully expressed with a 65 KDa specific band, and the solute composition of expressed protein is increased and has GST enzyme acitivy when inducement in low temperature.3. Expression optimization of pET50b-BmMOF and its interaction with recombinant MSL-3Different culture medium, IPTG concentration, and temperature were applied to optimize the expression of recombinate plasmid pET50b-BmMOF and the optimum condition was TB culture medium, 0.4 mM IPTG and 25℃.The co-expression of Bm-mof and MSL-3 homologs came true by transformation of recombinant pET50b-BmMOF and MSL-3 homologs to the same BL21(DE3) and screen by the culture medium with ampicillin and kanamycin sulfate. Expression proteins were copurification by His.Bind. SDS-PAGE, co-purification and detection of GST enzyme activity of the protein before and after purification were all carried out to identify the interaction between them. All the proteins has effective inducement and efficient expression by SDS-PAGE. But there is only recombinant protein pET50b-BmMOF and no recombinant MSL-3 homologs in the proteins after purification. The results of GST enzyme activity is the same except after purification, which has weak GST enzyme activity, too. All the results indicatd that there may be weak interaction between the two gene of silkworm.4. The analysis of silkworm CD genes and the intron and phylogenesis of CDSame to the homologs, most of silkworm CD genes has typical domain of the family which it is affiliated besides CD, except one, BmHP1γ, two CD contained and no CSD. Although the structure and residues composition between CD and CSD is similar, and both composed by 3β-sheets and oneα-helix, the function of them has distinct difference. The special structure of BmHP1γmay be related to chromosome shape of silkworm and the function of it needs further studies.The results of homologous search indicate that they are more similar to human homologs, especially BmCHD1,BmCHD6,BmHP1α,BmHP1β,Bm-mof, and the similarity to drosophila is lower, which is an interesting thing for the distance in evolution between silkworm(lepidopteran) and drosophila(dipteran) should be less than silkworm and human in general evolutionism, while it is the contrary in this event.The analysis results of intron of CD genes and CD domain shown that although Su(Var)3-9 and MOF gene of silkworm and drosophila are similar, no intron in them, while homologs of human has. To the CD domain of CHD family and HP1 family of silkworm and human, most of them have. While in drosophila, there are no except HP1α, one intron in the first CD domain, and no intron in the HP1βand HP1γgenes.