Analysis Of Polycomb Group And Functional Research Of BmRYBP In Silkworm, Bombyx Mori

Multicellular organisms generally originated from a single cell to differentiate into many types of cells, although each cell contains the same genetic information, cells in different organs execute completely different functions. Only 1%of the genetic information carried by cell can be coded for proteins, the remaining are inhibited by methylation of DNA. Polycomb group(PcG), a group of very important genes, maintain this established molecular mechanism in life. Although the members of PcG proteins have differences in structure and motifs, they form a common family due to perform the common function. PcG proteins regulate transcriptional repression of many important genes related to development, which are highly conserved among species. It has been confirmed in Drosophila that PcG execute function mainly through forming complex proteins. The modality of complex proteins identified at present mainly contain three forms:Ploycomb repression complex 1(PRC1), Ploycomb repression complex2(PRC2) and Pho repression complex(PhoRC). What is especially significant to define the mechanism of PcG proteins is that isolation and identification to the component of complex, as well as verification of function. New regulator of PcG proteins and the regulation mechanism have become to be a hot research field of developmental biology and epigenetics.Silkworm is a complete metamorphosis insect and the typical representive of Lepidoptera insects. The research of linkage analysis and so on in silkworm once kept pace with those in Drosophila and provided significant evidences for human to cognitive the regularity of heredity and variation in organisms. Silkworm is also a model in research of development,differentiation, growth and regulation, heredity, variation in animals. Stepping into this century, function of generous known and unknown genes are urgent to be resolved followed the fine silkworm genome map was finished. GAL4/UAS system is a useful tool to investigate gene function, which is generally under application in Drosophila but not in silkworm. In order to accomplish the application of this system in silkworm, we utilize the system to study the function of RYBP (Ring and YYl binding protein) which is a component of PcG proteins. On the one hand, we establish the transgenic interference system based on GAL4/UAS system in silkworm. On the other hand, we study the function of members in silkworm PcG proteins. The main results are as follows:1 bioinformatical analysis and expression profile of silkworm Polycomb group23 drosophila PcG homologs were indentified in silkworm genome by BLAST, all the members haven't been reported yet. Those genes were highly conserved between fruitfly and silkworm, especially the polycomb repressor complex 2 components, which were supposed to be related with its ancient function of transcriptional repression.18 of them were supported by EST data.18 out of 23 genes were detected based on whole genome microarray data.7 of them expressed in at least one tissue of day3 fifth instar larvae. PcG recruiter Dspl, PRC2 component E(z) and Nurf-55(Cafl) revealed high expression in all organs; Nurf-55 (Cafl), Esc, E(z), Sce/dRing, Dsp1, Rbf highly expressed in gonad, while rarely expressed in other organs; dSfmbt revealed low expression in head, body wall and midgut.11 out of 18 detectable genes were found to express in at least one stage during metamorphosis. Dspl, E(z) and Nuf-55(Cafl) persistently expressed in all stages, especially the chromosome assembly factor Cafl,which represented the highest expression level; Dspl, E(z), Nurf-55(Cafl), Rbf and Rpd3 exhibited a conspicuously different expression pattern between male and female moth, which was 2-fold in female than male, above all, sex-dependent differential expression pattern of Rpd3 and Rbf began at 8th day mounting, and continued till moth stage, which was notably expressed in female, rarely in male; the other three Pho, Sce/dRing and Psc were constrainedly detected in female.2 Cloning, sequence analysis of Ring and RYBPRing is a core member of the family of PRC1, which function as a subunit to catalyze the ubiquitination of histone protein H2A. RYBP is a protein reported both in Drosophila melanogaster and Mus musculus, which can interact with RING and predicted to be a member of PcG protein family, but its function remains undiscoved. Two homologous genes of Ring and RYBP of silkworm were identified from the silkworm genome database based on the amino acid sequence of Ring and RYBP reported from drosophila. Among these results, a sequence which is homologous to Ring and located on the nscaf2865 of No.17 chromosome with a number of BGIBMGA006985, was identified from silkworm prediction genome database, prediction of gene structure indicates that this gene consists of 4 exons and 3 introns, encodes a 41.8kDa protein with 377 amino acids, and is highly similar to the homologous gene from other species, the N-teminal of this protein contains a typical Ring-domain, and C-teminal also contains a conserved function domain which has not been named yet. Whereas, the sequence which is homologous to RYBP has not been identified from the silkworm prediction genome database, but we electronic-clone a gene sequence of 882nt from silkworm EST database in which contains a ORF of 420bp, encodes a 15.4kDa protein with 139 amino acids, this gene locates on the nscaf3097 of No.28 chromosome and its N-teminal zinc finger domain is high conserved to both RYBP/YEAF1 of human and YAF2 of human and mouse.The results of RT-PCR by which to investigate the expressions of Ring and RYBP of silkworm on 3^rd day fifth instar indicate that the Ring remarkably expressed in testis, ovary, head and hemocytes, and had a lower expression level in fat body, no signal was detected in other tissues, this was consistent with the mircroarray data. Meanwhile, RYBP expressed almost in all tissues with a high expression levels gonad and head and a lower expression levels in other tissues.3 Prokaryotic expression and preparing polyclonal antibody of the Ring and RYBPIn purpose of further functional analysis, we finished the prokaryotic expression of BmRing and BmRYBP. We first transformed the recombination plasmid Ring/p28and RYBP/p28 into the strain Rosetta(DE3), and then detected the expression of the two proteins under the different induced temperature and IPTG concentration. At last, we purified the proteins in the inclusion body to go on the backward experiments by IPTG inducement four hours under the temperature 37℃.We used the two purified proteins to immunize the adult male rabbits for five times, and then detected the antibody titer of serum by ELISA. When the antibody titer is up to 1:64000,we obtain the serum and purified the two polyclonal antibodies.4 Interference of BmRYBP by GAL4/UAS system in the transgenic silkwormGAL4/UAS system is a powerful way tool of founctional gene research. It can control the expression of the target genes in different tissues and different times and make gene overexpressed and transgentical interfered. With the broad application of RNAi technique in the reverse genetics, this system has more and more been used in the functional research. We have constructed the transgenic expression construct pBac[UASRYBP^IRSV40-3xp3EGFP], and by the strandard method of transgenic inject we have gotten the UAS-RYBP^IRtransgenic silkworm and established the separate silkworm transgenic lines. To induce the RNA interference and detect the founctional deletion phenotype of RYBP gene, we crossed the A4GAL4 lines marked by red fluorescence with UAS-RYBP^IRlines marked by green fluorescence and then successfully interfered the target gene in the double-fluorescence crossbreed by double-strand RNA forming by RNA hairpin structure.We used PCR, Southern blot and Inverse-PCR to detect the copy number and insertion sites of the transgenic individuals of UAS-RYBP^IR at the molecular level. Then, using RT-PCR, quantitative PCR and western blot to detect transcription and expression level of the hybrid progeny of UAS-RYBPIR and A4GAL4 idividuals, the results showed that the mRNA and protein expression of BmRYBP gene decreased in the hybrid progeny,which means specific interference of RYBP gene happened. Also, the mRNA and protein expression of BmRing decreased. It's speculated that BmRing may be regulated by BmRYBP. Besides, there are different phenotypes in hybrid offspring of different strains.Some of Gl embryos died during embryogenesis. A4GAL4/UAS-RYBPIR moths, which were screened at 5th instar according to the dual-fluorescence marker, formed relatively smaller wings compared with the wild-type, and also, they showed a low and unstable oviposition capacity, lots of the eggs were unfertilized; some of the embryos died before hatching or in the 1 st instar.10%of the dead larvae exhibited polymorphic abnormalities in their proleg, including absence of one or more prolegs and lacking of the suctorial disk. Some of the diastrophic larvae remained alive until eclosion. It's suspected that those diastrophic phenotypes of G2 silkworms may be related with the maternal expression of most PcG proteins, decreased PcGs in the G1 moth caused developmental disorder of its posterities.