Research On Differentially Expressed Genes From The Early Embryos Of Parthenogenesis In Silkworm, Bombyx Mori

As it is well known that Silkworm, Bombyx mori is an important economic insect, also an important organism model of lepidoperan insects,so the research background of silkworm genome and functional genome, which were achieved recently, is significant to explore the key crucial functional genes of lepidoperan insects related to life activities such as embryonic development, sex determination, sexual cell development.They not only provide a basis for the further molecular improvement and molecular design of silkworm breeding,will also be used for reference to control other lepidoperan pests.Silkworm individuals have the differences in economic value between male and female, and the male has high silk conversion efficiency.In general, the sex determination and sex differentiation of silkworm have been completed in the early embryos stages, but the research of molecular mechanism in sex determination of Bombyx mori has not yet been resolved.In particular, the functional genes related to sex determination, sex differentiation and development of silkworm in early development stage is a lack of any information.In order to explore the differentially expressed genes in early embryo between male and female of silkworm, in this experiment,the early embryos of silkworm parthenogenesis eggs were prepared by Ameiotic Parthenogenesis ( AMP ) and Bispermic Androgenesis ( BSA ) technology and the forward and reverse direction subtracted hybridization cDNA libraries between female and male of silkworm were constructed using Suppression Subtractive Hybridization ( SSH ).We also have done validation of differentially expressed sequences obtained from the two subtracted libraries.The main conclusions are as follows:1.The preparing of parthenogenesis eggs of silkwormThe early embryos of parthenogenesis eggs were prepared by AMP and BSA parthenogenesis technology in the jingsong×Haoyue silkworm varieties . After investigation, both the percentage of pigmented eggs are 76.43% and 76.56%, with higher rates and not very different.2.The construction of silkworm parthenogenesis SSH cDNA libraryIn order to explore the differentially expressed genes in early embryo between male and female of the silkworm, in this experiment, the forward and reverse direction subtracted hybridization cDNA libraries were constructed between female and male of the silkworm using SSH.The forward is AMP( 0 ~ 72 h) silkworm eggs as the experimental group against BSA ( 0 ~ 24 h ) eggs as a reference group to carry out SSH and construct a AMP cDNA subtracted library.The reverse is BSA (0 ~ 24 h) eggs for the experimental group against AMP( 0 ~ 72 h ) eggs as a reference group to carry out SSH and construct a BSA subtracted cDNA library.The products of two subtracted libraries are more between 200 bp and 750 bp.In the forward subtracted library 62 positive clones were selected to sequence and obtained 43 kinds of cDNA sequences between 88 bp and 479 bp.In the reverse subtracted library 46 positive clones were selected to sequence and obtained 29 kinds of cDNA sequences between 132 bp and 724 bp.3.Sequence analysis of cDNAs in SSH cDNA subtracted libraryThere are 12 sequences in the AMP subtracted library of 43 kinds of cDNA sequences appear two or more than two clones, with a total frequency of emergence of the total clones accounted for 50% . There are 26 sequences existing silkworm homology cDNA sequences,17 sequences could not be found silkworm high homology ESTs or cDNAs are the new cDNA sequences of Bombyx mori in our experiment.Through the BLAST analysis in NCBI and Silk DB database, there are 19 sequences have a high homology with the known functional genes (Unigenes), 7 sequences have a high homology with the Hypothetical protein genes, 17 sequences are unknown genes.The unigenes are Heat shock cognate protein, Ribosomal protein, Reverse transcriptase, Transposase enzyme, Transcription or Translation regulatory factors and Ransporter and so on.There are 11 sequences in the BSA subtracted library of 29 kinds of cDNA sequences appear two or more than two clones, with a total frequency of emergence of the total clones accounted for 60.87%.There are 21 sequences existing silkworm homology cDNA sequences.8 sequences could not be found silkworm high homology ESTs or cDNAs are the new cDNA sequence of Bombyx mori in our experiment.Through the BLAST analysis in NCBI and Silk DB database, there are 16 sequences have a high homology with the unigenes, 13 sequences are unknown genes . The unigenes are Cu / Zn SOD, Dehydrogenase class, Transposase, Glutathione S-transferase, Heat shock protein and Ribosomal protein and so on.4.Molecular cloning and analysis of Bombyx mori differentially expressed genesRapid Amplification of cDNA Ends ( RACE ) were separately carried out on the five sequences ( AMP05, AMP20, AMP25, AMP29, AMP50 ) of AMP subtracted cDNA library.Through the BLASTx analysis in NCBI of cDNA extended, AMP05 with Aedes aegypti and AMP29 with Pediculus humanus corporis have homology with the Hypothetical Protein gene.AMP20 have a homology gene in Tribolium castaneum , but failed to find in Bombyx mori.AMP25 and AMP50 don't have homology sequences to match.RACE also were separately carried out on the five sequences ( BSA12, BSA22, BSA23, BSA29, BSA30 ) of BSA subtracted cDNA library.Through the BLASTx analysis in NCBI of cDNA extended, BSA12 and BSA22 have high homology with silkworm Cu / Zn SOD and TCTP, which are confirmed these genes.The 3 'end of BSA29 is highly similar to the Cytosolic malate dehydrogenase (1 424 bp) by tBLASTx analysis, but the 5' end (140 bp) are not the same which worth exploring.BSA23 and BSA30 don't have homology sequences to match.5.Analysis of differentially expressed genes by semi-quantitative RT-PCRSix sequences from each subtracted library were chosen to do semi-quantitative RT-PCR in AMP and BSA silkworm egg samples.The results show that, AMP01 ( BmHSC70-4 ) is represented significantly differentially expressed genes in AMP silkworm egg.BSA05 and BSA30 are represented significantly differentially expressed genes in BSA silkworm egg.They are up-regulated expression.6.Analysis of differentially expressed genes by real-time fluorescence quantitative RT-PCRThe genes expression levels from the parts of differentially expressed sequences in the two subtracted library ware compared by real-time fluorescence quantitative RT-PCR in AMP and BSA silkworm eggs.The results show that, the expression of AMP01 in AMP silkworm eggs is 4.41 times different comparing to that in BSA silkworm eggs.But the expression of AMP05, AMP20, AMP39 and AMP50 in BSA silkworm eggs is not significant than in AMP eggs in 1 time difference.The expression of BSA05 and BSA30 in BSA silkworm eggs is 3.01 and 3.92 times different comparing to in AMP silkworm eggs.But the expression of BSA07, BSA12 and BSA29 ware not significant only 1 time difference.