Characterization Of The Sex-determination Gene Masc In The Silkworm, Bombyx Mori

Bombyx mori is a species of lepidoptera, as model organism to study the regulation mechanism. Its silk has important economic benefit in agricultural production, which is also classic material at developing a new generation of bioreactor and new insect industry. Farming support country. Sericulture and implanting mulberry has always been an important part of our country from ancient times to modern.China’s history of silkworm has been more than 5000 years. The Silk Road has become an important channel for Chinese and foreign economic development and cultural exchanges. By the end of 2003, We took the lead to complete the silkworm genome framework graph drawing work, access to the large amount of important information of Bombyx mori genome. It made China be in the lead of the world at functional genomics of silkworm, Bombyx mori. Through an important model organism for functional genomics research in silkworm, Bombyx mori, it will promote the development of silk industry, stimulating the agricultural and forestry related disciplines development, insect industry development at Bombyx mori as representative insect,and the basic research of life science.The sex control research for Silkworm is significant no matter in production or Pest Control. According to reports, the male silkworm does not participate in the oviposition, so their silk production capacity is much larger than the female. Therefore the adjusting sex ratio of silkworm in the production, making the number of male silkworm increases, can greatly improve the silk production capacity, which has important significance for industrial production of silk. As a model insect silkworm is studied at the sex control which can control the number of male silkworm, and then achieve the purpose of Lepidoptera insect control.Studying molecular biology mechanism of sex determination in silkworm can provide the theoretical basis and reference for agricultural production. Therefore, it is particularly important for the study of sex determination gene. So far, a lot of sex related genes have been identified. The dominant gene at sex determination is Bmdsx gene. The research related interaction of Bmdsx gene with other genes has a role in promoting the further exploration of the sex determination in silkworm. Masc was found in recent years, which the gene can interact with Fem, PSI and Imp gene. It regulates alternative splicing of the Bmdsx gene at its downstream, which determines the sex differentiation of silkworm. That is to say, the research of Masc gene in Silkworm sex determination of lepidopteran has important significance.Therefore, we selected Masc as the research object of silkworm sex regulation network, analyzing the domain area by bioinformatics, cloning and expression of a prokaryotic protein contains two CCCH-type zinc finger structure and part of curl domain regional structure. Then the activated supernatant protein was analyzed at two level structure.We selected the protein with specific functional areas to prepare polyclonal antibody between these two proteins. The expression characteristics of Masc in different tissues of Bombyx mori was identified using RT-PCR and fluorescence quantitative PCR, while the expression of the protein in germline was measured. And then the expression in cells and tissues was located. Finally, the interaction of Masc and other proteins was identified by IP and mass spectrometry research, the main results are as follows:1.Bioinformatics analysis of Masc;After analysis, the full-length of Masc m RNA was 2075 bp, the length of CDS sequence was 1767 bp, located between 147 bp and 1913 bp on m RNA.This gene located on the Z chromosome with position: AB840788.1 in the NCBI. The homology of Masc and other species was low at the full length of CDS,which the homology of Masc and Danaus plexippus was the highest. However, the homology of the domain contains two zinc finger structure and other species was very high.The probe expression of gene in the silk DB was sw18123. The chip data showed Masc in testis and ovary had a strong signal, which the signal of testis was larger than the ovary. The analysis of Masc in the SMART indicated that the CDS sequences of48 th to 73 th and 79 th to 105 th were zinc finger domain containing two CCCH-type respectively. The analysis in Ex PASY showed that full-length of Masc encoded 588 amino acids, molecular weight is 64.71 k D and the isoelectric point was 9.69, whichbelongs to the basic protein.2. cloning, prokaryotic expression, two level structure analysis and preparation of antibody of Masc domain;The testis on the third day of fifth-instar larvae of silkworm were ground and extracted to the total RNA,and then inverted to the c DNA as a template. By sequence analysis of Masc’s CDS domain region,two regions from 135 th bp to 435 th bp, 900 th bp and 1305 th bp were cloned.The gene was ligated into the p ET-28 a prokaryotic expression vector, which resulted in the fusion of a six-histidine tag to the amino terminus of Masc.Then, the recombinant vector p ET-28a-Masc was transformed into Transetta(DE3) Escherichia coli competent cells.Containing beta- folding structure might be in expression of domain1 area of Masc protein by circular dichroism spectroscopy and gel filtration chromatography.This was consistent with the result made of zinc finger structure. The expression protein of domain 2 was prepared as antibody in the company, acquired polyclonal antibody of 0.62 mg / ml, which the titer is 1:5 00000. By western blotting, it showed that the unicity of the antibody was well and could be used for the subsequent experiments.3.Characteristics analysis of the organization expression of Masc in levels of RNA and protein;We examined the expression patterns of the Masc gene in different tissues. The RT-PCR and q PCR results showed that Masc was highly expressed in testes but weak in ovaries on the third day of fifth-instar larvae of silkworm.We performed a western blotting analysis of testes and ovaries using the anti-Masc antibody,which the results were consistent with RT-PCR and q PCR. These results imply that Masc may play important roles in testis development.4.Expression and localization analysis of Masc in cells and tissues;Subcellular localization experiments identified that Masc was connected with1180[Hrs1000-Bm Act4-LUC-Ser1PA] eukaryotic expression vector and transfected into Bm E cells. Results showed that the expression of Masc was high in the cytoplasm of the Bm E cells.Immunofluorescence experiments showed that Masc was located in spermatogonial cells, the nuclei of sperm bundles but in the cytoplasm of spermatotheca and connective tissue layer of testes, indicating that Masc played a variety of functions in the testis.5.Analysis of Masc interactions with other proteins;Masc-IP experiments and mass spectrometry identification results showed that Masc protein may be involved in the ping-pong mechanism containing piwi domain of Ago3 and Siwi protein combination in the sex control process.