| A dosage compensation mechanism related gene named msl-1(male-specific lethal-1) was cloned from freshwater mussle-Hyriopsis schlegelii, using RACE PCR and nested PCR.1,The full-length cDNA sequence of msl-1 is 3097 bp, consisting of a 5’-terminal untranslated region(UTR) of 638 bp and a 3’-terminal UTR of 1586 bp with a 29 bp poly(A) tail and a tailing signal(ATTAAA). The open reading frame of msl-1 is873 bp which encodes a polypeptide of 290 amino acids without signal peptide. The predicted molecular mass of the msl-1 is 33694.12 Da with a theoretical isoelectric point of 8.27. Analysising of protein domain showed that there is a complete PEHE domain in the 166 th to 297 th amino acids N-terminal which is a specificity to MSL-1.The prediction of secondary structure suggested that Random coil was the highest, αhelical were relatively less and β turn was the least. Phylogenetic tree revealed that Hyriopsis schlegelii msl-1 gene sequence had a low homology with other known animals msl-1 genes, and clustered a group with Crassostrea gigas and Lottia gigantea.2,The expression of msl-1 in Hyriopsis schlegelii was analyzed by fluorescent real-time quantitative PCR and the results showed that the expression of msl-1 had obvious tissue specificity. The expression level of msl-1 in spermary was the highest.Followed by the kidney, the intestine, the hepatopancreas, the blood, the adductor muscle, the ovary. The expression level of msl-1 was the lowest in the heart. We found the expression levels of msl-1 gene is in different trend from the expression of different ages Hyriopsis schlegelii gonad. In ovary, the expression of msl-1 gene is very low in 1-ages Hyriopsis schlegelii, and it goes up slightly in 2-ages. However,the expression of the 3-ages Hyriopsis schlegelii dropped back, and in age 4 the expression level has increased significantly again to 6.48 times and dropped back again in 5-ages; but in spermary, the expression of msl-1 gene is very high and rising trend in 1,2ages, and dropped back in 3,4-ages.In 5-ages the expression level raise upfar more than the level in 1,2-ages.Moreover,the expression in spermary is higher than that in ovary except 4-ages expression.3,pEASY-msl-1 prokaryotic expression systems were constructed successfully. The correct prokaryotic expression plasmids confirmed by sequencing was then transformed into E.coli(DE3). After IPTG induction, the recombinant proteins was successfully expressed. MSL-1 recombinant protein is mainly in the form of soluble proteins, which access to the single target recombinant protein directly by nickel column.. Then the recombinant protein was puri?ed by using the native Ni2+ af?nity chromatography.Rabbit polyclonal antibody was prepared using purified MSL-1 recombinant protein. The serum antibody titers measured by ELISA was higher than 512000,indicating a high-titer antisera obtained for MSL-1. Western blotting showed that MSL-1 recombinant protein was able to combine with rabbit polyclonal antibody specifically. We also prepared frozen sections of gonad for immunofluorescent analysis of MSL-1 using the rabbit polyconal antisera. We found MSL-1 protein was mainly expressed in the karyotheca of the gonad cells. |
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