| Ubiquitin-proteasome pathway is the protein degradation pathway widely found in all organisms, it is almost involved in all the life activities of plant and animals. In order to study the functions of ubiquitin family in Penaeus monodon ovarian development,28 ubiquitin genes were cloned and validated in this research,including a ubiquitin precursor, 5 ubiquitin-like,2 ubiquitin-activating enzymes,14 ubiquitin-conjugating enzymes,3 Ubiquitin protein ligases and 3 ubiquitin carboxyl terminal hydrolytic enzymes. On this basis, the expression of 28 genes in hepatopancreas and ovary in different periods of gonad development of Penaeus monodonwere detected by q-PCR, and the clustering analysis was carried on by heatmap. The results showed that expression in ovarian trend can be broadly divided into three branches: The expression trendof 8 genes(UBE3S,UBE2 N,UBFD1, UBE2G1,UBE2J2,UFM,UFM1,UBE2H1) were conraise; The expression trend of 7 genes(UMAE5,UBE,UPL,UCHIL5,UBS30,UDCP2,ULP3)kept decreasing;10 genes’ expressions showed a trend of rising at first and lower later(UCH14,UBE2V2,UBE2Q1,UBE2 A, UBE2G2,UBA1,UBE2 H,UBE3B1F1,UBE2 C,UBE2H5b). In the hepatopancreas, The expression trend of 2 genes(UBE2V2,UBE2H1) were conraise;12 genes’ expressions of the general trend descended before they rised(UBE3S,UBE2 N,UFM,UBFD1,UBE,UBE2 A, UPL,UBE2G1,UBA1,UBE3B1F1);14 genes’ expressions showed a corrugated floating curve(UCH14,UBE2Q1,UBE2G2,UBE2 H,UBE2C,UBE2H5 b,UMAE5,UCHIL5,UBS30, UDCP2,ULP3,UBE2J2,UFM1,UBE2).According to the analysis result above,we screen out 6 genes to studying their protein function,like the ubiquitin precursor(UBS30),ubiquitin-activating enzyme(UBA1),ubiquitin-conjugating enzymes(UBE2A, UBE2G1, UBE2G2 and UBE2H).The c DNA of Pm UBS30 was 933 bp, including an open reading frame(ORF) of 396 bp, encoding a polypeptide of 131 amino acids. The results of q PCR showed that Pm UBS30 expressmainly in gastric and cardiac. Obtained the Pm UBS30 recombinant proteins by prokaryotic expression, and analyze it by Western Blot. The Purified protein is about 33.5 k D,and the concentration of it was 3.13 μg/ μl.The c DNA of Pm UBA1 was 3822 bp, including an ORF of 3120 bp encoding a polypeptide of 1039 amino acids.The results of q PCR showed that Pm UBA1 express mainly in lymphoid tissue and stomach, gill second. Obtained the Pm UBA1 recombinant proteins by prokaryotic expression, and analyze it by Western Blot. The Purified protein is about 34 k D, and the concentration of it was 3.20 μg/ μl.Immunohistochemical results show that Pm UBA1 mainly located in the cytoplasm, and there were different expression in different development period of Penaeus monodon ovary.The c DNA of Pm UBE2 A was 941 bp, including an ORF of 498 bp encoding a polypeptide of 165 amino acids. The results of q PCR showed that Pm UBE2 A expression mainly in lymphoid tissue and stomach. Obtained the Pm UBE2 A recombinant proteins by prokaryotic expression, and analyze it by Western Blot. The Purified protein is about 38 k D, and the concentration of it was 2.08 μg/ μl.The c DNA of Pm UBE2G1 was 911 bp, including an ORF of 501 bp encoding a polypeptide of 166 amino acids. The results of q PCR showed that Pm UBE2 Aexpression mainly in lymphoid tissue, gill and stomach. Obtained the Pm UBE2G1 recombinant proteins(about 38 k D) by prokaryotic expression, and analyze it by Western Blot. The c DNA of Pm UBE2G2 was 620 bp, including an ORFof 507 bp encoding a polypeptide of 168 amino acids.The results of q PCR showed that Pm UBE2G2 expression mainly in lymphoid tissue and stomach, the hepatopancreas second. Obtained the Pm UBE2G2 recombinant proteins by prokaryotic expression, and analyze it by Western Blot. The Purified protein is about 38 k D, and the concentration of it was 2.66 μg/ μl. The c DNA of Pm UBE2 H was 1010 bp, including an ORF of 555 bp encoding a polypeptide of 184 amino acids. The results of q PCR showed that Pm UBE2 H expression mainly in lymphoid tissue and gill which played an important role in immune. Obtained the Pm UBE2 H recombinant proteins by prokaryotic expression, and analyze it by Western Blot. The Purified protein is about 39 k D, and the concentration of it was 2.92 μg/ μl.This study researched the expressions of ubiquitin family in Penaeus monodon ovaries and hepatopancreas which were in different development period of gonad,it can concluded that these genes played a important role in ovarian development of Penaeus monodon.And then 6 proteins were screened out for further function study.This research can provide a reference for the further study of ubiquitin family regulatory mechanism ofovarian development in Penaeus monodon. |
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