| The oriental river prawn, Macrobrachium nipponense, is an economically important species cultured in China in the last few decades. In recent ten years, the natural resources and the germplasm of M. nipponense began to decline, e.g., precociousness have emerged, threating the sustainability of the aquacultured populations of the shrimp, severely affecting its quanlity and quantity. Female precociousness catches more attention since more nutrition is consumed in the development of ovary than testis. The reason for female precociousness is still unknown. Some work has doned by controlling the environment condition and the nutrition. However there is little work on the methanism of the precociousness Ovary development mechanism will provide theoretical base for the problem solve.During crustacean ovarian development, the most significant change is that the diameter of oocytes dramatically increase, causing by yolk protein and other nutrients accumulation. The vitellogenin (Vg), yolk protein precursor, starts to synthesis in ovary early development of in crustacean. Then Vg is hydrolyzed to yolk protein depositing in oocytes. The molecular mechanism of Vg synthesis and hydrolysis remains unknown in crustacean. In oviparous vertebrates, Vg, synthesized on the control of many hormones and other protein, such as heat shock protein 90 (HSP90) and ecoestrogen bisphenol A (BPA), is hydrolyzed by cathepsin B, cathepsin L and so on.To study the molecular regulation of Vg synthesis and hydrolysis in M. nipponense, Vg, heat shock protein 90 (HSP90), cathepsin L (CL) and cathepsin B (CB) gene cDNA were cloned and their expression during the ovary development were detected in this paper. The effect of BPA, as a coestrogen, on the ovarian development was also studied in the paper. These results would provide a theoretical basis to the precocious research in M. nipponense.Vg, HSP90, CL, CB cDNA in ovary of M. nipponense were cloned by rapid amplification of cDNA ends (RACE) methods, denoted as MnVg, MnHSP90, MnCL and MnCB, respectively. And real-time quantitative reverse transcription-PCR (qPCR) was used to measure the expression of four genes mRNA in the ovary and other tissues during the ovary development and BPA exposed. 1 The fragement sequence of MnVg in M. nipponense, was cloned by the primer designed according to the highly conserved regions of Vg in Macrobrchium rosenbergii. Nucleotide sequence analysis revealed that the MnVg cDNA fragment was 567 bp in length, encoding a 183 amino acid polypeptide. Sequence alignments showed that the MnVg shared 95%,76%,52%,52%,51%identity with M. rosenbergii, Pandalus hypsinotus, Litopenaeus vannamei, Marsupenaeus japonicus and Penaeus monodon, respectively. Fluorescent real-time quantitative PCR (qPCR) was performed to examine the expression profile of MnVg mRNA. The MnVg was ubiquitously expressed in the tested tissued, with the highest in thoracic ganglia. MnVg mRNA expressions were differentially expressed in ovary and hepatopancreas during ovarian maturation. The level of MnVg mRNA in the hepatopancreas and ovary reached a maximum value at the ovary yolk grannle stage (â…£stage), then decreased with further ovarian development. The level of MnVg expression in hepatopancreas was signigicantly higher than that in ovary at the same developmental stage (p<0.05). The results showed hepatopancreas was the main resource of Vg in M. nipponense.2 It is well known that HSP90 is a functional protein whose expression is increased when animals are exposed to elevated temperatures or other stresses. But recent reports show that HSP90 is also involved in regulating ovarian development in vertebrates. In the absence of estrogens, estrogen receptors bind with HSP90 in oviparous vertebrates. HSP90 functions as an enhancer through increasing the activity of estrogen hormone-receptor complex to Vg genes. The HSP90 cDNA was cloned from oriental river prawn, Macrobrachium nipponense, designated as MnHSP90, by the methods of degenerated oligonucleotide primers and rapid amplification of the cDNA ends (RACE). Nucleotide sequence analysis revealed that the MnHSP90 cDNA was 2,684 bp in length, containing a 126 bp 5'untranslated region (UTR), a 359 bp 3'UTR (GenBank access no:GU319963), and an open reading frame (ORF) of 2,199 bp encoding a 732 amino acid polypeptide with predicted molecular mass of 84.3 KDa. Sequence alignment showed that the MnHSP90 shared 72-79%identity with other animals. Phylogenetic analysis showed that the MnHSP90 was closely related to HSP90s of other crustaceans. Fluorescent real-time quantitative PCR (qPCR) was performed to examine the expression profile of MnHSP90 mRNA. The MnHSP90 was differentially expressed in ovary and hepatopancreas during ovarian maturation. The level of MnHSP90 mRNA in the hepatopancreas and ovary reached a maximum value at the oil globule ovary stage (III stage) and yolk granule ovary stage (IV stage), respectively, and then gradually decreased with further ovarian development. The level of MnHSP90 expression in hepatopancreas was higher than that in ovary at the same developmental stage.3 M. nipponense were exposed for 19 days in BPA of a serial levels (5.01mg/L,7.76mg/L,12.056mg/L,18.62mg/L,28.84mg/L), set according to the safe concertration of BPA on M. nipponense 12.06mg/L get from the results of acute tocity experiment. The results showed that low BPA concentration could induce MnVg and MnHSP90 expression while high BPA concentration restrained their expressions. And MnVg and MnHSP90 expression got the maxium value at the safe concertration of BPA.4 Vitellogenin is hydrated to yolk protein, the nutrition for ovarian maturation in vertebrate. Cathepsins play an important role in the hydration. In order to take more view in the hydrate of MnVg, MnCL and MnCB were cloned form the ovary in M. nipponense in the study.The full-length cDNA of MnCL genes consists of 31 bp 5'untranslated region (UTR), a 650 bp 3'UTR, and an open reading frame (ORF) of 1,026 bp encoding a 342 amino acid polypeptide with predicted molecular mass of 37.7KDa (GenBank access no:HM134078). The polypeptide is composed of a 18 amino acid signal peptide, a 61 amino acid propeptide and a 253 amino acid mature peptide. The polypeptide sequence comparison showed that the MnCL shares 73%,66%and 66% identity with that of Penaeus monodon, Tenebrio molitor and Sitophilus zeamais, respectively. MnCL transcripts were detected in all the examined tissues with the highest level in heart and muscle. The level of MnCL mRNA in ovary reached a maximum value at the yolk granule ovary stage (IV stage), then decreased with further ovarian development. And the level in hepatopancreas was high in yolk granule ovary stage (IV stage), with the highest in fusion nucleolus ovary stage (â…¡stage). The full-length MnCB gene cDNA comprised of 1,710 bp, containing 12 bp in the 5'-UTR,993 bp in the ORF,702 bp in 3'-UTR (GenBank access no:HM134079). The ORF encodes a polypeptide of 331 amino acids including a 16 amino acid signal peptide, a 40 amino acid propeptide and a 247 amino acid mature peptide. Sequence comparison of the MnCB deduced amino acid showed similarity of 84%to that of Penaeus monodon,65%to that of Aedes aegypti,64%to that of Diabrotica virgife. Phylogenetic analysis showed that the MnCB was closely related to CB of crustaceans, Pandalus borealis and P. monodon. The mRNAs of MnCB was detected in haemocyte, hepatopancreas, muscle gill, intestis, heart and thoracic ganglia with different levels of expression. mRNAs of the gene showed the highest expression levels in heart, the medicate in muscle, hepatopancreas and thoracic ganglia, the lowest in gill, intestis and haemocyte. The level of MnCL mRNA in ovary reached a maximum value at the yolk granule ovary stage (â…£stage), then decreased with further ovarian development. However there was no significantly difference in the MnCL expression in ovary between the oil globule ovary stage (â…¢stage) and yolk granule ovary stage (â…£stage) (p>0.05).All these results showed that the hepatopancreans is the main synthesis resource of vitellogenin in M. nipponense. Its expression reached the maxmum in the yolk granule ovary stage (â…£stage) during the ovary development. MnHSP90, MnCL and MnCB were involed in the ovary maturation. MnCL and MnCB may the vitellogenin hydrolase in M. nipponense. BPA could affect the synthesis of MnVg and MnHSP90. |
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