Cloning, Sequence And MRNA Quantitative Analysis Of Vitellogenin Gene Of Fish Gobiocypris Rarus

As a new mode of experimental fish,Gobiocypris rarus,a small cyprinids,hasbeen used in genetics, diseases, environmental science, as well as transgenicornamental fish and other research areas. Vitellogenin (VG) is a high molecularprotein with polysaccharide、adipose and phosphorus,exist in the female animal bloodduring the oviparous animals yolk formation process.In this study, according topublished in the NCBI website Gobiocypris rarus yolk protein cDNA sequence,design primers by ordinary PCR technology6299bp sequence; using6299bp in thesequence of the5’end as a template, design two primer for genome walking to clone1285bp sequence.7573bp sequence after Seqman software, which vitellogenin genesequences of6595bp, upstream regulatory sequences of978bp. Gobiocypris rarusvitellogenin gene contains25exons and25introns, encoding1299amino acidsequence, CDS length3900bp after open reading frame analysis. After BLAST(http://blast.ncbi.nlm.nih.gov/Blast.cgi) homology analysis, Gobiocypris rarusvitellogenin cDNA gene sequence was found that: from the sequence of Daniorerio,Cirrhinus molitorella, Catla catla, Tanichthys albonubes, Cryprinus carpiodvitellogenin gene cDNA sequence similarity, respectively69%,88%,87%,63%,70%;Gobiocypris rarus vitellogenin predicted amino acid sequence, and Daniorerio,Cirrhinus molitorella, Catla catla, Tanichthys albonubes, Cryprinus carpiodvitellogenin amino acid sequence comparison, the similarity of76%,85%,91%,82%,88%, respectively. Open reading frame analysis also found that, compared to Daniorerio and Gobiocypris rarus vitellogenin inserted a sequence length of21bp, locatedbetween exon1and exon3, could lead to seven amino acid sequence of the insert;22outside of Gobiocypris rarus vitellogenin exon (252bp) less than Danio reriovitellogenin21exon (312bp) period of60bp of sequence. Gobiocypris rarus andDanio rerio vitellogenin gene sequences to remove introns, respectively the4212bp (Gobiocypris rarus) and4256bp (Danio rerio) vitellogenin gene coding region, bothcompared and found a similarity of69percent. Found by BLAST,220bp-838bpGobiocypris rarus vitellogenin upstream regulatory sequence2857bp-3144bpinterval80%similarity with the Acheilognathus yamatsutae vitellogenin genepromoter sequences.According to Gobiocypris rarus ovarian development, Gobiocypris rarus’ovarian can be divided into six. The ovaries of three experiments to Gobiocypris rarus(Phase I, Phase III, Phase V) for the study, go to the liver tissue was extracted RNA byquantitative PCR technique to detect Gobiocypris rarus vitellogenin m RNA in threequantitative expression period. The results found in the liver, with the development ofthe ovary, the relative expression level of vitellogenin presents the s-shaped curve, thethird period Vitellogenin m RNA expression highest. Vitellogenin has a role topromote oocyte growth, in which three Phase III oocytes were most abundant. Thusthe present experimental results and vitellogenin this feature match.