Study On Transcription, Induce Transcription And Eukaryotic Expression Of Porcine RBP4 Gene

Retinol-Binding Protein is a transporter protein, it specifically binds retinol (Vitamin A) andcarries it from the liver to target tissues, helps VA to come true the transport and metabolism in cell,and is important in educing physiologic function of VA. At first, RBP forms a complex with VA,and then with transthyretin (TTR), formed VA-RBP-TTR complex, it through blood circulation totarget tissues, combines with recognition receptors (Retinoic acid receptor, RAR; retinoid Xreceptor, RXR), and transports VA to intracellular receptors, it completes its carry function then.RBP4 gene is not only a candidate gene of reproductive performance, but also a candidate gene ofresearch on VA metabolic block. This study was designed to investigate transcription characteristicof RBP4 genes by RT-PCR in heart, liver, stomach, spleen, kidney, lung, large intestine, smallintestine, muscle, uterus and ovarian of different days pigs; to investigate transcription changed ofRBP4 mRNA, RARs mRNA, RXRs mRNA and TTR mRNA in pig's fibroblast cell lines; toinvestigate the content of RBP and VA in serum and their relationships with young pig one monthweight; to construct the methanol induced expression vector in Pichia pastoris. Study result mainlyas follows:1. RBP4 mRNA is not transcribe in spleen and stomach of Large white pig; but keep on hightranscribing in liver, and the difference is significant among different days of uterus in transcriptionlevel (P＜0.05), lowest on 90 days. The transcription level in ovaries is increases subsequently days,it reaches the highest on 180 days (sexual maturity) and then descends, until 360 days reaches thelowest. It also transcribes in heart, kidney, lung, large intestine, small intestine and muscle, but hasspace and tissue specificity.2. RARαmRNA is keep on transcribing in liver, spleen, kidney, large intestine, small intestine,uterus and ovaries, among them spleen, large intestine and small intestine are continue hightranscribing; on 180 days, transcription level are lower in all tissues; on 360 days, transcriptionlevel are highest in 11 tissues. RARβmRNA is keep on transcribing in heart, kidney, large intestine,uterus and ovaries, among them large intestine, uterus and ovaries are continue high transcribing;on 1 day and 360 days, 11 tissues are all transcribe this gene and levels are higher; as it is continuehigher level in uterus and ovaries, maybe it can be researched as a candidate for reproductiveperformance. RARγmRNA is keep on transcribing in uterus and ovaries, and continue high levelsin uterus; on 360 days, 11 tissues are all transcribe this gene and levels are higher. 3. RXRαmRNA is keep on transcribing in kidney, lung, small intestine and muscle, but lowerlevel in heart and uterus, on 1 day, the tissue transcribe this gene is most, at the same time, the levelis the highest. RXRβmRNA is keep on transcribing in spleen, uterus and ovaries, the level is alsohighest on 1 day. RXRγmRNA is keep on transcribing in heart, spleen, large intestine, smallintestine and muscle, and the level is continue higher, but is not transcribe in kidney, and liver isscarcely.4. TTR mRNA is keep on transcribing in liver, uterus and ovaries, the highest in liver, andcoincidence with RBP4 mRNA status. 11 tissues are all transcription and higher on 1 day, but lowerin 90 days except heart, liver and stomach.5. Presume the possible nuclear receptor type which combine with RBP4 in 11 tissues,according to the change law of RBP4, RARs and RXRs.6. Excess VA can positive regulate the transcription of RBP4 among 24-72h in pig's fibroblastcell line; among the 72 culture period, RARα,RARβand RARγmRNA are all highest on 12h and 48h,but lowest on 24h and 72h, and are all under the control, just say, excess VA may negative regulatethe transcription of RARs mRNA. Compared with control, excess VA can positive regulate thetranscription of RXRγmRNA, but negative regulate the transcription of RXRαand RXRβmRNA.7. There is a significant correlation between RBP and VA in serum (P＜0.01), but is not linearcorrelation; content of RBP and VA in serum have not significant correlation with one monthweight of young pig (P＞0.05).8. Amplified the encoding maturation protein nucleotide sequence by PCR in Large whitepig's liver cDNA, produce length 549 bp, is the maturation protein of RBP4 gene encoding 183amino acid.9. Successful construction the eucaryon transcription vector pPICZaC-RBP4, it is determinedby PCR, enzyme digested and sequenced.10. Successful induced the expression of pPICZaC-RBP4 in GSll5 yeast fungus by methanol,and detected the expression of RBP4 maturation protein in protein level.