【作者】 张冬杰；

【导师】 刘娣；

【作者基本信息】 东北农业大学 ， 动物遗传育种， 2007， 博士

【摘要】 视黄醇结合蛋白(Retinol-Binding Proteins，RBPs)是体内一类将维生素A(VA)从肝中转运至靶组织以实现VA的细胞内转运代谢的特异的运载蛋白，在协助VA发挥生理功能中起着不可替代的作用。它先与VA结合再与甲状腺素运载蛋白(transthyretin，TTR)结合形成VA-RBP-TTR复合体，经血液流经靶组织后，与RBP的可识别受体(Retinoic acid receptor，RAR；retinoid X receptor，RXR)结合后，将VA转运至细胞内的受体，至此RBP完成其运载功能。RBP4既是一个影响猪的繁殖性状的候选基因，同时也是一个研究VA代谢障碍的候选基因。本研究以不同日龄大白猪为研究对象，采用RT-PCR法研究了RBP4 mRNA、RARs mRNA、RXRs mRNA和TTR mRNA在心脏、肝脏、胃、脾、肾脏、肺、大肠、小肠、肌肉、子宫和卵巢的转录情况；以猪的成纤维细胞系为研究对象，采用RT-PCR法研究了添加过量的VA后，RBP4 mRNA、RARs mRNA、RXRs mRNA和TTR mRNA的转录变化情况；采用ELISA和HPLC法检测了血清中RBP和VA的含量及与1月龄仔猪体重的关系；采用毕赤酵母真核表达系统构建了可被甲醇诱导表达的载体，主要研究结果如下：1．RBP4 mRNA在大白猪的脾脏和胃中不转录；在肝脏中是持续高转录；子宫中的转录水平在不同日龄间差异显著(P＜0.05)，且在90日龄时最低；卵巢中的转录情况是随着日龄的增加，转录水平也逐渐增加，180日龄时(性成熟期)达到最高，而后开始下降，至360日龄时最低。在心脏、肾脏、肺、大肠、小肠、肌肉也转录，但存在空间和时间的特异性。2．RARa mRNA在大白猪的肝脏、脾脏、肾脏、大肠、小肠、子宫和卵巢中持续转录，其中脾脏、大肠和小肠是持续高转录；180日龄时，所有组织的RARαmRNA的转录量普遍降低；360日龄时，所检的11个组织均高转录该基因。RARβmRNA在大白猪的心脏、肾脏、大肠、子宫和卵巢中持续转录，其中大肠、子宫和卵巢是持续高转录，在1日龄和360日龄的时候，所检的11个组织均转录该基因，且转录量较高；因RARβmRNA在子宫和卵巢的持续高表达，也可以考虑将其作为一个影响繁殖性状的候选基因来加以研究。RARγmRNA在大白猪的子宫和卵巢中持续转录，子宫中该基因的转录量一直持续较高，在360日龄时所检的11个组织均转录该基因，且转录量较高。3．RXRαmRNA在大白猪的肾脏、肺、小肠和肌肉组织中持续转录，而在心脏和子宫转录水平较低，1日龄时转录RXRαmRNA的组织最多，同时转录量也最高。RXRβmRNA在大白猪的脾脏、子宫和卵巢中持续转录，也是在1日龄时转录的组织最多且转录水平较高。RXRγmRNA在大白猪的心脏、脾脏、大肠、小肠和肌肉组织中持续转录，并且转录量一直持续较高，而肾脏不转录该基因。4．TTR mRNA在大白猪的肝脏、子宫和卵巢持续转录，且在肝脏中持续高转录，这与RBP4 mRNA的转录情况一致；1日龄时所检的11个组织均转录，且转录量较高，而90日龄时除了心脏、肝脏和胃外，其余组织的转录水平均较低。5．根据分析不同日龄不同组织中RBP4 mRNA的变化规律与RARs和RXRs mRNA的变化规律，推测出了所检的11个组织中，每个组织内可能与RBP4结合的核受体类型。6．在猪的成纤维细胞系中添加过量的VA后，在24-72h内可正调节RBP4 mRNA的转录；在72h的培养期内，RARα、RARβ和RARγmRNA的转录变化均是在12h和48h时最高，24h和72h时最低，但总体基本都处于对照组之下，也就是说过量的VA会负调节RARs mRNA的转录；与对照组相比，过量的VA正调节了RXRγmRNA的转录，而负调节RXRα和RXRβmRNA的转录。7．血清中RBP和VA的含量存在显著相关(P＜0.01)，但不存在线性关系；血清中RBP和VA的含量与1月龄体重均不存在显著性相关(P＞0.05)。8．从大白猪的肝脏组织的cDNA中，PCR扩增出猪RBP4成熟蛋白编码核苷酸序列，产物全长549bp，即为猪RBP4成熟蛋白183个氨基酸序列。9．构建了大白猪RBP4成熟蛋白编码序列的真核转录载体pPICZaC-RBP4，经PCR、酶切和测序检测分析，片段大小与理论相符，证明构建成功。10．成功的利用甲醇诱导真核转录质粒pPICZaC-RBP4在GS115酵母菌中的表达，并在蛋白水平上检测到了RBP4成熟蛋白的表达。更多还原

【Abstract】 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.更多还原