节点文献
广西巴马小型猪生长激素受体基因时空表达与转录调控的初步研究
Characterization of the Expression and Transcription Regulation of Ghr Gene in Guangxi Bama-mini Pig
【作者】 杨慧;
【导师】 兰干球;
【作者基本信息】 广西大学 , 动物遗传育种与繁殖, 2014, 硕士
【摘要】 动物体生长发育主要受GH-GHR-IGFs生长调节轴的影响,而生长激素受体(GHR)在其中处于中心地位。GHR除了介导IGF-I依赖性的生长调控作用,还可以直接作用于靶器官,介导非IGF-I依赖性的糖类、脂类和蛋白质代谢过程,从组织系统上参与到生物体整个生命代谢调控进程中,对生物体生长发育、形态建成和机体稳态的维持起着十分重要的作用。为探讨猪GHR基因及其表达调控与体型性状的关系,本试验选取体型矮小的广西巴马小型猪为研究对象,并以体型高大的猪品种长白猪为对照,对广西巴马小型猪和长白猪的GHR血清含量、基因编码区序列、mRNA时空表达和转录调控等方面进行了比较研究,试验结果如下:1.在1和180日龄,长白猪血清GHR和IGF-I含量显著高于巴马小型猪(P<0.05);但在30日龄,二者血清GHR和IGF-I含量没有显著差异(P>0.05);各日龄猪的血清GHR含量与IGF-I含量的变化趋势一致。血清GH含量在各日龄猪中变化不大,也没有表现出品种差异(P>0.05)。2.克隆得到的巴马小型猪GHR基因编码区序列发现有6处碱基突变,其中有2处导致氨基酸发生改变:第1225位点由丙氨酸→丝氨酸;第1801位点由缬氨酸→异亮氨酸,其它突变为同义突变。分别构建了巴马小型猪和长白猪GHR过表达载体并转染细胞,定量检测GHR调控的下游基因JAK2的表达量,发现长白猪GHR过表达载体组JAK2的表达量极显著高于巴马小型猪组(P<0.01),说明巴马小型猪与长白猪GHRCDS序列差异可能是造成二者GHR基因作用差异的一个原因。3.在巴马×长白猪F2资源家系中对G1248A和A1801G两个SNPs位点进行与生长性状的关联分析,结果表明pGHR/1248位点2月龄GG和AG基因型个体胸围较AA型大,4月龄GG和AG基因型个体体重显著大于AA型个体(P<0.05);pGHR/1801位点3月龄和6月龄GG和AG基因型个体胸围较AA型大,1月龄和6月龄AA基因型个体体高较AG、GG型高,且达显著水平(P<0.05);结合两位点进行haplotype分析,发现1月龄-AAAA-和-AGAG-型个体体高较-GGGG-型高,4月龄-GGGG-型个体体重较-AAAA-和-AGAG-型重,6月龄-GGGG-型个体胸围较-AAAA-和-AGAG-型大,且达显著水平(P<0.05)。4.实时荧光定量PCR检测巴马小型猪和长白猪mRNA的时空表达:不同日龄心、肝、肌肉、脾、肾和脑组织中GHR和IGF-I的表达量存在品种差异;GHR和IGF-I的表达量在各个组织中符合生长发育规律,并且IGF-I的表达水平受GHR影响,与之相平行。推测GHR表达水平低可能与巴马小型猪表现出的生长缓慢和体型矮小有密切关系。5.克隆得到GHR基因5’-1A和1B两种mRNA和巴马小型猪3 ’ cDNA全长序列,序列比对发现这些5’和3’调控序列不存在品种差异,而180日龄巴马小型猪GHR-1A表达量低于长白猪;分段扩增获得巴马小型猪ATG上游启动子区2700 bp调控序列,长白猪2696 bp调控序列;序列分析发现二者在存在较大差异:巴马小型猪在733-735 bp缺失3个碱基,1909-1915 bp插入7个碱基,并且还存在很多差异SNP位点,形成差异转录因子结合位点从而影响GHR和相关基因的转录调控和表达;构建广西巴马小型猪启动子区活性检测载体GHR-P1-EGFP和GHR-P2-EGFP,通过细胞转染荧光检测表明GHR基因启动子区1507 bp和1603 bp片段是具有基因转录启动活性的;巴马小型猪和长白猪不同日龄肝脏和肌肉启动子区CpG岛甲基化程度存在品种差异,其中在180日龄肝脏表现差异极显著(P<0.01),推测GHR基因甲基化程度主要与肝脏mRNA的转录调控相关。
【Abstract】 Animal growth and development were mainly regulated by GH-GHR-IGF-Ⅰaxis,in which the growth hormone receptor(GHR)played a central role.Except IGF-I depended pathway,GHR can also act directly on the target organs to regulate the sugar,lipid and protein metabolism process.Thus it took a significant part in animal growth,morphogenesis and maintaining body steady state from the levels of organization system.In order to verify the relationship between pGHR including its regulation and the trait of body size in pig,we choose Bama-mini pig as our research object owing to its dwarf body size,and select Landrace pig,a large body size breed,as control,to do the comparative study on such aspects as serum GHR levels,gene coding sequence SNP analysis,gene expression and regulation of its transcription.The researching results were shown as follows:1.In Landrace the serum levels of GHR and IGF-I were significantly higher than those in Guangxi Bama-mini pig on day 1,180 of age(P<0.01).However,on day 30 the differences were not significant(P>0.05).The serum content of IGF-I and GHR had the same changing trends in both pig breeds.The serum of GH kept relative constant levels in different ages,and no significant difference in serum GH level was detected in the two different breeds(P>0.05).2.The cloned Bama-mini pig GHR cDNA contained 6 SNPs,in which two of them are missense mutation lead to A1225S and V1801I,others were synonymous mutations.The Bama-mini pig and Landrace GHR over expression vectors were constructed and used to transfected PK15 cells,and then the expression of JAK2 gene in the transfected PK15 cells was detected by QRT-PCR.It was found that the expression level of JAK2 gene was significantly higher in the cells transfected with Landrace GHR over expression vector(P<0.01).The result indicated that the difference in CDS sequences of GHR between Bama-mini pig and Landrace may cause different regulation function to its downstream gene.3.Two pGHR SNPs(A1248G and A1801G)loci were verified in F2 generation of "Bama×Landrace" resources population,and the correlation between these SNPs loci with growth traits was determined.The results showed that 2 months old pigs with 1248GG and 1248AG genotypes had larger chest circumference compared to 1248AA genotype and 4 months old pigs with 1248GG and 1248AG genotypes had heavier body weight(P<0.05).The 1801GG and 1801 AG genotypes were also found having lager chest circumference in 3 and 6 months of age.However,the highest withers height in 1 and 6 months of age was found in pigs with 1801AA genotype(P<0.05).Two loci haplotype analysis found that in 1 months old pig,withers height of-AAAA-and-AGAG-genotype was higher compaired with-GGGG-genotypes,heavier body weight in 4 months of age was found in-GGGG-genotypes compaired with-AAAA-and-AGAG-genotype and chest circumference of 6 months old pig was larger in-GGGG-genotypes compaired to-AAAA-and-AGAG-genotype(P<0.05).4.In Bama-mini pig and Landrace QRT-PCR was used to analyze the expression of GHR and IGF-I on day 1,180 of age.The expression of GHR gene was detected in tissues including heart,liver,muscle,spleen,kidney and brain.It was found that the expression of GHR and IGF-I showed different levels owing to different pig breeds.The expression level of GHR and IGF-I were significantly higher in liver and muscle from Landrace on 180d of age(P<0.05).Thus,it was speculated that the low expression level of GHR may be related to the slower growth rate and dwarf body size of Bama-mini pig.5.The sequence of cloned mRNA 5’-1A,5’-1B and 3’ cDNA sequence of GHR showed no differences between Bama-mini pig and Landrace.However,Bama-mini pig showed lower expression level of GHR 5’-1A than Landrace in liver on day 180 of age.Segments of 2700 and 2696 bp from ATG upstream were obtained by segmented amplification of GHR promoter region in Bama-mini pig and Landrace respectively.Sequence analysis of the obtained GHR promoter region from Bama-mini pig found that there are 3 bp deletion mutation in loci 733-735 bp and 7 bp insertion mutation in loci 1909-1915 bp.In addition,several SNPs were also found in the GHR promoter region in Bama-mini pig.The deletion and insertion mutation and other SNPs found may created different combination sites for relevant transcription factors,in this way they could influence expression and transcriptional regulation in GHR and related genes.Bama-GHR-P1-EGFP and Bama-GHR-P2-EGFP that the active detection vectors involved in GHR gene promoter region were constructed and used to transfected PK15 cells,The fluorescence detection results indicated that the transcription of GHR gene could be activated by 1507 bp and 1603 bp fragments involved in the promoter region.The CpG island in GHR gene promoter region also showed different methylation levels between Bama-mini pig and Landrace.The methylation level in CpG island in GHR gene promoter region from liver of Bama-mini pig was significantly higher on day 180 of age(P<0.01).It was thus suggested that methylation level in GHR gene promoter region may be involved with the liver GHR gene transcription regulation.
【Key words】 Guangxi Bama-mini pig; Growth hormone receptor gene; Expression; Transcriptional regulation;