【作者】 白莹；

【导师】 吴常信； 方美英；

【作者基本信息】 中国农业大学 ， 动物遗传育种与繁殖， 2014， 博士

【摘要】 胰岛素样生长因子结合蛋白7(IGFBP7)是一种既能携带转运IGFs,又能调控IGFs生物活性的重要蛋白。同时,IGFBP7能独立调节细胞生长、分化和发育。已有研究结果表明IGFBP7基因在双肌臀猪肌肉组织RNA池中的表达量高于非双肌臀,说明IGFBJD7基因与猪肌肉生长发育相关。但关于该基因是如何影响猪肌肉生长发育以及其促生长的调控机制目前未见详细报道。本研究对IGFBP7基因遗传特性及其在猪种间背最长肌发育过程中的表达规律进行了分析,同时鉴定了以IGFBP7基因为靶标的microRNAs,以期阐述IGFBP-基因在肌肉生长发育中潜在的分子作用机制和调控机理。本研究首先分析了猪IGFBP7基因的遗传结构特性,序列分析结果显示IGFBP7基因编码区不存在品种间突变,只在内含子1、3、4中检测到了品种间序列变异。我们进而采用荧光定量PCR技术检测了第1、30、90、120、150和180日龄间大白猪和金华猪背最长肌组织中IGFBP7基因、胰岛素样生长因子1和2(IGF1,IGF2)基因mRNA表达变化,定量结果显示IGFBP7.IGF1和IGF2基因表达模式在品种和不同发育阶段存在显著差异。第1、30和180日龄,金华猪IGFBP7mRNA表达量高于大白猪,而第90、120、150日龄,大白猪IGFBP7mRNA表达高于金华猪,且在90日龄达到差异极显著水平(P<0.01)。大白猪IGFBP7mRNA表达量在90日龄时达到最高,且显著高于其它日龄(P<0.05),而金华猪IGFBP7mRNA表达量在1、30和90日龄间差异不显著,但显著高于120、150、和180日龄(P<0.05):IGF1和IGF2基因表达也呈时序性变化,但与IGFBP7基因表达具有不同的发育表达模式。以上结果表明IGFBP7基因表达可能与不同品种猪肌肉生长发育差异有关,且IGFBP7可能通过一种不依赖IGFs的方式来影响肌肉的发育。Western blot分析结果显示90日龄和180日龄IGFBP7蛋白的表达与此阶段mRNA的表达模式不一致,表明猪IGFBP7基因可能存在转录后蛋白水平的调控。通过生物信息学对IGFBP7基因启动子特性分析显示,IGFBP7基因启动子区有多个与肌肉生长发育相关的转录因子的结合位点,如MVoD、YY1等。缺失载体构建和瞬时转染实验结果表明：启动子的-333～+3区段活性最高,在-349--315之间存在至少一个增强子序列,-585--349之间至少存在一个沉默子序列。遗传特性分析结果显示IGFBP7基因ATG上游-687bp处有一个300bp片段的插入,但我们的研究证实300bp片段的插入并没有影响启动子的活性。而同时在启动子区发现的7个SNPs位点,其中5个位点引起了结合的转录因子的改变,这可能对IGFBP7启动子的转录调控具有作用。本研究还分析了猪IGFBP7基因潜在的可变剪接体模式,结果显示猪IGFBP7存在两个可变剪接体,共有三个转录本,其中IGFBP7完整的mRNA序列为主转录本。随后利用microRNA表达谱芯片筛选了90日龄大白猪与金华猪背最长肌组织中差异表达的microRNAs,结果显示：金华猪相对于大白猪有4个microRNAs表达显著上调,16个microRNAs表达显著下调。差异表达microRNAs靶基因预测和双荧光报告系统实验的结果显示ssc-miR-487b和ssc-miR-142-5p的靶基因可能是IGFBP7。ssc-miR-142-5p和ssc-miR-487b的表达在不同猪种不同日龄存在差异,这两个microRNAs可能在转录后水平调控IGFBP7基因的表达。更多还原

【Abstract】 The insulin-like growth factor binding-protein7(IGFBP7) has binding affinities to IGFs and been found to be able to etiher positively or negatively regulate the IGFs signaling pathway, and also plays a crucial role on cell growth, differentiation and development in an IGF-independent manner. It has been reported that the expression of IGFBP7in double-muscled pig RNA pool was higher than that in non-double-muscled pig RNA pool, and IGFBP7was a potential candidate gene associated for pig muscle growth and development. However, the regulatory mechanism of IGFBP7gene behind it is still unclear. In order to elucidate molecular mechanism of IGFBP7in promoting muscle growth, the genetic variations of IGFBP7and the expression pattern of IGFBP7in diverse pig breeds were investigated. Moreover the microRNAs were identified, which have IGFBP7as target gene.Firstly, the genetic structure and variation of IGFBP7gene among different pig breeds were analyzed. There were no sequences variation found in exons region but some sequences variation found in introns1,3and4. Subsequently real-time fluorescence quantitavite PCR was applied to investigate the developmental expression pattern of IGFBP7, IGF1and IGF2at the age of day1,30,90,120,150and180with longissimus muscle tissue from Yorkshire and Jinhua pigs. Results showed that IGFBP7, IGF1and IGF2mRNA expression exhibited different patterns between these two pig breeds. Furthermore, IGFBP7mRNA expression at day1,30and180was higher in Yorkshire pigs than that in Jinhua pigs, however IGFBP7mRNA expression was higher in Jinhua pigs at the rest investigated days. In Yorkshire pigs, IGFBP7expression was significantly higher at day90than at other ages. In Jinhua pigs, there were no significant differences among IGFBP7mRNA expression at day1,30and90, however, which were higher than that at day120,150,180days. The mRNA expression patterns among IGFBP7, IGF1and IGF2showed that IGFBP7may have function on muscle growth in an IGF-independent manner. The IGFBP7protein levels at day90and180were higher in Jinhua pigs than in Yorkshire pigs by western blot was inconsistent with the qPCR results, which may be due to post-transcriptional gene regulation.Sequence analysis by bioinformatics revealed that several putative transcription factors associated with muscle development existed in this region, such as MyoD, YY1. Transient transfection assay showed that the fragment at the region-333bp to+3bp exhibited the highest luciferase response and the-349～-315region might have positive elements binding sequences while the-585～-349region might have inhibitor binding sequences. The results of genetic variation analysis in the promoter region showed that a300bp-fragment insertion was identified at the-687bp site, however, which had no influence on the promoter activity. Moreover, seven genetic variations were found in IGFBP7promoter region and resulted in the change of transcription factors in the alternative alleles, which might have a role in IGFBP7transcriptional regulation. In the study, the potential alternative splicing pattern of IGFBP7gene was investigated. Two novel splice variants were identified. And the transcript was the primary transcript. The microRNA microarray was used to identify and characterize the differentially expressed miRNAs in Jinhua pigs relative to Yorkshire pigs. Four up-and sixteen down-regulated miRNAs were identified and had more than2-fold differential expression between Jinhua pigs and Yorkshire pigs (P value <0.05). Further, ssc-miR-487b and ssc-miR-142-5p were predicted targeting to IGFBP7gene and were validated by Dual-Glo Luciferase Assay System. ssc-miR-487b and ssc-miR-142-5p might regulated IGFBP7gene at post-transcriptional level.更多还原