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Smad信号转导通路相关基因表达与湖羊高繁殖力关系的研究
Gene Expressions Related to Smad Signaling Molecule and Its Relationship with Fecundity in Hu Sheep
【作者】 徐业芬;
【导师】 谢庄;
【作者基本信息】 南京农业大学 , 动物遗传育种与繁殖, 2010, 博士
【摘要】 湖羊是世界著名的多胎绵羊品种,Smad信号通路中的很多信号分子与绵羊的生殖和卵泡发育有密切关系,参与调控绵羊排卵数、产羔数等多胎性状,为了探讨湖羊多胎与Smad信号通路的关系,我们利用RT-PCR技术首次系统检测了Smad信号通路中相关信号分子基因的组织表达谱,并进一步利用实时荧光定量PCR的方法研究了繁殖力较低湖羊(经产单羔:单羔组)和繁殖力较高湖羊(经产三、四羔:多羔组)与Smad信号转导通路相关基因在垂体和卵巢组织以及有腔卵泡中的表达差异,并分析了Smad言号通路中的有关基因表达量与排卵数的相关性。我们的研究结果提供了Smad言号通路有关基因在mRNA表达水平对湖羊排卵数的可能调控机理,同时为揭示湖羊高繁多胎机理和筛选影响湖羊多胎性状的特有候选基因提供了参考。1.湖羊BMP/Smad言号通路相关基因组织表达特征用RT-PCR法检测湖羊BMP/Smad信号通路中信号分子基因组织表达特征,结果成功克隆得到了BMP/Smad信号通路中BMP2、BMP4、BMP6、BMP7、BMP15、BMPRIA、BMPRIB、BMPRII、Smad1、Smad5、Smad4等信号分子基因部分cDNA片段,组织表达谱研究表明,BMP2、BMP4、BMP7、BMPRII、BMPRIA、BMPRIB、Smad1知Smad4基因在成年发情期湖羊母羊下丘脑、垂体、卵巢、子宫、输卵管、心脏、肝脏、脾脏、肺脏、肾脏、肌肉组织中均有表达,而BMP6基因仅在成年湖羊母羊卵巢、输卵管、’肾脏、肌肉组织中有表达;Smad5在子宫、输卵管、肺脏和肌肉组织中没有表达;BMP15仅在卵巢组织表达,为卵巢组织特异表达基因。从结果看,BMP/Smad信号转导通路相关基因mRNA表达与湖羊垂体和卵巢功能可能有密切联系2.高、低产湖羊垂体组织BMP/Smad信号通路分子基因表达差异及与排卵数相关性的研究我们首次利用荧光定量PCR技术对湖羊BMP/Smad信号通路中信号分子基因(包括BMP蛋白家族基因BMP2、BMP4、BMP7; BMP受体基因BMPRIA、BMPRIB、BMPRII;细胞内Smad蛋白家族基因Smadl、Smad4、Smad5) mRNA在高繁殖力(经产三、四羔:多羔组)和低繁殖力(经产单羔:单羔组)湖羊母羊发情后24-36小时排卵时期在垂体组织中的表达水平进行分析。结果显示,多羔组卵巢排卵数极显著高于单羔组(P<0.01);在垂体组织中,BMP2、BMP7、BMPRIA、BMPRII.Smadl知Smad4基因表达量在单羔组和多羔组垂体组织内没有显著差异(P>0.05),但是,多羔组BMP4、BMPRIB知Smad5基因表达量极显著低于单羔组(P<0.01),且与卵巢排卵数均分别呈不同程度的负相关(P<0.05或P<0.01),说明湖羊垂体组织BMP4.BMPRIB知Smad5基因表达量差异可能是引起湖羊较高产羔数的原因之一。3.高、低产湖羊卵巢组织BMP/Smad信号通路分子基因表达差异及与排卵数相关性的研究我们利用荧光定量PCR技术分析了高繁殖力(经产三、四羔:多羔组)和低繁殖力(经产单羔:单羔组)湖羊母羊发情后24-36小时排卵时期卵巢组织中BMP/Smad通路中的BMP蛋白家族基因(BMP2、BMP4、BMP6、BMP7\BMP15), BMP受体基因(BMPRIA、BMPRIB、BMPRII)和细胞内Smad蛋白家族基因(Smadl、Smad5, Smad4)表达水平差异及其与卵巢排卵数间的相关性,结果显示:多羔组湖羊BMP4、BMPRIB知Smad4基因mRNA表达极显著高于单羔组(P<0.01),BMP15知BMPRII基因mRNA表达显著高于单羔组(P<0.05),而BMP2、BMP6、BMP7、BMPRIA、Smadl知Smad5基因mRNA表达在单羔组和多羔组间没有显著差异(P>0.05);相关分析表明卵巢组织中BMP4、BMP15、BMPRIB、BMPRII、Smad5和Smad4基因mRNA表达与排卵数呈正相关,表明BMP4、BMP15、BMPRIB、BMPRII知Smad4基因在mRNA水平对湖羊排卵数有影响,可能是影响湖羊高产、多胎性状的候选基因。4.不同繁殖力湖羊有腔卵泡BMP/Smad信号通路基因表达水平研究为了进一步探讨湖羊多胎与BMP/Smad信号通路的关系,我们以机械法获取高繁殖力(经产三、四羔:多羔组)和低繁殖力(经产单羔:单羔组)湖羊母羊发情后24-36小时排卵时期卵巢组织中的部分有腔卵泡,利用荧光定量PCR技术对BMP/Smad信号通路基因(包括BMP蛋白家族基因BMP2、BMP4、BMP6、BMP7、BMP15; BMP受体基因BMPRIA、BMPRIB、BMPRII;细胞内Smad蛋白家族基因Smadl、Smad4、Smad5) mRNA在有腔卵泡中的表达水平进行分析,结果显示:多羔组湖羊BMP4,BMPRIB和BMPRIl基因mRNA表达显著高于单羔组(P<0.05), Smad4基因mRNA表达极显著高于单羔组(P<0.01), BMP15 mRNA显著低于单羔组(P<0.05), BMP2、BMP6、BMP7、BMPRIA、Smad1知Smad5基因mRNA表达在单羔组和多羔组湖羊有腔卵泡中的表达没有显著差异(P>0.05)。结果提示单羔组和多羔组湖羊BMP4、BMP15、BMPRIB、BMPRIⅠ和Smad4基因表达差异可能是湖羊多胎原因之一。5.湖羊TGF-β/Smad言号通路相关基因组织表达特征用RT-PCR法检测湖羊TGF-β/Smad信号通路中信号分子基因组织表达特征,结果成功克隆得到了TGF-β/Smad信号通路中TGF-β1、TGF-β2、TGF-β3、TGF-βRⅠ、TGF-βRⅡ、Smad2、Smad7等信号分子基因部分cDNA片段,组织表达谱研究表明,TGF-β1、TGF-β2、TGF-β3、TGF-βRⅠ、TGF-βRⅡ、Smad2知Smad7在湖羊母羊卵巢、子宫、输卵管等生殖器官有表达外,在下丘脑、垂体、心脏、肝脏、脾脏、肾脏、肌肉组织中也均有表达,但是TGF-β1、TGF-β2、TGF-β3、TGF-βRⅠ在肺脏组织中没有表达,而TGF-βRⅡ、Smad2知Smad7在肺脏中有表达。从结果看,TGF-β/Smad信号通路相关基因在湖羊体内广泛表达,而在卵巢组织均可检测到其明显表达,说明TGF-β/Smad信号通路相关基因对湖羊繁殖功能可能有一定的影响。6.高、低产湖羊卵巢组织TGF-β/Smad信号通路分子基因表达差异及与排卵数相关性的研究对高繁殖力(经产三、四羔:多羔组)和低繁殖力(经产单羔:单羔组)湖羊母羊发情后24-36小时卵巢组织中TGF-β/Smad信号通路的TGF-β蛋白基因(TGF-β1、TGF-β2、TGF-β3), TGF-P受体基因(TGF-βRⅠ知TGF-βRⅡ)和细胞内Smad蛋白家族基因(Smad2、Smad7)在卵巢组织的表达水平及其与卵巢排卵数间的相关性进行研究,发现多羔组湖羊卵巢组织TGF-β1知TGF-β2基因表达显著高于单羔组(P<0.05), TGF-β3、TGF-βRⅡ知TGF-βRⅠ基因表达极显著高于单羔组(P<0.01);Smad7基因表达则显著低于单羔组(P<0.01);Smaad2基因在多羔组和单羔组卵巢组织间没有显著差异(P>0.05)。进一步的相关分析结果表明TGF-β1、TGF-β2、TGF-β3知TGF-βRⅠ与排卵数无相关,TGF-βRⅡ与卵巢排卵数存在显著正相关(P<0.05),Smad7与卵巢排卵数存在显著负相关(P<0.01)。表明TGF-PRⅡ和Smad7基因在mRNA水平对湖羊排卵数有影响,可能是影响湖羊高产、多胎性状的候选基因。
【Abstract】 Hu sheep is famous for its hyper-prolificacy in the world, and the members of Smad pathway are shown to have close relationships with reproduction and follicular development with roles of regulating ovulation and litter size in sheep. In order to investigate the mechanism of high fecundity in Hu sheep and its relationships with Smad pathway, tissue mRNA distributions of genes encoding Smad pathway molecules were detected by RT-PCR, and the differential expression levels in ovaries, pituitaries and antral follicles of high-fecundity (HF) and low-fecundity (LF) Hu sheep were further investigated by real-time PCR. In addition, SPSS software was used to analyze the relationship between the gene expression level and ovulation number in Hu sheep. Our data show the first evidence that genes of Smad pathway in the mRNA levels may be correlated with regulation of ovulation number in Hu sheep and provide the reference for finding candidate genes of high fecundity and understanding the molecular mechanism of hyper-prolificacy in Hu sheep.1. Tissue Expression Profiles of BMP/Smad Pathway Molecule Gene mRNAs in Hu SheepTissue expression profiles of BMP/Smad pathway molecule gene mRNAs in Hu sheep were detected using RT-PCR. The results showed that cDNA fragments were successfully cloned at expected sizes for genes encoding BMP/Smad pathway molecules including BMPs (BMP2, BMP4, BMP6, BMP7 and BMP 15), BMP receptors (BMPRIA, BMPRIB and BMPRII) and intracellular transducers (Smadl, Smad 4, Smad 5 and Smad 7), and tissue mRNA distributions indicated that BMP2, BMP4, BMP7, BMPRIA, BMPRII, Smadl, Smad4 and Smad7 mRNA exited in the ovary and other tissues, including hypothalamus, pituitary, uterus, heart, liver, spleen, lung, kidney, muscle and oviduct, but BMP6 mRNA was only detected in the ovary, kidney, muscle and oviduct, and Smad5 mRNA in uterus, lung, muscle and oviduct were not detected, but BMP15 mRNA was only found in the ovary.lt is suggested that BMP/Smad pathway might have roles in pituitary-ovary axis for prolificacy of Hu sheep.2. BMP/Smad Pathway Gene Expression Levels in Pituitaries and Associations with Ovulation Number in Hu Sheep The present study was performed to determine the expressional differences of mRNAs encoding BMP/Smad pathway molecules, including BMP2, BMP4, BMP7, BMPRIA,BMPRIB, BMPRII, Smadl, Smad5 and Smad4 in pituitaries of Hu sheep during 24 and 36 hours after estrus diagnosis between high-fecundity (HF) and low-fecundity (LF) and to further investigate the relationships between their expressions and ovulation number. The results showed that there were no significant differences in BMP2,BMP7, BMPRIA,BMPRII,Smadl and Smad4 mRNA abundance in the pituitary between HF and LF group (P>0.05) respectively. But BMP4, BMPRIB and Smad5 mRNA abundances in the pituitary were respectively higher in HF group than those in LF group (P<0.01) and negatively correlated with ovulation number. It is suggested that the expressional differences of BMP4, BMPRIB and Smad5 in the pituitary between high-fecundity and low-fecundity group might be one of factors for high fecundity in Hu sheep.3. BMP/Smad Pathway Gene Expression Levels in Ovaries and Associations with Ovulation Number in Hu SheepThe mRNA transcript expression differences for members of the BMP/Smad pathway molecules including BMPs (BMP2, BMP4, BMP6, BMP7 and BMP 15), BMP receptors (BMPRIA, BMPRIB and BMPRII) and intracellular transducers (Smadl, Smad5 and Smad4) in the ovary of High- fecundity(HF group,) and Low-fecundity (LF) Hu Sheep between 24 and 36 hours after estrus diagnosis were measured by real-time PCR. The results showed that BMP4, BMP15, BMPRIB, BMPRII and Smad4 mRNA abundances in the ovary of HF group were respectively higher than those in LF group (P<0.05 or P <0.01) and also correlated with ovulation number(P<0.05 or P<0.01). But there were no significant differences in BMP2, BMP6, BMP7, BMPRIA, Smadl and Smad5 mRNA abundances in the ovary between LF and HF group (P>0.05). It is suggested that BMP4、BMP15、BMPRIB、BMPRII and Smad4 might play roles in ovulation number and might be candidate genes for high fecundity in Hu sheep. These findings may help to improve the molecular breeding of Hu sheep.4.Differential Expression of mRNAs Encoding BMP/Smad Pathway Molecules in Antral Follicles of High-and Low-fecundity Hu SheepIn order to further investigate the mechanism of high fecundity in Hu sheep and its relationship with the BMP/Smad pathway, antral follicles were punctured for high-fecundity (HF) and low-fecundity (LF) animals. The gene expression levels of mRNAs encoding BMP/Smad pathway molecules including BMPs (BMP2, BMP4, BMP6, BMP7 and BMP15), BMP receptors (BMPRIA, BMPRIB and BMPRⅡ) and intracellular transducers (Smad1, Smad5 and Smad4) in antral follicles were detected by real-time PCR. The results showed that BMP4, BMPRIB, BMPRⅡand Smad4 mRNAs were more abundant in the antral follicles of HF animals than those of LF animals (P<0.05 or P<0.01), but BMP15 mRNA was less abundant (P<0.05).The expression levels of BMP2, BMP6, BMP7, BMPRIA, Smad1 and Smad5 did not differ between the HF and LF groups (P>0.05).This suggests that the expressional differences of BMP4, BMP 15, BMPRIB, BMPRⅡand Smad4 in the antral follicles between high-fecundity and low-fecundity group might be one of factors for high fecundity in Hu sheep.5. Tissue Expression Profiles of TGF-β/Smad Pathway Molecule Gene mRNAs in Hu SheepTissue expression profiles of TGFβ/Smad pathway molecule gene mRNAs encoding TGF-β1,TGF-β2,TGF-β3,TGF-βRⅠ,TGF-βRⅡ,Smad2 and Smad7 in Hu sheep were detected using RT-PCR. The results showed that cDNA fragments were successfully cloned at expected sizes for the genes and tissue mRNA distributions indicated that TGF-β1, TGF-β2,TGF-β3,TGF-βRⅠ,TGF-βRⅡ,Smad2 and Smad7 mRNA exited in the ovary and other tissues, including hypothalamus, pituitary, uterus, heart, liver, spleen, kidney, muscle and oviduct, and TGF-β1, TGF-β2,TGF-β3 and TGF-βRⅠmRNA were not only detected in the lung, but TGF-βRⅡ,Smad2 and Smad7 mRNA exited in lung. It is suggested that genes related toTGF-β/Smad pathway expressed extensively in Hu sheep and the obvious existences of their mRNAs in the ovary might play roles in the ovary for prolificacy in Hu sheep.6. TGF-β/Smad Pathway Gene Expression Levels in Ovaries and Associations with Ovulation Number in Hu SheepThe mRNA transcript expression differences for members of the TGFβ/Smad pathway molecules including TGF-β1、TGF-β2、TGF-β3 and their receptors (TGF-βRⅠand TGF-βRⅡ) and intracellular transducers (Smad2 and Smad7) in the ovary of High-fecundity(HF) and Low-fecundity (LF) Hu Sheep between 24 and 36 hours after estrus diagnosis were measured by real-time PCR. The results showed that TGF-β1,TGF-β2, TGF-β3,TGF-βRⅠand TGF-βRⅡmRNA abundances in the ovary of HF group were respectively higher but Smad7 was lower than LF group (P<0.05 or P<0.01), and there were no significant differences in Smad2 mRNA abundances in the ovary between HF and LF group (P>0.05), and the correlation analysis also indicated that TGF-βRⅡpositively and Smad7 negatively correlated with ovulation number(P<0.05 and P<0.01). It is suggested that TGF-βRⅡand Smad7 might play roles in ovulation number and might be candidate genes for high fecundity in Hu sheep.These findings may help to improve the molecular breeding of Hu sheep.