猪Pit-1基因分子遗传多态性研究
摘要
催乳素(Prolactin，PRL)、生长激素(Growth Hormone，GH)、促甲状腺素(Thyroid-stimulatingHormone，TSH)是动物垂体前叶分泌的具有重要生理功能的激素。而垂体特异性转录因子(Pituitary-Specific Transcription Factor)Pit-1则是动物垂体前叶特异表达的一种具有重要功能的转录因子，它与垂体中PRL基因、GH基因、TSH基因以及Pit-1自身的启动子结合，调控这些基因的转录和表达，从而影响动物的生长、发育和繁殖。
本研究的目的是通过分子生物技术手段，寻找猪Pit-1基因序列中的多态位点，为后续深入研究提供理论依据。试验选用北京黑猪、沂蒙黑猪、莱芜猪、巴马小型猪、滇南小耳猪、五指山猪、香猪7个中国地方猪品种以及英系大白猪、长白猪、法系大白猪、杜洛克4个国外引入品种(系)共11个试验动物群体508头个体。采用PCR-SSCP和PCR-RFLP分析方法研究猪Pit-1基因的遗传变异，研究结果如下：
1．应用PCR-SSCP技术，利用引物P_1，在7个中国地方猪种Pit-1基因第4外显子上发现了一个突变位点。序列测定结果表明，其单核苷酸变异是由碱基C→T的替换造成的，但并没有导致编码氨基酸的改变，均编码组氨酸。对7个中国地方猪群体共计347头个体的基因型进行了检测与统计分析，Hardy-Weinberg平衡适合性检验结果表明：在这7个群体中，沂蒙黑猪、巴马小型猪群体的基因频率和基因型频率都处于平衡状态(P＞0.05)，而北京黑猪、莱芜猪、滇南小耳猪、五指山猪、香猪群体则未达到平衡状态(P＜0.05)；另由多态信息含量(PIC)值的计算可知此基因座位在莱芜猪和五指山猪群体中多态性不高，遗传变异不大，而在北京黑猪、沂蒙黑猪、巴马小型猪、滇南小耳猪和香猪群体中则呈现中度多态。
2．应用PCR-RFLP技术，利用引物P_3，在4个国外引入猪种(系)Pit-1基因的包含部分内含子4、全部外显子5、内含子5、外显子6及部分3′侧翼的1747bp的扩增片段中检测到多个突变位点，并验证了一个RsaI-RFLP多态位点，在此位点上产生了A、B两个等位基因。测序分析表明：其多态性的产生是由于第542位碱基G→C的替代造成的。该切点存在时产生的片段为等位基因A，消失时产生的片段为等位基因B。在英系大白猪、长白猪、法系大白猪、杜洛克4个国外引入品种(系)群体共计161头个体中分析了此突变位点的基因型情况，Hardy-Weinberg平衡适合性检验结果表明：英系大白猪和杜洛克猪群体处于平衡状态(P＞0.05)，而长白猪和法系大白猪群体未处于平衡状态(P＜0.05)；另由多态信息含量(PIC)值的计算可见，4个国外引入品种(系)猪群体均处于中度多态，说明此基因座位在这4个国外引入品种(系)猪群体中多态性高，遗传变异大。同时，经过预测，发现其中的一个SNP可能使猪内含子5中出现了新的转录因子结合位点。
3．利用引物P_4成功地克隆出猪Pit-1基因的部分外显子3及全部内含子3的序列。从而为猪Pit-1基因的进一步研究奠定了基础。
本试验在Pit-1基因已知序列中检测到的突变可以进一步与生产性状相结合，进行更深入地研究。而所克隆的猪Pit-1基因部分外显子3及全部内含子3的序列则可被用作探针，通过构建基因组DNA文库，筛选猪Pit-1基因的全序列以及调控区的信息。此外，该段序列的克隆还为进一步寻求基因内的遗传学变异提供了基础。
Prolactin(PRL),Growth Hormone(GH) and Thyriod-Stimulating Hormone(TSH),secreting by animals' anterior pituitary, are essential hormones in physiology. Pituitary-Specific Transcription Factor(Pit-l) is one of the important transcription factors of GH, PRL and TSH gene. Pit-1 plays an important role in animals' growth, development and reproduction via regulating the expression of these genes.
The purpose of this study is to detect the genetic variations of the pit-1 gene. Seven Chinese local pig breeds, including Beijing Black pig, Yimeng Black pig, Laiwu pig, Bama Small pig, Diannan Small-ear pig, Wuzhishan pig, Xiang pig, and four foreign breeds, which are Large White(England), Landrace, Large White(France) and Duroc were used as experimental examples. PCR-SSCP and PCR-RFLP analyses were used to analyze genetic variations in Pit-1 gene. At the same time, intron3 of pig Pit-1 gene had been cloned successfully. The results were as follows:
1. Specific primer PI was designed to amplify fragment in exon4 of pig Pit-1 gene. Then one PCR-SSCP locus was discovered, it had two alleles, designated as A and B. Sequence analysis revealed that PI locus was generated because of C-T transilation, but they both code Histidine. The distribution of corresponding genotypes and alleles at this PCR-SSCP locus was analyzed in seven Chinese local breeds. A chi-square analysis suggested that Beijing Black pig and Bama Small pig reached Hardy-Weinberg equilibrium(P>0.05), the other five Chinese local breeds were in disequilibrium(P<0.05). Laiwu pig and Wuzhishan pig had low polymorphism(PIC<0.25), Beijing Black pig, Yimeng Black pig, Bama Small pig, Diannan Small-ear pig and Xiang pig had intermediate polymorphism(0.25 2. Using specific primer PS identified a Rsal-RFLP polymorphism site in 1747bp amplified fragment of pig Pit-1 gene. This site had two alleles, designated as A and B. The G-C transition was revealed at 542 position by sequencing analysis. The distribution of different genotypes and alleles was analyzed in four foreign breeds. A chi-square analysis suggested that Large White(England) and Duroc reached Hardy-Weinberg equiIibrium(P>0.05), Landrace and Large White(France)were in disequilibrium(P<0.05). All of these four foreign breeds had intermediate polymorphism(0.25 3. Parts of exon3 and wholly intron3 of pig Pit-1 gene were cloned successfully by using primer P4, which provide some of basal information to research the pig Pit-1 gene further.
Mutations that detected in this study should be associated with relative performance traits, which is worthy of being further studied. Parts of exon3 and wholly intron3 sequence of pig Pit-1 gene that we cloned, can be used as probe to select the whole sequence or the regulatory domian of pig Pit-1 gene through screen genomic DNA library. At the same time, we can use those sequences that we obtained to find more molecular mutations in pig Pit -1 gene.
本研究的目的是通过分子生物技术手段，寻找猪Pit-1基因序列中的多态位点，为后续深入研究提供理论依据。试验选用北京黑猪、沂蒙黑猪、莱芜猪、巴马小型猪、滇南小耳猪、五指山猪、香猪7个中国地方猪品种以及英系大白猪、长白猪、法系大白猪、杜洛克4个国外引入品种(系)共11个试验动物群体508头个体。采用PCR-SSCP和PCR-RFLP分析方法研究猪Pit-1基因的遗传变异，研究结果如下：
1．应用PCR-SSCP技术，利用引物P_1，在7个中国地方猪种Pit-1基因第4外显子上发现了一个突变位点。序列测定结果表明，其单核苷酸变异是由碱基C→T的替换造成的，但并没有导致编码氨基酸的改变，均编码组氨酸。对7个中国地方猪群体共计347头个体的基因型进行了检测与统计分析，Hardy-Weinberg平衡适合性检验结果表明：在这7个群体中，沂蒙黑猪、巴马小型猪群体的基因频率和基因型频率都处于平衡状态(P＞0.05)，而北京黑猪、莱芜猪、滇南小耳猪、五指山猪、香猪群体则未达到平衡状态(P＜0.05)；另由多态信息含量(PIC)值的计算可知此基因座位在莱芜猪和五指山猪群体中多态性不高，遗传变异不大，而在北京黑猪、沂蒙黑猪、巴马小型猪、滇南小耳猪和香猪群体中则呈现中度多态。
2．应用PCR-RFLP技术，利用引物P_3，在4个国外引入猪种(系)Pit-1基因的包含部分内含子4、全部外显子5、内含子5、外显子6及部分3′侧翼的1747bp的扩增片段中检测到多个突变位点，并验证了一个RsaI-RFLP多态位点，在此位点上产生了A、B两个等位基因。测序分析表明：其多态性的产生是由于第542位碱基G→C的替代造成的。该切点存在时产生的片段为等位基因A，消失时产生的片段为等位基因B。在英系大白猪、长白猪、法系大白猪、杜洛克4个国外引入品种(系)群体共计161头个体中分析了此突变位点的基因型情况，Hardy-Weinberg平衡适合性检验结果表明：英系大白猪和杜洛克猪群体处于平衡状态(P＞0.05)，而长白猪和法系大白猪群体未处于平衡状态(P＜0.05)；另由多态信息含量(PIC)值的计算可见，4个国外引入品种(系)猪群体均处于中度多态，说明此基因座位在这4个国外引入品种(系)猪群体中多态性高，遗传变异大。同时，经过预测，发现其中的一个SNP可能使猪内含子5中出现了新的转录因子结合位点。
3．利用引物P_4成功地克隆出猪Pit-1基因的部分外显子3及全部内含子3的序列。从而为猪Pit-1基因的进一步研究奠定了基础。
本试验在Pit-1基因已知序列中检测到的突变可以进一步与生产性状相结合，进行更深入地研究。而所克隆的猪Pit-1基因部分外显子3及全部内含子3的序列则可被用作探针，通过构建基因组DNA文库，筛选猪Pit-1基因的全序列以及调控区的信息。此外，该段序列的克隆还为进一步寻求基因内的遗传学变异提供了基础。
Prolactin(PRL),Growth Hormone(GH) and Thyriod-Stimulating Hormone(TSH),secreting by animals' anterior pituitary, are essential hormones in physiology. Pituitary-Specific Transcription Factor(Pit-l) is one of the important transcription factors of GH, PRL and TSH gene. Pit-1 plays an important role in animals' growth, development and reproduction via regulating the expression of these genes.
The purpose of this study is to detect the genetic variations of the pit-1 gene. Seven Chinese local pig breeds, including Beijing Black pig, Yimeng Black pig, Laiwu pig, Bama Small pig, Diannan Small-ear pig, Wuzhishan pig, Xiang pig, and four foreign breeds, which are Large White(England), Landrace, Large White(France) and Duroc were used as experimental examples. PCR-SSCP and PCR-RFLP analyses were used to analyze genetic variations in Pit-1 gene. At the same time, intron3 of pig Pit-1 gene had been cloned successfully. The results were as follows:
1. Specific primer PI was designed to amplify fragment in exon4 of pig Pit-1 gene. Then one PCR-SSCP locus was discovered, it had two alleles, designated as A and B. Sequence analysis revealed that PI locus was generated because of C-T transilation, but they both code Histidine. The distribution of corresponding genotypes and alleles at this PCR-SSCP locus was analyzed in seven Chinese local breeds. A chi-square analysis suggested that Beijing Black pig and Bama Small pig reached Hardy-Weinberg equilibrium(P>0.05), the other five Chinese local breeds were in disequilibrium(P<0.05). Laiwu pig and Wuzhishan pig had low polymorphism(PIC<0.25), Beijing Black pig, Yimeng Black pig, Bama Small pig, Diannan Small-ear pig and Xiang pig had intermediate polymorphism(0.25
Mutations that detected in this study should be associated with relative performance traits, which is worthy of being further studied. Parts of exon3 and wholly intron3 sequence of pig Pit-1 gene that we cloned, can be used as probe to select the whole sequence or the regulatory domian of pig Pit-1 gene through screen genomic DNA library. At the same time, we can use those sequences that we obtained to find more molecular mutations in pig Pit -1 gene.
引文
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