【作者】 程金花；

【导师】 陈国宏；

【作者基本信息】 扬州大学 ， 动物遗传育种与繁殖， 2009， 博士

【摘要】 垂体特异性转录因子(Pit-1)由PIT-1基因编码,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。PIT-1与垂体中PRL、GH、TSH-β以及自身的启动子结合,调控这些基因的转录,通过调节这些基因的表达而在机体的生长、发育、繁殖和免疫等很多方面起着重要作用。本文对鹅PIT-1基因进行了系统的研究,以狮头鹅、皖西白鹅和籽鹅为对象,首次获得了PIT-1基因的cDNA序列,对其编码区及氨基酸序列进行了生物信息学分析;检测了狮头鹅、皖西白鹅、籽鹅、浙东白鹅、四季鹅和朗德鹅群体PIT-1基因序列的遗传变异,比较不同群体内的基因型的分布规律,并分析了基因遗传变异对早期体重和屠宰性状的影响;同时采用荧光定量PCR技术首次分析了皖西白鹅PIT-1基因早期的发育性表达规律,分析了GH基因和PRL基因的表达模式,并首次对不同基因间表达规律的关系进行了初探。主要研究结果如下:1.以皖西白鹅、籽鹅和狮头鹅为研究对象,采用RT - PCR和克隆测序的方法,获得了鹅PIT-1基因2条长为1014 bp的cDNA序列以及内含子2和2a的全部序列;其中编码区长度为1011 bp,与人类、鼠、火鸡、鸡和鸭的同源性分别为63.2%、68.2%、83.5%、84.3%和97.2%;内含子2和内含子2a的长度分别为819 bp和333 bp,与鸭的内含子序列的同源性分别为83%和89.2%;2条编码区序列分别在485位和746位存在差异。2.采用生物信息学方法分析得到的编码区序列, 2条序列的差异引起了其编码氨基酸162位和250位的变化,氨基酸序列与火鸡、鸡和鸭的同源性较高。所编码的氨基酸序列有15个磷酸化位点,且大多为亲水性氨基酸,无跨膜螺旋结构;氨基酸序列的二级结构包含了alpha螺旋(154AA)、延伸片段(16AA)和随机螺旋(166AA)三种结构,具有POU蛋白家族特有的保守结构,确定为鹅Pit-1氨基酸序列。3.在鹅PIT-1基因内含子1内检测到2处插入/缺失变异,该变异的分布在不同群体内差异显著,在外来品种朗德鹅内表现为单态;统计结果显示该遗传变异与皖西白鹅早期体重显著相关,插入了片段的B等位基因有利于早期体重的增加,可作为辅助分子标记对鹅早期体重性状进行选育;同时该遗传变异与狮头鹅的屠体性状有显著的基因型效应,AB型的心率显著高于AA型。表明对不同类型群体而言,该变异所影响的性状可能不一致。采用PCR-SSCP技术在6个群体内检测到了3个SNPs,其中非编码区以及364位的变异只存在于朗德鹅群体内;基因型在群体内的分布差异极显著,EE基因型只存在于朗德鹅群体内;统计结果显示,狮头鹅、籽鹅群体内DD型早期体重显著高于CD型,浙东白鹅群体内CD型体重显著高于CC型,表明D等位基因对于体重而言可能为有利基因;狮头鹅群体内的DD型腹脂率显著高于CD型,与体重的基因型效应一致,而籽鹅群体内DD型屠体率高于CD型,但差异不显著,CC型屠体率显著高于CD,说明就不同类型的群体而言,基因型对性状的影响可能会存在差异。在检测到的2种遗传变异的分布上,朗德鹅与国内5个群体存在极显著的差异,在这2种变异上所有个体表现为单态,这可能与群体的不同起源有关,也可能与群体长期适应不同生产性能的选育有关。插入/缺失变异的AA基因型和SNPs变异的EE基因型可能更利于朗德鹅的肥肝性能,可作为其选育的辅助分子标记。4.采用荧光定量PCR技术检测PIT-1基因、GH基因和PRL基因的早期发育性表达模式,结果表明,公母之间垂体内PIT-1基因和GH基因在0 - 4周龄内的变化趋势存在差异,其余阶段变化趋势一致;公母之间下丘脑内GH基因的表达出现高峰和低谷的阶段不一致,其余大体相同,表明PIT-1基因和GH基因的表达在性别间表现出二态性,对不同性别性状的影响可能会不一致。同一性别内,GH基因在公鹅垂体内和下丘脑内的表达模式一致,而母鹅在0 - 2周龄及30周龄的变化存在差异;表明不同性别间GH基因表达受调控的机制可能会存在一些差别。母鹅PRL基因不同组织内表达的模式不一致,垂体和下丘脑在2 - 10周龄内变化趋势一致,而在15周龄前,卵巢与下丘脑的变化趋势相反,说明对PRL基因而言,不同组织内基因的表达可能因不同因素的调节而存在差异。对PIT-1、GH和PRL 3种基因垂体内的表达模式进行比较发现,无论公母,垂体内GH基因的表达模式与PIT-1基因完全相同,说明GH基因的表达随着PIT-1基因的表达变化而变化,受到PIT-1基因的遗传调控;而PRL基因的表达模式和PIT-1基因并不完全一致,暗示PRL基因的表达还可能受到PIT-1基因与其它因子的共同影响。这一结果与前人的相关报道一致。更多还原

【Abstract】 Pituitary-specific factor 1 (Pit-1) was encoded by PIT-1 gene, it is a transcription factor expressed in cephalic of Pituitary gland with essential function. PIT-1 regulates the transcription of PRL, GH, TSH-βand itself by binding to the promoter of them, and plays important roles in the growth, development, reproduction and immunity by regulates the expression of these genes.In this study, the cDNA sequence of PIT-1 gene was first cloned in Shitou goose, Wanxi white goose and Zi goose, and the bioinformatics analysis on the CDS and amino acid sequences was conducted; then analysis of genetic variation of PIT-1 was carried out in Shitou goose, Wanxi white goose, Zi goose, Zhedong white goose, Siji goose and landosie goose, the genotypes distribution in different populations was compared and the effects of genotypes on early bodyweight and carcass traits was analyzed; further, gene expression of PIT-1 gene, PRL gene and GH gene in different developmental stages in Wanxi white goose was investigated by Real-time PCR technology to explore the expression characteristics. The main results were as follows:1. Two cDNA sequences of 1014 bp size and the sequences of intron 2 and intron 2a was gained by the methods of RT - PCR and sequencing in Wanxi white goose, Shitou goose and Zi goose; the CDS of PIT-1 gene was 1011 bp in length, and the identity with human, mouse, turkey, chicken and duck were 63.2%, 68.2%, 83.5%, 84.3% and 97.2%, respectively; the size of intron 2 was 819 bp, and intron 2a was 333 bp, their identity with duck introns were 83% and 89.2%; the bases in position 485 and 746 was different in two cDNA sequences.2. Bioinformatic analysis was conducted on CDS sequences, the differences in nucleotides changed the amino acids at position 162 and 250, there were high identity between the amino acids sequence with turkey, chicken and duck. There were 15 phosphorlated sites in amino acids sequence, most of the amino acids were hydrophilic, the there was no transmenbrane helices in the sequence. The second structure of amino acids was consisted of alpha-helix (154AA), extended strand (16AA) and random coil (166AA). The structure of amino acids was characterized with POU specific conserved region, and make sure that this is the sequence of Pit-1.3. Two insertion/deletion were detected in intron 1 of goose PIT-1 gene, the distribution of this variation was significantly different in different populations, and the landosie goose showed as monomorphsim. Statistic analysis showed that, this variation was remarkable correlated with the early bodyweight of Wanxi white goose, allele B ,which has the inserted fragments was benefit to the gain of early bodyweight; at the same time, this variation significantly affected the heart ratio of Shitou goose, AB was higher than AA. This conclusion indicated that, the variation may affect different traits in different type of populations. Three SNPs of PIT-1 gene were detected in 6 populations, among them the SNP in intron and of position 364 were only appeared in landosie goose; genotype distribution was significant between populations, and EE genotype was detected only in landosie goose. Statistic analysis showed that the early bodyweight of DD genotype in Shitou and Zi goose was significantly higher than CD, and bodyweight of CD genotype in Zhedong white goose was significant than that of CC, which suggested that allele D was positive for bodyweight; ratio of abdominal fat of DD genotype was remarkable higher than CD in Shitou goose, however the difference of carcass ratio was not significant between DD and CD genotype in Zi goose, and CC was significantly higher than CD, which may indicated that, the effects of genotypes on traits depend on the commercial type of populations. Landosie goose shown extreme remarkable differences with other 5 populations on the variations, it shown monomorphism in both variations, which may due to the different originations or related to the long time breeding for specific productions; the AA genotype of insertion/deletion variation and the EE genotype of SNPs variation may be more beneficial to Foie Gras trait, and could be used as the assisted molecular marker for breeding.4. Ontogeny expression of PIT-1 gene, GH gene and PRL gene was conducted by Real-time PCR in Wanxi white goose, the results showed that, expression of PIT-1 gene and GH gene in pituitary of male shown differences with that of female during 0 - 4 weeks’age, and the peak and valley of GH gene expression in hypothalamus appeared at different time of male and female, which suggested sexual dimorphism of gene expression during 0 - 4week, and different effects on traits in male and female. Within the sex, GH gene expression patterns of male in Pituitary and hypothalamus was identical, and differences existed in two tissues in female at 0 - 2 and 30 week’s age, which suggested that there could be some differences in the mechanism of regulation on GH gene in male and female tissues. The expression patterns of PRL gene shown differences between tissues during 2 - 10 weeks, changes in pituitary and hypothalamus was the same, and before 15 weeks, changes in ovary was opposite with hypothalamus, this may due to the regulation of PRL gene by different factors in different tissues. Expression characteristics of PIT-1, GH and PRL in Pituitary were also compared in this study, and we found that, the expression pattern of GH gene was the same as PIT-1 gene in both sexes, which notifies that the expression of GH gene changed in response to PIT-1 gene, and it was under the genetic regulation of PIT-1. However, the pattern of PRL was not the same as PIT-1 at all stages, this result was similar to previous study, and it could be concluded that the expression of PRL may under the combined effects of PIT-1 and other factors.更多还原