【作者】 张晶晶；

【导师】 李祥龙；

【作者基本信息】 河北农业大学 ， 遗传学， 2010， 硕士

【摘要】 无角间性综合征(Polled Intersex Syndrome, PIS)的无角性状与间性性状紧密连锁。研究发现,导致山羊无角间性综合征产生的主要原因是一段长约11.7kb片段(PIS区域)的完全缺失。该缺失片段同时还影响着Forkhead box L2(FOXL2)基因的表达。FOXL2基因是一个单外显子基因,可编码一个376aa的蛋白序列。该蛋白序列在哺乳动物中除了含有一个110aa组成的DNA结合区外,还有一段14个丙氨酸残基组成的polyAla区。FOXL2基因突变不仅能引发人类I型和II型睑裂狭小综合征(blepharophimosis/ptosis/epicanthus inversus syndrome,BPES)的发生,同时还是XX型性反转山羊的候选基因。为了进一步了解无角间性个体中FOXL2基因及PIS区域变异情况,本文首先对已提交的FOXL2基因序列进行生物信息学分析,从整体上把握该基因的特征,初步预测该基因的蛋白结构,同时运用扩增,测序,比对及PCR-RFLP技术,对34个无角间性奶山羊、34个无角正常奶山羊及30个有角奶山羊进行研究,探讨FOXL2基因在无角间性奶山羊中的变异。另外,通过扩增、测序,检测PIS区域完整缺失是否存在于无角间性奶山羊中,PIS区域在间性个体之间是否存在差异及PIS区域在正常个体与间性个体之间的差异。通过对22个物种的43条FOXL2的CDS序列及其相应氨基酸序列进行分析,该基因密码子偏爱性不显著,但其编码序列的GC含量相对较高,其中哺乳动物的GC含量高达70%。哺乳动物FOXL2基因偏爱终止密码子TGA。DNA多态性分析检测到260个多态位点中,其中有61个单一多态位点,199个简约多态位点。哺乳动物和非哺乳动物之间的序列长度存在着很大的差异,结合氨基酸比对结果证明这种差异主要是由于非哺乳动物中不含有多聚丙氨酸编码区。哺乳动物中除了含有高度保守的多聚丙氨酸簇外,还含有甘氨酸重复区和脯氨酸重复区,这些重复序列在不同的哺乳动物中的数量有所差异;哺乳动物低复杂性区域的含量明显高于非哺乳动物。基序(motif)分析发现了10个高度保守位点,可能是蛋白的功能位点通过对FOXL2基因扩增、测序和比对,发现两个突变位点,分别为g.3911C >G和g.4289G >T(根据AY112725定位位点)。经PCR-RFLP分析及独立卡方检测发现尽管正常和间性山羊中都含有这两种突变,但其基因频率在正常个体与间性个体之间存在着显著的差异;同时这种基因频率的差异也表现在无角个体及有角个体之间。表明两个突变位点与无角间性性状之间存在着紧密联系。通过设计引物PIS1(26931～39428bp)、PIS2 (26931～27667bp)、PIS3 (27637～29012bp)、PIS4(38148～39044bp)及PIS5(35532～36425bp)对PIS区域(27015～38775bp)变异进行检测(根据AF404302)。在34个无角间性奶山羊中发现了PIS区域的部分缺失,但未检测到11.7kb的完全缺失。PIS5扩增片段序列比对结果显示间性个体在35532～36425 bp区段内不但存在部分片段的缺失、同时还含有大量碱基变异。与正常个体相比,在35607～36198 bp区段内间性个体中存在193个碱基突变位点和18处缺失。PIS2及PIS3的扩增测序结果证实间性个体PIS区域内部大量碱基突变并非存在于整个PIS区域,推测可能这些碱基突变只存在于部分片段,并影响着PIS区域的调节功能。间性性状的产生可能是由PIS部分区域内片段的缺失和碱基突变引起的。更多还原

【Abstract】 Hornlessness and intersex are closely linked in the Polled Intersex Syndrome (PIS), Study showed that it was the missing of one 11.7kb long fragment (PIS region) that induced the Polled Intersex Syndrome. This fragment’s missing also affected the expression of Forkhead box L2 gene (FOXL2). FOXL2 is one single exon gene, which encodes one 376aa long protein sequence. This protein possesses one polyAla tract, except for the 110aa DNA-binding domain. Mutations of FOXL2 not only could induce the blepharophimosis syndrome (blepharophimosis syndrome, BPES) type I and type II in humans, but also is a candidate gene for PIS in goats. In order to examine the variations of FOXL2 and the PIS region in the hornless intersex goats, we did comprehensive bioinformatics analysis of the submitted FOXL2 gene sequences and have grasped its basic features on the whole, including the structure of corresponding protein sequences. Meanwhile, variation of FOXL2 was also studied in 34 hornless intersex dairy goat individuals, 34 hornless normal dairy goat individuals and 30 horned normal goat individuals, via amplification, direct sequencing and PCR-RFLP technique. In addition, the polymorphism of PIS region was also studied within and among the above-mentioned three types of goat individuals.Analysis of the 43 FOXL2 gene CDS sequences and corresponding amino acid sequences showed that there was no significant codon bias among them, and relatively higher GC content existed, especially in mammals (70%). TGA is FOXL2’s favorite stop codon in mammals. DNA polymorphism analysis indicated that there were 260 variable sites, including 61 singleton variable sites and 199 parsimony informative sites, in the selected region (1–1229bp) of these 43 sequences. There was great sequence length variation between mammals and non-mammals, which was mainly due to, based on the amino acid sequences alignment, the missing of polyAla tract coding region in non-mammals. In mammals exists not only the highly conserved polyAla tract, but also the glycine repeat region and the proline repeat region, which has some variation among different mammals. There are more low complexity regions in mammals than in non-mammals. Ten highly conserved motifs were detected, which could be important functional sites. With the help of direct sequencing and alignment of FOXL2 gene, two SNPs were detected (g.3911C >G and g.4289G >T, based on AY112725). PCR-RFLP analysis and chi-square test showed that there were significant differences in gene frequencies of both the two SNP sites between normal individuals and intersex individuals and between hornless individuals and horned individuals, indicating some correlation between these two SNP sites and hornless and intersex phenotypes.Pairs of primers (PIS1, 26931～39428bp; PIS2, 26931～27667bp; PIS3, 27637～29012bp; PIS4, 38148～39044bp; PIS5, 35532～36425bp) (based on AF404302) were used to amplify the PIS region and screen its variation in dairy goats. Results showed that all those 34 hornless intersex dairy goats missed partial PIS, no complete missing was found. Alignment of PIS5 fragments (35532～36425bp) showed that there was not only partial fragment missing, but also a great deal of base mutations in those intersex individuals; 193 mutation locuses and 18 deletions between normal dairy goat and PIS dairy goat were detected in the region of PIS5 fragment (35607～36198bp). The alignment results of PIS2 and PIS3 fragment showed these base mutations might existed only in some fragments and could affect the regulatory function of the PIS region. It was inferred a great deal of base mutations and partial fragment missing of PIS could cause intersex character.更多还原