【作者】 李勇；

【导师】 彭克美； 李奎；

【作者基本信息】 华中农业大学 ， 基础兽医学， 2010， 博士

【摘要】 泛素-蛋白酶体途径作为一种高效的蛋白降解途径,能够选择性地降解细胞内蛋白质,使各种蛋白质的降解受到精确的调控,这个途径对于维持细胞的许多重要生理功能如细胞周期的调控、抗原呈递、转录调控、细胞凋亡和细胞内信号转导等有着极其重要的作用。其中泛素连接酶E3在泛素-蛋白酶体途径中可特异性地识别靶蛋白,具有连接靶蛋白和特定泛素结合酶E2的重要作用。本研究采用生物信息学和分子生物学技术相结合的方法分离克隆了E3连接酶SCF复合物中的底物蛋白识别组分F-box蛋白中FBXL亚家族4个基因的部分基因组和DNA序列,并对其进行了组织表达谱、SNP检测及初步的免疫性状关联分析,获得以下结果：(1)利用人的同源基因mRNA序列查询获得猪新基因的EST,分离获得猪SKP2基因第一和第六内含子、FBXL4基因第二内含子、FBXL8基因第二内含子和jBXL11基因第18、19及第20内含子的序列,分离获得SKP2FBXL4基因的部分cDNA及完整CDS序列,并查询与分析了FBXL11的cDNA序列。(2)采用实时荧光定量PCR方法检测了猪SKP2mRNA和FBXL4mRNA在10种重要器官中的表达情况。其中猪SKP2mRNA在这些器官中均表达明显,但表达水平波动范围较大,胸腺具有最高表达水平,而背腰最长肌表达水平最低。猪FBXL4mRNA同样均有明显的表达水平及差异较大,肺表达水平最高,而心脏表达水平最低。(3)对猪SKP2、FBXL4、FBXL8和FBXL114个基因的相关内含子进行了SNP检测。结果发现有以下SNP位点：猪SKP2第一内含子的第819位存在SNP-Cfr42I(T／C)；猪FBXL4第二内含子包括分别位于第49位(C／T)、157位(A／T)、187位(A／G)、227位(G／T)、321位(G／A)、371位(G／A)、381位(C／T)和569位(C／A)的8个SNP位点。有趣的是,前4个位点和后4个位点属于同一个内含子中的一种连锁突变。因此仅选取分别代表前后4个位点的两个做验证分析,即第49位SNP-Tail和第381位SNP-BsaJI;猪FBXL11第18内含子的第377位存在SNP-TspEI(A/G)；另外猪FBXL8第二内含子在第325位、361位和493位分别具有G与A、C与T和A与G碱基突变。(4)对猪SKP2、FBXL4和FBXL113个基因的SNPs运用PCR-RFLP进行群体遗传学分析。从猪SKP2基因Cfr42I-RFLP在莱芜、巴马、五指山及贵州4种中国传统小型猪和温氏集团长白猪种得出的结果可知：等位基因A在所检测的五个猪种中占有绝对的优势,4种小型猪几乎属于纯合AA基因型；而长白猪种既有纯合AA也有杂合AG基因型。对于猪FBXL4基因,其中根据Tail-RFLP,长白猪种中纯合TT基因型占有绝对的优势,在其它3种中国传统小型猪种中,贵州和五指山两品种的纯合TT基因型个体具有明显的优势,但纯合CC基因型在莱芜猪种占有优势。而BsaJI-RFLP的结果表明,在贵州和五指山两小型猪及长白猪种群体中,纯合CC基因型具有显著的优势。猪FBXL11基因的TspEI-RFLP在温氏集团长白猪种资源群体的分析结果表明：等位基因G在长白猪种中占有绝对的优势,并包括有杂合AG和纯合GG两种基因型。(5)对猪SKP2和FBXL4两个基因的多态性与免疫性状进行了初步的关联分析。从猪SKP2基因的关联分析结果可知,第一内含子的SNP-Cfr42I与0日龄新生仔猪的红细胞分布宽度存在显著相关性,纯合AA基因型个体的红细胞分布宽度明显高于杂合AG基因型个体的红细胞分布宽度(P=0.027)对于猪FBXL4基因SNP-Tail的关联分析,结果显示纯合CC基因型0日龄新生仔猪的淋巴细胞百分数显著低于其它两种基因型淋巴细胞百分数(P=0.0166)。而另一个SNP-BsaJI的关联分析结果表明,具有纯合TT基因型的0日龄新生仔猪的中性粒白细胞百分数明显要高于其它两种基因型,具有极显著意义(P=0.0005)；纯合TT基因型32日龄个体的淋巴细胞百分数显著低于其它两种基因型(P=0.0351)。然而对于纯合CC基因型的0日龄新生仔猪,其绝对淋巴细胞数要显著高于其它两种基因型(P=0.0458),杂合TC型的0日龄新生仔猪的中间细胞数高于其它两种基因型的中间细胞数,具有极显著差异性(P=0.0010)。(6)运用clustalW2在线软件对猪SKP2、FBXL4和FBXL113个基因进行了系统进化树的构建与分析。3种基因的系统进化树的结果都表明：这些基因在物种间具有非常保守的分子特性；同时在这些比较的人和各种动物间,仅有猪和牛均属于系统树结构中的同一组,具有最近的进化亲缘关系特征。(7)通过运用motif scan网站对猪SKP2和FBXL4两个基因进行了推导蛋白结构域的鉴定和分析。猪SKP2蛋白的结果显示：位于第90至140个氨基酸间,具有一个含51个氨基酸的保守F-box结构域,这表明其属于F-box蛋白,预测还可能具有其它8个相似的富含亮氨酸的重复序列。而猪FBXL4基因的结果同样显示该蛋白具有一个含56个氨基酸的保守F-box结构域,其分布位置在第277至332个氨基酸之间,说明其同样属于F-box蛋白的成员。更多还原

【Abstract】 As a greatly effective pathway of protein degradation, ubiquitin-proteasome pathway can degrade selectively intra cellular protein; And make various proteins to be precisely degraded. It is very important for maintaining much important physiological function, such as control of cell cycle, angtigen presentation, transcriptional control, apoptosis, and signal transduction. However, ubiquitin-protein ligases (E3s) can especially recognize target protein, with a role of linking the target protein and ubiquitin-conjugating enzymes (E2s).In this study, the bioinformatics and molecular biology technology was applied to isolate and clone the partial genomic fragments and cDNA of four genes in FBXL sub family belonging to F-box proteins which play a role as the recognizing components for subtrate protein in ubiquitin-protein ligases components. The tissue expression, SNP detection and the association analysis with immune traits were conducted as followings:1. The human homologous gene mRNAs were searched and used to obtain porcine new gene ESTs. The first and sixth intron of porcine SKP2 gene, the second intron of porcine FBXL4 gene, the second intron of FBXL8 gene and the eighteenth, nineteenth and twentieth intron of porcine FBXL11 gene were isolated. As the same time, the cDNAs and CDSs of porcine SKP2 and FBXL4 gene were obtained, cDNA of porcine FBXL11 gene also was searched and analyzed.2. Real Time-PCR was carried out in analysis on the tissue expression for porcine SKP2 and FBXL4 mRNA in ten important tissues. In conclusion, porcine SKP2 mRNA expresses fully and evidently in these tissues, but the expression levels fluctuated in a large range. The highest level was expressed in thymus, and the lowest level was found in longissimus dorsi muscle. As similarly, porcine FBXL4 mRNA expresses visibly in all of tissues with great difference levels, the highest level was expressed in lung, and the lowest level was located in heart.3. SNP detections in these introns related to porcine SKP2, FBXL4, FBXL11 and FBXL8 gene were completed, the following results was got:the SNP-Cfr42I (T/C) was located in the 819th in the first intron of porcine SKP2 gene; the second intron of porcine FBXL4 gene contains eight SNPs, including the 49th (C/T)、157th (A/T)、187th (A/G)、227th(G/T)、321st (G/A)、371st (G/A)、381st (C/T) and 569th (C/A). Interestingly, the fore and post four SNPs belong to a kind of linkage mutations in the same intron. Therefore, two SNPs representing respectively the fore and post four SNPs which were the 49th SNP-Tail and the 381st SNP-BsaJI were conducted to detection and analysis; SNP-TspEI (A/G) was found at the 377th in the eighteenth intron of porcine FBXL11 gene; Furthermore, there were three mutations with G/A、C/T and A/G at the 325th,361st and 493rd in the second intron of porcine FBXL8 gene respectively.4. PCR-RFLP technique was applied in the population genetics analysis of porcine SKP2, FBXL4 and FBXL11 gene. According to porcine SKP2 Cfr42I-RFLP analysis on Bama, Laiwu, Guizhou, Wuzhishan four kinds of unrelated Chinese indigenous miniature pig breeds and pure Landrace pig breed:Allele A was in dominance absolutely in all detected pig breeds. Four kinds of unrelated Chinese indigenous miniature pig breeds almost belong to homozygous AA genotype, while introduced pure Landrace pig breed has two different genotypes containing homozygous AA and heterozygous AG genotype.For porcine FBXL4 gene, according to the results on Tail-RFLP, homozygous TT genotype owned absolute dominance in introduced Landrace pig breed. Simultaneously, for unrelated three kinds of Chinese miniature pig breeds, homozygous TT genotype was also more than two other genotypes in Guizhou and Wuzhishan breeds, but homozygous CC genotype was in absolute dominance in Laiwu breed. Meanwhile, in another genotyping analysis on BsaJI-RFLP, homozygous CC genotype was obviously more than other genotypes in two kinds of Chinese miniature pig breeds including Guizhou and Wuzhishan and introduced Landrace pig breed.The analysis results on TspEI-RFLP of porcine FBXL11 gene in pure Landrace pig resources shows that allele G was in dominance absolutely and introduced pure Landrace pig breed had two different genotypes including heterozygous AG and homozygous GG genotype.5. The preliminary association analysis with immune traits was conducted for porcine SKP2 and FBXL4 gene. According to the results on porcine SKP2 gene, SNP-Cfr42I in intron 1 was significantly associated with RDW of neonate piglets at 0 day. RDW of the newborn piglets with homozygous AA genotype was obviously higher than that of newborn piglets with heterozygous AG genotype (P=0.027).With regard to SNP-Tail analysis of porcine FBXL4, it shows that lymphocyte percentage of neonate piglets at 0 day with homozygous CC genotype was obviously lower than those with other two genotypes (P=0.0166). while another results of SNP-BsaJI revealed that neutrophil percentage of neonate piglets at 0 day with homozygous TT genotype was visibly higher than those with other two genotypes (P=0.0005). Lymphocyte percentage of individuals at 32-day with homozygous TT genotype was notably lower than those with other two genotypes (P=0.0351). However, absolute lymphocyte count of neonate piglets at 0 day with homozygous CC genotype was obviously higher than those with other two genotypes (P=0.0458). The mixed cells of neonate piglets at 0 day with heterozygous TC genotype were markedly higher than those with other two genotypes (P=0.0010).6. The clustalW2 software online was applied to construct and analyse the phylogenetic trees for porcine SKP2、FBXL4 and FBXL11 genes. The analyses on the phylogenetic tree show that these genes share the quite conserved molecular characteristic among these species. Meanwhile, compared with human and other various species, only Sus Scrofa and Bos Taurus always belong to the same group according to these trees, as shows that they have most relative relation characteristic in their evolution.7. The detection and analysis on motif of the putative proteins of porcine SKP2 and FBXL4 gene were employed by motif scan online. The results on porcine SKP2 gene show that a conserved motif of F-box protein was found at position between the 90th and 140th amino acid, as indicated that the putative protein belongs to F-box proteins; By comparison, it was predictable that porcine SKP2 protein has probably other similar 8 leucine-rich repeats.While another results on porcine FBXL4 gene similarly revealed that this putative protein has a conserved motif of F-box protein with 56 amino acids, which located between the 277th and 332nd amino acid, as demonstrated that it also belongs to a member of F-box proteins.更多还原