【作者】 王志伟；

【导师】 樊斌； 李奎；

【作者基本信息】 华中农业大学 ， 动物遗传育种与繁殖， 2008， 硕士

【摘要】 第一部分猪F-box基因家族部分基因的分子特征、SNP检测与性状关联分析从早期追求猪生产性能的提高到现在生产性能与免疫抗性并重,人们已经认识到单纯的提高猪的生产性能会导致抗性降低。一般来说,动物的免疫力决定了动物的健康状况,它与动物的生产性能是密切相关的。这就使得研究者们相继提出了在分子水平揭示疾病发生的原因,并利用现代生物技术从遗传本质上提高动物对病原的抗性,增强动物自身的抵抗力。E3泛素连接酶F-box基因家族参与众多的生物学过程,包括信号转导、细胞周期、细胞凋亡、转录激活和MHCⅠ类抗原提呈等（Bai et al,1996;Craig and Tyers,1999）,与肿瘤的发生和机体免疫密切相关,已逐渐成为一个新研究热点。本研究采用生物信息学、群体遗传学和分子生物学等相结合的方法,对E3泛素连接酶F-box基因家族中3个基因进行了深入的研究,取得了如下结果:1)利用猪EST数据库的信息,分离了猪E3泛素连接酶F-box基因家族中FBXO5和FBXO7基因的完整的编码区（coding sequence,CDS）和部分基因组序列,并进行了蛋白质序列的推导和基因组结构的初步分析,所有的内含子和外显子的拼接位点都符合“GT-AG”规则;分离了猪FBXO40基因的大部分cDNA序列。2)利用IMpRH（The INRA-University of Minnesota porcine radiation hybrid panel）克隆板,对猪FBXO40基因进行了染色体定位,将其定位于SSC13q4.1-4.6,与微卫星S0075和SW1876连锁,连锁的LOD值分别为12.99和7.12。3)采用ClustalW2程序分析了FBXO5、FBXO7两个基因编码氨基酸序列的保守性及FBXO40基因F-box结构域氨基酸序列的保守性,并根据氨基酸序列信息构建了不同物种间的进化树,进行了序列的分子进化分析。4)采用半定量PCR的方法分析了猪FBXO5,FBXO7和FBXO40三个基因在成年五指山猪心脏、骨骼肌、淋巴、脾脏和胸腺等组织中的表达情况。FBXO5、FBXO7和FBXO40基因分别在胸腺、甲状腺和骨骼肌中高表达。5)采用半定量PCR的方法分别检测了猪FBXO5,FBXO7和FBXO40三个基因在正常肾脏组织和癌化的PK15细胞中的表达变化。猪FBXO5基因仅在正常肾脏组织中可检测到表达,猪FBXO40基因仅在PK15细胞中检测到表达,猪FBXO7基因在二者之间基本无差异。6)对上述3个基因进行了SNPs检测,发现如下5个可用PCR-RELP检测的多态位点:FBXO5基因第三内含子第160位碱基的PstⅠ-RFLP(T/C¹⁶⁰)和第四内含子第56位碱基的TaqⅠ-RFLP(A/G⁵⁶);FBXO7基因第三内含子第71位碱基的XbaⅠ-RFLP(T/C⁷¹)和第七外显子第113位碱基的NruⅠ-RFLP(T/C¹¹³);FBXO40基因第四外显子第299位碱基的HindⅡ（HincⅡ）-RFLP(A/C²⁹⁹)。7)对上述5个SNPs用PCR-RFLP方法在五指山、巴马、莱芜、贵州、通城、大白猪和长白猪中进行群体遗传学分析,结果表明,所有基因座的基因型频率在不同的品种中存在较大差异。8)在我室与温氏集团合作的温氏群体中,对上述SNPs（除XbaⅠ-RFLP位点）与部分免疫相关性状（如:白细胞数、红细胞数和血红蛋白浓度、猪瘟抗体阻断率和血小板分布宽度等）进行了初步关联分析,结果发现,FBXO5基因pstⅠ-RFLP(T/C¹⁶⁰)和TaqⅠ-RFLP(A/G⁵⁶)两个位点FBXO5基因pstⅠ(T/C¹⁶⁰)和TaqⅠ(A/G⁵⁶)两个位点不同基因型的个体都与部分红细胞指标和血小板分布宽度显著相关;FBXO7基因NruⅠ-RFLP(T/C¹¹³)多态与32日龄时白细胞数（P＜0.05）和猪瘟抗体阻断率（P＜0.05）在不同基因型的个体间差异显著;FBXO40基因HindⅡ-RFLP(A/C²⁹⁹)不同基因型个体在17日龄时白细胞数（P＜0.01）和淋巴细胞绝对值（P＜0.01）差异极显著。9)在我室与湖北省通城县畜牧局合作组建的通城群体中,对上述SNPs（除NruⅠ-RFLP位点）与部分生产性状（如:初生至上市平均日增重、达90kg（75kg）日龄、屠宰率、肌肉颜色和肌肉pH值等）和部分免疫相关性状（如:白细胞数、红细胞数和血红蛋白浓度等）进行了初步关联分析,结果显示,FBXO40基因HindⅡ（HincⅡ）-RFLP(A/C²⁹⁹)位点,血红蛋白浓度（P＜0.01）、平均血细胞血红蛋白浓度（P＜0.01）、肌肉颜色评分（P＜0.05）和达90kg（75kg）日龄（P＜0.05）在三种基因型个体间存在极显著差异。第二部分FBXO40基因的功能初探肌萎缩是临床上较为常见的一种疾病,去神经支配、禁食、糖尿病和癌症等都可以引起肌肉萎缩。当蛋白质降解速率超过合成速率时,肌肉发生萎缩,已有越来越多的实验发现与泛素-蛋白酶体途径有着紧密的联系。同时,诸多重要的信号通路网络业已被证实彼此交织发挥着不可替代的作用。阐明肌萎缩过程中一些分子代谢调节机制的基本概况就显得尤为重要,这不仅可以加深对肌萎缩类疾病的认识,也可以为治疗该类疾病提供新的依据和促进新的药物靶点的筛选。FBXO40基因是一个与肌肉疾病相关的基因,在去神经肌萎缩中表达上调。探讨研究该基因在肌萎缩中的具体作用具有重要意义。本研究采用生物信息学和分子生物学相结合的手段,初步得到以下结果:1) mFBXO40基因编码一个较大的F-box蛋白,是一个骨骼肌特异性表达的基因,具有一个未知功能的Zinc finger TRAF-type结构域。2)生物信息学预测,mFBXO40蛋白没有发现信号肽和线粒体定位信号,但发现明显的内质网信号锚定序列,推测该蛋白定位在内质网。3)成功构建了该基因的荧光融合蛋白表达载体、超表达载体和功能域缺失超表达载体。4)利用融合蛋白表达技术完成了mFBXO40蛋白的亚细胞定位,脂质体包裹转染C2C12细胞并染色后通过激光共聚焦显微镜观察,发现该蛋白集中分布在靠近核外膜的细胞质区域。更多还原

【Abstract】 People have been conscious of boosting the porcine performance traits merely decreased its disease resistance traits from seeking the high growth rate et al over the past long periods to emphasize the performance traits and immune resistance together. Generally speaking,animal’s immune capability,which has closely relation with its performance traits,determines its disease resistance.Thus,the researchers want to explore the molecular mechanism underlying the development of diseases,therefore to improve the disease resistance using the genetic methods.Increasing reports indicated that F-box gene families,members of E3 ubiquitin ligases,are involved in many biological processes,including signal transduction,cell cycle,apoptosis,transcriptional activation,MHC classⅠantigens presentation et al（Bai et al,1996;Craig and Tyers,1999）,and tightly correlated with tumorigenesis and individual immunity.Researches on these gene families have become a new hot spot gradually.Combining of the bioinformatics,population genetics and the biotechnology, three genes which including FBXO5（F-box protein 5）,FBXO7 and FBXO40 gene were analyzed deeply.The main results are listed as follows:1) Utilizing the porcine EST（Expressed sequence tag） database information,complete CDS and partial genomic sequences of pig FBXO5 and FBXO7 gene were obtained, and then the deduced amino acid sequences and the genomic structure were analyzed. All splice sites of the exon/intron conformed to the GT/AG rule;in addition,we also isolated partial cDNA sequence of pig FBXO40 gene.2) IMpRH was employed to determine the precise location of FBXO40 gene.Statistical analysis showed that FBXO40 gene is located to SSC13q4.1-4.6 and closely linked to the microsatellites S0075 and SW1876 with LOD scores of 12.99 and 7.12 respectively.3) The ClustalW2 program was used to analyze the amino acid sequences’ conservation of FBXO5,FBXO7 gene and FBXO40 gene F-box domain,and inferred the phylogenetic relationship of them among different species respectively.4) Semi-QPCR（semi-quantitative PCR） was employed to estimate the expression level of the three genes among different tissues,such as heart,skeletal muscle,lymphonode, spleen,thymus et al,from adult wuzhishan pigs.These three genes FBXO5、FBXO7 and FBXO40 have different tissue distribution,which are relatively high in thymus, thyroid gland and skeletal muscle,respectively.5) Semi-QPCR was employed to analyze the difference of the three genes between the normal kidney tissue and PK15 cells.The results demonstrated that FBXO5 gene was merely detected in the normal kidney tissue,FBXO40 gene only in PK15 cells,and no difference for FBXO7 gene.6) Detection of SNPs（single nucleotide polymorphisms） in the amplified fragments of these three genes was carried out and five SNPs were identified for further genotype by PCR-RFLP（PCR-restriction fragment length polymorphism）,these loci are as follows:Pst I-RFLP(T/C¹⁶⁰) in the third intron and Taq I-RFLP(A/G⁵⁶) in the fourth intron of porcine FBXO5 gene;Xba I-RFLP(T/C⁷¹) and Nru I-RFLP(T/C¹¹³) site in the third intron and seventh exon of porcine FBXO7 gene respectively; HindⅡ（HincⅡ）-RFLP(A/C²⁹⁹) in the fourth exon of FBXO40 gene.7) The population genetics analysis of these five SNPs were performed by PCR-RFLP method in Wuzhishan,Bama,Laiwu,Guizhou,Tongcheng,Large White and Landrace pigs,and showed that all genotype frequencies of all the loci were significant difference among the different populations.8) These four polymorphism sites except Xba I-RFLP(T/C⁷¹) site were analyzed in wens experimental population with some immune associated traits.The association analysis results revealed that these two sites of FBXO5 gene have the effects on partial erythrocyte indexes and platelet distribution width.The polymorphism of the FBXO7 gene(Nru I-RFLP(T/C¹¹³) site) prominently has the effects on leukocyte counts （P＜0.05,32 days） and blockade rate of swine plague antibody（P＜0.05,32 days）.The different genotypes of FBXO40 HindⅡ-RFLP(A/C²⁹⁹) site have the very significant difference on leukocyte counts（P＜0.01,days） and Lymphocyte modulus（P＜0.01,17 days）.9) The four SNPs(except the Nru I-RFLP(T/C¹¹³) site) were genotyped in the tongcheng experimental population which constructed by our lab.The association analysis between these SNPs and some traits,partial economic traits（including Avg. daily gain from birth to market,days of age at an ideal market weight,dressing percent,meat color score et al） and partial immune associated traits（leukocyte counts, total erythrocytes,hemoglobin et al）,were performed.The results indicated that the different genotypes of FBXO40 locus have the significant difference on hemoglobin （P＜0.01）,mean corpuscular volume hemoglobin concentration（P＜0.01）,Avg.daily gain from birth to market（P＜0.05） and meat color score（P＜0.05）. Muscle atrophy,caused by denervation,fasting,diabetes and cancer et al,is a fairly frequent disease clinically and also characterized that the rate of speed for protein degradation exceeds that of protein synthesis.Increasing experiment evidences indicated that the ubiquitin-proteasome pathway would participate in muscle atrophy;meanwhile, numerous signal pathways play an essential role through interacting with each other.It is especially important to elucidate the molecular mechanism of muscle atrophy processes, which not only could facilitate to deeply understand these diseases of muscle atrophy,but also provide foundation for therapeutics and promote the screening of new drug targets.FBXO40,a muscle disease related gene,is up-regulated in muscle atrophy.It is significant to study the concrete effects of FBXO40 gene in muscle atrophy.In our research,the bioinformatics and molecular biology methods were utilized to study this gene;the main results are listed as follows:1) mFBXO40,encoding a large F-box protein and possessing unknown function of Zinc finger TRAF-type domain,is a muscle-specific expression gene.2) By bioinformatics,the prediction results of mFBXO40 protein showed that the protein didn’t have signal peptide and mitochondrial signal peptide,but the endoplasmic reticulum signal anchor sequence could be obviously found.On the basis of the forecast results,we presumed that the mFBXO40 protein may localize in endoplasmic reticulum.3) Successfully constructed the mFBXO40 gene eukaryotic expression vector:the vector for subcellular localization pEGFP-N1-FBXO40,the vector for overexpression （complete CDS）:pcDNA3.1（+）-FBXO40,the vector for overexpression（functional domain-depleted）:pcDNA3.1（+）-FBXO40DF and pcDNA3.1（+）-FBXO40DT.4) The intracellular distribution of mFBXO40 protein in C2C12 cells was analyzed by the confocal analysis of transiently transfected with pEGFP-N1-FBXO40.After stained with Hoechst33342 and MitoTracker Red CM-H2Xros,the fusion proteins were obviously detected in cytoplasm surrounding the perinuclear membrane.更多还原