【作者】 李德臻；

【导师】 熊远著；

【作者基本信息】 华中农业大学 ， 动物遗传育种与繁殖， 2010， 硕士

【摘要】 随着分子生物学的研究进展,动物新品种的培育技术也得到了快速的发展,寻找有价值的分子标记或主效基因作为分子标记辅助选择的基础,结合传统的表型性状进行选择是目前猪遗传改良工程所采用的主要方法之一。同时,科技的发展和人们生活水平的提高也使猪育种的目标发生了变化,育种的重点也已由原来的降低背膘厚、提高生长速度等转变为提高瘦肉组织的生长效率、改良肉质等各项指标的改善。肌内脂肪含量能直接影响猪肉的嫩度和风味,脂肪性状作为影响猪肉质的主要指标之一越来越引起科学家们的关注,2009年启动的国家转基因新品种培育重大专项就把培育高肌内脂肪肉质新品种猪种列为其中一个主要目标。在人体中,脂滴(lipid droplets)积累过量就会导致肥胖,而在所有的真核细胞生命中,从最简单的酵母到最复杂的人类,细胞内脂滴储存脂肪的能力都是最基本的过程,之前科学家已经确定了很多负责合成脂肪的基因,但是控制将脂肪“打包”成脂滴的基因一直未被发现。07年12月17日出版的《PNAS》上,美国艾伯特·爱因斯坦医学院的David L Silver和同事发现了两个将脂肪打包成脂滴的关键基因,分别命名为FIT1和FIT2 (fat-inducing transcript,诱导脂肪转录基因)。文献报道通过多种卖验方法均证明FIT基因在人、小鼠和斑马鱼等生物中有重要功能,鉴于在猪中脂滴方面研究的不足,很有必要在猪中进行该基因的研究,进而了解脂滴的生物合成机制,探讨瘦肉型猪和脂肪型猪差异形成的分子机理,为人类肥胖提供更细致的解释,为从分子水平上选育高瘦肉率且肉质较好的猪种提供理论依据。本试验对FIT1和FIT2基因主要进行了基因克隆及其遗传效应的研究。初步结果如下：1.充分利用NCBI数据库和猪基因组计划公布的数据,克隆鉴定了猪FIT1和FIT2基因的cDNA序列,并进行生物信息学预测和分析,发现两基因均只由两个外显子和一个内含子构成,猪FIT1基因编码290个氨基酸,位于7号染色体,包含一个肌糖磷脂合成蛋白超家族结构域,物种间同源性比较发现猪FIT1编码的氨基酸序列与人、牛、大鼠和小鼠有很高的相似性(>93%),且遗传进化关系和人的距离最近。FIT2基因编码262个氨基酸,位于17号染色体。2.猪FIT2基因第二外显子的559bp处存在T/C突变,引起内切酶BcnI酶切位点的改变,利用PCR-BcnI-RFLP进行基因型分析,发现在梅山猪中A等位基因占优势,大白猪中B等位基因占优势,但在170头“大白×梅山”F2资源群体中进行与胴体性状的关联分析,结果发现该突变位点仅与肌肉pH值(SC)呈显著相关(P<0.05),与肌肉pH值(BF)接近显著相关,但是与其它性状关联不大。3.猪FIT1基因第一外显子72bp处G/A突变,引起内切酶FspBI酶切位点的改变,利用PCR-FspBI-RFLP进行基因型分析,发现在梅山猪中A等位基因占优势,大白猪中B等位基因占优势,在279头“大白×梅山”F2资源群体中进行性状关联分析表明,该突变位点对骨率、肥肉率、臀部平均膘厚、肋骨数、板油重的效应达到极显著水平(P<0.01)；对皮率、平均背膘厚的效应达到显著水平(P<0.05)。4.在猪FIT1基因第二外显子590-595bp处存在CACTCC的插入和缺失,在梅山猪中为插入型,在大白猪中为缺失型,而且在该突变前9个碱基,即581bp处存在C／A突变,共引起3个氨基酸的改变,利用直接测序和PCR-SSCP方法证明两位点连锁,用PCR-SSCP进行基因型分析发现梅山猪中C等位基因占优势,大白猪中D等位基因占优势,在314头“大白×梅山”F2资源群体中进行性状关联分析,表明该位点与肥肉率、6-7腰椎间膘厚、臀部平均膘厚、板油重、内脂合计、内脂率(%)等脂肪沉积性状的相关性达到极显著水平(P<0.01)。5.获得FIT1基因的启动子序列,利用CPGPLOT, NNPP, SignalScan MotifFinder及TFSEARCH等在线软件预测启动子的顺式元件和转录因子结合位点,预测仅发现一处可能的转录起始位点,即位于ATG上游-310bp处的C,且分值较高,为0.91。同时该段序列还具备启动子的一些特征元件,如帽子信号,TATA box, CCAAT box等,另外还有促使脂肪细胞分化的转录因子C/EBP和与调控成肌细胞分化相关的转录因子MyoD的结合位点。6.利用RT-PCR技术对FIT1和FIT2基因进行了不同组织的表达谱分析。表达谱显示两基因在肌肉和心脏中均高表达,总体趋势是FIT1基因只在特异组织中表达,而FIT2基因在多数组织中均有表达。7.由于大白和梅山猪的FIT1基因存在氨基酸的不同,我们分别构建了不同DNA序列的融合蛋白表达载体,转染猪成纤维细胞,在细胞水平初步研究其功能的差异。更多还原

【Abstract】 With the fast development in molecular biology, the technology of cultivate new animal breeds are making great progress. To determine valuale molecular markers and efficiency genes are the first step to do molecular marker-assisted selection(MAS). The combination of MAS and traditional breeding methods is one and the most popular means in the swine genetic improvement engineering. At the same time, with the progress made in science and improvement of people’s living standand, the focus of breeding is changing from reducing the fat thickness, increasing the grow speed to enhancing the grow efficience of lean meat, improve the meat taste, and homogeneous and better other indicators. Intramuscular fat (IMF) can effect tenderness and flavor of the meat, while fatty trait, one of the major index to judge pork quality, is being given more and more focus. In 2009, our state started an importantly special-purpose project, one of its objectives is to improve the IMF.In human, if excessive lipid droplets are cumulated, it may results in obesity. While in all eukaryotes, from the simplest yeast to the most complicated human, it is a basic course that fat is depoted as lipid droplets in cells. Scientists have determined many genes regard with fat synthesis, but a controling gene to package fat into lipid droplets is yet not found. David L Silver and his colleagues from Albert Einstein Medicine Institute detected two key genes that can package fat into lipid droplets, named FIT1and FIT2(fat-inducing transcript)respectively; and their reseach result was published in PNAS in December 17th 2007. Important functions of these two genes were proved in Homo sapien, Mus musculus and Zebra fish by multiple experimental methods. In view of their importance and little knowledge about lipid droplet, it’s necessary to research deeply in porcine to get the message about biosynthesis of lipid droplet, and the molecure difference among pork type pigs and fatty type ones. To some extent, we want to give more detailed explaination to human obesity, and give some theoretical support to select superordinary pigs possessing higher lean meat percentage and better tasted meat quality.Based on these, we cloned FIT1 and FIT2, and studied their genetic effects, the main results are as follows:1. Isolation and characterization of cDNA sequence of FIT1 and FIT2, with full use of database published in NCBI and swine genome project, and bioinformatical prediction and analysis the cloned sequences. It’s found that both of FIT1 and FIT2 contain two exones and one intron, respectively. Porcine FIT1 located at SSC7, codes 290 amino acids, including Inositol phospholipid synthesis protein Scs3p superfamily. Porcine FIT1 protein has high homology with corresponding protein in homo sapien, cattle, rat and mus musculus(>93%). As for FIT2, it is located at SSC17, codes 262 amino acid.2. The T559C mutation in exon 2 of FIT2 gene was detected, and PCR-BcnI-RFLP was used to detect the polymorphism of the genotypes among Large White×MeiShan F2 resource population. The result shows that Meishan breed mainly possess A allele, while Large white possess B allele, but T/C polymorphism is not significant associated with traits except Meat PH (m.Semispinalis Capitis) (P<0.05).3. An A/G mutation in exon one of FIT1 gene was detected, and PCR-FspBI-RFLP was developed to detect the A/G polymorphism. Results show that Meishan breed mainly possess A allele, while Large white possess B allele. And FspBI-RFLP polymorphism was extremely significantly associated with Bone percentage(BP), Fat percentage (FW), Buttock fat thickness(BFT) and Leaf fat weight(LFW)(P<0.01),while significantly correlated with Skin percentage(SP) and Average bakefat thickness(ABF) (P<0.05).4. An CACTCC insertion/deletion mutation exists at 590-595bp of exon 2 in FIT1 gene, and at 581bp, namely,9bp before the insertion/deletion mutation exists C/A mutation, and the two mutations changed three amino acid in all. We carried out PCR-SSCP analysis followed by association analysis in F2 "Large white×Meishan" resource family. The results are as follow:in all tested individuals, all Meishan pigs harbor the insertion, which was designated type C. All Large white pigs harbor the deletion and was named as type D. Association analysis in F2 resource family showed that this site was highly significantly associated with Fat percentage (FP),6-7rib fat thickness(RFT), Buttock average fat thickness(BAFT), Leaf fat weigh(LFW), Total internal fat weigh(TFW) and Internal fat rate(FP)(P<0.01).5. FIT1 promoter was isolated and NNPP, CPGPLOT, SignalScan, MotiFinder and TFSEARCH softwares were used for prediction of initial position of transcription, distribution of CpG island and transfactors banding sites. It is predicted to contain a sole transcription startpoint at the site of-310bp, with score 0.91. Also, some potential promoter characteristic elements exist in this sequence predictively, eg:Cap signal, TATA box, CCAAT box, et al. Interestingly, some transfactors banding locations were predicted that some C/EBP transfactors related with adipose cell differentiation, and some MyoD transfactor related with muscle differentiation were exist.6. The semi-quantitative RT-PCR was performed to detect the expression of porcine FITI and FIT2 in different tissues. It’s found that these two genes are mainly expressed in the longest back muscle and heart. And in total view, FIT1 is only expressed in special tissue, while FIT2 in most tissues.7. In addition, recombined plasmids were constructed, considering the mutations existing between Meishan and Large White breeds. And then transfected transiently into fibroblast to study its function difference in cellular level.更多还原