Molecular Biological Analysis Of Porcine Fat-Inducing Transcript Gene

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.