【作者】 高妍；

【导师】 张嘉保；

【作者基本信息】 吉林大学 ， 动物遗传育种与繁殖， 2011， 博士

【摘要】 过去30-40年间猪育种工作的主要目标是提高生长速度、饲料转化率和增加胴体瘦肉率,在获得较大遗传进展的同时也给猪的肉质带来了一定的负面影响,这种现象促使遗传学家们力图通过对动物体内重要调控基因的遗传操纵,改良猪种的肉用性状。RNA表达模式的研究对于深入探讨猪肉质调控的分子机理尤为关键。传统的RNA水平检测方法不能对基因表达进行全面系统分析,而表达谱基因芯片具有大规模、高通量、缩微和平行处理等优点,能够分析全基因组范围内基因表达的变化。瘦肉型的大白猪与脂肪型的东北民猪相比具有较低的肌内脂肪含量和较差的食用品质,它们是研究肉质差异的理想实验动物。有研究表明背最长肌的特征在肉质的测定方面发挥着关键作用。因此,本研究利用高通量芯片构建了两个猪种背最长肌组织的差异基因表达谱,系统筛选了与猪肉质相关的差异表达基因,采用蛋白质印迹、RNA干扰和单链构象多态性技术对这些基因进行了研究,有助于更全面地阐明肉质差异背后的生物学机制。研究结果如下：按照《猪肉质评定方法》对东北民猪和大白猪的肉质性状进行了测定和比较,结果表明东北民猪的肉质显著地优于大白猪。利用Affymetrix表达谱芯片进行了两个猪种背最长肌差异表达基因的检测。研究发现1134个基因发生了差异表达,其中401个为已知基因,733个为未知基因；以大白猪作为对照,136个为上调基因,998个为下调基因；并筛选出22个与猪肉品质相关的候选基因。应用Cluster 3.0和Treeview软件进行了差异表达基因的聚类分析,结果显示3头东北民猪和3头大白猪背最长肌的表达水平分别具有相似性。利用MAS对差异表达表达基因进行GO分析,结果表明这些基因参与骨骼肌再生、横纹肌发育的正向调节、脂肪酸氧化、肌动蛋白丝结合以及脂肪酸合酶活性。利用MAS进行KEGG Pathway分析,检测到5条有意义通路,即脂肪细胞因子信号通路、脂肪酸代谢、肌动蛋白细胞骨架调节、谷氨酸代谢和促分裂原活化蛋白激酶信号通路。硬脂酰辅酶A去饱和酶(SCD)和脂肪酸合酶(FASN)基因是由表达谱芯片筛选出的与脂肪代谢相关的基因。采用蛋白质印迹技术分别检测了这两个基因在东北民猪和大白猪背最长肌中的蛋白表达情况。结果发现它们在两个猪种的背最长肌中均有表达；以β-actin的表达作为参照,获得SCD和FASN的蛋白表达水平,分析表明它们在两个猪种中的蛋白表达水平差异均显著,与大白猪相比,SCD和FASN在东北民猪背最长肌中的表达均显著降低(P<0.05),此结果为进一步探讨它们在猪脂肪沉积中的作用提供了理论依据。构建了猪前体脂肪细胞体外培养体系,将合成的SCD-siRNA转染到细胞中,对SCD基因进行特异性沉默,采用荧光定量PCR和Western blot检测转染后SCD在猪前体脂肪细胞中的mRNA、蛋白表达的变化。结果表明转染后,SCD的mRNA和蛋白表达水平均显著降低(p<0.01),说明SCD基因在猪前体脂肪细胞中的表达被有效沉默,这为揭示其对猪脂肪沉积的效应及肉质调控机制奠定了基础。芯片实验表明A-FABP基因参与脂肪酸代谢、脂类生物合成和脂类代谢。L-FABP基因参与脂肪酸的摄取和转运,维持体内脂肪酸代谢的相对平衡。研究通过单链构象多态性和测序的方法调查了它们的遗传多态性,均出现3种基因型。测序发现在A-FABP基因第1内含子3481bp处发生A→C的碱基突变,在L-FABP基因第1内含子1740bp处发生T→C的碱基替换。χ2适合性检验表明,实验猪群均处于Hardy-Weinberg平衡(P>0.05)。利用SPSS13.0进行了不同基因型与肉质性状的关联分析,发现A-FABP和L-FABP基因的遗传变异均对大理石纹、IMF含量有显著效应(P<0.05),对其它肉质性状无显著影响(P>0.05)。采用蛋白质印迹检测了A-FABP在不同基因型个体背最长肌中的蛋白表达水平,发现A-FABP在DD型中的表达显著低于CC型(P<0.05),结果证实了它们对肉质性状的效应,为猪肉质的选种选育和遗传改良奠定了理论基础。更多还原

【Abstract】 The main goal of porcine breeding work has been raising growth velocity and feeder transformation efficiency, increasing carcass lean content in the last 30-40 years. However, selection toward leaner carcasses has resulted in a decrease on pork quality. Geneticists are working to improve meat quality traits in pigs using genetic manipulation to control important genes in vivo. It is very important to study RNA expression mode for approaching molecular mechanism regulating pork quality. Traditionary RNA level detection fails to analyse systematicly gene expression. Gene chip technique possesses the advantage of large scale、high-flux、demagnification and parallel handling, which has an important role in analyzing alterations in gene expression across the whole genome.The lean-type Large White breed has lower IMF content and inferior eating quality than the fat-type Northeastern Indigenous. They are ideal experimental animals used to study the difference of meat quality. Previous studies have indicated that characteristics of longissimus dorsi play a key role in determining meat quality. Therefore, the present study constructed differences in the expression profiles of genes in the longissimus dorsi of the two pig breeds by use of high-throughput microarray. Differentially expressed genes associated with meat quality were screened. These genes were investigated by western blot、RNA interference and single-strand conformational polymorphism. It helps to elucidate roundly the biological mechanism of different meat quality.The results are as follows:meat quality traits of the two pig breeds were determined according to evaluation method of pork quality. The result indicated that meat from Northeastern Indigenous pigs was of superior quality to that of Large White animals. Differentially expressed genes in the longissimus dorsi of the two pig breeds were detected by the gene chip technique. Microarray analysis demonstrated the differential expression of 1134 genes of which 401 have a known function. A total of 136 were up-regulated and 998 down-regulated in Northeastern Indigenous compared to Large White pigs. We screened 22 genes that were candidate genes associated with pork quality. Cluster analysis of differentially expressed genes was performed by Cluster 3.0 and Treeview software. The result indicated the transcriptome levels in the longissimus dorsi of 3 Northeastern Indigenous and 3 Large White pigs are similar, respectively. GO analysis of differentially expressed genes was performed by MAS. The result indicated these genes involved in skeletal muscle regeneration、positive regulation of striated muscle development、fatty acid oxidation、actin filament binding and fatty-acid synthase activity. KEGG Pathway analysis was performed using MAS. Five significant pathways were detected, I.E. adipocytokine signaling pathway、fatty acid metabolism、regulation of actin cytoskeleton、glutamate metabolism and MAPK signaling pathway.The gene SCD and FASN that were screened through microarray are associated with fatty metabolism. Protein expressions of SCD and FASN in longissimus dorsi of the Northeastern Indigenous and Large White pigs were detected by western blot. The result indicated protein expression of them were both observed. Protein expression levels of them were obtained compared with the expression ofβ-actin, respectively. The analysis indicated protein expression levels of SCD and FASN between the two pig breeds were both different significantly. The expression of them were both markedly reduced when the Northeastern Indigenous pig was compared with Large White animal (P<0.05). It helps to investigate the contribution to fatty deposition from the two genes.Porcine preadipocype culture system in vitro was constructed. SCD-siRNA was transfected into cell. SCD gene was silenced specificly. SCD mRNA and protein expression level in porcine preadipocytes transfected by siRNA were detected by real-time PCR and western blot. The result indicated SCD mRNA and protein expression was markedly reduced when transfected was compared with non-transfected (P<0.01). It is thus clear that expression of SCD gene in porcine preadipocytes was silenced effectively. It helps to reveal the contribution to fatty depositon from SCD and comprehend molecular mechanism regulating pork quality.Microarray experiment indicated the gene A-FABP involed in fatty acid metabolism、lipid biosynthesis and lipid metabolism. The gene L-FABP contributes to uptake and conveying of fatty acid. It maintains the relative balance of fatty acid metabolism in vivo. Genetic polymorphisms of the two genes were investigated by SSCP and sequencing. The results showed there were three kinds of genotypes. The sequencing results found that the polymorphisms of the gene A-FABP and L-FABP were due to a point mutation in position 3481bp and 1740bp, respectively. The x test showed that the pig populations were in Hardy-Weinberg equilibrium for the polymorphism both in the A-FABP and L-FABP gene (P>0.05). The association analysis between different genotypes and meat quality traits was performed by SPSS 13.0. The results revealed that the polymorphisms of the two genes had both a significant effect on IMF content and marbling (P<0.05). However, no significant conclusion can be drawn concerning other traits (P>0.05). Western blot was carried out to detect protein expression levels of A-FABP in three kinds of genotypes individuals’longissimus dorsi. The result found the expression of A-FABP was markedly reduced when genotype DD was compared with genotype CC (P<0.05). The results ascertained the effect on meat quality traits from the two genes. It helps to establish theoretical basis for selection and genetic improvement of pork quality.更多还原