摘要
猪肉是人类摄取动物蛋白的重要来源,优质猪肉细嫩多汁、口感丰富、肉色鲜红,而肌内脂肪(Intramuscular fat, IMF)含量与其有着极高的关联,因此成为评定肉质的重要指标。人类对猪的长期遗传选育投入了很大的人力和物力,更偏重于降低背膘厚度和提高瘦肉率,然而造成了肌内脂肪含量的大幅下降,甚至高瘦肉型猪IMF含量降到了1.5%(建议范围：2.5%-3%),导致猪肉品质下降、口感变差,因此今后的选育方向转变成在确保瘦肉率的同时增加IMF含量。研究表明,肌内脂肪含量的提高即意味着甘油三酯(triglycerol, TAG)含量的提高。DGAT1限速酶催化甘油三酯合成反应的最后一步,在哺乳动物的脂类代谢中发挥着重要作用。因此,本研究以二酰基甘油酰基转移酶1作为候选基因,构建了肌肉特异性真核表达载体sMCK-sDGAT1,通过显微注射和体细胞克隆分别获得了转基因小鼠和转基因猪,以研究肌肉超表达DGAT1的效果和影响,为今后的研究提供材料并奠定基础。取得的主要研究成果有：
1、根据基因重组的原理,从猪MCK基因的细菌人工染色体中,获得了MCK基因长度为7261个碱基的启动子序列。以真核表达载体pEGFP-N1为框架去除GFP基因后,连接MCK基因启动子和人工合成的DGAT1基因全长CDS,构建肌肉特异性超表达DGAT1的真核表达载体sMCK-sDGAT1。采用PCR、酶切和测序的方式进行鉴定,证明最终构建的载体准确无误。
2、通过显微注射法将线性化sMCK-sDGAT1注射进入小鼠受精卵的雄原核,我们得到了16只阳性转基因Founder鼠(委托上海南方模式生物科技有限公司),阳性公鼠与阴性C57BL/6J母鼠交配后获得F1代阳性转基因小鼠。采用半定量检测二月龄和四月龄小鼠的DGAT1基因表达量,进行组织表达谱的研究,结果表明相较于阴性小鼠,DAGT1基因在阳性小鼠的骨骼肌和肾脏内高表达,在心肌内中等表达,而在脑,肺,肠等组织内很少表达。上述结果说明MCK基因启动子能够驱动DGAT1基因在哺乳动物体内表达,与对哺乳动物各个组织内MCK基因表达的研究结果是一致的。
3、我们通过实时定量PCR检测了骨骼肌中参与脂肪合成和转运等相关基因的表达情况,发现部分基因的表达发生变化,如质体乙酰辅酶羧化酶1(ACC1)的mRNA增加34%(p<0.01),脂肪酸结合蛋白(FABP4)增加53%(p<0.01),脂肪酸合成酶(FAS)增加74%(p<0.01),乙烯前体ACC合成酶1(ACS1)增加62%(p<0.01),骨骼肌中的棕色脂肪特异基因UCP1也增加了74%(p<0.01)。Westen Blot的实验结果表明,DGAT1、ACC1、FABP4和UCP1的蛋白水平也相应的提高了。连续十周测定转DGAT1阳性小鼠与阴性小鼠的体重,数据显示二者差异不显著,说明特异性增加IMF含量,并不会导致小鼠其它组织脂肪的过度沉积和肥胖。测定转DGAT1阳性小鼠骨骼肌中的甘油三酯和脂肪酸,发现二者相对于阴性小鼠分别增加了35.5%(p<0.01)和20.3%(p<0.01)。
4、培养了猪30日龄胚胎的原代成纤维细胞系,用电击转染法将线性化的sMCK-sDGAT1片段稳定转染进猪的成纤维细胞,经G418筛选并进行PCR检测后获得阳性单克隆,作为体细胞核移植的供体细胞制备了转基因猪。
5、通过PCR及Southern Blot检测确定获得了一头原代阳性转基因猪,内参基因为GAPDH,使用实时荧光定量PCR法检测外源基因DGAT1基因的拷贝数,并通过计算绘制标准曲线,公式为Log2N(拷贝数)=-0.6497ΔCt+4.9372(R2=0.9835,p(0.01),计算得到原代转基因猪外源基因拷贝数为32.893±1.99。
Pork is the important source of human intake of animal protein. High quality pork performs is tender and juicy. Intramuscular fat (IMF) has a very high correlation with those important economic traits and becomes the significant index for assessing pork quality. In last century, the target of pig breeding was increasing lean meat content and reducing backfat thickness, which caused dramatic decrease of IMF content (dropped to1.5%, recommended range:2.5-3%) and had negative influence on pork palatability and quality. In future, without reducing the lean meat content, the goal of swine breeding has been changed into increasing IMF content which means the augmentation of triglyceride (TAG) content. DGAT1is not only the rate-limiting enzyme which catalyzes the final step of triglyceride synthesis, but also the important role that involved in the regulation of mammalian lipid metabolism. Therefore, we took DGAT1as a candidate gene and constructed the eukaryotic expression vector sMCK-sDGAT1. We obtained transgenic mice and pigs by micro injection and somatic cell cloning, to study the effect of muscle-specific overexpression of DGAT1gene. The results are as follows:
1. We arrested swine MCK gene's promoter sequence from BAC (bacterial artificial chromosome) by recombinant DNA technology. Eukaryotic expression vector pEGFP-N1(as a framework) and the synthesized full-length swine DGAT1coding sequence were used to construct muscle-specific expression vector sMCK-sDGAT1. The vector sMCK-sDGATl was correct which proved by polymerase chain resction、restriction enzyme assay and sequence analysis.
2.16positive transgenic Fouder mice were made in Shanghai Bio model Technology Co., Ltd by pronuclei microinjection. We obtained F1generation (positive male mice mating with negative C57BL/6J female mice). The results of real time PCR show that DGAT1gene of transgenic mice is highly expressed in skeletal muscle and kidney, medium expressed in cardiac muscle and rarely expressed in the brain, lung, intestine and other tissues. Those results are consistent with the expression in various tissues of mammalian MCK gene.
3. We detect genes expression which involved in fat synthesis and transport of skeletal muscle by real-time PCR. The results showed that related gene expression had increased, such as the plastid acetyl-CoA carboxylase1increased34%(p<0.01), fatty acid binding protein (53%, p<0.05), fatty acid synthase (74%, p<0.01), the ethylene precursor ACC synthase (62%, p<0.05) and the brown fat-specific genen uncoupling protein (74%, p<0.01). Western Blot improved that protein level of DGAT1、ACC1、FABP4and UCP1had increased. The difference of weight between positive mice and negative mice was not significant by continuous determination, indicating the increase of IMF will not cause the mouse obesity. Determination of triglycerides and fatty acids in skeletal muscle showed the DGAT1positive mice increased35.5%(p<0.01) and20.3%(p<0.01), respectively.
4. We cultured porcine primary fibroblast cell lines. Then vector sMCK-sDGAT1was linearized and stably transfected into fibroblast by electric shock. We obtained positive monoclonal cells after G418selection using PCR assay. Finally, we obtained transgenic pig by somatic cell nuclear transfer.
5. PCR and Southern Blot were used to identify the founder transgenic pigs and one founder transgenic pig was detected. Absolute quantitative PCR was employed to calculate the transgene copy number. The parameters of the standard curve was:Log2N=-0.6497△Ct+4.9372(R2=0.9835, p<0.01), and copy number is32.893±1.99.
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