猪LMNA基因对于肌肉和脂肪发育及分化的调控研究
发布时间：2018-10-29 09:59
【摘要】：猪肉一直是我国最主要的肉类消费品,随着人们生活水平的提高,瘦肉型猪已不能满足人们对于猪肉口感的要求了,因此近年来研究人员将科研重点转向了提高猪肉品质和口感。通过对具有优良性状的种猪进行主效基因、QTL、SNP、MAS的研究,筛选出影响肌肉或脂肪发育的相关基因。研究表明,LMNA基因能够影响肌肉和脂肪的发育及生成。LMNA即核纤层蛋白,是结合于内层核膜上参与核膜的形成,并维持细胞核的形态,能够调控进出核孔的通道,影响相关转录因子和细胞信号通路的激活与作用。在人类中LMNA基因突变会导致肌肉和脂肪发育,修复和生成相关的遗传病(Laminopathies),在小鼠中LMNA突变和敲除也会引起相似的肌肉或脂肪发育障碍。因此本研究以LMNA基因作为研究对象,构建pc DNA3.1-s LMNA超表达载体和合成LMNA的干扰si RNA序列,分别对C2C12和3T3-L1细胞进行超表达,干扰及分化的实验,以研究LMNA基因对于肌肉和脂肪的发育及生成的影响,为以后实际生产中的猪育种打下一定的基础。取得的主要研究成果如下:1、根据基因重组原理,利用猪的c DNA为模版,将LMNA基因连接到p MD18-T载体上,再以重组p MD18-T-s LMNA载体为模版,以内切酶NheⅠ和KpnⅠ酶切目的片段和pc DNA3.1载体,构建重组超表达载体pc DNA3.1-s LMNA。分别对载体进行测序验证,PCR和酶切验证。分别将超表达载体转染C2C12和3T3-L1细胞并在细胞内稳定表达。2、利用构建的pc DNA3.1-s LMNA超表达载体和苏州吉玛公司合成的LMNA基因si RNA干扰序列分别转染C2C12成肌细胞,另外在超表达和干扰转染同时分别做C2C12细胞的0、2、4、6天分化,收集细胞提取RNA和蛋白做定量PCR和Western Blot实验。结果显示LMNA超表达会引起肌肉相关基因的下调,LMNA干扰会引起肌肉相关基因的上调,C2C12分化过程中干扰LMNA表达会抑制细胞分化。3、利用超表达载体pc DNA3.1-s LMNA和LMNA si RNA干扰序列分别转染3T3-L1细胞,之后超表达和干扰转染后同时分别作3T3-L1细胞的0、2、4、6天分化,收集细胞提RNA和蛋白做定量PCR和Western Blot实验。结果显示超表达LMNA会导致脂肪相关基因表达下降,干扰LMNA会导致脂肪相关基因表达上升,干扰LMNA基因会抑制3T3-L1前体脂肪细胞的分化。4、在C2C12细胞和3T3-L1细胞的超表达LMNA分化实验中,肌肉和脂肪分化相关基因在分化第2天时表达量都出现了不同程度的上升,然而在分化第4、6天表达量又保持平稳或者降低,说明在成肌细胞和前体脂肪细胞分化初期LMNA可能具有一定程度的促进作用,但在分化中后期的结果却相反,具体原因有待进一步研究。
[Abstract]:Pork has always been the most important meat consumer in China. With the improvement of people's living standard, lean pig can no longer meet the requirements of pork taste. So in recent years researchers have shifted their focus to improving pork quality and taste. Through the study of the main effect gene and QTL,SNP,MAS of the breeding pigs with excellent traits, the genes related to muscle or fat development were screened out. Studies have shown that LMNA gene can affect the development and production of muscle and fat. LMNA, the nuclear laminin, is bound to the inner nuclear membrane to participate in the formation of nuclear membrane, maintain the shape of the nucleus, and regulate the channels in and out of the nuclear pore. Affect the activation and action of related transcription factors and cellular signaling pathways. In humans, LMNA gene mutations can lead to muscle and fat development, repair and production of related genetic disease (Laminopathies), in mice LMNA mutation and knockout will also cause similar muscle or fat development disorders. Therefore, in this study, LMNA gene was used as the research object to construct pc DNA3.1-s LMNA overexpression vector and synthesize LMNA interference si RNA sequence, and to conduct the experiments of superexpression, interference and differentiation of C2C12 and 3T3-L1 cells, respectively. In order to study the effect of LMNA gene on the development and production of muscle and fat, this study will lay a foundation for pig breeding in practical production. The main research results are as follows: 1. According to the principle of gene recombination, the LMNA gene is connected to p MD18-T vector by using the c DNA of pig as template, and then the recombinant p MD18-T-s LMNA vector is used as template. Recombinant superexpression vector pc DNA3.1-s LMNA. was constructed by digesting the target fragment and pc DNA3.1 vector of endonuclease Nhe 鈪，
本文编号：2297375
[Abstract]:Pork has always been the most important meat consumer in China. With the improvement of people's living standard, lean pig can no longer meet the requirements of pork taste. So in recent years researchers have shifted their focus to improving pork quality and taste. Through the study of the main effect gene and QTL,SNP,MAS of the breeding pigs with excellent traits, the genes related to muscle or fat development were screened out. Studies have shown that LMNA gene can affect the development and production of muscle and fat. LMNA, the nuclear laminin, is bound to the inner nuclear membrane to participate in the formation of nuclear membrane, maintain the shape of the nucleus, and regulate the channels in and out of the nuclear pore. Affect the activation and action of related transcription factors and cellular signaling pathways. In humans, LMNA gene mutations can lead to muscle and fat development, repair and production of related genetic disease (Laminopathies), in mice LMNA mutation and knockout will also cause similar muscle or fat development disorders. Therefore, in this study, LMNA gene was used as the research object to construct pc DNA3.1-s LMNA overexpression vector and synthesize LMNA interference si RNA sequence, and to conduct the experiments of superexpression, interference and differentiation of C2C12 and 3T3-L1 cells, respectively. In order to study the effect of LMNA gene on the development and production of muscle and fat, this study will lay a foundation for pig breeding in practical production. The main research results are as follows: 1. According to the principle of gene recombination, the LMNA gene is connected to p MD18-T vector by using the c DNA of pig as template, and then the recombinant p MD18-T-s LMNA vector is used as template. Recombinant superexpression vector pc DNA3.1-s LMNA. was constructed by digesting the target fragment and pc DNA3.1 vector of endonuclease Nhe 鈪，
本文编号：2297375
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