【摘要】：豬肉一直是我國最主要的肉類消費品,隨著人們生活水平的提高,瘦肉型豬已不能滿足人們對于豬肉口感的要求了,因此近年來研究人員將科研重點轉(zhuǎn)向了提高豬肉品質(zhì)和口感。通過對具有優(yōu)良性狀的種豬進行主效基因、QTL、SNP、MAS的研究,篩選出影響肌肉或脂肪發(fā)育的相關(guān)基因。研究表明,LMNA基因能夠影響肌肉和脂肪的發(fā)育及生成。LMNA即核纖層蛋白,是結(jié)合于內(nèi)層核膜上參與核膜的形成,并維持細胞核的形態(tài),能夠調(diào)控進出核孔的通道,影響相關(guān)轉(zhuǎn)錄因子和細胞信號通路的激活與作用。在人類中LMNA基因突變會導致肌肉和脂肪發(fā)育,修復和生成相關(guān)的遺傳病(Laminopathies),在小鼠中LMNA突變和敲除也會引起相似的肌肉或脂肪發(fā)育障礙。因此本研究以LMNA基因作為研究對象,構(gòu)建pc DNA3.1-s LMNA超表達載體和合成LMNA的干擾si RNA序列,分別對C2C12和3T3-L1細胞進行超表達,干擾及分化的實驗,以研究LMNA基因?qū)τ诩∪夂椭镜陌l(fā)育及生成的影響,為以后實際生產(chǎn)中的豬育種打下一定的基礎。取得的主要研究成果如下:1、根據(jù)基因重組原理,利用豬的c DNA為模版,將LMNA基因連接到p MD18-T載體上,再以重組p MD18-T-s LMNA載體為模版,以內(nèi)切酶NheⅠ和KpnⅠ酶切目的片段和pc DNA3.1載體,構(gòu)建重組超表達載體pc DNA3.1-s LMNA。分別對載體進行測序驗證,PCR和酶切驗證。分別將超表達載體轉(zhuǎn)染C2C12和3T3-L1細胞并在細胞內(nèi)穩(wěn)定表達。2、利用構(gòu)建的pc DNA3.1-s LMNA超表達載體和蘇州吉瑪公司合成的LMNA基因si RNA干擾序列分別轉(zhuǎn)染C2C12成肌細胞,另外在超表達和干擾轉(zhuǎn)染同時分別做C2C12細胞的0、2、4、6天分化,收集細胞提取RNA和蛋白做定量PCR和Western Blot實驗。結(jié)果顯示LMNA超表達會引起肌肉相關(guān)基因的下調(diào),LMNA干擾會引起肌肉相關(guān)基因的上調(diào),C2C12分化過程中干擾LMNA表達會抑制細胞分化。3、利用超表達載體pc DNA3.1-s LMNA和LMNA si RNA干擾序列分別轉(zhuǎn)染3T3-L1細胞,之后超表達和干擾轉(zhuǎn)染后同時分別作3T3-L1細胞的0、2、4、6天分化,收集細胞提RNA和蛋白做定量PCR和Western Blot實驗。結(jié)果顯示超表達LMNA會導致脂肪相關(guān)基因表達下降,干擾LMNA會導致脂肪相關(guān)基因表達上升,干擾LMNA基因會抑制3T3-L1前體脂肪細胞的分化。4、在C2C12細胞和3T3-L1細胞的超表達LMNA分化實驗中,肌肉和脂肪分化相關(guān)基因在分化第2天時表達量都出現(xiàn)了不同程度的上升,然而在分化第4、6天表達量又保持平穩(wěn)或者降低,說明在成肌細胞和前體脂肪細胞分化初期LMNA可能具有一定程度的促進作用,但在分化中后期的結(jié)果卻相反,具體原因有待進一步研究。
[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，

本文編號：2297375