CuZnSOD轉(zhuǎn)基因豬的制備與研究
發(fā)布時(shí)間：2018-10-14 10:37
【摘要】：近年來,豬的遺傳改良在豬的生長速度、飼料轉(zhuǎn)化率及胴體瘦肉率上已經(jīng)取得了很大的進(jìn)步,但伴隨著我國養(yǎng)豬集約化、規(guī)�；陌l(fā)展,商品豬的氧化應(yīng)激時(shí)有發(fā)生,對(duì)豬群的健康、生長及肉質(zhì)等造成了嚴(yán)重?fù)p害。CuZnSOD(Copper Zinc Superoxide Dismutase)是超氧化物歧化酶(SODs)家族的成員之一,是機(jī)體防御氧化損傷的重要金屬酶,能轉(zhuǎn)移性地清除超氧陰離子自由基,提高機(jī)體抗氧化能力,進(jìn)而改善健康與肉質(zhì)。為探索通過磁性納米顆粒介導(dǎo)精子載體法制備轉(zhuǎn)基因豬的可行性,進(jìn)一步研究CuZnSOD基因?qū)寡趸阅艿热赓|(zhì)特性的影響機(jī)理,本研究通過磁性納米顆粒介導(dǎo)精子載體法制備了轉(zhuǎn)基因豬。本研究應(yīng)用PCR、qRT-PCR、western blot等技術(shù)對(duì)F1代和F2代豬進(jìn)行檢測(cè),通過絕對(duì)定量PCR對(duì)F1代4頭轉(zhuǎn)基因陽性豬進(jìn)行拷貝數(shù)鑒定,與此同時(shí),本研究還驗(yàn)證了CuZnSOD對(duì)肉質(zhì)抗氧化性能等肉質(zhì)性狀的影響。主要研究結(jié)果如下:1、通過將實(shí)驗(yàn)室前期成功構(gòu)建的重組表達(dá)載體先后與磁性納米顆粒polyMAG和精液共孵育,然后對(duì)3頭試驗(yàn)大白母豬進(jìn)行人工授精,3頭母豬共產(chǎn)下F1代仔豬43頭,其中,陽性豬13頭,陽性率為30.23%。F1代轉(zhuǎn)基因豬中,陽性組在肌肉、肝臟、背膘、肺臟及腎臟組織中CuZnSOD基因mRNA表達(dá)量均高于陰性組,并且在肌肉和肺臟組織中達(dá)到差異顯著水平(P0.05);陽性組在肌肉、肝臟、背膘、肺臟及腎臟組織中CuZnSOD蛋白的表達(dá)量都高于陰性組,并且在肌肉組織和肝臟組織中達(dá)到差異顯著水平(P0.05)。絕對(duì)定量PCR對(duì)F1代轉(zhuǎn)基因陽性豬進(jìn)行拷貝數(shù)鑒定,4頭陽性豬中外源基因拷貝數(shù)分別為:25.89、80.61、106.69、83.03。2、3頭F1代轉(zhuǎn)基因陽性母豬共產(chǎn)F2代仔豬44頭,其中,陽性豬11頭,陰性豬33頭,陽性率為25.00%。F2代轉(zhuǎn)基因豬中,陽性組在肌肉、肝臟及背膘組織中CuZnSOD基因mRNA表達(dá)量均高于陰性組,并且在肌肉組織和肝臟組織中達(dá)到差異顯著水平(P0.05);陽性組肌肉組織中CuZnSOD蛋白的表達(dá)量顯著高于陰性組(P0.05)。3、對(duì)F1代轉(zhuǎn)基因豬進(jìn)行抗氧化性等肉質(zhì)測(cè)定,結(jié)果表明,轉(zhuǎn)基因陽性豬滴水損失顯著性低于陰性豬(P0.05);陽性組SOD活性始終高于陰性組,并且陽性組SOD1d、SOD2d顯著高于陰性組(P0.05);陽性組MDA含量始終低于陰性組,并且陽性組MDA1d、MDA2d顯著低于陰性組(P0.05)。對(duì)F2代轉(zhuǎn)基因豬進(jìn)行抗氧化性等肉質(zhì)測(cè)定,結(jié)果表明,轉(zhuǎn)基因陽性豬滴水損失及烹飪損失顯著性小于陰性豬(P0.05);陽性組SOD活性始終高于陰性組,并且陽性組SOD1d、SOD2d及SOD3d顯著高于陰性組(P0.05);陽性組MDA含量始終低于陰性組,并且陽性組MDA1d、MDA2d顯著低于陰性組(P0.05)。對(duì)F2代轉(zhuǎn)基因豬的血清生化指標(biāo)進(jìn)行測(cè)定,結(jié)果表明,陽性組的堿性磷酸酶(ALP)活性顯著低于陰性組(P0.05);陽性組的總蛋白(TP)含量顯著低于陰性組(P0.05)。本研究通過磁性納米顆粒介導(dǎo)精子載體法成功制備了CuZnSOD轉(zhuǎn)基因豬,且外源基因成功地遺傳給了F2代并在F2代體內(nèi)成功表達(dá)。轉(zhuǎn)基因陽性豬的部分肉質(zhì)性狀有所改善,主要表現(xiàn)在陽性豬肌肉具有較高的抗氧化性能及持水能力。
[Abstract]:In recent years, pig genetic improvement has made great progress in pig growth speed, feed conversion rate and carcass lean meat rate, but with the intensive and large scale development of pig raising in our country, the oxidative stress of commercial pigs sometimes happens, and the pig population is healthy. Growth and meat quality have caused serious damage. CuZnSOD is one of the members of the family of superoxide dismutase (SODs). It is an important metal enzyme for body defense oxidative damage. It can remove superoxide anion free radicals and improve the anti-oxidation ability of the organism, thus improving the health and meat quality. In order to explore the feasibility of preparing transgenic pigs by magnetic nanoparticle-mediated sperm vector method, the mechanism of the effects of CuZnSOD gene on meat quality, such as oxidation resistance, was studied, and transgenic pigs were prepared by magnetic nanoparticle-mediated sperm vector method. In this study, PCR, qRT-PCR, Western blot were used to detect F1 and F2 generation pigs, and the copy number of four transgenic positive pigs was identified by absolute quantitative PCR. Meanwhile, the effects of CuZnSOD on meat quality traits such as meat quality and oxidation resistance were also validated. The main research results are as follows: 1, incubating the recombinant expression vector successfully constructed in the prior period of the laboratory with the magnetic nanoparticle polyMAG and the semen, and then performing artificial insemination on the three test white sows, The positive rate was 30. 23%. In F1 generation transgenic pigs, the expression level of CuZnSOD mRNA in muscle, liver, dorsal pituitary, lung and kidney was higher than that in the negative group, and the difference was significantly higher in muscle and lung tissues (P0.05). The expression level of CuZnSOD protein in lung and kidney was higher than that in the negative group, and the difference was significantly higher in muscle tissue and liver tissue (P0.05). The copy number of F1 generation transgenic positive pigs was identified by absolute quantitative PCR, and the copy number of Chinese and foreign gene was 25. 89, 80. 61, 106. 69, 83. 03. 2, and 3 F1 generation transgenic positive sows had 44 piglets. Among them, the positive rate was 25. 00%. The expression of CuZnSOD mRNA in muscle, liver and liver was higher than that in the negative group (P <0.05). The expression of CuZnSOD protein in the muscle tissue of positive group was significantly higher than that in the negative group (P0.05). The results showed that the water loss of transgenic pigs was significantly lower than that in the negative group (P0.05), and the SOD activity of the positive group was higher than that of the negative group (P0.05). The content of MDA in the positive group was always lower than that of the negative group, and the MDA1d and MDA2d of the positive group were significantly lower than that in the negative group (P0.05). The results showed that there was no significant difference between the water loss of the transgenic pig and that of the negative pig (P0.05), and the SOD activity of the positive group was higher than that of the negative group (P0.05). The content of MDA in the positive group was always lower than that of the negative group, and the MDA1d and MDA2d of the positive group were significantly lower than that in the negative group (P0.05). The results showed that the activity of alkaline phosphatase (ALP) in the positive group was significantly lower than that in the negative group (P0.05), and the total protein (TP) content of the positive group was significantly lower than that of the negative group (P0.05). CuZnSOD transgenic pig was successfully prepared by magnetic nanoparticle-mediated sperm vector method, and the exogenous gene was successfully inherited to F2 generation and expressed successfully in F2 generation. Some meat quality traits of transgenic positive pigs were improved, which mainly showed higher oxidation resistance and water holding ability in the muscle of positive pigs.
【學(xué)位授予單位】：山東農(nóng)業(yè)大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2015
【分類號(hào)】：S828
本文編號(hào)：2270197
[Abstract]:In recent years, pig genetic improvement has made great progress in pig growth speed, feed conversion rate and carcass lean meat rate, but with the intensive and large scale development of pig raising in our country, the oxidative stress of commercial pigs sometimes happens, and the pig population is healthy. Growth and meat quality have caused serious damage. CuZnSOD is one of the members of the family of superoxide dismutase (SODs). It is an important metal enzyme for body defense oxidative damage. It can remove superoxide anion free radicals and improve the anti-oxidation ability of the organism, thus improving the health and meat quality. In order to explore the feasibility of preparing transgenic pigs by magnetic nanoparticle-mediated sperm vector method, the mechanism of the effects of CuZnSOD gene on meat quality, such as oxidation resistance, was studied, and transgenic pigs were prepared by magnetic nanoparticle-mediated sperm vector method. In this study, PCR, qRT-PCR, Western blot were used to detect F1 and F2 generation pigs, and the copy number of four transgenic positive pigs was identified by absolute quantitative PCR. Meanwhile, the effects of CuZnSOD on meat quality traits such as meat quality and oxidation resistance were also validated. The main research results are as follows: 1, incubating the recombinant expression vector successfully constructed in the prior period of the laboratory with the magnetic nanoparticle polyMAG and the semen, and then performing artificial insemination on the three test white sows, The positive rate was 30. 23%. In F1 generation transgenic pigs, the expression level of CuZnSOD mRNA in muscle, liver, dorsal pituitary, lung and kidney was higher than that in the negative group, and the difference was significantly higher in muscle and lung tissues (P0.05). The expression level of CuZnSOD protein in lung and kidney was higher than that in the negative group, and the difference was significantly higher in muscle tissue and liver tissue (P0.05). The copy number of F1 generation transgenic positive pigs was identified by absolute quantitative PCR, and the copy number of Chinese and foreign gene was 25. 89, 80. 61, 106. 69, 83. 03. 2, and 3 F1 generation transgenic positive sows had 44 piglets. Among them, the positive rate was 25. 00%. The expression of CuZnSOD mRNA in muscle, liver and liver was higher than that in the negative group (P <0.05). The expression of CuZnSOD protein in the muscle tissue of positive group was significantly higher than that in the negative group (P0.05). The results showed that the water loss of transgenic pigs was significantly lower than that in the negative group (P0.05), and the SOD activity of the positive group was higher than that of the negative group (P0.05). The content of MDA in the positive group was always lower than that of the negative group, and the MDA1d and MDA2d of the positive group were significantly lower than that in the negative group (P0.05). The results showed that there was no significant difference between the water loss of the transgenic pig and that of the negative pig (P0.05), and the SOD activity of the positive group was higher than that of the negative group (P0.05). The content of MDA in the positive group was always lower than that of the negative group, and the MDA1d and MDA2d of the positive group were significantly lower than that in the negative group (P0.05). The results showed that the activity of alkaline phosphatase (ALP) in the positive group was significantly lower than that in the negative group (P0.05), and the total protein (TP) content of the positive group was significantly lower than that of the negative group (P0.05). CuZnSOD transgenic pig was successfully prepared by magnetic nanoparticle-mediated sperm vector method, and the exogenous gene was successfully inherited to F2 generation and expressed successfully in F2 generation. Some meat quality traits of transgenic positive pigs were improved, which mainly showed higher oxidation resistance and water holding ability in the muscle of positive pigs.
【學(xué)位授予單位】：山東農(nóng)業(yè)大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2015
【分類號(hào)】：S828
【共引文獻(xiàn)】
相關(guān)期刊論文 前2條
1 石元;陳偉;曾勇慶;祝洪磊;徐正剛;張哲;楊云;張?zhí)礻?;豬CuZnSOD基因啟動(dòng)子的克隆鑒定及分析[J];生物工程學(xué)報(bào);2014年02期
2 張哲;李同明;曾勇慶;陳偉;徐正剛;楊云;房國鋒;王守棟;楚青惠;;2A肽介導(dǎo)豬PID1與CuZnSOD雙基因真核共表達(dá)載體的構(gòu)建與細(xì)胞表達(dá)[J];生物學(xué)雜志;2014年06期
，本文編號(hào)：2270197
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