CuZnSOD转基因猪的制备与研究
发布时间：2018-10-14 10:37
【摘要】：近年来,猪的遗传改良在猪的生长速度、饲料转化率及胴体瘦肉率上已经取得了很大的进步,但伴随着我国养猪集约化、规模化的发展,商品猪的氧化应激时有发生,对猪群的健康、生长及肉质等造成了严重损害。CuZnSOD(Copper Zinc Superoxide Dismutase)是超氧化物歧化酶(SODs)家族的成员之一,是机体防御氧化损伤的重要金属酶,能转移性地清除超氧阴离子自由基,提高机体抗氧化能力,进而改善健康与肉质。为探索通过磁性纳米颗粒介导精子载体法制备转基因猪的可行性,进一步研究CuZnSOD基因对抗氧化性能等肉质特性的影响机理,本研究通过磁性纳米颗粒介导精子载体法制备了转基因猪。本研究应用PCR、qRT-PCR、western blot等技术对F1代和F2代猪进行检测,通过绝对定量PCR对F1代4头转基因阳性猪进行拷贝数鉴定,与此同时,本研究还验证了CuZnSOD对肉质抗氧化性能等肉质性状的影响。主要研究结果如下:1、通过将实验室前期成功构建的重组表达载体先后与磁性纳米颗粒polyMAG和精液共孵育,然后对3头试验大白母猪进行人工授精,3头母猪共产下F1代仔猪43头,其中,阳性猪13头,阳性率为30.23%。F1代转基因猪中,阳性组在肌肉、肝脏、背膘、肺脏及肾脏组织中CuZnSOD基因mRNA表达量均高于阴性组,并且在肌肉和肺脏组织中达到差异显著水平(P0.05);阳性组在肌肉、肝脏、背膘、肺脏及肾脏组织中CuZnSOD蛋白的表达量都高于阴性组,并且在肌肉组织和肝脏组织中达到差异显著水平(P0.05)。绝对定量PCR对F1代转基因阳性猪进行拷贝数鉴定,4头阳性猪中外源基因拷贝数分别为:25.89、80.61、106.69、83.03。2、3头F1代转基因阳性母猪共产F2代仔猪44头,其中,阳性猪11头,阴性猪33头,阳性率为25.00%。F2代转基因猪中,阳性组在肌肉、肝脏及背膘组织中CuZnSOD基因mRNA表达量均高于阴性组,并且在肌肉组织和肝脏组织中达到差异显著水平(P0.05);阳性组肌肉组织中CuZnSOD蛋白的表达量显著高于阴性组(P0.05)。3、对F1代转基因猪进行抗氧化性等肉质测定,结果表明,转基因阳性猪滴水损失显著性低于阴性猪(P0.05);阳性组SOD活性始终高于阴性组,并且阳性组SOD1d、SOD2d显著高于阴性组(P0.05);阳性组MDA含量始终低于阴性组,并且阳性组MDA1d、MDA2d显著低于阴性组(P0.05)。对F2代转基因猪进行抗氧化性等肉质测定,结果表明,转基因阳性猪滴水损失及烹饪损失显著性小于阴性猪(P0.05);阳性组SOD活性始终高于阴性组,并且阳性组SOD1d、SOD2d及SOD3d显著高于阴性组(P0.05);阳性组MDA含量始终低于阴性组,并且阳性组MDA1d、MDA2d显著低于阴性组(P0.05)。对F2代转基因猪的血清生化指标进行测定,结果表明,阳性组的碱性磷酸酶(ALP)活性显著低于阴性组(P0.05);阳性组的总蛋白(TP)含量显著低于阴性组(P0.05)。本研究通过磁性纳米颗粒介导精子载体法成功制备了CuZnSOD转基因猪,且外源基因成功地遗传给了F2代并在F2代体内成功表达。转基因阳性猪的部分肉质性状有所改善,主要表现在阳性猪肌肉具有较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.
【学位授予单位】：山东农业大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：S828
本文编号：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.
【学位授予单位】：山东农业大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：S828
【共引文献】
相关期刊论文 前2条
1 石元;陈伟;曾勇庆;祝洪磊;徐正刚;张哲;杨云;张天阳;;猪CuZnSOD基因启动子的克隆鉴定及分析[J];生物工程学报;2014年02期
2 张哲;李同明;曾勇庆;陈伟;徐正刚;杨云;房国锋;王守栋;楚青惠;;2A肽介导猪PID1与CuZnSOD双基因真核共表达载体的构建与细胞表达[J];生物学杂志;2014年06期
，本文编号：2270197
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