PiggyBac转座子介导的嗜热菌β-糖苷酶转基因牛研究
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摘要
牛奶作为营养丰富的全蛋白食品,对于补充机体营养物质,促进钙、磷吸收和增强人体免疫力具有重要作用。然而,在我国高达92%的人群对牛奶中的乳糖存在消化和吸收障碍,表现为“乳糖不耐症”。如何降低牛奶中乳糖含量,进而减少“乳糖不耐症”的发生,是奶牛育种研究中亟待解决的问题。嗜热菌B-糖苷酶(LacS)具有催化糖苷键水解进而分解乳糖的作用。通过转基因方法在牛奶中表达此酶,可以从根本上减少“乳糖不耐症”的发生。PiggyBac转座子作为近年来发现的转基因新技术,具有整合效率高、负载容量大、以单拷贝形式整合,更易模拟内源基因的表达状况等优点,在转基因研究中具有重要的应用前景。目前,利用PiggyBac进行的转基因牛研究未见有成功报道。因此,本研究构建了PiggyBac介导的牛乳腺特异性表达嗜热菌B-糖苷酶载体,结合体细胞克隆技术,以期降低牛奶中的乳糖含量,为今后培育低乳糖奶牛新品种进行探索性研究。
实验一将来源于古细菌的嗜热菌β-糖苷酶基因根据牛的密码子使用频率进行了密码子优化,并将优化后的基因在公司合成。随后分别克隆了牛乳球蛋白(BLG)启动子、PiggyBac转座元件,通过酶切连接等方法分别构建了不含转座子元件的普通载体plox-EGFP-LacS和含有转座元件的乳腺特异性表达嗜热菌糖苷酶的载体ZGL-LacS-EN。两个载体都添加了LOXP锚定的GFP和Neomycine双标记基因。目的在于进一步提高转基因的筛选效率和安全性。实验二将普通载体转染鼠乳腺癌细胞,并筛选得到单克隆。将数个单克隆混合后进行整合检测和RT-PCR检测。结果表明,BLG启动子能正确启动经密码子优化后和嗜热菌β-糖苷酶基因在乳腺细胞中的表达。且测序结果表明目的基因的编码区未发生突变。实验三将PiggyBac转座子载体与转座酶载体以5：1的比例共转染牛成纤维细胞。48h后以1：30、1：60、1：100的比例进行细胞传代和药物筛选。对筛选得到的单克隆进行Gimasa染色和计数。分析结果表明,上述传代比例下,添加／不添加转座酶的细胞克隆数分别为177/5、134/0.2、79/0。上述结果表明,PiggyBac在很大程度上提高了目的基因的整合效率。同时实验中发现,这种高效整合对于牛成纤维细胞的形态和生长状态产生了一些不利影响。对分离培养的单克隆提取基因组进行Tail PCR检测插入位点侧翼序列,并对40个有效测序结果分析表明,PiggyBac转座子介导的目的基因倾向于整合到牛染色体基因组内含子和基因间序列中。实验四将两种质粒转染母牛成纤维细胞,筛选得到含有目的基因的单克隆用于核移植实验。结果表明,普通载体转染的转基因胚胎卵裂率(83.2%)稍高于转座子介导的转基因胚胎(75.7%),而囊胚率(15.3%/14.9%)无明显差别,但两者均明显低于非转基因的胚胎(卵裂率88.2%,囊胚率36.7%)。将体外培养的绿色荧光的囊胚移植到3头经同期发情处理的代孕母牛子宫中,未见怀孕。
本研究首次成功构建了PiggyBac转座子介导的乳腺特异性表达嗜热菌B-糖苷酶的双标记转基因载体,证明了PiggyBac转座子在牛成纤维细胞中可以高效转座,并且这种整合具有内含子和基因间序列的倾向性。体细胞核移植实验结果进一步表明,转座子介导的转基因胚胎的囊胚率与普通载体并无明显差别。上述实验结果为今后培育低乳糖奶牛新品种进行了探索性研究,并为利用PiggyBac转座子技术进行高效和安全的转基因牛育种奠定了一定基础。
Milk as a nutritious whole protein food, plays an important role in the supplement nutrition, promote absorption of calcium, phosphorus and enhance human immunity. However, in our country as much as92%of the crowd has the obstacle to digestion and absorption of lactose in milk, which termed as "lactose intolerance ". How to reduce the content of lactose in the milk, and then reduce "lactose intolerance" should be anxious solved in dairy cows breeding research area. Sulfolobus Solfataricus β-Glycosidases has a function on catalytic hydrolysis glycosidic bond and hydrolysis of lactose. So, the "lactose intolerance" could be fundamentally solved through the genetically modified method to expression of Sulfolobus Solfataricus β-Glycosidases in the milk. Piggybac transposon as a new transgenic technology, because of the advantages such as high integration rate and load capacity, single copy integration, easy simulation endogenous gene expression environment, which has an important application prospect. At present, the piggybac transposon mediated transgenic cattle research have not been reported success. So, in this study, we constructed a transgenic vector which piggybac transposon mediated mammary gland specific expression of Sulfolobus Solfataricus β-Glycosidases, combined with somatic nuclear transfer technology (SCNT) to produce transgenic cattle, in order to reduce lactose content in the milk, and do the exploratory research for low lactose dairy cows breeding.
Experiment1, the Sulfolobus Solfataricus β-Glycosidases was codon optimized and synthetized based on the codon usage frequency of bovine. The bovine lactoglobulin and piggybac transposon element was cloned, digest and ligated to complete the mammary specific expression of Sulfolobus Solfataricus β-Glycosidases vector, which one do not contain the transposon element as a negative control; Another one contain the transposon element. All of them have the GFP and neomycine marker gene anchoraged by LOXP sequence, in order to enhance the screen efficiency and safety of transgenic. Experiment2, verification of expression vector by mammary gland cell. The PCR and RT-PCR analysis result showed that, the BLG promoter could direct the codon optimized Sulfolobus Solfataricus β-Glycosidases expressed in mammary gland cells, and there are no mutations in coding region of Sulfolobus Solfataricus β-Glycosidases verified by sequencing. Experiment3, piggybac transposon and transposase were co-transfected to fibroblasts at proportion of5:1. After48h, cells were passaged at1:30,1:60,1:100proportion and screened with G418. Gimasa and cell counting result showed that, the cell colonies was177/5、134/0.2and79/0under add with transposase or without transposase condition at above three passage proportion. These results showed that piggybac transposon could enhance integration rate greatly. However, the high integration rate may have some adverse effect on cell shape and growth condition.40available sequencing data of tailPCR results of colonies showed that, the piggybac transposon mediated gene integration is more tend to intron and intergenic of bovine chromosome. Experiment4, transfection and screening of fibroblasts with vector which with or without transposon element, then, SCNT with single colonies. Result showed that, the embryo cleavage rate of plox-EGF-LacS is slightly higher than ZGL-LacS-EN (83.2%/75.7%), and the blastosphere rate was no obvious differences between them (15.3%/14.9%), but both of them was obvious higher than non-modified embryos (cleavage rate88.2%, blastosphere rate36.7%). The blastosphere with green fluorescent were transfered to uterus of three receptor cattle treated with synchronization, no pregnant of them.
In this study, we first constructed piggybac mediated a double marker gene vector, which specific expression of Sulfolobus Solfataricus β-Glycosidases in mammary gland. Our result showed that the piggybac transposon could mediated transposition of target gene in fibroblasts efficiently, and the integration of targeting gene have more tend to intron and intergenic of bovine chromosome. Result of SCNT showed that, there are no obvious differences between plox-EGF-LacS and ZGL-LacS-EN of blastosphere rate. The above study do an exploratory research at cultivating of low lactose dairy cow new species, meanwhile, lays a foundation on more efficiency and safety of genetically cattle breeding use of piggybac transposon technology.
实验一将来源于古细菌的嗜热菌β-糖苷酶基因根据牛的密码子使用频率进行了密码子优化,并将优化后的基因在公司合成。随后分别克隆了牛乳球蛋白(BLG)启动子、PiggyBac转座元件,通过酶切连接等方法分别构建了不含转座子元件的普通载体plox-EGFP-LacS和含有转座元件的乳腺特异性表达嗜热菌糖苷酶的载体ZGL-LacS-EN。两个载体都添加了LOXP锚定的GFP和Neomycine双标记基因。目的在于进一步提高转基因的筛选效率和安全性。实验二将普通载体转染鼠乳腺癌细胞,并筛选得到单克隆。将数个单克隆混合后进行整合检测和RT-PCR检测。结果表明,BLG启动子能正确启动经密码子优化后和嗜热菌β-糖苷酶基因在乳腺细胞中的表达。且测序结果表明目的基因的编码区未发生突变。实验三将PiggyBac转座子载体与转座酶载体以5：1的比例共转染牛成纤维细胞。48h后以1：30、1：60、1：100的比例进行细胞传代和药物筛选。对筛选得到的单克隆进行Gimasa染色和计数。分析结果表明,上述传代比例下,添加／不添加转座酶的细胞克隆数分别为177/5、134/0.2、79/0。上述结果表明,PiggyBac在很大程度上提高了目的基因的整合效率。同时实验中发现,这种高效整合对于牛成纤维细胞的形态和生长状态产生了一些不利影响。对分离培养的单克隆提取基因组进行Tail PCR检测插入位点侧翼序列,并对40个有效测序结果分析表明,PiggyBac转座子介导的目的基因倾向于整合到牛染色体基因组内含子和基因间序列中。实验四将两种质粒转染母牛成纤维细胞,筛选得到含有目的基因的单克隆用于核移植实验。结果表明,普通载体转染的转基因胚胎卵裂率(83.2%)稍高于转座子介导的转基因胚胎(75.7%),而囊胚率(15.3%/14.9%)无明显差别,但两者均明显低于非转基因的胚胎(卵裂率88.2%,囊胚率36.7%)。将体外培养的绿色荧光的囊胚移植到3头经同期发情处理的代孕母牛子宫中,未见怀孕。
本研究首次成功构建了PiggyBac转座子介导的乳腺特异性表达嗜热菌B-糖苷酶的双标记转基因载体,证明了PiggyBac转座子在牛成纤维细胞中可以高效转座,并且这种整合具有内含子和基因间序列的倾向性。体细胞核移植实验结果进一步表明,转座子介导的转基因胚胎的囊胚率与普通载体并无明显差别。上述实验结果为今后培育低乳糖奶牛新品种进行了探索性研究,并为利用PiggyBac转座子技术进行高效和安全的转基因牛育种奠定了一定基础。
Milk as a nutritious whole protein food, plays an important role in the supplement nutrition, promote absorption of calcium, phosphorus and enhance human immunity. However, in our country as much as92%of the crowd has the obstacle to digestion and absorption of lactose in milk, which termed as "lactose intolerance ". How to reduce the content of lactose in the milk, and then reduce "lactose intolerance" should be anxious solved in dairy cows breeding research area. Sulfolobus Solfataricus β-Glycosidases has a function on catalytic hydrolysis glycosidic bond and hydrolysis of lactose. So, the "lactose intolerance" could be fundamentally solved through the genetically modified method to expression of Sulfolobus Solfataricus β-Glycosidases in the milk. Piggybac transposon as a new transgenic technology, because of the advantages such as high integration rate and load capacity, single copy integration, easy simulation endogenous gene expression environment, which has an important application prospect. At present, the piggybac transposon mediated transgenic cattle research have not been reported success. So, in this study, we constructed a transgenic vector which piggybac transposon mediated mammary gland specific expression of Sulfolobus Solfataricus β-Glycosidases, combined with somatic nuclear transfer technology (SCNT) to produce transgenic cattle, in order to reduce lactose content in the milk, and do the exploratory research for low lactose dairy cows breeding.
Experiment1, the Sulfolobus Solfataricus β-Glycosidases was codon optimized and synthetized based on the codon usage frequency of bovine. The bovine lactoglobulin and piggybac transposon element was cloned, digest and ligated to complete the mammary specific expression of Sulfolobus Solfataricus β-Glycosidases vector, which one do not contain the transposon element as a negative control; Another one contain the transposon element. All of them have the GFP and neomycine marker gene anchoraged by LOXP sequence, in order to enhance the screen efficiency and safety of transgenic. Experiment2, verification of expression vector by mammary gland cell. The PCR and RT-PCR analysis result showed that, the BLG promoter could direct the codon optimized Sulfolobus Solfataricus β-Glycosidases expressed in mammary gland cells, and there are no mutations in coding region of Sulfolobus Solfataricus β-Glycosidases verified by sequencing. Experiment3, piggybac transposon and transposase were co-transfected to fibroblasts at proportion of5:1. After48h, cells were passaged at1:30,1:60,1:100proportion and screened with G418. Gimasa and cell counting result showed that, the cell colonies was177/5、134/0.2and79/0under add with transposase or without transposase condition at above three passage proportion. These results showed that piggybac transposon could enhance integration rate greatly. However, the high integration rate may have some adverse effect on cell shape and growth condition.40available sequencing data of tailPCR results of colonies showed that, the piggybac transposon mediated gene integration is more tend to intron and intergenic of bovine chromosome. Experiment4, transfection and screening of fibroblasts with vector which with or without transposon element, then, SCNT with single colonies. Result showed that, the embryo cleavage rate of plox-EGF-LacS is slightly higher than ZGL-LacS-EN (83.2%/75.7%), and the blastosphere rate was no obvious differences between them (15.3%/14.9%), but both of them was obvious higher than non-modified embryos (cleavage rate88.2%, blastosphere rate36.7%). The blastosphere with green fluorescent were transfered to uterus of three receptor cattle treated with synchronization, no pregnant of them.
In this study, we first constructed piggybac mediated a double marker gene vector, which specific expression of Sulfolobus Solfataricus β-Glycosidases in mammary gland. Our result showed that the piggybac transposon could mediated transposition of target gene in fibroblasts efficiently, and the integration of targeting gene have more tend to intron and intergenic of bovine chromosome. Result of SCNT showed that, there are no obvious differences between plox-EGF-LacS and ZGL-LacS-EN of blastosphere rate. The above study do an exploratory research at cultivating of low lactose dairy cow new species, meanwhile, lays a foundation on more efficiency and safety of genetically cattle breeding use of piggybac transposon technology.
引文
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