基于fat—1转基因牛的自身安全评价

发布时间：2018-05-26 00:33

本文选题：n-3多不饱和脂肪酸 + 体细胞核移植　；参考：《内蒙古大学》2016年博士论文

【摘要】：n-3多不饱和脂肪酸(n-3 PUFAs)与人类的健康密切相关,但人体自身不能合成n-3 PUFAs。自1997年,Spychalla等在线虫中获得了一个脂肪酸脱氢酶基因fat-1,多种富含n-3 PUFAs的fat-1转基因动物相继被研制成功,使这些转基因动物的产品为人类提供必需的n-3 PUFAs成为了可能。随着fat-1转基因家畜的大量出生,涉及转基因家畜的生物安全成为了人类倍受关注的问题,其中转基因家畜自身健康是生物安全检测的一项重要内容。截止目前,转基因家畜自身健康和安全的评价虽已开展了一些研究,但多局限于动物健康的某个方面,系统而全面评价转基因家畜自身健康的研究还未见报道。本研究利用体细胞核移植技术最终获得了3头成活的fat-1转基因牛,高通量测序技术鉴定了其中一头转基因牛的fat-1因整合在16号染色体的15726078bp处。通过对3头fat-1转基因牛在血液生化水平、基因漂移、自身基因表达变化、血浆蛋白表达变化及肠道菌群变化结果的系统分析,发现fat-1基因的转入对转基因牛的脂类代谢、免疫、心血管系统、抗应激等方面均表现出调节作用。实验结果对于建立转基因家畜,特别是转基因大家畜的自身安全评价体系,为获得健康、安全的转基因家畜的相关研究提供了有价值的参考资料。主要研究结果如下：1.fat-1转基因牛的生产及常规分析通过转基因及体细胞核移植技术(SCNT)获得了9头犊牛,6头犊牛被鉴定为fat-1转基因阳性牛,脂肪酸检测证实了fat-1基因的转入可以提升牛体内n-3PUFAs的含量,降低n-6 PUFAs的含量。血液生化水平检测,发现fat-1基因的转入显著降低了犊牛ALT及成年牛AST、GLU、TC和LDL-C的水平。常规PCR检测转基因牛肠道粪便、圈舍中土壤及其周围200 m各方位土壤微生物,均未发现有fat-1基因的存在。2.fat-1转基因牛整合位点分析应用高通量测序技术对fat-1转基因牛(FD006)进行了外源基因整合位点分析,结果表明fat-1基因整合在牛16号染色体的15726078bp处,且为单拷贝,根据测序获得的插入位点及其附近reads的结果分析,显示FD006为杂合子转基因牛,PCR的验证结果证实了fat-1基因确实插入了牛16号染色体的15726078 bp处,而且确实为杂合子转基因牛。本研究为转基因牛的整合位点分析、外源基因定点整合以及稳定表达等研究提供了技术路线与理论依据。3.fat-1转基因牛基因表达变化研究为分析fat-1基因的转入对牛基因表达的影响,提取fat-1转基因牛和野生型牛血液的总RNA,进行基因表达谱芯片检测。结果表明,有2042个基因表达存在显著差异,其中797个基因在fat-1转基因牛中是上调表达的,其余1245个基因则下调表达。基于2042个差异基因进行GO富集分析,发现90个GO Terms被显著富集,这些GO Terms主要与机体的脂类代谢、细胞行为、免疫及神经系统发育密切相关,其中8个GO Terms中包含了36个发生显著变化的脂类代谢关键基因。KEG G富集分析进一步获得了与脂类代谢,特别是与多不饱和脂肪酸的代谢密切相关的“PPAR signaling pathway”代谢通路。应用Real-time PCR对芯片检测获得的16个脂类代谢关键基因进行验证,证实了芯片获得的结果是可信的。综合以上结果,发现fat-1基因的转入引起了牛自身基因表达水平的变化,这些变化的基因在机体的脂类代谢、免疫、神经发育等生物学通路表现出调节作用。4.fat-1转基因牛血浆蛋白组学研究利用2D-双向电泳及质谱检测技术对fat-1转基因牛血浆蛋白组学进行了分析。结果表明,共有15个血浆差异蛋白被鉴定；对这15个差异蛋白及其互作蛋白的GO和KEGG富集分析,发现这些蛋白主要参与了机体的脂类代谢、免疫、应激、神经发育和血液凝固等生物学通路的调节；在18个重要的脂类代谢生物学通路中,有12个通路都富集到了APOA1,表明fat-1基因参与对脂类代谢的调控可能与APOA1有密切关系；血浆APOA1检测发现,转基因牛血浆中APOA1含量显著高于野生型牛,其表达水平与LDL-C高度负相关(r==一0.90),与HDL-C/TC比值存在显著正相关(r=0.69)。综合以上结果,与野生型牛比较发现,fat-1基因的转入使牛血浆中的一些蛋白表达发生了变化,并发现fat-1基因可能会介导APOA1的表达参与转基因牛脂类代谢的调控。5.fat-1转基因牛肠道微生物菌群的研究采用高通量测序技术,分别对3头fat-1转基因牛和3头野生型牛的直肠粪便进行了基于16s rDNA V4可变区肠道菌群多样性及组成的比较分析。结果显示,转基因牛和野生型牛共获得9714个OTUs的分类,转基因牛的OTUs分类(8907个)明显少于野生型牛(9488个)；物种多样性指数分析发现,转基因牛的Chao和Shannon指数均显著低于野生型牛(p0.05)。进一步的分析发现,fat-1基因的转入也改变了牛肠道菌群的组成和表达丰度。其中物种注释丰度比较发现,转基因牛与野生型牛间有3个门(广古菌门、变形菌门、螺旋体门),9个属(紫单胞菌属、拟杆菌属、甲烷短杆菌、密螺旋体属、梭菌属、毛螺菌属、罗氏菌属、消化球菌属、丁酸弧菌属)存在物种丰度差异(p0-05)。结合肠道菌群的组成、物种丰度比较结果及血糖、血脂生化检测结果,发现Dorea、 Roseburia、Succinivibrio和Alistipes与血糖、血脂的变化存在一定的相关性。此外,也发现与应激有关的Odoribacte r菌属在转基因牛肠道中显著降低。综合本研究结果表明,fat-1基因的转入改变了牛肠道菌群的多样性、群落组成及表达丰度,发生变化的菌属主要与宿主的糖、脂代谢以及机体的抗应激有关,推测fat-1基因的转入可能会通过改变牛肠道菌群的组成或表达丰度参与机体脂类代谢调控的潜在机制。
[Abstract]:N-3 polyunsaturated fatty acids (n-3 PUFAs) are closely related to human health, but the human body itself can not synthesize n-3 PUFAs. from the 1997, Spychalla and other online insects, which have obtained a fatty acid dehydrogenase gene fat-1, and a variety of fat-1 transgenic animals rich in n-3 PUFAs have been developed successively to make the products of these transgenic animals provided for human beings. The necessary n-3 PUFAs has become possible. With the massive birth of fat-1 transgenic livestock, the biological safety of genetically modified livestock has become a problem of great concern, among which the health of genetically modified livestock is an important part of biosafety detection. By the time, the assessment of the health and safety of transgenic livestock has been carried out. Some studies, however limited to a certain aspect of animal health, have not yet been reported in a systematic and comprehensive assessment of the health of genetically modified animals. 3 of the fat-1 genetically modified cattle were finally obtained by somatic cell nuclear transplantation. The fat-1 of one of the transgenic cattle was identified by high throughput sequencing. Through systematic analysis of the blood biochemical level, gene drift, gene expression change, plasma protein expression change and intestinal flora change results of 3 fat-1 transgenic cattle, it was found that the transfer of fat-1 gene showed regulatory effect on lipid metabolism, immunity, cardiovascular system and anti stress of transgenic cattle. The results provide valuable reference for the establishment of the safety evaluation system of genetically modified livestock, especially the genetically modified people and livestock, and the relevant research for obtaining healthy and safe transgenic livestock. The main results are as follows: the production and routine analysis of 1.fat-1 transgenic cattle by transgenic and somatic cell nuclear transplantation (S CNT) 9 calves and 6 calves were identified as fat-1 transgenic cattle. The fatty acid test confirmed that the transfer of fat-1 gene could increase the content of n-3PUFAs in the bovine body, reduce the content of n-6 PUFAs, and the biochemical level of the blood. It was found that the transformation of the fat-1 gene significantly reduced the AST, GLU, TC and LDL-C levels of calf ALT and adult cattle. PCR detection of transgenic cattle intestinal feces, soil and around 200 m around the soil microbes, no fat-1 gene was found in the presence of.2.fat-1 transgenic cattle integration site analysis and high throughput sequencing technology for fat-1 transgenic cattle (FD006) analysis of exogenous gene integration site, the results showed that fat-1 gene integration in cattle No. 16 On the 15726078bp of the chromosome, and a single copy, according to the analysis of the insertion site and the reads near the sequence, the FD006 is a heterozygote transgenic cow. The verification of PCR confirmed that the fat-1 gene did insert the 15726078 BP of the bovine chromosome 16, and was indeed a heterozygote transgenic cattle. This study was the whole of transgenic cattle. Studies on locus analysis, extraneous gene fixed-point integration and stable expression provide a technical route and theoretical basis for the change of gene expression of.3.fat-1 transgenic cattle to analyze the effect of fat-1 gene transfer on bovine gene expression, extract the total RNA of the blood of fat-1 transgenic cattle and wild cattle, and carry out gene expression spectrum chip detection. The results showed that there were significant differences in the expression of 2042 genes, of which 797 genes were up-regulated in fat-1 transgenic cattle and the other 1245 genes were down regulated. Based on 2042 differentially expressed genes, GO enrichment analysis showed that 90 GO Terms were significantly enriched, and these GO Terms were mainly associated with lipid metabolism, cell behavior, immunity and nerve of the body. The phylogeny is closely related, of which 8 GO Terms contains 36 significant changes in the lipid metabolism key gene.KEG G enrichment analysis to further obtain the "PPAR signaling pathway" Xie Tong Road, which is closely related to the metabolism of polyunsaturated fatty acids, which is obtained by Real-time PCR to the chip detection of 1. 6 key genes of lipid metabolism were verified and confirmed that the results obtained by the chip were credible. Combined the results, it was found that the transfer of the fat-1 gene caused the changes in the gene expression level of the bovine itself, which showed the regulation of.4.fat-1 transgenic cattle in the biological pathways of lipid metabolism, immunity and nerve development in the body. Plasma proteomics was used to analyze the plasma proteomics of fat-1 transgenic cattle by 2D- two-dimensional electrophoresis and mass spectrometry. The results showed that a total of 15 plasma differential proteins were identified, and the accumulation and analysis of GO and KEGG of the 15 differential proteins and their interacted proteins showed that these proteins were mainly involved in the metabolism of lipids in the body. The regulation of biological pathways such as epidemic, stress, nerve development and blood coagulation; in 18 important lipid metabolic biologic pathways, 12 pathways were enriched in APOA1, indicating that the involvement of fat-1 genes in the regulation of lipid metabolism may be closely related to APOA1; plasma APOA1 detection found that the content of APOA1 in genetically modified bovine plasma was significantly higher than that of APOA1. The expression level of wild type cattle was negatively correlated with the LDL-C height (r== 0.90), and there was a significant positive correlation with the ratio of HDL-C/TC (r=0.69). In comparison with the wild type, it was found that the transfer of fat-1 gene changed some of the protein expression in the bovine plasma, and the present fat-1 gene may mediate the expression of APOA1 in genetically modified cattle. Lipid metabolism regulation of.5.fat-1 transgenic cattle intestinal microflora using high throughput sequencing technology, the rectal feces of 3 fat-1 transgenic cattle and 3 wild type cattle were compared and analyzed based on the diversity and composition of the intestinal microflora in the 16S rDNA V4 variable region. The results showed that 9714 of the transgenic cattle and the wild type cattle obtained a total of 9714. In the classification of OTUs, the OTUs classification of transgenic cattle (8907) was significantly less than that of wild type cattle (9488). The analysis of species diversity index found that the Chao and Shannon index of transgenic cattle were significantly lower than those of wild type cattle (P0.05). Further analysis found that the transfer of fat-1 gene also changed the composition and expression abundance of the bovine intestinal flora. A comparison of annotation abundances found that there are 3 gates between genetically modified cattle and wild type cattle (archaea, proteus, spiralum), 9 genera (einomonas, Pseudomonas, brachytobacilli, Clostridium, Spirillum, rochella, rochella, alimentary, and Vibrio butyric). There are differences in species abundance (p0-05). It was found that Dorea, Roseburia, Succinivibrio and Alistipes were related to the changes of blood glucose and blood lipids. Furthermore, it was also found that the Odoribacte r genus associated with stress decreased significantly in the intestinal tract of transgenic cattle. The results of this study showed that the fat-1 gene was transferred to the fat-1 gene. The diversity of the intestinal flora, the composition of the community and the abundance of the expression, and the variation of the bacteria are mainly related to the host's sugar, lipid metabolism and the anti stress of the body. It is speculated that the transfer of fat-1 gene may be involved in the potential mechanism of regulating the lipid metabolism in the body by changing the composition or expression abundance of the bovine intestinal flora.
【学位授予单位】：内蒙古大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：S823

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