本文選題：n-3多不飽和脂肪酸 + 體細胞核移植�。� 參考：《內蒙古大學》2016年博士論文

【摘要】：n-3多不飽和脂肪酸(n-3 PUFAs)與人類的健康密切相關,但人體自身不能合成n-3 PUFAs。自1997年,Spychalla等在線蟲中獲得了一個脂肪酸脫氫酶基因fat-1,多種富含n-3 PUFAs的fat-1轉基因動物相繼被研制成功,使這些轉基因動物的產(chǎn)品為人類提供必需的n-3 PUFAs成為了可能。隨著fat-1轉基因家畜的大量出生,涉及轉基因家畜的生物安全成為了人類倍受關注的問題,其中轉基因家畜自身健康是生物安全檢測的一項重要內容。截止目前,轉基因家畜自身健康和安全的評價雖已開展了一些研究,但多局限于動物健康的某個方面,系統(tǒng)而全面評價轉基因家畜自身健康的研究還未見報道。本研究利用體細胞核移植技術最終獲得了3頭成活的fat-1轉基因牛,高通量測序技術鑒定了其中一頭轉基因牛的fat-1因整合在16號染色體的15726078bp處。通過對3頭fat-1轉基因牛在血液生化水平、基因漂移、自身基因表達變化、血漿蛋白表達變化及腸道菌群變化結果的系統(tǒng)分析,發(fā)現(xiàn)fat-1基因的轉入對轉基因牛的脂類代謝、免疫、心血管系統(tǒng)、抗應激等方面均表現(xiàn)出調節(jié)作用。實驗結果對于建立轉基因家畜,特別是轉基因大家畜的自身安全評價體系,為獲得健康、安全的轉基因家畜的相關研究提供了有價值的參考資料。主要研究結果如下：1.fat-1轉基因牛的生產(chǎn)及常規(guī)分析通過轉基因及體細胞核移植技術(SCNT)獲得了9頭犢牛,6頭犢牛被鑒定為fat-1轉基因陽性牛,脂肪酸檢測證實了fat-1基因的轉入可以提升牛體內n-3PUFAs的含量,降低n-6 PUFAs的含量。血液生化水平檢測,發(fā)現(xiàn)fat-1基因的轉入顯著降低了犢牛ALT及成年牛AST、GLU、TC和LDL-C的水平。常規(guī)PCR檢測轉基因牛腸道糞便、圈舍中土壤及其周圍200 m各方位土壤微生物,均未發(fā)現(xiàn)有fat-1基因的存在。2.fat-1轉基因牛整合位點分析應用高通量測序技術對fat-1轉基因牛(FD006)進行了外源基因整合位點分析,結果表明fat-1基因整合在牛16號染色體的15726078bp處,且為單拷貝,根據(jù)測序獲得的插入位點及其附近reads的結果分析,顯示FD006為雜合子轉基因牛,PCR的驗證結果證實了fat-1基因確實插入了牛16號染色體的15726078 bp處,而且確實為雜合子轉基因牛。本研究為轉基因牛的整合位點分析、外源基因定點整合以及穩(wěn)定表達等研究提供了技術路線與理論依據(jù)。3.fat-1轉基因�；虮磉_變化研究為分析fat-1基因的轉入對牛基因表達的影響,提取fat-1轉基因牛和野生型牛血液的總RNA,進行基因表達譜芯片檢測。結果表明,有2042個基因表達存在顯著差異,其中797個基因在fat-1轉基因牛中是上調表達的,其余1245個基因則下調表達�；�2042個差異基因進行GO富集分析,發(fā)現(xiàn)90個GO Terms被顯著富集,這些GO Terms主要與機體的脂類代謝、細胞行為、免疫及神經(jīng)系統(tǒng)發(fā)育密切相關,其中8個GO Terms中包含了36個發(fā)生顯著變化的脂類代謝關鍵基因。KEG G富集分析進一步獲得了與脂類代謝,特別是與多不飽和脂肪酸的代謝密切相關的“PPAR signaling pathway”代謝通路。應用Real-time PCR對芯片檢測獲得的16個脂類代謝關鍵基因進行驗證,證實了芯片獲得的結果是可信的。綜合以上結果,發(fā)現(xiàn)fat-1基因的轉入引起了牛自身基因表達水平的變化,這些變化的基因在機體的脂類代謝、免疫、神經(jīng)發(fā)育等生物學通路表現(xiàn)出調節(jié)作用。4.fat-1轉基因牛血漿蛋白組學研究利用2D-雙向電泳及質譜檢測技術對fat-1轉基因牛血漿蛋白組學進行了分析。結果表明,共有15個血漿差異蛋白被鑒定；對這15個差異蛋白及其互作蛋白的GO和KEGG富集分析,發(fā)現(xiàn)這些蛋白主要參與了機體的脂類代謝、免疫、應激、神經(jīng)發(fā)育和血液凝固等生物學通路的調節(jié)；在18個重要的脂類代謝生物學通路中,有12個通路都富集到了APOA1,表明fat-1基因參與對脂類代謝的調控可能與APOA1有密切關系；血漿APOA1檢測發(fā)現(xiàn),轉基因牛血漿中APOA1含量顯著高于野生型牛,其表達水平與LDL-C高度負相關(r==一0.90),與HDL-C/TC比值存在顯著正相關(r=0.69)。綜合以上結果,與野生型牛比較發(fā)現(xiàn),fat-1基因的轉入使牛血漿中的一些蛋白表達發(fā)生了變化,并發(fā)現(xiàn)fat-1基因可能會介導APOA1的表達參與轉基因牛脂類代謝的調控。5.fat-1轉基因牛腸道微生物菌群的研究采用高通量測序技術,分別對3頭fat-1轉基因牛和3頭野生型牛的直腸糞便進行了基于16s rDNA V4可變區(qū)腸道菌群多樣性及組成的比較分析。結果顯示,轉基因牛和野生型牛共獲得9714個OTUs的分類,轉基因牛的OTUs分類(8907個)明顯少于野生型牛(9488個)；物種多樣性指數(shù)分析發(fā)現(xiàn),轉基因牛的Chao和Shannon指數(shù)均顯著低于野生型牛(p0.05)。進一步的分析發(fā)現(xiàn),fat-1基因的轉入也改變了牛腸道菌群的組成和表達豐度。其中物種注釋豐度比較發(fā)現(xiàn),轉基因牛與野生型牛間有3個門(廣古菌門、變形菌門、螺旋體門),9個屬(紫單胞菌屬、擬桿菌屬、甲烷短桿菌、密螺旋體屬、梭菌屬、毛螺菌屬、羅氏菌屬、消化球菌屬、丁酸弧菌屬)存在物種豐度差異(p0-05)。結合腸道菌群的組成、物種豐度比較結果及血糖、血脂生化檢測結果,發(fā)現(xiàn)Dorea、 Roseburia、Succinivibrio和Alistipes與血糖、血脂的變化存在一定的相關性。此外,也發(fā)現(xiàn)與應激有關的Odoribacte r菌屬在轉基因牛腸道中顯著降低。綜合本研究結果表明,fat-1基因的轉入改變了牛腸道菌群的多樣性、群落組成及表達豐度,發(fā)生變化的菌屬主要與宿主的糖、脂代謝以及機體的抗應激有關,推測fat-1基因的轉入可能會通過改變牛腸道菌群的組成或表達豐度參與機體脂類代謝調控的潛在機制。
[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.
【學位授予單位】：內蒙古大學
【學位級別】：博士
【學位授予年份】：2016
【分類號】：S823

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