本文選題：n-3多不飽和脂肪酸 + 體細(xì)胞核移植�。� 參考：《內(nèi)蒙古大學(xué)》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(guān),但人體自身不能合成n-3 PUFAs。自1997年,Spychalla等在線蟲(chóng)中獲得了一個(gè)脂肪酸脫氫酶基因fat-1,多種富含n-3 PUFAs的fat-1轉(zhuǎn)基因動(dòng)物相繼被研制成功,使這些轉(zhuǎn)基因動(dòng)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(zhuǎn)基因家畜的大量出生,涉及轉(zhuǎn)基因家畜的生物安全成為了人類倍受關(guān)注的問(wèn)題,其中轉(zhuǎn)基因家畜自身健康是生物安全檢測(cè)的一項(xiàng)重要內(nèi)容。截止目前,轉(zhuǎn)基因家畜自身健康和安全的評(píng)價(jià)雖已開(kāi)展了一些研究,但多局限于動(dòng)物健康的某個(gè)方面,系統(tǒng)而全面評(píng)價(jià)轉(zhuǎn)基因家畜自身健康的研究還未見(jiàn)報(bào)道。本研究利用體細(xì)胞核移植技術(shù)最終獲得了3頭成活的fat-1轉(zhuǎn)基因牛,高通量測(cè)序技術(shù)鑒定了其中一頭轉(zhuǎn)基因牛的fat-1因整合在16號(hào)染色體的15726078bp處。通過(guò)對(duì)3頭fat-1轉(zhuǎn)基因牛在血液生化水平、基因漂移、自身基因表達(dá)變化、血漿蛋白表達(dá)變化及腸道菌群變化結(jié)果的系統(tǒng)分析,發(fā)現(xiàn)fat-1基因的轉(zhuǎn)入對(duì)轉(zhuǎn)基因牛的脂類代謝、免疫、心血管系統(tǒng)、抗應(yīng)激等方面均表現(xiàn)出調(diào)節(jié)作用。實(shí)驗(yàn)結(jié)果對(duì)于建立轉(zhuǎn)基因家畜,特別是轉(zhuǎn)基因大家畜的自身安全評(píng)價(jià)體系,為獲得健康、安全的轉(zhuǎn)基因家畜的相關(guān)研究提供了有價(jià)值的參考資料。主要研究結(jié)果如下：1.fat-1轉(zhuǎn)基因牛的生產(chǎn)及常規(guī)分析通過(guò)轉(zhuǎn)基因及體細(xì)胞核移植技術(shù)(SCNT)獲得了9頭犢牛,6頭犢牛被鑒定為fat-1轉(zhuǎn)基因陽(yáng)性牛,脂肪酸檢測(cè)證實(shí)了fat-1基因的轉(zhuǎn)入可以提升牛體內(nèi)n-3PUFAs的含量,降低n-6 PUFAs的含量。血液生化水平檢測(cè),發(fā)現(xiàn)fat-1基因的轉(zhuǎn)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(cè)轉(zhuǎn)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(zhuǎn)基因牛整合位點(diǎn)分析應(yīng)用高通量測(cè)序技術(shù)對(duì)fat-1轉(zhuǎn)基因牛(FD006)進(jìn)行了外源基因整合位點(diǎn)分析,結(jié)果表明fat-1基因整合在牛16號(hào)染色體的15726078bp處,且為單拷貝,根據(jù)測(cè)序獲得的插入位點(diǎn)及其附近reads的結(jié)果分析,顯示FD006為雜合子轉(zhuǎn)基因牛,PCR的驗(yàn)證結(jié)果證實(shí)了fat-1基因確實(shí)插入了牛16號(hào)染色體的15726078 bp處,而且確實(shí)為雜合子轉(zhuǎn)基因牛。本研究為轉(zhuǎn)基因牛的整合位點(diǎn)分析、外源基因定點(diǎn)整合以及穩(wěn)定表達(dá)等研究提供了技術(shù)路線與理論依據(jù)。3.fat-1轉(zhuǎn)基因�；虮磉_(dá)變化研究為分析fat-1基因的轉(zhuǎn)入對(duì)牛基因表達(dá)的影響,提取fat-1轉(zhuǎn)基因牛和野生型牛血液的總RNA,進(jìn)行基因表達(dá)譜芯片檢測(cè)。結(jié)果表明,有2042個(gè)基因表達(dá)存在顯著差異,其中797個(gè)基因在fat-1轉(zhuǎn)基因牛中是上調(diào)表達(dá)的,其余1245個(gè)基因則下調(diào)表達(dá)�；�2042個(gè)差異基因進(jìn)行GO富集分析,發(fā)現(xiàn)90個(gè)GO Terms被顯著富集,這些GO Terms主要與機(jī)體的脂類代謝、細(xì)胞行為、免疫及神經(jīng)系統(tǒng)發(fā)育密切相關(guān),其中8個(gè)GO Terms中包含了36個(gè)發(fā)生顯著變化的脂類代謝關(guān)鍵基因。KEG G富集分析進(jìn)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(guān)的“PPAR signaling pathway”代謝通路。應(yīng)用Real-time PCR對(duì)芯片檢測(cè)獲得的16個(gè)脂類代謝關(guān)鍵基因進(jìn)行驗(yàn)證,證實(shí)了芯片獲得的結(jié)果是可信的。綜合以上結(jié)果,發(fā)現(xiàn)fat-1基因的轉(zhuǎn)入引起了牛自身基因表達(dá)水平的變化,這些變化的基因在機(jī)體的脂類代謝、免疫、神經(jīng)發(fā)育等生物學(xué)通路表現(xiàn)出調(diào)節(jié)作用。4.fat-1轉(zhuǎn)基因牛血漿蛋白組學(xué)研究利用2D-雙向電泳及質(zhì)譜檢測(cè)技術(shù)對(duì)fat-1轉(zhuǎn)基因牛血漿蛋白組學(xué)進(jìn)行了分析。結(jié)果表明,共有15個(gè)血漿差異蛋白被鑒定；對(duì)這15個(gè)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(jī)體的脂類代謝、免疫、應(yīng)激、神經(jīng)發(fā)育和血液凝固等生物學(xué)通路的調(diào)節(jié)；在18個(gè)重要的脂類代謝生物學(xué)通路中,有12個(gè)通路都富集到了APOA1,表明fat-1基因參與對(duì)脂類代謝的調(diào)控可能與APOA1有密切關(guān)系；血漿APOA1檢測(cè)發(fā)現(xiàn),轉(zhuǎn)基因牛血漿中APOA1含量顯著高于野生型牛,其表達(dá)水平與LDL-C高度負(fù)相關(guān)(r==一0.90),與HDL-C/TC比值存在顯著正相關(guān)(r=0.69)。綜合以上結(jié)果,與野生型牛比較發(fā)現(xiàn),fat-1基因的轉(zhuǎn)入使牛血漿中的一些蛋白表達(dá)發(fā)生了變化,并發(fā)現(xiàn)fat-1基因可能會(huì)介導(dǎo)APOA1的表達(dá)參與轉(zhuǎn)基因牛脂類代謝的調(diào)控。5.fat-1轉(zhuǎn)基因牛腸道微生物菌群的研究采用高通量測(cè)序技術(shù),分別對(duì)3頭fat-1轉(zhuǎn)基因牛和3頭野生型牛的直腸糞便進(jìn)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(jié)果顯示,轉(zhuǎn)基因牛和野生型牛共獲得9714個(gè)OTUs的分類,轉(zhuǎn)基因牛的OTUs分類(8907個(gè))明顯少于野生型牛(9488個(gè))；物種多樣性指數(shù)分析發(fā)現(xiàn),轉(zhuǎn)基因牛的Chao和Shannon指數(shù)均顯著低于野生型牛(p0.05)。進(jìn)一步的分析發(fā)現(xiàn),fat-1基因的轉(zhuǎn)入也改變了牛腸道菌群的組成和表達(dá)豐度。其中物種注釋豐度比較發(fā)現(xiàn),轉(zhuǎn)基因牛與野生型牛間有3個(gè)門(廣古菌門、變形菌門、螺旋體門),9個(gè)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(jié)合腸道菌群的組成、物種豐度比較結(jié)果及血糖、血脂生化檢測(cè)結(jié)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(guān)性。此外,也發(fā)現(xiàn)與應(yīng)激有關(guān)的Odoribacte r菌屬在轉(zhuǎn)基因牛腸道中顯著降低。綜合本研究結(jié)果表明,fat-1基因的轉(zhuǎn)入改變了牛腸道菌群的多樣性、群落組成及表達(dá)豐度,發(fā)生變化的菌屬主要與宿主的糖、脂代謝以及機(jī)體的抗應(yīng)激有關(guān),推測(cè)fat-1基因的轉(zhuǎn)入可能會(huì)通過(guò)改變牛腸道菌群的組成或表達(dá)豐度參與機(jī)體脂類代謝調(diào)控的潛在機(jī)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(xué)位授予單位】：內(nèi)蒙古大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2016
【分類號(hào)】：S823

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