【作者】 朱贵明；

【导师】 陈宏； 邓继先；

【作者基本信息】 西北农林科技大学 ， 动物遗传育种与繁殖， 2006， 博士

【摘要】 多不饱和脂肪酸(PUFAs)是一类被广泛研究和关注的脂肪酸。PUFAs主要包括ω-6和ω-3 PUFAs两种类型,每一种类型都包括多个从短碳链(18 C)到长碳链(22 C)的、且含有多个不饱和键的脂肪酸。越来越多的研究证明,PUFAs具有广泛的生物学功能,它们参与细胞膜的构成并作为许多细胞应答过程中的信号分子,与人类的多种疾病的发生紧密相关,其适宜的含量对于人类及其他哺乳动物的正常发育和保持良好的健康状况极其重要。从总体上来说,ω-6 PUFAs对人体意义不大,其过量的摄入反而会危害人体健康。而ω-3 PUFAs对人体却具有积极的意义,已被证实在预防和治疗心血管疾病、关节炎、癌症等多种疾病方面发挥着重要作用。由于哺乳动物及人类都不能在体内合成ω-3 PUFAs与ω-6PUFAs,因此必须从食物中摄取。但自然界中ω-6 PUFAs含量丰富而ω-3 PUFAs含量极少,导致人体中ω-6 PUFAs摄入过多而ω-3 PUFAs摄入严重不足,使人体中ω-6/ω-3之比高达16以上。因此,如何使人体增加ω-3 PUFAs的摄入量就成了合理利用脂肪酸的关键,也是当今世界人们所关心的一个问题。基因工程技术与转基因动物技术的快速发展,为体外生产更多的ω-3 PUFAs提供了新的契机,也逐渐成为科学家们努力的方向之一。ω-3脂肪酸去饱和酶基因是ω-3 PUFAs合成的关键基因,它们利用ω-6 PUFAs为底物,在脂肪酸碳链甲基端第三个碳催化生成一个不饱和键(烯键),从而合成相应的ω-3 PUFAs。近年来,各国科学家克隆了多个ω-3脂肪酸去饱和酶基因,但其中大部分基因只能合成18 C的ω-3 PUFAs,开发利用价值不大。虽然也有报道能够合成更长链的ω-3 PUFAs基因,如真菌Saprolegnia diclina的ω-3脂肪酸去饱和酶能够合成20 C的ω-3 PUFAs,另一种真菌Mortierella alpina 1S-4不但能合成20 C的ω-3 PUFAs,而且能合成18 C的ω-3 PUFAs,但迄今尚未见这些基因用于基因工程或转基因动物中生产ω-3 PUFAs的报道。真正用于此类研究的基因是来自线虫C.elegans的ω-3脂肪酸去饱和酶基因fat-1。该基因转入哺乳动物细胞及小鼠后能使从18 C到22 C的ω-3 PUFAs的含量增加,显示出很大的开发利用价值。但美中不足的是,所合成的ω-3 PUFAs中价值更大的22 C的ω-3 PUFA(s如docosapentaenoic acid,DPA和docosahexaenoic acid,DHA)水平仍然很低。因此,能否找到在转基因动物体内广泛表达各种ω-3 PUFAs,且其DPA和DHA的水平更高的类似fat-1基因,则成为该问题的关键。本研究通过GenBank检索和文献调研,发现线虫C.Briggsae具有类似fat-1基因的基因组序列(为C.Briggsae基因组的测序结果,GenBank登录号为CAAC01000009),其氨基酸编码较C.elegans少了两个氨基酸。二者的核苷酸序列同源性为71.5%,氨基酸同源性为86.1%,未见其进一步的研究报道。本研究建立了一种新的基因合成方法,即用PCR法结合酶切连接法拼接化学合成的寡聚核苷酸来合成线虫C.Briggsae具有类似fat-1基因的全序列,并把合成的该基因命名为sFat-1。整个过程仅用了3轮PCR(共7个反应)、2轮酶切连接(3个反应),便完成了1226 bp的sFat-1基因的克隆。基因克隆前经过密码子优化设计,以便更适合在哺乳动物中表达。将构建的哺乳动物细胞表达载体pcDNA3.1-sFat1-EGFP通过脂质体转染CHO细胞系并对其进行抗性筛选,获得了稳定转染的细胞株。对稳定转染sFat-1细胞株的RT-PCR分析及脂肪酸组成的气相色谱-质谱联用(GC-MS)的分析表明,sFat-1基因完全能在CHO细胞中表达并发挥其ω-3去饱和酶的作用,即催化ω-6系列不饱和脂肪酸转变为相应的ω-3系列不饱和脂肪酸。ω-6不饱和脂肪酸总量从48.97%(转染GFP的CHO细胞)下降到35.29%(转染sFat-1的CHO细胞),而ω-3不饱和脂肪酸总量则相应地从7.86%上升到24.02%。ω-6多不饱和脂肪酸和ω-3多不饱和脂肪酸的比值从正常细胞中的6.23下降到转染细胞中的1.47。这说明线虫C.Briggsae的ω-3脂肪酸去饱和酶基因sFat-1的克隆是成功的。更重要的是,与已有的研究结果相比,sFat-1基因转移到哺乳动物细胞CHO中可以产生更高含量的DPA(5.98%)和DHA(1.97%),初步证明该基因可能更有利于更长链的ω-3系列不饱和脂肪酸的合成。将载体pcDNA3.1-sFat1-EGFP作为转基因动物表达载体,通过显微注射法制备转基因小鼠,在出生的33只后代小鼠中,经PCR和Southern blot鉴定,最终获得5只转基因小鼠。经对后代小鼠的检测,证明其多种组织及乳汁中合成了LA(linolenic acid , 18:3n-3)、EPA(eicosapentaenoic acid, 20:5n-3)、DPA(docosapentaenoic acid, 22:5n-3)、DHA (docosahexaenoic acid, 22:6n-3)等各种ω-3 PUFAs。所检出的ω-3 PUFAs的种类与CHO细胞转染的情况类似,但转基因小鼠各种组织都表达和产生了极为丰富的更长链的DHA和DPA。在肌肉组织中DHA平均含量高达12.15%,ω-3 PUFAs总含量平均高达23.04%。这些数据远远超出了C.elegans的ω-3去饱和酶基因fat-1在一系列转基因试验的结果。这再次表明本研究所合成的sFat-1基因能够在转基因动物中产生更多、更长链的、且更有价值的DHA以及DPA。此外,本研究还构建了乳腺特异性表达载体pBC1-sFat1,并通过同样的显微注射法获得了5只转基因小鼠(共生了24只小鼠),对后代检测发现,其中1只转基因小鼠的乳汁中表达了ω-3 PUFAs产物,且略高于周身性表达的转基因小鼠乳腺中所合成的各种ω-3 PUFAs。这为sFat-1在牛、羊等家畜上利用乳腺特异性表达载体表达ω-3 PUFAs奠定了实验基础。最后,本研究还尝试了利用慢病毒载体法生产转基因小鼠的可行性。以sFat-1为目的基因构建了慢病毒表达载体及其完整表达系统,通过转染293FT细胞生产慢病毒颗粒,病毒滴度约为5.0×106 TU/mL,经感染NIH3T3细胞系,表明该慢病毒对哺乳动物细胞具有良好的感染能力。再用该慢病毒感染小鼠早期胚胎,结果表明,所获得的慢病毒颗粒完全可以侵染小鼠胚胎。该研究初步证实利用慢病毒载体法制备转基因动物是完全可行的,为sFat-1走向实际应用提供了科学依据。更多还原

【Abstract】 Polyunsaturated fatty acids (PUFAs) have been broadly investigated and have been paid much attention to by the public. PUFAs mainly includes two groups,ω-3 PUFAs andω-6 PUFAs, each also includes many fatty acids from 18 carbons to 22 carbons with two or more unsaturated bonds in their chains. More and more studies shows that PUFAs have broad biological functions, serving as structural components of membrane phospholipids and as precursors of the eicosanoids of signaling molecules. They closely related with many human being’s diseases, so their proper levels of content in human bodies is very important for human health. As a whole,ω-6 PUFAs is not crucial for human beings. Contrarily, their excess will bring many healthy problems. Butω-3 PUFAs are beneficial PUFAs, which have been proved to exert many preventive and therapeutic actions on many diseases such as cardiovascular diseases, arthritis, cancer and neuropathic diseases. Human and mammals can not synthesizeω-3 PUFAs in their bodies(so as toω-6 PUFAs), so the PUFA need is, to a great extent, dependent on dietary intake. However the source ofω-6 PUFAs is abundant but that ofω-3 PUFAs is very limited, which lead to the level ofω-3 PUFAs in mammals is relatively too low compared withω-6 PUFAs, the ratio ofω-6/ω-3 PUFAs as high as 16 or more. Therefore, the only thing need to solve is to increase the level ofω-3 PUFAs content in human’s rational utilizing of fatty acids, and how to obtain abundant source ofω-3 PUFAs is a focus in the world. Production ofω-3 PUFAs in transgenic animals by gene engineering is becoming the direction for researchers.ω-3 fatty acids desaturase play important role in the synthesis ofω-3 PUFAs , which desaturateω-6 PUFAs into theω-3 PUFA. In recent years, researchers cloned a lot ofω-3 fatty acids desaturase gene from many kinds of organisms. But most of this genes only can synthesize 18-carbonω-3 PUFAs from 18 carbonω-6 PUFAs , so they seems to have no great value of applications. A few genes reported can desaturase longer carbonω-6 PUFAs intoω-3 PUFAs, such as a fungusω-3 desaturase gene from Saprolegnia diclina, desaturates 20-carbonω-6 PUFAs, another fungusω-3desaturase gene from Mortierella alpina 1S-4, desaturates both 18-carbon and 20-carbonω-6 PUFAs. However, these genes have not yet been reported to generate transgenic animals for productionω-3 PUFAs. The only gene used in transgenic animals generation is fat-1 gene, anω-3 desaturase gene from Caenorhabditis elegans, which have been transferred into mammalian cells and mice and as a result, produced abundantω-3 PUFAs, from 18-carbon to 22-carbon. Data show that this gene have great potential for productionω-3 PUFAs. But the level of 22-carbonω-3 PUFAs produced from fat-1 transgenic animals is relatively very low. So the genes that can produce high level of 22-carbonω-3 PUFAs in transgenic animals will be much better for this fields of research.From GenBank search and the published articles, we found that the gene sequence from the worm C.Briggsae (as a result of gene sequencing, the GenBank accession number is CAAC01000009 ) is similar to fat-1 gene, with two amino acids missing compared to the latter. This gene sequence alignment with fat-1 gene revealed 71.5 % gene sequence identity and 86.1% amino acids similarity, and the further study of this gene is not reported. This study established a new method, which was referred to as PCR-restrict enzyme ligation method, to synthesize this Caenorhabditis briggssae gene .By this method, the gene (named as sFat-1) is successfully synthesized following 3 rounds of PCR (7 reactions) and 2 rounds of restrict enzyme ligation (3 reactions). In order to express this worm gene in mammals, its codons were modified and optimized for mammalian gene expression.The synthesized gene was constructed as a mammal expression vector pcDNA3.1-sFat1-EGFP. This vector was introduced into CHO cells by lipid-mediated transfection, and it’s expression quickly and effectively elevate the cellularω-3 PUFA contents and dramatically balance the ratio ofω-6/ω-3 PUFAs as GC-MS analysis of cellular lipids extracted from stably selected cells showed. The amount of totalω-6 PUFAs dropped from 48.97% (in GFP cells)to 35.29%(in sFat-1 transferred cells),whereas the amount of totalω-3 PUFAs increased from 7.86% to 24.02% respectively. Theω-6/ω-3 ratio also dropped from 6.23 to 1.47 accordingly. These data demonstrates the Caenorhabditis briggssae sFat1 was synthesized successfully and can be used for further study. What is important is that this gene transferred into CHO cells produced higher level of DPA (5.98%) and DHA(1.97%) compared to the former similar research with fat-1 gene.Then we use vector pcDNA3.1-sFat1-EGFP as transgenic vector to produce transgenic mice. The DNA was microinjected into mouse zygote and transferred into recipients, at last 33 pups born. Analysis by PCR and southern blot, five of them are transgenic. GC-MS analysis results show that many tissues of these transgenic mice produced all kinds ofω-3 PUFAs, such as LA(18:3n-3)、EPA(20:5n-3)、DPA(22:5n-3) and DHA(22:6n-3), which similar with that of in sFat-1 gene transferred CHO cells. But these mice produced higher level of DHA and DPA. For example, the tissue of the transgenic mice muscle produced 12.5% of DHA( averagely) and 23.04% of totalω-3 PUFAs, all of which far exceed the former studies ofω-3 desaturase transgenic anmals. Our research also produced transgenic mice for mammary expression of the sFat-1 gene by microinjecting the constructed vector of pBC1-sFat1, 5 from 24 born pups are transgenic. GC-MS analysis results show that only 1 of them expressed the sFat-1 gene in the milk (other tissues not detected).Moreover, the expression level slightly higher than that in the milk of pcDNA3.1-sFat1-EGFP transgenic mice. This shows it is possible for mammary expression of the sFat-1 gene in domestic animals to produceω-3 PUFAs.Lentiviral vectors are high efficient vectors for gene transfer. In order to investigate their potential for production of transgenic animals, we constructed a lentiviral vector for the expression of sFat-1,a gene from C.Briggsae encodingω-3 Fatty Acid Desaturase. By co-transfection of pLP1、pLP2、pLP/VSVG with this vector, 293 FT cells produced lentiviral particle, which can infect mammalian cells(tested by NIH3T3 cell line). The tilter estimated to about 5.0×106TU/ml. Also, the lentiviral particle proved can infect embryos when co-cultured with mouse embryos. This research means a further step to production lentiviral transgenic animal expression a functional gene for actual use.更多还原