鸡△~6-脂肪酸脱氢酶基因的克隆及其在不同组织中的表达
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摘要
多不饱和脂肪酸(PUFA)在机体内具有一系列重要的生理功能。Δ~6-脂肪酸脱氢酶则是形成多不饱和脂肪酸的限速酶。基于Δ~6-脂肪酸脱氢酶在多不饱和脂肪酸形成过程中的重要作用,本实验开展了对固始鸡Δ~6-脂肪酸脱氢酶基因的研究工作。本实验通过两次RT-PCR和一次3,RACE,获得了全长为2141bp的cDNA,该cDNA具有一个长度为1335bp、编码443个氨基酸的开放阅读框。这是Δ~6-脂肪酸脱氢酶基因首次在鸡上的成功克隆。以得到的鸡的Δ~6-脂肪酸脱氢酶基因序列为搜索对象,通过Contig分析,获得了鸡Δ~6-脂肪酸脱氢酶基因编码序列的内含子和外显子边界,序列分析表明该基因含有12个外显子和11个内含子。
通过半定量PCR,比较脂肪酸脱氢酶在不同组织中的表达,发现在肝脏中表达量最大。
该试验为进一步研究该基因在脂肪酸形成过程中的作用奠定了基础。
本研究共分两个部分:
试验一Δ~6-脂肪酸脱氢酶基因的克隆
1Δ~6-脂肪酸脱氢酶基因部分编码序列的克隆根据GenBank上预测的鸡的Δ~6-脂肪酸脱氢酶基因序列XM421053设计引物,进行RT-PCR,扩增出一个长度为1323bp的片段(D6D1)。该序列与人、大鼠、小鼠、牛等动物的核苷酸同源性达到74%以上。
2Δ~6-脂肪酸脱氢酶基因5,端的克隆将已获得的部分编码序列作为搜索对象,在NCBI网站进行Blast,获得了一段鸡EST(BI390105)。该序列与上述克隆的部分编码序列的5,端有一部分重复序列。根据该EST设计基因特异性引物进行5,端的扩增。获得了一个283bp的片段(D6D2)。该片段与部分编码序列有99bp的重叠区,表明所得到的片段为同一基因的部分序列。
3、Δ~6-脂肪酸脱氢酶基因的3,UTR的克隆为了获得固始鸡Δ~6-脂肪酸脱氢酶基因的3,非翻译区端,根据所获得的部分编码序列设计基因特异性引物,利用3,RACE试剂盒进行3,末端cDNA的扩增,得到一个706bp的片段(D6D3)。
该片段与部分编码序列的3,端有36bp的重叠区。
通过DNAMAN将三个克隆片段连接,得到了2141bp的cDNA全长。
试验二、利用半定量PCR检测鸡△6-脂肪酸脱氢酶在不同组织中的表达为了检测△6-脂肪酸脱氢酶在不同组织中的表达,利用半定量PCR检测△6-脂肪酸脱氢酶在肝脏、心脏、肺脏等组织中的mRNA的相对含量。结果表明,肝脏、脾脏、胰脏、肺脏中的Δ~6-脂肪酸脱氢酶mRNA相对含量差异不显著;肝脏显著高于胸肌;胸肌、心脏和腹脂的差异不显著;肺脏显著高于腹脂。而Δ~6-脂肪酸脱氢酶在肌胃中未见有表达。
鸡Δ~6-脂肪酸脱氢酶基因的克隆及在不同组织中的表达为进一步研究Δ~6-脂肪酸脱氢酶在多不饱和脂肪酸的形成过程中的作用奠定了一定的基础。
LC-PUFA play an important role in the body.Δ~6-desaturase is the limited enzyme in the formation of PUFA.Because of the important role in the formation of polyunsaturated fatty acids.In this experiment we carried out chickenΔ~6-desaturase research. A 2141bp cDNA was obtained by two RT-PCR and 3,RACE.Sequence analysis showed this cDNA sequence han an open reading frame(ORF) of 1335bp coding 444 amino acids .This is the first successful cloning of chickenΔ~6-desaturase.Intron and exon boundaries of coding sequence was obtained by sequence analysis.This gene includs 12 exons and 11 introns.
Through the semi-quantitative PCR, we compared fatty acid dehydrogenase in differ -ent tissues, we found expression ofΔ~6-desaturase in the liver is the most.
This experiment lay the foundation for further study of the gene in the formation of polyunsaturated fatty acids.
This study is divided into two parts :
一、The cloning of chickenΔ~6-desaturase
1、The cloning of partial code ofΔ~6-desaturase A 1323 bp DNA fragment was amplified from Gushi chicken based on the sequence information for predicted delta-6 fatty acid desaturase of gallus(XM421053) by RT-PCR (GenBank Accession: EF636888). The base sequence comparability was beyond 74%.
2、The cloning of 5,end ofΔ~6-desaturase
Using the nucleotide sequence of partial CDS,Blast on the NCBI,we find a EST(BI390105).This EST is partial lapped with partial CDS in the 5,end. According to the EST designing gene-specific primers for 5,end of the amplification,We gained a 283bp nucleotide(D6D2). The fragment is overlapped 99bp with partial CDS. It received the gene fragment was part of the same sequence.
3、The cloning of 3,UTR ofΔ~6-desaturase
In order to obtain the 3’untranslated region ofΔ~6-desaturase,we designed gene-specific primers for the 3,untranslated region amplifacation by using 3, RACE kit.We obtained a 705bp nucleotide(D6D3) finally. The fragment is overlapped 36bp with partial CDS.
A 2153bp cDNA was obtained through DNAMAN linking.
二、The detection for expression of chickenΔ~6-desaturase gene in different tissues by semi-quantitative PCR
In order to detect the expression of chickenΔ~6-desaturase gene in different tissues,we detected the mRNA relative content ofΔ~6-desaturase in liver,heart and lung etc.The result showed that the defference of theΔ~6-desaturase mRNA relative content of liver ,spleen, pancreas and lungs was not significant;liver was higher significantly than breast;the difference of breast,cardiac and abdominal fat was not significant; the lung was significantly higher than that of the bodiminal fat .we didn,t detectΔ~6-desaturase in the stomach muscle.
通过半定量PCR,比较脂肪酸脱氢酶在不同组织中的表达,发现在肝脏中表达量最大。
该试验为进一步研究该基因在脂肪酸形成过程中的作用奠定了基础。
本研究共分两个部分:
试验一Δ~6-脂肪酸脱氢酶基因的克隆
1Δ~6-脂肪酸脱氢酶基因部分编码序列的克隆根据GenBank上预测的鸡的Δ~6-脂肪酸脱氢酶基因序列XM421053设计引物,进行RT-PCR,扩增出一个长度为1323bp的片段(D6D1)。该序列与人、大鼠、小鼠、牛等动物的核苷酸同源性达到74%以上。
2Δ~6-脂肪酸脱氢酶基因5,端的克隆将已获得的部分编码序列作为搜索对象,在NCBI网站进行Blast,获得了一段鸡EST(BI390105)。该序列与上述克隆的部分编码序列的5,端有一部分重复序列。根据该EST设计基因特异性引物进行5,端的扩增。获得了一个283bp的片段(D6D2)。该片段与部分编码序列有99bp的重叠区,表明所得到的片段为同一基因的部分序列。
3、Δ~6-脂肪酸脱氢酶基因的3,UTR的克隆为了获得固始鸡Δ~6-脂肪酸脱氢酶基因的3,非翻译区端,根据所获得的部分编码序列设计基因特异性引物,利用3,RACE试剂盒进行3,末端cDNA的扩增,得到一个706bp的片段(D6D3)。
该片段与部分编码序列的3,端有36bp的重叠区。
通过DNAMAN将三个克隆片段连接,得到了2141bp的cDNA全长。
试验二、利用半定量PCR检测鸡△6-脂肪酸脱氢酶在不同组织中的表达为了检测△6-脂肪酸脱氢酶在不同组织中的表达,利用半定量PCR检测△6-脂肪酸脱氢酶在肝脏、心脏、肺脏等组织中的mRNA的相对含量。结果表明,肝脏、脾脏、胰脏、肺脏中的Δ~6-脂肪酸脱氢酶mRNA相对含量差异不显著;肝脏显著高于胸肌;胸肌、心脏和腹脂的差异不显著;肺脏显著高于腹脂。而Δ~6-脂肪酸脱氢酶在肌胃中未见有表达。
鸡Δ~6-脂肪酸脱氢酶基因的克隆及在不同组织中的表达为进一步研究Δ~6-脂肪酸脱氢酶在多不饱和脂肪酸的形成过程中的作用奠定了一定的基础。
LC-PUFA play an important role in the body.Δ~6-desaturase is the limited enzyme in the formation of PUFA.Because of the important role in the formation of polyunsaturated fatty acids.In this experiment we carried out chickenΔ~6-desaturase research. A 2141bp cDNA was obtained by two RT-PCR and 3,RACE.Sequence analysis showed this cDNA sequence han an open reading frame(ORF) of 1335bp coding 444 amino acids .This is the first successful cloning of chickenΔ~6-desaturase.Intron and exon boundaries of coding sequence was obtained by sequence analysis.This gene includs 12 exons and 11 introns.
Through the semi-quantitative PCR, we compared fatty acid dehydrogenase in differ -ent tissues, we found expression ofΔ~6-desaturase in the liver is the most.
This experiment lay the foundation for further study of the gene in the formation of polyunsaturated fatty acids.
This study is divided into two parts :
一、The cloning of chickenΔ~6-desaturase
1、The cloning of partial code ofΔ~6-desaturase A 1323 bp DNA fragment was amplified from Gushi chicken based on the sequence information for predicted delta-6 fatty acid desaturase of gallus(XM421053) by RT-PCR (GenBank Accession: EF636888). The base sequence comparability was beyond 74%.
2、The cloning of 5,end ofΔ~6-desaturase
Using the nucleotide sequence of partial CDS,Blast on the NCBI,we find a EST(BI390105).This EST is partial lapped with partial CDS in the 5,end. According to the EST designing gene-specific primers for 5,end of the amplification,We gained a 283bp nucleotide(D6D2). The fragment is overlapped 99bp with partial CDS. It received the gene fragment was part of the same sequence.
3、The cloning of 3,UTR ofΔ~6-desaturase
In order to obtain the 3’untranslated region ofΔ~6-desaturase,we designed gene-specific primers for the 3,untranslated region amplifacation by using 3, RACE kit.We obtained a 705bp nucleotide(D6D3) finally. The fragment is overlapped 36bp with partial CDS.
A 2153bp cDNA was obtained through DNAMAN linking.
二、The detection for expression of chickenΔ~6-desaturase gene in different tissues by semi-quantitative PCR
In order to detect the expression of chickenΔ~6-desaturase gene in different tissues,we detected the mRNA relative content ofΔ~6-desaturase in liver,heart and lung etc.The result showed that the defference of theΔ~6-desaturase mRNA relative content of liver ,spleen, pancreas and lungs was not significant;liver was higher significantly than breast;the difference of breast,cardiac and abdominal fat was not significant; the lung was significantly higher than that of the bodiminal fat .we didn,t detectΔ~6-desaturase in the stomach muscle.
引文
[1]齐光海.家禽多不饱和脂肪酸营养作用的研究进展[J](上).国外畜牧科技,1994,21 (4):11-14
[2]袁书林,金荣斌,王宵燕.必须脂肪酸营养研究进展[J].养殖与饲料, 2002,(5):3-5
[3]Simopoulos,A. P. and N. Salem, Jr., N-3 fatty acids in eggs from range-fed Greek chickens. New Engh. J. Med,1989,321:1412.
[4] Simopoulos,A. P., and N.Salem, Jr. Egg yolk as a source of long-chain polyunsaturated fatty acids in infant feeding. Am. J.Clin. Nutr,1992, 55: 411-414
[5] Meluzzi, A., F.Sirri,G.Manfreda, N.Tallarico, and A.Franchini, Effect of dietary vitamin E on the quality of table eggs enriched with n-3 long-chain fatty acids[J]. Poul.Sci,2000,79:539-545
[6] Farrell.UNE,S designer egg [J].Poultry International,1993,32(5):62-66.
[7]Hulan HW,Ackman RG,Ratnayake WM,Proudfoot FG.Omega-3 fatty acid levels and general performance of commercial broilers fed practical levels of redfish meal.Poult Sci.1989,Jan;68(1):153-162.
[8]Rule D C,Broughton K S,Shellito S M.et al.Comparison of muscle fatty acid profiles and cholesterol concentrations of bison,beef cattle,elk,and chicken[J].J Anim Sci,2002,80:1202-1211.
[9]Effects of dietary carbohydrate on delayed onset muscle soreness and reactive oxygen species after contraction induced muscle damage.Close GL,Ashton T,Cable T etc.Br J Sports Med.2005,39(12):948-953.
[10]Migdal W and Kacamarczyk J,Effect of fish oils in the diets for sows on chemical composition of milk and results of piglet managenment.Acta Agraria et Silveslria. 1991,29:47-58.
[11]余文三,多不饱和脂肪酸的研究概况[J].国外医学卫生学分册.1998,25(6):359-367
[11].Uauy R,Mena P,Rajas C.Essential fatty acids in early life:structural and functional role [J].Proc Nutr Soc,2000,59(1):3-15.
[13] Carlson S E, Werkman S H, Rhodes P G, et al. Visual- acuity development in healthy preterm infants: effect of marine-oil supplementation [J].Am. J. Clin. Nutr., 1993,58(1),35-42.
[14].Agostoni C, Massetto N, Biasucci G, et al.Effects of long-chain polyunasturated fatty acid supplementation on fatty acid status and visual function in treated children with hyperphenylalaninemia[J].J Pediatr,2000,137(4):504-509.
[15].蔡双莲,李敏.多不饱和脂肪酸的研究进展[J].生命科学研究.2003,7(4):289-394.
[16]ORDART H,GROSCOLAS R,CALGARI C,et al.Brown fat thermogernesis in rats fed high-fat diets enriched with n-polyunsaturated fatty acids[J].International Jovrnal of Obesity.1997,21:955-962.
[17]颜新春,汪以真。动物脂肪酸合成酶(FAS)基因表达的调控[J].动物营养学报,2002,14(2):1-4.
[18]Flick PK,Chen J,Vagelos PR. Effect of dietary linoleate on synthesis and degradation of fatty acid synthetase from rat liver[J]. J Biol Chem. 1977 Jun 25;252(12):4242-4249.
[19] James GW,Church MK. Hyperalgesia after treatment of mice with prostaglandins and arachido -nic acid and its antagonism by anti-inflammatory-analgesic compounds. Arzneimittelforschung. 1978: 28(5):804-807.
[20]Coordinate suppression of liver acetyl-CoA carboxylase and fatty acid synthetase by polyunsatura -ted fat.Toussant MJ,Wilson MD,Clarke SD.J Nutr.1981,111(1):146-153.
[21] Tomlinson JE,Nakayama R,Holten D. Repression of pentose phosphate pathway dehydrogenase synthesis and mRNA by dietary fat in rats. J Nutr. 1988;118(3):408-15.
[22]Kobatake K,K Sako,Mizawa,et al.Autoradiographic determination of brain pH following middle cerebral arteryocclusion in therat[J].Stroke,1984,15:540-547.
[23]周兴,林成仁,饶琳,王敏.n- 3多烯脂肪酸对血脂影响的实验研究[J].中国中医基础医学杂志,1997,3(4): 29- 31.
[24] Kobatake Y,Kuroda K,Jinnorchi H,Nishide E,Innami S.et al. Differential effects of dietary eicosa -pentaenoic and docosahexaenoic fatty acids on lowering of triglyceride and cholesterol levels in the serum of rats on hypercholesterolemic diet[J] Nutr Sci,1984,30:357-372.
[25]Penny Etherton.ω-3脂肪酸的研究进展[J].泰山卫生。2002,26(3):55-56.
[26] Arrington JL, Chapkin RS, Switzer KC, et al. Dietary n-3 polyunsaturated fatty acidsmodulate purified murine T-cell subset activation[ J ]. Clin Exp Immunol, 2001 , 125 (3) : 499-507.
[27] Arrington JL, McMurrayDN, Switzer KC, et al. Docosahexaenoic acid suppresses function of the CD-8 costimulatory membrane receptor in p rimary murine and J urkat T cells[ J ] . J Nutr, 2001,131 (4) : 1147-1153.
[28] McMurray DN, Jolly CA, Chapkin RS. Effects of dietary n-3 fatty acids on T cell activation and T cell recep tormediated signaling in a murine model[ J ] . J InfectDis, 2000, 182 :S103-S107.
[29] Pompos LJ ,Fritsche KL.Antigen2driven murine CD4 + T lymphocyte proliferation and interleuk -in-2 p roduction are diminished by dietary ( n-3) polyunsaturated fatty acids[ J ]. J Nutr, 2002,132 (11) : 3293-3300.
[30]Calder P C, Field C J. Fatty acids, inflammation and immunity∥Calder P C, Field C J, Gill H S. Nutrition and Immune Function[C ]. CAB International, 2002: 57-92
[31] KELLY V E,FERRETTO A,IZUI S,et al. A fish oil diet rich in eicosapentaenoic acid reduces cyclooxygenase metabolites, and suppresses lupus in MRL-I pr mice[J].J Immunol,1985 (134): 1914 -1919.
[32] PRICKEIT J D,ROBINSON D R,STEINBERG A D. Dietary enrichment with the polyunsaturate -d fatty acid eicosapentaenoic prevents proteinuria and prolongs survival in (NZB×NZW)F1 mice[J].J Clin Invest,1981(68):556-559.
[33] LESLIE C A,GONNERMAN W ULLMAN M D, et al.Dietary fish oil modulates macrophage fatty acids and decreases arthritis susceptibilily in mice[J]. J Exp Med, 1985(162): 1336-1349.
[34] CATHCART E S,MORTENSEN R F,LESLIE C A,et al. A fish oil diet inhibits amyloid P compo -nent (AP)acute phase responses in arthritis susceptible mice[J]. J Immuno1,1987(139):89-91.
[35] BOLCK K J,HO B,XU L L,et al. Effect of fish-fat or beef-fat supplemented diet on immune complex-inducedenteropathy in the rat[J]. Prostaglandins,1989(38):385-396.
[36]王清华,贺永惠,郑素玲. n一多不饱和脂肪酸对动物炎症·免疫和细胞因子的调节作用安徽农业科学,Journal of Anhui Agr.Sci. 2006,34(14):3367-3370.
[37]James MJ,Gibson RA,Cleland L G.Dietary polyunsaturated fatty acids and inflammatory mediator production[J]. Am J Clin Nutr,2000,71(1):343.
[39]Xiao Y-F,Gomez AM,Morgan JP,et al.Suppression of voltage-gated L-typeCa2+ currents By poluunasturated fatty acid in adult and neonatal rat ventricular myocytes[J].Proc Natl Acad Sci (USA),1997,94:4182.
[39]李秋荣,谭力,王暢等?嘉逑┧岣谋銳urkat T细胞膜脂肪微区域脂肪环境的研究。营养学报,2006,28(2):124-127
[40]LOMBARDE V R M,CAGIAO A,FERNANDEANOVOA L.et al.Short term food supplementat- ion effects of a fish derivved extract on the immrnological status of pregnant rats and their sucking pups[J].Nutuition Research,2001,21:1425-1434.
[41] Clarke S.D.,Jump D.B. Dietary polyunsaturated fats uniquely suppress rat liver fatty acid syntha- se and S14 mRNA cotent[J]. Nutr.,1993,120:225-231.
[42] Salati L.M., Clarke S.D. Fatty acid inhibition of hormonal induction of acetyl-coenzyme A carboxylase in hepatocyte monolayers[J]. Arch.Biochim. Biophys, 1986,246:82-89.
[43] Sessler A.M., Kaur N., Palta J.P.,et al. Regulation of stearoyl-COA desaturase 1mRNA stability by polyunsaturated fatty acid in 3T3-L1adipocytes[J]. Biol. Chem.,1996, 271:29854-29858.
[44] Chapman B., Morgan L.M., Murphy M.C. Maternal and early dietary fatty acid intake:changes in lipid metabolism and liver enzymes in adult rats[J]. Nutr., 2000,130:146-151.
[45] Tebbey D.M., Mcgowan K.M., Stephens J.M., et al.Arachidonic acid down-regulates the insulin- dependent glucose transporter gene in 3T3-L1 adipocytes ny inhibiting transcription and enchancing mRNA turnover[J]. Biol.Chem.,1994, 269:639～644.
[46]Wahle K W, Rotondo D , Heys S D. PUFA and gene expression in mammalian systems. Proc Nut -r Soc ,2003 , 62 :349～360
[47]钟耀光.功能食品.北京:化学工业出版社,2004.8:121-128.
[48]刘冀红,曹伟新.ω-3多不饱和脂肪酸在肿瘤防治中的意义.肠外与肠内营养,2004,11(1):54-56.
[49] Stoll AL , Severus WE , Freeman MP , et al . Omega-3 fatty acids in bipolar disorders. A preliminary double-blind , placebo-controlledtrial [ J ] . Arch Gen Psychiatry ,1999 ,56(5):407.
[50] Bartsch H, Nair J, Owen R W. Dietary polyunsaturated fatty acids and cancers of the breast and colorectum : emerging evidence for their role as risk modifiers[J].Carcinogenesis,1999,20(12):2209 -2218.
[51]Jennifer M,Parker-Bames,Das T,et al.Identification and characterization of and enzyme involved in the elongation of n-6 and n-3 polyunsaturated fatty acid[J].Proc Natl Acad Sci USA,2000,97(15):8284
[52] Beaudoin F,Mechaelson LV,Hey SJ,et al.Heterologous reconstitution in yeast of the polyunsaturated fatty acid biosynthetic pathway[J].Proc Natl Acad Sci USA,2000,97(15):6421
[53] Qi B,Fraser T,Mugford S,et al.Production of very long chain poly unsaturated n-3 and n-6 fatty acid in plants[J].Nat Biotechnol,2004,22(6):739.
[54]刘训言,孟庆伟,李滨。植物ω-3脂肪酸去饱和酸的研究进展[J].细胞生物学杂志,2004,26(1):34.
[55]蒋汉明,张凤珍,翟静.ω-3多不饱和脂肪酸与人类健康。预防医学论坛,2005, 11(1):65-66
[56] Daviglus M L, Stamler J, Orencia A J, et al. Fish consumption and the 30-year risk of fatal myocardial infarction [J].N. Engl. J. Med,1997,336(15):1046-1053.
[57] Yuan J M,Ross R K, Gao Y T, et al. Fish and shellfish consumption in relation to death from myocardial infarction among men in Shanghai,China[ J].Am. J. Epidemiol. ,2001,154(9) :809-816.
[58] Hu F B, Bronner L, Willett W C,et al. Fish and omega-3 fatty acid intake and risk of coronary heart disease in women[J].JAMA, 2002,287(14):1815-1821.
[59]郭宝海,马秋刚,计成,臧海燕.富含多不饱和脂肪酸鸡蛋营养保健功能研究.中国饲料2003 ,24,12-13.
[60] Kehja, B. S., S. M. Sadikot, R.B. Phatak, and M. B. Rao, Significance of the n-6/n-3 ratio for insulin action in diabetes. Ann.NY Acad.Sci.,1993,683:258-271
[61]Sink,J.D.Lipid-soluble componments of meat flavor/odors and their biochemical orign.J.Americ Oil Chem.Soc,1973.50(11):470-474.
[62]Ramarathnam,N.,L.J.Rubin and L.L.Diosady.Studies on meat flavor.J.Agric.Food Chem,1993.41 (6):939-94
[63]Fatty acid content in the lipid fraction of pork.Monon G,Miteva E,Veterinarno -Meditsinski Nauki.Vet Med Nauki.1980,17(3):56-59.
[64] Cameron N D ,Enser M B.Fatty acid composition of lipid in longissimus dorsi muscle of Duroc and British Landrance pigs and its relationship with eating quality[J] . Meat Science ,1991. 29:295-30 7.
[65]Macartney A,Maresca B,Cossins A R.Acyl-CoA desaturases and the adaptive regulation of membrane lipid composition,in:A.R.Cossins(Ed.),Temperature Adaptation of Biological Membranes[M],1994,129-139.
[66]Hong H P,Nagamani D,Darwin W R,et a1.High-level production ofγ-1inolenic acid in brassica juncer using aΔ6-desaturase from Pythium irregulare[J].Plant Physiology,2002,129:354-362
[67] Reddy AS,Nuccio ML,Rross LM,et al.Isolation of aΔ6-desaturase gene from the cyanobacterium Synechocystis sp. strain PCC 6803 by gain-of-function expression in Anabaena sp. strain PCC 7120[J]. Plant mol Biol,1993,22(2):293-300.
[68] Aki T,Shimada Y,Inaqaki K,et al.Molecular Cloning and Functional Characterization of Rat Delta-6 Fatty Acid Desaturase[J]. Biochem Biophys Res Commun.1999,255(3):575-579.
[69] Hyekyung P. Cho, Manabu T. Nakamura, and Steven D. Clarke. Cloning, Expression, and Nutritional Regulation of the Mammalian Delta-6 Desaturase[J]. J Bio Chem. 1999, 274(1):471–477.
[70] Zheng X,Seiliez I,Hastings N,et al. Characterization and comparison of fatty acyl Delta6 desaturase cDNAs from freshwater and marine teleost fish species[J]. Comp Biochem Physiol B Biochem Mol Bio. 2004,139(2):269-279.
[71] Khang NT,Jennen DG,Tholen E, et al. Association of the FADS2 Gene with omega-6 and omega-3 PUFA Concentration in the Egg Yolk of Japanese Quail[J]. Animal biotechnology. 2007,18(3):189-201.
[72]Libish B,Michaelson LV,Lewis MJ et al.Chimeras ofΔ6-Fatty acid desaturase andΔ8-Sphigolipid desaturase[J].Biochem Biophy .Res Commun,2000,279:779-785.
[73] Guillou H,D,Abdrea S,Rioux V,et al. Distinct roles of endoplasmic reticulum cytochrome b5 and fused cytochrome b5-like domain for rat Delta6-desaturase activity[J]. Lipid Res 2004,45(1): 32-40.
[74]Hastings N,Agaba M,Tocher D R,et al.A vertebrate fatty acid desaturase with delta 5 and delta 6 activities[J].Proc Natl Acad Sci,2001,98(25):14304-14309.
[75] D,andrea S,Guillou H,Jan S,et al. The same rat Delta6-desaturase not only acts on 18- but also on 24-carbon fatty acids in very-long-chain polyunsaturated fatty acid biosynthesis [J].Biochem.2002, 364(1):49-55.
[76] Guillou H,Rioux V,Catheline D,et al. Conversion of hexadecanoic acid to hexadecenoic acid by rat Delta 6-desaturase[J]. Lipid Res. 2003,44(3):450-454.
[77]张琦,李明春,孙红妍,等.△6-脂肪酸脱氢酶的分子生物学研究进展[J].生物工程学报,2004,20(3):10-14.
[78] Saether T,Tran TN,Rootwelt H,et al. Expression and regulation of delta5-desaturase, delta6-desaturase, stearoyl-coenzyme A (CoA) desaturase 1, and stearoyl-CoA desaturase 2 in rat testis[J]. Reproduction Biol Reprod. 2003,69(1):117-124.
[79] IcNeil CJ,Finch AI,Page KR,et al.The effect of fetal pig size and stage of gestation on tissue fatty acid metabolism and profile[J]. Reproduction.2005,129(6):757-63.
[80] Gallardo N,Bonzon-Kulichenko E,Ferndndez-Agullo T,et al. Tissue-specific effects of central leptin on the expression of genes involved in lipid metabolism in liver and white adipose tissue[J]. Endocrinology.2007,148(12):5604-5610.
[81] Manabu T. Nakamura, Hyekyung P.et al. Regulation of HepaticΔ-6 Desaturase Expression and Its Role in the Polyunsaturased Fatty Acid Inhibition of Fatty Acid Synthase Gene Expression in Mice[J].J Nutr. 2000,130(6):1561-1565.
[82] Nakamura IT,Nara TY. Gene regulation of mammalian desaturases[J]. Biochem Soc Trans. 2002,30(6):1076-1079.
[83]Cherian G,Sim JS. Maternal dietary alpha-linolenic acid (18:3n-3) alters n-3 polyunsaturated fatty acid metabolism and liver enzyme activity in hatched chicks[J].Poult sci.2001,80(7):901-905.
[84] Das Un. A defect in the activity ofΔ6 andΔ5 desaturases may be a factor in the initiation and progression of atherosclerosis[J].Prostaglandins Leukot Essent Fatty Acids.2007,76(5):251-268..
[85] Lane J,Iansel RE. Expression of human delta-6-desaturase is associated with aggressiveness of human breast cancer[J].Int J Mol Med. 2003,12(2):253-257.
[86]Cloning and characterization of mouse growth hormone-releasing hormone (GRH) complementary DNA: increased GRH messenger RNA levels in the growth hormone-deficient lit/lit mouse. Frohman M A,Downs TR,Chomczynski P etc.Mol Endocrinol.1989,3(10):1529-1536.
[87]Polymerase chain reaction with single-sided specificity: analysis of T cell receptor delta chain.Loh EY,Elliott JF,Cwirla S etc.Science.1989,243(4888):217-220.
[88]One-sided polymerase chain reaction: the amplification of cDNA.Ohara O,Dorit RL,Gilbert W.Proc Natl Acad Sci U S A.1989.86(15):5673-5677.
[89] Okayasu T,Nagao M,Ishibashi T,and Imai Y. Purification and partial characterization of linol -eoyl-CoA desaturase from rat liver microsomes.Archives of biochemistry and biophysics, 1981,206(1):21-28
[90]Taniguchi M ,Miura K,1wao H,et a1.Quantitative assessment of DNA microarrays: Comparison with northern blot analyses[J].Genomics,2001,71(1):34-39.
[91]Favre N,Bordmann G,Rudin W.Comparison of cytokine measurements using ELISA , ELISPOT and semi-quantitative RT-PCR[J].J Immunol Methods,1997,204(1):57-66.
[2]袁书林,金荣斌,王宵燕.必须脂肪酸营养研究进展[J].养殖与饲料, 2002,(5):3-5
[3]Simopoulos,A. P. and N. Salem, Jr., N-3 fatty acids in eggs from range-fed Greek chickens. New Engh. J. Med,1989,321:1412.
[4] Simopoulos,A. P., and N.Salem, Jr. Egg yolk as a source of long-chain polyunsaturated fatty acids in infant feeding. Am. J.Clin. Nutr,1992, 55: 411-414
[5] Meluzzi, A., F.Sirri,G.Manfreda, N.Tallarico, and A.Franchini, Effect of dietary vitamin E on the quality of table eggs enriched with n-3 long-chain fatty acids[J]. Poul.Sci,2000,79:539-545
[6] Farrell.UNE,S designer egg [J].Poultry International,1993,32(5):62-66.
[7]Hulan HW,Ackman RG,Ratnayake WM,Proudfoot FG.Omega-3 fatty acid levels and general performance of commercial broilers fed practical levels of redfish meal.Poult Sci.1989,Jan;68(1):153-162.
[8]Rule D C,Broughton K S,Shellito S M.et al.Comparison of muscle fatty acid profiles and cholesterol concentrations of bison,beef cattle,elk,and chicken[J].J Anim Sci,2002,80:1202-1211.
[9]Effects of dietary carbohydrate on delayed onset muscle soreness and reactive oxygen species after contraction induced muscle damage.Close GL,Ashton T,Cable T etc.Br J Sports Med.2005,39(12):948-953.
[10]Migdal W and Kacamarczyk J,Effect of fish oils in the diets for sows on chemical composition of milk and results of piglet managenment.Acta Agraria et Silveslria. 1991,29:47-58.
[11]余文三,多不饱和脂肪酸的研究概况[J].国外医学卫生学分册.1998,25(6):359-367
[11].Uauy R,Mena P,Rajas C.Essential fatty acids in early life:structural and functional role [J].Proc Nutr Soc,2000,59(1):3-15.
[13] Carlson S E, Werkman S H, Rhodes P G, et al. Visual- acuity development in healthy preterm infants: effect of marine-oil supplementation [J].Am. J. Clin. Nutr., 1993,58(1),35-42.
[14].Agostoni C, Massetto N, Biasucci G, et al.Effects of long-chain polyunasturated fatty acid supplementation on fatty acid status and visual function in treated children with hyperphenylalaninemia[J].J Pediatr,2000,137(4):504-509.
[15].蔡双莲,李敏.多不饱和脂肪酸的研究进展[J].生命科学研究.2003,7(4):289-394.
[16]ORDART H,GROSCOLAS R,CALGARI C,et al.Brown fat thermogernesis in rats fed high-fat diets enriched with n-polyunsaturated fatty acids[J].International Jovrnal of Obesity.1997,21:955-962.
[17]颜新春,汪以真。动物脂肪酸合成酶(FAS)基因表达的调控[J].动物营养学报,2002,14(2):1-4.
[18]Flick PK,Chen J,Vagelos PR. Effect of dietary linoleate on synthesis and degradation of fatty acid synthetase from rat liver[J]. J Biol Chem. 1977 Jun 25;252(12):4242-4249.
[19] James GW,Church MK. Hyperalgesia after treatment of mice with prostaglandins and arachido -nic acid and its antagonism by anti-inflammatory-analgesic compounds. Arzneimittelforschung. 1978: 28(5):804-807.
[20]Coordinate suppression of liver acetyl-CoA carboxylase and fatty acid synthetase by polyunsatura -ted fat.Toussant MJ,Wilson MD,Clarke SD.J Nutr.1981,111(1):146-153.
[21] Tomlinson JE,Nakayama R,Holten D. Repression of pentose phosphate pathway dehydrogenase synthesis and mRNA by dietary fat in rats. J Nutr. 1988;118(3):408-15.
[22]Kobatake K,K Sako,Mizawa,et al.Autoradiographic determination of brain pH following middle cerebral arteryocclusion in therat[J].Stroke,1984,15:540-547.
[23]周兴,林成仁,饶琳,王敏.n- 3多烯脂肪酸对血脂影响的实验研究[J].中国中医基础医学杂志,1997,3(4): 29- 31.
[24] Kobatake Y,Kuroda K,Jinnorchi H,Nishide E,Innami S.et al. Differential effects of dietary eicosa -pentaenoic and docosahexaenoic fatty acids on lowering of triglyceride and cholesterol levels in the serum of rats on hypercholesterolemic diet[J] Nutr Sci,1984,30:357-372.
[25]Penny Etherton.ω-3脂肪酸的研究进展[J].泰山卫生。2002,26(3):55-56.
[26] Arrington JL, Chapkin RS, Switzer KC, et al. Dietary n-3 polyunsaturated fatty acidsmodulate purified murine T-cell subset activation[ J ]. Clin Exp Immunol, 2001 , 125 (3) : 499-507.
[27] Arrington JL, McMurrayDN, Switzer KC, et al. Docosahexaenoic acid suppresses function of the CD-8 costimulatory membrane receptor in p rimary murine and J urkat T cells[ J ] . J Nutr, 2001,131 (4) : 1147-1153.
[28] McMurray DN, Jolly CA, Chapkin RS. Effects of dietary n-3 fatty acids on T cell activation and T cell recep tormediated signaling in a murine model[ J ] . J InfectDis, 2000, 182 :S103-S107.
[29] Pompos LJ ,Fritsche KL.Antigen2driven murine CD4 + T lymphocyte proliferation and interleuk -in-2 p roduction are diminished by dietary ( n-3) polyunsaturated fatty acids[ J ]. J Nutr, 2002,132 (11) : 3293-3300.
[30]Calder P C, Field C J. Fatty acids, inflammation and immunity∥Calder P C, Field C J, Gill H S. Nutrition and Immune Function[C ]. CAB International, 2002: 57-92
[31] KELLY V E,FERRETTO A,IZUI S,et al. A fish oil diet rich in eicosapentaenoic acid reduces cyclooxygenase metabolites, and suppresses lupus in MRL-I pr mice[J].J Immunol,1985 (134): 1914 -1919.
[32] PRICKEIT J D,ROBINSON D R,STEINBERG A D. Dietary enrichment with the polyunsaturate -d fatty acid eicosapentaenoic prevents proteinuria and prolongs survival in (NZB×NZW)F1 mice[J].J Clin Invest,1981(68):556-559.
[33] LESLIE C A,GONNERMAN W ULLMAN M D, et al.Dietary fish oil modulates macrophage fatty acids and decreases arthritis susceptibilily in mice[J]. J Exp Med, 1985(162): 1336-1349.
[34] CATHCART E S,MORTENSEN R F,LESLIE C A,et al. A fish oil diet inhibits amyloid P compo -nent (AP)acute phase responses in arthritis susceptible mice[J]. J Immuno1,1987(139):89-91.
[35] BOLCK K J,HO B,XU L L,et al. Effect of fish-fat or beef-fat supplemented diet on immune complex-inducedenteropathy in the rat[J]. Prostaglandins,1989(38):385-396.
[36]王清华,贺永惠,郑素玲. n一多不饱和脂肪酸对动物炎症·免疫和细胞因子的调节作用安徽农业科学,Journal of Anhui Agr.Sci. 2006,34(14):3367-3370.
[37]James MJ,Gibson RA,Cleland L G.Dietary polyunsaturated fatty acids and inflammatory mediator production[J]. Am J Clin Nutr,2000,71(1):343.
[39]Xiao Y-F,Gomez AM,Morgan JP,et al.Suppression of voltage-gated L-typeCa2+ currents By poluunasturated fatty acid in adult and neonatal rat ventricular myocytes[J].Proc Natl Acad Sci (USA),1997,94:4182.
[39]李秋荣,谭力,王暢等?嘉逑┧岣谋銳urkat T细胞膜脂肪微区域脂肪环境的研究。营养学报,2006,28(2):124-127
[40]LOMBARDE V R M,CAGIAO A,FERNANDEANOVOA L.et al.Short term food supplementat- ion effects of a fish derivved extract on the immrnological status of pregnant rats and their sucking pups[J].Nutuition Research,2001,21:1425-1434.
[41] Clarke S.D.,Jump D.B. Dietary polyunsaturated fats uniquely suppress rat liver fatty acid syntha- se and S14 mRNA cotent[J]. Nutr.,1993,120:225-231.
[42] Salati L.M., Clarke S.D. Fatty acid inhibition of hormonal induction of acetyl-coenzyme A carboxylase in hepatocyte monolayers[J]. Arch.Biochim. Biophys, 1986,246:82-89.
[43] Sessler A.M., Kaur N., Palta J.P.,et al. Regulation of stearoyl-COA desaturase 1mRNA stability by polyunsaturated fatty acid in 3T3-L1adipocytes[J]. Biol. Chem.,1996, 271:29854-29858.
[44] Chapman B., Morgan L.M., Murphy M.C. Maternal and early dietary fatty acid intake:changes in lipid metabolism and liver enzymes in adult rats[J]. Nutr., 2000,130:146-151.
[45] Tebbey D.M., Mcgowan K.M., Stephens J.M., et al.Arachidonic acid down-regulates the insulin- dependent glucose transporter gene in 3T3-L1 adipocytes ny inhibiting transcription and enchancing mRNA turnover[J]. Biol.Chem.,1994, 269:639～644.
[46]Wahle K W, Rotondo D , Heys S D. PUFA and gene expression in mammalian systems. Proc Nut -r Soc ,2003 , 62 :349～360
[47]钟耀光.功能食品.北京:化学工业出版社,2004.8:121-128.
[48]刘冀红,曹伟新.ω-3多不饱和脂肪酸在肿瘤防治中的意义.肠外与肠内营养,2004,11(1):54-56.
[49] Stoll AL , Severus WE , Freeman MP , et al . Omega-3 fatty acids in bipolar disorders. A preliminary double-blind , placebo-controlledtrial [ J ] . Arch Gen Psychiatry ,1999 ,56(5):407.
[50] Bartsch H, Nair J, Owen R W. Dietary polyunsaturated fatty acids and cancers of the breast and colorectum : emerging evidence for their role as risk modifiers[J].Carcinogenesis,1999,20(12):2209 -2218.
[51]Jennifer M,Parker-Bames,Das T,et al.Identification and characterization of and enzyme involved in the elongation of n-6 and n-3 polyunsaturated fatty acid[J].Proc Natl Acad Sci USA,2000,97(15):8284
[52] Beaudoin F,Mechaelson LV,Hey SJ,et al.Heterologous reconstitution in yeast of the polyunsaturated fatty acid biosynthetic pathway[J].Proc Natl Acad Sci USA,2000,97(15):6421
[53] Qi B,Fraser T,Mugford S,et al.Production of very long chain poly unsaturated n-3 and n-6 fatty acid in plants[J].Nat Biotechnol,2004,22(6):739.
[54]刘训言,孟庆伟,李滨。植物ω-3脂肪酸去饱和酸的研究进展[J].细胞生物学杂志,2004,26(1):34.
[55]蒋汉明,张凤珍,翟静.ω-3多不饱和脂肪酸与人类健康。预防医学论坛,2005, 11(1):65-66
[56] Daviglus M L, Stamler J, Orencia A J, et al. Fish consumption and the 30-year risk of fatal myocardial infarction [J].N. Engl. J. Med,1997,336(15):1046-1053.
[57] Yuan J M,Ross R K, Gao Y T, et al. Fish and shellfish consumption in relation to death from myocardial infarction among men in Shanghai,China[ J].Am. J. Epidemiol. ,2001,154(9) :809-816.
[58] Hu F B, Bronner L, Willett W C,et al. Fish and omega-3 fatty acid intake and risk of coronary heart disease in women[J].JAMA, 2002,287(14):1815-1821.
[59]郭宝海,马秋刚,计成,臧海燕.富含多不饱和脂肪酸鸡蛋营养保健功能研究.中国饲料2003 ,24,12-13.
[60] Kehja, B. S., S. M. Sadikot, R.B. Phatak, and M. B. Rao, Significance of the n-6/n-3 ratio for insulin action in diabetes. Ann.NY Acad.Sci.,1993,683:258-271
[61]Sink,J.D.Lipid-soluble componments of meat flavor/odors and their biochemical orign.J.Americ Oil Chem.Soc,1973.50(11):470-474.
[62]Ramarathnam,N.,L.J.Rubin and L.L.Diosady.Studies on meat flavor.J.Agric.Food Chem,1993.41 (6):939-94
[63]Fatty acid content in the lipid fraction of pork.Monon G,Miteva E,Veterinarno -Meditsinski Nauki.Vet Med Nauki.1980,17(3):56-59.
[64] Cameron N D ,Enser M B.Fatty acid composition of lipid in longissimus dorsi muscle of Duroc and British Landrance pigs and its relationship with eating quality[J] . Meat Science ,1991. 29:295-30 7.
[65]Macartney A,Maresca B,Cossins A R.Acyl-CoA desaturases and the adaptive regulation of membrane lipid composition,in:A.R.Cossins(Ed.),Temperature Adaptation of Biological Membranes[M],1994,129-139.
[66]Hong H P,Nagamani D,Darwin W R,et a1.High-level production ofγ-1inolenic acid in brassica juncer using aΔ6-desaturase from Pythium irregulare[J].Plant Physiology,2002,129:354-362
[67] Reddy AS,Nuccio ML,Rross LM,et al.Isolation of aΔ6-desaturase gene from the cyanobacterium Synechocystis sp. strain PCC 6803 by gain-of-function expression in Anabaena sp. strain PCC 7120[J]. Plant mol Biol,1993,22(2):293-300.
[68] Aki T,Shimada Y,Inaqaki K,et al.Molecular Cloning and Functional Characterization of Rat Delta-6 Fatty Acid Desaturase[J]. Biochem Biophys Res Commun.1999,255(3):575-579.
[69] Hyekyung P. Cho, Manabu T. Nakamura, and Steven D. Clarke. Cloning, Expression, and Nutritional Regulation of the Mammalian Delta-6 Desaturase[J]. J Bio Chem. 1999, 274(1):471–477.
[70] Zheng X,Seiliez I,Hastings N,et al. Characterization and comparison of fatty acyl Delta6 desaturase cDNAs from freshwater and marine teleost fish species[J]. Comp Biochem Physiol B Biochem Mol Bio. 2004,139(2):269-279.
[71] Khang NT,Jennen DG,Tholen E, et al. Association of the FADS2 Gene with omega-6 and omega-3 PUFA Concentration in the Egg Yolk of Japanese Quail[J]. Animal biotechnology. 2007,18(3):189-201.
[72]Libish B,Michaelson LV,Lewis MJ et al.Chimeras ofΔ6-Fatty acid desaturase andΔ8-Sphigolipid desaturase[J].Biochem Biophy .Res Commun,2000,279:779-785.
[73] Guillou H,D,Abdrea S,Rioux V,et al. Distinct roles of endoplasmic reticulum cytochrome b5 and fused cytochrome b5-like domain for rat Delta6-desaturase activity[J]. Lipid Res 2004,45(1): 32-40.
[74]Hastings N,Agaba M,Tocher D R,et al.A vertebrate fatty acid desaturase with delta 5 and delta 6 activities[J].Proc Natl Acad Sci,2001,98(25):14304-14309.
[75] D,andrea S,Guillou H,Jan S,et al. The same rat Delta6-desaturase not only acts on 18- but also on 24-carbon fatty acids in very-long-chain polyunsaturated fatty acid biosynthesis [J].Biochem.2002, 364(1):49-55.
[76] Guillou H,Rioux V,Catheline D,et al. Conversion of hexadecanoic acid to hexadecenoic acid by rat Delta 6-desaturase[J]. Lipid Res. 2003,44(3):450-454.
[77]张琦,李明春,孙红妍,等.△6-脂肪酸脱氢酶的分子生物学研究进展[J].生物工程学报,2004,20(3):10-14.
[78] Saether T,Tran TN,Rootwelt H,et al. Expression and regulation of delta5-desaturase, delta6-desaturase, stearoyl-coenzyme A (CoA) desaturase 1, and stearoyl-CoA desaturase 2 in rat testis[J]. Reproduction Biol Reprod. 2003,69(1):117-124.
[79] IcNeil CJ,Finch AI,Page KR,et al.The effect of fetal pig size and stage of gestation on tissue fatty acid metabolism and profile[J]. Reproduction.2005,129(6):757-63.
[80] Gallardo N,Bonzon-Kulichenko E,Ferndndez-Agullo T,et al. Tissue-specific effects of central leptin on the expression of genes involved in lipid metabolism in liver and white adipose tissue[J]. Endocrinology.2007,148(12):5604-5610.
[81] Manabu T. Nakamura, Hyekyung P.et al. Regulation of HepaticΔ-6 Desaturase Expression and Its Role in the Polyunsaturased Fatty Acid Inhibition of Fatty Acid Synthase Gene Expression in Mice[J].J Nutr. 2000,130(6):1561-1565.
[82] Nakamura IT,Nara TY. Gene regulation of mammalian desaturases[J]. Biochem Soc Trans. 2002,30(6):1076-1079.
[83]Cherian G,Sim JS. Maternal dietary alpha-linolenic acid (18:3n-3) alters n-3 polyunsaturated fatty acid metabolism and liver enzyme activity in hatched chicks[J].Poult sci.2001,80(7):901-905.
[84] Das Un. A defect in the activity ofΔ6 andΔ5 desaturases may be a factor in the initiation and progression of atherosclerosis[J].Prostaglandins Leukot Essent Fatty Acids.2007,76(5):251-268..
[85] Lane J,Iansel RE. Expression of human delta-6-desaturase is associated with aggressiveness of human breast cancer[J].Int J Mol Med. 2003,12(2):253-257.
[86]Cloning and characterization of mouse growth hormone-releasing hormone (GRH) complementary DNA: increased GRH messenger RNA levels in the growth hormone-deficient lit/lit mouse. Frohman M A,Downs TR,Chomczynski P etc.Mol Endocrinol.1989,3(10):1529-1536.
[87]Polymerase chain reaction with single-sided specificity: analysis of T cell receptor delta chain.Loh EY,Elliott JF,Cwirla S etc.Science.1989,243(4888):217-220.
[88]One-sided polymerase chain reaction: the amplification of cDNA.Ohara O,Dorit RL,Gilbert W.Proc Natl Acad Sci U S A.1989.86(15):5673-5677.
[89] Okayasu T,Nagao M,Ishibashi T,and Imai Y. Purification and partial characterization of linol -eoyl-CoA desaturase from rat liver microsomes.Archives of biochemistry and biophysics, 1981,206(1):21-28
[90]Taniguchi M ,Miura K,1wao H,et a1.Quantitative assessment of DNA microarrays: Comparison with northern blot analyses[J].Genomics,2001,71(1):34-39.
[91]Favre N,Bordmann G,Rudin W.Comparison of cytokine measurements using ELISA , ELISPOT and semi-quantitative RT-PCR[J].J Immunol Methods,1997,204(1):57-66.