产PUFAs转基因株的构建
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摘要
多不饱和脂肪酸(PUFAs)，尤其是长链多不饱和脂肪酸，具有重要的生理与保健功能。目前，ω6系列的多不饱和脂肪酸主要来源是微生物发酵，ω3系列多不饱和脂肪酸主要来源于鱼油，鱼油来源有限，脂肪酸提取工艺复杂且难以保证纯度。ω6系列与ω3系列多不饱和脂肪酸目前都存在价格昂贵、不能满足市场需求的缺点。利用基因工程手段提高微生物中长链多不饱和脂肪酸的产量以及在油料作物中生产长链多不饱和脂肪酸，是两种有效的方法。本研究使用三角褐指藻的Δ6脱饱和酶基因、Δ6延长酶基因与Δ5脱饱和酶基因，利用可以体外构建多拷贝表达盒的酵母表达载体pAO815，构建了含有Δ6脱饱和酶基因、Δ6延长酶基因与Δ5脱饱和酶基因的多价表达载体(pAO1×D6E6D5)，并构建了三个基因各有两个拷贝的表达载体(pAO2×D6E6D5)，分别转化毕赤酵母，构建成产长链多不饱和脂肪酸的基因工程菌(PpDED和Pp2D2E2D)；使用来自甘蓝型油菜的种子特异表达启动子-napin启动子，将三角褐指藻的Δ6脱饱和酶基因、Δ6延长酶基因与Δ5脱饱和酶基因构建入植物表达载体pCAMBIA1303，每个基因都处于napin启动子的控制下，构建好的载体转化拟南芥和甘蓝型油菜，得到了产长链多不饱和脂肪酸转基因拟南芥，并初步得到转基因的甘蓝型油菜苗。具体结果如下：
1．转基因酵母的Southern杂交结果显示，Δ6脱饱和酶基因、Δ6延长酶基因与Δ5脱饱和酶基因已经整合入酵母的基因组，用pAO1×D6E6D5转化的酵母其基因组中整合有单拷贝的D6E6D5，而用pAO2×D6E6D55转化的酵母其基因组中整合有双拷贝的D6E6D5。
2．对诱导表达的PpDED和Pp2D2E2D进行半定量RT-PCR分析，结果表明Δ6脱饱和酶、Δ5脱饱和酶和Δ6延长酶基因在Pp2D2E2D中的表达量分别是在PpDED中的1.87、1.92、1.74倍，表明增加基因的拷贝数，可以显著提高mRNA的表达量。
3．GC-MS分析诱导表达的转基因酵母的脂肪酸，在PpDED中，GLA、DGLA、AA占总脂肪酸的含量分别为3.5％，1.4％，0.1％；SDA、ETA和EPA占总脂肪酸的含量分别为0.6％，0.1％，0.05％。在Pp2D2E2D中，GLA、DGLA、AA占总脂肪酸的含量分别为4.2％，2.4％，0.3％；SDA、ETA和EPA占总脂肪酸的含量分别为0.6％，0.2％，0.1％。Pp2D2E2D中AA和EPA占总脂肪酸的含量分别是PpDED中的三倍和两倍。
4．分析转入的基因在重组酵母的转化效率，A6脱饱和酶的转化效率在PpDED中分别为22.7％(ω6)和20.7％(ω3)，而在Pp2D2E2D中则增加为33.9％(ω6)和33.3％(ω3)。Δ6延长酶的转化效率在PpDED中分别为28.6％(ω6)和14.3％(ω3)，在Pp2D2E2D中则增加为36.4％(ω6)和25％(ω3)。Δ5脱饱和酶的转化效率在ω3步骤中没有变化，均为33.3％，在ω6步骤中，转化效率则从6.7％提高到11.1％。。
5．得到了能够产长链多不饱和脂肪酸的转基因拟南芥，Southern杂交结果显示Δ6脱饱和酶、Δ5脱饱和酶和Δ6延长酶基因已整合入拟南芥基因组。转入的外源基因都只在种子中表达。转基因拟南芥种子中GLA，DGLA，AA的含量分别为6.2％、1.6％、0.5％，SDA，ETA，EPA的含量分别为0.9％、0.5％、0.05％。
6．通过潮霉素筛选，筛选到抗潮霉素的转基因甘蓝型油菜苗。
Polyunsaturated fatty acids (PUFAs), especially long-chain polyunsaturated fattyacids, were important in physiology and health. At present, the main resource ofω6polyunsaturated fatty acids was microbial fermentation while the main resource ofω3polyunsaturated fatty acids was fish oil. The source of fish oil was limited and theextraction of fish oil was complicated. Theω6 andω3 polyunsaturated fatty acids havetwo common disadvantages, expensive and cannot meet market demand. Using themethod of genetic engineering to improve the yield of long-chain polyunsaturated fattyacids of microbial and produce long-chain polyunsaturated fatty acids in oil crops isavailable solutions. In this study, we used the△6,△5 desaturase gene and△6 elongasegene from Phaeodactylum tricornutum and the Pichia pastoris expression vector pAO815to construct a vector contains△6,△5 desaturase gene and△6 elongase gene(pAO1×D6E6D5). Target genes were single copy in this vector pAO1×D6E6D5. Based onthe pAO1×D6E6D5, we constructed a vector pAO2×D6E6D5 in which target genes weredouble copy. Pichia pastoris were transformed with pAO1×D6E6D5 and pAO2×D6E6D5,we had obtained genetic engineering yeast (PpDED and Pp2D2E2D) producinglong-chain polyunsaturated fatty acids. In order to construct plant seed specific expressiontrivalent vector,△6,△5 desaturase gene and△6 elongase gene was respectively under thecontrol of napin promoter and target genes were ligated to vector pCAMBIA1303.Arabidopsis thaliana and Brassica napus were transformed with trivalent vector and wehad obtained transgenic Arabidopsis thaliana producing long-chain polyunsaturated fattyacids and transgenic Brassica napus seedlings. The detailed results are as follows:
1. The results of Southern blot of transgenic Pichia indicate that△6,△5 desaturasegene and△6 elongase gene have integrated into the genome of Pichia pastoris. Targetgenes were single copy respectively in transgenic Pichia which transformed withpAO1×D6E6D5, and were double copy respectively in transgenic Pichia whichtransformed with pAO2×D6E6D.
2. The results of semi-quantitative RT-PCR of transgenic Pichia indicate that theexpression level of△6,△5 desaturase gene and△6 elongase gene in Pp2D2E2D were1.87、1.92、1.74 times of in PpDED. This shows that increasing the copy number of targetgene could significantly improve the expression level of mRNA.
3. GC-MS was used to analyze the total fatty acids of induced transgenic Pichia.GLA, DGLA, AA, SDA, ETA, and EPA respectively account for 3.5%, 1.4%, 0.1%, 0.6%,0.1%, and 0.05% of total fatty acids in PpDED。In Pp2D2E2D, GLA, DGLA, AA, SDA,ETA, and EPA respectively account for 4.2%, 2.4%, 0.3%, 0.6%, 0.2%, and 0.1% of totalfatty acids in Pp2D2E2D. In Pp2D2E2D, the content of AA and EPA was 3 and 2 times ofin PpDED.
4. The conversion efficiency of△6 desaturase gene was 22.7% (ω6) and 20.7% (ω3)in PpDED, in Pp2D2E2D the efficiency was 33.9% (ω6) and 33.3% (ω3). The conversionefficiency of△6 elongase gene was 28.6% (ω6) and 14.3% (ω3), in PpDED, inPp2D2E2D the efficiency was 36.4% (ω6) and 25% (ω3). The conversion efficiency of△5desaturase gene was same in the pathway ofω3 (33.3%) and in the pathway ofω6,increased from 9.7% to 11.1%.
5. We had obtained the transgenic Arabidopsis thaliana seedlings producinglong-chain polyunsaturated fatty acids. The results of Southern blot indicated that△6,△5desaturase gene and△6 elongase gene had integrated into the genome of Arabidopsisthaliana. The target genes were especially expressed in seed. In the seeds of transgenic A.thaliana, the content of GLA, DGLA, AA, SDA, ETA and EPA respectively was 6.2%,1.6%, 0.5%,0.9%,0.5% and 0.05% of total fatty acids.
6. Through the screening by hygromycin, we had obtained transgenic Brassica napusseedlings.
1．转基因酵母的Southern杂交结果显示，Δ6脱饱和酶基因、Δ6延长酶基因与Δ5脱饱和酶基因已经整合入酵母的基因组，用pAO1×D6E6D5转化的酵母其基因组中整合有单拷贝的D6E6D5，而用pAO2×D6E6D55转化的酵母其基因组中整合有双拷贝的D6E6D5。
2．对诱导表达的PpDED和Pp2D2E2D进行半定量RT-PCR分析，结果表明Δ6脱饱和酶、Δ5脱饱和酶和Δ6延长酶基因在Pp2D2E2D中的表达量分别是在PpDED中的1.87、1.92、1.74倍，表明增加基因的拷贝数，可以显著提高mRNA的表达量。
3．GC-MS分析诱导表达的转基因酵母的脂肪酸，在PpDED中，GLA、DGLA、AA占总脂肪酸的含量分别为3.5％，1.4％，0.1％；SDA、ETA和EPA占总脂肪酸的含量分别为0.6％，0.1％，0.05％。在Pp2D2E2D中，GLA、DGLA、AA占总脂肪酸的含量分别为4.2％，2.4％，0.3％；SDA、ETA和EPA占总脂肪酸的含量分别为0.6％，0.2％，0.1％。Pp2D2E2D中AA和EPA占总脂肪酸的含量分别是PpDED中的三倍和两倍。
4．分析转入的基因在重组酵母的转化效率，A6脱饱和酶的转化效率在PpDED中分别为22.7％(ω6)和20.7％(ω3)，而在Pp2D2E2D中则增加为33.9％(ω6)和33.3％(ω3)。Δ6延长酶的转化效率在PpDED中分别为28.6％(ω6)和14.3％(ω3)，在Pp2D2E2D中则增加为36.4％(ω6)和25％(ω3)。Δ5脱饱和酶的转化效率在ω3步骤中没有变化，均为33.3％，在ω6步骤中，转化效率则从6.7％提高到11.1％。。
5．得到了能够产长链多不饱和脂肪酸的转基因拟南芥，Southern杂交结果显示Δ6脱饱和酶、Δ5脱饱和酶和Δ6延长酶基因已整合入拟南芥基因组。转入的外源基因都只在种子中表达。转基因拟南芥种子中GLA，DGLA，AA的含量分别为6.2％、1.6％、0.5％，SDA，ETA，EPA的含量分别为0.9％、0.5％、0.05％。
6．通过潮霉素筛选，筛选到抗潮霉素的转基因甘蓝型油菜苗。
Polyunsaturated fatty acids (PUFAs), especially long-chain polyunsaturated fattyacids, were important in physiology and health. At present, the main resource ofω6polyunsaturated fatty acids was microbial fermentation while the main resource ofω3polyunsaturated fatty acids was fish oil. The source of fish oil was limited and theextraction of fish oil was complicated. Theω6 andω3 polyunsaturated fatty acids havetwo common disadvantages, expensive and cannot meet market demand. Using themethod of genetic engineering to improve the yield of long-chain polyunsaturated fattyacids of microbial and produce long-chain polyunsaturated fatty acids in oil crops isavailable solutions. In this study, we used the△6,△5 desaturase gene and△6 elongasegene from Phaeodactylum tricornutum and the Pichia pastoris expression vector pAO815to construct a vector contains△6,△5 desaturase gene and△6 elongase gene(pAO1×D6E6D5). Target genes were single copy in this vector pAO1×D6E6D5. Based onthe pAO1×D6E6D5, we constructed a vector pAO2×D6E6D5 in which target genes weredouble copy. Pichia pastoris were transformed with pAO1×D6E6D5 and pAO2×D6E6D5,we had obtained genetic engineering yeast (PpDED and Pp2D2E2D) producinglong-chain polyunsaturated fatty acids. In order to construct plant seed specific expressiontrivalent vector,△6,△5 desaturase gene and△6 elongase gene was respectively under thecontrol of napin promoter and target genes were ligated to vector pCAMBIA1303.Arabidopsis thaliana and Brassica napus were transformed with trivalent vector and wehad obtained transgenic Arabidopsis thaliana producing long-chain polyunsaturated fattyacids and transgenic Brassica napus seedlings. The detailed results are as follows:
1. The results of Southern blot of transgenic Pichia indicate that△6,△5 desaturasegene and△6 elongase gene have integrated into the genome of Pichia pastoris. Targetgenes were single copy respectively in transgenic Pichia which transformed withpAO1×D6E6D5, and were double copy respectively in transgenic Pichia whichtransformed with pAO2×D6E6D.
2. The results of semi-quantitative RT-PCR of transgenic Pichia indicate that theexpression level of△6,△5 desaturase gene and△6 elongase gene in Pp2D2E2D were1.87、1.92、1.74 times of in PpDED. This shows that increasing the copy number of targetgene could significantly improve the expression level of mRNA.
3. GC-MS was used to analyze the total fatty acids of induced transgenic Pichia.GLA, DGLA, AA, SDA, ETA, and EPA respectively account for 3.5%, 1.4%, 0.1%, 0.6%,0.1%, and 0.05% of total fatty acids in PpDED。In Pp2D2E2D, GLA, DGLA, AA, SDA,ETA, and EPA respectively account for 4.2%, 2.4%, 0.3%, 0.6%, 0.2%, and 0.1% of totalfatty acids in Pp2D2E2D. In Pp2D2E2D, the content of AA and EPA was 3 and 2 times ofin PpDED.
4. The conversion efficiency of△6 desaturase gene was 22.7% (ω6) and 20.7% (ω3)in PpDED, in Pp2D2E2D the efficiency was 33.9% (ω6) and 33.3% (ω3). The conversionefficiency of△6 elongase gene was 28.6% (ω6) and 14.3% (ω3), in PpDED, inPp2D2E2D the efficiency was 36.4% (ω6) and 25% (ω3). The conversion efficiency of△5desaturase gene was same in the pathway ofω3 (33.3%) and in the pathway ofω6,increased from 9.7% to 11.1%.
5. We had obtained the transgenic Arabidopsis thaliana seedlings producinglong-chain polyunsaturated fatty acids. The results of Southern blot indicated that△6,△5desaturase gene and△6 elongase gene had integrated into the genome of Arabidopsisthaliana. The target genes were especially expressed in seed. In the seeds of transgenic A.thaliana, the content of GLA, DGLA, AA, SDA, ETA and EPA respectively was 6.2%,1.6%, 0.5%,0.9%,0.5% and 0.05% of total fatty acids.
6. Through the screening by hygromycin, we had obtained transgenic Brassica napusseedlings.
引文
[1] 李殿鑫，陈银基，周光宏，徐幸莲．n-3多不饱和脂肪酸分类、来源与疾病防治功能．中国食物与营养，2006，6：52-54
[2] Russell NJ. Mechanisms for thermal adaptation in bacteria: blueprint for survival.Trends Biochem Sci, 1984, 9:108-12
[3] Hazel JR. Thermal adaptation in biological membranes: is homeoviscous adaptation the explanation? Annu Rev Physiol, 1995, 57:19-42
[4] Heird WC, and Lapillonne A. The role of essential fatty acids in development. Annu Rev Nutr, 2005, 25:549-551
[5] Jump DB. The biochemistry of n-3 polyunsaturated fatty acids. J Biol Chem, 2002, 277:8755-8758
[6] Spector AA. Essentiality of fatty acids. Lipids, 1999, 34:S1-S3
[7] Benatti P, Peluso G, Nicolai R, Calvani M. Polyunsaturated fatty acids: biochemical, nutritional and epigenetic properties. J Am Coll Nutr, 2004, 23:281-302
[8] Das UN. Estrogen, statins, and polyunsaturated fatty acids: similarities in their actions and benefits-is there a common link? Nutrition, 2002, 18:178-188
[9] 戴传超，袁生，刘吉华．DHA和EPA的生理功能及其应用．微生物学杂志，1998，18：48-51
[10] Herald PM, Kinsella JE. Fish oil consumption and decreased risk of cardiovascular disease: a oomparison of findings from animal and human feeding trials. Am J Clin Nutr, 1986, 43:566
[11] Kang JX, Leaf A. Prevention of fatal cardiac arrhythmias by polyunsaturated fatty acids. Am J Clin Nutr, 2000, 71:202-207
[12] Bairati I, Roy L, Meyer F. Double-blind, randomized, controlled trial of fish oil supplements in prevention of recurrence of stenosis after coronary angioplasty. Circulation, 1992, 85:950 -956
[13] Capdevila JH, Falck JR, Harris RC. Cytochrome P450 and arachidonic acid bioactivation, Molecular and functional properties of the arachidonate monooxygenase. J Lipid Res, 2000, 41:163-181
[14] Guil-Guerrero JL, Garc(?)a Maroto FF, and Gim(?)nez A. Fatty acid profiles from forty-nine plant species that are potential new sources of γ-linolenic acid. J Am Oil Chem Soc, 2001, 78:677-684
[15] Hansen CE, Stoessel P, and Rossi P. Distribution of gamma-linolenic acid in the confrey (Symphytum officinalle) plant. J Sci Food Agric, 1991, 54:309-312
[16] Whipkey A, Simon JE, Janick J. In vivo and in vitro lipid accumulation in Borago officinalis L. J Am Oil Chem Soc, 1998, 65:979-984
[17] Erwin J, Bloch K. Lipid metabolism of ciliated protozoa. J Biol Chem, 1963, 238:1618-1624
[18] Cohen Z, Didi S, and Heimer YM. Overproduction of γ-linolenic and eicosapentaenoic acids by algae. Plant Physiol, 1992, 98:569-572
[19] Radwan SS. Sources of C20-Polyunsaturated fatty acids for bioteehnologieal use. Appl Mierobiol Bioteehnol, 1991, 35:421-430
[20] 梁英，麦康森．微藻EPA和DHA的研究现状及前景．水产学报，2000，24：289-296
[21] 曹春晖，孙世春，麦康森，梁英．30株海洋绿藻的总脂含量和脂肪酸组成．青岛海洋大学学报，2000，30：428-434
[22] Shimizu S, Jareonkitmongkol S, Kawashima H, Akimoto K, and Yamada H. Production of a novel ω-3 eicosapentaenoic acid by Mortierella alpina IS-4 grown on 1-hexadecene. Arch Microbiol, 1991, 156:163-166
[23] Yamada H, Shimizu S, Shinmen Y, Kawashima H, Akimoto K. Production of arachidonic acid and eicosapentaenoic acid by microorganisms. J Am Oil Chem Soc, 1987, 64:1254
[24] Gandi SR, Weete JD. Production of the polyunsturatured fatty acids arachidonic acid and eicosapentaenoic acid by fungus pythium ultimum. J Gen Microbiol, 1991, 137:1825-1830
[25] 刘吉华，袁生，戴传超．二十碳五烯酸等多不饱和脂肪酸高产菌的筛选．菌物系统，2000．19：407-409
[26] Stinson EE, Kwoczak R, Kurantz MJ. Effect of cultural conditions on production of eicosapentaenoic acid by pythium irregulare. J InD Microbiol, 1991, 8:171-178
[27] Bajpai PK, Bajpai P, and Ward OP. Arachidonic acid production by fungi. Appl Environ Microbiol, 1991, 57:1255-1258
[28] Bazan HE, Careaga MM, Spereher H, Bazan NG. Chain elongation and desaturation of eicosapentaenoate to docosahexaenoate and phospholipid labeling in the rat retina in vivo. Biochim Biophys Acta, 1982, 712:123-128
[29] Wallis JG, Watts JL, Browse J. Polyunsaturated fatty acid synthesis: What will they think of next? Trends Biochem Sci, 2002, 27:467-470
[30] Sprecher H, Luthria DL, Mohammed BS. Re-valuation of the pathways for the biosynthesis of polyunsaturated fatty acids. J Lipid Res, 1995, 36:2471-2477
[31] Lees AM, Korn ED. Metabolism of unsaturated fatty acids in protozoa. Biochemistry, 1966, 5:1457-1481
[32] Ulsamer AG, Smith FR, Korn ED. Composition and effects of phagocytosos on incorporation of radioactive precursors. J Cell Biol, 1969, 43:105-114
[33] Wallis JG, Browse J. The Δ8-desaturase of Euglena gracilis: an alternate pathway for synthesis of 20-carbon polyunsaturated fatty acids. Arch Biochem Biophys, 1999, 365:307-316
[34] Venegas-Caler(?)n M, Beaudoin F, Sayanova O, and Napier JA. Co-transcribed genes for long chain polyunsaturated fatty acid biosynthesis in the protozoon Perkinsus marinus include a plant-like FAE1 3-Ketoacyl Coenzyme A Synthase. J Biol Chem, 2007, 282:2996-3003
[35] Napier JA, Beaudoin F, Michaelson LV, Sayanova O. The production of long chain polyunsaturated fatty acids in transgenic plants by reverse-engineering.Biochimie, 2004, 86:785-793
[36] Qiu X. Biosynthesis of docosahexaenoic acid (DHA, 22:6-4,7,10,13,16,19) : two distinct pathways. Prostaglandins Leukot Essent Fatty Acids, 2003, 68:181-186
[37] Haruko T, Daisuke T, Kazunaga Y, Yamada A, Tadashi M. Expression of the eicosapentaenoic acid synthesis gene cluster from Shewanella sp in a transgenic marine cyanobacterium,Synechococcus sp. Microbiology, 1997, 143:2725-2731
[38] Metz JG, Roessler P, Facciott D, Levering C, Dittrich F, Lassner M, Valentine R,Lardizabal K, Domergue F, Yamada A, Yazawa K, Knauf V, Browse J. Production of polyunsaturated fatty acids by polyketide synthases in both prokaryotes and eukaryotes. Science, 2001, 293:290-293
[39] Napier JA. Plumbing the depths of PUFA biosynthesis: a novel polyketide synthase-like pathway from marine organisms. Trends Plant Sci, 2002, 7:51-54
[40] Heinz E. Biosynthesis of polyunsaturated fatty acids, in: TS Moore (Ed) , Lipid Metabolism in Plants. CRC Press, Boca Raton, FL, 1993, pp 33-89
[41] Borgeson CE, de Renobales M, Blomquist GJ. Characterization of the △12 desaturase in the American cockroach, Periplaneta americana: the nature of the substrate. Biochim Biophys Acta, 1990,1047:135-140
[42] Cripps C, Borgeson C, Blomquist GJ, de Renobales M. The △12-desaturease from the house cricket Acheta domesticus (Orthoptera: Gryllidae): characterization and form of the substrate. Arch Biochem Biophys, 1990, 278:46-51
[43] Spychalla JP, Kinney AJ, Browse J. Identification of an animal omega-3 fatty acid desaturase by heterologous expression in Arabidopsis. Proc Natl Acad Sci USA,1997,94:1142-1147
[44] Peyou-Ndi MM, Watts JL, Browse J. Identification and characterization of an animal △12 fatty acid desaturase gene by heterologous expression in Saccharomyces cerevisiae. Arch Biochem Biophys 2000, 376:399-408
[45] Meesapyodsuk D, Reed DW, Savile CK, Buist PH, Ambrose SJ and Covello PS.Characterization of the regiochemistry and cryptoregiochemistry of a Caenorhabditis elegans fatty acid desaturase (FAT-1) expressed in Saccharomyces cerevisiae. Biochemistry, 2000, 39:11948-11954
[46] Huang YS, Chaudhary S, Thurmond JM, Bobik EG. Cloning of △12- and △6 desarurases from Mortierella alpina and recombinant production of gamma-linolenic acid in Saccharomyces cerevisiae. Lipids, 1999, 34:649-659
[47] Sakuradani E, Kobayashi M, Ashikari T, Shimiz S. Identification of △-12 fatty acid desaturase from an arachidonic acidproducing Mortierella fungus by heterologous expression in the yeast Saccharomyces cerevisiae and the fungus Aspergillus oryzae. Eur J Biochem, 1999, 261:812-820
[48] Passorn S, Laoteng K, Rachadawong S, Tanticharoen M, Cheevadhanarak S.Heterologous expression of Mucor rouxii △ ( 12 ) - desaturase gene in Saccharomyces cerevisiae. Biochem Biophys Res Commun, 1999, 263:47-51
[49] Sato N, Fujiwar S, Kawaguchi A, Tsuzuki M. Cloning of a gene for chloroplast omega 6 desaturase of a green alga, Chlamydomonas reinhardtii. J Biochem, 1997,122:1224-1232
[50] Wilson RA, Calvo AM, Chang PK and Keller NP. Characterization of the Aspergillus parasiticus △12-desaturase gene: a role for lipid metabolism in the Aspergillus-seed interaction. Microbiology, 2004, 150:2881-2888
[51] Li MC, Li H, Wei DS, Xing LJ. Cloning and molecular characterization of △12-fatty acid desaturase gene from Mortierella isabellina. World J Gastroenterol,2006, 12:3373-3379
[52] Wei DS, Li MC, Zhang XX, Ren Y, Xing LJ. Identification and characterization of a novel △12-fatty acid desaturase gene from Rhizopus arrhizus. FEBS Lett, 2004,573:45-50
[53] Zhang S, Sakuradani E, Ito K, Shimizu S. Identification of a novel bifunctional △12/△15 fatty acid desaturase from a basidiomycete, Coprinus cinereus TD#822-2.FEBS Lett, 2007, 581:315-319
[54] Kainou K, Kamisaka Y, Kimura K and Uemura H. Isolation of △12 and ω3-fatty acid desaturase genes from the yeast Kluyveromyces lactis and their heterologous expression to produce linoleic and α-linolenic acids in Saccharomyces cerevisiae.Yeast, 2006, 23:605-612
[55] Pirtle IL, Kongcharoensuntorn W, Nampaisansuk M, Knesek JE, Chapman KD,Pirtle RM. Molecular cloning and functional expression of the gene for a cotton -△12 fatty acid desaturase (FAD2) . Biochim Biophys Acta, 2001,1522:122-129
[56] Domergue F, Spiekermann P, Lerchl J, Beckmann C, Kilian O, Kroth PG, Boland W, Z(?)hringer U, and Heinz E. New insight into Phaeodactylum tricornutum fatty acid metabolism cloning and functional characterization of plastidial and microsomal △12-fatty acid desaturases. Plant Physiol, 2003,131:1648-1660
[57] Oura T, Kajiwara S. Saccharomyces kluyveri FAD3 encodes an ω3 fatty acid desaturase. Microbiology, 2004,150:1983-1990
[58] Sakuradani E, Abe T, Iguchi K, Shimizu S. A novel fungal ω3-desaturase with wide substrate specificity from arachidonic acid-producing Mortierella alpina 1S-4.Appl Microbiol Biotechnol, 2005, 66:648-654
[59] Pereira SL, Huang Y-S, Bobik EG, Kinney AJ, Stecca KL, Packer JCL, and Mukerji P. A novel ω3-fatty acid desaturase involved in the biosynthesis of eicosapentaenoic acid. Biochem J, 2004, 378:665-671
[60] Damude HG, Zhang H-X, Farrall L, Ripp KG, Tomb J-F, Hollerbach D, and Yadav NS. Identification of bifunctional △12 ω3 fatty acid desaturases for improving the ratio of ω3 to ω6 fatty acids in microbes and plants. Proc Natl Acad Sci USA, 2006, 103:9446-9451
[61] Sayanova O, Shewry PR, Napier JA. Histidine-41 of cytochrome b5 domain of the borage △6 fatty acid desaturase is essential for enzyme activity. Plant Physiol,1999,121:641-646
[62] Sayanova O, Beaudoin F, Libisch B, Shewry P, Napier J. Mutagenesis of the borage △6 fatty acid desaturase. Biochem Soc Trans, 2000, 28:636-638
[63] Laoten K, Mannontarat R, Tanticharoen M, Cheevadhanarak S. △6-desaturase of Mucor rouxii with high similarity to plant △6-desaturse and its heterologous expression in Saccharomyces cerevisiae. Biochem Biophys Res Commun, 2000,279:17-22
[64] Domergue F, Abbadi A, Z(?)hringer U, Moreau H, and Heinz E. In vivo characterization of the first acyl-CoA △6-desaturase from a member of the plant kingdom, the microalga Ostreococcus tauri. Biochem J, 2005, 389:483-490
[65] Hoffmann M, Wagner M, Abbadi A, Fulda M, and Feussner I. Metabolic engineering of ω3-Very long chain polyunsaturated fatty acid production by an exclusively Acyl-CoA-dependent pathway. J Biol Chem, 2008, 283:22352-22362
[66] Pugh EL, Kates M. Direct desaturation of eicosatrienoyl lecithin to arachidonoyl lecithin by rat liver microsomes. J Biol Chem, 1977, 252:68-73
[67] Watts JL, Browse J. Isolation and characterization of a D5-fatty acid desaturase from Caenorhabditis elegans. Arch Biochem Biophys, 1999, 362:175-182
[68] Hornung E, Korfei M, Pernstich C, Struss A, Kindl H, Fulda M, and Feussner I.Specific formation of arachidonic acid and eicosapentaenoic acid by a front-end △5-desaturase from Phytophthora megasperma. Biochim Biophys Acta, 2005,1686:181-189
[69] Kaewsuwan S, Cahoon EB, Perroud P-F, Wiwat C, Panvisavas N, Quatrano RS,Cove DJ, and Bunyapraphatsara N. Identification and functional characterization of the moss Physcomitrella patens △5-desaturase gene involved in arachidonic and eicosapentaenoic acid biosynthesis. J Biol Chem, 2006, 281:21988-21997
[70] Saito T, Morio T, and Ochiai H. A second functional △5 fatty acid desaturase in the cellular slime mould Dictyostelium discoideum. Eur J Biochem, 2000,267:1813-1818
[71] Saito T, and Ochiai H. Identification of △5-fatty acid desaturase from the cellular slime mold Dictyostelium discoideum. Eur J Biochem, 1999, 265:809-814
[72] Dormergue F, Lercgl J, Z(?)hringer U, and Heinz E. Cloning and functional characterization of Phaeodactylum tricornutum front-end desaturases involved in eicosapentaenoic acid biosynthesis. Eur J Biochem, 2002, 269:4105-4113
[73] Cahoon EB, Marillia EF, Stecca KL, Hall SE, Taylor DC, and Kinney AJ.Production of fatty acid components of meadowfoam oil in somatic soybean embryos. Plant Physiol, 2000,124:243-251
[74] Tonon T, Harvey D, Larson TR, Graham IA. Identification of a very long chain polyunsaturated fatty acid △4-desaturase from the microalga Pavlova lutheri.FEBS Lett, 2003, 553:440-444
[75] Tonon T, Sayanova O, Michaelson LV, Qing R-W, Harvey D, Larson TR, Li Y,Napier JA, and Graham IA. Fatty acid desaturases from the microalga Thalassiosira pseudonana. FEBS J, 2005, 272:3401-3412
[76] Meyer A, Cirpus P, Ott C, Schlecker R, Z(?)hringer U, and Heinz E. Biosynthesis of docosahexaenoic acid in Euglena gracilis: biochemical and molecular evidence for the involvement of a △4-fatty acyl group desaturase. Biochemistry, 2003,42:9779-9788
[77] Pereira SL, Leonard AE, Huang Y-S, Chuang LT, and Mukerji P. Identification of two novel microalgal enzymes involved in the conversion of the ω3-fatty acid,eicosapentaenoic acid, into docosahexaenoic acid. Biochem J, 2004, 384:357-366
[78] Zhou X-R, Robert SS, Petrie JR, Frampton D MF, Mansour MP, Blackburn SI,Nichol PD, Green AG, Singh SP. Isolation and characterization of genes from the marine microalga Pavlova salina encoding three front-end desaturases involved in docosahexaenoic acid biosynthesis. Phytochemistry, 2007, 68:785-796
[79] Sayanova O, Haslam R, Qi BX, Lazarus CM, Napier JA. The alternative pathway C20 △8-desarurase from the non-photosynthetic organism Acanthamoeba castellanii is an atypical cytochrome b5-fusion desaturase. FEBS Lett, 2006,580:1946-1952
[80] Cinti DL, Cook L, Nagi MN, Suneja SK. The fatty acid chain elongation system of mammalian endoplasmic reticulum. Prog Lipids Res, 1992, 31:1-51
[81] Moon YA, Shah NA, Mohapatra S, Warrington JA, Horton JD. Identification of a mammalian long chain acyl elongase regulated by sterol regulatory element-binding proteins. J Biol Chem, 2001, 276:45358-45366
[82] Suneja SK, Osei P, Cook L, Nagi MN, Cinti DL. Enzyme site-specific changes in hepatic microsomal fatty acid chain elongation in streptozotocin-induced diabetic rats. Biochim Biophy Acta, 1990, 1042:81-85
[83] Toke DA, Martin CE. Isolation and characterization of a gene affecting fatty acid elongation in Saccharomyces cerevisiae. J Biol Chem, 1996, 271:18413-18422
[84] Leonard AE, Bobik EG, Dorado J, Croeger PE, Chuang LT, Thurmond JM,Parker-Barnes JM, Das T, Huang YS, Mukerji P. Cloning of a human cDNA encoding a novel enzyme involved in the elongation of long-chain polyunsaturated acids. Biochem J, 2000, 350:765-770
[85] Zank TK, Zahringer U, Beckmann C, Pohnert G, Boland W, Holtorf H, Reski R,Lerchl J,and Heinz E. Cloning and functional characterisation of an enzyme involved in the elongation of △6-polyunsaturated fatty acids from the moss Physcomitrella patens. Plant J, 2002, 31:255-268
[86] Kajikawa M, Yamato KT, Kohzu Y, Nojiri M, Sakuradani E, Shimizu S, Sakai Y,Fukuzawa H, and Ohyama K. Isolation and characterization of △6-desaturase, an ELO-like enzyme and △5-desaturase from the liverwort Marchantia polymorpha and production of arachidonic and eicosapentaenoic acids in the methylotrophic yeast Pichia pastoris. Plant Mol Biol, 2004, 54:335-352
[87] Parker-Barnes JM, Das T, Bobik E, Leonard AE, Thurmond JM, Chaung LT,Huang YS, and Mukerji P. Identification and characterization of an enzyme involved in the elongation of n-6 and n-3 polyunsaturated fatty acids. Proc Natl Acad Sci USA , 2000, 97:8284-8289
[88] Qi BX, Beaudoin F, Fraser T, Stobart AK, Napier JA, Lazarus CM. Identification of a cDNA encoding a novel C18-△9 polyunsaturated fatty acid-specific elongating activity from the docosahexaenoic acid (DHA)-producing microalga, Isochrysis galbana. FEBS Lett, 2002, 510:159-165
[89] Venegas-Caleron M, Beaudoin F, Sayanova O, and Napier JA. Co-transcribed genes for long chain polyunsaturated fatty acid biosynthesis in the protozoon Perkinsus marinus include a plant-like FAE1 3-ketoacyl coenzyme A synthase. J Biol Chem, 2007,282:2996-3003
[90] Niu Y, Kong J, Fu LY, Yang J, Xu Y. Identification of a novel C20-elongase gene from the marine microalgae Pavlova viridis and its expression in Escherichia coli.Mar Biotechnol, 2009,11:17-23
[91] Yazawa K. Production of eicosapentaenoic acid from marine bacteria. Lipids, 1996,31:S297-S300
[92] Nishida T, Orikasa Y, Ito Y, Yu R, Yamada A, Watanabe K, and Okuyama H.Escherichia coli engineered to produce eicosapentaenoic acid becomes resistant against oxidative damages. FEBS Lett, 2006, 580:2731-2735
[93] Orikas Y, Nishida T, Hase A, Watanabe K, Morita N, and Okuyama H. A phosphopantetheinyl transferase gene essential for biosynthesis of n-3 polyunsaturated fatty acids from Moritella marina strain MP-1. FEBS Lett, 2006,580:4423-4429
[94] Orikasa Y, Yamada A, Yu R, Ito Y, Nishida T, Yumoto I, Watanabe K, and Okuyama H. Characterization of the eicosapentaenoic acid biosynthesis gene cluster from Shewanella sp strain SCRC-2738. Cell Mol Biol, 2004, 50:625-630
[95] Tanaka M, Ueno A, Kawasaki K, Yumoto I, Ohgiya S, Hoshino T, Ishizaki K, Okuyama H, and Morita N. Isolation of clustered genes that are notably homologous to the eicosapentaenoic acid biosynthesis gene cluster from the docosahexaenoic acid-producing bacterium Vibrio marinus strain MP-1. Biotechnol Lett, 1999, 21:939-945
[96] 陈波，张玲，贺新生，李代发，王熙．用抗性筛选法选育γ-亚麻酸(GLA)高产菌株．微生物学通报， 2003， 30：53-56
[97] 袁成凌，王纪，姚建铭，余增亮，贡国鸿，尚耘，钱云．低能离子注入花生四烯酸产生菌诱变选育及其产业化研究．高技术通讯，2003，6：22-25
[98] 张玲，李植峰，赖炳森，沈晓京，谭亚芳，孙树秦．雅致枝霉高产γ-亚麻酸突变株的选育．生物技术，2000，10：22-24
[99] 周蓬蓬，余龙江，吴元喜，朱敏，李为．高山被孢霉产花生四烯酸发酵条件的研究．工业微生物，2003，33：41-45
[100] Takeyama H, Takeda D, Yazawa K, Yamada A, and Matsunaga T. Expression of the eicosapentaenoic acid synthesis gene cluster from Shewanella sp in a transgenic marine cyanobacterium, Synechococcus sp.. Microbiology, 1997, 143:2725-2731
[101] Yu R, Yamada A, Watanabe K, Yazawa K, Takeyama H, Matsunaga T, and Kurane R. Production of eicosapentaenoic acid by a recombinant marine cyanobacterium, Synechococeus sp.. Lipids, 2000, 35:1061-1064
[102] Orikasa Y, Ito Y, Nishida T, Watanabe K, Morita N, Ohwada T, Yumoto I, and Okuyama H. Enhanced heterologous production of eicosapentaenoic acid in Escherichia coli cells that co-express eicosapentaenoic acid biosynthesis pfa genes and foreign DNA fragments including a high-performance catalase gene, vktA. Biotechnol Lett, 2007, 29:803-809
[103] Orikasa Y, Nishida T, Yamada A, Yu R, Watanabe K, Hase A, Morita N, Okuyama H. Recombinant production of docosahexaenoic acid in a polyketide biosynthesis mode in Escherichia coli. Biotechnol Lett, 2006,28:1841-1847
[104] Beaudoin F, Michaelson LV, Hey SJ, Lewis MJ, Shewry PR, Sayanova O, and Napier JA. Heterologous reconstitution in yeast of the polyunsaturated fatty acid biosynthetic pathway. Proc Natl Acad Sci USA, 2000, 97:6421-6426
[105] Domergue F, Lerchl J, Zahringer U, and Heinz E. Cloning and functional characterization of Phaeodactylum tricornutum front-end desaturases involved in eicosapentaenoic acid biosynthesis. Eur J Biochem, 2002, 269:4105-4113
[106] Qi B-X, Fraser T, Mugford S, G Dobson, O Sayanova, J Butler, JA Napier, AK Stobart, and C M Lazarus. Production of very long chain polyunsaturated omega-3 and omega-6 fatty acids in plants. Nat Biotech, 2004,22:739-745
[107] Abbadi A, Domergue F, Bauer J, Napier JA, Welti R, Zahringer U, Cirpus P, and Heinz E. Biosynthesis of very-long-chain polyunsaturated fatty acids in transgenic oilseeds: constraints on their accumulation. Plant Cell, 2004,16:2734-2748
[108] Chen R, Matsui K, Ogawa M, Oe M, Ochiai M, Kawashima H, Sakuradani E,Shimizu S, Ishimoto M, Hayashi M, Murooka Y, Tanaka Y. Expression of △6, △5 desaturase and GLELO elongase genes from Mortierella alpina for production of arachidonic acid in soybean [Glycine max (L) Merrill] seeds. Plant Sci, 2006,170:399-406
[109] Kajikawa M, Matsui K, Ochiai M, Tanaka Y, Kita Y, Ishimoto M, Kohzu Y, Shoji S-I, Yamato KT, Ohyama K, Fukuzawa H, and Kohchi T. Production of arachidonic and eicosapentaenoic acids in plants using bryophyte fatty acid △6-desaturase, △6-elongase, and △5-desaturase genes. Biosci Biotechnol Biochem,2008, 72:435-444
[110] Kinney AJ, Cahoon EB, Darmude HG, Hitz WD, Kolar CW, Liu ZB. Production of very long chain polyunsaturated fatty acids in oilseed plants. WO 2004/071467 A2
[111] Robert SS, Singh SP, Zhou X-R, Petrie, JR, Blackburn SI, Mansour PM, Nichols PD, Liu Q, and Green AG Metabolic engineering of arabidopsis to produce nutritionally important DHA in seed oil. Funct Plant Biol, 2005, 32:473-479
[112] Wu GH, Truksa M, Datla N, Vrinten P, Bauer J, Zank T, Cirpus P, Heinz E, and Qiu X. Stepwise engineering to produce high yields of very long-chain polyunsaturated fatty acids in plants. Nat Biotechnol, 2005, 23,1013-1017
[113] Metz JG, Flatt JH, Kuner JM. PUFA Polyketide syntha systems and uses. WO 2006/135866 A2
[114] Metz JG, Kuner JM, Lippmeier JC. Polyunsaturated fatty acid production in genetically modified organisms using PUFA polyketide synthase systems. WO2007/106903
[115] Truksa M, Wu GH, Vrinten P, and Qiu X. Metabolic engineering of plants to produce very long-chain polyunsaturated fatty acids. Transgenic Res, 2006, 15:131-137
[116] Singh SP, Zhou XR, Liu Q, Stymne S, and Green AG. Metabolic engineering of new fatty acids in plants. Curr Opin Plant Biol, 2005, 8:197-203
[117] Yongmanitchai W, and Ward OP. Growth of and omega-3 fatty acid production by Phaeodaetylum tricornutum under different culture conditions. Appl Environ Microbiol, 1991, 57:419-425
[118] Ausubel FM，Kingston RE，Seidman JG等．精编分子生物学实验指南(第四版)[M]．北京：科学出版社，2005，22-24，26，55-58
[119] Thompson J, Register E, Curotto J, Kurtz M, Kelly R. An improved protocol for the preparation of yeast cells for transformation by electroporation.Yeast, 1998, 14:565-571
[120] Wu SX, Letchworth GJ. High efficiency transformation by electroporation of Pichia pastoris pretreated with lithium acetate and dithiothreitol. BioTechniques, 2004, 36:152-154
[121] Harju S, Fedosyuk H and Peterson KR. Rapid isolation of yeast genomic DNA: Bustn' Grab. BMC Biotechnol, 2004, 4:8
[122] Zhu M, Yu LJ, Liu Z, Xu HB. Isolating Mortierella alpina strains of high yield of arachidonic acid. Lett Appl Microbiol, 2004, 39:332-335
[123] Sreekrishna K, Brankamp RG, Kropp KE, Blankenship DK, Tsay JT, Smith PL,Wierschke JD, Subramaniam A, Birkenberger LA. Strategies for optimal synthesis and secretion of heterologous proteins in the methylotrophic yeast Pichia pastoris.Gene, 1997,190:55-62
[124] Chiou SY, Su WW, Su YC. Optimizing production of polyunsaturated fatty acids in Marchantia polymorpha cell suspension culture. J Biotechnol, 2001,85:247-257
[125] Duplus E, Glorian M, Foresl C. Fatty acid regulation of gene transcription. J Biol chem, 2000,275: 30749-30752
[126] Choi JY, Stukey J, Hwang S-Y, and Martin CE. Regulatory elements that control transcription activation and unsaturated fatty acid-mediated repression of the Saccharomyces cerevisiae OLE1 gene. J Biol Chem, 1996, 271: 3581 - 3589
[127] Estelle MA, Somerville CR. Auxin-resistant mutants of Arabidopsis thaliana with an altered morphology. Mol Gen Genet, 1987,206: 200-206
[128] Zhang S, Raina S, Li H, Li J, Dec E, Ma H, Huang H, and NV Fedoroff. Resources for targeted insertional and deletional mutagenesis in Arabidopsis. Plant Mol Biol,2003,53:133-150
[129] Bechtold N, Pelletier G In planta Agrobacterium mediated transformation of adult Arabidopsis thaliana plants by vacuum infiltration. Meth Mol Biol, 1998, 82:59-256
[130] Chung M-H, Chen M-K, and Pan S-M. Floral spray transformation can efficiently generate Arabidopsis transgenic plants. Transgenic Res, 2000, 9:471-476
[131] Bechtold N, Ellis J, Pelletier G In plant Agrobacterium-mediated gene transfer by infiltration of adult Arabidopsis thaliana plants. C R Acad Sci Paris, Life Sci, 1993,316:1194-1199
[132] Qing CM, Fan L, Yao L, David B, Colette T, Li Y and Christophe R.Transformation of Pakchoi (Brassica raga L.ssp.chinensis) by Agrobacterium infiltration. Mol Breed, 2000, 6: 67-72
[133] Trieu AT, Burleigh SH, Kardailsky ?. Transformation of Medicago truncatula via infiltration of seedlings or flowering plants with Agrobacterium Plant J, 2000,22:531-541
[134] Clough SJ, Bent AF. Floral dip: a simplified method for Agrobacterium mediated transformation of Arahidopsis thaliana. Plant J, 1998, 16:735-742
[2] Russell NJ. Mechanisms for thermal adaptation in bacteria: blueprint for survival.Trends Biochem Sci, 1984, 9:108-12
[3] Hazel JR. Thermal adaptation in biological membranes: is homeoviscous adaptation the explanation? Annu Rev Physiol, 1995, 57:19-42
[4] Heird WC, and Lapillonne A. The role of essential fatty acids in development. Annu Rev Nutr, 2005, 25:549-551
[5] Jump DB. The biochemistry of n-3 polyunsaturated fatty acids. J Biol Chem, 2002, 277:8755-8758
[6] Spector AA. Essentiality of fatty acids. Lipids, 1999, 34:S1-S3
[7] Benatti P, Peluso G, Nicolai R, Calvani M. Polyunsaturated fatty acids: biochemical, nutritional and epigenetic properties. J Am Coll Nutr, 2004, 23:281-302
[8] Das UN. Estrogen, statins, and polyunsaturated fatty acids: similarities in their actions and benefits-is there a common link? Nutrition, 2002, 18:178-188
[9] 戴传超，袁生，刘吉华．DHA和EPA的生理功能及其应用．微生物学杂志，1998，18：48-51
[10] Herald PM, Kinsella JE. Fish oil consumption and decreased risk of cardiovascular disease: a oomparison of findings from animal and human feeding trials. Am J Clin Nutr, 1986, 43:566
[11] Kang JX, Leaf A. Prevention of fatal cardiac arrhythmias by polyunsaturated fatty acids. Am J Clin Nutr, 2000, 71:202-207
[12] Bairati I, Roy L, Meyer F. Double-blind, randomized, controlled trial of fish oil supplements in prevention of recurrence of stenosis after coronary angioplasty. Circulation, 1992, 85:950 -956
[13] Capdevila JH, Falck JR, Harris RC. Cytochrome P450 and arachidonic acid bioactivation, Molecular and functional properties of the arachidonate monooxygenase. J Lipid Res, 2000, 41:163-181
[14] Guil-Guerrero JL, Garc(?)a Maroto FF, and Gim(?)nez A. Fatty acid profiles from forty-nine plant species that are potential new sources of γ-linolenic acid. J Am Oil Chem Soc, 2001, 78:677-684
[15] Hansen CE, Stoessel P, and Rossi P. Distribution of gamma-linolenic acid in the confrey (Symphytum officinalle) plant. J Sci Food Agric, 1991, 54:309-312
[16] Whipkey A, Simon JE, Janick J. In vivo and in vitro lipid accumulation in Borago officinalis L. J Am Oil Chem Soc, 1998, 65:979-984
[17] Erwin J, Bloch K. Lipid metabolism of ciliated protozoa. J Biol Chem, 1963, 238:1618-1624
[18] Cohen Z, Didi S, and Heimer YM. Overproduction of γ-linolenic and eicosapentaenoic acids by algae. Plant Physiol, 1992, 98:569-572
[19] Radwan SS. Sources of C20-Polyunsaturated fatty acids for bioteehnologieal use. Appl Mierobiol Bioteehnol, 1991, 35:421-430
[20] 梁英，麦康森．微藻EPA和DHA的研究现状及前景．水产学报，2000，24：289-296
[21] 曹春晖，孙世春，麦康森，梁英．30株海洋绿藻的总脂含量和脂肪酸组成．青岛海洋大学学报，2000，30：428-434
[22] Shimizu S, Jareonkitmongkol S, Kawashima H, Akimoto K, and Yamada H. Production of a novel ω-3 eicosapentaenoic acid by Mortierella alpina IS-4 grown on 1-hexadecene. Arch Microbiol, 1991, 156:163-166
[23] Yamada H, Shimizu S, Shinmen Y, Kawashima H, Akimoto K. Production of arachidonic acid and eicosapentaenoic acid by microorganisms. J Am Oil Chem Soc, 1987, 64:1254
[24] Gandi SR, Weete JD. Production of the polyunsturatured fatty acids arachidonic acid and eicosapentaenoic acid by fungus pythium ultimum. J Gen Microbiol, 1991, 137:1825-1830
[25] 刘吉华，袁生，戴传超．二十碳五烯酸等多不饱和脂肪酸高产菌的筛选．菌物系统，2000．19：407-409
[26] Stinson EE, Kwoczak R, Kurantz MJ. Effect of cultural conditions on production of eicosapentaenoic acid by pythium irregulare. J InD Microbiol, 1991, 8:171-178
[27] Bajpai PK, Bajpai P, and Ward OP. Arachidonic acid production by fungi. Appl Environ Microbiol, 1991, 57:1255-1258
[28] Bazan HE, Careaga MM, Spereher H, Bazan NG. Chain elongation and desaturation of eicosapentaenoate to docosahexaenoate and phospholipid labeling in the rat retina in vivo. Biochim Biophys Acta, 1982, 712:123-128
[29] Wallis JG, Watts JL, Browse J. Polyunsaturated fatty acid synthesis: What will they think of next? Trends Biochem Sci, 2002, 27:467-470
[30] Sprecher H, Luthria DL, Mohammed BS. Re-valuation of the pathways for the biosynthesis of polyunsaturated fatty acids. J Lipid Res, 1995, 36:2471-2477
[31] Lees AM, Korn ED. Metabolism of unsaturated fatty acids in protozoa. Biochemistry, 1966, 5:1457-1481
[32] Ulsamer AG, Smith FR, Korn ED. Composition and effects of phagocytosos on incorporation of radioactive precursors. J Cell Biol, 1969, 43:105-114
[33] Wallis JG, Browse J. The Δ8-desaturase of Euglena gracilis: an alternate pathway for synthesis of 20-carbon polyunsaturated fatty acids. Arch Biochem Biophys, 1999, 365:307-316
[34] Venegas-Caler(?)n M, Beaudoin F, Sayanova O, and Napier JA. Co-transcribed genes for long chain polyunsaturated fatty acid biosynthesis in the protozoon Perkinsus marinus include a plant-like FAE1 3-Ketoacyl Coenzyme A Synthase. J Biol Chem, 2007, 282:2996-3003
[35] Napier JA, Beaudoin F, Michaelson LV, Sayanova O. The production of long chain polyunsaturated fatty acids in transgenic plants by reverse-engineering.Biochimie, 2004, 86:785-793
[36] Qiu X. Biosynthesis of docosahexaenoic acid (DHA, 22:6-4,7,10,13,16,19) : two distinct pathways. Prostaglandins Leukot Essent Fatty Acids, 2003, 68:181-186
[37] Haruko T, Daisuke T, Kazunaga Y, Yamada A, Tadashi M. Expression of the eicosapentaenoic acid synthesis gene cluster from Shewanella sp in a transgenic marine cyanobacterium,Synechococcus sp. Microbiology, 1997, 143:2725-2731
[38] Metz JG, Roessler P, Facciott D, Levering C, Dittrich F, Lassner M, Valentine R,Lardizabal K, Domergue F, Yamada A, Yazawa K, Knauf V, Browse J. Production of polyunsaturated fatty acids by polyketide synthases in both prokaryotes and eukaryotes. Science, 2001, 293:290-293
[39] Napier JA. Plumbing the depths of PUFA biosynthesis: a novel polyketide synthase-like pathway from marine organisms. Trends Plant Sci, 2002, 7:51-54
[40] Heinz E. Biosynthesis of polyunsaturated fatty acids, in: TS Moore (Ed) , Lipid Metabolism in Plants. CRC Press, Boca Raton, FL, 1993, pp 33-89
[41] Borgeson CE, de Renobales M, Blomquist GJ. Characterization of the △12 desaturase in the American cockroach, Periplaneta americana: the nature of the substrate. Biochim Biophys Acta, 1990,1047:135-140
[42] Cripps C, Borgeson C, Blomquist GJ, de Renobales M. The △12-desaturease from the house cricket Acheta domesticus (Orthoptera: Gryllidae): characterization and form of the substrate. Arch Biochem Biophys, 1990, 278:46-51
[43] Spychalla JP, Kinney AJ, Browse J. Identification of an animal omega-3 fatty acid desaturase by heterologous expression in Arabidopsis. Proc Natl Acad Sci USA,1997,94:1142-1147
[44] Peyou-Ndi MM, Watts JL, Browse J. Identification and characterization of an animal △12 fatty acid desaturase gene by heterologous expression in Saccharomyces cerevisiae. Arch Biochem Biophys 2000, 376:399-408
[45] Meesapyodsuk D, Reed DW, Savile CK, Buist PH, Ambrose SJ and Covello PS.Characterization of the regiochemistry and cryptoregiochemistry of a Caenorhabditis elegans fatty acid desaturase (FAT-1) expressed in Saccharomyces cerevisiae. Biochemistry, 2000, 39:11948-11954
[46] Huang YS, Chaudhary S, Thurmond JM, Bobik EG. Cloning of △12- and △6 desarurases from Mortierella alpina and recombinant production of gamma-linolenic acid in Saccharomyces cerevisiae. Lipids, 1999, 34:649-659
[47] Sakuradani E, Kobayashi M, Ashikari T, Shimiz S. Identification of △-12 fatty acid desaturase from an arachidonic acidproducing Mortierella fungus by heterologous expression in the yeast Saccharomyces cerevisiae and the fungus Aspergillus oryzae. Eur J Biochem, 1999, 261:812-820
[48] Passorn S, Laoteng K, Rachadawong S, Tanticharoen M, Cheevadhanarak S.Heterologous expression of Mucor rouxii △ ( 12 ) - desaturase gene in Saccharomyces cerevisiae. Biochem Biophys Res Commun, 1999, 263:47-51
[49] Sato N, Fujiwar S, Kawaguchi A, Tsuzuki M. Cloning of a gene for chloroplast omega 6 desaturase of a green alga, Chlamydomonas reinhardtii. J Biochem, 1997,122:1224-1232
[50] Wilson RA, Calvo AM, Chang PK and Keller NP. Characterization of the Aspergillus parasiticus △12-desaturase gene: a role for lipid metabolism in the Aspergillus-seed interaction. Microbiology, 2004, 150:2881-2888
[51] Li MC, Li H, Wei DS, Xing LJ. Cloning and molecular characterization of △12-fatty acid desaturase gene from Mortierella isabellina. World J Gastroenterol,2006, 12:3373-3379
[52] Wei DS, Li MC, Zhang XX, Ren Y, Xing LJ. Identification and characterization of a novel △12-fatty acid desaturase gene from Rhizopus arrhizus. FEBS Lett, 2004,573:45-50
[53] Zhang S, Sakuradani E, Ito K, Shimizu S. Identification of a novel bifunctional △12/△15 fatty acid desaturase from a basidiomycete, Coprinus cinereus TD#822-2.FEBS Lett, 2007, 581:315-319
[54] Kainou K, Kamisaka Y, Kimura K and Uemura H. Isolation of △12 and ω3-fatty acid desaturase genes from the yeast Kluyveromyces lactis and their heterologous expression to produce linoleic and α-linolenic acids in Saccharomyces cerevisiae.Yeast, 2006, 23:605-612
[55] Pirtle IL, Kongcharoensuntorn W, Nampaisansuk M, Knesek JE, Chapman KD,Pirtle RM. Molecular cloning and functional expression of the gene for a cotton -△12 fatty acid desaturase (FAD2) . Biochim Biophys Acta, 2001,1522:122-129
[56] Domergue F, Spiekermann P, Lerchl J, Beckmann C, Kilian O, Kroth PG, Boland W, Z(?)hringer U, and Heinz E. New insight into Phaeodactylum tricornutum fatty acid metabolism cloning and functional characterization of plastidial and microsomal △12-fatty acid desaturases. Plant Physiol, 2003,131:1648-1660
[57] Oura T, Kajiwara S. Saccharomyces kluyveri FAD3 encodes an ω3 fatty acid desaturase. Microbiology, 2004,150:1983-1990
[58] Sakuradani E, Abe T, Iguchi K, Shimizu S. A novel fungal ω3-desaturase with wide substrate specificity from arachidonic acid-producing Mortierella alpina 1S-4.Appl Microbiol Biotechnol, 2005, 66:648-654
[59] Pereira SL, Huang Y-S, Bobik EG, Kinney AJ, Stecca KL, Packer JCL, and Mukerji P. A novel ω3-fatty acid desaturase involved in the biosynthesis of eicosapentaenoic acid. Biochem J, 2004, 378:665-671
[60] Damude HG, Zhang H-X, Farrall L, Ripp KG, Tomb J-F, Hollerbach D, and Yadav NS. Identification of bifunctional △12 ω3 fatty acid desaturases for improving the ratio of ω3 to ω6 fatty acids in microbes and plants. Proc Natl Acad Sci USA, 2006, 103:9446-9451
[61] Sayanova O, Shewry PR, Napier JA. Histidine-41 of cytochrome b5 domain of the borage △6 fatty acid desaturase is essential for enzyme activity. Plant Physiol,1999,121:641-646
[62] Sayanova O, Beaudoin F, Libisch B, Shewry P, Napier J. Mutagenesis of the borage △6 fatty acid desaturase. Biochem Soc Trans, 2000, 28:636-638
[63] Laoten K, Mannontarat R, Tanticharoen M, Cheevadhanarak S. △6-desaturase of Mucor rouxii with high similarity to plant △6-desaturse and its heterologous expression in Saccharomyces cerevisiae. Biochem Biophys Res Commun, 2000,279:17-22
[64] Domergue F, Abbadi A, Z(?)hringer U, Moreau H, and Heinz E. In vivo characterization of the first acyl-CoA △6-desaturase from a member of the plant kingdom, the microalga Ostreococcus tauri. Biochem J, 2005, 389:483-490
[65] Hoffmann M, Wagner M, Abbadi A, Fulda M, and Feussner I. Metabolic engineering of ω3-Very long chain polyunsaturated fatty acid production by an exclusively Acyl-CoA-dependent pathway. J Biol Chem, 2008, 283:22352-22362
[66] Pugh EL, Kates M. Direct desaturation of eicosatrienoyl lecithin to arachidonoyl lecithin by rat liver microsomes. J Biol Chem, 1977, 252:68-73
[67] Watts JL, Browse J. Isolation and characterization of a D5-fatty acid desaturase from Caenorhabditis elegans. Arch Biochem Biophys, 1999, 362:175-182
[68] Hornung E, Korfei M, Pernstich C, Struss A, Kindl H, Fulda M, and Feussner I.Specific formation of arachidonic acid and eicosapentaenoic acid by a front-end △5-desaturase from Phytophthora megasperma. Biochim Biophys Acta, 2005,1686:181-189
[69] Kaewsuwan S, Cahoon EB, Perroud P-F, Wiwat C, Panvisavas N, Quatrano RS,Cove DJ, and Bunyapraphatsara N. Identification and functional characterization of the moss Physcomitrella patens △5-desaturase gene involved in arachidonic and eicosapentaenoic acid biosynthesis. J Biol Chem, 2006, 281:21988-21997
[70] Saito T, Morio T, and Ochiai H. A second functional △5 fatty acid desaturase in the cellular slime mould Dictyostelium discoideum. Eur J Biochem, 2000,267:1813-1818
[71] Saito T, and Ochiai H. Identification of △5-fatty acid desaturase from the cellular slime mold Dictyostelium discoideum. Eur J Biochem, 1999, 265:809-814
[72] Dormergue F, Lercgl J, Z(?)hringer U, and Heinz E. Cloning and functional characterization of Phaeodactylum tricornutum front-end desaturases involved in eicosapentaenoic acid biosynthesis. Eur J Biochem, 2002, 269:4105-4113
[73] Cahoon EB, Marillia EF, Stecca KL, Hall SE, Taylor DC, and Kinney AJ.Production of fatty acid components of meadowfoam oil in somatic soybean embryos. Plant Physiol, 2000,124:243-251
[74] Tonon T, Harvey D, Larson TR, Graham IA. Identification of a very long chain polyunsaturated fatty acid △4-desaturase from the microalga Pavlova lutheri.FEBS Lett, 2003, 553:440-444
[75] Tonon T, Sayanova O, Michaelson LV, Qing R-W, Harvey D, Larson TR, Li Y,Napier JA, and Graham IA. Fatty acid desaturases from the microalga Thalassiosira pseudonana. FEBS J, 2005, 272:3401-3412
[76] Meyer A, Cirpus P, Ott C, Schlecker R, Z(?)hringer U, and Heinz E. Biosynthesis of docosahexaenoic acid in Euglena gracilis: biochemical and molecular evidence for the involvement of a △4-fatty acyl group desaturase. Biochemistry, 2003,42:9779-9788
[77] Pereira SL, Leonard AE, Huang Y-S, Chuang LT, and Mukerji P. Identification of two novel microalgal enzymes involved in the conversion of the ω3-fatty acid,eicosapentaenoic acid, into docosahexaenoic acid. Biochem J, 2004, 384:357-366
[78] Zhou X-R, Robert SS, Petrie JR, Frampton D MF, Mansour MP, Blackburn SI,Nichol PD, Green AG, Singh SP. Isolation and characterization of genes from the marine microalga Pavlova salina encoding three front-end desaturases involved in docosahexaenoic acid biosynthesis. Phytochemistry, 2007, 68:785-796
[79] Sayanova O, Haslam R, Qi BX, Lazarus CM, Napier JA. The alternative pathway C20 △8-desarurase from the non-photosynthetic organism Acanthamoeba castellanii is an atypical cytochrome b5-fusion desaturase. FEBS Lett, 2006,580:1946-1952
[80] Cinti DL, Cook L, Nagi MN, Suneja SK. The fatty acid chain elongation system of mammalian endoplasmic reticulum. Prog Lipids Res, 1992, 31:1-51
[81] Moon YA, Shah NA, Mohapatra S, Warrington JA, Horton JD. Identification of a mammalian long chain acyl elongase regulated by sterol regulatory element-binding proteins. J Biol Chem, 2001, 276:45358-45366
[82] Suneja SK, Osei P, Cook L, Nagi MN, Cinti DL. Enzyme site-specific changes in hepatic microsomal fatty acid chain elongation in streptozotocin-induced diabetic rats. Biochim Biophy Acta, 1990, 1042:81-85
[83] Toke DA, Martin CE. Isolation and characterization of a gene affecting fatty acid elongation in Saccharomyces cerevisiae. J Biol Chem, 1996, 271:18413-18422
[84] Leonard AE, Bobik EG, Dorado J, Croeger PE, Chuang LT, Thurmond JM,Parker-Barnes JM, Das T, Huang YS, Mukerji P. Cloning of a human cDNA encoding a novel enzyme involved in the elongation of long-chain polyunsaturated acids. Biochem J, 2000, 350:765-770
[85] Zank TK, Zahringer U, Beckmann C, Pohnert G, Boland W, Holtorf H, Reski R,Lerchl J,and Heinz E. Cloning and functional characterisation of an enzyme involved in the elongation of △6-polyunsaturated fatty acids from the moss Physcomitrella patens. Plant J, 2002, 31:255-268
[86] Kajikawa M, Yamato KT, Kohzu Y, Nojiri M, Sakuradani E, Shimizu S, Sakai Y,Fukuzawa H, and Ohyama K. Isolation and characterization of △6-desaturase, an ELO-like enzyme and △5-desaturase from the liverwort Marchantia polymorpha and production of arachidonic and eicosapentaenoic acids in the methylotrophic yeast Pichia pastoris. Plant Mol Biol, 2004, 54:335-352
[87] Parker-Barnes JM, Das T, Bobik E, Leonard AE, Thurmond JM, Chaung LT,Huang YS, and Mukerji P. Identification and characterization of an enzyme involved in the elongation of n-6 and n-3 polyunsaturated fatty acids. Proc Natl Acad Sci USA , 2000, 97:8284-8289
[88] Qi BX, Beaudoin F, Fraser T, Stobart AK, Napier JA, Lazarus CM. Identification of a cDNA encoding a novel C18-△9 polyunsaturated fatty acid-specific elongating activity from the docosahexaenoic acid (DHA)-producing microalga, Isochrysis galbana. FEBS Lett, 2002, 510:159-165
[89] Venegas-Caleron M, Beaudoin F, Sayanova O, and Napier JA. Co-transcribed genes for long chain polyunsaturated fatty acid biosynthesis in the protozoon Perkinsus marinus include a plant-like FAE1 3-ketoacyl coenzyme A synthase. J Biol Chem, 2007,282:2996-3003
[90] Niu Y, Kong J, Fu LY, Yang J, Xu Y. Identification of a novel C20-elongase gene from the marine microalgae Pavlova viridis and its expression in Escherichia coli.Mar Biotechnol, 2009,11:17-23
[91] Yazawa K. Production of eicosapentaenoic acid from marine bacteria. Lipids, 1996,31:S297-S300
[92] Nishida T, Orikasa Y, Ito Y, Yu R, Yamada A, Watanabe K, and Okuyama H.Escherichia coli engineered to produce eicosapentaenoic acid becomes resistant against oxidative damages. FEBS Lett, 2006, 580:2731-2735
[93] Orikas Y, Nishida T, Hase A, Watanabe K, Morita N, and Okuyama H. A phosphopantetheinyl transferase gene essential for biosynthesis of n-3 polyunsaturated fatty acids from Moritella marina strain MP-1. FEBS Lett, 2006,580:4423-4429
[94] Orikasa Y, Yamada A, Yu R, Ito Y, Nishida T, Yumoto I, Watanabe K, and Okuyama H. Characterization of the eicosapentaenoic acid biosynthesis gene cluster from Shewanella sp strain SCRC-2738. Cell Mol Biol, 2004, 50:625-630
[95] Tanaka M, Ueno A, Kawasaki K, Yumoto I, Ohgiya S, Hoshino T, Ishizaki K, Okuyama H, and Morita N. Isolation of clustered genes that are notably homologous to the eicosapentaenoic acid biosynthesis gene cluster from the docosahexaenoic acid-producing bacterium Vibrio marinus strain MP-1. Biotechnol Lett, 1999, 21:939-945
[96] 陈波，张玲，贺新生，李代发，王熙．用抗性筛选法选育γ-亚麻酸(GLA)高产菌株．微生物学通报， 2003， 30：53-56
[97] 袁成凌，王纪，姚建铭，余增亮，贡国鸿，尚耘，钱云．低能离子注入花生四烯酸产生菌诱变选育及其产业化研究．高技术通讯，2003，6：22-25
[98] 张玲，李植峰，赖炳森，沈晓京，谭亚芳，孙树秦．雅致枝霉高产γ-亚麻酸突变株的选育．生物技术，2000，10：22-24
[99] 周蓬蓬，余龙江，吴元喜，朱敏，李为．高山被孢霉产花生四烯酸发酵条件的研究．工业微生物，2003，33：41-45
[100] Takeyama H, Takeda D, Yazawa K, Yamada A, and Matsunaga T. Expression of the eicosapentaenoic acid synthesis gene cluster from Shewanella sp in a transgenic marine cyanobacterium, Synechococcus sp.. Microbiology, 1997, 143:2725-2731
[101] Yu R, Yamada A, Watanabe K, Yazawa K, Takeyama H, Matsunaga T, and Kurane R. Production of eicosapentaenoic acid by a recombinant marine cyanobacterium, Synechococeus sp.. Lipids, 2000, 35:1061-1064
[102] Orikasa Y, Ito Y, Nishida T, Watanabe K, Morita N, Ohwada T, Yumoto I, and Okuyama H. Enhanced heterologous production of eicosapentaenoic acid in Escherichia coli cells that co-express eicosapentaenoic acid biosynthesis pfa genes and foreign DNA fragments including a high-performance catalase gene, vktA. Biotechnol Lett, 2007, 29:803-809
[103] Orikasa Y, Nishida T, Yamada A, Yu R, Watanabe K, Hase A, Morita N, Okuyama H. Recombinant production of docosahexaenoic acid in a polyketide biosynthesis mode in Escherichia coli. Biotechnol Lett, 2006,28:1841-1847
[104] Beaudoin F, Michaelson LV, Hey SJ, Lewis MJ, Shewry PR, Sayanova O, and Napier JA. Heterologous reconstitution in yeast of the polyunsaturated fatty acid biosynthetic pathway. Proc Natl Acad Sci USA, 2000, 97:6421-6426
[105] Domergue F, Lerchl J, Zahringer U, and Heinz E. Cloning and functional characterization of Phaeodactylum tricornutum front-end desaturases involved in eicosapentaenoic acid biosynthesis. Eur J Biochem, 2002, 269:4105-4113
[106] Qi B-X, Fraser T, Mugford S, G Dobson, O Sayanova, J Butler, JA Napier, AK Stobart, and C M Lazarus. Production of very long chain polyunsaturated omega-3 and omega-6 fatty acids in plants. Nat Biotech, 2004,22:739-745
[107] Abbadi A, Domergue F, Bauer J, Napier JA, Welti R, Zahringer U, Cirpus P, and Heinz E. Biosynthesis of very-long-chain polyunsaturated fatty acids in transgenic oilseeds: constraints on their accumulation. Plant Cell, 2004,16:2734-2748
[108] Chen R, Matsui K, Ogawa M, Oe M, Ochiai M, Kawashima H, Sakuradani E,Shimizu S, Ishimoto M, Hayashi M, Murooka Y, Tanaka Y. Expression of △6, △5 desaturase and GLELO elongase genes from Mortierella alpina for production of arachidonic acid in soybean [Glycine max (L) Merrill] seeds. Plant Sci, 2006,170:399-406
[109] Kajikawa M, Matsui K, Ochiai M, Tanaka Y, Kita Y, Ishimoto M, Kohzu Y, Shoji S-I, Yamato KT, Ohyama K, Fukuzawa H, and Kohchi T. Production of arachidonic and eicosapentaenoic acids in plants using bryophyte fatty acid △6-desaturase, △6-elongase, and △5-desaturase genes. Biosci Biotechnol Biochem,2008, 72:435-444
[110] Kinney AJ, Cahoon EB, Darmude HG, Hitz WD, Kolar CW, Liu ZB. Production of very long chain polyunsaturated fatty acids in oilseed plants. WO 2004/071467 A2
[111] Robert SS, Singh SP, Zhou X-R, Petrie, JR, Blackburn SI, Mansour PM, Nichols PD, Liu Q, and Green AG Metabolic engineering of arabidopsis to produce nutritionally important DHA in seed oil. Funct Plant Biol, 2005, 32:473-479
[112] Wu GH, Truksa M, Datla N, Vrinten P, Bauer J, Zank T, Cirpus P, Heinz E, and Qiu X. Stepwise engineering to produce high yields of very long-chain polyunsaturated fatty acids in plants. Nat Biotechnol, 2005, 23,1013-1017
[113] Metz JG, Flatt JH, Kuner JM. PUFA Polyketide syntha systems and uses. WO 2006/135866 A2
[114] Metz JG, Kuner JM, Lippmeier JC. Polyunsaturated fatty acid production in genetically modified organisms using PUFA polyketide synthase systems. WO2007/106903
[115] Truksa M, Wu GH, Vrinten P, and Qiu X. Metabolic engineering of plants to produce very long-chain polyunsaturated fatty acids. Transgenic Res, 2006, 15:131-137
[116] Singh SP, Zhou XR, Liu Q, Stymne S, and Green AG. Metabolic engineering of new fatty acids in plants. Curr Opin Plant Biol, 2005, 8:197-203
[117] Yongmanitchai W, and Ward OP. Growth of and omega-3 fatty acid production by Phaeodaetylum tricornutum under different culture conditions. Appl Environ Microbiol, 1991, 57:419-425
[118] Ausubel FM，Kingston RE，Seidman JG等．精编分子生物学实验指南(第四版)[M]．北京：科学出版社，2005，22-24，26，55-58
[119] Thompson J, Register E, Curotto J, Kurtz M, Kelly R. An improved protocol for the preparation of yeast cells for transformation by electroporation.Yeast, 1998, 14:565-571
[120] Wu SX, Letchworth GJ. High efficiency transformation by electroporation of Pichia pastoris pretreated with lithium acetate and dithiothreitol. BioTechniques, 2004, 36:152-154
[121] Harju S, Fedosyuk H and Peterson KR. Rapid isolation of yeast genomic DNA: Bustn' Grab. BMC Biotechnol, 2004, 4:8
[122] Zhu M, Yu LJ, Liu Z, Xu HB. Isolating Mortierella alpina strains of high yield of arachidonic acid. Lett Appl Microbiol, 2004, 39:332-335
[123] Sreekrishna K, Brankamp RG, Kropp KE, Blankenship DK, Tsay JT, Smith PL,Wierschke JD, Subramaniam A, Birkenberger LA. Strategies for optimal synthesis and secretion of heterologous proteins in the methylotrophic yeast Pichia pastoris.Gene, 1997,190:55-62
[124] Chiou SY, Su WW, Su YC. Optimizing production of polyunsaturated fatty acids in Marchantia polymorpha cell suspension culture. J Biotechnol, 2001,85:247-257
[125] Duplus E, Glorian M, Foresl C. Fatty acid regulation of gene transcription. J Biol chem, 2000,275: 30749-30752
[126] Choi JY, Stukey J, Hwang S-Y, and Martin CE. Regulatory elements that control transcription activation and unsaturated fatty acid-mediated repression of the Saccharomyces cerevisiae OLE1 gene. J Biol Chem, 1996, 271: 3581 - 3589
[127] Estelle MA, Somerville CR. Auxin-resistant mutants of Arabidopsis thaliana with an altered morphology. Mol Gen Genet, 1987,206: 200-206
[128] Zhang S, Raina S, Li H, Li J, Dec E, Ma H, Huang H, and NV Fedoroff. Resources for targeted insertional and deletional mutagenesis in Arabidopsis. Plant Mol Biol,2003,53:133-150
[129] Bechtold N, Pelletier G In planta Agrobacterium mediated transformation of adult Arabidopsis thaliana plants by vacuum infiltration. Meth Mol Biol, 1998, 82:59-256
[130] Chung M-H, Chen M-K, and Pan S-M. Floral spray transformation can efficiently generate Arabidopsis transgenic plants. Transgenic Res, 2000, 9:471-476
[131] Bechtold N, Ellis J, Pelletier G In plant Agrobacterium-mediated gene transfer by infiltration of adult Arabidopsis thaliana plants. C R Acad Sci Paris, Life Sci, 1993,316:1194-1199
[132] Qing CM, Fan L, Yao L, David B, Colette T, Li Y and Christophe R.Transformation of Pakchoi (Brassica raga L.ssp.chinensis) by Agrobacterium infiltration. Mol Breed, 2000, 6: 67-72
[133] Trieu AT, Burleigh SH, Kardailsky ?. Transformation of Medicago truncatula via infiltration of seedlings or flowering plants with Agrobacterium Plant J, 2000,22:531-541
[134] Clough SJ, Bent AF. Floral dip: a simplified method for Agrobacterium mediated transformation of Arahidopsis thaliana. Plant J, 1998, 16:735-742