深黄被孢霉高产花生四烯酸的制备及其调控机制
|
摘要
花生四烯酸是一种人体必须的不饱和脂肪酸,具有广泛的生物活性和重要的营养作用。其传统制备方法成本较高、提取率低,微生物发酵法生产花生四烯酸可降低成本、提高产量。△5-、△6-脱饱和酶是花生四烯酸合成途径上的关键酶,评价脱饱和酶与花生四烯酸产量的关系,可进一步确立该酶在花生四烯酸生物合成中的作用。本研究的目的是选育花生四烯酸高产菌株,优化花生四烯酸提取和纯化的工艺参数,研究深黄被孢霉诱变菌株脱饱和酶的基因序列变化,探讨脱饱和酶的活性及mRNA表达量与花生四烯酸产量的关系。
以深黄被孢霉为出发菌株,分别采用微波诱变、紫外诱变、原生质体再生诱变和紫外诱变原生质体等方法进行诱变,利用气相色谱分析花生四烯酸含量,最终获得四株花生四烯酸高产菌株,其中YZ-124花生四烯酸产量最高,为4.72 g/L,比出发菌株提高576 %,并且遗传性能稳定。采用CO2超临界萃取技术提取微生物油脂中的花生四烯酸,花生四烯酸提取率为45.7%,花生四烯酸含量为19.34%。
采用尿素包合法纯化花生四烯酸,最终得到产品中花生四烯酸含量为36.84%。以深黄被孢霉原始菌株及高产花生四烯酸的诱变菌株为基础,利用RT-PCR及基因克隆方法获得了5株深黄被孢霉的△5、△6-脱饱和酶基因的完整cDNA,对其进行序列分析,结果表明:诱变菌株与原始菌株的核苷酸及氨基酸序列同源性较高。利用TTC-脱氢酶活性测定法及荧光定量PCR方法测定了5株深黄被孢霉的同一培养时间及高产花生四烯酸菌株YZ-124在不同培养时间的活性及mRNA表达量,结果显示:脱饱和酶的活性及表达量与培养物油脂中花生四烯酸含量呈正相关关系。
Arachidonic acid is an essential dietary component for human beings,and has various physiological functions and plays an important role in infant nutrition. Arachidonic acid(ARA), widely existing in the animal kingdom,can be isolated from the lipids extracted from pig adrenal gland or pig liver and sardines as well; however, the yield of it is only 0.2%(w/w) or lower, so it is very dificult to be applied in a industrial scale and the research and application are restricted.It has been focused on the study of ARA fermented by microbe in the whole world.△5-、△6-desaturase is the key enzyme of arachidonic acid synthetic pathway. Evaluating the relation of desaturase and ARA production can further establish its function on biological Synthesis.The aim of this study is to selectively breed the high-yield ARA strain, to optimize the technology parameter of extraction and purification for ARA ,to study the gene sequence variation of Mortierlla isabellina mutant strain desaturase, to explore the activity of desaturase and the relationship between the expressed quantity and ARA production.
Mortierella isabellina3.3410 was used as initial strain, microwave mutation, ultraviolet mutation, protoplast mutation and ultraviolet mutation protoplast were used to mutate, and then the content of the ARA was measured by GC. The ultimum four strains who had the most ARA yield and genetic stability were obtained. Under powe of microwave at 700W and heating time of 35s,lethality of spores of Mortierella isabellina was 78.3%.After two-time microwave mutation and repeated screen, a mutation of Mortierella isabellina W35s2-153 was obtained,whose ARA yield was 2.61g/L, 3.18 times of the control strains.The power of viltalight lamp was 20W,exposure distance was 30cm and exposure time was 80s,lethality of spores of Mortierella isabellina was 76.4%.After two-time ultraviolet mutation and repeated screen,a mutation of Mortierella isabellina Z80s2-109 was obtained, whose ARA yield was 2.74g/L, 3.77 times of the control strains.After protoplast mutation, enzyme concentration at 4%,enzymolysis temperature at 30℃and enzymolysis time at 7.5h,the rate of protoplast was 78.4%.After mutation and repeated screen,a mutation of Mortierella isabellina Y-69 was obtained whose ARA yield was 2.92g/L and the concentration in biomass were 3.56 times of the control strains. After ultraviolet mutation protoplast, the power of viltalight lamp at 20W ,exposure distance at 30cm and exposure time at 80s,lethality of spores of Mortierella isabellina was 76.4%,a mutation of YZ-124 was obtained,whose ARA yield was 4.72 g/L , 5.76 times of the control strains.
The process condition optmization of arachidonic acid extraction from microbial oil Was carried out by means of supercritical CO2 fluid technology.It was used to increase the extraction rate of arachidonic acid in microbial oil. The supercritical CO2 fluid technology was used to extract the ARA from microbial oil; an orthogonal, rotatable, central composite was employed to determine the optimum conditions for extraction; GC was used to measure the content of the ARA. The results indicated that extraction temperature of 32℃, extraction pressure of 16mPa,extraction time of 101min and Taking 10% methanol as entrainer were the most Effective conditions, Which could obtain extraction rate of 44.6% and purity of 29.34%.Through the variance analysis and verification test ,it showed that mathematical model was reliable.
An orthogonality experiment was employed to determine the optimum conditions for purification of ARA by urea clathration.GC was used to measure the content of the ARA. The results indicated that urea:fatty acid of 4:1, fatty acid:ethanol of 1:12, clathration temperature of -15℃and clathration time of 12h, the purity of ARA was 36.84%.
The clone of△5-、△6-desaturase gene. Full cDNA of Mortierlla isabellina AS3.3410、W35s2-153、Z80s2-109、Y-69、YZ-124 5 strain△5-、△6 -desaturase gene was obtained by RT-PCR and gene clone based on the original strain of Mortierlla isabellina and the mutant strain of high-yield AA and its sequence was analyzed. The result showed that the full-length cDNA sequence of 5 strain Mortierlla isabellina△6-desaturase gene was 1374bp and 457 amino acids were coded.△5-desaturase gene was 1341bp and 446 amino acids were coded. The cDNA and amino acid sequence were analysed with biological information software. The results showed that the△5-、△6-desaturase gene sequence of Mortierlla isabellina AS3.3410 original strain had higher homology with Genebank published sequence, while nucleotide sequences of the mutant strain had low variation. Animo acid sequences of desaturase gene of the mutant strain had lower homology Compared with original strain sequence, which could show the protein of mutant strain probable had a certain variation.
The relation on activity and expression levels of desaturase and ARA quantity. The activities of 5 strain Mortierlla isabellina at the same culture time and high-yield ARA strain YZ-124 at the different culture time were determined by TTC-desaturase activity determining method and the experimental condition was established by using fluorescence quantitative PCR method to determine the mRNA expression levels of Mortierlla isabellina△5-、△6 -desaturase gene. The results showed that the desaturase activities of different strain and different cultured time were different and the desaturase activity of original strain AS3.3410 was the lowest while the mutant strain YZ-124 was the highest. The activities of desaturase cultured in 3-5d increased obviously and were basicly stable after 5d.The results of fluorescence quantitative PCR testing showed the mRNA expression level of different strain△5-、△6 -desaturase gene were different.The desaturase gene mRNA expression quantity of Mortierlla isabellina YZ-124 at different culture time was different, the△5、△6 -desaturase mRNA expression quantity of 3-8d cultures gradually increased with bacterium age increasing, and the expression quantity reached the highest at 8d.The activity and expression levels of desaturase and ARA quantity in cultured oil presented a positive correlation with ARA acquisition.
以深黄被孢霉为出发菌株,分别采用微波诱变、紫外诱变、原生质体再生诱变和紫外诱变原生质体等方法进行诱变,利用气相色谱分析花生四烯酸含量,最终获得四株花生四烯酸高产菌株,其中YZ-124花生四烯酸产量最高,为4.72 g/L,比出发菌株提高576 %,并且遗传性能稳定。采用CO2超临界萃取技术提取微生物油脂中的花生四烯酸,花生四烯酸提取率为45.7%,花生四烯酸含量为19.34%。
采用尿素包合法纯化花生四烯酸,最终得到产品中花生四烯酸含量为36.84%。以深黄被孢霉原始菌株及高产花生四烯酸的诱变菌株为基础,利用RT-PCR及基因克隆方法获得了5株深黄被孢霉的△5、△6-脱饱和酶基因的完整cDNA,对其进行序列分析,结果表明:诱变菌株与原始菌株的核苷酸及氨基酸序列同源性较高。利用TTC-脱氢酶活性测定法及荧光定量PCR方法测定了5株深黄被孢霉的同一培养时间及高产花生四烯酸菌株YZ-124在不同培养时间的活性及mRNA表达量,结果显示:脱饱和酶的活性及表达量与培养物油脂中花生四烯酸含量呈正相关关系。
Arachidonic acid is an essential dietary component for human beings,and has various physiological functions and plays an important role in infant nutrition. Arachidonic acid(ARA), widely existing in the animal kingdom,can be isolated from the lipids extracted from pig adrenal gland or pig liver and sardines as well; however, the yield of it is only 0.2%(w/w) or lower, so it is very dificult to be applied in a industrial scale and the research and application are restricted.It has been focused on the study of ARA fermented by microbe in the whole world.△5-、△6-desaturase is the key enzyme of arachidonic acid synthetic pathway. Evaluating the relation of desaturase and ARA production can further establish its function on biological Synthesis.The aim of this study is to selectively breed the high-yield ARA strain, to optimize the technology parameter of extraction and purification for ARA ,to study the gene sequence variation of Mortierlla isabellina mutant strain desaturase, to explore the activity of desaturase and the relationship between the expressed quantity and ARA production.
Mortierella isabellina3.3410 was used as initial strain, microwave mutation, ultraviolet mutation, protoplast mutation and ultraviolet mutation protoplast were used to mutate, and then the content of the ARA was measured by GC. The ultimum four strains who had the most ARA yield and genetic stability were obtained. Under powe of microwave at 700W and heating time of 35s,lethality of spores of Mortierella isabellina was 78.3%.After two-time microwave mutation and repeated screen, a mutation of Mortierella isabellina W35s2-153 was obtained,whose ARA yield was 2.61g/L, 3.18 times of the control strains.The power of viltalight lamp was 20W,exposure distance was 30cm and exposure time was 80s,lethality of spores of Mortierella isabellina was 76.4%.After two-time ultraviolet mutation and repeated screen,a mutation of Mortierella isabellina Z80s2-109 was obtained, whose ARA yield was 2.74g/L, 3.77 times of the control strains.After protoplast mutation, enzyme concentration at 4%,enzymolysis temperature at 30℃and enzymolysis time at 7.5h,the rate of protoplast was 78.4%.After mutation and repeated screen,a mutation of Mortierella isabellina Y-69 was obtained whose ARA yield was 2.92g/L and the concentration in biomass were 3.56 times of the control strains. After ultraviolet mutation protoplast, the power of viltalight lamp at 20W ,exposure distance at 30cm and exposure time at 80s,lethality of spores of Mortierella isabellina was 76.4%,a mutation of YZ-124 was obtained,whose ARA yield was 4.72 g/L , 5.76 times of the control strains.
The process condition optmization of arachidonic acid extraction from microbial oil Was carried out by means of supercritical CO2 fluid technology.It was used to increase the extraction rate of arachidonic acid in microbial oil. The supercritical CO2 fluid technology was used to extract the ARA from microbial oil; an orthogonal, rotatable, central composite was employed to determine the optimum conditions for extraction; GC was used to measure the content of the ARA. The results indicated that extraction temperature of 32℃, extraction pressure of 16mPa,extraction time of 101min and Taking 10% methanol as entrainer were the most Effective conditions, Which could obtain extraction rate of 44.6% and purity of 29.34%.Through the variance analysis and verification test ,it showed that mathematical model was reliable.
An orthogonality experiment was employed to determine the optimum conditions for purification of ARA by urea clathration.GC was used to measure the content of the ARA. The results indicated that urea:fatty acid of 4:1, fatty acid:ethanol of 1:12, clathration temperature of -15℃and clathration time of 12h, the purity of ARA was 36.84%.
The clone of△5-、△6-desaturase gene. Full cDNA of Mortierlla isabellina AS3.3410、W35s2-153、Z80s2-109、Y-69、YZ-124 5 strain△5-、△6 -desaturase gene was obtained by RT-PCR and gene clone based on the original strain of Mortierlla isabellina and the mutant strain of high-yield AA and its sequence was analyzed. The result showed that the full-length cDNA sequence of 5 strain Mortierlla isabellina△6-desaturase gene was 1374bp and 457 amino acids were coded.△5-desaturase gene was 1341bp and 446 amino acids were coded. The cDNA and amino acid sequence were analysed with biological information software. The results showed that the△5-、△6-desaturase gene sequence of Mortierlla isabellina AS3.3410 original strain had higher homology with Genebank published sequence, while nucleotide sequences of the mutant strain had low variation. Animo acid sequences of desaturase gene of the mutant strain had lower homology Compared with original strain sequence, which could show the protein of mutant strain probable had a certain variation.
The relation on activity and expression levels of desaturase and ARA quantity. The activities of 5 strain Mortierlla isabellina at the same culture time and high-yield ARA strain YZ-124 at the different culture time were determined by TTC-desaturase activity determining method and the experimental condition was established by using fluorescence quantitative PCR method to determine the mRNA expression levels of Mortierlla isabellina△5-、△6 -desaturase gene. The results showed that the desaturase activities of different strain and different cultured time were different and the desaturase activity of original strain AS3.3410 was the lowest while the mutant strain YZ-124 was the highest. The activities of desaturase cultured in 3-5d increased obviously and were basicly stable after 5d.The results of fluorescence quantitative PCR testing showed the mRNA expression level of different strain△5-、△6 -desaturase gene were different.The desaturase gene mRNA expression quantity of Mortierlla isabellina YZ-124 at different culture time was different, the△5、△6 -desaturase mRNA expression quantity of 3-8d cultures gradually increased with bacterium age increasing, and the expression quantity reached the highest at 8d.The activity and expression levels of desaturase and ARA quantity in cultured oil presented a positive correlation with ARA acquisition.
引文
[1]刘长征,田安思,陈陵.不饱和脂肪酸与脑功能关系的研究进展[J].医学综述,2003,9(7):401-403.
[2]黄绍重,秦振华.ω-3多不饱和脂肪酸对不同群体的保健价值及疾病防治[J].医药产业资讯,2006,3(3):41-43.
[3]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):289-292.
[4]姚昕,秦文,齐春梅,等.花生四烯的生理活性及其应用[J].粮油加工与食品机械,2004,(5):57-59.
[5]秦振华.多不饱和脂肪酸及其保健价值[J].实用医技杂志,2006,13(5):777-778.
[6]毛峰,秦振华.长链多不饱和脂肪酸与人体健康[J].医药产业资讯,2006,3(3):40-41.
[7]杨朝霞,张丽,李朝阳.花生四烯酸的营养保健功能[J].食品与药品,2005,7(1):69-71.
[8]王啸,邱树毅.微生物发酵生产花生四烯酸的研究进展[J].中国油脂,2004,29(9):37-40.
[9]符嫦娥,韩伟,卞进发,等.花生四烯的研究进展[J].云南化工, 2004,31(5):31-34.
[10]于长青,李丽娜.花生四烯酸研究进展[J].农产品加工(学刊),2007,(4):10-12.
[11]张永刚,印遇龙,黄瑞林.等.多不饱和脂肪酸的营养作用及其基因表达调控[J].中国饲料,2006,13:9-12.
[12]张伟利,吴圣楣,钱继红,等.母乳中二十二碳六烯酸及花生四烯酸含量的观察[J].中华围产医学杂志,2002,5(1):51-55.
[13]Brick E E,Garfield S,Hoffman DR,et al.A randomized controlled trial of early dietary supply of long chain polyunsaturated fatty acids and mental development in term infants. Developmental Medicine and Child Neurology.2000,42:173-181
[14]施东奎,胡春梅.花生四烯酸的主要作用及提取方法[J].中国中药杂志,2007,32(11):1009-1011.
[15]钱继红,吴圣楣,张伟利,等.含二十二碳六烯酸和花生四烯酸的配方奶对婴儿生长发育的研究[J].实用儿科临床杂志,2000,15(5):256-257.
[16]何平,汪志明,何卫加.花生四烯酸在婴幼儿配方食品中的应用[J].加工技术,2003,1:52.
[17]何平.花生四烯酸在奶粉中的应用[J].中国乳品工业,2003,31(3):61-62.
[18]张小平,葛庆丰,顾瑞霞,等.配方乳粉中花生四烯酸(ARA)添加工艺条件研究[J].添加剂,2004,8:31-35.
[19]孙程,左曙辉.新型营养强化剂在液体乳制品中的应用[J].中国食品添加剂,2003,1:77-78.
[20]刘洋,谢飞.被孢霉发酵生产花生四烯酸的研究进展[J].江苏食品与发酵,2007,2:12-16..
[21]Nigam S, Fiore S, Luscinskas F W et al. J Cell Physiol,1990, 143(5):511-523.
[22]汪志明.花生四烯酸生产及应用[J].中国食品添加剂,2001,1:30-33.
[23]汤世华,陈明锴,杨建斌,等.深黄被孢霉产脂的研究[J].中国油脂,2007,32(12):35-37.
[24]黄建忠,施巧琴,周晓兰,等.高产脂微生物-深黄被孢霉M018变株的选育及其油脂合成条件的研究[J].药物生物技术,1998,5(4):232-237.
[25]黄惠琴,鲍时翔,朱法科,等.被孢霉发酵生产花生四烯酸工艺的研究[J].华南热带农业大学学报,1999,5(2):1-4
[26]刘吉华,袁生,戴传超.轮枝霉(Diasporangiun sp.)产生EPA、ARA发酵条件的初步研究[J].食品与发酵工业,2000,26(4):9-12.
[27]王志举,尚耘,李翔宇,等.花生四烯酸油脂的产业化生产何工艺改进[J].中国乳品工业,2005,33(10):21-24.
[28]周蓬蓬,余龙江,吴元喜,等.高山被孢霉产花生四烯酸发酵条件的研究[J].工业微生物,2003,33(2):41-44.
[29]贡国鸿,尚耘,高翔,等.微生物发酵法生产花生四烯酸油脂[J].中国油脂,2002,27(1):42-44.
[30]尚耘,贡国鸿,高翔,等.发酵法生产新型营养强化剂-花生四烯酸[J].中国食品添加剂,2002,1:61-65.
[31]王啸,邱树毅,叶丹,等.花生四烯产生菌的选育[J].贵州工业大学学报(自然科学版),2005,34(1):56-59.
[32]赵沫,戴传超,王安琪,等.高产花生四烯酸菌的原生质体诱变育种研究[J].食品科学,2008,29(2):275-279.
[33]黄娟,陈正杰,冯军,等.紫外和微波结合诱变选育Lipstatin产生菌[J].微生物药物与生物技术,2006,37(1):11-14.
[34]何东平.高产PUFAs菌株的选育及其制备工艺的研究[C].武汉:中国科学院武汉病毒研究所,2001.
[35]Chen Zhi,Lin Wen,Yu Ping,et al.Enhancement of monacolin K production via intergeneric protoplast fusion between aspergillus terreus and monascus anka[J].Pro Gress in Natural Science,2007,17(6):703707.
[36]Kazuhiro Miyazaki,Hajime Maeda,Masahid Sunagawa,et al.Screening of heterozygous DNA markers in shitake(Lentinula edodes)using de-dikaryotization via preparation of protoplasts and isolation of four meiotic monokaryons from one basidium[J].The Japan Wood Research Society,2000,46:395-400.
[37]Zhang Ying,Gong Sufang,Wang Jingang,et al.Research on protoplast preparation and culture in gladiolus hybridus hort[J].Journal of Northeast Agricultural University,2007,14(1):5-8.
[38]Andrew J.Morgan,Aurora Brunner,Peter A.Whittaker.Protoplast fusion in a petite-negative yeast,kluyveromyces lactis[J].Current Genetics,1980,2:87-93.
[39]袁成凌,王纪,姚建铭,等.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[J].高技术通讯,2003,6:21-25.
[40]周蓬蓬,余龙江,李为,等.YAG激光照射对高山被孢霉花生四烯产量的影响[J].激光生物学报,2002,(05):371-376.
[41]朱敏,刘智,余龙江,等.高山被孢霉Δ5脱饱和酶基因的分离与验证[J].遗传学报,2005,32(9):986-992.
[42]朱法科,林炜铁,鲍时翔,等.花生四烯酸高产菌株的选育[J].工业微生物,1999,29(2):1-4.
[43]王安琪,戴传超,赵沫,等.轮梗霉高产花生四烯酸的诱变育种研究[J].食品科学,2007,28(2):158-162.
[44]咸漠,康亦兼,孙岩,等.深黄被孢霉的化学诱变[J].吉林大学自然科学学报,2001,1:92-95.
[45]Eiji Sakuradani,Yuriko Hirano,Nozomu Kamada et al.Improvement of arachidonic acid production by mutants with lower n-3 desaturation activity derived from Mortierella alpine IS-4[J].Appl Microbiol Biotechnol, 2004, (66):242-248
[46]Eiji S,Takahiro A,Ketta I,et al.A novel fungalω2-desaturase with wide substrate specificity from arachidonic acid-producting Mortierella alpina IS-4[J],Appl Microbiol Biotechnol,2005,66:648-654.
[47]杨革,王玉萍,李翔太,等.花生四烯酸高产突变株的选育及其发酵调控[J].菌物系统,1998,17(1):86-90.
[48]王啸,邱树毅,何腊平,等.UV、LiCl复合诱变深黄被孢霉选育多不饱和脂肪酸高产菌株[J].食品科学,2004,25(4):50-52.
[49]周蓬蓬,余龙江,朱敏,等.花生四烯产生菌的原生质体诱变育种[J].华中理工大学学报,2000,28(07):105-110.
[50]Chengling Yuan,Ji Wang,Yun Shang.Production of arachidonic acid by mortierella alpina I49-N18[J].Food Technology and Biotechnology,2002,40(4):311-315.
[51]袁成凌,姚建铭,王纪,等.低能离子注入在花生四烯酸(ARA)高产菌株选育中的研究[J].辐射研究与辐射工艺学报,2003,21(04):237-242.
[52]周蓬蓬,余龙江,汪建华,等.微波等离子体溅射诱变选育花生四烯高产菌及补料工艺研究[J].激光生物学报,2003,12(01):59-62.
[53]周蓬蓬,秦文敏,余龙江,等.毛霉油对被孢霉产花生四烯酸的影响[J].生命科学研究,2002,6(3):246-249.
[54]周蓬蓬,秦文敏,余龙江,等.KLa对高山被孢霉产花生四烯酸的影响[J].生命科学研究,2002,6(4):339-342.
[55]周蓬蓬,秦文敏,余龙江,等.表面活性剂对被孢霉产花生四烯酸的影响[J].华中科技大学学报(自然科学版),2003,31(5):98-100.
[56]马雪瑞.Co60辐射诱变深黄被孢霉高产多不饱和脂肪酸突变株的选育[C].武汉:中国科学院研究生院,2004.
[57]朱敏,余龙江,肖靓,等.高山被孢霉的红四氮唑染色程度与菌体油脂中花生四烯酸量的关系[J].生命科学研究,2004,8(4):339-343.
[58]Seki Takeno, Eiji Sakuradani,Akiko Tomi et al.Trasformation of Oil-Producting Fungus,Mortierella alpina IS-4.Using Zeocin,and Application to Arachidonic Acid Production[J].Journal of Bioscience and Bioengingeering 2005,100(6):617-622.
[59]朱敏,余龙江,刘智,等.花生四烯酸高产菌M6的鉴定[J].菌物学报,2004,23(4):508-513.
[60]赵广民,郝书杰.花生四烯酸生物提取和纯化[J].肉类工业,2000,7:31-32.
[61]袁成凌,余增亮,贺敬翠,等.富含花生四烯酸的微生物油脂的室内精炼及其成分测定[J].中国油脂,2001,26(4):17-18.
[62]杨小花,苏宝根,杨亦文,等.花生四烯酸分离纯化方法研究进展[J].粮食与油脂,2006,(2):12-16.
[63]袁成凌,余增亮,刘昌伟,等.脲包法富集微生物油脂中花生四烯酸的研究[J].营养研究,2002,(5):67-69.
[64]陈文利.尿素包合法纯化油脂中花生四烯酸的工艺研究[J].浙江化工,2003,34(8):28-29.
[65]黄和,李学坤,张昆,等.应用尿素包合法提纯花生四烯酸[J].化学与生物工程,2006,23(4):29-31.
[66]陈文利,余龙江,欧阳贻德,等.银离子络合萃取法纯化油脂中花生四烯酸[J].武汉化工学院学报,2003,25(2):16-17.
[67]袁成凌,余增亮,刘昌伟等.CO2超临界萃取技术富集微生物油脂中花生四烯酸的研究[J].中国油脂,2002,27(6):48-50.
[68]陈文利,朱敏,于龙江,等.超临界CO2萃取被抱霉菌体油脂及花生四烯酸的研究[J].精细化工中间体,2003, 33(1):33-37.
[69]袁成凌,张鉴,王相勤,等.高效液相色谱法分离纯化微生物油脂中的花生四烯酸[J].色谱,2003, 21(1):60-62.
[70]王明霞,黄凤洪.多不饱和脂肪酸萃取中超临界技术的应用进展[J].中国油脂,2005,30(8):60-64.
[71]王宪达,黄梅,杨亦文,等.超临界流体色谱法制备EPA-EE和DHA-EE[J].化工学报,2003,54(11):1558-1562.
[72] Muphy D J,Piffaneth P.Fatty acid desaturase:structure mechanism and regulation In Plant Lipid Biosynthesis[J].Recent Advances of Agri-cultural importance,1998,11:95-130.
[73] Gill l,Valivety R.Polyunsaturated fatty acids,part l:occurrence,biological activities and application[J].Trends Biotechnol,1997,15:401-409.
[74] Brick E E,Garfield S,Hoffman D R,Uauy R,Birch D G.A randomized controlled trial of early dietary supply of long chain polyunsaturated fatty acids and mental development in term infants[J].Dev Med Child Neuro1, 2000,42:174-181.
[75] Gunstone F D.Gamma linolenic acid occurrence and physical and chemical properties[J].Prog Lipid Res,1992,31(2):145-161.
[76] Yung-Sheng H,Sunita C,Jennifer M T, et a1.Cloning of△12-and△6 -desaturase from Mortierella aplina and recombinant production ofγ-linolenic acid in Saccharomyces cerevisiae[J].Lipids,1999,34(7):649-659.
[77] Sayanova O,Smith M A,Lapinskas P, et a1.Expression of a borage desaturase cDNA containing an N-terminal cytochrome b5 domain resuits in the accumulation of high levels of△6-desaturated fatty acids in transgenic tobacco[J].Proc Natl Acad Sci USA ,1997,94:421l-4216.
[78] Mount S M.A catalogue of splice junction sequences[J].Nucleic Acids Research,1982,10:459-472.
[79] Robin R,Tom M.1ntron sequence involved in lariat formation during pre-mRNA splicing[J].Cell,1985,41:95-105.
[80] Kajikawa M,Yamato K T,Kohzu Y.Isolation and characte rization of△6-desatu rase,an ELO-Like enzyme and△5-desaturase from the Liverwort Marchantia Polymorpha and production of arachidonic and elcosapentaenoic acids in the methylotrophic Yeast Pichia Pastoris[J].Plant Mo1 Bio1,2004,54:335-352.
[81] Nicola H,Morris A,Douglas R T,et a1.A vertebrate fatty acid desaturase with△5 an d△6 activities[J]. Biochemistry,2001,25:14304-14309.
[82] Ll M C,LIU L,ZHANG L ,et a1.Cloning and sequencing analysis of△6-fatty acid desaturase gene from Mortierella isabellina[J]. Mycosystema, 2001, 20(1):44-50.
[83] LIU L,Ll M C,HU G W,et a1.Identification of Mortierella isabellina M6-22△6-fatty acid desaturase by heterologous expression in Saccharomyces cerevisiae[J].Acta Microbiologica Sinica,2001,41(4):397-401.
[84] Reddy A S,Nuccio M L,Gross L M ,et a1.Isolation of a△6-desaturase gene from the cyanobactefium Synechocystis sp.strain PCC6803 by gain-of-function expression in Anabacna SP.Strain PCC7120[J].Plant Molecular Biology, 1993, 27:293-300.
[85] Napicr J A,Sandru J H,Dominic J L et a1.Identification of a Caenorhabditiselegans△6-fatty-acid-desaturese by heterolagous expression in Saecharomyces cerevisiae.Biochem J,1998,330:61l-614.
[86] Tsunehiro A,Yayoi S,Katsuya Iet a1.Molecular cloning and functional characterization of rat△6-fatty acid desaturase.Biochemca1 and Biophysica1 Research Communications,1999,255:575-579.
[87] Sayanova O,Smith M A,Lapinskas P,et a1.Expression of a borage desaturase cDNA containing an N-terminalcytochrome b5 domain results in the accumulation of high levels of△6-desaturated fatty acids in trangenic tobacco[J].Proc Natl Acad Sci USA,1997,94:4211-4216.
[88] Liu Li,Li Mingchun,Hu Guowu,et a1.Identification of Mortierella isabellina M6-22△6 -fatty acid desaturase gene by heterologous expression in Saccharomyces cerevisiae[J].Acta Microbiologica Sinica,2001,41(4):397-401.
[89]李明春,刘莉,胡国武,等.深黄被孢霉△6-脂肪酸脱氢酶基因在转基因烟草中的表达.生物工程学报.2003,19(2):178-184.
[90] Sakuradani E,Kobayashi M,Shimizu S.△6-fatty acid desaturase from an arachidonic acid-producing Mortierella fungus Gene cloning and its heterologous expression in a fungus Aspergillus[J].Gene,1999,238:445-453.
[91] Huang Y S H,Sunita C,Jennifer M T.et al.Cloning of△6 2-and△6 -desaturase from Mortierella aplina and recombinant production ofγ-linolenic acid in saccharomyces cerevisiae[J].Lipids,1999,34(7):649-659.
[92] Napier J A,Sandra J H,Dominic J L,et a1.Identification of a Caenorhabditis elegans△6-fatty-acid-desaturase by heterelogous expression in Saccharomyces cerevisiae[J].Biochem J,1998,330:611-614.
[93] Petra S,Michael L,Thomas G,et a1.A bifuncfional△6-fatty acyl acetylenase /desaturase from the moss Ceratodon purpureus.Eur JBiachem, 2000, 267: 3801-3811.
[94] Sakuradani E,Kobayashi M,Shimizu S.△6-Fatty acid desaturase from an arachidonic acid-producing Mortierella fungus gene cloning and its heterologous expression in a fungus[J].Aspergillus.Gene.1999,238:445-453.
[95] Botha A,Paul I,Roux c,et a1.An isolation procedure for arachidonic acid producing Monierella species[J].Amonie van Leeuwenhoek.199l9,75,253-256.
[96] Botha A,Paul l,Roux C,Kock J L F,Coetzee D J,Strauss T,Maree C.An isolation procedure for arachidonic acid producing Mortierella species[J].Antonie van Leeuwenhoek,1999,75:253-256.
[97]肖靓,刘智,朱敏,等.实时PCR分析△5脱饱和酶在花生四烯酸合成中的作用[J].中国生物工程杂志.2006,26(8):57-61.
[98] Shimada T,Somlyo A P.Modulation of voltage-dependent Ca channel current by arachidonic acid and other long-chain fatty acids in rabbit intestinal smooth muscle[J].J Gen Physio1,1992,100:27-44.
[99] ChenH C,ChangC C,Chen CX.Optimization of arachidonic acid production by Mortierella alpina Wuji-H4 isolate[J].Joumal of the American Oil Chemists’Society,1997,74:569-578.
[100] Zhu M,Yu L J,Liu Z,et a1.Isolating Mortierella alpina strains of high yield of arachidonic acid[J].Leters in Applied Microbiology,2004,39:332-335.
[101] Zhu M,Yu L J,Liu Z,et a1.Identification of arachidonic acid high yielding strain M6[J].Mycosystem,2004,23(4):508-513.
[102] Watts J L,Browse J.Isolation and characterization of a△5-fatty acid desaturase from Caenorhabditis elegans[J].Arch Biochem Biophys,1999,362(1):175-182.
[103]朱敏,刘智,余龙江,等.高山被孢霉△5脱饱和酶基因的分离与验证[J].遗传学报2005,32(9):986-992.
[104] Eroshin V K,Dedyukhina E G,Satroutdinov A D.G rowthcoupled lipid synthesis in Mortierella alpina LPM 301 a producer of arachidonic acid[J]. Microbiology, 2002,71:1 69-172.
[105] Zhu M,Yu L J,Wu Y X.An inexpensive medium for production of arachidonic acid by MortiereIla alpine[J].J Ind Microbiol Biot,2003,30:75-79.
[106] Chung HW.Reverse transcriptase PCR (RT-PCR) and quantitative-competitive PCR (QC-PCR) [J].Exp Mol Med,2001,33(1 Suppl): 85-97.
[107] Cheng B. Comparing RNA PCR test [J] .PI Perspect .,1995,(17):15-18.
[108] L Wen,Effect of primer purity on the banding patterns of differential display polymerase chain reaction[J].J.Biomol.Tech.,2000,11:87-91.
[109] Kim BR,Nam HY,Kim SU.Normalization of reverse transcription quantitative- PCR with housekeeping genes in rice[J].Biotechnol Lett., 2003,25(21):1869-1872.
[110] Ke LD,Chen Z,Yung WK.A reliability test of standard-based quantitative PCR:exogenous vs endogenous standards[J].Mol.Cell Probes, 2000, 14(2): 127-135 .
[111]张维铭主编,现代分子生物学实验手册[M].北京:科学出版社出版.2003.
[112] Goidin D,Mamessier A,Staguet T M,et a1.Ribosomal 18S RNA prevails over glyceraldehyde-3-phosphate dehydrogenase andβ-actin genes as internal standard for quantitative comparison of mRNA levels in invasive and noninvasive human melanoma cell subpopulations[J].Analytical Biachemistry, 2001,295:l7-2l.
[113]胥保华,许梓荣.肉雏鸡肝细胞谷胱甘肽过氧化物酶RT-PCR定量分析[J].中国兽医科技,2003,33(2):24-26. [114 ]Annemarie Berger,Wolfgang Preiser. Viral genomequantification as a tool for improving patient management :the example of HIV,HBV,HCV and CMV[J].J.Antimicrob.Chemother,2002,49:713-721.
[115] James C.Paton and Adrienne W.Paton.Pathogenesis and Diagnosis of Shiga Toxin-Producing Escherichia coli Infections[J]. Clin. Microbiol. Rev., 1998, 11:450-479
[116]肖白,刘敬忠,王建秋.半定量PCR检测致龋性变形链球菌的技术及应用[J].中国实验诊断学,2003,7(2):109-112
[117] Schnell S,Mendoza C.Enzymological considerations for a theoretical description of the quantitative competitive polymerase chain reaction (QC-PCR) [J].J Theor.Biol.,1997,184(4):433-440.
[118]马中富,黄帆,高劲松.以cDNA为内参标RT-PCR定量检测心肌细胞内p53和Bcl-2基因mRNA表达量的方法研究[J].中国危重病急救医学.2002,14(10):597-601.
[119]程正江.用统计学方法解释和处理临床定量PCR实验结果[J]数理医药杂志,2003,1:18-21.
[120] F Ferre. Quantitative or semi-quantitative PCR: reality versus myth[J].Genome Res,1992,2:1-9.
[121] Actor JK,Limor JR,Hunter RL.A flexible bioluminescent-quantitive polymerasereaction assay for analysis of compeitive PCR amplicons[J].J Clin Lab Anal.,1999,13(1):40-47.
[122] Anderson KM,Cheung PH,Kell MD,Rapid generation of homologous internal standards and evaluation of data for quantitaion of messenger RNA by competitive polymerase chain reaction[J].J pharmacol Toxicol Methods, 1997,38:133-140.
[123] Tartaglia M,Fragale A,Battaglia PA.A competitive PCR-based method to measure human fibroblast growth factor receptor 1-4 gene expression[J].Cell Biol.,2001,20(6):367-379.
[124] Montagne A,Grepinet O,Peloille M,et al.Quantification of ovine cytokine gene expressiont by a competitive RT-PCR method[J].J Immunol Methods, 2001,253(1-2):83-93.
[125] AI-Robaiy S,Rupf S,Eschrich K.Raoid competitive PCR using melting curve analysis for DNA quantification[J].Biotechniques,2001,31(6):1382-1388.
[126]王宁.定量PCR技术研究进展[J].国外医学:临床生物化学与检查学分册.2000,21(6):314-316.
[127] Wuhu A,Waiztu M,Koch A.A rapid and sensitive protocol for competitive reverse transcriprase (CRT)PCR analysis of cellular genes[J].Brain Pathol., 1998,8:13-18.
[128] Liljekvist-Larsson I,Torngren M,Abrahamson M.Johansson K.Growth of the postnatal rat retina in vitro:quantitative RT-PCR analyses of mRNA expression for photoreceptor protein[J].Mol Vis.,2003,9:657-64.
[129]牛艾如,陈培佳.定量DNA和Igm抗性检测CMV感染[J].中国优生与遗传杂志,2002,10(6):40-41.
[130]张立国,张琚.实时定量PCR技术的介绍[J].生物技术,2003,13(2):39-40.
[131] Kazutoshi Kaji,Kazuhiro Ueda,Takashi Aoyama.Development of a real-time jump-ratio imaging system equipped with a stem[J].Journal of Electron Microscopy 2001,50:15-21.
[132] Ke LD,Chen Z,Yung WK.A reliability test of standard-based quantitative PCR:exogenous VS endogenous standards[J].Mol Cell Probes,2000,14(2):127.
[133] Becker KD, Pan,Whitely CB.Real-time quantitative pelymerase chain reactionto assess gene transfer[J]. Hum Gene Ther.,1999,10:2559.
[134] Higgis JA.Nuclease PCR assay to detect Yersinia pesits[J].J Clin Microbiol.,1998,36:2284.
[135]阳成波,印遇龙,龚建华,等.定时定量PCR研究进展及其应用[J].中国预防兽医学报,2003,25(5):395.
[136] Gibson U E.A novel method for real time quantitative RT-PCR[J]. Genome-Res,1996,6:995-1001.
[137] Wei S F,Wang SL,CHANG J. Influence of ultraviolet radiation on the expression of preliferating cell nuclear antigen and DNA polymerase alpha in Skeletonema costatum (Bacillariophyceae)[J].J Phycol,2004,40(4):655-663.
[138] Hahnen S,Joeris T, Kreuzaler F, et al.Quantification of photosynthetic gene expression in maize C3 and C4 tissues by real time PCR[J]. Photosynthesis Research,2003,75(2):183-192.
[139]翟新验.实时TaqMan技术在兽医学中的应用[J].实验动物科学与管理,2002,19(2):50.
[140]罗长保,鱼海琼,林志雄.应用荧光RT-PCR技术检测口蹄疫病毒[J].中国兽医杂志,2003,39(12):31.
[141] Mason G,Provero P.Varia A M,et al.Estimating the number of integrations in transformed plants by quantitative real-time PCR[J].BMC Biotechnic Res,2002,11(1):43-48.
[142] Tesson L,Heslan J M, Menoret S,et al.Rapid and ac. curate determination of zygosity in transgenic an imals by real-time quantitative PCR[J].Transgenic Trend ,2003,7(1):76-79.
[143] Shah C A,Bon J,Bisset L R,et al.Ultra-sensitive and specific detection of porcine endogenous retrovirus using a sequence-capture real-time PCR approach[J].J Virol Methods,2002,109(2):209-216.
[144] Park S B,Popovich B W,Press R D .Real-time polymerase chain reaction with fluorescent hybridization pmbes for for the detection of prevalent mutations causing common thrombophilic and iron overload phenotypes[J].Am J Clin Pathol.,2001,115:439-447.
[145]徐秋英,彭胜权,刘亚敏,等.荧光定量PCR技术在评价中医药治疗乙型肝炎中的价值[J].新中医,2002,34(10):71-72.
[146] Funato T,Fujimaki S,Fujiwara J,et al.Genetic diagnosis for drug resistance in cancers[J].Rinsho Byori.,2000,48(2):162-166.
[147]范瑞强,陈信生,棚国维,等.荧光定量PCR观察中药抗病毒胶囊对豚鼠HSV-2系统感染的影响[J].中华皮肤科杂志,2003,36(5):276-278.
[148] Higashiyama K, Fujikawa S,Park EY, et al.Production of arachidonic acid by Mortierella fungi[J]. Biotechnology and Bioprocess Engineering. 2002, 7(5): 251-262.
[149] Jin MJ,Huang H, Xiao AH,et al.A novel two-step fermentation process for improved arachidonic acid production by Mortierella alpina[J]. Biotechnology Letters.2008,30(6):87-91.
[150]Kenichi Higashiyama,Shigeaki Fujikawa,Enoch Y.Park et al.Production of Arachidonic Acid by Mortierella Fungi[J].Biotechnol.Bioprocess Eng. 2002,7(5):251-262.
[151]Takeno S, Sakuradani E, Tomi A, et al.Trasformation of oil-producting fungus,Mortierella alpina IS-4.using zeocin,and application to arachidonic acid production.Journal of Bioscience and Bioengingeering.2005,100(6):617-622.
[152]James G M,Paul R,Daniel F.et al.Production of polyunsaturated fatty acids by polyketide synthases in both prokaryotes and eukaryotes[J]. science, 2001,293:290-293.
[153]吕晴,余德顺,雷邦星,等.被孢霉菌丝体脂肪酸组成的气相色谱-质谱分析[J].分析测试学报,2003,22(2):21-24.
[154]邹耀洪,陆云琦.婴儿奶粉中的二十二碳六烯酸和二十碳四烯酸气相色谱分析[J].常熟高专学报,2004,18(2):33-37.
[155]葛庆丰,张小平,顾瑞霞,等.乳粉中花生四烯酸(ARA)测定方法研究[J].添加剂,2004,10-38-39.
[156]薛刚,张进,张均媚,等.婴儿配方奶粉中花生四烯酸含量的测定[J].食品研究与开发,2006,27(5):119.
[157]王相勤,姚建铭,袁成凌,等.高产花生四烯酸产生菌干菌体中脂肪酸成分的气相色谱-质谱分析[J].分析化学,2001,29(3):287-289.
[158]K Goodrum J.W.,Kilgo M.B.Peanut oil extraction with SC- CO2:solubility andkinetic functions[J].Transactins of the ASAE,1987,30(6):1865-1868.
[159]麻成金,张永康,马美湖,等.微波和超临界CO2萃取杜仲籽油工艺研究[J].食品科学,2006,27(6):131-135.
[160]苏香萍,宋必卫,陈振华,等. CO2超临界萃取金银花挥发油工艺及抗炎活性研究[J].天然产物研究与开发,2006,18:662-666.
[161]Toshihiko Osaki,Toshiki Mori.Kinetic studies of CO2 dissociation on supported ni catalysts[J].Akademiai kiado,budapest and springer, dordrecht. 2006, 87(1): 149-156.
[162]Olivera R.,Rodrigues M.F.,Bernardo-Gil M.G.Charactrerization and supercritical carbon dioxide extraction of walnut oil[J].Journal of the american oil chemists` society,2002,79(3):225-230.
[163]闫志芳,刘必旺,赵水平. CO2超临界萃取蛇床子中蛇床子素的工艺研究[J].世界中西医结合杂志,2009,4(1):20-22.
[164]赵淑英,王秋芬,宋湛谦. CO2超临界萃取印楝素的研究[J].福建林学院学报,2005,25(2):152-156.
[165]吕晓玲,李肇奖. CO2超临界萃取油茶皂苷的研究[J].食品与发酵工业,2005,31(1):22-26.
[166]黄纪念,屠鹏飞,蔡同一.超临界CO2流体萃取迷迭香中抗氧化活性成分的工艺研究[J].中草药,2004,35(2):150-153.
[167]邓亦峰,梁念.半边旗中二萜类化合物的超临界CO2萃取及HPLC-MS分析[J].中药.2004,35(2):145-148.
[168]黄欣,苏乐群,邵伟,等.白芷香豆素类化合物的成分分析及CO2超临界萃取工艺的研究[J].中国药房.2005,16(11):823-826.
[169]Povh NP,Marques MO,Meireles MA.Supercritical CO2 extraction of essential oil and oleresin from chamomile(Chamomilla recutia L.Rauschert).J Supercritical Fluids,2001,21:245-256.
[170]梁呈元,李维林,夏冰.等.薄荷油超临界CO2萃取条件的优化和筛选[J].植物资源与环境学报,2006,15(4):38-41.
[171]罗登林,丘泰球,王睿瑞.不同强化方法对超临界CO2萃取人参皂甙的影响[J].精细化工,2006,23(3):269-272.
[172]陈文利,余龙江,朱敏,等.被孢霉菌体油脂的提取及其花生四烯酸的纯化[J].武汉化工学院学报,2001,23(3):29-31.
[173]周蓬蓬,余龙江,朱敏,等.被孢霉产n-6多不饱和脂肪酸的分离纯化[J].华中师范大学学报(自然科学版),2002,36(3):341-344.
[174]崔振坤,杨国龙,毕艳兰.α-亚麻酸的纯化[J].油脂工程技术,2008,12:82-85.
[175]HAN XJ,XU P,MENG XL,etal. Preraration ofhigh-purity lino-lenic acid from oil of Lithospermum Erythrohizon by urea inclu-sion and column chromatography[J].JChin Pharm Sci,2004,13(1): 52-57.
[176]张娟梅,柯崇榕,黄建忠. DHA单细胞油脂的萃取与浓缩[J].中国油脂,2008,33(10):42-46.
[177]杨倩,王四旺,王剑波,等.椒目中α-亚麻酸的提取与鉴定[J].中国新药杂志,2008,17(4):318-320.
[178]刘书成,李德涛,欧冠强.等.尿素包合法富集蛇鲻鱼油中EPA和DHA的研究[J].广东海洋大学学报, 2008,28(3):61-65.
[179]Gamez,Meza N.Concentration of eicosapentaenoic acid and docosahexaenoic acid from fish oil by hydrolysis and urea complexation[J].Food research international,2003,36(7):721-727.
[180]李清光,万惠萍.尿素包合法富集松油酸的研究[J].食品与机械,2008,24(4):52-55.
[181]张桂梅,何美莹,邹建云.尿素包合法富集橡胶籽油亚麻酸的工艺试验[J].热带农业科技,2008,31(1):1-3.
[182]Liu Shucheng,Zhang Chaohua,Hong Pengzhi,et al.Concentration of docosahexaenoic acid(DHA)and eicosapentaenoic acid(EPA)of tuna oil by urea complexation: optimization of process parameters [J].Journal of food engineering,2006,73:202-207.
[183]陈雅,孟德胜,吴畏,等.松籽油中γ-亚麻酸的纯化研究[J].药物研究,2007,16(18):25.
[184]沈彩英.乌贼油中多不饱和脂肪酸的提取[J].中国油脂,2008,33(2):76-79.
[185]顾小红,孙素玲,汤坚,等.尿素包合法富集海狗油中的多不饱和脂肪酸[J].食品与机械,2005,22(4): 15-18.
[186]张氽,阚建全,陈宗道,等.尿素包合法分离花椒籽油中的α-亚麻酸的正交试验研究[J].中国食品添加剂, 2004, 17(2):28-31.
[187]刘凤霞,薛刚,高秋华,等.高纯度γ-亚麻酸包合工艺研究Ⅰ-皂化值及皂化反应[J].中国中药杂志, 2005, 30(5): 343~346.
[188]刘凤霞,薛刚,高秋华,等.高纯度GLA包合工艺研究Ⅱ-包合工艺的单因素、2因素试验[J].中国中药杂志,2005,30(6): 433~435.
[189] F奥斯伯,R布伦特,R E金斯顿,等.精编分子生物学实验指南[M].北京:科学出版社,2001,523.
[190] J萨姆布鲁克,E F佛里奇,T曼尼阿蒂斯(金冬雁等译).分子克隆实验指南.第二版[M].北京:科学出版社,1993,463-468.
[191]朱敏,余龙江,肖靓,等.高山被孢霉的红四氮唑染色程度与菌体油脂中花生四烯酸含量的关系[J].2004,8(4):339-344.
[2]黄绍重,秦振华.ω-3多不饱和脂肪酸对不同群体的保健价值及疾病防治[J].医药产业资讯,2006,3(3):41-43.
[3]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):289-292.
[4]姚昕,秦文,齐春梅,等.花生四烯的生理活性及其应用[J].粮油加工与食品机械,2004,(5):57-59.
[5]秦振华.多不饱和脂肪酸及其保健价值[J].实用医技杂志,2006,13(5):777-778.
[6]毛峰,秦振华.长链多不饱和脂肪酸与人体健康[J].医药产业资讯,2006,3(3):40-41.
[7]杨朝霞,张丽,李朝阳.花生四烯酸的营养保健功能[J].食品与药品,2005,7(1):69-71.
[8]王啸,邱树毅.微生物发酵生产花生四烯酸的研究进展[J].中国油脂,2004,29(9):37-40.
[9]符嫦娥,韩伟,卞进发,等.花生四烯的研究进展[J].云南化工, 2004,31(5):31-34.
[10]于长青,李丽娜.花生四烯酸研究进展[J].农产品加工(学刊),2007,(4):10-12.
[11]张永刚,印遇龙,黄瑞林.等.多不饱和脂肪酸的营养作用及其基因表达调控[J].中国饲料,2006,13:9-12.
[12]张伟利,吴圣楣,钱继红,等.母乳中二十二碳六烯酸及花生四烯酸含量的观察[J].中华围产医学杂志,2002,5(1):51-55.
[13]Brick E E,Garfield S,Hoffman DR,et al.A randomized controlled trial of early dietary supply of long chain polyunsaturated fatty acids and mental development in term infants. Developmental Medicine and Child Neurology.2000,42:173-181
[14]施东奎,胡春梅.花生四烯酸的主要作用及提取方法[J].中国中药杂志,2007,32(11):1009-1011.
[15]钱继红,吴圣楣,张伟利,等.含二十二碳六烯酸和花生四烯酸的配方奶对婴儿生长发育的研究[J].实用儿科临床杂志,2000,15(5):256-257.
[16]何平,汪志明,何卫加.花生四烯酸在婴幼儿配方食品中的应用[J].加工技术,2003,1:52.
[17]何平.花生四烯酸在奶粉中的应用[J].中国乳品工业,2003,31(3):61-62.
[18]张小平,葛庆丰,顾瑞霞,等.配方乳粉中花生四烯酸(ARA)添加工艺条件研究[J].添加剂,2004,8:31-35.
[19]孙程,左曙辉.新型营养强化剂在液体乳制品中的应用[J].中国食品添加剂,2003,1:77-78.
[20]刘洋,谢飞.被孢霉发酵生产花生四烯酸的研究进展[J].江苏食品与发酵,2007,2:12-16..
[21]Nigam S, Fiore S, Luscinskas F W et al. J Cell Physiol,1990, 143(5):511-523.
[22]汪志明.花生四烯酸生产及应用[J].中国食品添加剂,2001,1:30-33.
[23]汤世华,陈明锴,杨建斌,等.深黄被孢霉产脂的研究[J].中国油脂,2007,32(12):35-37.
[24]黄建忠,施巧琴,周晓兰,等.高产脂微生物-深黄被孢霉M018变株的选育及其油脂合成条件的研究[J].药物生物技术,1998,5(4):232-237.
[25]黄惠琴,鲍时翔,朱法科,等.被孢霉发酵生产花生四烯酸工艺的研究[J].华南热带农业大学学报,1999,5(2):1-4
[26]刘吉华,袁生,戴传超.轮枝霉(Diasporangiun sp.)产生EPA、ARA发酵条件的初步研究[J].食品与发酵工业,2000,26(4):9-12.
[27]王志举,尚耘,李翔宇,等.花生四烯酸油脂的产业化生产何工艺改进[J].中国乳品工业,2005,33(10):21-24.
[28]周蓬蓬,余龙江,吴元喜,等.高山被孢霉产花生四烯酸发酵条件的研究[J].工业微生物,2003,33(2):41-44.
[29]贡国鸿,尚耘,高翔,等.微生物发酵法生产花生四烯酸油脂[J].中国油脂,2002,27(1):42-44.
[30]尚耘,贡国鸿,高翔,等.发酵法生产新型营养强化剂-花生四烯酸[J].中国食品添加剂,2002,1:61-65.
[31]王啸,邱树毅,叶丹,等.花生四烯产生菌的选育[J].贵州工业大学学报(自然科学版),2005,34(1):56-59.
[32]赵沫,戴传超,王安琪,等.高产花生四烯酸菌的原生质体诱变育种研究[J].食品科学,2008,29(2):275-279.
[33]黄娟,陈正杰,冯军,等.紫外和微波结合诱变选育Lipstatin产生菌[J].微生物药物与生物技术,2006,37(1):11-14.
[34]何东平.高产PUFAs菌株的选育及其制备工艺的研究[C].武汉:中国科学院武汉病毒研究所,2001.
[35]Chen Zhi,Lin Wen,Yu Ping,et al.Enhancement of monacolin K production via intergeneric protoplast fusion between aspergillus terreus and monascus anka[J].Pro Gress in Natural Science,2007,17(6):703707.
[36]Kazuhiro Miyazaki,Hajime Maeda,Masahid Sunagawa,et al.Screening of heterozygous DNA markers in shitake(Lentinula edodes)using de-dikaryotization via preparation of protoplasts and isolation of four meiotic monokaryons from one basidium[J].The Japan Wood Research Society,2000,46:395-400.
[37]Zhang Ying,Gong Sufang,Wang Jingang,et al.Research on protoplast preparation and culture in gladiolus hybridus hort[J].Journal of Northeast Agricultural University,2007,14(1):5-8.
[38]Andrew J.Morgan,Aurora Brunner,Peter A.Whittaker.Protoplast fusion in a petite-negative yeast,kluyveromyces lactis[J].Current Genetics,1980,2:87-93.
[39]袁成凌,王纪,姚建铭,等.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[J].高技术通讯,2003,6:21-25.
[40]周蓬蓬,余龙江,李为,等.YAG激光照射对高山被孢霉花生四烯产量的影响[J].激光生物学报,2002,(05):371-376.
[41]朱敏,刘智,余龙江,等.高山被孢霉Δ5脱饱和酶基因的分离与验证[J].遗传学报,2005,32(9):986-992.
[42]朱法科,林炜铁,鲍时翔,等.花生四烯酸高产菌株的选育[J].工业微生物,1999,29(2):1-4.
[43]王安琪,戴传超,赵沫,等.轮梗霉高产花生四烯酸的诱变育种研究[J].食品科学,2007,28(2):158-162.
[44]咸漠,康亦兼,孙岩,等.深黄被孢霉的化学诱变[J].吉林大学自然科学学报,2001,1:92-95.
[45]Eiji Sakuradani,Yuriko Hirano,Nozomu Kamada et al.Improvement of arachidonic acid production by mutants with lower n-3 desaturation activity derived from Mortierella alpine IS-4[J].Appl Microbiol Biotechnol, 2004, (66):242-248
[46]Eiji S,Takahiro A,Ketta I,et al.A novel fungalω2-desaturase with wide substrate specificity from arachidonic acid-producting Mortierella alpina IS-4[J],Appl Microbiol Biotechnol,2005,66:648-654.
[47]杨革,王玉萍,李翔太,等.花生四烯酸高产突变株的选育及其发酵调控[J].菌物系统,1998,17(1):86-90.
[48]王啸,邱树毅,何腊平,等.UV、LiCl复合诱变深黄被孢霉选育多不饱和脂肪酸高产菌株[J].食品科学,2004,25(4):50-52.
[49]周蓬蓬,余龙江,朱敏,等.花生四烯产生菌的原生质体诱变育种[J].华中理工大学学报,2000,28(07):105-110.
[50]Chengling Yuan,Ji Wang,Yun Shang.Production of arachidonic acid by mortierella alpina I49-N18[J].Food Technology and Biotechnology,2002,40(4):311-315.
[51]袁成凌,姚建铭,王纪,等.低能离子注入在花生四烯酸(ARA)高产菌株选育中的研究[J].辐射研究与辐射工艺学报,2003,21(04):237-242.
[52]周蓬蓬,余龙江,汪建华,等.微波等离子体溅射诱变选育花生四烯高产菌及补料工艺研究[J].激光生物学报,2003,12(01):59-62.
[53]周蓬蓬,秦文敏,余龙江,等.毛霉油对被孢霉产花生四烯酸的影响[J].生命科学研究,2002,6(3):246-249.
[54]周蓬蓬,秦文敏,余龙江,等.KLa对高山被孢霉产花生四烯酸的影响[J].生命科学研究,2002,6(4):339-342.
[55]周蓬蓬,秦文敏,余龙江,等.表面活性剂对被孢霉产花生四烯酸的影响[J].华中科技大学学报(自然科学版),2003,31(5):98-100.
[56]马雪瑞.Co60辐射诱变深黄被孢霉高产多不饱和脂肪酸突变株的选育[C].武汉:中国科学院研究生院,2004.
[57]朱敏,余龙江,肖靓,等.高山被孢霉的红四氮唑染色程度与菌体油脂中花生四烯酸量的关系[J].生命科学研究,2004,8(4):339-343.
[58]Seki Takeno, Eiji Sakuradani,Akiko Tomi et al.Trasformation of Oil-Producting Fungus,Mortierella alpina IS-4.Using Zeocin,and Application to Arachidonic Acid Production[J].Journal of Bioscience and Bioengingeering 2005,100(6):617-622.
[59]朱敏,余龙江,刘智,等.花生四烯酸高产菌M6的鉴定[J].菌物学报,2004,23(4):508-513.
[60]赵广民,郝书杰.花生四烯酸生物提取和纯化[J].肉类工业,2000,7:31-32.
[61]袁成凌,余增亮,贺敬翠,等.富含花生四烯酸的微生物油脂的室内精炼及其成分测定[J].中国油脂,2001,26(4):17-18.
[62]杨小花,苏宝根,杨亦文,等.花生四烯酸分离纯化方法研究进展[J].粮食与油脂,2006,(2):12-16.
[63]袁成凌,余增亮,刘昌伟,等.脲包法富集微生物油脂中花生四烯酸的研究[J].营养研究,2002,(5):67-69.
[64]陈文利.尿素包合法纯化油脂中花生四烯酸的工艺研究[J].浙江化工,2003,34(8):28-29.
[65]黄和,李学坤,张昆,等.应用尿素包合法提纯花生四烯酸[J].化学与生物工程,2006,23(4):29-31.
[66]陈文利,余龙江,欧阳贻德,等.银离子络合萃取法纯化油脂中花生四烯酸[J].武汉化工学院学报,2003,25(2):16-17.
[67]袁成凌,余增亮,刘昌伟等.CO2超临界萃取技术富集微生物油脂中花生四烯酸的研究[J].中国油脂,2002,27(6):48-50.
[68]陈文利,朱敏,于龙江,等.超临界CO2萃取被抱霉菌体油脂及花生四烯酸的研究[J].精细化工中间体,2003, 33(1):33-37.
[69]袁成凌,张鉴,王相勤,等.高效液相色谱法分离纯化微生物油脂中的花生四烯酸[J].色谱,2003, 21(1):60-62.
[70]王明霞,黄凤洪.多不饱和脂肪酸萃取中超临界技术的应用进展[J].中国油脂,2005,30(8):60-64.
[71]王宪达,黄梅,杨亦文,等.超临界流体色谱法制备EPA-EE和DHA-EE[J].化工学报,2003,54(11):1558-1562.
[72] Muphy D J,Piffaneth P.Fatty acid desaturase:structure mechanism and regulation In Plant Lipid Biosynthesis[J].Recent Advances of Agri-cultural importance,1998,11:95-130.
[73] Gill l,Valivety R.Polyunsaturated fatty acids,part l:occurrence,biological activities and application[J].Trends Biotechnol,1997,15:401-409.
[74] Brick E E,Garfield S,Hoffman D R,Uauy R,Birch D G.A randomized controlled trial of early dietary supply of long chain polyunsaturated fatty acids and mental development in term infants[J].Dev Med Child Neuro1, 2000,42:174-181.
[75] Gunstone F D.Gamma linolenic acid occurrence and physical and chemical properties[J].Prog Lipid Res,1992,31(2):145-161.
[76] Yung-Sheng H,Sunita C,Jennifer M T, et a1.Cloning of△12-and△6 -desaturase from Mortierella aplina and recombinant production ofγ-linolenic acid in Saccharomyces cerevisiae[J].Lipids,1999,34(7):649-659.
[77] Sayanova O,Smith M A,Lapinskas P, et a1.Expression of a borage desaturase cDNA containing an N-terminal cytochrome b5 domain resuits in the accumulation of high levels of△6-desaturated fatty acids in transgenic tobacco[J].Proc Natl Acad Sci USA ,1997,94:421l-4216.
[78] Mount S M.A catalogue of splice junction sequences[J].Nucleic Acids Research,1982,10:459-472.
[79] Robin R,Tom M.1ntron sequence involved in lariat formation during pre-mRNA splicing[J].Cell,1985,41:95-105.
[80] Kajikawa M,Yamato K T,Kohzu Y.Isolation and characte rization of△6-desatu rase,an ELO-Like enzyme and△5-desaturase from the Liverwort Marchantia Polymorpha and production of arachidonic and elcosapentaenoic acids in the methylotrophic Yeast Pichia Pastoris[J].Plant Mo1 Bio1,2004,54:335-352.
[81] Nicola H,Morris A,Douglas R T,et a1.A vertebrate fatty acid desaturase with△5 an d△6 activities[J]. Biochemistry,2001,25:14304-14309.
[82] Ll M C,LIU L,ZHANG L ,et a1.Cloning and sequencing analysis of△6-fatty acid desaturase gene from Mortierella isabellina[J]. Mycosystema, 2001, 20(1):44-50.
[83] LIU L,Ll M C,HU G W,et a1.Identification of Mortierella isabellina M6-22△6-fatty acid desaturase by heterologous expression in Saccharomyces cerevisiae[J].Acta Microbiologica Sinica,2001,41(4):397-401.
[84] Reddy A S,Nuccio M L,Gross L M ,et a1.Isolation of a△6-desaturase gene from the cyanobactefium Synechocystis sp.strain PCC6803 by gain-of-function expression in Anabacna SP.Strain PCC7120[J].Plant Molecular Biology, 1993, 27:293-300.
[85] Napicr J A,Sandru J H,Dominic J L et a1.Identification of a Caenorhabditiselegans△6-fatty-acid-desaturese by heterolagous expression in Saecharomyces cerevisiae.Biochem J,1998,330:61l-614.
[86] Tsunehiro A,Yayoi S,Katsuya Iet a1.Molecular cloning and functional characterization of rat△6-fatty acid desaturase.Biochemca1 and Biophysica1 Research Communications,1999,255:575-579.
[87] Sayanova O,Smith M A,Lapinskas P,et a1.Expression of a borage desaturase cDNA containing an N-terminalcytochrome b5 domain results in the accumulation of high levels of△6-desaturated fatty acids in trangenic tobacco[J].Proc Natl Acad Sci USA,1997,94:4211-4216.
[88] Liu Li,Li Mingchun,Hu Guowu,et a1.Identification of Mortierella isabellina M6-22△6 -fatty acid desaturase gene by heterologous expression in Saccharomyces cerevisiae[J].Acta Microbiologica Sinica,2001,41(4):397-401.
[89]李明春,刘莉,胡国武,等.深黄被孢霉△6-脂肪酸脱氢酶基因在转基因烟草中的表达.生物工程学报.2003,19(2):178-184.
[90] Sakuradani E,Kobayashi M,Shimizu S.△6-fatty acid desaturase from an arachidonic acid-producing Mortierella fungus Gene cloning and its heterologous expression in a fungus Aspergillus[J].Gene,1999,238:445-453.
[91] Huang Y S H,Sunita C,Jennifer M T.et al.Cloning of△6 2-and△6 -desaturase from Mortierella aplina and recombinant production ofγ-linolenic acid in saccharomyces cerevisiae[J].Lipids,1999,34(7):649-659.
[92] Napier J A,Sandra J H,Dominic J L,et a1.Identification of a Caenorhabditis elegans△6-fatty-acid-desaturase by heterelogous expression in Saccharomyces cerevisiae[J].Biochem J,1998,330:611-614.
[93] Petra S,Michael L,Thomas G,et a1.A bifuncfional△6-fatty acyl acetylenase /desaturase from the moss Ceratodon purpureus.Eur JBiachem, 2000, 267: 3801-3811.
[94] Sakuradani E,Kobayashi M,Shimizu S.△6-Fatty acid desaturase from an arachidonic acid-producing Mortierella fungus gene cloning and its heterologous expression in a fungus[J].Aspergillus.Gene.1999,238:445-453.
[95] Botha A,Paul I,Roux c,et a1.An isolation procedure for arachidonic acid producing Monierella species[J].Amonie van Leeuwenhoek.199l9,75,253-256.
[96] Botha A,Paul l,Roux C,Kock J L F,Coetzee D J,Strauss T,Maree C.An isolation procedure for arachidonic acid producing Mortierella species[J].Antonie van Leeuwenhoek,1999,75:253-256.
[97]肖靓,刘智,朱敏,等.实时PCR分析△5脱饱和酶在花生四烯酸合成中的作用[J].中国生物工程杂志.2006,26(8):57-61.
[98] Shimada T,Somlyo A P.Modulation of voltage-dependent Ca channel current by arachidonic acid and other long-chain fatty acids in rabbit intestinal smooth muscle[J].J Gen Physio1,1992,100:27-44.
[99] ChenH C,ChangC C,Chen CX.Optimization of arachidonic acid production by Mortierella alpina Wuji-H4 isolate[J].Joumal of the American Oil Chemists’Society,1997,74:569-578.
[100] Zhu M,Yu L J,Liu Z,et a1.Isolating Mortierella alpina strains of high yield of arachidonic acid[J].Leters in Applied Microbiology,2004,39:332-335.
[101] Zhu M,Yu L J,Liu Z,et a1.Identification of arachidonic acid high yielding strain M6[J].Mycosystem,2004,23(4):508-513.
[102] Watts J L,Browse J.Isolation and characterization of a△5-fatty acid desaturase from Caenorhabditis elegans[J].Arch Biochem Biophys,1999,362(1):175-182.
[103]朱敏,刘智,余龙江,等.高山被孢霉△5脱饱和酶基因的分离与验证[J].遗传学报2005,32(9):986-992.
[104] Eroshin V K,Dedyukhina E G,Satroutdinov A D.G rowthcoupled lipid synthesis in Mortierella alpina LPM 301 a producer of arachidonic acid[J]. Microbiology, 2002,71:1 69-172.
[105] Zhu M,Yu L J,Wu Y X.An inexpensive medium for production of arachidonic acid by MortiereIla alpine[J].J Ind Microbiol Biot,2003,30:75-79.
[106] Chung HW.Reverse transcriptase PCR (RT-PCR) and quantitative-competitive PCR (QC-PCR) [J].Exp Mol Med,2001,33(1 Suppl): 85-97.
[107] Cheng B. Comparing RNA PCR test [J] .PI Perspect .,1995,(17):15-18.
[108] L Wen,Effect of primer purity on the banding patterns of differential display polymerase chain reaction[J].J.Biomol.Tech.,2000,11:87-91.
[109] Kim BR,Nam HY,Kim SU.Normalization of reverse transcription quantitative- PCR with housekeeping genes in rice[J].Biotechnol Lett., 2003,25(21):1869-1872.
[110] Ke LD,Chen Z,Yung WK.A reliability test of standard-based quantitative PCR:exogenous vs endogenous standards[J].Mol.Cell Probes, 2000, 14(2): 127-135 .
[111]张维铭主编,现代分子生物学实验手册[M].北京:科学出版社出版.2003.
[112] Goidin D,Mamessier A,Staguet T M,et a1.Ribosomal 18S RNA prevails over glyceraldehyde-3-phosphate dehydrogenase andβ-actin genes as internal standard for quantitative comparison of mRNA levels in invasive and noninvasive human melanoma cell subpopulations[J].Analytical Biachemistry, 2001,295:l7-2l.
[113]胥保华,许梓荣.肉雏鸡肝细胞谷胱甘肽过氧化物酶RT-PCR定量分析[J].中国兽医科技,2003,33(2):24-26. [114 ]Annemarie Berger,Wolfgang Preiser. Viral genomequantification as a tool for improving patient management :the example of HIV,HBV,HCV and CMV[J].J.Antimicrob.Chemother,2002,49:713-721.
[115] James C.Paton and Adrienne W.Paton.Pathogenesis and Diagnosis of Shiga Toxin-Producing Escherichia coli Infections[J]. Clin. Microbiol. Rev., 1998, 11:450-479
[116]肖白,刘敬忠,王建秋.半定量PCR检测致龋性变形链球菌的技术及应用[J].中国实验诊断学,2003,7(2):109-112
[117] Schnell S,Mendoza C.Enzymological considerations for a theoretical description of the quantitative competitive polymerase chain reaction (QC-PCR) [J].J Theor.Biol.,1997,184(4):433-440.
[118]马中富,黄帆,高劲松.以cDNA为内参标RT-PCR定量检测心肌细胞内p53和Bcl-2基因mRNA表达量的方法研究[J].中国危重病急救医学.2002,14(10):597-601.
[119]程正江.用统计学方法解释和处理临床定量PCR实验结果[J]数理医药杂志,2003,1:18-21.
[120] F Ferre. Quantitative or semi-quantitative PCR: reality versus myth[J].Genome Res,1992,2:1-9.
[121] Actor JK,Limor JR,Hunter RL.A flexible bioluminescent-quantitive polymerasereaction assay for analysis of compeitive PCR amplicons[J].J Clin Lab Anal.,1999,13(1):40-47.
[122] Anderson KM,Cheung PH,Kell MD,Rapid generation of homologous internal standards and evaluation of data for quantitaion of messenger RNA by competitive polymerase chain reaction[J].J pharmacol Toxicol Methods, 1997,38:133-140.
[123] Tartaglia M,Fragale A,Battaglia PA.A competitive PCR-based method to measure human fibroblast growth factor receptor 1-4 gene expression[J].Cell Biol.,2001,20(6):367-379.
[124] Montagne A,Grepinet O,Peloille M,et al.Quantification of ovine cytokine gene expressiont by a competitive RT-PCR method[J].J Immunol Methods, 2001,253(1-2):83-93.
[125] AI-Robaiy S,Rupf S,Eschrich K.Raoid competitive PCR using melting curve analysis for DNA quantification[J].Biotechniques,2001,31(6):1382-1388.
[126]王宁.定量PCR技术研究进展[J].国外医学:临床生物化学与检查学分册.2000,21(6):314-316.
[127] Wuhu A,Waiztu M,Koch A.A rapid and sensitive protocol for competitive reverse transcriprase (CRT)PCR analysis of cellular genes[J].Brain Pathol., 1998,8:13-18.
[128] Liljekvist-Larsson I,Torngren M,Abrahamson M.Johansson K.Growth of the postnatal rat retina in vitro:quantitative RT-PCR analyses of mRNA expression for photoreceptor protein[J].Mol Vis.,2003,9:657-64.
[129]牛艾如,陈培佳.定量DNA和Igm抗性检测CMV感染[J].中国优生与遗传杂志,2002,10(6):40-41.
[130]张立国,张琚.实时定量PCR技术的介绍[J].生物技术,2003,13(2):39-40.
[131] Kazutoshi Kaji,Kazuhiro Ueda,Takashi Aoyama.Development of a real-time jump-ratio imaging system equipped with a stem[J].Journal of Electron Microscopy 2001,50:15-21.
[132] Ke LD,Chen Z,Yung WK.A reliability test of standard-based quantitative PCR:exogenous VS endogenous standards[J].Mol Cell Probes,2000,14(2):127.
[133] Becker KD, Pan,Whitely CB.Real-time quantitative pelymerase chain reactionto assess gene transfer[J]. Hum Gene Ther.,1999,10:2559.
[134] Higgis JA.Nuclease PCR assay to detect Yersinia pesits[J].J Clin Microbiol.,1998,36:2284.
[135]阳成波,印遇龙,龚建华,等.定时定量PCR研究进展及其应用[J].中国预防兽医学报,2003,25(5):395.
[136] Gibson U E.A novel method for real time quantitative RT-PCR[J]. Genome-Res,1996,6:995-1001.
[137] Wei S F,Wang SL,CHANG J. Influence of ultraviolet radiation on the expression of preliferating cell nuclear antigen and DNA polymerase alpha in Skeletonema costatum (Bacillariophyceae)[J].J Phycol,2004,40(4):655-663.
[138] Hahnen S,Joeris T, Kreuzaler F, et al.Quantification of photosynthetic gene expression in maize C3 and C4 tissues by real time PCR[J]. Photosynthesis Research,2003,75(2):183-192.
[139]翟新验.实时TaqMan技术在兽医学中的应用[J].实验动物科学与管理,2002,19(2):50.
[140]罗长保,鱼海琼,林志雄.应用荧光RT-PCR技术检测口蹄疫病毒[J].中国兽医杂志,2003,39(12):31.
[141] Mason G,Provero P.Varia A M,et al.Estimating the number of integrations in transformed plants by quantitative real-time PCR[J].BMC Biotechnic Res,2002,11(1):43-48.
[142] Tesson L,Heslan J M, Menoret S,et al.Rapid and ac. curate determination of zygosity in transgenic an imals by real-time quantitative PCR[J].Transgenic Trend ,2003,7(1):76-79.
[143] Shah C A,Bon J,Bisset L R,et al.Ultra-sensitive and specific detection of porcine endogenous retrovirus using a sequence-capture real-time PCR approach[J].J Virol Methods,2002,109(2):209-216.
[144] Park S B,Popovich B W,Press R D .Real-time polymerase chain reaction with fluorescent hybridization pmbes for for the detection of prevalent mutations causing common thrombophilic and iron overload phenotypes[J].Am J Clin Pathol.,2001,115:439-447.
[145]徐秋英,彭胜权,刘亚敏,等.荧光定量PCR技术在评价中医药治疗乙型肝炎中的价值[J].新中医,2002,34(10):71-72.
[146] Funato T,Fujimaki S,Fujiwara J,et al.Genetic diagnosis for drug resistance in cancers[J].Rinsho Byori.,2000,48(2):162-166.
[147]范瑞强,陈信生,棚国维,等.荧光定量PCR观察中药抗病毒胶囊对豚鼠HSV-2系统感染的影响[J].中华皮肤科杂志,2003,36(5):276-278.
[148] Higashiyama K, Fujikawa S,Park EY, et al.Production of arachidonic acid by Mortierella fungi[J]. Biotechnology and Bioprocess Engineering. 2002, 7(5): 251-262.
[149] Jin MJ,Huang H, Xiao AH,et al.A novel two-step fermentation process for improved arachidonic acid production by Mortierella alpina[J]. Biotechnology Letters.2008,30(6):87-91.
[150]Kenichi Higashiyama,Shigeaki Fujikawa,Enoch Y.Park et al.Production of Arachidonic Acid by Mortierella Fungi[J].Biotechnol.Bioprocess Eng. 2002,7(5):251-262.
[151]Takeno S, Sakuradani E, Tomi A, et al.Trasformation of oil-producting fungus,Mortierella alpina IS-4.using zeocin,and application to arachidonic acid production.Journal of Bioscience and Bioengingeering.2005,100(6):617-622.
[152]James G M,Paul R,Daniel F.et al.Production of polyunsaturated fatty acids by polyketide synthases in both prokaryotes and eukaryotes[J]. science, 2001,293:290-293.
[153]吕晴,余德顺,雷邦星,等.被孢霉菌丝体脂肪酸组成的气相色谱-质谱分析[J].分析测试学报,2003,22(2):21-24.
[154]邹耀洪,陆云琦.婴儿奶粉中的二十二碳六烯酸和二十碳四烯酸气相色谱分析[J].常熟高专学报,2004,18(2):33-37.
[155]葛庆丰,张小平,顾瑞霞,等.乳粉中花生四烯酸(ARA)测定方法研究[J].添加剂,2004,10-38-39.
[156]薛刚,张进,张均媚,等.婴儿配方奶粉中花生四烯酸含量的测定[J].食品研究与开发,2006,27(5):119.
[157]王相勤,姚建铭,袁成凌,等.高产花生四烯酸产生菌干菌体中脂肪酸成分的气相色谱-质谱分析[J].分析化学,2001,29(3):287-289.
[158]K Goodrum J.W.,Kilgo M.B.Peanut oil extraction with SC- CO2:solubility andkinetic functions[J].Transactins of the ASAE,1987,30(6):1865-1868.
[159]麻成金,张永康,马美湖,等.微波和超临界CO2萃取杜仲籽油工艺研究[J].食品科学,2006,27(6):131-135.
[160]苏香萍,宋必卫,陈振华,等. CO2超临界萃取金银花挥发油工艺及抗炎活性研究[J].天然产物研究与开发,2006,18:662-666.
[161]Toshihiko Osaki,Toshiki Mori.Kinetic studies of CO2 dissociation on supported ni catalysts[J].Akademiai kiado,budapest and springer, dordrecht. 2006, 87(1): 149-156.
[162]Olivera R.,Rodrigues M.F.,Bernardo-Gil M.G.Charactrerization and supercritical carbon dioxide extraction of walnut oil[J].Journal of the american oil chemists` society,2002,79(3):225-230.
[163]闫志芳,刘必旺,赵水平. CO2超临界萃取蛇床子中蛇床子素的工艺研究[J].世界中西医结合杂志,2009,4(1):20-22.
[164]赵淑英,王秋芬,宋湛谦. CO2超临界萃取印楝素的研究[J].福建林学院学报,2005,25(2):152-156.
[165]吕晓玲,李肇奖. CO2超临界萃取油茶皂苷的研究[J].食品与发酵工业,2005,31(1):22-26.
[166]黄纪念,屠鹏飞,蔡同一.超临界CO2流体萃取迷迭香中抗氧化活性成分的工艺研究[J].中草药,2004,35(2):150-153.
[167]邓亦峰,梁念.半边旗中二萜类化合物的超临界CO2萃取及HPLC-MS分析[J].中药.2004,35(2):145-148.
[168]黄欣,苏乐群,邵伟,等.白芷香豆素类化合物的成分分析及CO2超临界萃取工艺的研究[J].中国药房.2005,16(11):823-826.
[169]Povh NP,Marques MO,Meireles MA.Supercritical CO2 extraction of essential oil and oleresin from chamomile(Chamomilla recutia L.Rauschert).J Supercritical Fluids,2001,21:245-256.
[170]梁呈元,李维林,夏冰.等.薄荷油超临界CO2萃取条件的优化和筛选[J].植物资源与环境学报,2006,15(4):38-41.
[171]罗登林,丘泰球,王睿瑞.不同强化方法对超临界CO2萃取人参皂甙的影响[J].精细化工,2006,23(3):269-272.
[172]陈文利,余龙江,朱敏,等.被孢霉菌体油脂的提取及其花生四烯酸的纯化[J].武汉化工学院学报,2001,23(3):29-31.
[173]周蓬蓬,余龙江,朱敏,等.被孢霉产n-6多不饱和脂肪酸的分离纯化[J].华中师范大学学报(自然科学版),2002,36(3):341-344.
[174]崔振坤,杨国龙,毕艳兰.α-亚麻酸的纯化[J].油脂工程技术,2008,12:82-85.
[175]HAN XJ,XU P,MENG XL,etal. Preraration ofhigh-purity lino-lenic acid from oil of Lithospermum Erythrohizon by urea inclu-sion and column chromatography[J].JChin Pharm Sci,2004,13(1): 52-57.
[176]张娟梅,柯崇榕,黄建忠. DHA单细胞油脂的萃取与浓缩[J].中国油脂,2008,33(10):42-46.
[177]杨倩,王四旺,王剑波,等.椒目中α-亚麻酸的提取与鉴定[J].中国新药杂志,2008,17(4):318-320.
[178]刘书成,李德涛,欧冠强.等.尿素包合法富集蛇鲻鱼油中EPA和DHA的研究[J].广东海洋大学学报, 2008,28(3):61-65.
[179]Gamez,Meza N.Concentration of eicosapentaenoic acid and docosahexaenoic acid from fish oil by hydrolysis and urea complexation[J].Food research international,2003,36(7):721-727.
[180]李清光,万惠萍.尿素包合法富集松油酸的研究[J].食品与机械,2008,24(4):52-55.
[181]张桂梅,何美莹,邹建云.尿素包合法富集橡胶籽油亚麻酸的工艺试验[J].热带农业科技,2008,31(1):1-3.
[182]Liu Shucheng,Zhang Chaohua,Hong Pengzhi,et al.Concentration of docosahexaenoic acid(DHA)and eicosapentaenoic acid(EPA)of tuna oil by urea complexation: optimization of process parameters [J].Journal of food engineering,2006,73:202-207.
[183]陈雅,孟德胜,吴畏,等.松籽油中γ-亚麻酸的纯化研究[J].药物研究,2007,16(18):25.
[184]沈彩英.乌贼油中多不饱和脂肪酸的提取[J].中国油脂,2008,33(2):76-79.
[185]顾小红,孙素玲,汤坚,等.尿素包合法富集海狗油中的多不饱和脂肪酸[J].食品与机械,2005,22(4): 15-18.
[186]张氽,阚建全,陈宗道,等.尿素包合法分离花椒籽油中的α-亚麻酸的正交试验研究[J].中国食品添加剂, 2004, 17(2):28-31.
[187]刘凤霞,薛刚,高秋华,等.高纯度γ-亚麻酸包合工艺研究Ⅰ-皂化值及皂化反应[J].中国中药杂志, 2005, 30(5): 343~346.
[188]刘凤霞,薛刚,高秋华,等.高纯度GLA包合工艺研究Ⅱ-包合工艺的单因素、2因素试验[J].中国中药杂志,2005,30(6): 433~435.
[189] F奥斯伯,R布伦特,R E金斯顿,等.精编分子生物学实验指南[M].北京:科学出版社,2001,523.
[190] J萨姆布鲁克,E F佛里奇,T曼尼阿蒂斯(金冬雁等译).分子克隆实验指南.第二版[M].北京:科学出版社,1993,463-468.
[191]朱敏,余龙江,肖靓,等.高山被孢霉的红四氮唑染色程度与菌体油脂中花生四烯酸含量的关系[J].2004,8(4):339-344.