油菜长链脂酰CoA合成酶基因的克隆与功能鉴定
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摘要
在油料作物中,种子油脂是对人类最具有经济价值的生活必需品和工业原料,提高种子油脂含量也就是提升油料作物的生产效益。本文主要通过克隆长链酰基辅酶A合成酶基因并鉴定其在油脂合成途径中的生物学功能,从而期望通过基因工程手段提高油料作物种子含油量。
长链酰基辅酶A合成酶(LACS)把游离脂肪酸活化成为长链酰基辅酶A,从而在脂肪酸的合成与分解代谢中起着重要作用。这类重要的酶参与若干种与脂肪酸相关的代谢,如脂肪酸链的活化和延伸,磷脂、甘油三酯、茉莉酸的生物合成等。最近我们实验室从油菜油脂合成旺盛部位(花和发育期的种子)分离出四个长链脂酰辅酶A合成酶基因(BnLACS1、BnLACS2、BnLACS3和BnLACS4)。生物信息学分析显示这四个长链脂酰辅酶A合成酶(BnLACSs)氨基酸序列都具有三个Block,初步被认为属于LACS家族。分子进化树分析显示,BnLACS1与拟南芥AtLACS1具有较高的同源性,BnLACS2和BnLACS3与拟南芥AtLACS2具有较高的同源性,AtLACS1和AtLACS2定位于内质网上,在植物角质主要成分长链脂肪酸的合成中起着重要作用。BnLACS4与定位于叶绿体的AtLACS9有较高的同源性。
通过酵母LACS缺陷型补救实验,进一步证明四个BnLACSs都具有LACS活性。油菜BnLACSs蛋白都偏好利用长链脂肪酸。在水解蛋白酶缺陷型酵母pep4中异源表达,结果显示BnLACS1和BnLACS2能够使宿主酵母的脂肪酸和油脂含量显著提高。以上在酵母真核表达系统中的结果初步确定BnLACS1和BnLACS2参与油脂合成,并且能够使酵母油脂含量提高。
利用RT-PCR技术分别分析了BnLACS1、BnLACS2、BnLACS3和BnLACS4在不同含油量油菜品系种子(35DAP)中的表达差异,结果显示在高含油量品系中这些基因表达更为强烈。研究这四个基因在高含油量油菜(50%)和低含油量油菜(30%)两个油菜品系的种子发育时期的表达丰度动态变化,结果显示BnLACS1和BnLACS2表达量变化趋势与种子发育期油脂合成速率的S型曲线趋势一致,并且结果还进一步验证BnLACS1和BnLACS2表达量在高含油量油菜品系种子明显高于低含油量油菜品系种子。以上结果显示BnLACSs,尤其是BnLACS1和BnLACS2在油菜种子的油脂合成途径中起着重要作用。
为了鉴定油菜BnLACSs的生物学功能,我们还构建了各个基因的双元植物表达载体,载体中利用在种子中特异表达的Napin启动子,然后通过农杆菌介导转化拟南芥和甘蓝型油菜。目前已经得到BnLACS1和BnLACS4的拟南芥转化株系,并通过筛选得到转基因纯系。通过浸花法和抗生素筛选法得到油菜转化株系,利用近红外和核磁共振技术初步分析显示:BnLACS1油菜转基因植株的种子含油量提高约5%。
In oil crops, the oil (i.e. triacylglycerols) is the most valuable product of the seeds. Enhancing the quantity of oil per weight basis in oil crops would increase the value of the crop. As part of our effort to identify enzymes that is critical for producing large amounts of plant oil in the seed of oil crops.
Long-chain acyl-coenzyme A synthetases (LACSs) activate free fatty acid to acyl-CoA thioesters and play an important role in fatty acid anabolism and catabolism. These classes of important enzymes are involved in several fatty acid-derived metabolic pathways, such as the activation and elongation of fatty acids, biosynthesis of phospholipids, triacylglycerol and jasmonate. In this study, four cDNAs encoded long chain fatty acyl-CoA synthetase have been isolated from rapeseed (Brassica napus) flower or seeds, designated as BnLACS1, BnLACS2, BnLACS3 and BnLACS4, respectively. Sequence analysis indicated that the four LACSs possessed typical molecular characteristics of LACS. The phylogenetic analysis showed that AtLACS1, AtLACS2, AtLACS9 and 4 BnLACSs were in group I. AtLACS1 has overlapping functions with AtLACS2 in plant wax and cutin synthesis.
The function of genes BnLACSs was confirmed to encode LACS activity by yeast complementation tests. LACS substrate specificity assays indicated that the BnLACSs preferentially activate long chain fatty acids and saturated fatty acids. Yeast strain cells pep4 containing pYES2-BnIACS1 or pYES2-BnLACS2 can improve fatty acids and lipid content than the control yeast. These studies suggest that this enzymes increase lipid stored in yeast and have potential use in plant seed oil and bio-energy produced algea.
Compared with low oil content varieties seed, the four genes is strongly expressed in high oil content varieties seeds at 35 days after pollination (DAP). The expression pattern suggested that the four BnLACSs might be involved in the biosynthesis of lipids and oil accumulation in rapeseed. Real-time PCR analysis reveals that BnLACS1 and BnLACS2 were strongly expressed in lipid developmental stages of seeds. The trend is similar to the 'S' trend of lipid synthesis in seed. In high oil content varieties, the two genes expressed more stronger.
To study the biological functions of BnLACSs, the plant expression vectors were constructed, and the positive transformants of Agrobacterium Tumefaciens have infected Brassica napus, respectively. At present, we obtained over-expression of BnLACS1 in transgenic Arabidopsis thaliana and Brassica napus plants. Preliminary results showed that 5% increased in transgenic Brassica napus plants seed oil content.
长链酰基辅酶A合成酶(LACS)把游离脂肪酸活化成为长链酰基辅酶A,从而在脂肪酸的合成与分解代谢中起着重要作用。这类重要的酶参与若干种与脂肪酸相关的代谢,如脂肪酸链的活化和延伸,磷脂、甘油三酯、茉莉酸的生物合成等。最近我们实验室从油菜油脂合成旺盛部位(花和发育期的种子)分离出四个长链脂酰辅酶A合成酶基因(BnLACS1、BnLACS2、BnLACS3和BnLACS4)。生物信息学分析显示这四个长链脂酰辅酶A合成酶(BnLACSs)氨基酸序列都具有三个Block,初步被认为属于LACS家族。分子进化树分析显示,BnLACS1与拟南芥AtLACS1具有较高的同源性,BnLACS2和BnLACS3与拟南芥AtLACS2具有较高的同源性,AtLACS1和AtLACS2定位于内质网上,在植物角质主要成分长链脂肪酸的合成中起着重要作用。BnLACS4与定位于叶绿体的AtLACS9有较高的同源性。
通过酵母LACS缺陷型补救实验,进一步证明四个BnLACSs都具有LACS活性。油菜BnLACSs蛋白都偏好利用长链脂肪酸。在水解蛋白酶缺陷型酵母pep4中异源表达,结果显示BnLACS1和BnLACS2能够使宿主酵母的脂肪酸和油脂含量显著提高。以上在酵母真核表达系统中的结果初步确定BnLACS1和BnLACS2参与油脂合成,并且能够使酵母油脂含量提高。
利用RT-PCR技术分别分析了BnLACS1、BnLACS2、BnLACS3和BnLACS4在不同含油量油菜品系种子(35DAP)中的表达差异,结果显示在高含油量品系中这些基因表达更为强烈。研究这四个基因在高含油量油菜(50%)和低含油量油菜(30%)两个油菜品系的种子发育时期的表达丰度动态变化,结果显示BnLACS1和BnLACS2表达量变化趋势与种子发育期油脂合成速率的S型曲线趋势一致,并且结果还进一步验证BnLACS1和BnLACS2表达量在高含油量油菜品系种子明显高于低含油量油菜品系种子。以上结果显示BnLACSs,尤其是BnLACS1和BnLACS2在油菜种子的油脂合成途径中起着重要作用。
为了鉴定油菜BnLACSs的生物学功能,我们还构建了各个基因的双元植物表达载体,载体中利用在种子中特异表达的Napin启动子,然后通过农杆菌介导转化拟南芥和甘蓝型油菜。目前已经得到BnLACS1和BnLACS4的拟南芥转化株系,并通过筛选得到转基因纯系。通过浸花法和抗生素筛选法得到油菜转化株系,利用近红外和核磁共振技术初步分析显示:BnLACS1油菜转基因植株的种子含油量提高约5%。
In oil crops, the oil (i.e. triacylglycerols) is the most valuable product of the seeds. Enhancing the quantity of oil per weight basis in oil crops would increase the value of the crop. As part of our effort to identify enzymes that is critical for producing large amounts of plant oil in the seed of oil crops.
Long-chain acyl-coenzyme A synthetases (LACSs) activate free fatty acid to acyl-CoA thioesters and play an important role in fatty acid anabolism and catabolism. These classes of important enzymes are involved in several fatty acid-derived metabolic pathways, such as the activation and elongation of fatty acids, biosynthesis of phospholipids, triacylglycerol and jasmonate. In this study, four cDNAs encoded long chain fatty acyl-CoA synthetase have been isolated from rapeseed (Brassica napus) flower or seeds, designated as BnLACS1, BnLACS2, BnLACS3 and BnLACS4, respectively. Sequence analysis indicated that the four LACSs possessed typical molecular characteristics of LACS. The phylogenetic analysis showed that AtLACS1, AtLACS2, AtLACS9 and 4 BnLACSs were in group I. AtLACS1 has overlapping functions with AtLACS2 in plant wax and cutin synthesis.
The function of genes BnLACSs was confirmed to encode LACS activity by yeast complementation tests. LACS substrate specificity assays indicated that the BnLACSs preferentially activate long chain fatty acids and saturated fatty acids. Yeast strain cells pep4 containing pYES2-BnIACS1 or pYES2-BnLACS2 can improve fatty acids and lipid content than the control yeast. These studies suggest that this enzymes increase lipid stored in yeast and have potential use in plant seed oil and bio-energy produced algea.
Compared with low oil content varieties seed, the four genes is strongly expressed in high oil content varieties seeds at 35 days after pollination (DAP). The expression pattern suggested that the four BnLACSs might be involved in the biosynthesis of lipids and oil accumulation in rapeseed. Real-time PCR analysis reveals that BnLACS1 and BnLACS2 were strongly expressed in lipid developmental stages of seeds. The trend is similar to the 'S' trend of lipid synthesis in seed. In high oil content varieties, the two genes expressed more stronger.
To study the biological functions of BnLACSs, the plant expression vectors were constructed, and the positive transformants of Agrobacterium Tumefaciens have infected Brassica napus, respectively. At present, we obtained over-expression of BnLACS1 in transgenic Arabidopsis thaliana and Brassica napus plants. Preliminary results showed that 5% increased in transgenic Brassica napus plants seed oil content.
引文
[1]Scarth R,Tang J.Modification of Brassica Oil Using Conventional and Transgenic Approaches.Crop Sci,2006.46:1225-1236.
[2]柴国华,白泽涛,蔡丽,郭学兰,张学江,石磊,董彩华,刘胜毅.油菜基因BnWRI1的克隆及RNAi对种子含油量的影响.中国农业科学,2009.42(5):1512-1518.
[3]王汉中.我国油菜产需形势分析及产业发展对策.中国油料作物学报,2007.29(1):101-105.
[4]朱福各,谭小力,崇保强,周佳,袁伟伟.油菜脂酰CoA合成酶基因pXT166的鉴定和功能分析.中国油料作物学报,2009.31(3):274-278.
[5]李军,吴平治,李美茹,吴国江.能源植物的研究进展及其发展趋势.Chinese Journal of Nature,2007.29(1):21-25.
[6]张壵,李云昌,梅德圣,胡琼.油菜油脂研究进展.植物学通报,2007.24(4):435-443.
[7]官春云.油菜生产情况与科研进展.作物研究,2001.4:4-8.
[8]程红焱,宋松泉.种子萌发过程中贮藏油脂的动员.云南植物研究,2007.29(1):67-73.
[9]Si P,Rodney JM,Nick G,David WT.Influence of genotype and environment on oil and protein concentrations of canola(Brassica napus L.) grown across southern Australia.Aust J Agric Res,2003.54:397-407.
[10]Gutierrez Boem FH,Lavado RS,Porcelli CA.Note on the effects of winter and spring watedogging on growth,chemical composition and yield of rapeseed.Field Crops Res,1996.47:175-179.
[11]Wu JG,Shi CH,Zhang HZ.Partitioning genetic effects due to embryo,cytoplasm and maternal parent for oil content in oilseed rape(Brassica napus L.).Genet Mol Biol,2006.29:533-538.
[12]Hom NH,Girke A,M(o|¨)llers C,Becker HC.Influence of pollen genotype on rapeseed quality-analyses of single seeds by near-infrared reflectance spectroscopy(NIRS).Proceedings of the 11th International Rapeseed Congress.2003.266-269.
[13]张洁夫,戚存扣,浦惠明,陈松,陈锋,陈新军,高建芹,傅寿仲.甘蓝型油菜主要脂肪酸的主基因+多基因遗传分析.中国油料作物学报,2007.29(4):259-364.
[14]Ohlrogge J,Browse J.Lipid biosynthesis.Plant Cell,1995.7:957-970.
[15]Mu J,Tan H,Zheng Q,Fu F,Liang Y,Zhang J,Yang X,Wang T,Chong K,Wang X,Zuo J.Leafy Cotyledon1 is a key regulator of fatty acid biosynthesis in Arabidopsis thaliana.Plant Physiol,2008.148:1042-1054.
[16]Harwood JL.Recent advances in the biosynthesis of plant fatty acids.Biochim Biophys Acta,1996.1301:7-56.
[17]周奕华,陈正华.植物种子中脂肪酸代谢途径的遗传调控与基因工程.植物学通报, 1998.15(5): 16-23.
[18] Hugly S, Somerville C. A Role for Membrane Iipid Polyunsaturation in Chloroplast Biogenesis at Low Temperature. Plant Physiol, 1992. 99(1): 197-202.
[19] Weselake RJ. Storage lipids. Plant lipids: biology, utilization and manipulation. 2005.162-225.
[20] Olsen JA, Lusk K. Acyl-CoA synthetase activity associated with rapeseed lipid body membranes. Phytochemistry, 1994. 36: 7-9.
[21] Huang AH. Oleosins and Oil Bodies in Seeds and Other Organs. Plant Physiol, 1996. 110(4):1055-1061.
[22] Ecke W, Uzunova M, Weissleder K. Mapping the genome of rapeseed (Brassica napus L.) II Localization of genes controlling erucic acid synthesis and seed oil content. Theor Appl Genet, 1995.91: 972-977.
[23] Cheung WY, Landry BS, Raney P, Pakow GFW. Molecular mapping of seed quality traits in Brassica juncea (L.) Czern and Coss. Acta Hort, 1998.459:139-147.
[24] Burns MJ, Barnes SR, Bowman JG, Clarke MHE, Werner CP, Kearsey MJ . QTL analysis of an intervarietal set of substitution lines in Brassica napus: (i) seed oil content and fatty acid composition. Heredity, 2003. 90: 39-48.
[25] Zhao JY, Heiko CB, Zhang DQ. Oil content in a EuropeanxChinese rapeseed population: QTL with additive and epistatic effects and their genotype-environment interactions. Crop Sci, 2005. 45: 51-59.
[26] Delourme R, Falentin C, Huteau V, Clouet V. Genetic control of oil content in oilseed rape (Brassica napus L.). Theor Appl Genet, 2006.113: 1331-1345.
[27] Ohlrogge J, Jaworski JG Regulation of fatty acid synthesis. Annu Rev Plant Physiol Plant Mol Biol, 1997. 48: 109-136.
[28] Roesler K, Shintani D, Savage L, Boddupalli S, Ohlrogge J. Targeting of the Arabidopsis homomeric acetyl-coenzyme A carboxylase to plastids of rapeseeds. Plant Physiol, 1997.113: 75-81.
[29] Matthew J H. Control of storage-product synthesis in seeds. Curr Opin Plant Biol, 2004. 7:302-308.
[30] Weselake RJ, Pomeroy MK, Furukawa TL, Golden JL, Little DB, Laroche A Developmental profile of diacylglycerolacyltransferase in maturing seeds of oilseed rape and safflower and micro-spore-derived cultures of oilseed rape. Plant Physiol, 1993.102: 565-571.
[31] Perry HY, Bligny R, Gout E, Harwood JL. Changes in Kennedy pathway intermediates associated with increased triacylglycerol synthesis in oil-seeds rape. Phytochemistry, 1999.52: 799-804.
[32] Dahlqvist A, St(?)hl U, Lenman M, Banas A, Lee M, Sandager L, Ronne H, Stymne S. Phospholipid: diacylglycerol acyltransferase: An enzyme that catalyzes the acyl-CoA-independent formation of triacylglycerol in yeast and plants. PNAS, 2000. 97(12) : 6487-6492.
[33]Jako C,Kumar A,Wei YD,Zou JT,Barton DL,Giblin ME,Covello PS,Taylor DC.Seed-specific over-expression of an Arabidopsis cDNA encoding a diacylglycerol acyltransferase enhances seed oil content and seed weight.Plant Physiol,2001.126:861-874.
[34]官春云,李旬.转基因油菜应用研究.细胞生物学杂志,1997.19(1):18-23.
[35]Vesna K,Winnie F,Dennis LB,Kalie KG,Giblin EM,Tammy L,Samuel LM,Wilfred AK,Daryl M,Taylor DC.Improving erucic acid content in rape seed through biotechnology:what can the Arabidopsis FAE1 and the yeast SLC1-1 genes contribute.Crop Sci,2001.41:739-747.
[36]Focks N,Benning C.wrinkled1:A novel,low-seed-oil mutant of Arabidopsis with a deficiency in the seed-specific regulation of carbohydrate metabolism.Plant Physiol,1998.118(1):91-101.
[37]Cernac A,Benning C,WRINKLED1 encodes an AP2/EREB domain protein involved in the control of storage compound biosynthesis in Arabidopsis.Plant J,2004.40(4):575-585.
[38]石淑稳,周永明,孙学成.甘蓝型油菜遗传转化体系的研究.华中农业大学学报,1998.17(3):205-210.
[39]Santarem ER,Trick HN,Essig JS,Finer JJ.Sonication-assisted Agrobacterium-mediated transformation of soybean immature cotyledons:optimization of transient expression.Plant Cell Rep,1998.17:752-759.
[40]谭小力,孔凡明,张丽丽,李娟.蓝细菌血红蛋白基因的克隆及其向甘蓝型油菜中的转化.作物学报,2009.35(1):66-70.
[41]Kong F,Li J,Tan X.A new time-saving transformation system for Brassica napus.African Journal of Biotechnology,2009.8(11):2497-2502.
[42]Clough SJ,Bent AF.Floral dip:A simplified method for Agrobacterium-mediated transformation of Arabidopsis thaliana.Plant J,1998.16:735-743.
[43]徐光硕,饶永强,陈雁.用in planta方法转化甘蓝型油菜.作物学报,2004.30(1):125.
[44]Juan Li,Xiaoli Tan.A rapid and simple method for Brassica napus floral-dip transformation and selection of transgenic plantlets.International Journal of Biology,2010,2(1):127-131.
[45]韦献雅,牛应泽,张其坤,3种筛选NPT-Ⅱ标记转基因油菜方法研究.中国农学通报,2009.25(14):37-41.
[46]牛应泽,韦献雅,郭世星.利用卡那霉素浸种法筛选转基因油菜的方法.中华人民共和国国家知识产权局,2008.CN101236197A.
[47]谭小力,张志燕,伍娟,李家洋.油菜种子特异表达启动子NAPIN的功能鉴定.中国油料作物学报,2005.27(4):85-88.
[48]Watkins PA.Fatty acid activation.Prog Lipid Res,1997.36:55-83.
[49]Groot P,Scholte H,Hulsmann W.Fatty acid activation:specificity,localization,and function.Adv Lipid Res,1976.14:75-126.
[50]Bibb MJ,Sherman DH,Omura S,Hopwood DA.Cloning,sequencing and deduced functions of a cluster of Streptomyces genes probably encoding biosynthesis of the polyketide antibiotic frenolicin.Gene,1994.142(1):31-39.
[51]Conti E,Franks NP,Brick P.Crystal structure of firefly luciferase throws light on a superfamily of adenylate-forming enzymes.Structure,1996.4(3):287-298.
[52]Conti E,Stachelhaus T,Marahiel MA,Peter B.Structural basis for the activation of phenylalanine in the non-ribosomal biosynthesis of gramicidin S.The EMBO J,1997.16(4):4174-4183.
[53]Babbitt PC,Kenyon GL,Martin BM,Charest H,Slyvestre M,Scholten JD,Chang KH,Liang PH,Dunaway MD.Ancestry of the 4-chlorobenzoate dehalogenase:analysis of amino acid sequence identities among families of acyl:adenyl ligases,enoyl-CoA hydratases/isomerases,and acyl-CoA thioesterases.Biochemistry,1992.31(24):5594-5604.
[54]Steinberg SJ,Morgenthaler J,Heinzer AK,Smith KD,Watkins PA.Very long-chain acyl-CoA synthetases.Human "Bubblegum"represents a new family of proteins capable of activating very long-chain fatty acids.J Biol Chem,2000.275(45):35162-35169.
[55]Iijima H,Fujino T,Minekura H,Suzuki H,Kang M J,Yamamoto T.Biochemical studies of two rat acyl-CoA synthetases,ACS1 and ACS2.Eur J Biochem,1996.242(2):186-190.
[56]Chong B,Tan X,Zhou J,Yuan W.In Silicon Cloning and Analysis of a LACS Gene in Brassica napus.2008 IEEE,2009.140-143.
[57]崇保强,谭小力.油菜LACS4基因的电子克隆和功能分析.江苏农业学报,2008.25(1):38-43.
[58]Black P,DiRusso C,Metzger A,Heimert T.Cloning,sequencing,and expression of the fadD gene of Escherichia coli encoding acyl coenzyme A synthetase.J Biol Chem,1992.267(35):25513-25520.
[59]Faergeman NJ,Black PN,Zhao XD,Knudsen J,DiRusso CC.The Acyl-CoA synthetases encoded within FAA1 and FAA4 in Saccharomyces cerevisiae function as components of the fatty acid transport system linking import,activation,and intracellular Utilization.J Biol Chem,2001.276:37051-37059.
[60]Tonon T,Qing R,Harvey D,Li Y,Larson TR,Graham IA.Identification of a Long-Chain Polyunsaturated Fatty Acid Acyl-Coenzyme A Synthetase from the Diatom Thalassiosira pseudonana.Plant Physiol,2005.138:402-408.
[61]Choi JY,Martin CE.The Saccharomyces cerevisiae FAT1 gene encodes an acyl-CoA synthetase that is required for maintenance of very long chain fatty acid levels.J Biol Chem,1999.274:4671-4683.
[62]Mashek DG,Bornfeldt KE.Revised nomenclature for the mammalian long-chain acyl-CoA synthetase gene family.J Lipid Res,2004.45:1958-1961.
[63] Ohlrogge JB, Browse J, Somerville CR. The genetics of plant lipids. Biochim Biophys Acta,1991.1082:1-26.
[64] Shockey JM, Fulda MS, Browse JA. Arabidopsis contains nine long-chain acyl-coenzyme A synthetase genes that participate in fatty acid and glycerolipid metabolism. Plant Physiol,2002.129(4): 1710-1722.
[65] Schnurr J, Shockey J, Browse J. The acyl-CoA synthetase encoded by LACS2 is essential for normal cuticle development in Arabidopsis. Plant Cell, 2004.16(3): 629-642.
[66] Shiyou Lu¨, Song T, Kosma DK, Parsons EP, Rowland O, Jenks MA. Arabidopsis CER8 encodes long-chain acyl-CoA synthetase 1 (LACS1) that has overlapping functions with LACS2 in plant wax and cutin synthesis. Plant J, 2009. (Doi:10.1111/j.1365-313X.2009.03892.x).
[67] Fulda M, Shockey J. Two long-chain acyl-CoA synthetases from Arabidopsis thaliana involved in peroxisomal fatty acid beta-oxidation. Plant J, 2002. 32(1): 93-103.
[68] Schnurr JA, Shockey JM, De Boer GJ, Browse JA. Fatty Acid export from the chloroplast.Molecular characterization of a major plastidial acyl-Coenzyme A synthetase from Arabidopsis. Plant Physiol, 2002.129(4): 1700-1709.
[69] Li XB, Wang XL. The GhACSl gene encodes an acyl-CoA synthetase which is essential for normal microsporogenesis in early anther development of cotton. Plant J, 2008. 57(3):473-486.
[70] He XH, Chen GQ, Kang ST, McKeon TA. Ricinus communis Contains an Acyl-CoA Synthetase that Preferentially Activates Ricinoleate to Its CoA Thioester. Lipids, 2007. 42:931-938. (Doi: 10.1007/sll745-007-3090-0).
[71] Fulda M, Heinz E, Wolter FP. Brassica napus cDNAs encoding fatty acyl-CoA synthetase. Plant Mol Biol, 1997. 33: 911-922.
[72] Hills M, Pongdontri P. Characterization of a novel plant acyl-CoA synthetase that is expressed in lipogenic tissues of Brassica napus L. Plant Mol Biol, 2001. 47: 717-726.
[73] Browse J, Walk's JG Mutants of Arabidopsis reveal many roles for membrane lipids. Prog Lipid Res, 2002. 41(3): 254-278.
[74] Huang AH. Oleosins and Oil Bodies in Seeds and Other Organs. Plant Physiol, 1996.110(4):1055-1061.
[75] Napier JA, Graham IA. Tailoring plant lipid composition: designer oilseeds come of age. Cur Opin Plant Biol, 2010.13:1-8. (Doi 10.1016/j.pbi.2010.01.008)
[76] Lepiniec L, Baud S. Regulation of de novo fatty acid synthesis in maturing oilseeds of Arabidopsis. Plant Physiol Biochem, 2009. 47(6): 448-455
[77] Wang HW, Zhang B, Hao YJ, Huang J, Tian AG, Liao Y, Zhang JS, Chen SY. The soybean Dof-type transcription factor genes, GmDof4 and GmDofll, enhance lipid content in the seeds of transgenic Arabidopsis plants. Plant J, 2007.52: 716-729.
[78] Ichihara KI, Kobayashi N, Saito K. lipid synthesis and acyl-CoA synthetase in developing rice seeds.Lipids,2003.38(8):881-884.
[79]Rubio S,Whitehead L,Larson TR,Graham IA,Rodriguez PL The coenzyme A biosynthetic enzyme phosphopantetheine adenylyltransferase plays a crucial role in plant growth,salt/osmotic stress resistance,and seed lipid storage.Plant Physiology 2008.148:546-556.(Doi:10.1104/pp.108.124057)
[80]Cooper TG,Beevers H.Mitochondria and glyoxysomes from castor bean endosperm.Enzyme constitutents and catalytic capacity.J Biol Chem,1969.244:3507-3513.
[81]Zolman BK,Silva ID,Bartel B.The Arabidopsis pxal Mutant is defective in an ATP-binding cassette transporter-like protein required for peroxisomal fatty acid beta -oxidation.Plant Physiol.2001.127(3):1266-1278.
[82]Footitt S,Slocombe SP,Larner V,Kurup S,Wu Y,Larson T,Graham I,Baker A,Holdsworth M.Control of germination and lipid mobilization by COMATOSE,the Arabidopsis homologue of human ALDP.Plant Biology,2002.21:2912-2922.
[83]Slocombe SP,Cornah J,Pinfield H,Soady K,Zhang QY,Gilday A,Dyer JM,Graham IA.Oil accumulation in leaves directed by modification of fatty acid breakdown and lipid synthesis pathways.Plant Biotechnol J,2009.7(7):694-703.
[84]Fuldaa M,Schnurra J,Abbadib A,Heinzb E,Browsea J.Peroxisomal Acyl-CoA synthetase activity is essential for seedling development in Arabidopsis thaliana.Plant Cell,2004.16(2):394-405.
[85]Fulda M,Shockey J,Werber M,Wolter FP,Heinz E.Two long-chain acyl-CoA synthetases from Arabidopsis thaliana involved in peroxisomal fatty acid β-oxidation.Plant J,2002.32(1):93-103.
[86]徐靖,李辉亮,朱家红,彭世清,植物脂肪酸β-氧化的研究进展.生物技术通讯,2008.19(1):141-144.
[87]Farmer EE,Weber H,Vollenweider S.Fatty acid signaling in Arabidopsis.Planta,1998.206(2):167-174.
[88]Weng H,Molina I,Shockey J,Browse J.Organ fusion and defective cuticle function in a lacs1 lacs2 double mutant of Arabidopsis.Planta,2010.231:1089-1100.
[89]董欣荣,曹健,赵斌和许伟华,几种真菌油脂的检测与提取.郑州工程学院学报,2002.23(1):1-5.
[2]柴国华,白泽涛,蔡丽,郭学兰,张学江,石磊,董彩华,刘胜毅.油菜基因BnWRI1的克隆及RNAi对种子含油量的影响.中国农业科学,2009.42(5):1512-1518.
[3]王汉中.我国油菜产需形势分析及产业发展对策.中国油料作物学报,2007.29(1):101-105.
[4]朱福各,谭小力,崇保强,周佳,袁伟伟.油菜脂酰CoA合成酶基因pXT166的鉴定和功能分析.中国油料作物学报,2009.31(3):274-278.
[5]李军,吴平治,李美茹,吴国江.能源植物的研究进展及其发展趋势.Chinese Journal of Nature,2007.29(1):21-25.
[6]张壵,李云昌,梅德圣,胡琼.油菜油脂研究进展.植物学通报,2007.24(4):435-443.
[7]官春云.油菜生产情况与科研进展.作物研究,2001.4:4-8.
[8]程红焱,宋松泉.种子萌发过程中贮藏油脂的动员.云南植物研究,2007.29(1):67-73.
[9]Si P,Rodney JM,Nick G,David WT.Influence of genotype and environment on oil and protein concentrations of canola(Brassica napus L.) grown across southern Australia.Aust J Agric Res,2003.54:397-407.
[10]Gutierrez Boem FH,Lavado RS,Porcelli CA.Note on the effects of winter and spring watedogging on growth,chemical composition and yield of rapeseed.Field Crops Res,1996.47:175-179.
[11]Wu JG,Shi CH,Zhang HZ.Partitioning genetic effects due to embryo,cytoplasm and maternal parent for oil content in oilseed rape(Brassica napus L.).Genet Mol Biol,2006.29:533-538.
[12]Hom NH,Girke A,M(o|¨)llers C,Becker HC.Influence of pollen genotype on rapeseed quality-analyses of single seeds by near-infrared reflectance spectroscopy(NIRS).Proceedings of the 11th International Rapeseed Congress.2003.266-269.
[13]张洁夫,戚存扣,浦惠明,陈松,陈锋,陈新军,高建芹,傅寿仲.甘蓝型油菜主要脂肪酸的主基因+多基因遗传分析.中国油料作物学报,2007.29(4):259-364.
[14]Ohlrogge J,Browse J.Lipid biosynthesis.Plant Cell,1995.7:957-970.
[15]Mu J,Tan H,Zheng Q,Fu F,Liang Y,Zhang J,Yang X,Wang T,Chong K,Wang X,Zuo J.Leafy Cotyledon1 is a key regulator of fatty acid biosynthesis in Arabidopsis thaliana.Plant Physiol,2008.148:1042-1054.
[16]Harwood JL.Recent advances in the biosynthesis of plant fatty acids.Biochim Biophys Acta,1996.1301:7-56.
[17]周奕华,陈正华.植物种子中脂肪酸代谢途径的遗传调控与基因工程.植物学通报, 1998.15(5): 16-23.
[18] Hugly S, Somerville C. A Role for Membrane Iipid Polyunsaturation in Chloroplast Biogenesis at Low Temperature. Plant Physiol, 1992. 99(1): 197-202.
[19] Weselake RJ. Storage lipids. Plant lipids: biology, utilization and manipulation. 2005.162-225.
[20] Olsen JA, Lusk K. Acyl-CoA synthetase activity associated with rapeseed lipid body membranes. Phytochemistry, 1994. 36: 7-9.
[21] Huang AH. Oleosins and Oil Bodies in Seeds and Other Organs. Plant Physiol, 1996. 110(4):1055-1061.
[22] Ecke W, Uzunova M, Weissleder K. Mapping the genome of rapeseed (Brassica napus L.) II Localization of genes controlling erucic acid synthesis and seed oil content. Theor Appl Genet, 1995.91: 972-977.
[23] Cheung WY, Landry BS, Raney P, Pakow GFW. Molecular mapping of seed quality traits in Brassica juncea (L.) Czern and Coss. Acta Hort, 1998.459:139-147.
[24] Burns MJ, Barnes SR, Bowman JG, Clarke MHE, Werner CP, Kearsey MJ . QTL analysis of an intervarietal set of substitution lines in Brassica napus: (i) seed oil content and fatty acid composition. Heredity, 2003. 90: 39-48.
[25] Zhao JY, Heiko CB, Zhang DQ. Oil content in a EuropeanxChinese rapeseed population: QTL with additive and epistatic effects and their genotype-environment interactions. Crop Sci, 2005. 45: 51-59.
[26] Delourme R, Falentin C, Huteau V, Clouet V. Genetic control of oil content in oilseed rape (Brassica napus L.). Theor Appl Genet, 2006.113: 1331-1345.
[27] Ohlrogge J, Jaworski JG Regulation of fatty acid synthesis. Annu Rev Plant Physiol Plant Mol Biol, 1997. 48: 109-136.
[28] Roesler K, Shintani D, Savage L, Boddupalli S, Ohlrogge J. Targeting of the Arabidopsis homomeric acetyl-coenzyme A carboxylase to plastids of rapeseeds. Plant Physiol, 1997.113: 75-81.
[29] Matthew J H. Control of storage-product synthesis in seeds. Curr Opin Plant Biol, 2004. 7:302-308.
[30] Weselake RJ, Pomeroy MK, Furukawa TL, Golden JL, Little DB, Laroche A Developmental profile of diacylglycerolacyltransferase in maturing seeds of oilseed rape and safflower and micro-spore-derived cultures of oilseed rape. Plant Physiol, 1993.102: 565-571.
[31] Perry HY, Bligny R, Gout E, Harwood JL. Changes in Kennedy pathway intermediates associated with increased triacylglycerol synthesis in oil-seeds rape. Phytochemistry, 1999.52: 799-804.
[32] Dahlqvist A, St(?)hl U, Lenman M, Banas A, Lee M, Sandager L, Ronne H, Stymne S. Phospholipid: diacylglycerol acyltransferase: An enzyme that catalyzes the acyl-CoA-independent formation of triacylglycerol in yeast and plants. PNAS, 2000. 97(12) : 6487-6492.
[33]Jako C,Kumar A,Wei YD,Zou JT,Barton DL,Giblin ME,Covello PS,Taylor DC.Seed-specific over-expression of an Arabidopsis cDNA encoding a diacylglycerol acyltransferase enhances seed oil content and seed weight.Plant Physiol,2001.126:861-874.
[34]官春云,李旬.转基因油菜应用研究.细胞生物学杂志,1997.19(1):18-23.
[35]Vesna K,Winnie F,Dennis LB,Kalie KG,Giblin EM,Tammy L,Samuel LM,Wilfred AK,Daryl M,Taylor DC.Improving erucic acid content in rape seed through biotechnology:what can the Arabidopsis FAE1 and the yeast SLC1-1 genes contribute.Crop Sci,2001.41:739-747.
[36]Focks N,Benning C.wrinkled1:A novel,low-seed-oil mutant of Arabidopsis with a deficiency in the seed-specific regulation of carbohydrate metabolism.Plant Physiol,1998.118(1):91-101.
[37]Cernac A,Benning C,WRINKLED1 encodes an AP2/EREB domain protein involved in the control of storage compound biosynthesis in Arabidopsis.Plant J,2004.40(4):575-585.
[38]石淑稳,周永明,孙学成.甘蓝型油菜遗传转化体系的研究.华中农业大学学报,1998.17(3):205-210.
[39]Santarem ER,Trick HN,Essig JS,Finer JJ.Sonication-assisted Agrobacterium-mediated transformation of soybean immature cotyledons:optimization of transient expression.Plant Cell Rep,1998.17:752-759.
[40]谭小力,孔凡明,张丽丽,李娟.蓝细菌血红蛋白基因的克隆及其向甘蓝型油菜中的转化.作物学报,2009.35(1):66-70.
[41]Kong F,Li J,Tan X.A new time-saving transformation system for Brassica napus.African Journal of Biotechnology,2009.8(11):2497-2502.
[42]Clough SJ,Bent AF.Floral dip:A simplified method for Agrobacterium-mediated transformation of Arabidopsis thaliana.Plant J,1998.16:735-743.
[43]徐光硕,饶永强,陈雁.用in planta方法转化甘蓝型油菜.作物学报,2004.30(1):125.
[44]Juan Li,Xiaoli Tan.A rapid and simple method for Brassica napus floral-dip transformation and selection of transgenic plantlets.International Journal of Biology,2010,2(1):127-131.
[45]韦献雅,牛应泽,张其坤,3种筛选NPT-Ⅱ标记转基因油菜方法研究.中国农学通报,2009.25(14):37-41.
[46]牛应泽,韦献雅,郭世星.利用卡那霉素浸种法筛选转基因油菜的方法.中华人民共和国国家知识产权局,2008.CN101236197A.
[47]谭小力,张志燕,伍娟,李家洋.油菜种子特异表达启动子NAPIN的功能鉴定.中国油料作物学报,2005.27(4):85-88.
[48]Watkins PA.Fatty acid activation.Prog Lipid Res,1997.36:55-83.
[49]Groot P,Scholte H,Hulsmann W.Fatty acid activation:specificity,localization,and function.Adv Lipid Res,1976.14:75-126.
[50]Bibb MJ,Sherman DH,Omura S,Hopwood DA.Cloning,sequencing and deduced functions of a cluster of Streptomyces genes probably encoding biosynthesis of the polyketide antibiotic frenolicin.Gene,1994.142(1):31-39.
[51]Conti E,Franks NP,Brick P.Crystal structure of firefly luciferase throws light on a superfamily of adenylate-forming enzymes.Structure,1996.4(3):287-298.
[52]Conti E,Stachelhaus T,Marahiel MA,Peter B.Structural basis for the activation of phenylalanine in the non-ribosomal biosynthesis of gramicidin S.The EMBO J,1997.16(4):4174-4183.
[53]Babbitt PC,Kenyon GL,Martin BM,Charest H,Slyvestre M,Scholten JD,Chang KH,Liang PH,Dunaway MD.Ancestry of the 4-chlorobenzoate dehalogenase:analysis of amino acid sequence identities among families of acyl:adenyl ligases,enoyl-CoA hydratases/isomerases,and acyl-CoA thioesterases.Biochemistry,1992.31(24):5594-5604.
[54]Steinberg SJ,Morgenthaler J,Heinzer AK,Smith KD,Watkins PA.Very long-chain acyl-CoA synthetases.Human "Bubblegum"represents a new family of proteins capable of activating very long-chain fatty acids.J Biol Chem,2000.275(45):35162-35169.
[55]Iijima H,Fujino T,Minekura H,Suzuki H,Kang M J,Yamamoto T.Biochemical studies of two rat acyl-CoA synthetases,ACS1 and ACS2.Eur J Biochem,1996.242(2):186-190.
[56]Chong B,Tan X,Zhou J,Yuan W.In Silicon Cloning and Analysis of a LACS Gene in Brassica napus.2008 IEEE,2009.140-143.
[57]崇保强,谭小力.油菜LACS4基因的电子克隆和功能分析.江苏农业学报,2008.25(1):38-43.
[58]Black P,DiRusso C,Metzger A,Heimert T.Cloning,sequencing,and expression of the fadD gene of Escherichia coli encoding acyl coenzyme A synthetase.J Biol Chem,1992.267(35):25513-25520.
[59]Faergeman NJ,Black PN,Zhao XD,Knudsen J,DiRusso CC.The Acyl-CoA synthetases encoded within FAA1 and FAA4 in Saccharomyces cerevisiae function as components of the fatty acid transport system linking import,activation,and intracellular Utilization.J Biol Chem,2001.276:37051-37059.
[60]Tonon T,Qing R,Harvey D,Li Y,Larson TR,Graham IA.Identification of a Long-Chain Polyunsaturated Fatty Acid Acyl-Coenzyme A Synthetase from the Diatom Thalassiosira pseudonana.Plant Physiol,2005.138:402-408.
[61]Choi JY,Martin CE.The Saccharomyces cerevisiae FAT1 gene encodes an acyl-CoA synthetase that is required for maintenance of very long chain fatty acid levels.J Biol Chem,1999.274:4671-4683.
[62]Mashek DG,Bornfeldt KE.Revised nomenclature for the mammalian long-chain acyl-CoA synthetase gene family.J Lipid Res,2004.45:1958-1961.
[63] Ohlrogge JB, Browse J, Somerville CR. The genetics of plant lipids. Biochim Biophys Acta,1991.1082:1-26.
[64] Shockey JM, Fulda MS, Browse JA. Arabidopsis contains nine long-chain acyl-coenzyme A synthetase genes that participate in fatty acid and glycerolipid metabolism. Plant Physiol,2002.129(4): 1710-1722.
[65] Schnurr J, Shockey J, Browse J. The acyl-CoA synthetase encoded by LACS2 is essential for normal cuticle development in Arabidopsis. Plant Cell, 2004.16(3): 629-642.
[66] Shiyou Lu¨, Song T, Kosma DK, Parsons EP, Rowland O, Jenks MA. Arabidopsis CER8 encodes long-chain acyl-CoA synthetase 1 (LACS1) that has overlapping functions with LACS2 in plant wax and cutin synthesis. Plant J, 2009. (Doi:10.1111/j.1365-313X.2009.03892.x).
[67] Fulda M, Shockey J. Two long-chain acyl-CoA synthetases from Arabidopsis thaliana involved in peroxisomal fatty acid beta-oxidation. Plant J, 2002. 32(1): 93-103.
[68] Schnurr JA, Shockey JM, De Boer GJ, Browse JA. Fatty Acid export from the chloroplast.Molecular characterization of a major plastidial acyl-Coenzyme A synthetase from Arabidopsis. Plant Physiol, 2002.129(4): 1700-1709.
[69] Li XB, Wang XL. The GhACSl gene encodes an acyl-CoA synthetase which is essential for normal microsporogenesis in early anther development of cotton. Plant J, 2008. 57(3):473-486.
[70] He XH, Chen GQ, Kang ST, McKeon TA. Ricinus communis Contains an Acyl-CoA Synthetase that Preferentially Activates Ricinoleate to Its CoA Thioester. Lipids, 2007. 42:931-938. (Doi: 10.1007/sll745-007-3090-0).
[71] Fulda M, Heinz E, Wolter FP. Brassica napus cDNAs encoding fatty acyl-CoA synthetase. Plant Mol Biol, 1997. 33: 911-922.
[72] Hills M, Pongdontri P. Characterization of a novel plant acyl-CoA synthetase that is expressed in lipogenic tissues of Brassica napus L. Plant Mol Biol, 2001. 47: 717-726.
[73] Browse J, Walk's JG Mutants of Arabidopsis reveal many roles for membrane lipids. Prog Lipid Res, 2002. 41(3): 254-278.
[74] Huang AH. Oleosins and Oil Bodies in Seeds and Other Organs. Plant Physiol, 1996.110(4):1055-1061.
[75] Napier JA, Graham IA. Tailoring plant lipid composition: designer oilseeds come of age. Cur Opin Plant Biol, 2010.13:1-8. (Doi 10.1016/j.pbi.2010.01.008)
[76] Lepiniec L, Baud S. Regulation of de novo fatty acid synthesis in maturing oilseeds of Arabidopsis. Plant Physiol Biochem, 2009. 47(6): 448-455
[77] Wang HW, Zhang B, Hao YJ, Huang J, Tian AG, Liao Y, Zhang JS, Chen SY. The soybean Dof-type transcription factor genes, GmDof4 and GmDofll, enhance lipid content in the seeds of transgenic Arabidopsis plants. Plant J, 2007.52: 716-729.
[78] Ichihara KI, Kobayashi N, Saito K. lipid synthesis and acyl-CoA synthetase in developing rice seeds.Lipids,2003.38(8):881-884.
[79]Rubio S,Whitehead L,Larson TR,Graham IA,Rodriguez PL The coenzyme A biosynthetic enzyme phosphopantetheine adenylyltransferase plays a crucial role in plant growth,salt/osmotic stress resistance,and seed lipid storage.Plant Physiology 2008.148:546-556.(Doi:10.1104/pp.108.124057)
[80]Cooper TG,Beevers H.Mitochondria and glyoxysomes from castor bean endosperm.Enzyme constitutents and catalytic capacity.J Biol Chem,1969.244:3507-3513.
[81]Zolman BK,Silva ID,Bartel B.The Arabidopsis pxal Mutant is defective in an ATP-binding cassette transporter-like protein required for peroxisomal fatty acid beta -oxidation.Plant Physiol.2001.127(3):1266-1278.
[82]Footitt S,Slocombe SP,Larner V,Kurup S,Wu Y,Larson T,Graham I,Baker A,Holdsworth M.Control of germination and lipid mobilization by COMATOSE,the Arabidopsis homologue of human ALDP.Plant Biology,2002.21:2912-2922.
[83]Slocombe SP,Cornah J,Pinfield H,Soady K,Zhang QY,Gilday A,Dyer JM,Graham IA.Oil accumulation in leaves directed by modification of fatty acid breakdown and lipid synthesis pathways.Plant Biotechnol J,2009.7(7):694-703.
[84]Fuldaa M,Schnurra J,Abbadib A,Heinzb E,Browsea J.Peroxisomal Acyl-CoA synthetase activity is essential for seedling development in Arabidopsis thaliana.Plant Cell,2004.16(2):394-405.
[85]Fulda M,Shockey J,Werber M,Wolter FP,Heinz E.Two long-chain acyl-CoA synthetases from Arabidopsis thaliana involved in peroxisomal fatty acid β-oxidation.Plant J,2002.32(1):93-103.
[86]徐靖,李辉亮,朱家红,彭世清,植物脂肪酸β-氧化的研究进展.生物技术通讯,2008.19(1):141-144.
[87]Farmer EE,Weber H,Vollenweider S.Fatty acid signaling in Arabidopsis.Planta,1998.206(2):167-174.
[88]Weng H,Molina I,Shockey J,Browse J.Organ fusion and defective cuticle function in a lacs1 lacs2 double mutant of Arabidopsis.Planta,2010.231:1089-1100.
[89]董欣荣,曹健,赵斌和许伟华,几种真菌油脂的检测与提取.郑州工程学院学报,2002.23(1):1-5.