枸杞叶片、果实cDNA文库的构建及LycB、IPI基因的分离
|
摘要
本研究以宁夏枸杞叶片及果实为材料，采用异硫氰酸胍—酚—氯仿法提取完整
的总RNA，利用PolyATract mRNA Isolation System(Promega公司)分离mRNA，
然后利用Universal Riboclone ~RcDNA Synthesis System (Promega公司)合成双链
cDNA，在cDNA末端连接上EcoRⅠ接头，并与λExCell载体连接，利用Packagene Lambda DNA Packaging System进行了包装，在国内外首次构建出滴度分别为2.78×
10~5 pfu／ml、8.4×10~4 pfu/ml，重组效率为88.1％、86.9％的枸杞叶片及果实cDNA文库。
用Digoxigenin(DIG)标记龙胆草LycB探针、IPI探针、PSY探针、ZDS探针、
LycE探针，并对枸杞叶片及果实cDNA文库进行筛选，获得8个LycB cDNA阳性
克隆及7个IPI cDNA阳性克隆。释放质粒后，进行酶切鉴定，LycB阳性克隆cDNA
插入片段的大小为2.1kb左右，IPI阳性克降cDNA插入片段的大小约为1.5kb。本
研究为利用基因工程手段来调控类胡萝卜素的生物合成奠定了基础。
Total RNA of leaf and fruit Lycium Barbarum L. was isolated using guanidinium isothioccyanate and phenol hloroform. mRNA was prepared by PolyATract mRNA Isolation Systems (Promega). Full條ength cDNA was synthesized by Universal RiboClone cDNA Synthesis System(Promega) and then cloned into phage # Excell vector, After Pachage in Packagene lambda DNA Packaging System, the capacities of the libraries were measured. The capacities of the libraries were 2.78 X 105pfu/ml and 8.4 X 104 pfu/ml. the recombination rates reached to 88.1% and 86.9%.
Heterologous probe of Lye-. IPI PSY, ZDS, LycE originate from Getina lutea were labeled with digoxingenin-11-dUTP (DIG), were further used to screen the cDNA libraries of leaf and fruit of lycium barbarm. Eiight lycB positive clones were obtained and seven IPI positive clones were obtained. The recombinants with the cDNA insert fragment were released from the positive clones and were digested with EcoR I , The cDNA fragment were 2.1kb and 1.5kb in lycB and IPI positive clones respectively. This
study will lay foundations for regulating carotenoid biosynthesis via genetic engineening.
的总RNA，利用PolyATract mRNA Isolation System(Promega公司)分离mRNA，
然后利用Universal Riboclone ~RcDNA Synthesis System (Promega公司)合成双链
cDNA，在cDNA末端连接上EcoRⅠ接头，并与λExCell载体连接，利用Packagene Lambda DNA Packaging System进行了包装，在国内外首次构建出滴度分别为2.78×
10~5 pfu／ml、8.4×10~4 pfu/ml，重组效率为88.1％、86.9％的枸杞叶片及果实cDNA文库。
用Digoxigenin(DIG)标记龙胆草LycB探针、IPI探针、PSY探针、ZDS探针、
LycE探针，并对枸杞叶片及果实cDNA文库进行筛选，获得8个LycB cDNA阳性
克隆及7个IPI cDNA阳性克隆。释放质粒后，进行酶切鉴定，LycB阳性克隆cDNA
插入片段的大小为2.1kb左右，IPI阳性克降cDNA插入片段的大小约为1.5kb。本
研究为利用基因工程手段来调控类胡萝卜素的生物合成奠定了基础。
Total RNA of leaf and fruit Lycium Barbarum L. was isolated using guanidinium isothioccyanate and phenol hloroform. mRNA was prepared by PolyATract mRNA Isolation Systems (Promega). Full條ength cDNA was synthesized by Universal RiboClone cDNA Synthesis System(Promega) and then cloned into phage # Excell vector, After Pachage in Packagene lambda DNA Packaging System, the capacities of the libraries were measured. The capacities of the libraries were 2.78 X 105pfu/ml and 8.4 X 104 pfu/ml. the recombination rates reached to 88.1% and 86.9%.
Heterologous probe of Lye-. IPI PSY, ZDS, LycE originate from Getina lutea were labeled with digoxingenin-11-dUTP (DIG), were further used to screen the cDNA libraries of leaf and fruit of lycium barbarm. Eiight lycB positive clones were obtained and seven IPI positive clones were obtained. The recombinants with the cDNA insert fragment were released from the positive clones and were digested with EcoR I , The cDNA fragment were 2.1kb and 1.5kb in lycB and IPI positive clones respectively. This
study will lay foundations for regulating carotenoid biosynthesis via genetic engineening.
引文
[1] 陶俊，张上降，炎胡萝卜素合成的相关基因及其基因工程，生物工程学报，2002，5：276—281
[2] 韩雅珊，类胡萝卜素的功能研究进展，中国农业大学学报，1999，4(1)：5—9
[3] Bartleyg E, Scolnik P A. Plant carotenoids:Pigments for photoprotection, visual attraction and human health. Plant Cell, 1995, 7: 1027-1038
[4] Rock C D, Zeevaart J A D. The ABAmutant of Arabidopsis thaliana is impaired in epoxy-carotenoid biosynthesis. Proc Natl Aead Sci USA, 1991, 88:7496-7499
[5] goodwin T W, Brittong. Distribution and analysis ofcarotenoids. In:Goodwin FW(Ed), Plant Pigment. Academic Press, London, 1988, 61—132
[6] 徐昌杰，张上隆，植物类胡萝卜素的生物合成及其调控，植物生理学通讯．2000，2 16(1)：64—70
[7] Bouvier. F., Keller. Y., Xanthophy 11 biosynthesis molecular and functional characterization of carotenoid hydroxylases from pepper fruits Biochim. Biophs 1998 1391(3): 320—828
[8] Pecker I, gabbay R, Francis X et al. Cloning and characterization of the cDNA for β-cyclasc from tomatoreveals decrease in ins expression during fruit ripening. Plant Mol Biol, 1996, 30: 807—819
[9] Fraser PD, TruesdalemR, Bird CR et al. Carotenoid biosynthesis during tomato fruit development Plant Physioi, 1994, 105:379—387
[10] Albrechtm, Sandanng. Light-stimulated carotenoid biosynthesis during transformation ofmaize etioplasts is regulated by increased activity of isopentenyl pyrophosphate isomerase. Plant Physiol, 1994, 105:529—534
[11] Giulianog, BartleygE. Scolnik PA. Regulation of carotenold biosynthesis during tomato development. Plant Cell, 1993, 5:379—387
[12] Von Lintig J, Welsch R , Bonkm et al. Tight dependent regulation of carotenoid biosynthesis occurs at the level of phytoene synthase expression and ismediated by phytochrome in Sinapis alba and Arabidopsis thaliana seedlings. Plant J, 1997, 12:625—634
[13] Yamamoto HY Xanthophyll cycles, method Enzymol, 1985, 110:303—312
[14] Dogbo O, Laferriere A, d' Harlingue A et al. Carotenoid biosynthsis:Isolation and characterization of a bifunctional enzyme catalyzing the synthesis of phytoene. Proc Natl Acad Scid Sci USA, 1988, 85:7054--7058
[15] JefferyD, SmithC, Goodenough P et al. Ethylene independent and ethylene- dependent biochemical changes inripening tomatoes. Plant Physiol, 1984, 74:32—38
[16] BartleygE, Scolnik PA. Plantcarotenoids: Pigments for photoprotection, visual attraction, and human health. Plant Cell, 1995, 7:1027—1038
[17] BartleygE, ScolnikPA. Molecular biology of carotenoid biosynthesis in plants. Annu Rev Plant Physiol Plantmol Biol, 1994, 45:287—301
[18] SteWard I, Wheaton TA. Carotenoids in Citrus: Their accumulation induced by ethylene. J Agric Food Chem , 1972, 20:448—449
[19] Perucka I. Ethephon induced changes in accumulation of carotenoids in red pepper fruit (Capsicum annuum L.) Hortic Abstr, 1997, 67:4997—5001
[20] Babczinski P , Heinemann U , Sandmanng et al. Inhibition of carotenoid biosynthesis by interaction of 2, 6- diphenylpyridine derivatives with phytoene desaturation. J Agric Food Chem, 1992, 40:2497—2499
[21] gamble PE, Mullet JE. Inhibition of carotenoid accumulation and abscisic acid biosynthesis in fiuridone- treated dark-grown barley. Eur J Biochem, 1986, 160:117—121
[22] Bouvier F, d' Harlingue A , Camara B .Molecular analysis of carotenoid cyclase inhibition. Arch Biochem Biophys, 1997, 346:53—64
[23] Coggins CW Jr, HenninggL, Yokoyama H. Lycopene accumulation induced by 2-(4
-chlorophenylthio)-triethylaminc hydrochloride. Sci,1970. 168: 1589—1590
[24] Ye X, Al Babili S, Kloti A et al. Engineering the provitamin A (β-carotene) biosynthetic pathway into (carotenoid-free) rice endosperm. Science, 2000. 287:303—305
[25] Shewmaker C K , Sheehy J A , Daleym et al. Seed specific overexpressein of phytoene synthase:increade in carotenoids and othermetabolic effectd, plant J, 1999, 20:401—412
[26] Bramley P, Teulicres C, Blain i et al. Biochemical characterization of transgenic tomato plantsin which carotenoid synthesis has been inhibited through the expression of antisense RNA topTOM5. Plant J. 1992, 2:343—349
[27] gray Rg, Grierson D. Identification andgenetic analysis of normal andmutant phytoene synthasegenes of tomato by sequencing, complemcntation and co-suppressoon. Plantmol Biol, 1993, 22:589—602
[28] Rosati C, Apuilanti R, Dharmapuri S et al. metabolie engineering of beta-carotene and lycopene content on tomato fruit. Plant J, 2000, 24:413—420
[29] misawa N, Yamano S, Linden H et al. Funetional expression of the Erwinoa uredevora carotwnoid biosynthesis activity and resistance to the blwaching herbicidemorflurazon. Plant J, 1993, 4:833—840
[30] Kumagaim H, Donson J.della-Cioppag et al. Cytoplasmic inhibition of carotenoid biosynthesis with viral-derived RNA. Proc Natl Acad Sci USA, t995, 92:1679—1683
[31] Dogbo O, Camara B. Purification of isopentenylpyrophosphate isomerase andgeranylgeranyl pyrophosphate synthase from Capsicum chromoplasts by affinity chromatography. Biochim BiophysActa, t987, 920:140—148
[32] Kuntzm, Romer S, Suite C et al. Identification of a Cdna for the plastid-locatedgeranylgeranyl pyrophosphate synthase from Capsicum annum: Correlative increase in enzyme activity and transcript level during fruit ripening. Plant J, 1992, 2:25—34
[33] Scolnik PA, BartleygE. Nucleotide sequence of anArabidopsis cDNA forgeranylgeranyl
pyrophosphate synthase. Plant Physiol, 1994, 104:1469—1470
[34] Ray JA, Bird CR, Manndersm et al. Sequence of pTOM5, a ripening related cDNA from tomato. Nucl Acid Res, 1987, 24:10587—10590
[35] ArmstronggA, Albertim, Hearst JE. Conserved enzymesmediate the early reactions of carotenoid biosynthesis in non-photosynthetic and photosynthetic procaryotes. Proc Natl Acad Sci USA, 1990, 87:9975—9979
[36] Bird CR, Ray JA, Fletcher JD et al. Using antisense RNA to studygene function :Inhibition of carotenoid biosynthesis in transgenic tomatoes. Bio/Technol, 1991, 9:635—639
[37] Bramley P, Teulieres C, Blain I et al. Biochemical characterization of transgenic tomato plants in which carotenoid synthesis has been inhibited through the expression of antisense RNA to pTOM5 Plant J, 1992, 2:343—349
[38] BartleygE, Viitanen PV , Bacot KO et al. A tomatogene expressed during fruit ripening encodes an enzyme of the carotenoid biosynthesis pathway .J Biol Chem, 1992, 267:5056—5039
[39] von Lintig J, Welsch R , Bonkm et al. Light- dependent regulation of carotenoid biosynthesis occurs at the level of phytoene synthase expression and ismediated by phytochrome in Sinapis alba and Arabidopsis thaliana seedlings. Plant J , 1997, 12:625—634
[40] BartleygE , Scolnik PA. cDNA cloning, expression during development andgenomemapping of PSY2 , a second tomatogene encoding phytoene synthase. J Biol Chem, 1993, 268: 25718—25721
[41] Chamovitz D, Pecker I, Hirschberg J. Themolecular basis of resistance to the herbicide norflurazon. Plantmol Biol, 1991, 16:967—974
[42] BartleygE, Viitanen PV, Pecker I et al. molecular cloning and expression in photosynthetic bacteria of asoybean cDNA coding fo rphytoene desaturase, an enzyme of the carotenoid biosynthesis pathway. Proc Natl Acad Sci USA, 1991, 88:6532—6536
[43] Pecker I, Chamovitz D, Linden H et al. A single polypeptide catalyzing the conversion of
phytoene to ζ- carotene is transcriptionally regulated during tomato fruit ripening. Proc Natl Acad Sci USA. 1992, 89: 4962—4966
[44] Albrechtm , Klein A, Hugueney P et al. molecular cloning and functional expression in E .cell of a novel plant enzymemediating zeta- carotene desaturation. FEBS Lett, 1995, 372: 199—202
[45] Cunningham FX Jr, Pogson B, Sun Z et al. Functional analysis of the β and ε lycopene cyclase enzymes of Arabidopsis reveals amechanism for control of cyclic carotenoid formation. Plant Cell, 1996, 8:1613—1626
[46] 刘宪华，宋文华，欧文氏菌AEBL002菌株八氢番茄红素脱氢酶基因保守序列的克隆与同源性分析．南开大学学报(自然科学)，2001，9，34(3)：102—107
[47] 季静 山村三郎，5个克隆自龙胆(Getina Iutea)的类胡萝卜素生物合成酶基因在烟草中对β—胡萝卜素和叶黄素生物合成量的影响规律，待发表
[48] 陈一明，薛颖，胡萝卜茄素β—环化酶cDNA的分离及其在肉质根中的差异表达．植物学报，2001 43(12)：265—1270
[49] Bouvier, F., Keller, Y., Xanthophy 11biosynthesis:motecular and functional characterization of carotenoid hydroxylases from peper fruits (Capsicum annuum L.) Biochim. Biophys. Acta 1998 1391(3): 320—328
[50] Bouvier F, Hugueney P, d' Harlingue A et al. Xanthophyll biosynthesis in chromoplasts: Isolation andmolecular cloning of an enzyme catalyzing the conversion of 5, 6- epoxycarotenoid intoketocarotenoid. Plant J, 1994, 6:45—54
[51] Newman, T, eBrruijn. F.J. genesgalore: a summary ofmethods for accessing results from large—scale partial sequencing of anonymous Arabidopsis cDNA clones. Plant Physiol 1994, 106:1241—1255
[52] uvierF, d' Harlingue A , Hugueney P et al. Xanthophyll biosynthesis .Cloning, expression, functional reconstruction and regulation of β cyclohexenyl carotenoid epoxidase from pepper (Capsicumannuum). J Biol Chem, 1996, 271:28861—28867
[53] 吴乃虎，基因工程原理，科学技术出版社
[54] 王劲，袁旭，芥蓝，cDNA文库的构建及文库质量检测．四川大学学报(自然科学版)，2000，10(37)：76—79
[55] 李明刚，刘讯，植酸酶基因表达与调控机制研究(Ⅱ)—番茄幼苗cDNA文库的构建与植酸酶基因的筛选．烟台大学学报(自然科学与工程版)，1999 1 12(1)：34—41
[56] 陈绍荣，水稻原胚和刚启动分化的幼胚cDNA文库的构建与分析．植物学报，2000 42(2)：214—216
[57] 刘长征，玉米叶片cDNA文库的构建及Cab基因克隆．山东农业大学学报，1995 26(4)：447—451
[58] 周兆斓，水稻cDNA文库的构建及巯基抑制剂cDNA的分离．中国科学(C辑)，1996 26(2)：149—155
[59] 江树业，光敏核不育籼稻Hs—1可育和不育幼穗cDNA义库的构建．高技术通讯，1998 10：45—48
[60] 周立，应用cDNA文库快速构建法克隆高粱肌动蛋白基因．生物化学杂志，199713(1)：7—13
[61] 叶志彪，与油梨成熟有关的mRNA的分离及cDNA文库的构建．华中农业大学学报，1993 12(5):485—489
[62] 叶秋，水稻成熟花粉cDNA文库的构建与分析．四川大学学报(自然科学版)，2001 4 38(4)251—253
[63] 苟小平，水稻精细胞cDNA文库的构建与分析．植物学报，2001 48(10)：1093—1096
[64] 胡汉桥，白粉菌诱导的小黑麦异多附加系M17特异cDNA克隆的研究．博士学位论文，1998
[65] 张领兵，向日葵恢复系R9花蕾cDNA文库的构建及Rfl恢复基因的研究．硕士学位论文，2001
[66] 印莉萍，缺铁玉米根cDNA文库的构建及蛋白和cDNA差异比较．首都师范大学学报(自然科学版)，1999 12 20(4)：54—59
[67] 江昌俊，欧洲油菜花粉cDNA文库的构建．安徽农业科学，1995 9 23(3)：199—200
[68] ADAMSETAI. D, KELLEY JM, GOCAYNIE J O, et al. Complementary DNA sequencing: Expressed sequence tags and human gcnome project[J]. Science 1991, 251: 1651—1656
[69] LCULESCU VE, ZHANG L, VOGELSTEIN B, et al. Serial analysis of gene expression[J]. Science 1995, 270:484—487
[70] 朱乾浩，转座子在植物基因分离中的应用研究进展．生物工程进展，1996 16(2)：22—25
[71] Hugueney P, Romer S, Kuntz M. Characterization and molecular cloning of fiavoprotein catalyzing the synthesis of phytofiuene and zeta-carolene in capsicum chromoplasts. Eun J Biochem. 1992, 209:399—407
[72] Kajiwara S, Eraser PD, Kondo K, Misawa N. Expression of an exogenous isopentenyl diphosphate isomerase gene enhances isoprcnoid biosynthesis in Escherichia coli Biochem. J. 1997 324:421—426
[2] 韩雅珊，类胡萝卜素的功能研究进展，中国农业大学学报，1999，4(1)：5—9
[3] Bartleyg E, Scolnik P A. Plant carotenoids:Pigments for photoprotection, visual attraction and human health. Plant Cell, 1995, 7: 1027-1038
[4] Rock C D, Zeevaart J A D. The ABAmutant of Arabidopsis thaliana is impaired in epoxy-carotenoid biosynthesis. Proc Natl Aead Sci USA, 1991, 88:7496-7499
[5] goodwin T W, Brittong. Distribution and analysis ofcarotenoids. In:Goodwin FW(Ed), Plant Pigment. Academic Press, London, 1988, 61—132
[6] 徐昌杰，张上隆，植物类胡萝卜素的生物合成及其调控，植物生理学通讯．2000，2 16(1)：64—70
[7] Bouvier. F., Keller. Y., Xanthophy 11 biosynthesis molecular and functional characterization of carotenoid hydroxylases from pepper fruits Biochim. Biophs 1998 1391(3): 320—828
[8] Pecker I, gabbay R, Francis X et al. Cloning and characterization of the cDNA for β-cyclasc from tomatoreveals decrease in ins expression during fruit ripening. Plant Mol Biol, 1996, 30: 807—819
[9] Fraser PD, TruesdalemR, Bird CR et al. Carotenoid biosynthesis during tomato fruit development Plant Physioi, 1994, 105:379—387
[10] Albrechtm, Sandanng. Light-stimulated carotenoid biosynthesis during transformation ofmaize etioplasts is regulated by increased activity of isopentenyl pyrophosphate isomerase. Plant Physiol, 1994, 105:529—534
[11] Giulianog, BartleygE. Scolnik PA. Regulation of carotenold biosynthesis during tomato development. Plant Cell, 1993, 5:379—387
[12] Von Lintig J, Welsch R , Bonkm et al. Tight dependent regulation of carotenoid biosynthesis occurs at the level of phytoene synthase expression and ismediated by phytochrome in Sinapis alba and Arabidopsis thaliana seedlings. Plant J, 1997, 12:625—634
[13] Yamamoto HY Xanthophyll cycles, method Enzymol, 1985, 110:303—312
[14] Dogbo O, Laferriere A, d' Harlingue A et al. Carotenoid biosynthsis:Isolation and characterization of a bifunctional enzyme catalyzing the synthesis of phytoene. Proc Natl Acad Scid Sci USA, 1988, 85:7054--7058
[15] JefferyD, SmithC, Goodenough P et al. Ethylene independent and ethylene- dependent biochemical changes inripening tomatoes. Plant Physiol, 1984, 74:32—38
[16] BartleygE, Scolnik PA. Plantcarotenoids: Pigments for photoprotection, visual attraction, and human health. Plant Cell, 1995, 7:1027—1038
[17] BartleygE, ScolnikPA. Molecular biology of carotenoid biosynthesis in plants. Annu Rev Plant Physiol Plantmol Biol, 1994, 45:287—301
[18] SteWard I, Wheaton TA. Carotenoids in Citrus: Their accumulation induced by ethylene. J Agric Food Chem , 1972, 20:448—449
[19] Perucka I. Ethephon induced changes in accumulation of carotenoids in red pepper fruit (Capsicum annuum L.) Hortic Abstr, 1997, 67:4997—5001
[20] Babczinski P , Heinemann U , Sandmanng et al. Inhibition of carotenoid biosynthesis by interaction of 2, 6- diphenylpyridine derivatives with phytoene desaturation. J Agric Food Chem, 1992, 40:2497—2499
[21] gamble PE, Mullet JE. Inhibition of carotenoid accumulation and abscisic acid biosynthesis in fiuridone- treated dark-grown barley. Eur J Biochem, 1986, 160:117—121
[22] Bouvier F, d' Harlingue A , Camara B .Molecular analysis of carotenoid cyclase inhibition. Arch Biochem Biophys, 1997, 346:53—64
[23] Coggins CW Jr, HenninggL, Yokoyama H. Lycopene accumulation induced by 2-(4
-chlorophenylthio)-triethylaminc hydrochloride. Sci,1970. 168: 1589—1590
[24] Ye X, Al Babili S, Kloti A et al. Engineering the provitamin A (β-carotene) biosynthetic pathway into (carotenoid-free) rice endosperm. Science, 2000. 287:303—305
[25] Shewmaker C K , Sheehy J A , Daleym et al. Seed specific overexpressein of phytoene synthase:increade in carotenoids and othermetabolic effectd, plant J, 1999, 20:401—412
[26] Bramley P, Teulicres C, Blain i et al. Biochemical characterization of transgenic tomato plantsin which carotenoid synthesis has been inhibited through the expression of antisense RNA topTOM5. Plant J. 1992, 2:343—349
[27] gray Rg, Grierson D. Identification andgenetic analysis of normal andmutant phytoene synthasegenes of tomato by sequencing, complemcntation and co-suppressoon. Plantmol Biol, 1993, 22:589—602
[28] Rosati C, Apuilanti R, Dharmapuri S et al. metabolie engineering of beta-carotene and lycopene content on tomato fruit. Plant J, 2000, 24:413—420
[29] misawa N, Yamano S, Linden H et al. Funetional expression of the Erwinoa uredevora carotwnoid biosynthesis activity and resistance to the blwaching herbicidemorflurazon. Plant J, 1993, 4:833—840
[30] Kumagaim H, Donson J.della-Cioppag et al. Cytoplasmic inhibition of carotenoid biosynthesis with viral-derived RNA. Proc Natl Acad Sci USA, t995, 92:1679—1683
[31] Dogbo O, Camara B. Purification of isopentenylpyrophosphate isomerase andgeranylgeranyl pyrophosphate synthase from Capsicum chromoplasts by affinity chromatography. Biochim BiophysActa, t987, 920:140—148
[32] Kuntzm, Romer S, Suite C et al. Identification of a Cdna for the plastid-locatedgeranylgeranyl pyrophosphate synthase from Capsicum annum: Correlative increase in enzyme activity and transcript level during fruit ripening. Plant J, 1992, 2:25—34
[33] Scolnik PA, BartleygE. Nucleotide sequence of anArabidopsis cDNA forgeranylgeranyl
pyrophosphate synthase. Plant Physiol, 1994, 104:1469—1470
[34] Ray JA, Bird CR, Manndersm et al. Sequence of pTOM5, a ripening related cDNA from tomato. Nucl Acid Res, 1987, 24:10587—10590
[35] ArmstronggA, Albertim, Hearst JE. Conserved enzymesmediate the early reactions of carotenoid biosynthesis in non-photosynthetic and photosynthetic procaryotes. Proc Natl Acad Sci USA, 1990, 87:9975—9979
[36] Bird CR, Ray JA, Fletcher JD et al. Using antisense RNA to studygene function :Inhibition of carotenoid biosynthesis in transgenic tomatoes. Bio/Technol, 1991, 9:635—639
[37] Bramley P, Teulieres C, Blain I et al. Biochemical characterization of transgenic tomato plants in which carotenoid synthesis has been inhibited through the expression of antisense RNA to pTOM5 Plant J, 1992, 2:343—349
[38] BartleygE, Viitanen PV , Bacot KO et al. A tomatogene expressed during fruit ripening encodes an enzyme of the carotenoid biosynthesis pathway .J Biol Chem, 1992, 267:5056—5039
[39] von Lintig J, Welsch R , Bonkm et al. Light- dependent regulation of carotenoid biosynthesis occurs at the level of phytoene synthase expression and ismediated by phytochrome in Sinapis alba and Arabidopsis thaliana seedlings. Plant J , 1997, 12:625—634
[40] BartleygE , Scolnik PA. cDNA cloning, expression during development andgenomemapping of PSY2 , a second tomatogene encoding phytoene synthase. J Biol Chem, 1993, 268: 25718—25721
[41] Chamovitz D, Pecker I, Hirschberg J. Themolecular basis of resistance to the herbicide norflurazon. Plantmol Biol, 1991, 16:967—974
[42] BartleygE, Viitanen PV, Pecker I et al. molecular cloning and expression in photosynthetic bacteria of asoybean cDNA coding fo rphytoene desaturase, an enzyme of the carotenoid biosynthesis pathway. Proc Natl Acad Sci USA, 1991, 88:6532—6536
[43] Pecker I, Chamovitz D, Linden H et al. A single polypeptide catalyzing the conversion of
phytoene to ζ- carotene is transcriptionally regulated during tomato fruit ripening. Proc Natl Acad Sci USA. 1992, 89: 4962—4966
[44] Albrechtm , Klein A, Hugueney P et al. molecular cloning and functional expression in E .cell of a novel plant enzymemediating zeta- carotene desaturation. FEBS Lett, 1995, 372: 199—202
[45] Cunningham FX Jr, Pogson B, Sun Z et al. Functional analysis of the β and ε lycopene cyclase enzymes of Arabidopsis reveals amechanism for control of cyclic carotenoid formation. Plant Cell, 1996, 8:1613—1626
[46] 刘宪华，宋文华，欧文氏菌AEBL002菌株八氢番茄红素脱氢酶基因保守序列的克隆与同源性分析．南开大学学报(自然科学)，2001，9，34(3)：102—107
[47] 季静 山村三郎，5个克隆自龙胆(Getina Iutea)的类胡萝卜素生物合成酶基因在烟草中对β—胡萝卜素和叶黄素生物合成量的影响规律，待发表
[48] 陈一明，薛颖，胡萝卜茄素β—环化酶cDNA的分离及其在肉质根中的差异表达．植物学报，2001 43(12)：265—1270
[49] Bouvier, F., Keller, Y., Xanthophy 11biosynthesis:motecular and functional characterization of carotenoid hydroxylases from peper fruits (Capsicum annuum L.) Biochim. Biophys. Acta 1998 1391(3): 320—328
[50] Bouvier F, Hugueney P, d' Harlingue A et al. Xanthophyll biosynthesis in chromoplasts: Isolation andmolecular cloning of an enzyme catalyzing the conversion of 5, 6- epoxycarotenoid intoketocarotenoid. Plant J, 1994, 6:45—54
[51] Newman, T, eBrruijn. F.J. genesgalore: a summary ofmethods for accessing results from large—scale partial sequencing of anonymous Arabidopsis cDNA clones. Plant Physiol 1994, 106:1241—1255
[52] uvierF, d' Harlingue A , Hugueney P et al. Xanthophyll biosynthesis .Cloning, expression, functional reconstruction and regulation of β cyclohexenyl carotenoid epoxidase from pepper (Capsicumannuum). J Biol Chem, 1996, 271:28861—28867
[53] 吴乃虎，基因工程原理，科学技术出版社
[54] 王劲，袁旭，芥蓝，cDNA文库的构建及文库质量检测．四川大学学报(自然科学版)，2000，10(37)：76—79
[55] 李明刚，刘讯，植酸酶基因表达与调控机制研究(Ⅱ)—番茄幼苗cDNA文库的构建与植酸酶基因的筛选．烟台大学学报(自然科学与工程版)，1999 1 12(1)：34—41
[56] 陈绍荣，水稻原胚和刚启动分化的幼胚cDNA文库的构建与分析．植物学报，2000 42(2)：214—216
[57] 刘长征，玉米叶片cDNA文库的构建及Cab基因克隆．山东农业大学学报，1995 26(4)：447—451
[58] 周兆斓，水稻cDNA文库的构建及巯基抑制剂cDNA的分离．中国科学(C辑)，1996 26(2)：149—155
[59] 江树业，光敏核不育籼稻Hs—1可育和不育幼穗cDNA义库的构建．高技术通讯，1998 10：45—48
[60] 周立，应用cDNA文库快速构建法克隆高粱肌动蛋白基因．生物化学杂志，199713(1)：7—13
[61] 叶志彪，与油梨成熟有关的mRNA的分离及cDNA文库的构建．华中农业大学学报，1993 12(5):485—489
[62] 叶秋，水稻成熟花粉cDNA文库的构建与分析．四川大学学报(自然科学版)，2001 4 38(4)251—253
[63] 苟小平，水稻精细胞cDNA文库的构建与分析．植物学报，2001 48(10)：1093—1096
[64] 胡汉桥，白粉菌诱导的小黑麦异多附加系M17特异cDNA克隆的研究．博士学位论文，1998
[65] 张领兵，向日葵恢复系R9花蕾cDNA文库的构建及Rfl恢复基因的研究．硕士学位论文，2001
[66] 印莉萍，缺铁玉米根cDNA文库的构建及蛋白和cDNA差异比较．首都师范大学学报(自然科学版)，1999 12 20(4)：54—59
[67] 江昌俊，欧洲油菜花粉cDNA文库的构建．安徽农业科学，1995 9 23(3)：199—200
[68] ADAMSETAI. D, KELLEY JM, GOCAYNIE J O, et al. Complementary DNA sequencing: Expressed sequence tags and human gcnome project[J]. Science 1991, 251: 1651—1656
[69] LCULESCU VE, ZHANG L, VOGELSTEIN B, et al. Serial analysis of gene expression[J]. Science 1995, 270:484—487
[70] 朱乾浩，转座子在植物基因分离中的应用研究进展．生物工程进展，1996 16(2)：22—25
[71] Hugueney P, Romer S, Kuntz M. Characterization and molecular cloning of fiavoprotein catalyzing the synthesis of phytofiuene and zeta-carolene in capsicum chromoplasts. Eun J Biochem. 1992, 209:399—407
[72] Kajiwara S, Eraser PD, Kondo K, Misawa N. Expression of an exogenous isopentenyl diphosphate isomerase gene enhances isoprcnoid biosynthesis in Escherichia coli Biochem. J. 1997 324:421—426