摘要
小麦是重要的粮食作物之一,利用基因工程进行遗传改良的研究一直倍受人们关注。小麦的遗传转化还处于不断发展和完善阶段。以Alondra为材料,用直径0.6μm、1μm的金粉作为包裹介质,以直径小于0.5mm、1mm、大于1.5mm的幼胚作受体材料,1-7mg/L浓度的Basta筛选培养,以此建立起良好的再生休系和基因枪转化体系。
赖氨酸为人体必须氨基酸,小麦中赖氨酸含量较匮乏,因此降低了小麦的营养品质。而其他禾谷类作物赖氨酸含量也较低,通过远源杂交的方法获得富含赖氨酸的小麦潜力不大,因此可通过基因工程的方法导入相关基因,如富含赖氨酸的蛋白基因,赖氨酸合成关键酶基因,赖氨酸分解酶基因干扰序列,以提高结合态或游离态赖氨酸含量。本研究把从辣椒中克隆了富含赖氨酸的蛋白基因cflr,(Genbank基因登录号:EU367999),以PAHC-25为基本载体,构建PAHC-cflr载体,通过基因枪法将其转入小麦。Cflr基因总氨基酸数223个,赖氨酸数40个,赖氨酸含量为21.2%(w/w),苏氨酸数24个,苏氨酸含量10.3%(w/w)。
主要的结果:在质粒包裹过程中,采用直径为0.6μm的金粉作为质粒包裹的介质时,不易结块,分散较好,转化效率较高,而直径为1μm时,颗粒较大,容易沉淀,在载体膜上不易分散。用1mm左右的幼胚作为受体时,形成再生苗较好,较小或较大不易形成再生植株。用Basta筛选时,培养基中Basta含量3-4mg/L时筛选效果较好。通过Basta筛选,共获得T0代筛选植株144棵,其中成活并结实的阳性植株5株。T1代共获得33株,通过RNA表达水平PCR检测,有28株阳性,PCR产物测序证明了阳性植株中扩出的片段即为cflr基因。赖氨酸含量检测阳性植株中赖氨酸的含量比对照提高了7.4%。
Being one of important food crops, the application of genetic engineering on wheat improvement has been attracted great attention recently. In contrast to rice, maize and other crops of agronomic importance, genetic transformation of wheat is still in the exploratory stage. In the study, biolistics-mediated transformation was employed. The transformation, development and regeneration systems are as the following: immature embryos of common wheat cv. Alondra was used as the receptor with the diameter less than 0.5mm, 1mm or greater than 1.5mm, coated by 0.6 and/or 1μm gold particles; screening and selection were performed on 1-7mg/L“Basta”culture medium.
As one of the essential amino acids on human nutrient, lysine content is relatively low in wheat which reduces its nutritional quality. Since the situation is the same to other cereal crops, it seems insignificant to perform traditional crossing with the distal wheat varieties. Biolistics-mediated transformation can make it possible through the introgression of genes encoding lysine-rich proteins, a key enzyme in lysine biosynthesis and the proteins and/or enzymes inhibiting or decreasing lysine biolysis process.
cflr(accession number: EU367999), one gene coding high-lysine protein isolated from pepper, was constructed into the vector pAHC25 and then transformed into wheat immature embryos by gold particle bombardment. CFLR consists of 223 amino acids, of them the number of lysine 40, 21.2% (w / w), and the number of threonine 24, 10.3% (w / w), respectively.
Main results: In the process of“bullet”preparation, 0.6μm gold powder can make good results with hard caking, good dispersion, high transformation efficiency, while 1μm or larger particles can precipitate easily and not be dispersed easily in the carrier membrane. Plantlets can be regenerated easily using 1mm immature embryos while smaller or larger ones make bad results. A Basta concentration of 3-4mg/L was more favorable for screening. In total, 144 T0 generation positive plants were obtained. Of them, 5 had produced offsprings. 28 of 33 lines in total were positive in T1 generation using PCR assay at RNA level. Sequencing confirmed the occurrence of cflr transcripts. The content of lysine increased by 7.4% compared to the control.
引文
Alpteper F,Vasil V,srivastava V,Eva St?ger,Indra K,Vasil. Celerated production of transgenic wheat (Triticum aestivum L.) plants. Plant Cell Reports,1996,16:12-17
Amoah B K,Wu H,Sparks C,and Jones H D. ctors influencing Agrobacterium- ediated transient expression of uidA in wheat inflorescence tissue. Journal of Experimental Botany,2001,52(385):1135-1142
Arao F J L,Grossi de Sa M F,Davey M R,Brasileiro A C M,Faria J C,Rech E L. Factors influencing transient gene expression in bean(phaseolus vulgaris L) using an electrical Particle acceleration device. Plant Cell Report,1993,12:483-490
Becker D,Brettschneider R,and Lorz H. Fertile transgenic wheat from microprojectile bombardment of scutellar tissue. Plant Journal,1994 5:299-307
Blechl A E,Anderson O D Expression of anovel high molecular-weight glutenin subunit gene in transgenic wheat. Nature Biotechnology, 1996,14:875-879
Bloek M,Moens T, Debrouwer D. The development of a nuclear male sterility system in wheat-Expression of the barnase gene under the control of tapetum specific promoters,Theoretieal and Applied Genetics ,1997,95:125-131
Brettschneider R,Lorz H,Beeker D. Efficient transformation of scutellar tissue of immature maize embryos. Theoretical and Applied Genetics,1997,94:737-748
Carman J G,Jefferson N E,Campbell W F. Induction of embryogenic Triticum aestivum L.calli. Quantification of genotype and culture medium effects. Plant Cell Tissue and Organ Culture,1987,10(2):101-108
Chen W P,Gu X,Liang G H ,Muthukrishnan S,Chen P D,Liu D J,Gill B S. Introduction and constitutive expression of a rice chitinase gene in bread wheat using biolistic bombardment and the bar gene as a selectable marker. Theoretical and Applied Geneties,1998,97:1296-1306
Chen W P,Liu D J,Kynast R,Friebe B,Velazhahan R,Muthukrishnan S,Gill B S,Chen P D . Development of wheat scab symptoms is delayed in transgenic wheat plants that constitutively express a rice thaumatin-like protein gene. Theoretical and Applied Genetics,1999,99:755-760
Cheng M,Fry J E,Pang S,Zhou H,Hironaka C M,Duncan D R,Conner T W,Wan Y. Genetic transformation of wheat mediated by Agrobacterrium tumefaciens. Plant Physiol,1997, 115: 971~980
Demeke T,Hucl P,Baga M,Caswell K,Leung N,Chibbar R N . Transgene inheritance and silencing in Hexaploid spring wheat. Theoreticaland Applied Genetics, 1999,99:947-953
Halluin D K,Bonne E,Bossut M, De Beuckeleer M,Leemans J. Transgenic maize Plants by tissue electroporation. The Plant Cell,1992,4,1495-1505
Hauptmann R M,Ozias-Akins P,Vasil V,Tabaeizadeh Z,Rogers S G,Horsch R B,Vasil I K,Fraley R T. Transient expression of electroporated DNA in monocotyledonous and dicotyledonous species. Plant Cell Reports,2003,6:265-270
Hunold R ,Bronner R ,Hahne G . Early events in microprojectile bombardment: cell viability and particle location. The plant Journal,1994,5:593-604
Iida A,seki M,Kamada M,Yamada Y,Morikawa H. Gene delivery into cultured plant cells by DNA-coated gold particles accelerated by a pneumatic particle gun. Theoretical and Applied Genetics,1990,80:813-816
Ishide Y,Saito H, Ohta S,HieiY,Hiei Y,Komari T,Kumashiro T. High efficiency transformation of maize(Zea mays L.) mediated by Agrobacteium tumefaciens. Nature biotechnology,1996 14:745-749
Khanna H K,Daggard G E. Agrobacterium tumefaciens-mediated transformation of wheat using a superbinary vector and a polyamine-supplemented regeneration medium. Plant Cell RePorts,2003,21:429-436
Klein T M,Gradziel T,Fromm M E,Sanford. Factors Influencing Gene Delivery into Zea Mays Cells by High?Velocity Microprojectiles. Bio/Technology,1988,6:559-563
Klein T M,Wolf E D,Wu R,Sanford J C. High-velocity microprojectiles for delivering nucleic acids into living cells . Nature,1987,327:70-73
Kohli A,Leech M,Vain P,Laurie D A,Christou P. Transgene organization in rice engineered through direct DNA transfer supports a two-phase integration mechanism mediated by the establishment of integration hot spots. Proceedings of theNational Academy of Sciences of the United States of America,1998,95(12):7203-7209
Lacorte C,Almeida E R,Mansur E,Rech E L. Transient expression of GUS and the 2s albumin gene from Brazil nut in peanut(Arachis hypogaeal)seed explants using particle bombardment. Plant Cell Reports,1997,16:619-623
Leekband G,Lorz H. Transformation and expression of a stilbene synthase gene of Vitis vinifera L. in barley and wheat for increased fungal resistance.Theoretical and Applied Geneties ,1998,96:1004-1012
Mccabe D E,Swain W F,Martineli B J,Christou p. Stable transformation of soybean(glycine max) by particle acceleration. Bio/Technol,1988,6:923-926
Nehra N A,Chibbar R N,Leuns N,Caswell K,Mallard C,Steinbauer L,Baga M,Kartha K K. Self-fertile transgenic wheat Plants regenerated from isolated scutellar tissues following microprojectile bombardment with two distant gene constructs. the Plant Journal,1994,5:285-297
Neuhaus G,Spangenberg G,Mittelsten Scheid O Schweiger H G . Transgenic rapeseed Plants obtained by the microinjection of DNA into microspore-derived embryoids. Theoretical and Applied Genetics,1987,75,30-36
Nikovies K,Simidjieva J,Peres A,Ayaydin F. Cell-cycle,Phase-specific activation of maize streak virus Promoter ..Molecular Plant Microbe Interact,2001,14(5):609-617
Ortiz J P A,Rggiardo M I,Ravizzini R A,Altabe S G. Hygomycin restance as an efficient selectable marker for wheat stable transformation. Plant Cell Reports,1996,15:877-881
Pedersen C,Zimny J,Becker D,Jahne-Gartner A. Localization of introduced genes on the chromosomes of transgenic barley,wheat and triticale by fluorescence in situ hybridization. Theoretical and Applied Genetics,1997,94:749-757
Pena L,Cervera M, Juarez J,Navarro A,Pina J A,Navarro L. Genetic transformation of lime(Citrus aurantifolia Swing):factors affecting transformation and regeneration. Plant Cell Reports,1997,16:731-737
Perl A,Kless H,Blumenthal A,Galili G,Galun E. Improvement of plant regeneration and GUS expression in scutell wheat calli optimization of cumture conditions and DNA -microjectile delivery procedures. Molecularand General genetics,1992,235:279-284
Potrykus I,Saum M W,Petruska J,Paszkski J,Shillito R D. Direct gene transfer to cells of a gramimaceous monocot. Molecular Genetics and Genomics,1985,199:183-188
Raizada M N,Brewer K V,Walbot V. A maize MuDR transposon Promoter shows limited autoregulation. Molecular Genetics and Genomics,2001,265(14):82-94
Rajasekaran K,Hudspeth R L,Cary J W,Anderson D M,and Cleveland T E. High-frequency stable transformation of cotton (Gossypium hirsutum L.) by particle bombardment of embryogenic cell suspension cultures. Plant Cell Reports,2000,19:539-545
Rhodes C A,Pierce D A,Mettler I L,Mascarenhas,Detmer J. Genetically transformed maize. Plants from Protoplasts,1988,240:204-207
Sanford J V, Smith F D, Russell J A. Optimizing the biolistic process for different biological applications. Methods in Eezymology,1993,217:483-509
Serik O,Ainur l,Murat K,Tetsno M,Masaki I. Silicon carbide fiber-mediated DNA delivery into cells of wheat(Triticum acstivm L) mature embryos. Plant Cell Reperts,1996,16:133-135
Singh N,Chawla H S. Use of silicon carbide fibers for Agrobacterium-mediaed transformation in wheat. Current Science,1999,76(11):1483-1485
Srivastava V,Vail V,Vail I K. Molecumar characterization of the fate ofTransgenes in transformed wheat. Tlieor Appl Genet,1996,92:1031-1037
Stepansky A,Less H,Angelovici R,Aharon R,Zhu X,Galili G . Lysine catabolism, an effective versatile regulator of lysine level in plants. Amino Acids ,2006,30: 121–125
Stoger E,Williams S,Keen D,Christou P. Molecμmar characteristics of transgenic wheat and the effect on transgene expresaion. Transgenic Research,1998,7:463-471
Sun X H,Ao G M,Yu J J,Zhao Q.Transfer of high lysine-rich gene into maize inbred lines and the detection of transgenic plants,J Agfic Biotech,2001,9:156-158 (in Chinese with English abstact)
Svitashev S,Ananiev E,Pawlowski W P,Somers D A. Association of transgene integration sites with chromosome rearrangements in hexaploid oat,Theoretical and Applied Genetics,2000,100:872-880
Tang K X,Fei J,Sun X F,Yao J H,Wan B L,Qi H X, Lu X G. The development of transgenic rice homozygous plnats containing mumtiple genes via genetic transformation. Jounral of Fudan Univesrity(Natural Scienee),2001,40(5):483-490
Thomas J C,wasann C C,Echt C,Dunn H J. Introduction and expression of an insect proteinase inhibitor in alfalfa,Plant Cell Reports,1994,14:31-36
Vain P,Mcmullen M D,Finer J J. Osmotic treament enhances particle bombardment -mediated transient and stable transformation of maize,Plant Cell Reports,1993,12:84-88
Vasil V,Castillo A M,Fromm M E,Vasil I K. Herbicide resistant fertile transgenic wheat plants obtained by microprojectile bombardment of regenerable embryogenic callus ,Nature Biotechnology,1992,10,667-674
Vasil V,Srivastava V,Castillo A M,Fromm M E. Rapid production of transgonic wheat plants by direct bombardment of cumtured immature embryos,Nature Biotechnology,1993 11:1553-1558
Weeks J T,Anderson O D,Blechl A E. Rapid Production of multiple independent lines of fertile transgenic wheat. Plant Physiology,1993,102(4):1077-1084
Yu J J,Peng P,Zhang X,Zhao Q,Zhu D Y,Sun X,Liu J Q,Ao G M.Seed-specific expression of a lysine rich protein sb401 gene significantly increases both lysine and total protein content in nlaize seeds,Molecular Breeding,2004,14(1):1-7
Zambryski P,Joos H,Genetello C,Leemans J,Van M. Ti plasmid wector for the introduction of DNA into plant cells without alteration of their normal regeneration capacity,EMBO Journal,1983,3:2143-2150
zengliang Yu, Jianbo Y, Yuejin W,Beiju C,Yuping Huo. Transferring GUS gene into rice cells by low energy ion beam, Nuclear Instruments and Methods in Physics Research,B80/81,1993,1328-1331
Zheng Z,Sumi K,Tanaka K,Murai N.The bean seed storage protein [beta]-phaseolin is synfheized,processed,and accumumated in the vacuolar type-Ⅱprotein bodies of transgenic rice endosperm,Plant Physiology,1995,109(3):777-786
Ziberstein A,Schuster S,Flaistman Metal. International Congress of Plant,Moleudar Biology Amsterdm , 1994
蔡润,中田和男,平井八十一.小麦根愈伤组织诱导再生植株.上海农业学报,1999,15(4):13-17
蔡体树,田惠琴,林书康.基因型和胚龄对小麦未成熟胚离体培养的影响.遗传学报,1989,16(2):81-88
陈惠民,腾世云,于家驹.小麦幼叶愈伤组织的诱导及器官的形成.植物学报,1980,11(2):111-115
陈梁鸿,王新望,张晓东.基因枪转化小麦不同受体的研究华北农学报. 1998,13(l):l-5
陈柳等.农杆菌介导法的高赖氨酸基因在油菜中的转化.扬州大学优秀硕士论文.2008
成卓敏,何小源,陈彩层.大麦黄矮病毒外壳蛋白基因合成及用花粉管途径获得小麦转基因植株.自然科学进展,1993,3(6):560-564
丁群星,谢友菊,戴景瑞.用子房注射法将Bt毒蛋白基因导入玉米.中国科学,1993,23(l):707-713
傅荣昭,曹光诚,马江生.用基因枪法将人工雄性不育基因导入小麦的研究初报.遗传学报,1997,24(4):358-361
高越峰,荆玉祥.高赖氨酸蛋白基因导入水稻及可育转基因植株的获得.遗传学报遗传学报2001,遗传学报遗传学报43(5): 506-511
关畅,曾寒冰,李文雄.小麦未成熟胚离体培养的研究-愈伤组织发生及器官建成.东北农学院学报,1993,24(2):107-116
侯文胜,郭三堆,路明.利用花粉管通道法获得转雪花莲凝集素基因(sgna)小麦.植物学通报, 2003,20(2):198-204
华志华,黄大年.转基因植物中外源基因的遗传学行为.植物学报,1999,41(l):l-5
冀俊丽,盛长忠,石明.通过负压花粉管法将耐盐基因HVAI转入小麦的研究.麦类作物学报,2002,22(2):10-13
蒋家焕,刘峰,许明.高赖氨酸蛋白基因遗传转化水稻的研究.福建农林大学学报(自然科学版) 2006,35(6):615-618
蓝海燕,田颖川,王长海.表达l一3葡聚糖酶及几丁质酶基因的转基因烟草及其抗真菌病的研究.遗传学报二,2000,27(l):70-77
郎志宏,于静娟,朱登云,赵倩,敖光明.高赖氨酸蛋白基因SBgLR的克隆及其对提高玉米种子中蛋白质和赖氨酸含量的作用.农业生物技术学报,2004,12(5):487-492.
李科,王世全,吴发强,李双成,邓其明,王玲霞,梁越洋,李平.农杆菌介导的转高赖氨酸蛋白基因(sb401)水稻T4代分析.中国水稻科学,2008,22(2):131-136
李宏潮,胡道芬,王虹.小麦成熟胚培养因素研究.植物细胞工程与育种.北京工业大学出版社, 1990a,308-313
李宏潮,胡道芬,王虹.小麦成熟胚培养因素研究.华北农学报1990b,5(1)22-27
李兴涛,李霞,张金文高赖氨酸蛋白基因在转基因生菜中的表达和遗传转化.应用与环境生物学报,2006,12(4):472-475
李兴涛.高赖氨酸蛋白基因在转基因生菜中的表达和遗传研究.甘肃农业大学优秀硕士学位论文,2006
李学宝,秦明辉.施荣华芥菜型油菜抗虫转基因植株及其后代株系的研究.生物工程学报,1999,15(4),482-488
梁辉,赵铁汉,李良材.影响基因枪法转化小麦幼胚的几个因素的研究.遗传学报,1998,25(5):443-448
刘博林,荆玉祥,匡廷云.高赖氨酸植物种的筛选及其蛋白质纯化与鉴定.植物学报,1993,35(1):22-25
刘岩,王国英,刘俊君.大肠杆菌gutD基因转入玉米及耐盐转墓因植株的获得.中国科学,C辑,1998,28(6):542-547
吕德扬,范云六,俞梅敏.苜蓿高含硫氨基酸蛋白基因植株再生.遗传学报,2000,27(4):331-337
孟超敏,陈绪清,梁荣奇,杨凤萍,张立全,张晓东,陈天佑,辛世文.高赖氨酸含量基因在转基因小麦的表达及其赖氨酸含量分析.科学通报,2004,49(19):1731-1736.
牟红梅,刘树俊,周文娟.慈菇蛋白酶抑制剂通过花粉管途径对小麦的导入及转基因植株分析.遗传学报,1999,26(6):634-642
闰新甫,刘文轩,林秋萍,外源DNA导入小麦后代变异株系的醋酶同工醉分析.华北农学报,1999,19(1):39-43
孙晓波,房瑞,于桂红,刘全兵,马鸿翔,辣椒高赖氨酸蛋白基因cflr全长cDNA的克隆及其组织表达特征.园艺学报,2008,35(9):1310-1316
田苗英,吴茂森,陈彩层等.大麦黄矮病毒缺失复制酶基因植物表达载体的构建及转基因小麦的获得.中国农业科学, 1999,32(5):49-54
唐俐,刘巧泉,邓晓湘,武小金,辛世文.无抗性选择标记的转高赖氨酸蛋白(LRP)基因籼稻恢复系的获得.作物学报,2006,32(8):1248-1251
王常云,王作全,李小亮等.小麦幼胚离体培养育种技术研究.麦类作物学报. 1999,19 (1):14-16
王关林,方宏筠(主编).植物基因工程(第二版).北京科学出版社,2002,517-532
王关林,方宏药.植物基因工程原理与技术.科学出版社.北京,1998
王国英,杜天兵,张宏.用基因枪将Bt毒蛋白基因转入玉米及转基因植株的再生.中国科学, 1995,25(1):71-76
王景雪,孙毅,崔贵梅.花粉介导法获得玉米转基因植株,植物学报,2001,43(3):275-279
王睿辉,陈耀锋,梁虹.胚龄和基因型对小麦幼胚体细胞胚性无性系的诱导,西北农林科技大学学报. 2002,30(4):17-20
王为民,赵倩,朱登云,于静娟,敖光明.高赖氨酸蛋白质基因和bar基因导人水稻及转基因植株的检测.农业生物技术学报,2004,12(4):363-368
吴超,傅亚萍等,转高赖氨酸蛋白基因脆茎水稻的收获指数及秸秆赖氨酸含量的研究.浙江农业学报,2008,20(4):225-230
吴丽芳,李红,冯慧云,吴李君,余增亮.用低能氢离子柬介导将水稻几丁质酶基因导入小麦.科学通报,.2000,45(21):2316-2321
吴丽芳,李红,宋道君.建立低能离子束介导小麦转基因方法并获得转GUS植株.遗传学报,2000,27(11):982-991
夏光敏,李忠谊,贺晨霞.根癌农杆菌介导的小麦转基因植株再生.植物生理学报,1999,25(1):22-28
徐明良,杨金水,葛扣麟.植物转基因的整合机制.植物生理学通讯,1996,32(3):234-240
徐子勤.重要禾本类植物转基因研究.生物工程进展,2001,21(l):59-74
徐子勤,Becker D,Lorz H.质粒DNA物理形态和其它因素对获得可育转基因小麦的影响.实验生物学报,2001,34(3):183-188
颜泽洪,万永芳,刘坤凡.一种新型高分子量麦谷蛋白亚基的鉴定及其同源蛋白间氨基酸序列的差异.科学通报,2001,46(17):1454-1459
杨剑波.应用低能离子束介导法获得水稻转基因植株.科学通报,1994,39(16):1530-1534
姚华建,李大伟,于嘉林.甜菜坏死黄脉病毒外壳蛋白基因在天灾转基因植株中的表达.生物工程学报,1997,13(4):440一442
叶兴国,Shriley Sato,徐惠君.小麦农杆菌介导转基因植株的稳定获得和检测.中国农业科学,2001,34(5):469-474
于洪欣,柳建军,冯兆礼.通过花粉管途径将抗虫基因(CPTI)导入小麦的研究.山东农业科学,1999,(l):5-8
于晓红,朱祯,付志明.提高小麦愈伤组织分化频率的因素.植物生理学报,1999,25(4):388-394
云月,胡道芬,刘敏.不同基因型冬小麦花药出愈率及其愈伤组织的蛋白质电泳分析.华北农学报,1994,9(3):34-38
曾君祉,王东江,吴有强.花粉管途径获得小麦转基因植株.中国科学(B辑). 1993,23 (3): 256-261
曾君祉.花粉管通道(或运载)法转化的植株后代遗传表现及转化机理的探讨.科学通报,1998,43(6):561-566
张宏,王国英,谢友菊.超声波介导法转化玉米愈伤组织及可育转基因植株的获得.中国科学,C辑,1997,27(2):162-167
张茂银,刘庆昌,王子霞.用花粉管通道法将新疆大赖草DNA导入普通小麦的研究.农业生物技术学报,2000,8(2):165-168
张鹏,霍艳,马鸿翔.外援添加物对小麦幼胚成苗质量的影响.麦类作物学报,2009,29(1):31-34
张秀君,刘俊起.用基因枪将高赖氨酸基因导入玉米及转基因植株的检测.农业生物技术学报,1999,7(4):363-367
周淼平,余桂红,任丽娟,朱伟芳,马鸿翔.转抗除草剂基因小麦植株的筛选方法研究.麦类作物学报,2008,28(6):34-38
周岩,田颖川,陶慧中,莽克强.雪花莲外源凝集素基因的克隆及序列分析.生物工程学报,1996,12(4):495-498