杨树转AtGolS2和SRK2C基因的遗传转化研究
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摘要
非生物胁迫如干旱、极端温度和盐胁迫,严重影响林木生长和产量。我国有三亿亩属于盐碱地,且干旱面积在不断增加,所以如何解决非生物胁迫是林木植物研究迫切需要解决的重要问题。AtGolS2基因(肌醇半乳糖苷酶基因)是拟南芥中GolS基因家族中的一员,受干旱和盐胁迫诱导。SRK2C基因是SnRK基因家族中的一员,SnRK(蔗糖非发酵相关蛋白激酶)通过多种复杂的信号传递途径对干旱、高盐、低温胁迫作出响应,激发抗逆基因表达,提高植物的抗逆性。
本研究以南林895杨为材料,运用农杆菌介导法,开展了抗逆相关基因AtGolS2基因和SRK2C基因的遗传转化研究,主要研究结果如下:
1.遗传转化中选择压的确定。本研究采用的筛选标记基因是潮霉素磷酸转移酶(hpt)基因,在遗传转化中采用潮霉素作为筛选转化植株的选择剂。经过分析比对试验,结果表明采用Hyg2.5mg/L作为筛选时的选择压。
2.遗传转化体系的优化:分析比较了影响农杆菌转化的多个因素(预培养时间、菌液浓度、侵染时间、共培养时间等),获得杨树合适的遗传优转化条件:预培养3d,菌液浓度OD6000.8左右,侵染时间15～20min,共培养时间4d。
3.通过遗传转化条件的优化及潮霉素筛选获得了一批转AtGolS2基因和SRK2C基因的南林895杨植株,并对转基因植株进行了分子生物学鉴定和耐盐性试验。
4.分子生物学鉴定(包括PCR和RT-PCR分析)结果显示目的基因已经整合进杨树基因组中。耐盐性初步试验表明转基因植株耐盐性有一定提高。
Abiotic stresses such as drought,extreme temperature,salt injury which can affect the growth and quantity of forest trees sharply.In China,there are three million hectares are saline land,and arid area is increasing.So it is urgent to solve the abiotic stresses in forest research.At least seven GolS-related genes are found in the Arabidopsis genome.AtGolS2 is a member of GolS gene family in Arabidopsis thaliana.AtGolS2 were induced by drough and high-salinity stresses,but not by cold stress.SRK2C gene is a member of SnRK gene family.SnRK genes are capable of mediating signals initiated during drought,salt and cold stress,play important roles in transducing stress signals,increasing protein synthesization and regulating signal transduction.
In this study,Populus×euramericana cv.‘Nanlin895’was used as experiment material,adapting to Agrobacterium-mediated to introduce separately two resistance gene of AtGolS2 gene and SRK2C gene.The main conclusion of this experiment was as following:
1.Determination of selective pressure of transformation:the selectable marker gene of AtGolS2 and SRK2C were both htp , so adopting Hyg as selective agent in transformation.Through contrast experiment,it is 2.5mg/L Hyg as selective pressure.
2.Establishment of the efficient system of transformation:we studied several factors affecting Agrobacterium-mediated transformation,drawing the best transform condition: pre-culture time were 3 days, bacterial concentration OD600 0.8,infection time were 15～20 minute,coculture time were 4 days.
3.Transgenic poplars were selected to survive on the culture medium with antibiotics,which includes Populus×euramericana cv.‘Nanlin895’with single AtGolS2 gene and SRK2C gene.The molecular biological tests and salt stress experiment were carried out to verity these transgenic poplar.
4.The two genes had been integrated into poplar genome and could express,which verified by PCR and RT-PCR.The result of salt treatment showed that salt-resistance ability of transgenic poplars with single AtGolS2 gene and SRK2C gene were enhanced.
本研究以南林895杨为材料,运用农杆菌介导法,开展了抗逆相关基因AtGolS2基因和SRK2C基因的遗传转化研究,主要研究结果如下:
1.遗传转化中选择压的确定。本研究采用的筛选标记基因是潮霉素磷酸转移酶(hpt)基因,在遗传转化中采用潮霉素作为筛选转化植株的选择剂。经过分析比对试验,结果表明采用Hyg2.5mg/L作为筛选时的选择压。
2.遗传转化体系的优化:分析比较了影响农杆菌转化的多个因素(预培养时间、菌液浓度、侵染时间、共培养时间等),获得杨树合适的遗传优转化条件:预培养3d,菌液浓度OD6000.8左右,侵染时间15～20min,共培养时间4d。
3.通过遗传转化条件的优化及潮霉素筛选获得了一批转AtGolS2基因和SRK2C基因的南林895杨植株,并对转基因植株进行了分子生物学鉴定和耐盐性试验。
4.分子生物学鉴定(包括PCR和RT-PCR分析)结果显示目的基因已经整合进杨树基因组中。耐盐性初步试验表明转基因植株耐盐性有一定提高。
Abiotic stresses such as drought,extreme temperature,salt injury which can affect the growth and quantity of forest trees sharply.In China,there are three million hectares are saline land,and arid area is increasing.So it is urgent to solve the abiotic stresses in forest research.At least seven GolS-related genes are found in the Arabidopsis genome.AtGolS2 is a member of GolS gene family in Arabidopsis thaliana.AtGolS2 were induced by drough and high-salinity stresses,but not by cold stress.SRK2C gene is a member of SnRK gene family.SnRK genes are capable of mediating signals initiated during drought,salt and cold stress,play important roles in transducing stress signals,increasing protein synthesization and regulating signal transduction.
In this study,Populus×euramericana cv.‘Nanlin895’was used as experiment material,adapting to Agrobacterium-mediated to introduce separately two resistance gene of AtGolS2 gene and SRK2C gene.The main conclusion of this experiment was as following:
1.Determination of selective pressure of transformation:the selectable marker gene of AtGolS2 and SRK2C were both htp , so adopting Hyg as selective agent in transformation.Through contrast experiment,it is 2.5mg/L Hyg as selective pressure.
2.Establishment of the efficient system of transformation:we studied several factors affecting Agrobacterium-mediated transformation,drawing the best transform condition: pre-culture time were 3 days, bacterial concentration OD600 0.8,infection time were 15～20 minute,coculture time were 4 days.
3.Transgenic poplars were selected to survive on the culture medium with antibiotics,which includes Populus×euramericana cv.‘Nanlin895’with single AtGolS2 gene and SRK2C gene.The molecular biological tests and salt stress experiment were carried out to verity these transgenic poplar.
4.The two genes had been integrated into poplar genome and could express,which verified by PCR and RT-PCR.The result of salt treatment showed that salt-resistance ability of transgenic poplars with single AtGolS2 gene and SRK2C gene were enhanced.
引文
[1]王明庥.[M]林木育种学概论.北京:中国林业出版社,1989.
[2]胥猛,潘惠新,张博,王树东,黄敏仁.林木遗传改良中的分子生物学研究进展[J].林业科学,2009,45(1):136～143.
[3]Parons T J,Sinkar V P,Stettler R F,et al.Transformation of Poplar by Agrobacterium tumefaciens[J].Biotechnology,1986,4(6):533～536.
[4]苏晓华,张冰玉,黄烈健,等.转基因林木研究进展[J].林业科学研究,2003,16(1):95～103.
[5]赵鑫,詹立平,刘桂丰.杨树基因工程抗性育种研究进展[J].东北林业大学学报,2004,32(6):76～78.
[6]陆德如,陈永青主编.[M]基因工程.化学工业出版社,2004.
[7]陈英,何秋伶,诸葛强,等.林木基因工程研究进展[J].分子植物育种,2006,4(1):1～7.
[8]陈凌娜,曲延英,陈永坤,等.基因枪介导外源基因转化棉花幼胚方法的研究[J].新疆农业大学学报,2006,29(1):15～18.
[9]信金娜,韩烈保,刘君,等.基因枪转化法获得草地早熟禾(Poapm tensis L.)转基因植株[J].中国生物工程杂志.2006,26(8):10～14.
[10]宫本贺,熊和平,马雄风,等.基因枪介导法转化苎麻获得转基因植株的研究[J].作物杂志.2010,1:87～90.
[11]刘海坤,卫志明.大豆遗传转化研究进展[J].植物生理与分子生物学学报.2005,31(2):126～134.
[12]胡贻椿,陈天金,朴建华,等.转基因水稻及安全性的研究进展[J].中国食物与营养.2009(8):19～22.
[13]Santarém E.R.,Trick H.N.,Essig J.S.,et al. Sonicationassisted Agrobacterium-mediated transformation of soybean immature cotyledons:optimization of transient expression.Plant Cell Rep.1998,17:752～759.
[14]王荃,潘琪芳,袁芳,等.超声波辅助农杆菌介导的长春花(Catharanthus roseus)遗传转化[J].上海交通大学学报(农业科学版).2009,28(6):615～618
[15]刘国花,韩素英,齐力旺.植物抗旱耐盐基因工程研究及应用前景[J].世界农业.2003,7(291):44～46.
[16]潘瑞炽主编.植物生理学(第四版).高等教育出版社,2004.
[17]张积森,陈由强,李伟,等.甘蔗吡咯啉-5-羧酸合成酶基因(P5CS)的克隆及其表达分析[J].热带作物学报.2009,9(30):1337～1344.
[18] Delauney A.J.,Verma D.P.S.. A soybean gene encoding delta-1-pyrroline-5-carboxylate reductase was isolated by functional complementation in Escherichia coil and is found to be osmoregulated.Mol Gen Genet.1990,221:299～305.
[19]刘振林,尹伟伦,戴思兰.菠菜甜菜碱醛脱氢酶基因同源片段的克隆与序列分析.分子育种学,2006,4(1):35～40.
[20]Weretilnyk E.A.,and Hanson A.D.,Molecular cloning of a plant betaine-aldehyde dehydrogenase, an enzyme implicated in adaptation to salinity and drought, Proc.Natl. Acad.Sci..1990,87(7):2745～2749.
[21]Rathinasabathi B.,Mccue K.F.,Gago D.A.,et al.Metabolic engineering of giycine betaine synthesis:plant betaine aldehyde dehydrogenase lacking thpical transit peptides are targeted to tobacco chloroplasts where they confer betaine aldehyde resistance.Planta,193(2):155-162.
[22]梁峥,马德钦,汤岚,等.菠菜甜菜碱醛脱氢酶基因在烟草中的表达[J].生物工程学报.1997,13(3):236～240.
[23]舒卫国,艾万东,陈受宜.菠菜甜菜碱醛脱氢酶基因全序列分析.科学通报.1997,22(42):2441～2445.
[24]刘凤华。郭岩,谷冬梅,等.转甜菜碱醛脱氢酶基因植物的耐盐性研究.遗传学报.1997,24(1):54～58.
[25]樊军锋,李嘉瑞,韩一凡,等.mulD/gutD双价耐盐基因转化秦美猕猴桃的研究.西北农林科技大学学报(自然科学版).2002,6(3):53～58.
[26]王慧中,刘俊军,卢尔德赵,等.1-磷酸甘露醇脱氢酶基因转化水稻的研究.中国水稻科学.2003,17(1):6～10.
[27]王智华,张凌云,徐德芳.转BADH/mtlD基因耐盐旱稻生理指标的测定.安徽农业科学.2007,35(34):10944～10986.
[28]董云洲.表达肌醇甲基转移酶基因载体的构建及转基因烟草的耐盐性研究.植物学报.1999,41(2):146～149.
[29]Ritsemat,Smeekenss C.M..Engineering fructan metabolism in plants[J].Journal of plant Physioloyg.2003,160(7):811～820.
[30]Steinmetz M.,Le C.D.,Mymerich S.,et al.The DNA sequence of the gene for the secreted Bacillus subtilis enzyme leavansucrase and its genetic control sites[J].Mol Gen Genet.1985,200(2):220～228.
[31]杨晓红,陈晓阳,理守菊,等.盐胁迫对转SacB、BADH基因的美丽胡枝子部分逆境生理指标的影响.北京林业大学学报.2009,31(4):36～40.
[32]李义良,苏晓华,张冰玉,等.外源SacB基因在银腺杂种杨基因组中的表达及抗旱性分析.北京林业大学学报.2007,29(2):1～6.
[33]Teruaki Taji,Chieko Ohsumi,Satoshi luchi,et al.Important roles of drought- and cold-inducible genes for galactinol sunthase in stress tolerance in Arabidopsis thaliana.The Plant Journal.2002,29(4):417～426.
[34]崔润丽,刁现民.植物耐盐相关基因克隆与转化研究进展[J].中国生物工程杂志.2005,25(8):25～30.
[35]Akkapeddi A.S.,Shin D.I.,Stanek M.T.,et al.cDNA and derived amino acid sequence of the chloroplastic copper/zinc-superoxide dismutase from aspen(Populus tremuloides).Plant Physiol.1994,106:1231～1232.
[36]赵风云,郭善利,王增兰,等.耐盐转基因植物研究进展.植物生理与分子生物学报.2003,29(3):171～178.
[37]奉斌,代其林,王劲.非生物胁迫下植物体内活性氧清除酶系统的研究进展.绵阳师范学院学报.2009,28(11):50～54.
[38]赵凤云,徐忠俊.转GST和CAT1基因水稻根系抗氧化系统对干旱高温胁迫的响应.西北植物学报.2009,29(10):1980～1987.
[39]毕毓芳,诸葛强.林木非生物胁迫抗性基因工程研究进展.世界林业研究.2008,21(5):30～36.
[40]李颖,马锋旺.坏血酸过氧化物酶基因转化苹果的研究.西北农业学报.2010,19(1):140～143,163.
[41]Tanaka K.,Masuda R.,Sugimoto T.,et al.Water deficiency induced changes in the contenes of denfensive substances against active oxygen in spinach leaves[J].Agric Biol chem..1990,54:26～29.
[42]段苏然,施卫明,王俊儒.过量表达pAPX基因提高水稻对镉胁迫的耐性.土壤学报.2006,43(1):111～116.
[43]孙卫红,李风,束德峰,等.西红柿正义抗坏血酸过氧化物酶基因提高烟草耐盐能力.中国农业科学.2009,42(4):1165～1171.
[44]Mizoguchi T.,Ichimura K.,Shinozaki K..Enviromental stress response in plants:the role of mitogenactivated protein kinases.Trends Biotech.1997,15:15～19.
[45]杨洪强,梁小娥.蛋白激酶与植物逆境信号传递途径.植物生理学通讯.2001,37(3):185～191.
[46]Pestenaacz A.,Erdei L..Calcium-dependent protein kinase in maize and sorghum induced by polyethylene glycol.Physiol plant.1996,97:360～364.
[47]Urao T.,Katagir T.,Mizoguchi T.,et al.Two gene that encode Ca2+-dependent protein kinase are induced by drought and high-salt stress in Arabidopsis thaliana.Mol Gen Genet.1994,244:331～340.
[48] Hong S.W.,Jon J.H.,Kwak J.M.,et al.Identification of a receptor-like protein kinases gene rapidly induced by absicisic acid,dehydration,high salt and cold treatments in Arabidopsis thaliana.Plant physiol.1997,113:1203～1212.
[49]Lee J.H.,Montagu M.V.,Verbruggen N..A highly conserved kinase is an essential component for stress tolerance in yeast and plant cells.Proc Natl Acad Sci.1999,96:5873～5877.
[50]Liu H.Y.,Toyn J.H.,Chiang Y.C.,et al.DBF2,a cell cycle-regulated protein kinase,is physically and functionally associated with the CCR4 transcriptional regulatory complex.EMBO J.1997,17:5289～5289.
[51] Taishi Umezawa, Riichiro Yoshida, Kyonoshin Maruyama,et al. SRK2C, a SNF1-related protein kinase 2, improves drought tolerance by controlling stress-responsive gene expression in Arabidopsis thaliana.PNAS.2004,101(49):17306～17311.
[52]张钰,吴加林,黄永芬,等.转美洲拟鲽抗冻蛋白基因(afp)西红柿过氧化物酶、过氧化氢酶、超氧化物歧化酶活性测定.哈尔滨师范大学自然科学学报.1998,14(4):85～88.
[53]Worrall D.,Elias L.,Ashford D.,et al.A carrot leucine-rich-repeat protein tha inhibits ice recrystallization[J].Science.1998,282(2):11～13.
[54]Meyer K.,et al.A leucine-rich-repeat protein of carrot that exhibits antifreeze activity[J].FEBS Letters.1999,447:171～178.
[55]郝凤,刘晓静,毛娟,等.抗冻蛋白基因AFP表达载体构建及转化紫花苜蓿初报.草地学报.2009,17(6):779～783.
[56]徐春香,何勇强,尉义明,等.抗动蛋白(afp)基因表达载体的构建及对香蕉胚性细胞悬浮系的转化.广西植物.29(5):664～668.
[57]Dure L.,Greenway S.C.,Galau G.A..Developmental biochemistry of cottonseed embroyogenesis and germination changing messenger ribonucleic acid populations as shown by in vitro and in vivo protein synthesis[J].Biochemistry.1981,20(14):4162～4168.
[58]陈雷、李磊、李金花,等.植物LEA蛋白及其功能.中国农学通报.2009,25(24):143～146.
[59]Xu D.,Duan X.,Wang B.,et al.Expression of a late embryogenesis abundant protein gene,HVA1,from barley confers tolerance to water desicit and salt stress in transgenic rice[J].Plant Physiol.1996,110(1):249～257.
[60]刘甜甜,于影,赵鑫,等.转LEA基因烟草的NaHCO3抗性分析[J].分子植物育种.2006,4(2):216～222.
[61]Zhang H.,Zhou R.,Zhang L.,et al.CbLEA,a novel LEA gene from Chorispora Bungeana,congers cold tolerance in transgenic tobacco[J].Journal of Plant Biology.2007,50(3):336～343.
[62]李芳,李先文,黎定军.AP2/EREBP转录因子及其在植物抗逆中的作用.江西农业学报.2008,20(1):44～47.
[63]Wilson Staci , Krizek B.A. . DNA binding properties of the Arabidopsis floral development protein AINTEGUMENTA[J].Nucleic Acids Research.2000,28(21):4076～4082.
[64]孙静文.构树DREB转录因子及木质素合成代谢相关基因的克隆和功能分析[D].北京:中国科学院研究生院(植物研究所),2006.
[65]张勇,张守攻,齐力旺,等.杨树——林木基因组学研究的模式物种.植物学通报.2006,23(3):286～293.
[66]Taylor G..Populus:Arabidopsis for forestry.Do we need a model tree?Annu.Bot.2002,90:677～687.
[67]Fillatti J.J.,Sellmerj,Mccownb.Agrobacteriun mediated transformation and regeneration of Populus.Mol Gene Genet,1987,206:192～199.
[68]Mccown B.H.,Mccabede,Russelldr,et al.Stable transformation of Populus and incorporation of pest resistance by electricdischarge particle acceleration.Plant Cell Report.1991,9:590～594.
[69]伍宁丰,获云六.含苏云金杆菌晶体蛋白基因的杨树基因工程植株的建立.科学通报.1991,9:705～708.
[70]田颖川,韩一凡.抗虫转基因欧洲黑杨的培育.生物工程学报.1993,9 (4):29～297.
[71]郑均宝,张玉满.741杨离体叶片再生及抗虫基因转化.河北农业大学学报.1995,18(3) :20～25.
[72]胡彦师,梁一池.我国林业遗传改良的研究现状及展望.林业科技.2002,27(5):8～12.
[73]李玮,陈颖,李铃,等.抗菌肽LCI基因转化杨树的阶段研究.林业科学研究.1996,9(6):646～649.
[74]饶红宇,陈英,黄敏仁,等.杨树NL-80106转Bt基因植株的获得及抗虫性.植物资源与环境学报.2000,9(2):1～5.
[75] Augustin S.,Courtin C.,et al.Genetics of resistance to transgenic Bacillus thuringiensis poplars in Chrysomela tremulae (Coleoptera:Chrysomelidae).J Econ Entomol.2004,97(3):1058～1064.
[76]McNabb H.S..A field trial of transgenic hybrid Poplar trees:establishment and growth the second season.Agroculture Research institute.1991,9:155～159.
[77]郝贵霞,朱祯,朱之悌.豇豆胰蛋白酶抑制基因转化毛白杨的研究.植物学报.1999,41(12) :1276～1282.
[78]田颖川,郑均宝,虞红梅,等.转双抗虫基因杂种741白毛杨的研究.植物学报.2000,42(3) :263～268.
[79]李明亮,张辉,胡建军,等.转Bt基因和蛋白酶抑制剂基因杨树抗虫性的研究.林业科学.2000,36(12):93～97.
[80]许农,黄敏仁,陈道明.杨树基因工程研究进展.南京林业大学学报.1993,17(4):78～82.
[81]Mentag R.,Luckevich M.,Morency M.J.,et al.Bacterial disease resistance of transgenic hybrid poplar expressing the synthetic antimicrobial peptide D4E1.Tree Physiol.2003,23 (6):11.
[82]Liang H.Y.,Catharine M.,Catranis,et al.Enhanced resistance to the poplar fungal pathogen,Septoria musiva,in hybrid poplar clones transformed with genes encoding antimicrobial peptides.Biotechnology Letters.2002,24:383～389.
[83]Mohamed R.,Meilan R.,Ostry M.E.,et al..Bacterio-opsin gene overexpression fails to elevate fungal disease resistance in transgenic poplar (populus).Can.J.For.Res.2001,31:268～275.
[84]Baucher M.,Chabbert B.,Pilate G.,et al.Red xylem and higher lignin extractability by down-regulating acennamyl alcohol dehydrogenase in poplar.Plant Physiol.1996,112:1479～1490.
[85]魏建华,赵华燕,张景昱,等.毛果杨CCoAOMT cDNA片段的克隆与转基因杨木质素含量的调控.植物学报.2001,43(11):1179～1183.
[86]田晓明,谭晓风.转基因杨树的研究进展及展望.湖南林业科技.2009,36(2):71～74.
[87]刘凤华,孙仲序,崔德才,等.细菌mtl-D基因的克隆及在转基因八里庄杨中的表达.遗传学报.2000,27(5):428～433.
[88]樊军锋,韩一凡,李玲,等.mtlD/gutD双价基因转化美洲黑杨×青杨的研究.林业科学.2002,38(6):30～35.
[89]樊军锋,韩一凡,李玲,等.84K杨树耐盐基因转化研究.西北林学院学报.2002,17(4):33～37.
[90]杨春霞,李火根,程强,等.南林895杨抗旱耐盐基因DREB1C的转化.林业科学.2009,45(2):17～21.
[91]温尚昆,董春海,伊承俏,等.由根癌农杆菌介导向毛白杨导入激素合成基因建立遗传转化系统.生物技术.1997 ,7(2) :11～14.
[92]李春霞.抗冻蛋白基因对山杨等植物遗传转化的研究[D].哈尔滨:东北林业大学,2003.
[93]Arisi A C,Cornic G,Jouanin L,et al.Overexpression of Fe-SOD in transformed poplar modifies the regulation of photo synthesis at low CO2 partial pressures or following to the prooxident herbicide methyl viologen.Plant Physiol.1998,117:565～574.
[94]Rugh C.L. , Senecoff J.F. , Meagher R.B. , et al . Development of transgenic yellow poplar for mercuryphytoremediation.Nat.Biotechnol.1998,16:925～928.
[95]Joan M.H.,Roy French.The polymerase chain reaction and Plant disease diagnosis[J].Ann,Rev.Phyto Pathol.1993,31:81～109.
[96]吕瑞玲,吴小凤,刘敏超.分子标记技术及在水稻遗传研究中的应用.中国农学通报.2009,25(04):65～73.
[97]陶生策,张治平.PCR技术研究进展[J].生物工程进展.2001,21(4):26～29.
[98]Ramsay G..DNA chips:state of the art[J].Nature Biotechnology.1998,16(1):40～44.
[99]邹锋,袁德义,张琳.基因芯片技术及其在果树上的应用.南方园艺.2009,20(2):54～56.
[100]王世绩主编.杨树研究进展.北京:中国林业出版社.1995.
[101]张新春,庄炳昌,李自超,等.植物耐盐性研究进展.玉米科学.2002,10(1):50～56.
[102]Horsch R.B.,Fry J.E.,Hoffmann N.L.,et al.A simple and general method for transferring genes into plants.Science.1985,227(4691):1229～1231.
[103]王彩霞,吴殿星,沈圣泉,等.转基因及常规水稻幼苗对潮霉素敏感性的研究.核农学报.2005,19(3):168～171.
[2]胥猛,潘惠新,张博,王树东,黄敏仁.林木遗传改良中的分子生物学研究进展[J].林业科学,2009,45(1):136～143.
[3]Parons T J,Sinkar V P,Stettler R F,et al.Transformation of Poplar by Agrobacterium tumefaciens[J].Biotechnology,1986,4(6):533～536.
[4]苏晓华,张冰玉,黄烈健,等.转基因林木研究进展[J].林业科学研究,2003,16(1):95～103.
[5]赵鑫,詹立平,刘桂丰.杨树基因工程抗性育种研究进展[J].东北林业大学学报,2004,32(6):76～78.
[6]陆德如,陈永青主编.[M]基因工程.化学工业出版社,2004.
[7]陈英,何秋伶,诸葛强,等.林木基因工程研究进展[J].分子植物育种,2006,4(1):1～7.
[8]陈凌娜,曲延英,陈永坤,等.基因枪介导外源基因转化棉花幼胚方法的研究[J].新疆农业大学学报,2006,29(1):15～18.
[9]信金娜,韩烈保,刘君,等.基因枪转化法获得草地早熟禾(Poapm tensis L.)转基因植株[J].中国生物工程杂志.2006,26(8):10～14.
[10]宫本贺,熊和平,马雄风,等.基因枪介导法转化苎麻获得转基因植株的研究[J].作物杂志.2010,1:87～90.
[11]刘海坤,卫志明.大豆遗传转化研究进展[J].植物生理与分子生物学学报.2005,31(2):126～134.
[12]胡贻椿,陈天金,朴建华,等.转基因水稻及安全性的研究进展[J].中国食物与营养.2009(8):19～22.
[13]Santarém E.R.,Trick H.N.,Essig J.S.,et al. Sonicationassisted Agrobacterium-mediated transformation of soybean immature cotyledons:optimization of transient expression.Plant Cell Rep.1998,17:752～759.
[14]王荃,潘琪芳,袁芳,等.超声波辅助农杆菌介导的长春花(Catharanthus roseus)遗传转化[J].上海交通大学学报(农业科学版).2009,28(6):615～618
[15]刘国花,韩素英,齐力旺.植物抗旱耐盐基因工程研究及应用前景[J].世界农业.2003,7(291):44～46.
[16]潘瑞炽主编.植物生理学(第四版).高等教育出版社,2004.
[17]张积森,陈由强,李伟,等.甘蔗吡咯啉-5-羧酸合成酶基因(P5CS)的克隆及其表达分析[J].热带作物学报.2009,9(30):1337～1344.
[18] Delauney A.J.,Verma D.P.S.. A soybean gene encoding delta-1-pyrroline-5-carboxylate reductase was isolated by functional complementation in Escherichia coil and is found to be osmoregulated.Mol Gen Genet.1990,221:299～305.
[19]刘振林,尹伟伦,戴思兰.菠菜甜菜碱醛脱氢酶基因同源片段的克隆与序列分析.分子育种学,2006,4(1):35～40.
[20]Weretilnyk E.A.,and Hanson A.D.,Molecular cloning of a plant betaine-aldehyde dehydrogenase, an enzyme implicated in adaptation to salinity and drought, Proc.Natl. Acad.Sci..1990,87(7):2745～2749.
[21]Rathinasabathi B.,Mccue K.F.,Gago D.A.,et al.Metabolic engineering of giycine betaine synthesis:plant betaine aldehyde dehydrogenase lacking thpical transit peptides are targeted to tobacco chloroplasts where they confer betaine aldehyde resistance.Planta,193(2):155-162.
[22]梁峥,马德钦,汤岚,等.菠菜甜菜碱醛脱氢酶基因在烟草中的表达[J].生物工程学报.1997,13(3):236～240.
[23]舒卫国,艾万东,陈受宜.菠菜甜菜碱醛脱氢酶基因全序列分析.科学通报.1997,22(42):2441～2445.
[24]刘凤华。郭岩,谷冬梅,等.转甜菜碱醛脱氢酶基因植物的耐盐性研究.遗传学报.1997,24(1):54～58.
[25]樊军锋,李嘉瑞,韩一凡,等.mulD/gutD双价耐盐基因转化秦美猕猴桃的研究.西北农林科技大学学报(自然科学版).2002,6(3):53～58.
[26]王慧中,刘俊军,卢尔德赵,等.1-磷酸甘露醇脱氢酶基因转化水稻的研究.中国水稻科学.2003,17(1):6～10.
[27]王智华,张凌云,徐德芳.转BADH/mtlD基因耐盐旱稻生理指标的测定.安徽农业科学.2007,35(34):10944～10986.
[28]董云洲.表达肌醇甲基转移酶基因载体的构建及转基因烟草的耐盐性研究.植物学报.1999,41(2):146～149.
[29]Ritsemat,Smeekenss C.M..Engineering fructan metabolism in plants[J].Journal of plant Physioloyg.2003,160(7):811～820.
[30]Steinmetz M.,Le C.D.,Mymerich S.,et al.The DNA sequence of the gene for the secreted Bacillus subtilis enzyme leavansucrase and its genetic control sites[J].Mol Gen Genet.1985,200(2):220～228.
[31]杨晓红,陈晓阳,理守菊,等.盐胁迫对转SacB、BADH基因的美丽胡枝子部分逆境生理指标的影响.北京林业大学学报.2009,31(4):36～40.
[32]李义良,苏晓华,张冰玉,等.外源SacB基因在银腺杂种杨基因组中的表达及抗旱性分析.北京林业大学学报.2007,29(2):1～6.
[33]Teruaki Taji,Chieko Ohsumi,Satoshi luchi,et al.Important roles of drought- and cold-inducible genes for galactinol sunthase in stress tolerance in Arabidopsis thaliana.The Plant Journal.2002,29(4):417～426.
[34]崔润丽,刁现民.植物耐盐相关基因克隆与转化研究进展[J].中国生物工程杂志.2005,25(8):25～30.
[35]Akkapeddi A.S.,Shin D.I.,Stanek M.T.,et al.cDNA and derived amino acid sequence of the chloroplastic copper/zinc-superoxide dismutase from aspen(Populus tremuloides).Plant Physiol.1994,106:1231～1232.
[36]赵风云,郭善利,王增兰,等.耐盐转基因植物研究进展.植物生理与分子生物学报.2003,29(3):171～178.
[37]奉斌,代其林,王劲.非生物胁迫下植物体内活性氧清除酶系统的研究进展.绵阳师范学院学报.2009,28(11):50～54.
[38]赵凤云,徐忠俊.转GST和CAT1基因水稻根系抗氧化系统对干旱高温胁迫的响应.西北植物学报.2009,29(10):1980～1987.
[39]毕毓芳,诸葛强.林木非生物胁迫抗性基因工程研究进展.世界林业研究.2008,21(5):30～36.
[40]李颖,马锋旺.坏血酸过氧化物酶基因转化苹果的研究.西北农业学报.2010,19(1):140～143,163.
[41]Tanaka K.,Masuda R.,Sugimoto T.,et al.Water deficiency induced changes in the contenes of denfensive substances against active oxygen in spinach leaves[J].Agric Biol chem..1990,54:26～29.
[42]段苏然,施卫明,王俊儒.过量表达pAPX基因提高水稻对镉胁迫的耐性.土壤学报.2006,43(1):111～116.
[43]孙卫红,李风,束德峰,等.西红柿正义抗坏血酸过氧化物酶基因提高烟草耐盐能力.中国农业科学.2009,42(4):1165～1171.
[44]Mizoguchi T.,Ichimura K.,Shinozaki K..Enviromental stress response in plants:the role of mitogenactivated protein kinases.Trends Biotech.1997,15:15～19.
[45]杨洪强,梁小娥.蛋白激酶与植物逆境信号传递途径.植物生理学通讯.2001,37(3):185～191.
[46]Pestenaacz A.,Erdei L..Calcium-dependent protein kinase in maize and sorghum induced by polyethylene glycol.Physiol plant.1996,97:360～364.
[47]Urao T.,Katagir T.,Mizoguchi T.,et al.Two gene that encode Ca2+-dependent protein kinase are induced by drought and high-salt stress in Arabidopsis thaliana.Mol Gen Genet.1994,244:331～340.
[48] Hong S.W.,Jon J.H.,Kwak J.M.,et al.Identification of a receptor-like protein kinases gene rapidly induced by absicisic acid,dehydration,high salt and cold treatments in Arabidopsis thaliana.Plant physiol.1997,113:1203～1212.
[49]Lee J.H.,Montagu M.V.,Verbruggen N..A highly conserved kinase is an essential component for stress tolerance in yeast and plant cells.Proc Natl Acad Sci.1999,96:5873～5877.
[50]Liu H.Y.,Toyn J.H.,Chiang Y.C.,et al.DBF2,a cell cycle-regulated protein kinase,is physically and functionally associated with the CCR4 transcriptional regulatory complex.EMBO J.1997,17:5289～5289.
[51] Taishi Umezawa, Riichiro Yoshida, Kyonoshin Maruyama,et al. SRK2C, a SNF1-related protein kinase 2, improves drought tolerance by controlling stress-responsive gene expression in Arabidopsis thaliana.PNAS.2004,101(49):17306～17311.
[52]张钰,吴加林,黄永芬,等.转美洲拟鲽抗冻蛋白基因(afp)西红柿过氧化物酶、过氧化氢酶、超氧化物歧化酶活性测定.哈尔滨师范大学自然科学学报.1998,14(4):85～88.
[53]Worrall D.,Elias L.,Ashford D.,et al.A carrot leucine-rich-repeat protein tha inhibits ice recrystallization[J].Science.1998,282(2):11～13.
[54]Meyer K.,et al.A leucine-rich-repeat protein of carrot that exhibits antifreeze activity[J].FEBS Letters.1999,447:171～178.
[55]郝凤,刘晓静,毛娟,等.抗冻蛋白基因AFP表达载体构建及转化紫花苜蓿初报.草地学报.2009,17(6):779～783.
[56]徐春香,何勇强,尉义明,等.抗动蛋白(afp)基因表达载体的构建及对香蕉胚性细胞悬浮系的转化.广西植物.29(5):664～668.
[57]Dure L.,Greenway S.C.,Galau G.A..Developmental biochemistry of cottonseed embroyogenesis and germination changing messenger ribonucleic acid populations as shown by in vitro and in vivo protein synthesis[J].Biochemistry.1981,20(14):4162～4168.
[58]陈雷、李磊、李金花,等.植物LEA蛋白及其功能.中国农学通报.2009,25(24):143～146.
[59]Xu D.,Duan X.,Wang B.,et al.Expression of a late embryogenesis abundant protein gene,HVA1,from barley confers tolerance to water desicit and salt stress in transgenic rice[J].Plant Physiol.1996,110(1):249～257.
[60]刘甜甜,于影,赵鑫,等.转LEA基因烟草的NaHCO3抗性分析[J].分子植物育种.2006,4(2):216～222.
[61]Zhang H.,Zhou R.,Zhang L.,et al.CbLEA,a novel LEA gene from Chorispora Bungeana,congers cold tolerance in transgenic tobacco[J].Journal of Plant Biology.2007,50(3):336～343.
[62]李芳,李先文,黎定军.AP2/EREBP转录因子及其在植物抗逆中的作用.江西农业学报.2008,20(1):44～47.
[63]Wilson Staci , Krizek B.A. . DNA binding properties of the Arabidopsis floral development protein AINTEGUMENTA[J].Nucleic Acids Research.2000,28(21):4076～4082.
[64]孙静文.构树DREB转录因子及木质素合成代谢相关基因的克隆和功能分析[D].北京:中国科学院研究生院(植物研究所),2006.
[65]张勇,张守攻,齐力旺,等.杨树——林木基因组学研究的模式物种.植物学通报.2006,23(3):286～293.
[66]Taylor G..Populus:Arabidopsis for forestry.Do we need a model tree?Annu.Bot.2002,90:677～687.
[67]Fillatti J.J.,Sellmerj,Mccownb.Agrobacteriun mediated transformation and regeneration of Populus.Mol Gene Genet,1987,206:192～199.
[68]Mccown B.H.,Mccabede,Russelldr,et al.Stable transformation of Populus and incorporation of pest resistance by electricdischarge particle acceleration.Plant Cell Report.1991,9:590～594.
[69]伍宁丰,获云六.含苏云金杆菌晶体蛋白基因的杨树基因工程植株的建立.科学通报.1991,9:705～708.
[70]田颖川,韩一凡.抗虫转基因欧洲黑杨的培育.生物工程学报.1993,9 (4):29～297.
[71]郑均宝,张玉满.741杨离体叶片再生及抗虫基因转化.河北农业大学学报.1995,18(3) :20～25.
[72]胡彦师,梁一池.我国林业遗传改良的研究现状及展望.林业科技.2002,27(5):8～12.
[73]李玮,陈颖,李铃,等.抗菌肽LCI基因转化杨树的阶段研究.林业科学研究.1996,9(6):646～649.
[74]饶红宇,陈英,黄敏仁,等.杨树NL-80106转Bt基因植株的获得及抗虫性.植物资源与环境学报.2000,9(2):1～5.
[75] Augustin S.,Courtin C.,et al.Genetics of resistance to transgenic Bacillus thuringiensis poplars in Chrysomela tremulae (Coleoptera:Chrysomelidae).J Econ Entomol.2004,97(3):1058～1064.
[76]McNabb H.S..A field trial of transgenic hybrid Poplar trees:establishment and growth the second season.Agroculture Research institute.1991,9:155～159.
[77]郝贵霞,朱祯,朱之悌.豇豆胰蛋白酶抑制基因转化毛白杨的研究.植物学报.1999,41(12) :1276～1282.
[78]田颖川,郑均宝,虞红梅,等.转双抗虫基因杂种741白毛杨的研究.植物学报.2000,42(3) :263～268.
[79]李明亮,张辉,胡建军,等.转Bt基因和蛋白酶抑制剂基因杨树抗虫性的研究.林业科学.2000,36(12):93～97.
[80]许农,黄敏仁,陈道明.杨树基因工程研究进展.南京林业大学学报.1993,17(4):78～82.
[81]Mentag R.,Luckevich M.,Morency M.J.,et al.Bacterial disease resistance of transgenic hybrid poplar expressing the synthetic antimicrobial peptide D4E1.Tree Physiol.2003,23 (6):11.
[82]Liang H.Y.,Catharine M.,Catranis,et al.Enhanced resistance to the poplar fungal pathogen,Septoria musiva,in hybrid poplar clones transformed with genes encoding antimicrobial peptides.Biotechnology Letters.2002,24:383～389.
[83]Mohamed R.,Meilan R.,Ostry M.E.,et al..Bacterio-opsin gene overexpression fails to elevate fungal disease resistance in transgenic poplar (populus).Can.J.For.Res.2001,31:268～275.
[84]Baucher M.,Chabbert B.,Pilate G.,et al.Red xylem and higher lignin extractability by down-regulating acennamyl alcohol dehydrogenase in poplar.Plant Physiol.1996,112:1479～1490.
[85]魏建华,赵华燕,张景昱,等.毛果杨CCoAOMT cDNA片段的克隆与转基因杨木质素含量的调控.植物学报.2001,43(11):1179～1183.
[86]田晓明,谭晓风.转基因杨树的研究进展及展望.湖南林业科技.2009,36(2):71～74.
[87]刘凤华,孙仲序,崔德才,等.细菌mtl-D基因的克隆及在转基因八里庄杨中的表达.遗传学报.2000,27(5):428～433.
[88]樊军锋,韩一凡,李玲,等.mtlD/gutD双价基因转化美洲黑杨×青杨的研究.林业科学.2002,38(6):30～35.
[89]樊军锋,韩一凡,李玲,等.84K杨树耐盐基因转化研究.西北林学院学报.2002,17(4):33～37.
[90]杨春霞,李火根,程强,等.南林895杨抗旱耐盐基因DREB1C的转化.林业科学.2009,45(2):17～21.
[91]温尚昆,董春海,伊承俏,等.由根癌农杆菌介导向毛白杨导入激素合成基因建立遗传转化系统.生物技术.1997 ,7(2) :11～14.
[92]李春霞.抗冻蛋白基因对山杨等植物遗传转化的研究[D].哈尔滨:东北林业大学,2003.
[93]Arisi A C,Cornic G,Jouanin L,et al.Overexpression of Fe-SOD in transformed poplar modifies the regulation of photo synthesis at low CO2 partial pressures or following to the prooxident herbicide methyl viologen.Plant Physiol.1998,117:565～574.
[94]Rugh C.L. , Senecoff J.F. , Meagher R.B. , et al . Development of transgenic yellow poplar for mercuryphytoremediation.Nat.Biotechnol.1998,16:925～928.
[95]Joan M.H.,Roy French.The polymerase chain reaction and Plant disease diagnosis[J].Ann,Rev.Phyto Pathol.1993,31:81～109.
[96]吕瑞玲,吴小凤,刘敏超.分子标记技术及在水稻遗传研究中的应用.中国农学通报.2009,25(04):65～73.
[97]陶生策,张治平.PCR技术研究进展[J].生物工程进展.2001,21(4):26～29.
[98]Ramsay G..DNA chips:state of the art[J].Nature Biotechnology.1998,16(1):40～44.
[99]邹锋,袁德义,张琳.基因芯片技术及其在果树上的应用.南方园艺.2009,20(2):54～56.
[100]王世绩主编.杨树研究进展.北京:中国林业出版社.1995.
[101]张新春,庄炳昌,李自超,等.植物耐盐性研究进展.玉米科学.2002,10(1):50～56.
[102]Horsch R.B.,Fry J.E.,Hoffmann N.L.,et al.A simple and general method for transferring genes into plants.Science.1985,227(4691):1229～1231.
[103]王彩霞,吴殿星,沈圣泉,等.转基因及常规水稻幼苗对潮霉素敏感性的研究.核农学报.2005,19(3):168～171.