桃再生体系的优化与反义PG基因遗传转化的研究
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摘要
桃(Prunus persica(L. ) Batsch)是世界性重要果树,我国是桃主产国,产量居世界之首,甘肃省是桃的原产地和最适栽培区之一。桃是典型的呼吸跃变型果实,成熟果实采后很快出现呼吸高峰,果肉迅速变软,给贮藏运输带来极大不便,这是长期制约桃生产的主要问题之一。而通常采用的低温冷藏等方法易造成桃果冷害,结果十分不理想。因此,通过植物基因工程技术控制果实软化,选育出耐贮运的桃新品种是从根本上解决桃贮运难题的有效途径之一。本研究通过反义基因技术特异性抑制果实成熟后PG的表达,有望延迟桃果实的软化,获得耐贮性硬溶质桃新种质。
本研究以甘肃省常栽品种白粉桃为试材,建立了桃遗传转化再生体系,并以白粉桃茎段为材料,在优化桃遗传转化再生体系的基础上,通过根癌农杆菌介导反义PG基因转化,得到了转化植株并用PCR进行了检测。研究主要取得如下结果:
1.以白粉桃幼茎和花后不同时间成熟的果实种胚为外植体,进行桃遗传转化再生体系优化。其幼茎最佳诱导培养基为MS+6-BA 1.0mg/L+ NAA 0.5mg/L,诱导率可达93.5%。开花75d后的种胚能在MS+6-BA 0.5mg/L+NAA 0.05mg/L上直接诱导分化成苗,诱导分化率可达91.5%。将不定芽用100mg/L IBA浸蘸其基部转移到不含激素的1/2MS培养基中诱导生根,生根率达到78%。
2.用携带有PG基因反义片段和35S启动子的表达载体pCAMBIA2300,转化白粉桃茎段。受体材料预培养3d后,经根癌农杆菌菌液(OD600值0.4)浸染10min后共培养60h,转移至分化培养基MS+6-BA 1.0mg/L +NAA 0.5 mg/L+kan 50mg/L+Cef 250mg/L上诱导产生不定芽,将抗kan的不定芽先后置于含有kan 50mg/L的生长、增殖和生根培养基上继续选择,获得完整植株。经PCR检测初步证明,反义PG基因已导入桃中。
Peach〔Prunuspersica (L.) Batsch.〕is an important fruit in the worldwide. China is a major producing country of peach, whose output ranks first in the world, and Gansu Province is the original and the most appropriate cultivation area of peach. Peach is a tipical respiratory climacteric fruit. Soon ripe fruits picked appeared the respiratory peak and flesh became soft rapid, which caused great inconvenience in transport and storage and it is a major problem of restraining the production in peach .While it is unsatisfactory that the common method such as cold storage usually leads to chilling injury of peach. Therefore, using plant genetic engineering technology can control fruit softening, and changing fundamentally storage and transport properties in peach has an important significance.
In this study, Baifen peach often planted in Gansu Province was used as test materials, and then established a peach regeneration system. with Baifen peach stem segments as materials, based on the optimization genetic transformation system of peach, taking advantage of Agrobacterium mediated transformed into antisense PG gene and obtained transformed plants, and then tested by PCR. The main results obtained are as follows:
1. With tender stem and matural fruit in different time of Baifen after flowering as explants, optimize the genetic transformation system. The best medium for induction was MS+1.0mg/L6-BA+0.5mg/LNAA, and induction frequency was up to 93.5%. After flowering 75d embryo can directly differentiate into seedlings, induction frequency was up to 91.5%. The shoots dipped its base with 100mg/L1IBA were shifted to 1/2MS medium without hormone to induce roots whose rate was up to 78%.
2. Transform into the stem of Baifen peach by expressive vector pCAMBIA2300 carrying the antisense PG gene fragments and 35S promoter. Receptor materials precultivated for 3 days, were co-cultured for 60h by Agrobacterium bacteria (OD600 value of 0.4) after being disseminated, transferred into the differentiative medium MS +1.0 mg/L6-BA +0.5 mg / LNAA +50 mg / L km +250 mg / LCef to induce adventitious buds, and anti-km adventitious buds were placed in the 50mg/Lkm mediums of growth, proliferation and rooting. Ultimately the whole plants were obtained by The PCR test which showed that the antisense PG gene has been introduced into the plants.
本研究以甘肃省常栽品种白粉桃为试材,建立了桃遗传转化再生体系,并以白粉桃茎段为材料,在优化桃遗传转化再生体系的基础上,通过根癌农杆菌介导反义PG基因转化,得到了转化植株并用PCR进行了检测。研究主要取得如下结果:
1.以白粉桃幼茎和花后不同时间成熟的果实种胚为外植体,进行桃遗传转化再生体系优化。其幼茎最佳诱导培养基为MS+6-BA 1.0mg/L+ NAA 0.5mg/L,诱导率可达93.5%。开花75d后的种胚能在MS+6-BA 0.5mg/L+NAA 0.05mg/L上直接诱导分化成苗,诱导分化率可达91.5%。将不定芽用100mg/L IBA浸蘸其基部转移到不含激素的1/2MS培养基中诱导生根,生根率达到78%。
2.用携带有PG基因反义片段和35S启动子的表达载体pCAMBIA2300,转化白粉桃茎段。受体材料预培养3d后,经根癌农杆菌菌液(OD600值0.4)浸染10min后共培养60h,转移至分化培养基MS+6-BA 1.0mg/L +NAA 0.5 mg/L+kan 50mg/L+Cef 250mg/L上诱导产生不定芽,将抗kan的不定芽先后置于含有kan 50mg/L的生长、增殖和生根培养基上继续选择,获得完整植株。经PCR检测初步证明,反义PG基因已导入桃中。
Peach〔Prunuspersica (L.) Batsch.〕is an important fruit in the worldwide. China is a major producing country of peach, whose output ranks first in the world, and Gansu Province is the original and the most appropriate cultivation area of peach. Peach is a tipical respiratory climacteric fruit. Soon ripe fruits picked appeared the respiratory peak and flesh became soft rapid, which caused great inconvenience in transport and storage and it is a major problem of restraining the production in peach .While it is unsatisfactory that the common method such as cold storage usually leads to chilling injury of peach. Therefore, using plant genetic engineering technology can control fruit softening, and changing fundamentally storage and transport properties in peach has an important significance.
In this study, Baifen peach often planted in Gansu Province was used as test materials, and then established a peach regeneration system. with Baifen peach stem segments as materials, based on the optimization genetic transformation system of peach, taking advantage of Agrobacterium mediated transformed into antisense PG gene and obtained transformed plants, and then tested by PCR. The main results obtained are as follows:
1. With tender stem and matural fruit in different time of Baifen after flowering as explants, optimize the genetic transformation system. The best medium for induction was MS+1.0mg/L6-BA+0.5mg/LNAA, and induction frequency was up to 93.5%. After flowering 75d embryo can directly differentiate into seedlings, induction frequency was up to 91.5%. The shoots dipped its base with 100mg/L1IBA were shifted to 1/2MS medium without hormone to induce roots whose rate was up to 78%.
2. Transform into the stem of Baifen peach by expressive vector pCAMBIA2300 carrying the antisense PG gene fragments and 35S promoter. Receptor materials precultivated for 3 days, were co-cultured for 60h by Agrobacterium bacteria (OD600 value of 0.4) after being disseminated, transferred into the differentiative medium MS +1.0 mg/L6-BA +0.5 mg / LNAA +50 mg / L km +250 mg / LCef to induce adventitious buds, and anti-km adventitious buds were placed in the 50mg/Lkm mediums of growth, proliferation and rooting. Ultimately the whole plants were obtained by The PCR test which showed that the antisense PG gene has been introduced into the plants.
引文
[1]沙立勋.我国桃产销状况和当前市场分析[N].中国食品报,2006-08-09(6).
[2]张晓宇,王春生,赵桂芳,等.桃果实采后生理研究及贮藏保鲜技术应用进展[J].中国农学通报.2008,24 (5):117～120.
[3] A.Gentile,S. Monticelli,C.Damiano.Adventitious shoot regeneration in peach [Prunus persica (L) Batsch][J].Plant Cell Rep,2002,20:1011～1016.
[4] Hammerschlag F.A,Bauchan G,Scorza R.Regeneration of peach plants from callus drived from immature embryos[J].Theoretical and Apple Genetics,1985, (70): 248～251.
[5]张永庆,陈大明,金勇丰,等.桃离体组织分化再生植株的研究[J].园艺学报,2001,28(4):342～344.
[6]吴延军,张上隆,谢鸣,等.桃幼胚及幼胚子叶再生的研究[J].林业科学,2005, 41(5):45～51.
[7] Rosa M.Pérez-Clemente, Amparo Perez-Sanjuan, Lorenzo García-Ferriz,et.al.Transgenic peach plants (Prunus persica L.) produced by genetic transformation of embryo sections using the green fluorescent protein(GFP) as an in vivo marker[J]. Molecular Breeding,2004, 14: 419～427.
[8]吴延军,张上隆,谢鸣,等.桃ACO基因反义转化桃幼胚子叶的研究[J].遗传,2006,28(1):65～70.
[9]李曜东,魏玉凝.肥城桃组培苗诱导、基因转化及其增殖[J].果树学报,2003,20(1):70～72.
[10] Isabel M.G.Padilla, Agnieszka Golis,Adele Gentile, Carmine Damiano,Ralph Scorza. Evaluation of transformation in peach Prunus persica explants using green fluorescent protein (GFP) and beta-glucuronidase (GUS) reporter genes[J].Plant Cell,Tissue and Organ Culture 2006 (84): 309～314.
[11] Ye X, Brown SK.Genetic transformuation of peach tissues by particle bormbardment[J].J Amer Soc Hort Sci,1994,119(2):367～373.
[12] Scorza R, Morgens PH, Cordts JM, Mante Callahan AM.Agrobacterium-mediated transformation of peach (Prunus persica Batsch) leaf segments, immature embryos and long-term embryogenic callus[J]. In Vitro Cell Dev. Biol,1990,26: 829～834.
[13]吴延军,徐昌杰,张上隆.桃组织培养和遗传转化研究现状及展望[J].果树学报,2002,19(2):123～127.
[14] Byrne DH. Peach breeding trends a worldwide perspective[J].ActaHort, 2002,592:49~58.
[15] Smigicki.AC,Owins LD,Hammerschlag FA,In vitro transformation of peach,Prunus persica L.)Batsch.using A grobacterium tumefaciens[J].HortScience,1987,(22): 5131～5135.
[16] Hammerschlag F.A,Bauchan G,Scorza R.Regeneration of peach plants from callus drived from immature embryos[J].Theoretical and Apple Genetics,1985, (70): 248~251.
[17]吴延军,张上隆,徐昌杰,等.根癌农杆菌介导的桃幼胚转化实验参数研究[J].果树学报,2004,21(5):477~479.
[18] Hobson GE. The fiemness of tomato fruit in relation to polygalacturonase activity[J].Biochem,1963,86: 365~385.
[19]李正国.果实成熟的基因调控[J].生物工程进展,2000,20(3):30~34.
[20] Dawson D M, L D Melton, C B Watkins. Cell wallchanges in nectarine [J]. Plant Physiol, 1992, 100:1203~1210.
[21] Fry SC,Smith Rc,Renwick KF,et al.Xyloglucan endotransglycosylase, a new wall- loosening enzyme activity from plant[J].Biochem J,1992,282:821～828.
[22]陈昆松,李方,张上隆.猕猴桃果实成熟进程中木葡聚糖内糖基转移酶mRNA的变化[J].植物学报,1999,41(11):1231~1234.
[23] Huber D.J.The role of cell wall hydrolases in fruit softening [J].Horticultural Reviews, 1983, 5:169~219.
[34]吴敏,陈昆松,贾惠娟,等.桃果实采后软化过程中内源IAA,ABA和乙烯的变化[J].果树学报,2003,20(3):157~160.
[25]刘彩莉,霍君生.贮藏条件对桃果实的生理影响[J].河北农业大学学报,1992,15(1):88~91.
[26] Anderson P.E.Long-tern storage of peaches and nectarines intemittendy warned during controlled atmosphere storage.J.Am.Soc.Hort.Sci, 1982, 107(2):214-216.
[27] Anderson P.E.The influenceof storage temperature and warning during storage on peach and nectarine fruit quality .J.Am.Soc.Hoit.Sci. 1979, 104(4):459~461.
[28]吕昌文,齐灵,修德仁.桃波动温度贮藏及其机理研究[J].华北农学报,1994.9(1):75~80.
[29] Anderson R.E. Nectarines stored in a controoled atmosphere or air [J]. Am.Soc.Hort.Sci.1975, 100:151~153.
[30]石海燕,冯双庆.气调贮藏对紫花芒果PG、纤维素酶及果实硬度的影响[J].园艺学报,1997,24 (4):407-409.
[31] Wang CY. Physiological and Biochemical Effects of CA on Fruit and Vegelables.Calderorn M, Barkai G(Eds). Food Preservation by Modified Atmosphere[J]. Boca Raton FL: CRC Press. 1990, 101~129..
[32] Xue YB,Kubo Y,Inaba A,el al.Effect of humidify on ripening and texture in banana fruit.[J].Japan Soc Hort Sci, 1995,64(3):657-669.
[33] Pressey R.Exopolygalacturonase in tomato fruit[J].Phytochemistry,1987,26(7):1867~1870.
[34] PhaffH J, Demain AL.J.Biological Chemistry [J].1956, 218:875.
[35] Crooks P R, Grierson D.Ultrastructure oftomato fruit ripening and the role of polygalacturonase isoenzymes in cellwall degradation [J].PlantPhysiology, 1983, 72:248～254.
[36]薛炳烨,束怀瑞.应用基因工程改良果实成熟特性研究进展[J].山东农业科学,2002,4:49～52.
[37] Lee E,Speirs J,GrayJ,et al.Homologies to the tomato endopolygalacturonase gene in the peach genome[J].Plant Cell and Environment,1990,13:513～521.
[38] Fischer R L, Bennett A B.Role of cell wall hydrolases in fruitripening [J].Annu.Rev.Plant Physiol.Plant Mol.Biol., 1991, 42:675～703.
[39] Ali Z M, Brady C J.Purification and characterization of the polygalacturonases of tomato fruits [J].Australia J.Plant Physiology, 1982, 9:155～169.
[40] Tucker G A,Robertson NG,Grierson D.Changes in polygalacturonase isoenzymes during the ripening of normal and mutant tomato fruit[J].Eur.J.B chemistry,1980,112:119～124.
[41] Moshrefi M, Luth B S.Carbohydrate composition and electrophoretic properties of tomato polygalacturonase isoenzymes [J].Eur.J.Biochemistry, 1983, 135:511～514.
[42] Pressey R, Avants J K.Two forms of polygalacturonase in tomatoes [J].Biochemistry Biophysics Acta, 1973, 309:363～369.
[43] Dellapenna D,Danny C,etal.Molecular cloning oftomato fruit PG analysis of PG mRNA levets during ripening proc[J]. Natl Acad Sci USA, 1986, (83):6420～6424.
[44] Smith CJS,Watson CF,Ray J,et al.Antisense RNA inhibition of polygalacturonase gene expression in transgenic tomatoes[J].Nature,1988,334:724～726.
[45] Sheehy RE, Kramer M, and Hiatt WR.Reduction of polygalacturonase activity in tomato fruit byantisense RNA [J] .Proc Natl Acad Sci USA, 1988, 85:8805～8809.
[46] Cooley MB, Yodor J I.Insertional inactivation of the tomato polygalacturonasegene [J].Plant MolecularBiology, 1998, 38:521~530.
[47] Crook PR, GriersonD.Ultrasturctureoftomatofruitripeningandroleofpolygalacturonaseisoenzymes [J].Plant Physiol, 1983, 72:1 088～1093.
[48] Sitrit Y, Bent AB.Regulation of tomato fruit polyg-alacturoase mRNA accumulation by ethylenrare-ex-amination [J]. PlantPhysiol, 1998, 116:1145～1150.
[49]周玉蝉,唐友林,谭兴杰,等.栗后ABA、GA3对芒果细胞壁降解酶、类胡萝卜索含量的作用[J].植物生理学报,1996,22(4):421~426.
[50] Lieberman M, Baker J E, Sloger M.Influence of plant hormones on ethylene production in apple tomatoand avocado slices during maIuration and senescene [J].Plant Physiol, 1977, 60:214~217.
[51] Simons R,and Kleckner N,Translation control of IS10 transposition,Cell,1983,34,683～691.
[52] Izant J·G,and Weintraub H,Constitutive and conditionalsuppression of exogenous and endogenous gene by antisense RNA,Science,1985,229(4711),345～351.
[53] Izant J·G·,and Weintraub H,Inhibition of thymidine kinasegene expression by antisense RNA:a molecular approach to genetic analysis,Cell,1984,36,1007～1015.
[54] Sheehy RE,Kramer M,Hiatt WR.Reduction of polygalacturonase activity in tomato fruit byantisense RNA[J].Proc Natl Acad Sci USA,1988,85:8805～8809
[55] Smith CJS,Watson CF,Ray J,et al.Antisense RNA inhibition of polygalacturonase gene expression in transgenic tomatoes[J].Nature.1988,334:724～726
[56]鞠戎,田颖川,沈全光,等.番茄多聚半乳糖醛酸酶cDNA的克隆及其对番茄中PG表达的反义抑制[J].生物工程学报.1994.10(2):96～102
[57]陈正华主编.木本植物组织培养及其应用[M].北京:高等教育出版社,1986,290～314.
[58]孙俊,孙其宝,俞飞飞.桃快速繁殖技术体系的研究[J].安徽农业科学,2003,31(5):731～732.
[59] Chen ZhiLing,Zhang ChengHe,Lu ZengRen,et.al.Excised root culture of peach and apricot[J].Advances in Horticulture,1998,2:150～153.
[60]庄思及,姚强,吴钰良,等.早熟和特早熟桃胚珠培养研究[J].园艺学报,1991,18(4):303～308.
[61]孟新法,周维燕.桃胚乳离体培养再生植株的研究[J].北京农业大学学报,1981,7(4):95~98.
[62]刘用生,胡霓云,路广明.早熟桃胚珠离体培养研究[M].西北农业大学学报,1999,19(3):37~42.
[63] Abou E Nasr NM,Rabie KAE.Production and embryo culture of some new interspecific hybrids of almond and peach[J].Annals of Agricultural Science Cairo.1996,41(2):931～949.
[64] Declerck V, Korban SS. Influence of growth regulators and carbon sources on callus induction, growth and morphogenesis from leaf tissues of peach [Prunrs persica (L.) Batsch] [J].Journal of Horticultural Science, 1996, 71(1):49~55.
[65] Chen ZL, Zhang CH, Lu ZR, et al. Excised root culture of peach and apricot [J]. Advances in Horiculture, 1998, (2):150~153.
[66] Hammsehlag F.A,Bauehan G,Seorza R, Regeneration of Peach Plant from cellus drived from immature embryos[J]. Theoretieal and APPlied Genetie,1985,70:248~25.
[67]姚强,胡霓云,路广明.早熟桃幼胚愈伤组织再生植株技术研究[J].果树科学,1989,6(2):85~90.
[68] A.Gentile, S. Monticelli, C.Damiano.Adventitious shoot regeneration in peach [Prunus persica (L.) Batsch] [J].Plant Cell Rep, 2002, 20:1011～1016.
[69]闰国华,周宇.桃幼胚离体培养再生植株的研究[J].园艺学报,2002,29(5):480～482.
[70]孙仲序,刘静,王玉军,等.果树组培苗瓶外滤纸桥生根技术研究[J].园艺学报,2001,28(4): 345～347.
[71]孙其宝,俞飞飞,孙俊,等.安农水蜜的离体培养[J].安徽农业科学,2002,30(6):950~964.
[72]孙俊,孙其宝,俞飞飞,桃快速繁殖技术体系的研究[J].安徽农业科学,2003,31(5):731～732.
[73]李子银,胡会兴.农杆菌介导的植物遗传转化进展[J].生物工程进展,1998,l8(1):22～26.
[74] De Bfock M.Expression of foreign genes in regenerated plants and their progeny[J].EMBOJ.1984,3:1681~1684.
[75] Smigochi AC, Owins LD, Hammerschlag FA, In vitro transformation of peach, Prunus persica L.)Batsch.using A grobacterium tumefaciens[J].HortScience,1987,(22):745～749.
[76]周玉碧,李唯.晚熟桃微繁技术的研究[J].甘肃农业大学学报, 2005, 40(6):745～749.
[77]石丽娜.桃遗传转化再生体系的建立及E8启动子的克隆[D].甘肃农业大学硕士学位论文, 2007.
[78]陈子聋,曹孜义,田福平,等.扁桃砧木Nemaguard和Lovell的组培快繁[J].甘肃农业大学学报,2004,39(5):524~528.
[79]黄学林,李筱菊.高等植物组织离体培养得形态建成及其调控[M].北京:科学出版社,1995,101~146.
[80]余云舟,杜娟,王罡等.重组质粒导入根癌农杆菌冻融法的研究[J].吉林农业大学学报,2003,25(3): 257~259,262.
[81]单世华,李春娟.农杆菌质粒DNA提取方法的改良与鉴定[J].生物技术,2003,13(2):19~20.
[82]陈绍宁,崔继哲.快速提取番茄总DNA应用于规模化PCR检测[J].北方园艺,2004,(3):46～47.
[83]王关林,方宏筠.植物基因工程[M].北京:科学出版社,2002:345~346.
[84]彭细桥.番茄高效离体再生体系的建立与番茄C011同源基因的转化[D].湖南农业大学硕士学位论文,2005:33~34.
[85] Fray A. Earle ED examination of factors affecting the efficiency of Agrobacterium-mediated transformation of tomato [J]. Plant Cell Reports, 1996, 16:235~240.
[86]张赛群,叶志彪,吴昌银,等.异戊烯基转移酶基因导入番茄及转基因植株再生[J].园艺学报,1999,26(6):376~379.
[87]贾士荣,王志兴.农杆菌介导的植物遗传转化[A].莽克强主编.农业生物技术[C].北京:化学工业出版社,1998.85~115.
[88]那杰.植物基因转化受体系统的建立[A].王关林,方宏筠主编.植物基因工程(第二版[C].北京:科学出版社,2002.388~389.
[89]周春丽,郭卫东,路梅.农杆菌介导佛手遗传转化主要影响因素的研究[J].热带亚热带植物学报,2006,14(5):374~381.
[90]贾小明,樊军锋.影响农杆菌介导的河北杨遗传转化的因素[J].西北林学院学报,2006,21(5):102~105.
[91]程振东,卫志明等.根癌农杆菌对甘蓝型油菜的转化及转基因植株的再生[J].植物学报,1994,36(9):657~663.
[92]蔡小宁,余建明.建立青菜农杆菌介导法转化体系[J].江苏农业学报,1997,13(2):110~114.
[93]张建民.利用农杆菌介导将外源基因导入番茄的研究[J].东北农业大学学报,1999,27:42~44.
[94]莽克强.农业生物工程[M].化学工业出版社.2001, 91~106.
[95]张勇,周小云,何江,等.影响根癌农杆菌介导甜瓜转化NP-1基因的外部因子研究[J].生物技术,2004,14(4):9~11.
[96]黄永红.甜瓜ACC氧化酶反义基因植物表达载体的构建及其转化甜瓜品种GT-1的研究[D].甘肃农业大学硕士学位论文.2005.
[97] Godwin I. The effects of acetosyringone and pH on Agro bacterium-mediated transformation vary according to plant species [J]. Plant Mol Biol Rep, 1992, 10:12~36.
[2]张晓宇,王春生,赵桂芳,等.桃果实采后生理研究及贮藏保鲜技术应用进展[J].中国农学通报.2008,24 (5):117～120.
[3] A.Gentile,S. Monticelli,C.Damiano.Adventitious shoot regeneration in peach [Prunus persica (L) Batsch][J].Plant Cell Rep,2002,20:1011～1016.
[4] Hammerschlag F.A,Bauchan G,Scorza R.Regeneration of peach plants from callus drived from immature embryos[J].Theoretical and Apple Genetics,1985, (70): 248～251.
[5]张永庆,陈大明,金勇丰,等.桃离体组织分化再生植株的研究[J].园艺学报,2001,28(4):342～344.
[6]吴延军,张上隆,谢鸣,等.桃幼胚及幼胚子叶再生的研究[J].林业科学,2005, 41(5):45～51.
[7] Rosa M.Pérez-Clemente, Amparo Perez-Sanjuan, Lorenzo García-Ferriz,et.al.Transgenic peach plants (Prunus persica L.) produced by genetic transformation of embryo sections using the green fluorescent protein(GFP) as an in vivo marker[J]. Molecular Breeding,2004, 14: 419～427.
[8]吴延军,张上隆,谢鸣,等.桃ACO基因反义转化桃幼胚子叶的研究[J].遗传,2006,28(1):65～70.
[9]李曜东,魏玉凝.肥城桃组培苗诱导、基因转化及其增殖[J].果树学报,2003,20(1):70～72.
[10] Isabel M.G.Padilla, Agnieszka Golis,Adele Gentile, Carmine Damiano,Ralph Scorza. Evaluation of transformation in peach Prunus persica explants using green fluorescent protein (GFP) and beta-glucuronidase (GUS) reporter genes[J].Plant Cell,Tissue and Organ Culture 2006 (84): 309～314.
[11] Ye X, Brown SK.Genetic transformuation of peach tissues by particle bormbardment[J].J Amer Soc Hort Sci,1994,119(2):367～373.
[12] Scorza R, Morgens PH, Cordts JM, Mante Callahan AM.Agrobacterium-mediated transformation of peach (Prunus persica Batsch) leaf segments, immature embryos and long-term embryogenic callus[J]. In Vitro Cell Dev. Biol,1990,26: 829～834.
[13]吴延军,徐昌杰,张上隆.桃组织培养和遗传转化研究现状及展望[J].果树学报,2002,19(2):123～127.
[14] Byrne DH. Peach breeding trends a worldwide perspective[J].ActaHort, 2002,592:49~58.
[15] Smigicki.AC,Owins LD,Hammerschlag FA,In vitro transformation of peach,Prunus persica L.)Batsch.using A grobacterium tumefaciens[J].HortScience,1987,(22): 5131～5135.
[16] Hammerschlag F.A,Bauchan G,Scorza R.Regeneration of peach plants from callus drived from immature embryos[J].Theoretical and Apple Genetics,1985, (70): 248~251.
[17]吴延军,张上隆,徐昌杰,等.根癌农杆菌介导的桃幼胚转化实验参数研究[J].果树学报,2004,21(5):477~479.
[18] Hobson GE. The fiemness of tomato fruit in relation to polygalacturonase activity[J].Biochem,1963,86: 365~385.
[19]李正国.果实成熟的基因调控[J].生物工程进展,2000,20(3):30~34.
[20] Dawson D M, L D Melton, C B Watkins. Cell wallchanges in nectarine [J]. Plant Physiol, 1992, 100:1203~1210.
[21] Fry SC,Smith Rc,Renwick KF,et al.Xyloglucan endotransglycosylase, a new wall- loosening enzyme activity from plant[J].Biochem J,1992,282:821～828.
[22]陈昆松,李方,张上隆.猕猴桃果实成熟进程中木葡聚糖内糖基转移酶mRNA的变化[J].植物学报,1999,41(11):1231~1234.
[23] Huber D.J.The role of cell wall hydrolases in fruit softening [J].Horticultural Reviews, 1983, 5:169~219.
[34]吴敏,陈昆松,贾惠娟,等.桃果实采后软化过程中内源IAA,ABA和乙烯的变化[J].果树学报,2003,20(3):157~160.
[25]刘彩莉,霍君生.贮藏条件对桃果实的生理影响[J].河北农业大学学报,1992,15(1):88~91.
[26] Anderson P.E.Long-tern storage of peaches and nectarines intemittendy warned during controlled atmosphere storage.J.Am.Soc.Hort.Sci, 1982, 107(2):214-216.
[27] Anderson P.E.The influenceof storage temperature and warning during storage on peach and nectarine fruit quality .J.Am.Soc.Hoit.Sci. 1979, 104(4):459~461.
[28]吕昌文,齐灵,修德仁.桃波动温度贮藏及其机理研究[J].华北农学报,1994.9(1):75~80.
[29] Anderson R.E. Nectarines stored in a controoled atmosphere or air [J]. Am.Soc.Hort.Sci.1975, 100:151~153.
[30]石海燕,冯双庆.气调贮藏对紫花芒果PG、纤维素酶及果实硬度的影响[J].园艺学报,1997,24 (4):407-409.
[31] Wang CY. Physiological and Biochemical Effects of CA on Fruit and Vegelables.Calderorn M, Barkai G(Eds). Food Preservation by Modified Atmosphere[J]. Boca Raton FL: CRC Press. 1990, 101~129..
[32] Xue YB,Kubo Y,Inaba A,el al.Effect of humidify on ripening and texture in banana fruit.[J].Japan Soc Hort Sci, 1995,64(3):657-669.
[33] Pressey R.Exopolygalacturonase in tomato fruit[J].Phytochemistry,1987,26(7):1867~1870.
[34] PhaffH J, Demain AL.J.Biological Chemistry [J].1956, 218:875.
[35] Crooks P R, Grierson D.Ultrastructure oftomato fruit ripening and the role of polygalacturonase isoenzymes in cellwall degradation [J].PlantPhysiology, 1983, 72:248～254.
[36]薛炳烨,束怀瑞.应用基因工程改良果实成熟特性研究进展[J].山东农业科学,2002,4:49～52.
[37] Lee E,Speirs J,GrayJ,et al.Homologies to the tomato endopolygalacturonase gene in the peach genome[J].Plant Cell and Environment,1990,13:513～521.
[38] Fischer R L, Bennett A B.Role of cell wall hydrolases in fruitripening [J].Annu.Rev.Plant Physiol.Plant Mol.Biol., 1991, 42:675～703.
[39] Ali Z M, Brady C J.Purification and characterization of the polygalacturonases of tomato fruits [J].Australia J.Plant Physiology, 1982, 9:155～169.
[40] Tucker G A,Robertson NG,Grierson D.Changes in polygalacturonase isoenzymes during the ripening of normal and mutant tomato fruit[J].Eur.J.B chemistry,1980,112:119～124.
[41] Moshrefi M, Luth B S.Carbohydrate composition and electrophoretic properties of tomato polygalacturonase isoenzymes [J].Eur.J.Biochemistry, 1983, 135:511～514.
[42] Pressey R, Avants J K.Two forms of polygalacturonase in tomatoes [J].Biochemistry Biophysics Acta, 1973, 309:363～369.
[43] Dellapenna D,Danny C,etal.Molecular cloning oftomato fruit PG analysis of PG mRNA levets during ripening proc[J]. Natl Acad Sci USA, 1986, (83):6420～6424.
[44] Smith CJS,Watson CF,Ray J,et al.Antisense RNA inhibition of polygalacturonase gene expression in transgenic tomatoes[J].Nature,1988,334:724～726.
[45] Sheehy RE, Kramer M, and Hiatt WR.Reduction of polygalacturonase activity in tomato fruit byantisense RNA [J] .Proc Natl Acad Sci USA, 1988, 85:8805～8809.
[46] Cooley MB, Yodor J I.Insertional inactivation of the tomato polygalacturonasegene [J].Plant MolecularBiology, 1998, 38:521~530.
[47] Crook PR, GriersonD.Ultrasturctureoftomatofruitripeningandroleofpolygalacturonaseisoenzymes [J].Plant Physiol, 1983, 72:1 088～1093.
[48] Sitrit Y, Bent AB.Regulation of tomato fruit polyg-alacturoase mRNA accumulation by ethylenrare-ex-amination [J]. PlantPhysiol, 1998, 116:1145～1150.
[49]周玉蝉,唐友林,谭兴杰,等.栗后ABA、GA3对芒果细胞壁降解酶、类胡萝卜索含量的作用[J].植物生理学报,1996,22(4):421~426.
[50] Lieberman M, Baker J E, Sloger M.Influence of plant hormones on ethylene production in apple tomatoand avocado slices during maIuration and senescene [J].Plant Physiol, 1977, 60:214~217.
[51] Simons R,and Kleckner N,Translation control of IS10 transposition,Cell,1983,34,683～691.
[52] Izant J·G,and Weintraub H,Constitutive and conditionalsuppression of exogenous and endogenous gene by antisense RNA,Science,1985,229(4711),345～351.
[53] Izant J·G·,and Weintraub H,Inhibition of thymidine kinasegene expression by antisense RNA:a molecular approach to genetic analysis,Cell,1984,36,1007～1015.
[54] Sheehy RE,Kramer M,Hiatt WR.Reduction of polygalacturonase activity in tomato fruit byantisense RNA[J].Proc Natl Acad Sci USA,1988,85:8805～8809
[55] Smith CJS,Watson CF,Ray J,et al.Antisense RNA inhibition of polygalacturonase gene expression in transgenic tomatoes[J].Nature.1988,334:724～726
[56]鞠戎,田颖川,沈全光,等.番茄多聚半乳糖醛酸酶cDNA的克隆及其对番茄中PG表达的反义抑制[J].生物工程学报.1994.10(2):96～102
[57]陈正华主编.木本植物组织培养及其应用[M].北京:高等教育出版社,1986,290～314.
[58]孙俊,孙其宝,俞飞飞.桃快速繁殖技术体系的研究[J].安徽农业科学,2003,31(5):731～732.
[59] Chen ZhiLing,Zhang ChengHe,Lu ZengRen,et.al.Excised root culture of peach and apricot[J].Advances in Horticulture,1998,2:150～153.
[60]庄思及,姚强,吴钰良,等.早熟和特早熟桃胚珠培养研究[J].园艺学报,1991,18(4):303～308.
[61]孟新法,周维燕.桃胚乳离体培养再生植株的研究[J].北京农业大学学报,1981,7(4):95~98.
[62]刘用生,胡霓云,路广明.早熟桃胚珠离体培养研究[M].西北农业大学学报,1999,19(3):37~42.
[63] Abou E Nasr NM,Rabie KAE.Production and embryo culture of some new interspecific hybrids of almond and peach[J].Annals of Agricultural Science Cairo.1996,41(2):931～949.
[64] Declerck V, Korban SS. Influence of growth regulators and carbon sources on callus induction, growth and morphogenesis from leaf tissues of peach [Prunrs persica (L.) Batsch] [J].Journal of Horticultural Science, 1996, 71(1):49~55.
[65] Chen ZL, Zhang CH, Lu ZR, et al. Excised root culture of peach and apricot [J]. Advances in Horiculture, 1998, (2):150~153.
[66] Hammsehlag F.A,Bauehan G,Seorza R, Regeneration of Peach Plant from cellus drived from immature embryos[J]. Theoretieal and APPlied Genetie,1985,70:248~25.
[67]姚强,胡霓云,路广明.早熟桃幼胚愈伤组织再生植株技术研究[J].果树科学,1989,6(2):85~90.
[68] A.Gentile, S. Monticelli, C.Damiano.Adventitious shoot regeneration in peach [Prunus persica (L.) Batsch] [J].Plant Cell Rep, 2002, 20:1011～1016.
[69]闰国华,周宇.桃幼胚离体培养再生植株的研究[J].园艺学报,2002,29(5):480～482.
[70]孙仲序,刘静,王玉军,等.果树组培苗瓶外滤纸桥生根技术研究[J].园艺学报,2001,28(4): 345～347.
[71]孙其宝,俞飞飞,孙俊,等.安农水蜜的离体培养[J].安徽农业科学,2002,30(6):950~964.
[72]孙俊,孙其宝,俞飞飞,桃快速繁殖技术体系的研究[J].安徽农业科学,2003,31(5):731～732.
[73]李子银,胡会兴.农杆菌介导的植物遗传转化进展[J].生物工程进展,1998,l8(1):22～26.
[74] De Bfock M.Expression of foreign genes in regenerated plants and their progeny[J].EMBOJ.1984,3:1681~1684.
[75] Smigochi AC, Owins LD, Hammerschlag FA, In vitro transformation of peach, Prunus persica L.)Batsch.using A grobacterium tumefaciens[J].HortScience,1987,(22):745～749.
[76]周玉碧,李唯.晚熟桃微繁技术的研究[J].甘肃农业大学学报, 2005, 40(6):745～749.
[77]石丽娜.桃遗传转化再生体系的建立及E8启动子的克隆[D].甘肃农业大学硕士学位论文, 2007.
[78]陈子聋,曹孜义,田福平,等.扁桃砧木Nemaguard和Lovell的组培快繁[J].甘肃农业大学学报,2004,39(5):524~528.
[79]黄学林,李筱菊.高等植物组织离体培养得形态建成及其调控[M].北京:科学出版社,1995,101~146.
[80]余云舟,杜娟,王罡等.重组质粒导入根癌农杆菌冻融法的研究[J].吉林农业大学学报,2003,25(3): 257~259,262.
[81]单世华,李春娟.农杆菌质粒DNA提取方法的改良与鉴定[J].生物技术,2003,13(2):19~20.
[82]陈绍宁,崔继哲.快速提取番茄总DNA应用于规模化PCR检测[J].北方园艺,2004,(3):46～47.
[83]王关林,方宏筠.植物基因工程[M].北京:科学出版社,2002:345~346.
[84]彭细桥.番茄高效离体再生体系的建立与番茄C011同源基因的转化[D].湖南农业大学硕士学位论文,2005:33~34.
[85] Fray A. Earle ED examination of factors affecting the efficiency of Agrobacterium-mediated transformation of tomato [J]. Plant Cell Reports, 1996, 16:235~240.
[86]张赛群,叶志彪,吴昌银,等.异戊烯基转移酶基因导入番茄及转基因植株再生[J].园艺学报,1999,26(6):376~379.
[87]贾士荣,王志兴.农杆菌介导的植物遗传转化[A].莽克强主编.农业生物技术[C].北京:化学工业出版社,1998.85~115.
[88]那杰.植物基因转化受体系统的建立[A].王关林,方宏筠主编.植物基因工程(第二版[C].北京:科学出版社,2002.388~389.
[89]周春丽,郭卫东,路梅.农杆菌介导佛手遗传转化主要影响因素的研究[J].热带亚热带植物学报,2006,14(5):374~381.
[90]贾小明,樊军锋.影响农杆菌介导的河北杨遗传转化的因素[J].西北林学院学报,2006,21(5):102~105.
[91]程振东,卫志明等.根癌农杆菌对甘蓝型油菜的转化及转基因植株的再生[J].植物学报,1994,36(9):657~663.
[92]蔡小宁,余建明.建立青菜农杆菌介导法转化体系[J].江苏农业学报,1997,13(2):110~114.
[93]张建民.利用农杆菌介导将外源基因导入番茄的研究[J].东北农业大学学报,1999,27:42~44.
[94]莽克强.农业生物工程[M].化学工业出版社.2001, 91~106.
[95]张勇,周小云,何江,等.影响根癌农杆菌介导甜瓜转化NP-1基因的外部因子研究[J].生物技术,2004,14(4):9~11.
[96]黄永红.甜瓜ACC氧化酶反义基因植物表达载体的构建及其转化甜瓜品种GT-1的研究[D].甘肃农业大学硕士学位论文.2005.
[97] Godwin I. The effects of acetosyringone and pH on Agro bacterium-mediated transformation vary according to plant species [J]. Plant Mol Biol Rep, 1992, 10:12~36.