摘要
甘蔗花叶病是一种世界性的病害,目前病毒基因工程在甘蔗抗花叶病育种中得到广泛的应用。农业部甘蔗生理生态与遗传改良实验室于2005年利用基因枪技术成功的将SrMV-P1基因转入受体材料FN95-1702中,获得6个转基因无性系。本研究以上述转SrMV-P1基因为材料,对T3代转基因甘蔗的遗传稳定性、产质量性状进行评价,并研究在病毒胁迫下转基因甘蔗的活性氧代谢和酚类代谢的变化。结果表明:
SrMV-P1基因能够有效地提高甘蔗抗花叶病能力。对转基因甘蔗3个世代的田间发病率进行调查,转基因甘蔗的发病率都低于5%,转基因甘蔗田间发病率明显低于受体材料与空载P7,其中TF64表现高抗,三年间均没有发病。对T3代转SrMV-P1基因甘蔗的SrMV-P1基因进行PCR检测,在转基因甘蔗中仍然能检测到SrMV-P1基因,而FN95-1702未能检测到目的基因。说明SrMV-P1基因在转基因甘蔗中能够稳定遗传。
对T3代转SrMV-P1基因甘蔗产质量性状进行评价,结果表明:转基因甘蔗的生长速度明显比非转基因甘蔗快,转基因无性系TF37、TF50、TF53、TF63、TF64和TF67的株高显著高于转基因无性系TF50和对照受体材料与空载P7。除转基因无性系TF64外,其他转基因甘蔗的茎径均与受体材料和空载对照无显著差异。6个转基因无性系的产量均比P7高,且高出5.51%～43.30%,其中TF50、TF53、TF63、TF67的产量比FN95-1702高出了8.22%～21.38%。在工艺性状方面上,6个转基因株系的锤度、转光度、蔗汁糖分、视纯度、重力纯度、甘蔗纤维分分别比FN95-1702高出5.00%～24.36%、10.15%～36.52%、9.61%～37.07%、4.65%～9.73%、3.29%～10.25%、3.01%～21.54%和0.37%～26.26%。对转基因甘蔗T1~T3代的产质量性状进行模糊数学隶属函数综合评价可以看出TF53,TF64在三个世代中始终表现优异,FN95-1702和空载P7的表现较差。
对转SrMV-P1基因中蔗糖酸性转化酶在甘蔗工艺成熟期前活性较高,有助于甘蔗的营养生长,随着工艺成熟期的到来,蔗糖中性转化酶的活性逐渐上升,有助于甘蔗的糖分积累。FN95-1702与P7由于受到病毒的侵染,甘蔗糖代谢较为混乱。
通过对转SrMV-P1基因抗病的生理生化机制研究表明:在四次甘蔗花叶病毒接种过程中,转基因甘蔗TF53、TF64的H2O2、O2-均呈一定规律性变化,并在接种后期其含量处于一个较低的水平;受体材料FN95-1702和P7在整个接种过程中,H2O2、O2-的变化极不规则,并在接种后期其含量维持在一个较高水平。对比接种前后丙二醛含量,发现受体材料FN95-1702和P7在病毒接种后其丙二醛含量比接种前分别提高了58.59%和42.38%,而转基因无性系TF53和TF64的丙二醛含量并无明显提高。对保护酶的研究表明:转基因无性系TF53、TF64在接种病毒后,POD和SOD的活性维持在一个较高水平,有利于清除多余的活性氧,保护叶绿体细胞结构的完整。在酚类代谢研究中得出:接种病毒胁迫后,转基因无性系TF53和TF64的PAL、总酚、类黄酮最后都维持在一个较高的水平,这对甘蔗的抗病性具有积极地意义。
Sugarcane mosaic virus is one of the worldwide diseases in sugarcane production.Genetic engineering is used widely for the sugarcane breeding of resistance-disease.SrMV-P1 gene was transformed to sugarcane cv. FN95-1702 by particle bombardment in the Key Lab of Sugarcane Eco-Physiology& Genetic Improvement, Ministry of Agriculture in 2005, and six transgenic sugarcane clones were obtained. The genetic stability, yield and quality of characteristics of the above transgenic sugarcane were evaluated. Active oxygen metabolism and phenolic metabolism were analyzed in transgenic sugarcane inoculated with SCMV virus. The results were showed as followed:
Resistance to ScMV was improved effectively in the transgenic sugarcane mediated with SrMV-P1 gene. The SrMV incidence was investigated from T1 to T3 generation. The result was showed that ScMV incidences of all transgenic clones were 5% or less, significantly lower than the receptor cultivars (FN95-1702, CK1) and the transgenic clones without mediated with SrMV-P1 gene (P7, CK2). The trangenic sugarcane clone TF64 was not infected by SrMV virus. In T3 generation, the SrMV-P1 gene was detected in transgenic sugarcane, but not be detected in FN95-1702 and P7, showing that the SrMV-P1 gene could be genetic stability in transgenic sugarcane clones.
Yield and quality characteristics in transgenic sugarcane clones were evaluated in T3 generation. The results were showed thatgrowth rate of transgenic sugarcane clones was higher than the control FN95-1702 and P7. plant height of all the transgenic sugarcane except TF50, were higher than FN95-1702 and P7. Stalk diameter in the transgenic sugarcane clones had not significantly difference than those in FN95-1702 and P7. Yield of all the transgenic sugarcane increased by 5.51%～43.30% when compared with P7,and yield of TF50, TF53, TF63, TF67 increased by 8.22%～21.38% when compared with FN95-1702. According to the quality of characteristics in transgenic sugarcane clones, the results indicated that Brix of transgenic sugarcane increased by 5.00%～24.36%, pol by 10.15%～36.52%, sugar content in sugarcane juice by 9.61%～37.07%, apparent purity by 4.65%～9.73%, gravity purity by 3.29%～10.25%, sugarcane fiber by 0.37%～26.26%, Tonnage sugar content (TSC) by 0.37%～26.26%. The results from the fuzzy comprehensive analysis showed that transgenic sugarcane clones had great performance in T1-T3 generation, as compared with FN95-1702 And P7.
Acid invertase activity in transgenic sugarcane was higher before sugarcane technical maturing stage with comparison to non-transgenic sugarcane, which was helpful to nutritional growth. As technical maturing stage coming, neutral invertase activity in transgenic sugarcane increased, which was benefit to sucrose accumulation. The FN95-1702 and P7 were infected by SrMV, so the glucose metabolism was confused.
The studies on physiological and biochemical basis on virus resistance of transgenic sugarcane TF53, TF64 showed thatwhen sugarcane was inoculated with SrMV virus for four times,content of H2O2 and O2- in TF53 and TF64 were rhythmic change. The content of H2O2 and O2- had a low level at the end of inoculation. During the whole inoculation process, content of H2O2 and O2- in P7 and FN95-1702 were change irregularly, then at the end of inoculation process, the content of H2O2 and O2- had a high level. Comparison of MDA in transgenic sugarcane pre- and post inoculation, the result showed that after SrMV inoculation, the content of MDA in FN95-1702 and P7 increased by 58.59% and 42.38%. MDA content in transgenic sugarcane TF53 and TF64 had not significantly increased after inoculation. The research on protective enzyme system indicated thatthe activities of POD and SOD in transgenic sugarcane were maintained in high level, as compared to non-transgenic sugarcane P7 and FN95-1702, which was used to scavenger reactive oxygen species, to protect cell integrity. Vaiations of phenolic metabolism in transgenic sugarcane showed that:the content of PAL,total phenols,flavonoid in transgenic sugarcane TF53 and TF64 were maintained in high level after SrMV-virus inoculation, , as compared to non-transgenic sugarcane P7 and FN95-1702.
引文
[1]许东林,周国辉,沈万宽,等.侵染甘蔗的高梁花叶病毒遗传多样性分析[J].作物学报, 2008,34(11):1916-1920
[2]许莉萍,陈如凯,李跃平.利用愈伤组织培养和茎尖培养去除甘蔗花叶病毒[J].福建农业大学学报(自然科学版),1994,23(3):253～256
[3]周仲驹,林奇英,谢联辉.甘蔗病毒及类似病害的研究现状和进展[J].四川甘蔗,1988(2):28-30
[4]陈宇航,周仲驹,林奇.甘蔗花叶病毒株系的研究[J].福建农学院学报,1988,17(1):44-48
[5]蒋军喜,周雪平,洪健.杭州地区甘蔗花叶病病原鉴定.农业生物技术学报,2002,10(4377-380
[6]陈炯,杨建平,程晔.浙江甘蔗花叶病病原初步鉴定.植物病理学报,2001,31(3):271-273
[7]Shukla DD,Frenkel MJ,McKern NM,Ward CWJika J,Tosic M,Ford RE.Present status of the sugarcane mosaic subgroup of potyvFiruses. Arch Virol Suppl,1992,5:363-373
[8]Seifers D L,Salomon R,Marie J V,Alliot B,et al. Characterization of a novel potyvirus isolated from maize in Israel[J]. Phytopathology, 2000, 90:505-513.
[9]朱福成.玉米矮花叶病病株系和相关病毒侵染玉米的分离鉴定[J].甘肃农业科技,1986.(12):24-27.
[10]Powel-Abel. Delay of disease development in transgenic plants that express the tobacco mosaic virus coat protein gene. Science,1986,232:738-743
[11]Joyce P A, McQualter R B, Bernard M J, et al. Engineering for resistance to SCMV in sugarcane[J]. Acta Horticulture, 1998, 461:385-391
[12]Smith GR, Gambley RZ, Egan B T, et al. Progress in development of a sugarcane meristem transformation system and production of SCMV-resistant transgenics[J].Proceedings of the.Australian.Society of Sugar Cane Technologists.1994, 15: 237-243
[13]Ivan L. Ingelbrecht, James E. Irvine, et al.Posttranscriptional Gene Silencing in Transgenic Sugarcane. Dissection of Homology-Dependent Virus Resistance in a Monocot That Has a Complex Polyploid Genome [J]. Plant Physiol. 1999, 119: 1187-1198
[14]姚伟,余爱丽,徐景升,耿广良,张木清,陈如凯.转ScMV-CP基因甘蔗的分子生物学分析与鉴定[J].分子植物育种.2004,2(1):13-18
[15]阮妙鸿,转SCMV-CP基因甘蔗抗病的分子基础及环境安全性评价[D].福建:福建农林大学,2007.
[16]唐晓东转SCMV-CP基因抗花叶病甘蔗无性系的评价[D]福建:福建农林大学,2009
[17]郭莺我国四个蔗区甘蔗花叶病毒分子鉴定及其蛋白基因的分析[D].福建:福建农林大学,2008.
[18]郑碧霞转SrMV-P1基因甘蔗的抗病性分析及安全性评价[D]福建,福建农林大学2009
[19]Wilson TMA. Strategies to protect crop plants against viruses:Pathogen-derived resistance blossoms[J].Proc Natl Acad Sci USA,1993,903134-3141
[20]Neves-Borges AC,Collares WM,Pontes JA,etal. Coat protein RNA-mediated protection against Andean potato mottle virus in transgenic tobacco[J]. Plant Sci,2001,160:699-712
[21]Loesch-Fries LS, Merlo D, Zinnen T et al. Expresion of alfalfa mosaic virus RNA 4 in transgenic plants confers virus resistance[J]. EMBOJ, 1987,6:1845-1851.
[22]Hoekema A, Huisman MJ, Molendijk L et al. The Genetic engineering of two commercial potato cultivars for resistance to potato virus X [J],Bio/Technology, 1989,7:273-278.
[23]Hill KK, Jarvis-Eagan N, Haik EL et al. The development of virus-resistant alfalfa, Medicag sativa L [J]. Bio/Technology, 1991,9:373-377.
[24]Hayakawa T, Zhu Y, Itoh K et al. Genetically engineered rice resistant to rice stripe virus, in insect-transmitted virus [J]. Proc.Natl.Acad.Sci.USA, 1992,89:9865-9869.
[25]Kunik T, Salomon R, Zamin D et al. Transgenic tomato plants expressing the tomato yellow leaf curl virus capsid protein are resistant to the virus[J].Bio/Technology, 1994,12:500-504.
[26]Beachy RN In Plant Gene Research:Temporal and Spatial Regulation of Plant Genes(Eds.Verrna DPS,Goldberg RB),Springer-Verlag.New York, 1990,313-327.
[27]曹云侠,蒋世英RNA介导抗性研究进展[J]河南科技2006 12(6) 56-57
[28]高永珍,金奇RNA干扰抗病毒研究进展[J]病毒学报2005 21(4)314-317
[29] Lindbo J A,Douherty WG Mol Plant[J] Microbe Interact.1992,5(2):144-53
[30] Mourrain P,Beclin C,Elmayan T,et a1.Current Opinion in Genetics& Development,2000,10;638-643
[31] LOW P. S.MERIDA J. R. The oxidative burst in plant defense : Function and signal transduction [J]. Physiologia Plantarum 2006.96:533～542
[32]李合生.现代植物生理学[M]北京,高等教育出版社,1999:260-261
[33]田敏,饶龙兵,李纪元.植物细胞中的活性氧及其生理作用[J].植物生理学通讯,2005,41(2):235-241.
[34] Ingeborg Thoma, Christiane Loeffler, Alok K Sinha. Cyclopentenone isoprostanes induced by reactive oxygen species trigger defense gene activation and phytoalexin accumulation in plants[J].The Plant J, 2003 ,34(3) : 363-368.
[35] Peng, M., Kuc, J.: Peroxidase generated hydrogen peroxide as a source of antifungal activity in vitro and on tobacco leaf disks.[J]– Phytopathology 1992. 82: 696-699,.
[36]Baker C J,Orlandi EW.Active Oxygen in Plant Pathogenesis [J] .Annual Review of Phytopathology, 1995 ,33:299—321.
[37] WU,shortt.BJ,Lawrence EB et al .Disease resistance conferred by expressionof a gene encoding H2O2-generating glucose oxidase in transgenic potato plants[J] .Plant Cell ,1995 ,7(9) :1357 - 1368 .
[38]Svalheim, B Robertsen Induction of peroxidases in cucumber hypocotyls by wounding and fungal infection[J] Physiologia Plantarum, 2006,78(2) ,261-267
[39]DJ Bradley, P Kjellbom, CJ Lamb. Elicitor- and wound-induced oxidative cross-linking of a proline-rich plant cell wall protein: A novel, rapid defense response[J]. Cell.1992,70(1) 21-30
[40]R Tenhaken, A Levine, LF Brisson.Function of the oxidative burst in hypersensitive disease resistance lJ].Proc. Nat. Acad. Sci. USA 1995,92(1) 4158—4163.
[41]Baker CJ,Orlandi EW. Active Oxygen in Plant Pathogenesis,Annual Review of Phytopathology[J] 1995,33(2):299-321
[42] Levine A, Tenhaken R, Dixon R et al. H2O2 from the oxidative burst orchestrates the plant hypersensitive disease resistance response[J]. Cell, 1994,79: 583-593
[43] Chamnongpol S , Willekens H, Moeder W, et al. Defense activation andenh2an2cedpagyogen tolerance induced by H2O2 in transgenic plants[J]. Proceedings ofthe National Acadeny of Sciences , 1998 ,95 :5818-5823.
[44]何晨阳、王金生抗病植物的反应和机制[J]生物学通报1994 29(5)9-10
[45]欧阳光察、薛应龙棉花种子萌发过程中抗氰呼吸的变化及乙烯的诱导作用[J]植物生理通讯1986 3(9) 38-41
[46]郑世英植物抗病的物质基础[J]德州学院学报2004 17(4) 74-76
[47]VanceCP et al. Lignification as a mechanism of disease resistance[J] plant physiol 1980,18:171
[48]Hedrick S et al. Organization and differential activation of a gene family encoding the plantdefense enzyme chalcone synthase in Phaseolus vulgaris [J] plant physiol,1987,84:438
[49]李亚玲,龙书生,张宇宏玉米对镰刀菌茎腐病抗性的生化反应[J]中国农学通报2005 21(10) 299-302
[50] Zhou W, Eudes F, Laroche A, et al.Identification of differentially regulated proteins in response to a compatible interaction between the pathogen Fusarium graminearum and its host, Triticum aestivum. Proteomics. 2006 6(16):4599-609
[51]Tariq Mahmood, Asad Jan, Makoto Kakishima,et al.Proteomic analysis of bacterial-blight defense-responsive proteins in rice leaf blades[J]. Proteomics, 2006,6(22):6053-65.
[52] Smith JA, Blanchette RA, Burnes TA,et al.Proteomic comparison of needles from blister rust-resistant and susceptible Pinus strobus seedlings reveals upregulation of putative disease resistance proteins[J]. Mol Plant Microbe Interact. 2006 ,19(2):150-60.
[53] Rampitsch C, Bykova NV, McCallum B, et al.Analysis of the wheat and Puccinia triticina (leaf rust) proteomes during a susceptible host-pathogen interaction[J]. Proteomics. 2006,6(6):1897-907
[54] Kim ST, Kim SG, Hwang DH,et al.Proteomic analysis of pathogen-responsive proteins from rice leaves induced by rice blast fungus[J], Magnaporthe grisea. Proteomics. 2004,4(11):3569-78
[55]SHEN S,K0MATSU S.Characterization of proteins responsive to gibberelin in the leaf sheath of rice(Oryza sativa L.)seedling using proteome analysis[J].Biological and Pharmaceutical Bulletin,2003,26:129-136
[56]刘守伟.枯萎病菌对不同抗性黄瓜品种几种酶活性的影响[J].植物保护,2009,26:129-136
[57]姚伟,耿广良,余爱丽等.一种改良的转基因甘蔗基因组DNA进取方法[J].热带亚热带植物学报,2004,12(2):256-260
[58]Zhou Q(邹琦). Plant physiology Experiment Guide(植物生理学实验指导). Beijing: China Agriculture Press. 2000, 72-76(in Chinese)
[59]李杨瑞.不同基因型甘蔗的经济性状与伸长盛期叶片生化性状的多元回归分析[J].西南农业学报,1990,3(1):26-32
[60]李合生.植物生理生化实验原理和技术[M],北京,高等教育出版社,1999:260-261
[61]中国科学院上海植物生理研究所.现代植物生理学实验指南[M],上海,科学出版社.1999-12-1.
[62]李杨瑞.甘蔗组织中过氧化物酶活性及其与生长和工艺成熟的关系初探[J].广西农学院学报,1990,9(1):13-18.
[63]Giannoplitis CN,Sk Ries Superoxide dismutase 1.Purification and quantitative relationship with water-soluble protein in seeding[J]. Plant Physiol, 1977, 59: 315-318
[64]章骏德,刘国屏,等.编.植物生理实验法[M].南昌:江西人民出版社,1982,26-29
[65]沈文彪,徐朗莱,叶茂炳等.抗坏血酸过氧化物酶活性测定的探讨[J].植物生理学通讯,1996,32(3):203-205
[66]王敬文,薛应龙.植物苯丙氨酸解氨酶的研究II苯丙氨酸解氨酶在马铃薯晚疫病中的作用[J].植物生理学报,1982,8(1):35-41
[67] Bolwell GP, Butt VS, Davies DR, et al. The origin of the oxidative burst in plants[J]. Free Radical Res,1995,23:517-532
[68] Bradford,M.M. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding[J]. Anal.Biochem. 1976,72:248-254
[69]程亮.植物病毒基因介导的抗性研究进展[J].广西农业科学,2008,39(1):323-329
[70]吴祥华,胡宗利植物抗病毒基因工程研究进展[J].中国农学通报,2007,23(1):60-64
[71]金万梅,潘青华外源基因在转基因植物中的遗传稳定性及其转化研究[J] 2005,3(6)864-868
[72]潘有强甘蔗节间蔗糖含量与和蔗糖代谢相关的4种酶活性之间的关系剖析[J]植物生理通讯2007 43(5) 861-864
[73]董金皋,韩建民.植物与病原物互作中的活性氧代谢及其作用[J].沈阳农业大学学报, 2000,31(5):427-431.
[74] Potikha TS, Collins CC, Johnson DI et al. The Involvement of hydrogen peroxide in the differentiation of secondary walls in cotton fibers[J]. Plant Physiol, 1999,119: 849-858.
[75] Kiraly Z, El-Zahaby H. Golal A, Abdou S, Adam A et al. Effect of oxy free radicals on plant pathogenic bacteria and fungi and on some plant diseases[J]. See Ref, 1993, 77a, pp. 9-19.
[76] David H. Slaymaker , Duroy A. Navarre , Daniel Clark, et al. The tobacco salicylic acid-binding protein 3 (SABP3) is the chloroplast carbonic anhydrase, which exhibits antioxidant activity and plays a role in the hypersensitive defense response[J].pnas, 2002,99(18):11640-11645
[77]孙红炜,路兴波,杨崇良,等.不同抗性玉米品种接种甘蔗花叶病毒(SCMV)后4种防御酶活性变化研究[J].植物病理学报,200636(2):181-184
[78]杨辉,沈火林,朱鑫,等.防御酶活性、木质素和总酚含量与辣椒抗黄瓜花叶病毒的关系.中国农学通报, 2006,22(5):369-373
[79]朱宏波,腾冰,等.不同抗性大豆品种感染SMVl后若干生化变化[J].西北农业学报,2001,10(3):38-40.
[80]余清,刘勇,等.几种烟草抗病毒剂田间药效及对烟株抗病性的影响[J].云南农业大学学报,2001,16(1):9-12.
[81]滕冰,吴宗蹼,等.大豆感染SMV后种粒多酚类物质变化的研究[J].中国油料作物报,1999,21(3):41-43.
[82] Levine A, Tenhaken R, Dixon R et al. H2O2 from the oxidative burst orchestrates the plant hypersensitive disease resistance response[J]. Cell, 1994,79: 583-593
[83] Chamnongpol S , Willekens H, Moeder W, et al. Defense activation and enh2an2ced pagyogen tolerance induced by H2O2 in transgenic plants[J]. Proceedings ofthe National Acadeny of Sciences , 1998 ,95 :5818-5823.
[84]程艳丽,宋纯鹏.植物细胞H2O2的信号转导途径.中国科学C辑(生命科学)2005,35(6):480-489
[85]王爱国,邵从本,罗广华.1986. MDA作为植物过氧化指标的探讨。植物生理学通讯[J] 1986(2):55-57
[86]廖朝晖,朱必凤,刘安玲.水稻对白叶枯病菌抗性与活性氧关系的研究[J].中山大学学报(自然科学版),2002,41(4):78-81.
[87]吴晓丽,李建民,段留生,等.花椰菜幼苗叶片抗氧化酶系统与抗黑腐病关系的研究[J].植物病理学报,2005,35(6):509-513.
[88]宾金华,姜胜.茉莉酸甲酯诱导烟草幼苗抗炭疽病与PAL活性及细胞壁物质的关系[J].植物生理学报.2000,26(1):1-6.
[89]宋凤鸣,郑重.绿原酸和阿魏酸与棉花对枯萎病抗性的关系[J] .浙江农业大学学报.1996,22(3):236-240.
[90]范文艳,姜述军.植物抗病性及抗病的转导研究[J].中国农学通报.中国农学通报,2005,21(2)249-252
[91]罗广华,王爱国.几种外源因子对大豆幼苗SOD活性的影响[J].植物生理通讯,1990,9(1)239-244.
