转不同抗菌肽和几丁质酶基因烟草的抗病性研究
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摘要
真菌病害是导致作物减产的重要因素之一，利用广谱抗真菌新资源和新基因培育持久抗病新品种是农业食品安全的重要保证。建立在分子遗传学、分子生物学基础上的基因工程技术为之提供了一条可行的途径。利用转基因技术将抗病基因转入植物，来提高植物的抗病性是目前培育抗病品种的重要策略之一。
本实验以本明烟(Nicotiana benthamiana)的叶盘为受体，通过农杆菌介导法将不同来源的基因(SPCEMA：加有信号肽的CEMA基因，AFP：来源于苜蓿的防御素基因，CHI：来源于苦瓜的几丁质酶基因以及双价SPCEMA-CHI、AFP-CHI、AFP-SPCEMA基因)导入烟草，进行了抗病性检测，并比较了不同转基因植株的抗病效果，从而为它们的进一步利用提供依据。其主要结果如下：
1．抗病检测体系的建立
通过对培养疫霉菌的培养基配方和培养方法的比较，确定疫霉菌在芝麻培养基上生长较好，且获得了一种能产生大量致病力强的游动孢子囊的培养方法，使疫霉菌游动孢子囊的量从5×10~4个／ml提高到1.5×10~5个／ml。同时确定了疫霉菌和赤星病菌的接种浓度，疫霉菌浓度为1×10~4个／ml，赤星病菌浓度为5×10~5个／ml。
2．转目的基因烟草的获得
取烟草无菌苗叶片，切成0.5cm~2大小的叶盘，放入OD_(600)值为0.1～0.2的农杆菌菌液中，轻轻振荡侵染5min后，立刻倾去菌液，取出外植体转入表面铺有一层滤纸的共培养基MSB_1(MSB_0+2.0mg／L BA+0.1mg／L NAA)上，24℃，暗处共培养3d。共培养完成后，将外植体直接转入筛选培养基MSB_2(MSB_1+500mg／L Cef+100mg／L Km)中进行分化培养，每3周继代一次。待抗性芽长出，将其切下转入生根培养基MSB_3(MSB_0+200mg／L Cef+150mg／L Km)中筛选生根。
3．转基因植株分子生物学鉴定
首先对筛选到的抗性植株进行GUS组织化学检测，初步筛选确定已转化植株。对GUS
西南农业大学硕士学位论文摘要
阳性的植株做PCR扩增检测，结果显示每个GUS阳性株系均显示与阳性对照相同的特异带，
非转化植株未得到与阳性对照相同的特异带。初步证明6个基因已整合到烟草基因组中，获
得的GUS阳性植株是转基因烟草。
4.转基因植株抗病性检测
4.1转基因植株T。代疫霉菌接种实验
将T0代转基因烟草进行疫霉菌接种实验，结果表明:转基因烟草与对照相比抗性明显增
强，6个基因的病情指数从低到高依次为AFP-C川引.67%、APP一SPCEMA5833%、
SPCEMA一CH160.87%、AFP63.71%、SPCEMA63.89%、CHI70.54%，而对照的病情指数则为
100%。其中三个双价基因烟草的抗病性较好。
4.2转基因植株T:代赤星病菌接种实验
将T，代转基因烟草进行赤星病菌接种实验，结果表明:转基因烟草与对照相比抗性明显
增强，6个基因的病情指数从低到高依次为AFP一CH137.sl%、AFP一SPCEMA39.69%、
SPCEMA一CHI4O%、AFP51.25%、SPCEMA55.63%、CHI56.25%，而对照的病情指数则为100%.
说明这六个基因对真菌病都有一定的抗菌作用，并能在后代中稳定遗传与表达。我们又从每
个基因中随机选取了四个株系，接种烟草赤星病菌，比较各株系的抗病性。结果表明:不同
基因不同株系间抗病效果有差异，以AFP一CHI基因为最好，其各个株系的病情指数均较低。
5.转基因植株遗传稳定性鉴定
每个转基因烟草随机选取2个株系，萌发其T:代种子300粒左右，萌动后进行GUS染色，
x。2适合性测验结果表明其外源基因的传递符合孟德尔规律，呈3:1单基因显性的孟德尔式分
禺。
Fungal diseases are one of the major factors limiting crops production worldwide.The important guarantee of agriculture foodstuff safety is to breed new disease resistance variety by using new antifungal resource and genes.It is offered a feasible approach with gene engineering technique basing on molecular genetics and molecular biology.At present, one of the important strategies to breed disease resistance variety is transfered disease resistence genes into plant to enhance resistance.
In this paper, we selected the leaf dish of Nicotiana benthamiana as the explants and introduced six different genes (SPCEMA, AFP, CHI,SPCEMA-CHI, AFP-CH1, AFP-SPCEMA)into tobacco by Agrobacterium mediated transformation, we compared the resistant effect of different foreign genes in transgenic plants. The results are as following:
1. Development of examination method of disease resistance
From experiment,it was shown that sesame agar can be used to culture Phytophthora nicatianae which grew well on it.At the same time, a new culture method which can produce a lot of zoosporangia was obstained.The outputs of zoosporangia were increased from 5×104conidia/ml to 1.5×105conidia/ml.The inoculative concentration of Phytophthora nicatianae and Alternaria alternate were 1 ×104conidia/ml and 5×105 个/ml.respectively.
2. Acquirement of transgenic tobacco of different genes
Sterile tobacco leaves sliced into leaf dishes (0.5cm2/tablet) were immerged into the
Agrobacterium suspension of OD600 0.1-0.2 for 5min, and the leaf dishes were co-cultured on MSB1
(MSB0+ 2.0mg/L BA +0.1mg/L NAA) medium covered with a sterile filter paper disc for 3d in
darkness at 24℃. After co-culture was over, the exolants were directlv transferred to the selection
Medium MSB2 (MSB1+Cef500mg/L+Km100mg/L) for three weeks. The explants were selected twice. After green buds appeared, they were sliced and transferred to radication medium MSB3 (MSB|+ Cef500mg/L+Kml50mg/L). Finally, green seedlings were selected for further assays.
3. Molecular assays of transgenic plant
Explants from the Kanamycin-resistant regenerated plants were stained for assaying GUS activity using a protocol derived from Jefferson (1987). Expression of the GUS gene confirmed that the T-DNA had been transferred and integrated into the regenerated plants. At the same time, the introduction of the target genes were confirmed by PCR analysis of the DNA samples isolated from the GUS positive regenerated plants.
4. Resistance analysis
4.1 Inoculation with Phytophthora nicatianae of TO- transgenic plants
To-transgenic plants of different genes were inoculated with Phytophthora nicatianae in greenhouse. The results showed that the resistance of transgenic plants containing different g enes was strengthened distinctively, compared with control. The disease index of the six gen es were AFP-CHI41.67%, AFP-SPCEMA58.33%, SPCEMA-CHI60.87%, AFP63.71%,SPCEM A63.89%,CHI70.54%.
4.2 Inoculation with Alternaria alternata of T1-transgenic plants
T1-transgenic plants of different genes were inoculated with Alternaria alternat in green house.The results manifested that the resistance of transgenic plants containing different gene s was strengthened distinctively, compared with control.The disease index of the six genes w ere AFP-CHI37.81%, AFP-SPCEMA39.69%, SPCEMA-CHI40.00%, AFP51.25%,SPCEMA55.6 3%,CHI56.25%.From the above,we can see that all of the six genes had antifungal effect an d can be transmitted and expressed in offspring.
5. Assaying the stability of descendiblity in transgenic plants
we randomly selected two plants from transgenic plants of every gene. After germinated, they were stained for assaying GUS activity. The x c2 aptness workout indicated that the heritability of foreign gene accorded with the law of segregation.
本实验以本明烟(Nicotiana benthamiana)的叶盘为受体，通过农杆菌介导法将不同来源的基因(SPCEMA：加有信号肽的CEMA基因，AFP：来源于苜蓿的防御素基因，CHI：来源于苦瓜的几丁质酶基因以及双价SPCEMA-CHI、AFP-CHI、AFP-SPCEMA基因)导入烟草，进行了抗病性检测，并比较了不同转基因植株的抗病效果，从而为它们的进一步利用提供依据。其主要结果如下：
1．抗病检测体系的建立
通过对培养疫霉菌的培养基配方和培养方法的比较，确定疫霉菌在芝麻培养基上生长较好，且获得了一种能产生大量致病力强的游动孢子囊的培养方法，使疫霉菌游动孢子囊的量从5×10~4个／ml提高到1.5×10~5个／ml。同时确定了疫霉菌和赤星病菌的接种浓度，疫霉菌浓度为1×10~4个／ml，赤星病菌浓度为5×10~5个／ml。
2．转目的基因烟草的获得
取烟草无菌苗叶片，切成0.5cm~2大小的叶盘，放入OD_(600)值为0.1～0.2的农杆菌菌液中，轻轻振荡侵染5min后，立刻倾去菌液，取出外植体转入表面铺有一层滤纸的共培养基MSB_1(MSB_0+2.0mg／L BA+0.1mg／L NAA)上，24℃，暗处共培养3d。共培养完成后，将外植体直接转入筛选培养基MSB_2(MSB_1+500mg／L Cef+100mg／L Km)中进行分化培养，每3周继代一次。待抗性芽长出，将其切下转入生根培养基MSB_3(MSB_0+200mg／L Cef+150mg／L Km)中筛选生根。
3．转基因植株分子生物学鉴定
首先对筛选到的抗性植株进行GUS组织化学检测，初步筛选确定已转化植株。对GUS
西南农业大学硕士学位论文摘要
阳性的植株做PCR扩增检测，结果显示每个GUS阳性株系均显示与阳性对照相同的特异带，
非转化植株未得到与阳性对照相同的特异带。初步证明6个基因已整合到烟草基因组中，获
得的GUS阳性植株是转基因烟草。
4.转基因植株抗病性检测
4.1转基因植株T。代疫霉菌接种实验
将T0代转基因烟草进行疫霉菌接种实验，结果表明:转基因烟草与对照相比抗性明显增
强，6个基因的病情指数从低到高依次为AFP-C川引.67%、APP一SPCEMA5833%、
SPCEMA一CH160.87%、AFP63.71%、SPCEMA63.89%、CHI70.54%，而对照的病情指数则为
100%。其中三个双价基因烟草的抗病性较好。
4.2转基因植株T:代赤星病菌接种实验
将T，代转基因烟草进行赤星病菌接种实验，结果表明:转基因烟草与对照相比抗性明显
增强，6个基因的病情指数从低到高依次为AFP一CH137.sl%、AFP一SPCEMA39.69%、
SPCEMA一CHI4O%、AFP51.25%、SPCEMA55.63%、CHI56.25%，而对照的病情指数则为100%.
说明这六个基因对真菌病都有一定的抗菌作用，并能在后代中稳定遗传与表达。我们又从每
个基因中随机选取了四个株系，接种烟草赤星病菌，比较各株系的抗病性。结果表明:不同
基因不同株系间抗病效果有差异，以AFP一CHI基因为最好，其各个株系的病情指数均较低。
5.转基因植株遗传稳定性鉴定
每个转基因烟草随机选取2个株系，萌发其T:代种子300粒左右，萌动后进行GUS染色，
x。2适合性测验结果表明其外源基因的传递符合孟德尔规律，呈3:1单基因显性的孟德尔式分
禺。
Fungal diseases are one of the major factors limiting crops production worldwide.The important guarantee of agriculture foodstuff safety is to breed new disease resistance variety by using new antifungal resource and genes.It is offered a feasible approach with gene engineering technique basing on molecular genetics and molecular biology.At present, one of the important strategies to breed disease resistance variety is transfered disease resistence genes into plant to enhance resistance.
In this paper, we selected the leaf dish of Nicotiana benthamiana as the explants and introduced six different genes (SPCEMA, AFP, CHI,SPCEMA-CHI, AFP-CH1, AFP-SPCEMA)into tobacco by Agrobacterium mediated transformation, we compared the resistant effect of different foreign genes in transgenic plants. The results are as following:
1. Development of examination method of disease resistance
From experiment,it was shown that sesame agar can be used to culture Phytophthora nicatianae which grew well on it.At the same time, a new culture method which can produce a lot of zoosporangia was obstained.The outputs of zoosporangia were increased from 5×104conidia/ml to 1.5×105conidia/ml.The inoculative concentration of Phytophthora nicatianae and Alternaria alternate were 1 ×104conidia/ml and 5×105 个/ml.respectively.
2. Acquirement of transgenic tobacco of different genes
Sterile tobacco leaves sliced into leaf dishes (0.5cm2/tablet) were immerged into the
Agrobacterium suspension of OD600 0.1-0.2 for 5min, and the leaf dishes were co-cultured on MSB1
(MSB0+ 2.0mg/L BA +0.1mg/L NAA) medium covered with a sterile filter paper disc for 3d in
darkness at 24℃. After co-culture was over, the exolants were directlv transferred to the selection
Medium MSB2 (MSB1+Cef500mg/L+Km100mg/L) for three weeks. The explants were selected twice. After green buds appeared, they were sliced and transferred to radication medium MSB3 (MSB|+ Cef500mg/L+Kml50mg/L). Finally, green seedlings were selected for further assays.
3. Molecular assays of transgenic plant
Explants from the Kanamycin-resistant regenerated plants were stained for assaying GUS activity using a protocol derived from Jefferson (1987). Expression of the GUS gene confirmed that the T-DNA had been transferred and integrated into the regenerated plants. At the same time, the introduction of the target genes were confirmed by PCR analysis of the DNA samples isolated from the GUS positive regenerated plants.
4. Resistance analysis
4.1 Inoculation with Phytophthora nicatianae of TO- transgenic plants
To-transgenic plants of different genes were inoculated with Phytophthora nicatianae in greenhouse. The results showed that the resistance of transgenic plants containing different g enes was strengthened distinctively, compared with control. The disease index of the six gen es were AFP-CHI41.67%, AFP-SPCEMA58.33%, SPCEMA-CHI60.87%, AFP63.71%,SPCEM A63.89%,CHI70.54%.
4.2 Inoculation with Alternaria alternata of T1-transgenic plants
T1-transgenic plants of different genes were inoculated with Alternaria alternat in green house.The results manifested that the resistance of transgenic plants containing different gene s was strengthened distinctively, compared with control.The disease index of the six genes w ere AFP-CHI37.81%, AFP-SPCEMA39.69%, SPCEMA-CHI40.00%, AFP51.25%,SPCEMA55.6 3%,CHI56.25%.From the above,we can see that all of the six genes had antifungal effect an d can be transmitted and expressed in offspring.
5. Assaying the stability of descendiblity in transgenic plants
we randomly selected two plants from transgenic plants of every gene. After germinated, they were stained for assaying GUS activity. The x c2 aptness workout indicated that the heritability of foreign gene accorded with the law of segregation.
引文
1．陈瑞泰，朱贤朝，王智发，等．全国16个主产烟省(区)烟草侵染性病害调研报告[J]．中国烟草科学，1997，1(4)：1～7
2．邓晓梅，周根余．GUS基因导入烟草获得转基因植株及其组织特异性表达．上海农业学报．1996，12(4)：85～87
3．董汉松，王智发．烟草赤星病菌致病力测定方法的研究．山东农业大学学报，1989，(4)：1～8
4．范濂．农业试验统计方法．河南科学技术出版社，1983，第一版
5．方玉达，刘大钧．转水稻几丁质酶基因烟草植株及其对烟草赤星病(Alternaria alternata)的抗性．南京农业大学学报，2000，23(1)：5～9
6．傅荣昭．刘明，梁红健，等．通过根癌农杆菌介导法获得菊花转基因植株．植物学报，1998．24(1)：72～76
7．龚龙英，郑小波，等．烟草疫霉对烟草的致病性及生理分化研究．南京农业大学学报，1993，16(4)：68～72
8．顾红雅，瞿礼嘉，明小天，等．植物基因与分子操作．北京大学出版社 1995，第一版
9．郝转芳，李润植．抗真菌蛋白基因的应用．世界农业，2001(8)：38～40
10．胡苹，安成才等．原核表达的天花粉蛋白和另外两种蛋白具有体外抗真菌活性．微生物学报，1999，39(3)：234～239
11．贾士荣．植物遗传转化概论植物遗传转化技术手册．中国科学出版社1994，第一版
12．贾士荣，王志兴．农杆菌介导的植物遗传转化．农业生物工程 莽克强主编，化学工业出版社 1998，第一版
13．孔凡玉，朱贤朝，石金开等．我国烟草侵染性病害发生趋势及防治对策[J]．中国烟草，1995(1)：31～34
14．蓝海燕，陈正华．植物抗真菌基因工程研究进展．生物技术，8(5)：6～9，1998
15．蓝海燕，田颖川，王长海，等．表达β-1，3-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，2000，27(1)：70～77
16．李乃坚，袁四清，黄自然，等．抗菌肽B基因转化烟草及转基因植株抗青枯病的研究．农业生物技术学报，1998，6(2)：178～184
17．刘国胜，刘玉乐，等．病原细菌无毒基因avrD介导的抗赤星病转基因烟草．植物病理学报，1996，26(2)：165～170
18．吕晓波．转基因技术在抗植物真菌病害方面的研究策略．黑龙江农业科学，2001(3)：28～30
19．马国胜，高智谋，陈娟．烟草黑胫病研究进展．烟草科技，2001(9)：44～47
20．黎定军，赵开军，周清明．转几丁质酶基因和β-1，3-葡聚糖酶基因烟草的研究．湖南农业大学学报，2002，28(3)：211～213
21．石强，刘飞鹏．抗菌肽克隆基因的表达和转基因研究现状．生物工程进展，2000，20(1)：37～40
22．宋金耀，李松波，等．毛白杨嵌合体的研究：1．嵌合体的认证及生物学研究．河北农业技术师范学院学报，1998，12(3)：5～9
23．王关林，方宏筠．植物基因工程原理与技术科学出版社 1998，第一版
24．王晖，孙超，彭学贤．表达多肽抗生素apidaecin的转基因烟草及其抗病性的初步分析．生物工程学报，2001，17(4)：423～427
25．王卫东．抗菌肽及其应用前景．生物学教学，2002，27(6)：41
26．王智发．我国烟草黑胫病菌生理小种鉴定．山东农业大学学报，1987，18(1)：1～8
27．王革，郑小波，等．烟草黑胫病菌厚垣孢子和菌丝体在土壤中的存活状态．南京农业大学学报，1998，21(1)：41～45
28．吴中心，张同庆，等．外源DNA导入转移烟草抗赤星病性状的研究．河南农业大学学报，1995，29(1)：71～75
29．吴中心，姚根怀，等．烟草赤星病抗性鉴定方法的研究．河南农业科学，1994，(6)：15～17
30．谢成颂，王智发．烟草黑胫病菌(Phytophthora parasitica var．nicotianae)生理小种鉴定技术研究．山东农业大学学报，1989(1)：20～25
31．杨博，王永华．抗菌肽研究进展．生命科学，2002，14(3)：144～145
32．杨建卿．江彤．烟草疫霉菌的培养及大量产生游动孢子囊和游动孢子方法的研究．植物保护，2001，27(4)：12～14
33．杨玉范，华致甫，马贵龙．烟草赤星病菌的生长和孢子产生条件及寄主范围的研究．吉林农业科学．1994，(4)：55～59
34．易自力，周朴华，朱雅安．植物抗真菌病害基因工程研究进展．作物研究，2001(3)：81～84
35．张满朝，郑国铝．人防御素-1转基因烟草的获得及其TMV的初步观察．植物学报，1999，39(6)：574～576
36．赵世民，祖国诚，刘根齐等．通过农杆菌介导法将兔防御NP-1导入毛白杨(Ptomeatosa)．遗传学报，1999，26(6)：711～714
37．周霞，诸葛洪祥．抗菌肽的分子生物学研究进展．国外医学·流行病学传染病学分册，，2002，29(5)：310～313
38. Alexander D, R M Goodman, et al. Increased tolerance to two oomycete pathogens in transgenic tobacco expression pathogenesis-related protein. Proc.Nat.Acad.Sci.USA 1993, 90:7327～7331
39. Ben J C Comelissen, Leo S Melchers. Strategies for Control of Fungal Diseases with Transgenic Plants. Plant Physiol, 1993, 101:709～712
40. Broglie K, Chet I, Holliday M, et al. Transgenic Plants with Enhanced Resistance to the Fungal Pathogen Rhizoctonia solani. Science, 1991,254:1194～1197
41. Broglie K, Gaynor J, Broglie R, et al. Ethylene-Regulated Gene Expression: Molecular Cloning of the Genes Encoding an Endochitinase from Phaseolus volgaris. Proceeding of the National Academy of Sciences, 1986, 83:6820～6824
42. Bruce L Kagan, Michael E Selsted, et al. Antimicrobial defensin peptides from voltage- dependent ion-permeable channels in planar lipid bilayer membranes. Proc.Natl.Acad.Sci.USA, 1990, 87:210～214
43. Bruno P A CAMMUE, Miguel F C DE BOLLE, et al. Antimicrobial Peptides from M. jalapa L.Seeds.
44. De Wit PJGM. Molecular characterization of gene-for-gene systems in plant-fungus interactions and the application of avirulence genes in control of plant pathogens. Annu Rev Phytopathol, 1992, 30:391～399
45. Duncan Robertson, Geoffrey EMitchell, et al. Differential Extraction and Protein Sequencing Reveals Major Defferences in Patterns of Primary Cell Wall Proteins from Plants. The Journal of Biological Chemistry, 1997, 272(25): 15841～15848
46. Epple P, Apel K, Bohlmann H. Overexpression of an endogenous thionin gives enhanced resistance of Arabidopsis against Fusarium oxysporum. Plant Cell, 1991, 9:509～520
47. Flor H H. Current status of the gene-for-gene concept. Ann Rex Phytopathol, 1971, 9:275～296
48. Gao AG, Hakimi SM, et al. Fungal pathogen protection in potato by expression of a plant defensin peptide. Nature Biotechnology, 2000, 16:1307～1310
49. Gooding G V, Lucas G B. Effect of inoculum level on the severity of tobacco black shank. Phytopathology, 1959, 49:274～276
50. Hain R, Reif H J, et al. Disease resistance results from foreign phytoalexin expression in a novel plant. Nature, 1993, 361:153～156
51. Hain R, Stabel P, et al. Uptake, integration, expression and genetic transmission of a
selectable chimaeric gene by plant protoplasts. Mol Gen Genet, 1985, 199:161～168
52. Huang YRO, Nordeen RO,Di M, et al. Expression of an engineered cecropin gene cassette confer desease resistance to Pseudomonas syringae pv. Tabacci. Phytopathology, 1997, 87:494～495
53. Isabelle E J A Franoois, Miguel F C De Bolle, et al. Transgenic Expression in Arabidopsis of a Polyprotein Construct Leading to Production of Two Different Antimicrobial Proteins. Plant Physiology, 2002, 128:1346～1358
54. Jach G, Gǒrnhardt B. et al. Enhanced quantitative resistance against fungal disease by combinatorial expression of different barley antifungal proteins in transgenic tobacco. The plantJ, 1995, 8(1): 97～109
55. Jefferson R A. Assaying chimeric genes in plants: The GUS gene fusion system. Plant Mol Biol Rep, 1987, 5:387～392
56. Jefferson R A, Kavanagh T A, Bevan M. GUS fusions: β-glucuronidase as a senstitive and versatile gene fusion marker in higher plants. EMBO J, 1987, 6:3901～3907
57. Jefferson R A. The GUS reporter gene system. Nature, 1989, 342:837～838
58. Lee D G, Park Y, et al. Antifungal Mechanism of an Antiicrobial Peptide, HP(2-20), Derived from N-Terminus of Helicobacter pylori Ribosomal Protein L1 against Candida albicans. Biochemical and Biophysical Research Communications, 2002, 291:1006～1013
59. Lee D G, Shin S Y. et al. Antifungal mechanism of a cysteine-rich antimicrobial peptide, Ib-AMP1, from Impatiens balsamina against Candida albicans. Biotechnology Letters, 1999, 21:1047～1050
60. Lockey TD, et al. Formation of pores in Escherichia coil cell membranes by a cecropin isolated from hemolymph of Heliothis virescens larvae. Eur J Biochem, 1996, 236: 263～271
61. Logemann J, Jach G, Tommerup H, et al. Expression of a barley ribosome-inactivating protein leads to increased fungal protection in transgenic tobacco plants. Bio/Technology, 1992, 10:305～308
62. Marianne B Sela-Buurlage, Anne S Ponstein, et al. Only Specific Tobaeco(Nicotiana tabacum) Chitinases and β-1,3-Glucanases Exhibit Antifungal Activity. Plant Physiol, 1993, 101:857～863
63. Matteo Lortto, Sheridan L Woo, et al. Genes from mycoparasltic fungl as a source for improving plant resistance to fungal pathogens. Proc.Natl.Acad.Sci.USA, 1998, 95: 7860～7865
64. Neuhaus J-M, AhI-Goy P, Hinz U, et al. High-level expression of a tobacco chitinase
gene in Nicotiana sylcestris. Susceptibility of transgenic plants to Cercospora nicotianae infection. Plant Mol Biol,1991, 16:141～151
65. Osusky M., Zhou GQ et al. Transgenic plants expression cationic peptide chimeras exhibit broad-spectrum resistance to phytopathogens. Nature Biotechnology, 2000, 18:1162～1166
66. Qiu X, WU Y, Jaynes J, et al. Effect of a membrane interactive peptide on plant cells of canola (Brassica napus) and two fungal pathogens. Plant Cell Rep, 1995, 15(1/2): 115～118
67. Vacek M, et al. In Cell Culture and Somatic Cell Genetics of Plants. Academic Press Troy MI, 1987, 6:420～440
68. Wade D, Boman J. All D-amino acid containing channel-forming antibiotic peptides. PNAS, 1990, 87(12): 4761～4765
69. Woloshuk C P, et al. Pathogen-induced proteins with inhibitory activity toward Phytophthora infestans. Plant cell, 1991, 3(6): 619～628
70. Yoshikawa M, Tsuda M. Expression of soybean β-1,3-gluconase cDNA in transgenic tobacco plants. Molecular Strategies of pathogens and Host Plants. Springer-Verlag, 1992, 247
71. Zambryski P C, et al. Plant Mol Biol, 1992, 43:465
2．邓晓梅，周根余．GUS基因导入烟草获得转基因植株及其组织特异性表达．上海农业学报．1996，12(4)：85～87
3．董汉松，王智发．烟草赤星病菌致病力测定方法的研究．山东农业大学学报，1989，(4)：1～8
4．范濂．农业试验统计方法．河南科学技术出版社，1983，第一版
5．方玉达，刘大钧．转水稻几丁质酶基因烟草植株及其对烟草赤星病(Alternaria alternata)的抗性．南京农业大学学报，2000，23(1)：5～9
6．傅荣昭．刘明，梁红健，等．通过根癌农杆菌介导法获得菊花转基因植株．植物学报，1998．24(1)：72～76
7．龚龙英，郑小波，等．烟草疫霉对烟草的致病性及生理分化研究．南京农业大学学报，1993，16(4)：68～72
8．顾红雅，瞿礼嘉，明小天，等．植物基因与分子操作．北京大学出版社 1995，第一版
9．郝转芳，李润植．抗真菌蛋白基因的应用．世界农业，2001(8)：38～40
10．胡苹，安成才等．原核表达的天花粉蛋白和另外两种蛋白具有体外抗真菌活性．微生物学报，1999，39(3)：234～239
11．贾士荣．植物遗传转化概论植物遗传转化技术手册．中国科学出版社1994，第一版
12．贾士荣，王志兴．农杆菌介导的植物遗传转化．农业生物工程 莽克强主编，化学工业出版社 1998，第一版
13．孔凡玉，朱贤朝，石金开等．我国烟草侵染性病害发生趋势及防治对策[J]．中国烟草，1995(1)：31～34
14．蓝海燕，陈正华．植物抗真菌基因工程研究进展．生物技术，8(5)：6～9，1998
15．蓝海燕，田颖川，王长海，等．表达β-1，3-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，2000，27(1)：70～77
16．李乃坚，袁四清，黄自然，等．抗菌肽B基因转化烟草及转基因植株抗青枯病的研究．农业生物技术学报，1998，6(2)：178～184
17．刘国胜，刘玉乐，等．病原细菌无毒基因avrD介导的抗赤星病转基因烟草．植物病理学报，1996，26(2)：165～170
18．吕晓波．转基因技术在抗植物真菌病害方面的研究策略．黑龙江农业科学，2001(3)：28～30
19．马国胜，高智谋，陈娟．烟草黑胫病研究进展．烟草科技，2001(9)：44～47
20．黎定军，赵开军，周清明．转几丁质酶基因和β-1，3-葡聚糖酶基因烟草的研究．湖南农业大学学报，2002，28(3)：211～213
21．石强，刘飞鹏．抗菌肽克隆基因的表达和转基因研究现状．生物工程进展，2000，20(1)：37～40
22．宋金耀，李松波，等．毛白杨嵌合体的研究：1．嵌合体的认证及生物学研究．河北农业技术师范学院学报，1998，12(3)：5～9
23．王关林，方宏筠．植物基因工程原理与技术科学出版社 1998，第一版
24．王晖，孙超，彭学贤．表达多肽抗生素apidaecin的转基因烟草及其抗病性的初步分析．生物工程学报，2001，17(4)：423～427
25．王卫东．抗菌肽及其应用前景．生物学教学，2002，27(6)：41
26．王智发．我国烟草黑胫病菌生理小种鉴定．山东农业大学学报，1987，18(1)：1～8
27．王革，郑小波，等．烟草黑胫病菌厚垣孢子和菌丝体在土壤中的存活状态．南京农业大学学报，1998，21(1)：41～45
28．吴中心，张同庆，等．外源DNA导入转移烟草抗赤星病性状的研究．河南农业大学学报，1995，29(1)：71～75
29．吴中心，姚根怀，等．烟草赤星病抗性鉴定方法的研究．河南农业科学，1994，(6)：15～17
30．谢成颂，王智发．烟草黑胫病菌(Phytophthora parasitica var．nicotianae)生理小种鉴定技术研究．山东农业大学学报，1989(1)：20～25
31．杨博，王永华．抗菌肽研究进展．生命科学，2002，14(3)：144～145
32．杨建卿．江彤．烟草疫霉菌的培养及大量产生游动孢子囊和游动孢子方法的研究．植物保护，2001，27(4)：12～14
33．杨玉范，华致甫，马贵龙．烟草赤星病菌的生长和孢子产生条件及寄主范围的研究．吉林农业科学．1994，(4)：55～59
34．易自力，周朴华，朱雅安．植物抗真菌病害基因工程研究进展．作物研究，2001(3)：81～84
35．张满朝，郑国铝．人防御素-1转基因烟草的获得及其TMV的初步观察．植物学报，1999，39(6)：574～576
36．赵世民，祖国诚，刘根齐等．通过农杆菌介导法将兔防御NP-1导入毛白杨(Ptomeatosa)．遗传学报，1999，26(6)：711～714
37．周霞，诸葛洪祥．抗菌肽的分子生物学研究进展．国外医学·流行病学传染病学分册，，2002，29(5)：310～313
38. Alexander D, R M Goodman, et al. Increased tolerance to two oomycete pathogens in transgenic tobacco expression pathogenesis-related protein. Proc.Nat.Acad.Sci.USA 1993, 90:7327～7331
39. Ben J C Comelissen, Leo S Melchers. Strategies for Control of Fungal Diseases with Transgenic Plants. Plant Physiol, 1993, 101:709～712
40. Broglie K, Chet I, Holliday M, et al. Transgenic Plants with Enhanced Resistance to the Fungal Pathogen Rhizoctonia solani. Science, 1991,254:1194～1197
41. Broglie K, Gaynor J, Broglie R, et al. Ethylene-Regulated Gene Expression: Molecular Cloning of the Genes Encoding an Endochitinase from Phaseolus volgaris. Proceeding of the National Academy of Sciences, 1986, 83:6820～6824
42. Bruce L Kagan, Michael E Selsted, et al. Antimicrobial defensin peptides from voltage- dependent ion-permeable channels in planar lipid bilayer membranes. Proc.Natl.Acad.Sci.USA, 1990, 87:210～214
43. Bruno P A CAMMUE, Miguel F C DE BOLLE, et al. Antimicrobial Peptides from M. jalapa L.Seeds.
44. De Wit PJGM. Molecular characterization of gene-for-gene systems in plant-fungus interactions and the application of avirulence genes in control of plant pathogens. Annu Rev Phytopathol, 1992, 30:391～399
45. Duncan Robertson, Geoffrey EMitchell, et al. Differential Extraction and Protein Sequencing Reveals Major Defferences in Patterns of Primary Cell Wall Proteins from Plants. The Journal of Biological Chemistry, 1997, 272(25): 15841～15848
46. Epple P, Apel K, Bohlmann H. Overexpression of an endogenous thionin gives enhanced resistance of Arabidopsis against Fusarium oxysporum. Plant Cell, 1991, 9:509～520
47. Flor H H. Current status of the gene-for-gene concept. Ann Rex Phytopathol, 1971, 9:275～296
48. Gao AG, Hakimi SM, et al. Fungal pathogen protection in potato by expression of a plant defensin peptide. Nature Biotechnology, 2000, 16:1307～1310
49. Gooding G V, Lucas G B. Effect of inoculum level on the severity of tobacco black shank. Phytopathology, 1959, 49:274～276
50. Hain R, Reif H J, et al. Disease resistance results from foreign phytoalexin expression in a novel plant. Nature, 1993, 361:153～156
51. Hain R, Stabel P, et al. Uptake, integration, expression and genetic transmission of a
selectable chimaeric gene by plant protoplasts. Mol Gen Genet, 1985, 199:161～168
52. Huang YRO, Nordeen RO,Di M, et al. Expression of an engineered cecropin gene cassette confer desease resistance to Pseudomonas syringae pv. Tabacci. Phytopathology, 1997, 87:494～495
53. Isabelle E J A Franoois, Miguel F C De Bolle, et al. Transgenic Expression in Arabidopsis of a Polyprotein Construct Leading to Production of Two Different Antimicrobial Proteins. Plant Physiology, 2002, 128:1346～1358
54. Jach G, Gǒrnhardt B. et al. Enhanced quantitative resistance against fungal disease by combinatorial expression of different barley antifungal proteins in transgenic tobacco. The plantJ, 1995, 8(1): 97～109
55. Jefferson R A. Assaying chimeric genes in plants: The GUS gene fusion system. Plant Mol Biol Rep, 1987, 5:387～392
56. Jefferson R A, Kavanagh T A, Bevan M. GUS fusions: β-glucuronidase as a senstitive and versatile gene fusion marker in higher plants. EMBO J, 1987, 6:3901～3907
57. Jefferson R A. The GUS reporter gene system. Nature, 1989, 342:837～838
58. Lee D G, Park Y, et al. Antifungal Mechanism of an Antiicrobial Peptide, HP(2-20), Derived from N-Terminus of Helicobacter pylori Ribosomal Protein L1 against Candida albicans. Biochemical and Biophysical Research Communications, 2002, 291:1006～1013
59. Lee D G, Shin S Y. et al. Antifungal mechanism of a cysteine-rich antimicrobial peptide, Ib-AMP1, from Impatiens balsamina against Candida albicans. Biotechnology Letters, 1999, 21:1047～1050
60. Lockey TD, et al. Formation of pores in Escherichia coil cell membranes by a cecropin isolated from hemolymph of Heliothis virescens larvae. Eur J Biochem, 1996, 236: 263～271
61. Logemann J, Jach G, Tommerup H, et al. Expression of a barley ribosome-inactivating protein leads to increased fungal protection in transgenic tobacco plants. Bio/Technology, 1992, 10:305～308
62. Marianne B Sela-Buurlage, Anne S Ponstein, et al. Only Specific Tobaeco(Nicotiana tabacum) Chitinases and β-1,3-Glucanases Exhibit Antifungal Activity. Plant Physiol, 1993, 101:857～863
63. Matteo Lortto, Sheridan L Woo, et al. Genes from mycoparasltic fungl as a source for improving plant resistance to fungal pathogens. Proc.Natl.Acad.Sci.USA, 1998, 95: 7860～7865
64. Neuhaus J-M, AhI-Goy P, Hinz U, et al. High-level expression of a tobacco chitinase
gene in Nicotiana sylcestris. Susceptibility of transgenic plants to Cercospora nicotianae infection. Plant Mol Biol,1991, 16:141～151
65. Osusky M., Zhou GQ et al. Transgenic plants expression cationic peptide chimeras exhibit broad-spectrum resistance to phytopathogens. Nature Biotechnology, 2000, 18:1162～1166
66. Qiu X, WU Y, Jaynes J, et al. Effect of a membrane interactive peptide on plant cells of canola (Brassica napus) and two fungal pathogens. Plant Cell Rep, 1995, 15(1/2): 115～118
67. Vacek M, et al. In Cell Culture and Somatic Cell Genetics of Plants. Academic Press Troy MI, 1987, 6:420～440
68. Wade D, Boman J. All D-amino acid containing channel-forming antibiotic peptides. PNAS, 1990, 87(12): 4761～4765
69. Woloshuk C P, et al. Pathogen-induced proteins with inhibitory activity toward Phytophthora infestans. Plant cell, 1991, 3(6): 619～628
70. Yoshikawa M, Tsuda M. Expression of soybean β-1,3-gluconase cDNA in transgenic tobacco plants. Molecular Strategies of pathogens and Host Plants. Springer-Verlag, 1992, 247
71. Zambryski P C, et al. Plant Mol Biol, 1992, 43:465