几丁质酶产生菌的筛选及初步应用的研究
|
摘要
【研究背景和意义】水稻是世界上最重要的农作物之一，养育着世界近50％的人口。中国是世界最大的水稻生产和消费国，全国60％以上的人口以大米为主食，每年的总产量达2000亿公斤左右。虽然我国水稻生产已取得举世瞩目的成就，但也面临着新的挑战，粮食生产成本高、效益低、国际竞争力弱的情况依然存在。在生态环境中，影响水稻生长的因子多种多样，并且处于动态的变化中，这些环境因子一旦超出水稻忍耐的范围就成为了逆境胁迫因子，对水稻产生危害，导致了巨大的产量和经济损失。水稻病害是其主要的逆境胁迫因子之一，也是影响水稻产量和质量的重要因素之一。特别是真菌病害，具有传染快、危害大等特点。对水稻危害最大的三种病害为稻瘟病、水稻纹枯病、水稻白叶枯病，其中有两种是真菌病害，稻瘟病在流行年份可减产40％～50％，有的田块甚至颗粒无收。近几十年来，防治水稻病害的措施主要是化学防治，但是化学防治对土壤、空气及水体污染严重，破坏了生态环境的平衡。同时导致农药残留在农副产品中，严重影响了人类健康。为了达到既能有效的防治农作物病虫害，又能减少化学农药用量的目的，生物农药以及一些无害防治途径的研究与开发越来越引起广泛重视。
几丁质酶产生菌是一类能够利用几丁质作为碳源而生存、繁殖的特殊微生物种群，其生理生化方面的共同特征就是能够分泌几丁质酶，并通过这些酶的作用将几丁质降解为几丁质寡糖、几丁质二糖直至几丁质单糖。而几丁质酶和几丁寡糖在植物抗病上的应用已经被广泛的报道，而且有研究证实几丁寡糖还能促进植物的生长发育。几丁质酶产生菌是一类很有前途的抗真菌的微生物，因此，在这方面作更广泛更深入的研究是很有意义的工作。
【目的】获得对病原真菌有较好抑制效果的几丁质酶产生菌及其应用技术，初步探索拮抗菌的作用机理。
【内容】分离纯化对病原真菌有较好抑制作用的几丁质酶产生菌，优化产酶条件，研究拮抗菌株对一些病原真菌的抑菌效果，探讨拮抗菌发酵液对水稻幼苗保护酶活性的影响。
【方法】菌种筛选方法：初步分离：采用平板透明圈方法筛选几丁质酶产生菌菌株，
中文摘要
产生透明圈的培养基选用分离培养基。将土样（棉用土．稻田土、桔园土、池塘底泥、
水产养殖场的泥土等）或植物器官（水稻、棉花、玉米、烟叶及一些蔬菜的根、茎、
叶）用无菌水稀释（梯度稀释法人在稀释倍数为 10叶、10刁、10、时分别取 0．sml涂
于分离培养基中，30aC培养3～5天，观察透明圈是否产生。
二次分离：继续培养得到的菌株，同样用梯度稀释法在分离培养基中培养菌株单个菌
落，观察其在分离培养基中生长情况。根据生长情况和产生透明圈的大小选出较好的
菌株。
三次分离：在含稻瘟病病原菌细胞壁成分的培养基的平板上培养，在二次分离的基础
上，观察筛选菌株的生长情况，并检测菌株的几丁质酶话性。
微生物几丁质酶活性测定方法：参照 Ac e介绍的方法。
抑菌实验的方法：参照擅建新和李力介绍的方法。
过氧化氢酶活性测定：按照《植物生理生化实验》中介绍的方法进行。
过氧化物酶活性测定方法：按照《生化实验技术方法和技术》中介绍的方法进行。
苯丙氨酸解氨酶活性和植物几丁质酶活性测定方法：按照《现代植物生理学实验指南》
中介绍的方法进行。
【结果】
1．从自然界中筛选到两株产酶能力较强的几丁质酶产生菌，分别编号为H3和 Hg。初
步鉴定结果，H3菌株为苏云金芽抱杆菌，Hg可能为盲霉菌。
2．产酶条件：H3菌株以细粉几丁质和酵母粉为 C源和 N源，在 30aC、pH72下培养
72小时为最佳产酶条件＊ 菌株以细粉几丁质和酵母粉为 C源和 N源，在 35℃，pH6．9
下，培养84小时为最佳产酶条件。
3H3和 Hg菌株对几种病原真菌有明显抑制作用，H3对稻瘟病、水稻效枯病、棉花枯
萎病、小麦赤霉病、黄瓜枯萎病的抑菌率分别为 73．l％、44．3 ％、62．5 ％、50上％、56．3
％：＊9对它们的抑菌率分别为58．2％、忙＊％、56．3％、27．2％、36石％。
4．H3菌株发酵液处理水稻种子后，接种稻瘟病，培两优288出苗率、苗高、百苗干物
重分别为 31．5％，11．2上1．6cm、1．95土0 089；旱干 127出苗率、苗高、百苗干物重分
别为65 ％、12．5土2．Icm、1．67土0．119；新香优80出苗率、苗高、百苗干物重分别
为42％、13．2士2，ZCm、2．06土0．149。Hg菌株发酵液处理后，培两优288出苗率、苗
高、百苗干物重分别为 33％、8＊h石cm、1．87上0 069。旱干 127出苗率、苗高、百苗
干物重分别为 57．5％、11．4士2．Zcm、1．59上0 079；新香优 80出苗率、苗高、百苗干
物重分别为35％、11．5土3．ocm、2．10土0．15g。与对照相比均有明显提高。
5两株几丁质酶产生菌的产酶发酵液（含几丁质底物）能诱导水稻幼苗保护酶系活性的
改变，使其朝着有利于水稻幼苗生长和抵抗不利因素的方向发展，产酶发酵液作用比
[ Background ]Rice is one of the most important crops in the world.With nearly 50 % of the world's population depends on it.China is the biggest rice-producing and rice-cons uming country.More than 60% of the population in our country regard it as the staple f ood,The total output of every year reaches about 200 billion kilograms.Athough our cou ntry have made great achievement of worldwide interests in planting rice,still faces new challenge.The relative high costs,the low efficiency and the weak international competitiv eness still exists.In ecological environment,the factors influencing the growth of rice chan ges.Every time going beyond the range of rice-restrained, these environmental factors bee ome stress factors of adverse circumstances and do harm to rice,they will bring obvious negative effect on rice growth and cause enormous output and economical losses.The di sease of rice is one of the main adverse circumstance and important factors influencing on the output and quality of rice.Especially for fungi disease, it has the characteristics o f fast infection,great harm and so on.As far as the rice is concerned,three kinds of most harmful diseases are rice blast, rice sheath blight, rice bacterial leaf blight, two of whi ch are fungi disease.Among them, rice blast cause 40%-50% decreasing of production in prevailing year,even no harvest at all.In recent decades,the main measures of preventing and controlling the rice diseases is chemical control.However,chemical control gives rise to serious pollution on soil, atmosphere and water, and destroys the balance of the ecol ogical environment.At the same time it leads to the held-up pesticide in the agricultural byproducts and influences on the human's health seriously. In order to control the grain crop diseases and reduce the quantities of chemical pesticides effectively,the research on biological pesticides and biological controls were paid great attention in the world.
Chitinase forming strain is a kind of special microorganisms .This strain can utilize chitin as carbon source to survive and repoduce.And it has the common biochemical ch aracteristics of secreting chitinase.Chitinase can degrade chitin into chitin oligosaccharide, chitin disaccharide,and chitin monosaccharide.The application of chitinase and chitin oligo saccharide on plant resistance are extensively reported.Moreover researches verified that c hitin oligosaccharide can promot the growth of plant.So chitinase froming strain is a kin d of promising fungi-resistant microorgnanism.Therefore,it's a very meaningful work to d o more extensive and deeper researches in this respect.
[Objective] Obstain the chitinase producing strains inhibiting pathogen fungi and the a plicaion technology and explore the mechanism of antifungi strain
[Content] Obtained one or several chitinase secreting strains capable with inhibiting pa thogen fungi found out the premium conditions and measuring the effect of inhibiting to
pathogen fungi.This experiment explore the mechanism of strain's fermented liquids by examinng the effect of rice defensive enzymes activity.
[Method] The screening method of strains: preliminarily isolation: Chitinase producing strains are screened by the method of transparent zones and use selective medium.The s oil samples(cotton field soil,rice field soil,tangerine garden soil,pond bottom mud,aquacult ure mud ,etc.) and plant organs (roots,stems and leaves from rice,cotton,maize,tobacco le aves and some vegetables) are diluted with sterile water(serial dilution method).when the
samples are diluted to 10-4,10-5,10-6 times,o.5ml dilute solution are put into selective medium and are cultived for 3~5 days and observed wheather the transparent zones are produced. The second separation :Strain were obstained by continuing cultivation.single colony was
cultivated in separate medium by serial dilution method.,better strain with bisser of tran sparent zones will be choosed.
The third separation:Based on the second separation,the colony was cultivated in the pi ate containing blast p
几丁质酶产生菌是一类能够利用几丁质作为碳源而生存、繁殖的特殊微生物种群，其生理生化方面的共同特征就是能够分泌几丁质酶，并通过这些酶的作用将几丁质降解为几丁质寡糖、几丁质二糖直至几丁质单糖。而几丁质酶和几丁寡糖在植物抗病上的应用已经被广泛的报道，而且有研究证实几丁寡糖还能促进植物的生长发育。几丁质酶产生菌是一类很有前途的抗真菌的微生物，因此，在这方面作更广泛更深入的研究是很有意义的工作。
【目的】获得对病原真菌有较好抑制效果的几丁质酶产生菌及其应用技术，初步探索拮抗菌的作用机理。
【内容】分离纯化对病原真菌有较好抑制作用的几丁质酶产生菌，优化产酶条件，研究拮抗菌株对一些病原真菌的抑菌效果，探讨拮抗菌发酵液对水稻幼苗保护酶活性的影响。
【方法】菌种筛选方法：初步分离：采用平板透明圈方法筛选几丁质酶产生菌菌株，
中文摘要
产生透明圈的培养基选用分离培养基。将土样（棉用土．稻田土、桔园土、池塘底泥、
水产养殖场的泥土等）或植物器官（水稻、棉花、玉米、烟叶及一些蔬菜的根、茎、
叶）用无菌水稀释（梯度稀释法人在稀释倍数为 10叶、10刁、10、时分别取 0．sml涂
于分离培养基中，30aC培养3～5天，观察透明圈是否产生。
二次分离：继续培养得到的菌株，同样用梯度稀释法在分离培养基中培养菌株单个菌
落，观察其在分离培养基中生长情况。根据生长情况和产生透明圈的大小选出较好的
菌株。
三次分离：在含稻瘟病病原菌细胞壁成分的培养基的平板上培养，在二次分离的基础
上，观察筛选菌株的生长情况，并检测菌株的几丁质酶话性。
微生物几丁质酶活性测定方法：参照 Ac e介绍的方法。
抑菌实验的方法：参照擅建新和李力介绍的方法。
过氧化氢酶活性测定：按照《植物生理生化实验》中介绍的方法进行。
过氧化物酶活性测定方法：按照《生化实验技术方法和技术》中介绍的方法进行。
苯丙氨酸解氨酶活性和植物几丁质酶活性测定方法：按照《现代植物生理学实验指南》
中介绍的方法进行。
【结果】
1．从自然界中筛选到两株产酶能力较强的几丁质酶产生菌，分别编号为H3和 Hg。初
步鉴定结果，H3菌株为苏云金芽抱杆菌，Hg可能为盲霉菌。
2．产酶条件：H3菌株以细粉几丁质和酵母粉为 C源和 N源，在 30aC、pH72下培养
72小时为最佳产酶条件＊ 菌株以细粉几丁质和酵母粉为 C源和 N源，在 35℃，pH6．9
下，培养84小时为最佳产酶条件。
3H3和 Hg菌株对几种病原真菌有明显抑制作用，H3对稻瘟病、水稻效枯病、棉花枯
萎病、小麦赤霉病、黄瓜枯萎病的抑菌率分别为 73．l％、44．3 ％、62．5 ％、50上％、56．3
％：＊9对它们的抑菌率分别为58．2％、忙＊％、56．3％、27．2％、36石％。
4．H3菌株发酵液处理水稻种子后，接种稻瘟病，培两优288出苗率、苗高、百苗干物
重分别为 31．5％，11．2上1．6cm、1．95土0 089；旱干 127出苗率、苗高、百苗干物重分
别为65 ％、12．5土2．Icm、1．67土0．119；新香优80出苗率、苗高、百苗干物重分别
为42％、13．2士2，ZCm、2．06土0．149。Hg菌株发酵液处理后，培两优288出苗率、苗
高、百苗干物重分别为 33％、8＊h石cm、1．87上0 069。旱干 127出苗率、苗高、百苗
干物重分别为 57．5％、11．4士2．Zcm、1．59上0 079；新香优 80出苗率、苗高、百苗干
物重分别为35％、11．5土3．ocm、2．10土0．15g。与对照相比均有明显提高。
5两株几丁质酶产生菌的产酶发酵液（含几丁质底物）能诱导水稻幼苗保护酶系活性的
改变，使其朝着有利于水稻幼苗生长和抵抗不利因素的方向发展，产酶发酵液作用比
[ Background ]Rice is one of the most important crops in the world.With nearly 50 % of the world's population depends on it.China is the biggest rice-producing and rice-cons uming country.More than 60% of the population in our country regard it as the staple f ood,The total output of every year reaches about 200 billion kilograms.Athough our cou ntry have made great achievement of worldwide interests in planting rice,still faces new challenge.The relative high costs,the low efficiency and the weak international competitiv eness still exists.In ecological environment,the factors influencing the growth of rice chan ges.Every time going beyond the range of rice-restrained, these environmental factors bee ome stress factors of adverse circumstances and do harm to rice,they will bring obvious negative effect on rice growth and cause enormous output and economical losses.The di sease of rice is one of the main adverse circumstance and important factors influencing on the output and quality of rice.Especially for fungi disease, it has the characteristics o f fast infection,great harm and so on.As far as the rice is concerned,three kinds of most harmful diseases are rice blast, rice sheath blight, rice bacterial leaf blight, two of whi ch are fungi disease.Among them, rice blast cause 40%-50% decreasing of production in prevailing year,even no harvest at all.In recent decades,the main measures of preventing and controlling the rice diseases is chemical control.However,chemical control gives rise to serious pollution on soil, atmosphere and water, and destroys the balance of the ecol ogical environment.At the same time it leads to the held-up pesticide in the agricultural byproducts and influences on the human's health seriously. In order to control the grain crop diseases and reduce the quantities of chemical pesticides effectively,the research on biological pesticides and biological controls were paid great attention in the world.
Chitinase forming strain is a kind of special microorganisms .This strain can utilize chitin as carbon source to survive and repoduce.And it has the common biochemical ch aracteristics of secreting chitinase.Chitinase can degrade chitin into chitin oligosaccharide, chitin disaccharide,and chitin monosaccharide.The application of chitinase and chitin oligo saccharide on plant resistance are extensively reported.Moreover researches verified that c hitin oligosaccharide can promot the growth of plant.So chitinase froming strain is a kin d of promising fungi-resistant microorgnanism.Therefore,it's a very meaningful work to d o more extensive and deeper researches in this respect.
[Objective] Obstain the chitinase producing strains inhibiting pathogen fungi and the a plicaion technology and explore the mechanism of antifungi strain
[Content] Obtained one or several chitinase secreting strains capable with inhibiting pa thogen fungi found out the premium conditions and measuring the effect of inhibiting to
pathogen fungi.This experiment explore the mechanism of strain's fermented liquids by examinng the effect of rice defensive enzymes activity.
[Method] The screening method of strains: preliminarily isolation: Chitinase producing strains are screened by the method of transparent zones and use selective medium.The s oil samples(cotton field soil,rice field soil,tangerine garden soil,pond bottom mud,aquacult ure mud ,etc.) and plant organs (roots,stems and leaves from rice,cotton,maize,tobacco le aves and some vegetables) are diluted with sterile water(serial dilution method).when the
samples are diluted to 10-4,10-5,10-6 times,o.5ml dilute solution are put into selective medium and are cultived for 3~5 days and observed wheather the transparent zones are produced. The second separation :Strain were obstained by continuing cultivation.single colony was
cultivated in separate medium by serial dilution method.,better strain with bisser of tran sparent zones will be choosed.
The third separation:Based on the second separation,the colony was cultivated in the pi ate containing blast p
引文
1．邱立友．微生物几丁质酶与害虫防治[J]．河南农业大学学报．19995，29(2)：184～189
2．冯波．微生物几丁质酶的研究概况[J]．天津师范大学(自然科学版)．1998，18(3)：50～56
3．蓝海燕，陈正华．几丁质酶及其研究进展[J]．生命科学研究．1998，2(3)：163～171
4．欧阳石文，赵开军，冯兰香，谢丙炎．植物几丁质酶研究进展[J]．生物工程进展．2001，21(4)：30～34
5. Humphreys A M. Properties of chitinase activities from Macor mucedo. evidence for a me mbrane bound zymogenic form[J]. Gen. microbial. 1984, 130: 1359～1366
6．徐同，柳良好．木霉几丁质酶及其对植物病源真菌的拮抗作用[J]．植物病理学报 2002，32(2)：97～102
7. Vorgias C, Kingswell A, Dauter Z. Crystallisation of recombinant chiti-nase from the clo ned chiA gene of Serratia marcescents[J]. Mol. Biol. 1992, 226, 897～898
8. Suslow T. V., Matsubara D., Jones J., et al.. Effect of expression of bacterial chitinase on tobacco susceptibility to leaf brown spot[J]. Phytopathology, 1988, 78: 1556
9. Koby.s, Schickler. H, Chet. I, et al. The chitinase encloding Tn-7-based ChiA gen endows P sendomonas fluorescens with the capacity to control plant pathogens in soil[J]. gene. 1996, 147: 81～83
10．王益民，唐文华．几丁质酶基因和β-1，3-葡聚糖酶基因的克隆及双价基因在枯草芽孢杆菌B-908中的表达[J]。植物病理学报．1998，28(3)：288
11. Benhamou. N, Broglie. K, and Chet. I, et al. Antifungal effect of bean endochitinase on Rhizoctonia solani: ultrastructural changes and cytochemical aspects of chitin breakdown [J]. Canadian Journal of Microbiology 1993, 39: 318～328
12．蔡静平，李容启，王钦宏等．氨基寡糖素发酵生产的研究[J]．食品科学，2000，21(9)：21～24
13．王杨，娄永江，杨文鸽．酶法制备几丁寡糖和壳聚糖研究现状与进展[J]，东海海洋，2001，19(4)：40～44
14．竺国芳，赵鲁杭．几丁寡糖和壳聚糖研究进展[J]．中国海洋药物，2000，(1)：43～46
15. Einosuki M, Fumiko Y, Hiroyuki K. Preparation and crystallization of D-gluo-samine o ligosaccharides with dp 6-8. Carbohydrate Research, 1993, 239: 227
16．刘和从，刘东辉．几丁质在农业上的应用[J]．应用技术，2001，(3)11～13
17．郑志成，周美英，姚丙新．海洋链霉菌几丁质酶的研究[J]．厦门大学学报(自然科学版)，31(5)：543～547
18．陈三凤，李季伦．几丁质酶研究历史和发展前景[J]．微生物学通报，1993，20(3)：156～160
19. Beyer M, Diekmana H. The chitinases system of Streptomycessp. ATCC11238 and Its Si gnificance for Fungal Cell Wall Degradation[J]. Appl, Microbiol. Biotec-hmol. 1985, 23: 140-146
20. Reynolds D M. Exocellular Chitinase from a Streptomyces. sp[J]. Gen, Microbiol, 1954, 11: 150～159
21. Bleak H, Schrempf H. Binding Substrate. sp ecificities of a Streptomyces oliva-ceoviridis chitinase comparison with its proteolytically[J]. Eur Biochem, 1995, 229(1): 132～139
22. Trachuk L A, Revina L P, Shemyakina T M, et al. Chitinase of Bacillus Licheniformis B-6839: Isolation and Properties[J]. Canadian Microbiol, 1996, 42(4): 307～315
23．彭仁旺，黄秀梨．球孢白僵菌孢内几丁质酶的分离纯化及性质[J]．微生物学报，1995，35(6)：427～432
24. Fuchs R L. Purification and Properties of Chitinases from Sarratia Marcescens[J]. Envir on Microbiol, 1987, 53(7): 1718～1720
25. Phillips W R, Charles A, Barbara B. Cloning and Expression of a Streptomyces plicatus Chtinase in E.coli[J]. Biol Chem 1988, 263(1): 443～447
26. Saburo H, Yoshitaka Y, Yoko F, et al. Purification and Characterization of Chiti-nase Pro duced by Streptomyces erythraeus[J]. Biochem, 1989, 105: 484～489
27. Takeshi W, Wataru O, Kazushi S, et al. Chitinase System of Bacillus circulans WL-12 a nd importance of Chitinase A1 in Chtin Degradation[J]. Bacteriol, 1990, 172(7): 4017～4022
28. Jones. Isolation and Characterization of Genes Encoding Two Chitinase Enzymes from Serratia Macescens[J]. The EMBO Journal, 1986, 5: 467～473
29. T Araki, J Funatsu, M Kuramoto, H Konno, et al. The complete amino acid sequence of yam (Dioscorea japonica) chitinase. A newly identified acidic class I chitinase[J]. Biol. Chem., Oct 1992, 267: 19944～19947
30. T Ohno, S Armand, T Hata, et al. A modular family 19 chitinase found in the prokaryoti c organism Streptomyces griseus HUT 6037[J]. Bacteriol., Sep 1996, 178: 5065～5070
31. Z. Zhang, G. Y. Yuen, G. Sarath, et al. Chitinases from the Plant Disease Biocon-trol Agent Stenotrophomonas maltopilia Strain C3 [J]. Phytopathology 2001, 91(2): 1276～1288
32. Z. Zhang, G. Y. Yuen. The Role of Chitinase Production by Stenotrophomonas maltophili a Strain C3 in Biological Control of Bipolaris Sorokiniana[J] Phytopathology 2000, 90(4): 673～681
33. Ace M. Baty Ⅲ, Callie C, et al. Differentiation of Chitinase-Active and Non-Chitinase-Active Subpopulations of a Marine Bacterium during Chitin Degrada-tion[J]. Appl. Envir. Microbiol., Aug 2000, 66: 3566～3573
34. C. Sasaki, A. Yokoyama, Y. Itoh, et al. Comparative study of the reaction mec-hanism of family 18 chitinases from plants and microbes.[J]. Histochem. Cytoc-hem., Aug 2002, 50: 1081～1089
35．李君，曾中文，欧阳石文．微生物几丁质酶的特性、基因表达调控及应用[J]．微生物学通报，2001，28(4)：84～87
36．陈振明，王郑逸，郭泽建．等．绿色木酶内切几丁质酶基因的克隆及其毛壳菌的转化[J]．菌物系统，2002，21(3)：375～382
37. Ulhoa CJ, JF Peberdy. Purification and Some Properties of the Extracellular Chitinase Produced by Trichoderma Harzianum [J]. Enzyme and Microbial Tech-nology, 1992, 14(3): 236～240
38．唐亚雄，赵建，丁诗华等．产气肠杆菌几丁质酶的分离纯化及性质研究[J]．微尘物学报，2001，41(1)：82～86
39. T Watanabe, K Kimura, T Sumiya, et al. Genetic analysis of the chitinase syst-em of Serratia marcescens 2170[J]. Bacteriol., Nov 1997, 179: 7111～7117
40．李江，胡学军，苏乔等．水稻几丁质酶基因的克隆及序列分析[J]．农业生物技术学报，1998，6(4)：382～388
41．张世宏，李多川，魏毅．粉红聚端孢菌胞外几丁质酶纯化、特性及抗菌活性[J]．植物病理学报，2002，32(3)：262～266
42．冯波，郑津辉，魏艳敏．链霉菌酶发酵液对植物病原菌的拮抗作用[J]．河北农业大学学报，1998，21(4)：102～104
43．曾艳，赵南明，刘进元．几丁质酶与植物防卫反应[J]．生物工程进展，1997，17(4)：31～39
44．林稚兰，黄秀梨主编．现代微生物学与实验技术．北京：科学出版社，1998
45．谭周进，肖启明，何可佳等．土壤几丁质酶对土传性真菌病害的防治作用[J]．生物技术通讯，2003，14(1)：71～73
46. M. Lorito, L. Woo. Genes from mycoparasitic fungi as a source for improving plant resi stance to fungal pathogens[Jl. PNAS. Jul 1998. 95: 7860～7865
47．吴志刚，朱旭芬．几丁质酶的分子生物学特性及其在转基因植物中的应用[J]．生命科学，2002，14(2)：117～121
48．韩宝芹，余长缨，刘万顺，等．几丁质酶研究现状及展望[J]．中国海洋药物杂志，2001，83(5)：41～43
49．王华，周鹏，郭安平．几丁质酶基因与抗真菌蛋白基因、葡聚糖酶基因双价表达载体的构建及农杆菌工程菌株的重组[J]．西北植物学报，2002，22(2)：250～256
50．吴潇韫，朱旭芬．生物降解甲壳质的研究[J]．东海海洋，2001，19(2)：15～22
51．黄丽萍，刘宗明．几丁寡糖、壳糖素的应用与开发[J]．中国微生态学杂志，1998，10(3)：180～183
52．候春林，顾其胜主编．几丁质与医学[M]．上海：上海科学技术出版社 2001：1～5
53．冯培章．寡糖素对植物生长发育的调控[J]．生物学通讯，2001，36(3)：16～17
54．杜建玲．植物诱导抗性的理论及其应用前景[J]．北京林业大学学报，1994，16(2)：83～88
55. R. Bradley Day, Mitsuo Okada, Yuki Ito, et al. Binding Site for Chitin Oligos-accharides in the Soybean Plasma Membrane[J]. Plant Physiology, Jul 2001, 126: 1162-1173
56. HR Schlaman, AA Gisel, NE Quaedvlieg, et al. Chitin oligosaccharides can in-duce cort ical cell division in roots of Vicia sativa when delivered by ballistic mi-crotargeting[J]. Development, Dec 1997, 124: 4887～4895
57．林晓蓉，白雪芳，杜昱光．寡聚糖素诱导植物抗病性反应研究进展[J]．生物工程进展，1998，18(3)：26～31
58. Roby D, Cadelle A, Toppan A. Chitin oligosaccdarides as elicitors of chitinase activety in melon plants[J]. Biochemical and Biophysical Research Communicati-ons. 1987, 143(3): 885
59. Carlos E, Semino, Miguel L, et al. Expression of Rhizobium Chitin Oligosacch-aride Fucosyltransferase in Zebrafish Embryos Disrupts Normal Development[J], Ann. N. Y. Acad. Sci., Apr 1998, 842: 49～54
60．王克夷．寡糖素——类新的植物调节分子[J]．植物生理学通讯，1989(4)：56～58
61. BL Bassler, C Yu, YC Lee, et al. Chitin utilization by marine bacteria. Degradation and catabolism of chitin oligosaccharides by Vibrio furnissii[J]. Biol. Chem., Dec 1991, 266: 24276～24286
62．苏凤池．寡聚糖生物农药[J]．名产名品 2000，(2)：30
63. A. Souleimanov, B. Prithiviraj, D. L. Smith. The major Nod factor of Bradyrhizobium japonicum prom-otes early growth of soybean and corn[J]. Exp. Bot., September 1, 2002, 53(376): 1929～1934
64. W. D. Haeze, M. Holsters. Nod factor structures, responses, and perception d-uring initiation of nodule development[J]. Glycobiology, June 1, 2002, 12(6): 79～105
65．高丽锋，胡志昂．根瘤菌NOD因子的感知与信号传导[J]．中国生物工程杂志，2002，22(5)：65～68
66. A. Souleimanov, B. Prithiviraj, D. L. Smith. The major Nod factor of Bradyrh-izobium japonicum promotes early growth of soybean and corn[J]. Exp. Bot., S-eptember, 2002, 53(376): 1929～1934
67. Carlos E, Charles A, Alejandra Raimondi, et al. Homologs of the Xenopus dev-elopmental gene DG42 are present in zebrafish and mouse and are involved in the synthesis of No d-like chitin oligosaccharides during early embryogenesis[J]. PNAS, May 1996, 93: 4548～4553
68．罗志勇，胡维新．水稻基因图谱绘制成功对世界粮食和环境问题的意义[J]，湖南医科大学学报(社会科学版)，2002，4(3)：66～68
69．中央农业广播电视学校主编．水稻栽培与病虫害防治[M]．北京：中国农业出版社，1995
70．陈辉蓉，吴振斌，贺锋等．植物抗逆性的研究进展[J]．环境污染治理与设备，2001，2(3)：3～7
71．张正斌．植物对环境胁迫整体抗逆性研究的若干问题[J]．西北农林学报，2000，9(3)：112～116
72．丁克坚，檀根甲．稻瘟病为害损失研究[J]．植物保护学报．1999，26(1)：61～64
73．史清亮，孙树荣．生物农药的研究进展与发展对策[J]。山西农经，2000，2：35～37
74．郑爱萍，李平．微生物防治存在的问题及发展方向[J]．中国农学通报，2000，16(6)：28～29
75．刘开启．走向21世纪的植物保护[J]．农业新技术新方法，1997，3：23～30
76．陈志谊．微生物农药在植物病害防治中的应用及发展策略[J]．江苏农业科学，2001，4：39～42
77．万方浩，叶正楚，郭建英等．我国生物防治研究的进展及展望[J]．2000，37(2)：65～74
2．冯波．微生物几丁质酶的研究概况[J]．天津师范大学(自然科学版)．1998，18(3)：50～56
3．蓝海燕，陈正华．几丁质酶及其研究进展[J]．生命科学研究．1998，2(3)：163～171
4．欧阳石文，赵开军，冯兰香，谢丙炎．植物几丁质酶研究进展[J]．生物工程进展．2001，21(4)：30～34
5. Humphreys A M. Properties of chitinase activities from Macor mucedo. evidence for a me mbrane bound zymogenic form[J]. Gen. microbial. 1984, 130: 1359～1366
6．徐同，柳良好．木霉几丁质酶及其对植物病源真菌的拮抗作用[J]．植物病理学报 2002，32(2)：97～102
7. Vorgias C, Kingswell A, Dauter Z. Crystallisation of recombinant chiti-nase from the clo ned chiA gene of Serratia marcescents[J]. Mol. Biol. 1992, 226, 897～898
8. Suslow T. V., Matsubara D., Jones J., et al.. Effect of expression of bacterial chitinase on tobacco susceptibility to leaf brown spot[J]. Phytopathology, 1988, 78: 1556
9. Koby.s, Schickler. H, Chet. I, et al. The chitinase encloding Tn-7-based ChiA gen endows P sendomonas fluorescens with the capacity to control plant pathogens in soil[J]. gene. 1996, 147: 81～83
10．王益民，唐文华．几丁质酶基因和β-1，3-葡聚糖酶基因的克隆及双价基因在枯草芽孢杆菌B-908中的表达[J]。植物病理学报．1998，28(3)：288
11. Benhamou. N, Broglie. K, and Chet. I, et al. Antifungal effect of bean endochitinase on Rhizoctonia solani: ultrastructural changes and cytochemical aspects of chitin breakdown [J]. Canadian Journal of Microbiology 1993, 39: 318～328
12．蔡静平，李容启，王钦宏等．氨基寡糖素发酵生产的研究[J]．食品科学，2000，21(9)：21～24
13．王杨，娄永江，杨文鸽．酶法制备几丁寡糖和壳聚糖研究现状与进展[J]，东海海洋，2001，19(4)：40～44
14．竺国芳，赵鲁杭．几丁寡糖和壳聚糖研究进展[J]．中国海洋药物，2000，(1)：43～46
15. Einosuki M, Fumiko Y, Hiroyuki K. Preparation and crystallization of D-gluo-samine o ligosaccharides with dp 6-8. Carbohydrate Research, 1993, 239: 227
16．刘和从，刘东辉．几丁质在农业上的应用[J]．应用技术，2001，(3)11～13
17．郑志成，周美英，姚丙新．海洋链霉菌几丁质酶的研究[J]．厦门大学学报(自然科学版)，31(5)：543～547
18．陈三凤，李季伦．几丁质酶研究历史和发展前景[J]．微生物学通报，1993，20(3)：156～160
19. Beyer M, Diekmana H. The chitinases system of Streptomycessp. ATCC11238 and Its Si gnificance for Fungal Cell Wall Degradation[J]. Appl, Microbiol. Biotec-hmol. 1985, 23: 140-146
20. Reynolds D M. Exocellular Chitinase from a Streptomyces. sp[J]. Gen, Microbiol, 1954, 11: 150～159
21. Bleak H, Schrempf H. Binding Substrate. sp ecificities of a Streptomyces oliva-ceoviridis chitinase comparison with its proteolytically[J]. Eur Biochem, 1995, 229(1): 132～139
22. Trachuk L A, Revina L P, Shemyakina T M, et al. Chitinase of Bacillus Licheniformis B-6839: Isolation and Properties[J]. Canadian Microbiol, 1996, 42(4): 307～315
23．彭仁旺，黄秀梨．球孢白僵菌孢内几丁质酶的分离纯化及性质[J]．微生物学报，1995，35(6)：427～432
24. Fuchs R L. Purification and Properties of Chitinases from Sarratia Marcescens[J]. Envir on Microbiol, 1987, 53(7): 1718～1720
25. Phillips W R, Charles A, Barbara B. Cloning and Expression of a Streptomyces plicatus Chtinase in E.coli[J]. Biol Chem 1988, 263(1): 443～447
26. Saburo H, Yoshitaka Y, Yoko F, et al. Purification and Characterization of Chiti-nase Pro duced by Streptomyces erythraeus[J]. Biochem, 1989, 105: 484～489
27. Takeshi W, Wataru O, Kazushi S, et al. Chitinase System of Bacillus circulans WL-12 a nd importance of Chitinase A1 in Chtin Degradation[J]. Bacteriol, 1990, 172(7): 4017～4022
28. Jones. Isolation and Characterization of Genes Encoding Two Chitinase Enzymes from Serratia Macescens[J]. The EMBO Journal, 1986, 5: 467～473
29. T Araki, J Funatsu, M Kuramoto, H Konno, et al. The complete amino acid sequence of yam (Dioscorea japonica) chitinase. A newly identified acidic class I chitinase[J]. Biol. Chem., Oct 1992, 267: 19944～19947
30. T Ohno, S Armand, T Hata, et al. A modular family 19 chitinase found in the prokaryoti c organism Streptomyces griseus HUT 6037[J]. Bacteriol., Sep 1996, 178: 5065～5070
31. Z. Zhang, G. Y. Yuen, G. Sarath, et al. Chitinases from the Plant Disease Biocon-trol Agent Stenotrophomonas maltopilia Strain C3 [J]. Phytopathology 2001, 91(2): 1276～1288
32. Z. Zhang, G. Y. Yuen. The Role of Chitinase Production by Stenotrophomonas maltophili a Strain C3 in Biological Control of Bipolaris Sorokiniana[J] Phytopathology 2000, 90(4): 673～681
33. Ace M. Baty Ⅲ, Callie C, et al. Differentiation of Chitinase-Active and Non-Chitinase-Active Subpopulations of a Marine Bacterium during Chitin Degrada-tion[J]. Appl. Envir. Microbiol., Aug 2000, 66: 3566～3573
34. C. Sasaki, A. Yokoyama, Y. Itoh, et al. Comparative study of the reaction mec-hanism of family 18 chitinases from plants and microbes.[J]. Histochem. Cytoc-hem., Aug 2002, 50: 1081～1089
35．李君，曾中文，欧阳石文．微生物几丁质酶的特性、基因表达调控及应用[J]．微生物学通报，2001，28(4)：84～87
36．陈振明，王郑逸，郭泽建．等．绿色木酶内切几丁质酶基因的克隆及其毛壳菌的转化[J]．菌物系统，2002，21(3)：375～382
37. Ulhoa CJ, JF Peberdy. Purification and Some Properties of the Extracellular Chitinase Produced by Trichoderma Harzianum [J]. Enzyme and Microbial Tech-nology, 1992, 14(3): 236～240
38．唐亚雄，赵建，丁诗华等．产气肠杆菌几丁质酶的分离纯化及性质研究[J]．微尘物学报，2001，41(1)：82～86
39. T Watanabe, K Kimura, T Sumiya, et al. Genetic analysis of the chitinase syst-em of Serratia marcescens 2170[J]. Bacteriol., Nov 1997, 179: 7111～7117
40．李江，胡学军，苏乔等．水稻几丁质酶基因的克隆及序列分析[J]．农业生物技术学报，1998，6(4)：382～388
41．张世宏，李多川，魏毅．粉红聚端孢菌胞外几丁质酶纯化、特性及抗菌活性[J]．植物病理学报，2002，32(3)：262～266
42．冯波，郑津辉，魏艳敏．链霉菌酶发酵液对植物病原菌的拮抗作用[J]．河北农业大学学报，1998，21(4)：102～104
43．曾艳，赵南明，刘进元．几丁质酶与植物防卫反应[J]．生物工程进展，1997，17(4)：31～39
44．林稚兰，黄秀梨主编．现代微生物学与实验技术．北京：科学出版社，1998
45．谭周进，肖启明，何可佳等．土壤几丁质酶对土传性真菌病害的防治作用[J]．生物技术通讯，2003，14(1)：71～73
46. M. Lorito, L. Woo. Genes from mycoparasitic fungi as a source for improving plant resi stance to fungal pathogens[Jl. PNAS. Jul 1998. 95: 7860～7865
47．吴志刚，朱旭芬．几丁质酶的分子生物学特性及其在转基因植物中的应用[J]．生命科学，2002，14(2)：117～121
48．韩宝芹，余长缨，刘万顺，等．几丁质酶研究现状及展望[J]．中国海洋药物杂志，2001，83(5)：41～43
49．王华，周鹏，郭安平．几丁质酶基因与抗真菌蛋白基因、葡聚糖酶基因双价表达载体的构建及农杆菌工程菌株的重组[J]．西北植物学报，2002，22(2)：250～256
50．吴潇韫，朱旭芬．生物降解甲壳质的研究[J]．东海海洋，2001，19(2)：15～22
51．黄丽萍，刘宗明．几丁寡糖、壳糖素的应用与开发[J]．中国微生态学杂志，1998，10(3)：180～183
52．候春林，顾其胜主编．几丁质与医学[M]．上海：上海科学技术出版社 2001：1～5
53．冯培章．寡糖素对植物生长发育的调控[J]．生物学通讯，2001，36(3)：16～17
54．杜建玲．植物诱导抗性的理论及其应用前景[J]．北京林业大学学报，1994，16(2)：83～88
55. R. Bradley Day, Mitsuo Okada, Yuki Ito, et al. Binding Site for Chitin Oligos-accharides in the Soybean Plasma Membrane[J]. Plant Physiology, Jul 2001, 126: 1162-1173
56. HR Schlaman, AA Gisel, NE Quaedvlieg, et al. Chitin oligosaccharides can in-duce cort ical cell division in roots of Vicia sativa when delivered by ballistic mi-crotargeting[J]. Development, Dec 1997, 124: 4887～4895
57．林晓蓉，白雪芳，杜昱光．寡聚糖素诱导植物抗病性反应研究进展[J]．生物工程进展，1998，18(3)：26～31
58. Roby D, Cadelle A, Toppan A. Chitin oligosaccdarides as elicitors of chitinase activety in melon plants[J]. Biochemical and Biophysical Research Communicati-ons. 1987, 143(3): 885
59. Carlos E, Semino, Miguel L, et al. Expression of Rhizobium Chitin Oligosacch-aride Fucosyltransferase in Zebrafish Embryos Disrupts Normal Development[J], Ann. N. Y. Acad. Sci., Apr 1998, 842: 49～54
60．王克夷．寡糖素——类新的植物调节分子[J]．植物生理学通讯，1989(4)：56～58
61. BL Bassler, C Yu, YC Lee, et al. Chitin utilization by marine bacteria. Degradation and catabolism of chitin oligosaccharides by Vibrio furnissii[J]. Biol. Chem., Dec 1991, 266: 24276～24286
62．苏凤池．寡聚糖生物农药[J]．名产名品 2000，(2)：30
63. A. Souleimanov, B. Prithiviraj, D. L. Smith. The major Nod factor of Bradyrhizobium japonicum prom-otes early growth of soybean and corn[J]. Exp. Bot., September 1, 2002, 53(376): 1929～1934
64. W. D. Haeze, M. Holsters. Nod factor structures, responses, and perception d-uring initiation of nodule development[J]. Glycobiology, June 1, 2002, 12(6): 79～105
65．高丽锋，胡志昂．根瘤菌NOD因子的感知与信号传导[J]．中国生物工程杂志，2002，22(5)：65～68
66. A. Souleimanov, B. Prithiviraj, D. L. Smith. The major Nod factor of Bradyrh-izobium japonicum promotes early growth of soybean and corn[J]. Exp. Bot., S-eptember, 2002, 53(376): 1929～1934
67. Carlos E, Charles A, Alejandra Raimondi, et al. Homologs of the Xenopus dev-elopmental gene DG42 are present in zebrafish and mouse and are involved in the synthesis of No d-like chitin oligosaccharides during early embryogenesis[J]. PNAS, May 1996, 93: 4548～4553
68．罗志勇，胡维新．水稻基因图谱绘制成功对世界粮食和环境问题的意义[J]，湖南医科大学学报(社会科学版)，2002，4(3)：66～68
69．中央农业广播电视学校主编．水稻栽培与病虫害防治[M]．北京：中国农业出版社，1995
70．陈辉蓉，吴振斌，贺锋等．植物抗逆性的研究进展[J]．环境污染治理与设备，2001，2(3)：3～7
71．张正斌．植物对环境胁迫整体抗逆性研究的若干问题[J]．西北农林学报，2000，9(3)：112～116
72．丁克坚，檀根甲．稻瘟病为害损失研究[J]．植物保护学报．1999，26(1)：61～64
73．史清亮，孙树荣．生物农药的研究进展与发展对策[J]。山西农经，2000，2：35～37
74．郑爱萍，李平．微生物防治存在的问题及发展方向[J]．中国农学通报，2000，16(6)：28～29
75．刘开启．走向21世纪的植物保护[J]．农业新技术新方法，1997，3：23～30
76．陈志谊．微生物农药在植物病害防治中的应用及发展策略[J]．江苏农业科学，2001，4：39～42
77．万方浩，叶正楚，郭建英等．我国生物防治研究的进展及展望[J]．2000，37(2)：65～74