几丁质酶和内切葡聚糖酶编码基因的克隆及重组芽孢杆菌的构建
|
摘要
本研究旨在从两株生防菌中分别克隆几丁质酶和内切葡聚糖酶的编码基因，并进行测序。将所获得的基因同穿梭质粒连接，转化野生型植病生防芽孢杆菌菌株，通过平板拮抗实验和盆栽试验检测获得的转化子对病原菌的拮抗作用的大小。
本实验室从泰安郊区小麦根际及来自澳大利亚的小麦根际土样中，经分离筛选出两株芽孢杆菌，枯草芽孢杆菌(Bacillus subtilis)Ap113和巨大芽孢杆菌(B.megaterium)Ap25，两个菌株对小麦纹枯病和全蚀病都有防治效果；利用几丁质培养基和羧甲基纤维素培养基对这两株菌进行酶活性检测，已证实这两株菌分别具有几丁质酶和内切葡聚糖酶活性。
几丁质酶基因的克隆：根据GeneBank上已有的枯草芽孢杆菌几丁质酶基因的编码序列，设计一对简并引物，根据引物的Tm值和GC含量选择适宜的PCR条件进行PCR，PCR产物与pMD18-T载体连接，转化E.coliDH5α，蓝白斑筛选得到23株菌株，在几丁质平板上筛选阳性克隆子4株。质粒分析证明克隆子中含有重组质粒，外源插入片段大小为2.6kb左右，与PCR最初产物的大小一致。测序后在GeneBank上进行序列比较，该基因片段同编号为2634966的枯草芽孢杆菌全序列的2599451到2812870(功能未知)有85％的同源性，但同已发表的13种几丁质酶的基因(包括枯草芽孢杆菌几丁质酶基因)的同源性很低，只有30％。尽管同源性比较低，但酶活性检测发现该基因片段具有几丁质酶活性，认为此PCR片段含有几丁质酶编码基因的全序列或部分序列，此基因片段是一个新基因或基因片段。
内切葡聚糖酶基因的克隆采取基因文库方法。提取B.megateriumAp25的染色体DNA，Sau3A酶切，回收0.7-3.0kb的片段，与同Sau3A具有相同粘性末端的BamHI完全酶切并脱磷酸化的质粒pBluescriptⅡ连接，转化E.coli JM109，蓝白斑筛选得到108株菌株，通过羧甲基纤维素培养基筛选具有内切葡聚糖酶活性的菌株有74个，选取其中酶活性表达最强的阳性克隆子，质粒分析并进行测序，证明外源插入片段大小为0.7kb左右。测序并序列比较结果表明该基因片段同已发表的枯草芽孢杆菌
几丁质酶和内切葡聚糖酶编码幕因的克隆及重组芽抱杆菌的构建
glyB一aprE之间的同源性是最高的，为35%;同Bacz’了了us sP.BP23 ce1B、
B.p朋刀us内切葡聚糖酶和B.pol理vxap一1，4一内切葡聚糖酶的编码基
因的同源性只有27%。虽然同源性较低，但酶活性表达较强，认为该基因
是编码内切葡聚糖酶的一个新基因片段。
用EcoRI和Sall对含有几丁质酶基因的重组质粒TvCHI(含几丁质
酶编码基因的pMD 18一T载体)和穿梭质粒pHY300PLK进行双酶切并连
接，转化E.coli DHS。，通过几丁质培养基平板筛选得到2株转化子，提
取其中的一种，质粒酶切分析插入的外源片段同PCR产物大小接近一致。
将此重组质粒pHCHI转化巨大芽抱杆菌B.megaterium Ap25，得到两株转
化子，分别命名为p25 113一9，p251 13一10。转化子进行酶活性检测，同时
具有几丁质酶和葡聚糖酶的酶活性。根据水解圈的大小可以看到，几丁质
酶的表达活性不如E.coli DH5a中的表达活性高。平板拮抗实验同野生菌
株相比，转化子对麦长蠕抱菌的抑制作用最明显，抑制百分数最高可达
33.3%，而Apll3和Ap25分别是23.1%和25.6%，同时转化子对小麦纹
枯病菌、棉花立枯病菌、棉花枯萎病菌和小麦的全蚀病菌也具有较为明显
的抑制作用。
对小麦全蚀病和纹枯病的盆栽试验结果表明工程菌株p25 1 13一9的防
病作用强于野生菌株，尤其是对小麦全蚀病的防治作用，其防治效果同野
生菌株Ap 113和Ap25相比，分别增长了22 .54%和20%。
对芽抱杆菌几丁质酶和内切葡聚糖酶编码基因的克隆及其转化实验
表明植病生防芽袍杆菌的作用机制之一就是产生几丁质酶和葡聚糖酶这
两种抗菌物质，己证实这两种抗菌物质之间具有协同效应，这为今后的生
防菌遗传改良提供了一个新途径。
This article dealt with cloning and sequencing of chitinase and endoglucanase genes of Bacillus spp. and recombinant biocontrol isolates of Bacillus spp. by transformation with the genes.
Plant disease biocontrol bacteria Bacillus subtil is ApllS and B. megaterium Ap25 were isolated from wheat field soils collected from South Australia and Tai an. Enzyme activity analysis on chitin agar and ABP media showed that B. subtilis ApllS secreted chitinase and B. megaterium Ap25 secreted endoglucanase, respectively.
PCR methodology was adopted for cloning of chitinase encoding genes. Based on the sequences of chitinase gene from GeneBank, the primers for chitinase gene amplification were designed. PCR fragment was ligated with pMD18~T vector and transformed into E. coli DH5 a . Four colonies of transformed E. coli DH5 a with clear hydroiyzing zone on the chitin agar were obtained. The gene fragment in these isolates was identified by the methods of plasmid processing. DNA sequencing analysis showed that sequence homology between PCR fragment and chromosomal DNA of B. subtilis from 2599451 to 2812870 was 85%, and was 30% between the fragment and the genes encoding for chitinase of Bacillus (including B. subtilis) in GeneBank. It was thought to be a new gene encoding for chitinanse of B. subtilis.
Genome library methodology was adopted for cloning of endoglucanase encoding genes. Genoraic DNA of B. megaterium was partially digested by restriction enzyme Sau3A and was used to establish the genomic library in plasmid pBluscripts. Selection for endoglucanase positive clones from transformed E. coli was carried out on the CMC medium. Seventy-four clones that showed hydrolysis ability on the CMC plate were obtained. Sequencing analysis showed
that the sequence of endoglucanase fragment exhibits 35% homology with B. subtil is chromosomal DNA(from glyB to aprE), and 27% homology with Bacillus sp. BP23 celB genes, B.pwnilus endoglucanase and B. polymyxa beta-1, 4-endoglucanase 'genes, respectively. It was recognized as a new gene encoding for endoglucanase of B. mega terium.
The recombinant plasmid TvCHI (pMD18~T inserted with chitinase encoding gene from ApllS) and E. coli-Bacillus shuttle vector pHYSOOPLK were digested by EcoRI and Sail completely, and the chitinase gene was ligated with shuttle vector, and the recombinant vector was used to transform B. megaterium Ap25 competent cell. PCR and enzyme acitivity check on chitin agar showed that the chitinase gene fragment existed and expressed in the wildtype strains. Antagonistic activity test in vitro suggest that the transformants remained the ability to produce antibiotics. The recombinant strains showed an increased biocontrol efficacy against wheat take-all and Rhizoctonia sheath blight in greenhouse.
本实验室从泰安郊区小麦根际及来自澳大利亚的小麦根际土样中，经分离筛选出两株芽孢杆菌，枯草芽孢杆菌(Bacillus subtilis)Ap113和巨大芽孢杆菌(B.megaterium)Ap25，两个菌株对小麦纹枯病和全蚀病都有防治效果；利用几丁质培养基和羧甲基纤维素培养基对这两株菌进行酶活性检测，已证实这两株菌分别具有几丁质酶和内切葡聚糖酶活性。
几丁质酶基因的克隆：根据GeneBank上已有的枯草芽孢杆菌几丁质酶基因的编码序列，设计一对简并引物，根据引物的Tm值和GC含量选择适宜的PCR条件进行PCR，PCR产物与pMD18-T载体连接，转化E.coliDH5α，蓝白斑筛选得到23株菌株，在几丁质平板上筛选阳性克隆子4株。质粒分析证明克隆子中含有重组质粒，外源插入片段大小为2.6kb左右，与PCR最初产物的大小一致。测序后在GeneBank上进行序列比较，该基因片段同编号为2634966的枯草芽孢杆菌全序列的2599451到2812870(功能未知)有85％的同源性，但同已发表的13种几丁质酶的基因(包括枯草芽孢杆菌几丁质酶基因)的同源性很低，只有30％。尽管同源性比较低，但酶活性检测发现该基因片段具有几丁质酶活性，认为此PCR片段含有几丁质酶编码基因的全序列或部分序列，此基因片段是一个新基因或基因片段。
内切葡聚糖酶基因的克隆采取基因文库方法。提取B.megateriumAp25的染色体DNA，Sau3A酶切，回收0.7-3.0kb的片段，与同Sau3A具有相同粘性末端的BamHI完全酶切并脱磷酸化的质粒pBluescriptⅡ连接，转化E.coli JM109，蓝白斑筛选得到108株菌株，通过羧甲基纤维素培养基筛选具有内切葡聚糖酶活性的菌株有74个，选取其中酶活性表达最强的阳性克隆子，质粒分析并进行测序，证明外源插入片段大小为0.7kb左右。测序并序列比较结果表明该基因片段同已发表的枯草芽孢杆菌
几丁质酶和内切葡聚糖酶编码幕因的克隆及重组芽抱杆菌的构建
glyB一aprE之间的同源性是最高的，为35%;同Bacz’了了us sP.BP23 ce1B、
B.p朋刀us内切葡聚糖酶和B.pol理vxap一1，4一内切葡聚糖酶的编码基
因的同源性只有27%。虽然同源性较低，但酶活性表达较强，认为该基因
是编码内切葡聚糖酶的一个新基因片段。
用EcoRI和Sall对含有几丁质酶基因的重组质粒TvCHI(含几丁质
酶编码基因的pMD 18一T载体)和穿梭质粒pHY300PLK进行双酶切并连
接，转化E.coli DHS。，通过几丁质培养基平板筛选得到2株转化子，提
取其中的一种，质粒酶切分析插入的外源片段同PCR产物大小接近一致。
将此重组质粒pHCHI转化巨大芽抱杆菌B.megaterium Ap25，得到两株转
化子，分别命名为p25 113一9，p251 13一10。转化子进行酶活性检测，同时
具有几丁质酶和葡聚糖酶的酶活性。根据水解圈的大小可以看到，几丁质
酶的表达活性不如E.coli DH5a中的表达活性高。平板拮抗实验同野生菌
株相比，转化子对麦长蠕抱菌的抑制作用最明显，抑制百分数最高可达
33.3%，而Apll3和Ap25分别是23.1%和25.6%，同时转化子对小麦纹
枯病菌、棉花立枯病菌、棉花枯萎病菌和小麦的全蚀病菌也具有较为明显
的抑制作用。
对小麦全蚀病和纹枯病的盆栽试验结果表明工程菌株p25 1 13一9的防
病作用强于野生菌株，尤其是对小麦全蚀病的防治作用，其防治效果同野
生菌株Ap 113和Ap25相比，分别增长了22 .54%和20%。
对芽抱杆菌几丁质酶和内切葡聚糖酶编码基因的克隆及其转化实验
表明植病生防芽袍杆菌的作用机制之一就是产生几丁质酶和葡聚糖酶这
两种抗菌物质，己证实这两种抗菌物质之间具有协同效应，这为今后的生
防菌遗传改良提供了一个新途径。
This article dealt with cloning and sequencing of chitinase and endoglucanase genes of Bacillus spp. and recombinant biocontrol isolates of Bacillus spp. by transformation with the genes.
Plant disease biocontrol bacteria Bacillus subtil is ApllS and B. megaterium Ap25 were isolated from wheat field soils collected from South Australia and Tai an. Enzyme activity analysis on chitin agar and ABP media showed that B. subtilis ApllS secreted chitinase and B. megaterium Ap25 secreted endoglucanase, respectively.
PCR methodology was adopted for cloning of chitinase encoding genes. Based on the sequences of chitinase gene from GeneBank, the primers for chitinase gene amplification were designed. PCR fragment was ligated with pMD18~T vector and transformed into E. coli DH5 a . Four colonies of transformed E. coli DH5 a with clear hydroiyzing zone on the chitin agar were obtained. The gene fragment in these isolates was identified by the methods of plasmid processing. DNA sequencing analysis showed that sequence homology between PCR fragment and chromosomal DNA of B. subtilis from 2599451 to 2812870 was 85%, and was 30% between the fragment and the genes encoding for chitinase of Bacillus (including B. subtilis) in GeneBank. It was thought to be a new gene encoding for chitinanse of B. subtilis.
Genome library methodology was adopted for cloning of endoglucanase encoding genes. Genoraic DNA of B. megaterium was partially digested by restriction enzyme Sau3A and was used to establish the genomic library in plasmid pBluscripts. Selection for endoglucanase positive clones from transformed E. coli was carried out on the CMC medium. Seventy-four clones that showed hydrolysis ability on the CMC plate were obtained. Sequencing analysis showed
that the sequence of endoglucanase fragment exhibits 35% homology with B. subtil is chromosomal DNA(from glyB to aprE), and 27% homology with Bacillus sp. BP23 celB genes, B.pwnilus endoglucanase and B. polymyxa beta-1, 4-endoglucanase 'genes, respectively. It was recognized as a new gene encoding for endoglucanase of B. mega terium.
The recombinant plasmid TvCHI (pMD18~T inserted with chitinase encoding gene from ApllS) and E. coli-Bacillus shuttle vector pHYSOOPLK were digested by EcoRI and Sail completely, and the chitinase gene was ligated with shuttle vector, and the recombinant vector was used to transform B. megaterium Ap25 competent cell. PCR and enzyme acitivity check on chitin agar showed that the chitinase gene fragment existed and expressed in the wildtype strains. Antagonistic activity test in vitro suggest that the transformants remained the ability to produce antibiotics. The recombinant strains showed an increased biocontrol efficacy against wheat take-all and Rhizoctonia sheath blight in greenhouse.
引文
[1] 陈三风，李季伦 几丁质酶的研究历史和发展前景 1993 微生物通报，20(3)：156-160
[2] 陈三风，李季伦，微生物学报，黄杆菌(Flavobacterium sp．)几丁质酶的纯化和性质，1994，34(1)：14-19
[3] 程爱丽，王益民等，枯草芽孢杆菌 B-908 几丁质酶基因的转化及表达 1996 植物病理学报 26(3)：204
[4] 董继海，董海涛，李德葆，植物抗病基因研究进展 2001 植物病理学报 31(1)1-8
[5] 杜良成等，稻瘟菌诱导的水稻几丁酶、β-1，3-葡聚糖酶活性及分布 1992 植物生理学报 18(1)29-36
[6] 冯波 微生物几丁质酶的研究概论 1998 天津师大学报 (自然科学版) 18(3)50-56
[7] 高必达 植物几丁酶的分子生物学研究进展 1996 22(6)587-602
[8) 顾真荣 马承铸 韩长安产几丁质酶芽孢杆菌对病原真菌的抑菌作用 2001 上海农业学报 17(4) 88-92
[9] 黄玉杰，杨合同，丁爱云几丁质酶和葡聚糖酶生物学特性及其编码基因的克隆和转化 2002 山东科学 15(1)28-34
[10] 林毅 陈金辉 芽孢杆菌几丁质酶及其在植物病虫害生物防治中的应用 1998
[11) 刘伊强、王雅平等 芽孢杆菌原生质体的形成、再生及种间融合的研究 1994 微生物学报 34(1)：76-80
[12] 毛世宏 β-1，3-葡聚糖酶的纯化及基因克隆 1993．中国农业大学硕士学位论文
[13] 唐文华 植物病害生物防治与遗传工程 1995 全国生物防治学术讨论会 (论文摘要集) 北京 50-56
[14] 颜子颖 王海林译 精编分子生物学实验指南 1998 科学出版社出版
[15] 杨合同 黄玉杰等 木霉菌的几丁质酶与植病生防 2003 山东科学 16(1)1-8
[16] 杨智源 陈荣忠 杨丰等，短小芽孢杆菌葡聚糖酶内切酶基因的克隆及序列测定 2001 微生物学报 41(1)77-81
[17] 王金生(编著)，分子植物病理学．北京：中国农业出版社，1999，217-220
[18] 王益民 几丁质酶和β-1，3-葡聚糖酶基因的克隆及双价基因在枯草芽孢杆菌B908中的表达．1997 中国农业大学博士学位论文
[19] 吴东儒 糖类的生物化学 1987 北京：高等教育出版社 349-352
[20] 吴江 徐虹 陈代杰等 枯草芽孢杆菌突变种 CW-06 产生β-葡聚糖酶的纯化及其性质的初步研究 1995 中国医药工业杂志 26(1)：3-4
[21] 余健秀 李建华 庞义 芽孢杆菌原生质体融合技术．2000 生物技术6月 第10卷 第3期 45-47
[22] 余荔华 刘进元 赵南明 克氏固氮菌与枯草芽孢杆菌的原生质体电融合 199 9清华大学学报(自然科学版) 06期 46-48
[23] 郑洪武等 环状芽孢杆菌C-2几丁酶基因的克隆 生物工程学报 1998年1月14(1)28-32
[24] Alam, M.M.,T. Mizutani,M. Isono,N.et al.Three chitinase genes (chiA, chic, and chiD) comprise the chitinase system of Bacllus circulans WL-12.1996.J.Ferment,Bioeng.82:28-36
[25] Anne-Marie M. and A.Macalus.Expression of Bacillus thuriengiensis δ-Endotoxin Genes during vegetative Growth.Appliend and Environmental.Microbiology.Apr.1990,p. 1128-1134
[26] Anuradha Krishnan et al.Isolation,cloning and characterization of new chitinase stored in active form in chitin-linked renom reservoir 1994 The Journal of biological chemistry. Vol. 269, No. 33, Issue of August 19, pp. 20971-20976.
[27] Ben J.C. et al. Strategies for control of fungal diseases with transgenic plants.1993 Plant Physiol.101:709-712
[28] Berhard,Leubner-Metzger et al.Antisense-transformation
reveals roles for class I β-1, 3-glucanase in tobacco seed after ripening and photodormancy 2001 Journal of experimental Botany,vol 52, NO 362, 99, 1753-1759
[29] Boiler T. et al. Plant gene research genes involved in Plant defense Springer-Verlag Wien. 1992 New York 246-255
[30] Boniss R et al. Structure of the beta-1, 3-1, 4-glucanase gene of Bacillus macerans: homologies to other beta-glucanases. 1990 Mol c Genet, 222:278-283
[31] Borriss R. et al. Molecular cloning of a gene coding for thermostable β-glucanase from Bacillus macerans. 1988 J Basic Microbiol 28:3-10
[32] Bui B H et al. Expression of Bacillus subtilis endoglucanase gene in Lactobalillus acidophilus 1990 Biotechnol, Lett, 12:919-924
[33] Chang. S et al.High frequency transformation of Bacillus subtilis protoplasts by plasmid DNA 1979 Mol. Gen. Gent. 158:111-115
[34] Chih-Min , Chien-Sheng Tsang et al. Purification , characterization and cloning of a chitinase from Bacillus sp. 2002． NCTU2． Biotechnol Appl. Biochem 35: 213-219
[35] Christopher K. et al. Isolation and sequence of an endochitinase encoding gene from a cDNA library of Trichoderma harzianum. 1994 Gene 138 143-148
[36] Claeyssens, M., and P. Tomme. 1990． Structure-function relationships of cellulolytic protions from Trichoderma reesei, p. 1-11． In C. P. Kubicek, D. E. Eveleigh, H. Esterbauer, W.Steiner, and E. M. Kubicek-Prnz(ed.), Trichoderma reesei cellulases: biochemistry, genetics, physiology and application. The Royal Society of Chemistry, Chemistry, Cambridge, United Kingdom
[37] C. Tabernero et al. Cloning and DNA sequencing of bgaA, a gene encoding an endo-beta-1, 3-1, 4-glucanase, from an alkalophilic Bacillus strain(N137) . Applied and Environmental Microbiology, Apr. 1994 Vol.60, No. 4 1213-1220
[38] Davies, G & Henrissat, B. Structures and mechanisms of glycosyl hydrolases. 1995． Structure 3, 853-859
[39] De la Cruz J Hidalgo-Gallego A, Lora J M. et al. Isolation and characterization of three chitinases from Tricboderma harzianum[J].1992 EUROPEAN Journal of Giocbemistry. (206) :859-867
[40] D.F.Martin et al. Distribution of β-glucanases within the genus Bacillus. 1998 Applied and Environmental Microbiology,Apr.1998, p. 1442-1446
[41] Draborg H, Christgau S, Halkier T, et al. Secretion of an enzymatically active Tricboderma harzianum endochitinase by Saccbaromyces cerevisiae of two exochitinases from Tricboderma harzianum [J]. 1995 Biocbemistry and Molecular Biology Interactions, (36) :781-791
[42] Duncan W.A. M. et al. Enzyme systems in Marine algae, The carbohydrase activities of unfractionated extrade of Cladophora rupestris, Laminaria digetata. 1965 Rhodymenia palmate and Ulva lactuca Biochem, J. 63:44-51
[43] Felix G, et al.Planta, Purification, immunoassay and characterization of an abundant, cytokinin-regulated polypeptide in cultured tobacco tissues. Evidence the protein is a β-1, 3-glucanase 1985． 164:423-428
[44] Fujino T. et al. Purification and properties of an endoβ-1,4-glucanase translated from a Clostridium josui gene in E.coli. 1990 Appl Environ Microb 56:1173-1178
[45] Gemma M.Escott et al. Chitinase activity in Human serum and
leukocytes. Infection and immunity. Dec.1995, p. 4770-4773
[46] George Payne et al. Isolation of complementary DNA clones encoding pathogenesis-related proteins P and Q, two acidic chitinases from tobacoo. 1990 Proc. Natl. Acad. Sci. USA, 85: 5541-545
[47] Gonzalez R. et al. Cloning, sequence analysis and yeast expression of the eglI gene from Trichoderma longibrachiatum. 1992 Appl. Microbiol. Biotechnol. 38:370-375
[48] Henrissat, B. A classi cation of glycosyl hydrolases based on amino acid sequencesimilarities.1991 Biochem J 280, 309-316
[49] Henrissat, B. &Bairoch, A. New families in the classi-cation of glycosyl hydrolases based on amino acid sequence similarities. 1993 Biochem J293, 781-788
[50] Hirofumi Okada, Kohji Tada, Tadashi Sekiya et al. Molacular characterization and Heterlogous Expression of the Gene encoding a low-Molecular-Mass Endoglucanase from Trichoderma reesei QM9414 Applied and Environmental Microbiology, Feb. 1998, p.555-563
[51] Ho-seong Lim, et al. Psedomonas stutzeri YPL-1 genetic transformation and antifungal mechanism against Fusarium solani, an agent of plant root rot. 1991． Applied and Environmental Microbiology, 57(2) :510-516
[52] HO-Genu Yoon, Hee-Yun Kin et al. Thermostable chitosanase from Bacillus sp. Strain CK4:Cloning and expression of the gene and characterization of the enzyme. 2000 Applied and Environmental microbiology, Sept p.3727-3734
[53] Jongedijk et al., Melchers LS Synergetic activity of chitinases and β-1, 3-glucanases enhances fungal resistance in transgenic tomato plants. 1995． Euphytica 85: 173-180
[54] Karen E. B. et al. Ethylene-regulated gene expression:
Molecular cloning of the genes encoding an endochitinase from Phaseolus vulgar is. 1986． Proc. Natl. Acad. Sci. USA, 83: 6820-6824
[55] Knowles, J. , P. Lethtovaara, and T.Teeri. Cellulase families and their genes. 1987 Trends Biotechnol. 5:255-261
[56] Kullnig C M, Mach R L, Lorito M, et, al. Enzyme diffusion from Trichoderma atroviride(=T. harzianumP1) to Rhizoctonia solani is a prerequisite for triggering of Trichoderma ech42 gene expression before mycoparasitic contact [J]. 2000． Applied and Environemtal Microbiology, (66) :2232-2234
[57] Lesya A. T. Chitinases of Bacillus licheniformis B-6839: Isolation and properties. 1996 Can. J. Microbiol. 42:307-315
[58] Leubner-Metzger et al. Effects of gibberellins,darkness and osmotica on endosperm rupture and class I β-1, 3-glucanase induction in tobacco seed germination. 1996． Planta,199,282-288
[59] Leubner-Metzger et al. class I β-1, 3-glucanase in the endosperm of tobacco during germination. 1995． Plant Physiology,109:751-759
[60] Leubner-Metzger et al. Functions and regulation of plant β-1, 3-glucanases ( PR-2 ). In: Datta SK Muthuk rishnan S, eds. Pathogenesis-related proteins in plants. 1999 Boca Raton, Florida: CRC Press LLC,49-76
[61] Leubner-Metzger et al. Sense transformation reveals a novel role for class Iβ-1, 3-glucanase in tobacco seed germination. 2000 The Plant Journal, 23, 215-221
[62] Linthorst HJM et al. Analysis of gene families encoding acidic and basicβ-1,3-glucanases of tobacco. 1990 Proc. Natl. Aacad. Sci. USA 87:8756-8760
[63] Logemann, L. S. et al. Synergistic activity of chitinase and
β-1, 3-glucanases enhances Fusarium resistance in transgenic tomato plants 1994 J. Cell Biochem. Suppl. 18A:88
[64] Mark Swegle et al. Identification of endochitinase cDNA clone from barley aleurone cells 1989-Plant Molecular Biology 12: 403-412
[65] Masayuki Hashimoto et al. Expression and characterization of the clitin-Binding Domain of clutinase Al from Bacillus cerculans-12． Journal of Bacteriology. 2000 June P3045-3054
[66] Mauch F. et al. Antifungal hydrolases in pea tissue I, Purification and characterization of two chitinases and two β-1, 3-glucanase differentially regulated during development and in response to fungal infection. 1988． Plant physiol. 87:325-333
[67] Mauch F. et al. Antifungal hydrolases in pea tissue Ⅱ, Inhibition of fungal growth by combinations of chitinase and β-1,3-glucanase. 1988． Plant physiol. 88:936-942
[68] Men e ndez A. L. et al. Engineering protein thermal stability. 1989． Journal Molecular Biology 206:397-406
[69] Michael J. F. et al. Cloning of two genes from Bacillus circulans WL-12 which encode β-1, 3-Glucanase activity. 1990． Journal of General Microbiology, 136:2377-2383
[70] M. Lorito et al. Chitinolytic enzymes produced by Trichoderna harzianum: antifungal activity of purified endochitinase and chitobiosidase. 1993． Phytopathology P. 302-307
[71] Nicholas C. K. et al. Cloning and Expression of an endo-1, 3-1, 4-β-Glucanase gene from Bacillus macerans in Lactobacillus reuteri. 1997 Applied and Environmental Microbiology, Aug, 1997, p. 3336-3340
[72] Olsen O. et al. Improvement of bacterialβ-glucanase thermostability by glycosylation 1990
[73] Olsen O. et al. Hybrid bacillus (1-3,1-4) β-glucanase: engineering thermostable enzyme by construction of hybrid genes. 1991 Mol. Genet 225:177-185
[74] Ordentlich A. et al. Rhizosphere colonization by Serratia marasceas for the control of Sclerotium rolfsii. 1987 Soil Biol. Biochem. 747-751
[75] Pan Ferrer et al. Nucleotide sequence of β-1, 3-Glucanase Isoenzyme IIA gene for Oerskovia xanthineolytica LLG109(Cellulomonas cellulans) and Initial Characterization of the Recombinant Enzyme Expressed in Bacillus Subtilis. 1996 Journal of Bacteriology, Aug. p.4751-4754
[76] Paul A. et al. Bioconversion of shellfish chitin wastes: Process conception and selection of microorganisms. 1978 Journal of Food Science. 43: 1158-1161
[77] Payne G. et al. Evidence for a third structural class of β-1,3-glucanase in tobacco.1990 Plant Mol. Bio. 15:797-808
[78] Pegg G. F. et al. Purification and characterization of chitinase enzymes from healthy and Verticillium albo-atrum-infected tomato plants, and from V. albo-atrum. 1982 Physiological Plant Pathology, 21: 389-409
[79] Pelletier A, Sygusch J. Purification and characterization of Three chitinase activities from Bacillus megaterium PL Appl Environ Microbiol, 1990, 56(4) :844-848
[80] Penttila, M., P. Lehtovaara, H. Nevalainen, et al.Homology between cellulase genes of Trichoderma reesei: complete nucleotide sequence of the endoglucanase I gene. 1986． Gene 45:253-263
[81] Peterbauer C, Lorito M, Hayes C K, et al. Molecular cloning and expression of nagl(Nacetyl-β-D-glucosaminidase-encoding) gene from Tricboderma harzianum P1 [J] 1996 Current Gentics,
(29) :812-820．
[82] Phillips W.R. et al. Cloning and expression of a Strptomyces plicatus chitinase (chitinase-63) in Escherichia coli 1988 The Journal of Biological Chemistry, 233(1) :443-447
[83] Qunzhu et al. Enhanced protection against fungal attack by constitutiue co-expression of ditiese and glucanese genes in transgentic gobacco. 1994． Bio/Technology, 12:807-810．
[84] Ridout C. J. et al. Enzyme activity and electrophoretic profile of extracellular protein induced in Trichoderma spp. by cell walls of Rhizoctonia solani 1986 Journal of Gerneral Microbiology, 132:2345-2352
[85] R. L. Fuchs et al. Cloningof a Serratia marcescens gene encoding chitinase 1986 Applied and Environmental Microbiology, Mar. 1986, p. 504-509
[86] RM. Mackay, A.Lo, G.Willick et al. Structure of a Bacillus subtilis endo-beta-1,4-glucanase gene Nucleic Acids Research, Vol.14,Issue 22 9159-9170
[87] Robert Leah et al. Biochemical and molecular characterization of three barley seed proteins with antifungal properties, The Journal of Biological Chemistry. 1991 266(3) : 1564-1573
[88] Saloheimo, A., B. Henrissat, A.-M. Hoffren et al. A novel, small endoglucanase gene, eg15, from Trichoderma reesei isolated by expression in yesst. 1994． Mol. Microbiol. 13:219-228
[89] Saloheimo, M., P.Lehtovaara, M. Penttila et al. EGⅢ, a new endoglucanase from Trichoderma reesei: the characterization of both gene and enzyme. 1988． Gene 63:11-21
[90] Sela-Buurlage M. B. et al.Only specific tobacco(Nicotiana tabacum) chitinase and β-1,3-glucanase exhibit antifungal activity. 1993 Plant physiol. 101:857-863
[91] Shinshi et al. Evidence for N-and C-terminal processing of a
plant defense-related enzyme: Primary structure of tobacco prepro-β-1, 3-glucanase. 1988 Pro. Acad. Sci. USA 85:5541-5545
[92] Shoemaker, S. , V. Schweickaut, M. Ladner et al. Molecular cloning of exocellobiohydrolase I derived from Trichoderma reesei strain L27． 1983． Bio/Technology 1:691-696
[93] Shufen Liu et al. The study on endophytic Streptomyces of cotton advances in biological control of plant diseases. 1996． China Agricultureal University Press, Beijing China 212-213
[94] Sietsma J.H.,Wessels JGH . Evidence for covalent linkage between chitin and β-glucan in a fungal wall. 1979． J Gen Microbiol 114:99-108
[95] Sperisen C. et al. Comparison of cloned genes provides evidence for intergenomic exchange of DNA in the evolution of a tobacco glucan endo-1, 3-β-glucanase gene family. 1991． Proc. Natl. Acad. Sci. USA 88:1820-1824
[96] Takeshi Watanabe et al. Gene cloning of chitinase Al from Bacillus circulans WL-12 revealed its evolutionary relationship to Serratia chitinase and to the type Ⅲ homology units of fibronectin. The Journal of biological chemistry. Issue of Sept. 1990． Vol. 265, No. 26, pp.15659-15665
[97] Takeshi Watanabe et al. Family 19 chitinases of Streptomyces species:characterization and distribution. 1999． Microbiology, 145, 3353-3363
[98] Teeri, T. , M. Penttila, S. Keranen et al. Structure function and genetics of cellulases, 1999． p.419-447． in C.Ball and D. Finkelstein(ed.), Biotechnology of filamentous fungi. Butterworth, Uondon, United Kingdom.
[99] Teeri, T. T., P.Lehtovaara, S.Kauppinen et al. Homologous domain in Trichoderma reesei cellulolytic enzymes: gene
sequence and expression of cellobiohydrolase Ⅱ. 1987 Gene 51:43-52
[100] Thierry Lonhienne et al.Cloning, Sequences and characterization of two chitinase genes from the Antarctic Arthrobacter sp. Strain TAD20:Isolation and Partial characterization of the enzymes. 2001． Journal of Bacteriology, Mar. 2001, p. 1773-1779
[101] Thomas Keitel et al. Molecular and active-site structure of a Bacillus 1,3-1,4-β-glucanase. 1993 Proc. Natl. Acad. Sci.USA. Vol. 90, pp. 5287-5291, June
[102] Trachuk L A, Revina LP, Shemyakina TM et al. Chitinases of Bacillus licheniformis B-6839: Isolation and Properties. Canadian J Microbiol, 1996, 42(4) :307-305
[103] Van Arsdell, J. N., S. Kwok, V. L. Schweickart et al. Cloning, characterization and expression in Saccharomvces cerevisiae of endoglucanase I from Trichoderma reesei. 1987． Bio/Technology 5:60-64
[104] Watanabe, T., W. Oyanagi, K. Suzuki, et al. Chitinase system of Bacillus circulans WL-12 and importance of chitinase Al in chitin degradation. 1990． J. Bacterial. 172:4017-4022
[105] Watanabe, T., Oyanagi, W., Suzuki, K., Ohnishi, K., and Tanaka, H. Structure of the gene encoding chitinase D of Bacillus circulans WL-12 and possible homology of the enzyme to other prokaryotic chitinase and class Ⅲ plant chitinases. 1992． J. Bacteriol. 174:408-414．
[106] Watanabe T, Kobori K, Miyashita K, Fujii et al. Identification of glutamic acid 204 and aspartic acid 200 in chitinase Al of Bacillus circulans WL-12 as essential residues for chitinase activity. J Biol Chem 1993 Sep 5;268 (25) :18567-72
[107] Ward E. R. et al. Differential regulation of β-1, 3-glucanase messenger RNAs in response to pathogen infection. 1991． Plant Physiol. 96:390-397
[108] Zhu Q. et al. Enhanced protection against fungal attack by constitutive w-expression of chitinase and glucanase genes in transgenic tobacco. 1994． Bio/Techology, 12:807-812
[2] 陈三风，李季伦，微生物学报，黄杆菌(Flavobacterium sp．)几丁质酶的纯化和性质，1994，34(1)：14-19
[3] 程爱丽，王益民等，枯草芽孢杆菌 B-908 几丁质酶基因的转化及表达 1996 植物病理学报 26(3)：204
[4] 董继海，董海涛，李德葆，植物抗病基因研究进展 2001 植物病理学报 31(1)1-8
[5] 杜良成等，稻瘟菌诱导的水稻几丁酶、β-1，3-葡聚糖酶活性及分布 1992 植物生理学报 18(1)29-36
[6] 冯波 微生物几丁质酶的研究概论 1998 天津师大学报 (自然科学版) 18(3)50-56
[7] 高必达 植物几丁酶的分子生物学研究进展 1996 22(6)587-602
[8) 顾真荣 马承铸 韩长安产几丁质酶芽孢杆菌对病原真菌的抑菌作用 2001 上海农业学报 17(4) 88-92
[9] 黄玉杰，杨合同，丁爱云几丁质酶和葡聚糖酶生物学特性及其编码基因的克隆和转化 2002 山东科学 15(1)28-34
[10] 林毅 陈金辉 芽孢杆菌几丁质酶及其在植物病虫害生物防治中的应用 1998
[11) 刘伊强、王雅平等 芽孢杆菌原生质体的形成、再生及种间融合的研究 1994 微生物学报 34(1)：76-80
[12] 毛世宏 β-1，3-葡聚糖酶的纯化及基因克隆 1993．中国农业大学硕士学位论文
[13] 唐文华 植物病害生物防治与遗传工程 1995 全国生物防治学术讨论会 (论文摘要集) 北京 50-56
[14] 颜子颖 王海林译 精编分子生物学实验指南 1998 科学出版社出版
[15] 杨合同 黄玉杰等 木霉菌的几丁质酶与植病生防 2003 山东科学 16(1)1-8
[16] 杨智源 陈荣忠 杨丰等，短小芽孢杆菌葡聚糖酶内切酶基因的克隆及序列测定 2001 微生物学报 41(1)77-81
[17] 王金生(编著)，分子植物病理学．北京：中国农业出版社，1999，217-220
[18] 王益民 几丁质酶和β-1，3-葡聚糖酶基因的克隆及双价基因在枯草芽孢杆菌B908中的表达．1997 中国农业大学博士学位论文
[19] 吴东儒 糖类的生物化学 1987 北京：高等教育出版社 349-352
[20] 吴江 徐虹 陈代杰等 枯草芽孢杆菌突变种 CW-06 产生β-葡聚糖酶的纯化及其性质的初步研究 1995 中国医药工业杂志 26(1)：3-4
[21] 余健秀 李建华 庞义 芽孢杆菌原生质体融合技术．2000 生物技术6月 第10卷 第3期 45-47
[22] 余荔华 刘进元 赵南明 克氏固氮菌与枯草芽孢杆菌的原生质体电融合 199 9清华大学学报(自然科学版) 06期 46-48
[23] 郑洪武等 环状芽孢杆菌C-2几丁酶基因的克隆 生物工程学报 1998年1月14(1)28-32
[24] Alam, M.M.,T. Mizutani,M. Isono,N.et al.Three chitinase genes (chiA, chic, and chiD) comprise the chitinase system of Bacllus circulans WL-12.1996.J.Ferment,Bioeng.82:28-36
[25] Anne-Marie M. and A.Macalus.Expression of Bacillus thuriengiensis δ-Endotoxin Genes during vegetative Growth.Appliend and Environmental.Microbiology.Apr.1990,p. 1128-1134
[26] Anuradha Krishnan et al.Isolation,cloning and characterization of new chitinase stored in active form in chitin-linked renom reservoir 1994 The Journal of biological chemistry. Vol. 269, No. 33, Issue of August 19, pp. 20971-20976.
[27] Ben J.C. et al. Strategies for control of fungal diseases with transgenic plants.1993 Plant Physiol.101:709-712
[28] Berhard,Leubner-Metzger et al.Antisense-transformation
reveals roles for class I β-1, 3-glucanase in tobacco seed after ripening and photodormancy 2001 Journal of experimental Botany,vol 52, NO 362, 99, 1753-1759
[29] Boiler T. et al. Plant gene research genes involved in Plant defense Springer-Verlag Wien. 1992 New York 246-255
[30] Boniss R et al. Structure of the beta-1, 3-1, 4-glucanase gene of Bacillus macerans: homologies to other beta-glucanases. 1990 Mol c Genet, 222:278-283
[31] Borriss R. et al. Molecular cloning of a gene coding for thermostable β-glucanase from Bacillus macerans. 1988 J Basic Microbiol 28:3-10
[32] Bui B H et al. Expression of Bacillus subtilis endoglucanase gene in Lactobalillus acidophilus 1990 Biotechnol, Lett, 12:919-924
[33] Chang. S et al.High frequency transformation of Bacillus subtilis protoplasts by plasmid DNA 1979 Mol. Gen. Gent. 158:111-115
[34] Chih-Min , Chien-Sheng Tsang et al. Purification , characterization and cloning of a chitinase from Bacillus sp. 2002． NCTU2． Biotechnol Appl. Biochem 35: 213-219
[35] Christopher K. et al. Isolation and sequence of an endochitinase encoding gene from a cDNA library of Trichoderma harzianum. 1994 Gene 138 143-148
[36] Claeyssens, M., and P. Tomme. 1990． Structure-function relationships of cellulolytic protions from Trichoderma reesei, p. 1-11． In C. P. Kubicek, D. E. Eveleigh, H. Esterbauer, W.Steiner, and E. M. Kubicek-Prnz(ed.), Trichoderma reesei cellulases: biochemistry, genetics, physiology and application. The Royal Society of Chemistry, Chemistry, Cambridge, United Kingdom
[37] C. Tabernero et al. Cloning and DNA sequencing of bgaA, a gene encoding an endo-beta-1, 3-1, 4-glucanase, from an alkalophilic Bacillus strain(N137) . Applied and Environmental Microbiology, Apr. 1994 Vol.60, No. 4 1213-1220
[38] Davies, G & Henrissat, B. Structures and mechanisms of glycosyl hydrolases. 1995． Structure 3, 853-859
[39] De la Cruz J Hidalgo-Gallego A, Lora J M. et al. Isolation and characterization of three chitinases from Tricboderma harzianum[J].1992 EUROPEAN Journal of Giocbemistry. (206) :859-867
[40] D.F.Martin et al. Distribution of β-glucanases within the genus Bacillus. 1998 Applied and Environmental Microbiology,Apr.1998, p. 1442-1446
[41] Draborg H, Christgau S, Halkier T, et al. Secretion of an enzymatically active Tricboderma harzianum endochitinase by Saccbaromyces cerevisiae of two exochitinases from Tricboderma harzianum [J]. 1995 Biocbemistry and Molecular Biology Interactions, (36) :781-791
[42] Duncan W.A. M. et al. Enzyme systems in Marine algae, The carbohydrase activities of unfractionated extrade of Cladophora rupestris, Laminaria digetata. 1965 Rhodymenia palmate and Ulva lactuca Biochem, J. 63:44-51
[43] Felix G, et al.Planta, Purification, immunoassay and characterization of an abundant, cytokinin-regulated polypeptide in cultured tobacco tissues. Evidence the protein is a β-1, 3-glucanase 1985． 164:423-428
[44] Fujino T. et al. Purification and properties of an endoβ-1,4-glucanase translated from a Clostridium josui gene in E.coli. 1990 Appl Environ Microb 56:1173-1178
[45] Gemma M.Escott et al. Chitinase activity in Human serum and
leukocytes. Infection and immunity. Dec.1995, p. 4770-4773
[46] George Payne et al. Isolation of complementary DNA clones encoding pathogenesis-related proteins P and Q, two acidic chitinases from tobacoo. 1990 Proc. Natl. Acad. Sci. USA, 85: 5541-545
[47] Gonzalez R. et al. Cloning, sequence analysis and yeast expression of the eglI gene from Trichoderma longibrachiatum. 1992 Appl. Microbiol. Biotechnol. 38:370-375
[48] Henrissat, B. A classi cation of glycosyl hydrolases based on amino acid sequencesimilarities.1991 Biochem J 280, 309-316
[49] Henrissat, B. &Bairoch, A. New families in the classi-cation of glycosyl hydrolases based on amino acid sequence similarities. 1993 Biochem J293, 781-788
[50] Hirofumi Okada, Kohji Tada, Tadashi Sekiya et al. Molacular characterization and Heterlogous Expression of the Gene encoding a low-Molecular-Mass Endoglucanase from Trichoderma reesei QM9414 Applied and Environmental Microbiology, Feb. 1998, p.555-563
[51] Ho-seong Lim, et al. Psedomonas stutzeri YPL-1 genetic transformation and antifungal mechanism against Fusarium solani, an agent of plant root rot. 1991． Applied and Environmental Microbiology, 57(2) :510-516
[52] HO-Genu Yoon, Hee-Yun Kin et al. Thermostable chitosanase from Bacillus sp. Strain CK4:Cloning and expression of the gene and characterization of the enzyme. 2000 Applied and Environmental microbiology, Sept p.3727-3734
[53] Jongedijk et al., Melchers LS Synergetic activity of chitinases and β-1, 3-glucanases enhances fungal resistance in transgenic tomato plants. 1995． Euphytica 85: 173-180
[54] Karen E. B. et al. Ethylene-regulated gene expression:
Molecular cloning of the genes encoding an endochitinase from Phaseolus vulgar is. 1986． Proc. Natl. Acad. Sci. USA, 83: 6820-6824
[55] Knowles, J. , P. Lethtovaara, and T.Teeri. Cellulase families and their genes. 1987 Trends Biotechnol. 5:255-261
[56] Kullnig C M, Mach R L, Lorito M, et, al. Enzyme diffusion from Trichoderma atroviride(=T. harzianumP1) to Rhizoctonia solani is a prerequisite for triggering of Trichoderma ech42 gene expression before mycoparasitic contact [J]. 2000． Applied and Environemtal Microbiology, (66) :2232-2234
[57] Lesya A. T. Chitinases of Bacillus licheniformis B-6839: Isolation and properties. 1996 Can. J. Microbiol. 42:307-315
[58] Leubner-Metzger et al. Effects of gibberellins,darkness and osmotica on endosperm rupture and class I β-1, 3-glucanase induction in tobacco seed germination. 1996． Planta,199,282-288
[59] Leubner-Metzger et al. class I β-1, 3-glucanase in the endosperm of tobacco during germination. 1995． Plant Physiology,109:751-759
[60] Leubner-Metzger et al. Functions and regulation of plant β-1, 3-glucanases ( PR-2 ). In: Datta SK Muthuk rishnan S, eds. Pathogenesis-related proteins in plants. 1999 Boca Raton, Florida: CRC Press LLC,49-76
[61] Leubner-Metzger et al. Sense transformation reveals a novel role for class Iβ-1, 3-glucanase in tobacco seed germination. 2000 The Plant Journal, 23, 215-221
[62] Linthorst HJM et al. Analysis of gene families encoding acidic and basicβ-1,3-glucanases of tobacco. 1990 Proc. Natl. Aacad. Sci. USA 87:8756-8760
[63] Logemann, L. S. et al. Synergistic activity of chitinase and
β-1, 3-glucanases enhances Fusarium resistance in transgenic tomato plants 1994 J. Cell Biochem. Suppl. 18A:88
[64] Mark Swegle et al. Identification of endochitinase cDNA clone from barley aleurone cells 1989-Plant Molecular Biology 12: 403-412
[65] Masayuki Hashimoto et al. Expression and characterization of the clitin-Binding Domain of clutinase Al from Bacillus cerculans-12． Journal of Bacteriology. 2000 June P3045-3054
[66] Mauch F. et al. Antifungal hydrolases in pea tissue I, Purification and characterization of two chitinases and two β-1, 3-glucanase differentially regulated during development and in response to fungal infection. 1988． Plant physiol. 87:325-333
[67] Mauch F. et al. Antifungal hydrolases in pea tissue Ⅱ, Inhibition of fungal growth by combinations of chitinase and β-1,3-glucanase. 1988． Plant physiol. 88:936-942
[68] Men e ndez A. L. et al. Engineering protein thermal stability. 1989． Journal Molecular Biology 206:397-406
[69] Michael J. F. et al. Cloning of two genes from Bacillus circulans WL-12 which encode β-1, 3-Glucanase activity. 1990． Journal of General Microbiology, 136:2377-2383
[70] M. Lorito et al. Chitinolytic enzymes produced by Trichoderna harzianum: antifungal activity of purified endochitinase and chitobiosidase. 1993． Phytopathology P. 302-307
[71] Nicholas C. K. et al. Cloning and Expression of an endo-1, 3-1, 4-β-Glucanase gene from Bacillus macerans in Lactobacillus reuteri. 1997 Applied and Environmental Microbiology, Aug, 1997, p. 3336-3340
[72] Olsen O. et al. Improvement of bacterialβ-glucanase thermostability by glycosylation 1990
[73] Olsen O. et al. Hybrid bacillus (1-3,1-4) β-glucanase: engineering thermostable enzyme by construction of hybrid genes. 1991 Mol. Genet 225:177-185
[74] Ordentlich A. et al. Rhizosphere colonization by Serratia marasceas for the control of Sclerotium rolfsii. 1987 Soil Biol. Biochem. 747-751
[75] Pan Ferrer et al. Nucleotide sequence of β-1, 3-Glucanase Isoenzyme IIA gene for Oerskovia xanthineolytica LLG109(Cellulomonas cellulans) and Initial Characterization of the Recombinant Enzyme Expressed in Bacillus Subtilis. 1996 Journal of Bacteriology, Aug. p.4751-4754
[76] Paul A. et al. Bioconversion of shellfish chitin wastes: Process conception and selection of microorganisms. 1978 Journal of Food Science. 43: 1158-1161
[77] Payne G. et al. Evidence for a third structural class of β-1,3-glucanase in tobacco.1990 Plant Mol. Bio. 15:797-808
[78] Pegg G. F. et al. Purification and characterization of chitinase enzymes from healthy and Verticillium albo-atrum-infected tomato plants, and from V. albo-atrum. 1982 Physiological Plant Pathology, 21: 389-409
[79] Pelletier A, Sygusch J. Purification and characterization of Three chitinase activities from Bacillus megaterium PL Appl Environ Microbiol, 1990, 56(4) :844-848
[80] Penttila, M., P. Lehtovaara, H. Nevalainen, et al.Homology between cellulase genes of Trichoderma reesei: complete nucleotide sequence of the endoglucanase I gene. 1986． Gene 45:253-263
[81] Peterbauer C, Lorito M, Hayes C K, et al. Molecular cloning and expression of nagl(Nacetyl-β-D-glucosaminidase-encoding) gene from Tricboderma harzianum P1 [J] 1996 Current Gentics,
(29) :812-820．
[82] Phillips W.R. et al. Cloning and expression of a Strptomyces plicatus chitinase (chitinase-63) in Escherichia coli 1988 The Journal of Biological Chemistry, 233(1) :443-447
[83] Qunzhu et al. Enhanced protection against fungal attack by constitutiue co-expression of ditiese and glucanese genes in transgentic gobacco. 1994． Bio/Technology, 12:807-810．
[84] Ridout C. J. et al. Enzyme activity and electrophoretic profile of extracellular protein induced in Trichoderma spp. by cell walls of Rhizoctonia solani 1986 Journal of Gerneral Microbiology, 132:2345-2352
[85] R. L. Fuchs et al. Cloningof a Serratia marcescens gene encoding chitinase 1986 Applied and Environmental Microbiology, Mar. 1986, p. 504-509
[86] RM. Mackay, A.Lo, G.Willick et al. Structure of a Bacillus subtilis endo-beta-1,4-glucanase gene Nucleic Acids Research, Vol.14,Issue 22 9159-9170
[87] Robert Leah et al. Biochemical and molecular characterization of three barley seed proteins with antifungal properties, The Journal of Biological Chemistry. 1991 266(3) : 1564-1573
[88] Saloheimo, A., B. Henrissat, A.-M. Hoffren et al. A novel, small endoglucanase gene, eg15, from Trichoderma reesei isolated by expression in yesst. 1994． Mol. Microbiol. 13:219-228
[89] Saloheimo, M., P.Lehtovaara, M. Penttila et al. EGⅢ, a new endoglucanase from Trichoderma reesei: the characterization of both gene and enzyme. 1988． Gene 63:11-21
[90] Sela-Buurlage M. B. et al.Only specific tobacco(Nicotiana tabacum) chitinase and β-1,3-glucanase exhibit antifungal activity. 1993 Plant physiol. 101:857-863
[91] Shinshi et al. Evidence for N-and C-terminal processing of a
plant defense-related enzyme: Primary structure of tobacco prepro-β-1, 3-glucanase. 1988 Pro. Acad. Sci. USA 85:5541-5545
[92] Shoemaker, S. , V. Schweickaut, M. Ladner et al. Molecular cloning of exocellobiohydrolase I derived from Trichoderma reesei strain L27． 1983． Bio/Technology 1:691-696
[93] Shufen Liu et al. The study on endophytic Streptomyces of cotton advances in biological control of plant diseases. 1996． China Agricultureal University Press, Beijing China 212-213
[94] Sietsma J.H.,Wessels JGH . Evidence for covalent linkage between chitin and β-glucan in a fungal wall. 1979． J Gen Microbiol 114:99-108
[95] Sperisen C. et al. Comparison of cloned genes provides evidence for intergenomic exchange of DNA in the evolution of a tobacco glucan endo-1, 3-β-glucanase gene family. 1991． Proc. Natl. Acad. Sci. USA 88:1820-1824
[96] Takeshi Watanabe et al. Gene cloning of chitinase Al from Bacillus circulans WL-12 revealed its evolutionary relationship to Serratia chitinase and to the type Ⅲ homology units of fibronectin. The Journal of biological chemistry. Issue of Sept. 1990． Vol. 265, No. 26, pp.15659-15665
[97] Takeshi Watanabe et al. Family 19 chitinases of Streptomyces species:characterization and distribution. 1999． Microbiology, 145, 3353-3363
[98] Teeri, T. , M. Penttila, S. Keranen et al. Structure function and genetics of cellulases, 1999． p.419-447． in C.Ball and D. Finkelstein(ed.), Biotechnology of filamentous fungi. Butterworth, Uondon, United Kingdom.
[99] Teeri, T. T., P.Lehtovaara, S.Kauppinen et al. Homologous domain in Trichoderma reesei cellulolytic enzymes: gene
sequence and expression of cellobiohydrolase Ⅱ. 1987 Gene 51:43-52
[100] Thierry Lonhienne et al.Cloning, Sequences and characterization of two chitinase genes from the Antarctic Arthrobacter sp. Strain TAD20:Isolation and Partial characterization of the enzymes. 2001． Journal of Bacteriology, Mar. 2001, p. 1773-1779
[101] Thomas Keitel et al. Molecular and active-site structure of a Bacillus 1,3-1,4-β-glucanase. 1993 Proc. Natl. Acad. Sci.USA. Vol. 90, pp. 5287-5291, June
[102] Trachuk L A, Revina LP, Shemyakina TM et al. Chitinases of Bacillus licheniformis B-6839: Isolation and Properties. Canadian J Microbiol, 1996, 42(4) :307-305
[103] Van Arsdell, J. N., S. Kwok, V. L. Schweickart et al. Cloning, characterization and expression in Saccharomvces cerevisiae of endoglucanase I from Trichoderma reesei. 1987． Bio/Technology 5:60-64
[104] Watanabe, T., W. Oyanagi, K. Suzuki, et al. Chitinase system of Bacillus circulans WL-12 and importance of chitinase Al in chitin degradation. 1990． J. Bacterial. 172:4017-4022
[105] Watanabe, T., Oyanagi, W., Suzuki, K., Ohnishi, K., and Tanaka, H. Structure of the gene encoding chitinase D of Bacillus circulans WL-12 and possible homology of the enzyme to other prokaryotic chitinase and class Ⅲ plant chitinases. 1992． J. Bacteriol. 174:408-414．
[106] Watanabe T, Kobori K, Miyashita K, Fujii et al. Identification of glutamic acid 204 and aspartic acid 200 in chitinase Al of Bacillus circulans WL-12 as essential residues for chitinase activity. J Biol Chem 1993 Sep 5;268 (25) :18567-72
[107] Ward E. R. et al. Differential regulation of β-1, 3-glucanase messenger RNAs in response to pathogen infection. 1991． Plant Physiol. 96:390-397
[108] Zhu Q. et al. Enhanced protection against fungal attack by constitutive w-expression of chitinase and glucanase genes in transgenic tobacco. 1994． Bio/Techology, 12:807-812