【作者】 黄玉杰；

【导师】 丁爱云；

【作者基本信息】 山东农业大学 ， 植物病理学， 2003， 硕士

【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。更多还原

【Abstract】 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 showedthat 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.更多还原