通过导入几丁质酶基因提高巨大芽孢杆菌的生防效果  

Improving Biocontrol Effect of Bacillus megterium (Bacillaceae) on Plant Disease through Genetic Modification with Chitinase Gene

作　　者：张新建[1] 黄玉杰[1] 杨合同[1,2] 任艳[1] 陈凯[1] 

机构地区：[1]山东省科学院中日友好生物技术研究中心,山东省应用微生物重点实验室,山东济南250014 [2]山东理工大学生命科学学院,山东淄博255O49

出　　处：《云南植物研究》2007年第6期666-670,共5页Acta Botanica Yunnanica

基　　金：国家"863"计划现代农业技术领域重大项目资助(2006AA10A211)

摘　　要：以质粒pMSDChi113为模板,经PCR扩增,获得了几丁质酶基因1.8 kb DNA片段,将该基因与大肠杆菌-芽孢杆菌穿梭质粒pHY300PLK连接,获得重组质粒pHYChi113。重组质粒经芽孢杆菌感受态方法转入巨大芽孢杆菌(Bacillus megaterium) Ap25中,获得的工程菌株B.megateriums Ap25-chi113同时具有几丁质酶和内切葡聚糖酶酶活。平板对峙实验结果表明,工程菌株对病原真菌Rhizoctonia solani,Coniothyrium fuckellii,Fusarium graminearum,Macrophoma kawatsukai,Fusarium oxysporium,Botrytis cinerea,Rhizoctonia cerea-lis,Colletotrichum gloeosporioides,Bipolarls sorohlulana的拮抗性能明显提高,尤其是对Coniothyrium fuckellii的抑制率相对于野生菌Ap25提高了13%以上。盆栽防病实验结果表明,工程菌株显著降低纹枯病菌(Rhi-zotonia cerealis)对小麦(Triticum aestivum L.)及枯萎病菌(Fusarium oxysporum)对棉花(Gossypium hirsutumLinn.)的致病能力,对这两种病原菌的防效分别是70.34 %和58.51 %,同原始菌株相比,防效分别提高了27.54 %和21.28 %。Bacillus megaterium Ap25 which can produce endoglucanase was proved as a biocontrol agent of plant disease. The aim of this study is to enhance the biocontrol effect of this strain by introduction of a chitinase gene. A 1.8 kb DNA fragment containing chitinase gene and SD sequence was amplified with the template pMSDChill3 and was inserted into shuttle vector pHY300PLK to construct a new plasmid, pHYChill3. The recombinant plasmid was transformed into B. megterium, resulting in a new strain named Ap25-chi113. Chitin plate culture and PCR amplification confirmed that Ap25- chi113 contained a functional chitinase gene. Comparing with the wild type Ap25 in dural culture experiment, Ap25-chi113 increased its effect against the pathogenic fungi. Especially, the inhibition percentage against Coniothyrium fuckellii increased about 13%. In pot experiment, Ap25-chi113 also increased the effect on suppression of wheat sheath blight and cotton Fusarium wilt caused by Rhizotonia cerealis and Fusarium oxysporum respectively, comparing with the wild strain.

关 键 词：几丁质酶 芽孢杆菌 穿梭质粒 转化 

分 类 号：S476[农业科学—农业昆虫与害虫防治]