【作者】 郑爱萍；

【导师】 李平；

【作者基本信息】 四川农业大学 ， 作物遗传育种， 2004， 博士

【摘要】 微生物可作为研究生物学的理想材料，同时由于具有代谢能力强、功能各具特色、产物多种多样，在农业、医学、食品以及许多工业中的应用也历来受到人们的关注。特别是农用微生物资源，可进行微生物农药、微生物肥料的产业化。随着经济的发展，人们对绿色无公害食品的需求越来越大，化学农药的一些高残留、高毒性、破坏生态环境的特点被认识到，基于此种状况，所以有必要探求新型的生物农药，以期能为保持生态平衡，保护人类的健康，为农业可持续发展打下基础。 本研究在四川生态条件下，针对特有的农用微生物资源，进行了防病、杀虫因子的分离以及结构解析研究，希望能为四川生态条件下微生物源农药的产业化提供条件和理论基础。主要结果如下： 一、农用抗生素的研究 1．从土壤中筛选土壤链霉菌，通过反复纯化，以水稻稻瘟病、水稻纹枯病病原菌作为指示菌，在PDA平板上与病原菌进行对峙培养，共获得150株对病原菌有不同程度抑制作用的链霉菌株。根据对病原菌的抑制效果，共获得4株链霉菌菌株，分别编号为S-21、S-25、S-26、S-34。 2．通过生理、生化检测和形态鉴定，S-21、S-25、S34、S-26菌株分别被确定为Streptomyces flavescens、Streptomyces luteogriseus、Streptomyces lavendularectus、Streptomyces fradiae var．Sichuan。 3．抑菌谱、杀虫谱测定结果表明，菌株对14种病原菌具有不同程度的抑制作用，综合抑制效果，S-26对水稻病害和蔬菜病害病原菌具有比较明显的抑制优势，具有广泛的抑菌谱带，发酵菌液的上清液，饲喂害虫，利用发酵原液作为对照，S2-26、S2-25对于供试害虫具有较好的杀虫效果，特别是S-26处理后，草地蝗虫、十八星瓢虫、螨虫的校正死亡率均在82％以上，最高可达98.2％。 4．发酵液有效成分稳定性的检测结果表明，利用S2-26菌株在普通发酵培养基，收集发酵液，经过120℃30min，分别在PH8、PH6的作用，紫外线照射30min，发酵液与水稻纹枯病原菌拮抗作用，仍然具有强拮抗活性，抑菌圈直径为6.0-5.5cm，与对照差异不明显，表明S2-26的有效次生代谢产物具有稳定的特性。 5．发酵配方和发酵条件实验表明，菌株在碳源为10％淀粉、氮源为2％的花生饼粉，培养条件在28℃条件、转速为250r／min、5％接种量、起始PH为5时、加入优化培养基150ml、一级种子的培养时间为72h、发酵时间为144h的条件下，可以获得最好的抑菌效果，且整个发酵过程中以一次性加入各培养基成分为效果最好。 6．田间试验表明，应用上述优化配方进行发酵，收集发酵菌液，进行大田防治水稻纹枯病，田间防效达到50.2％，高出井冈霉素和清水对照，对茄十八星瓢虫试验表明，对瓢虫的防效达到78.5%。高于清水对照，并对平均结实个数和结实率具有促进作用，高于其它对照。针对编号为S一26的菌株并进行了发酵生物学特性的研究，并经过发酵产物的分离、纯化，通过IH一NMR、1 3C一MR、红外、紫外、质谱、H一HCOSY、H一CCOSY、HMBC等测定了asperufoside、pinoresino、eseuletin、magnolioside、52、536种化合物的结构，通过功能测定，52、S3化合物为功能性化合物，结构解析表明，是一类杀虫、防病的大环内醋类抗生素。经过三维结构模拟和功能预测，该类化合物可能通过阻断虫体神经信号传导而达到杀虫效果的。二、芽抱类杀虫基因的研究1.本研究在四力}生态条件下，对芽抱杆菌进行分离筛选，共获得279株，经过特异引物 对基因型进行分析，结果表明107株菌株具有明显的多态性2.多态性分析表明，所分析的资源含有4种复合基因型的比例高，对鞘翅目有特异作用 的芽抱杆菌资源最多，聚类分析表明，具有十分多样的基因分布特点。3.通过生理生化检测和形态指标的测定，确定了其中为非Bt芽抱杆菌2、4一14、39、D3 菌株分别为刀acizzusfasrs成osus、刀aeszzus刀rmus、刀aeillus cereos、Baeillus me卯terium菌 类，确定了s、sL、不SS菌株分别为Bacillus thringglensis var tolworthi、Bacillus thringiensis var刀nitimus、Baeillus thringiensis var entomocidus、Bacillus rhingiensis var galleriae4类苏云金芽抱杆菌的分类地位。4.对2种D3、39芽饱杆菌基因进行测序分析、比较，结果表明，cry1Ab类基因与报道 的基因一致，cry1Ac、cry1C类基因与报道的基因核昔酸具有一定的同源性，分别为 87%、89%和78%，氨基酸序列比较:同源性分别为76.5%、79.8%、51%，不同菌株 种类之间cry IA。类之间核昔酸同源性为90%，氨基酸的同源性为85%。5.通过PcR克隆方法获得cry1Ab基因的全长序列。6.通过饲喂、小区实验，测定了菌株的杀虫谱和毒力方程。大田防效测定表明，该类与 所报道的cry基因具有一定同源性的菌株具有比很好的防治效果，最高防虫效果可达 到98%，显著高于对照和Bt悬浮制剂，并显示对鳞翅目、鞘翅目害虫较宽的杀虫谱。7.对cry类基因进行了定位和毒性蛋白质的分离、纯化，同时，还进行生物学特性等方 面的研究。三、抑菌肤研究1.从黄瓜植株的根围土壤中分离到有拮抗性的细菌100多株。通过对峙法?更多还原

【Abstract】 Being their special characters, microorganisms are used as good research subjects. Recently the application about them in agriculture, food etc fields is paid more attention. Especially as agricultural resources, many relative industrial can be developed. With the development of economics, many green food, green pesticide, green fertilizer were needed. So, it is essential to do some research work about the pesticide from biology to keep the balance of nature, keep the health of human well and the agriculture continual development.On our research, many factors that control the plant diseases and pests from microorganisms were screened and researched about function and structure in Sichuan, the west area of China. We hope that the research can be as a special condition and theory basis for pesticide production from these special organisms.The main results are summarized as below.Study on the agricultural antibiotics1. Many streptomyces were screened from the soil. After purified, they were used to cultured against the Rhizoctonia solani AG-K Magnaporthe griesa B-13 on the PDA plate. 150 strains were got through this way. As to the controlling effects and growth conditions, 4 strains, S-21 S-25 S-26 S-34were used to researched more.2. From physiological and biochemical characters and configuration character of the strains, S-21 S-25 S-26 S-34 were identified as Streptomyces flavescens Streptomyces luteogriseus Streptomyces lavendularectus Streptomyces fradiae var. Sichuan respectively.3. It showed that the strains inhibit the growth against 14 common pathogens and pests through tests.S-26 strain showed the strong antagonistic abilities against diseases and had wider abilities controlling diseases and pests. The highest death rate could reach 98.2%. When heated in 120C for 30 min, done in PH8 and PH6 and under UV for 30min, the effective fermentations were tested stable and not changed. The inhibit diameter could be 6.0-5.5cm still and same to CK almost.4. S-26 strain could grow best under the fermentation conditions, t10%strach as carbon resource, 2% pea nut powder as nitrogen resource , 28C, 250r/m, PH5, 144h as fermentation time, 150ml as culturing were needed. At the same time, all nutrition should be put together only at the beginning.5. The result showed that the control effect against R Sonali can reach 50.2%, higher than ginggangmycin and CK. The control effect was 78.5% against ladybug. In addition, the yield could be added.6. Six compounds from fermentations were screened through 1H-NMR, 13C-NMR, EI, H-HCOSY, H-CCOS, HMBC, UV etc ways. Among them S2, S3 were as kinds of antibiotics just like tetranactin and nonactin. Through 3D and functional predicting, thecompounds effected on the pests by inhibiting nerve signal transduction probably.Study on the killing pests genes of Bacillus1. Two hundred and seventy-nine Bacillus strains were isolated from ninety-four soils samples collected from in Sichuan. Among them 107 strains showed the abilities against crop pests.2. Based on PCR and gene analysis, four genes were contained mostly in the all recourse. The genes against Cleoptera were the most, then Lvepidoptera. At the same time, it showed many genes types.3. From physiological and biochemical characters and configuration character of the strains, 2, 4-14, 39, D3 strains were defined as Bacillus fastidiosus Bacillus firmus Bacillus cereus Bacillus megaterium .S SL X SS were defined as Bacillus thringgiensis var tolworthi Bacillus thringiensis var finitimus Bacillus thringiensis var entomocidus Bacillus thingiensis var galleriae.4. Though the gene sequences testing, comparing with each other in NCBI blast and other software, crylAb from 39 was same to the report in gene bank, crylAc crylCgenes were same to the known at 87% 89% and 78% from 39and D3. Amino comparing tests showed that there were same at 76.5% 79.8% 51%. Between the crylAc from different strains 39 and D3, they were same at 90% in DNA, 85% in amino. The crylAb g更多还原