论文标题:水稻内生细菌的研究及其杀虫工程菌的构建
Study on Endophytic Bacteria in Rice and Construction of Pesticide Engineering Bacteria
论文作者 孙惠青
论文导师 李平,论文学位 硕士,论文专业 作物遗传育种
论文单位 四川农业大学,点击次数 87,论文页数 63页File Size6830k
2003-05-01论文网 http://www.lw23.com/lunwen_91841242/ 内生细菌;优势种群;ERIC-PCR;回接验证;重组
Endophytic bacteria;Dominant population;ERIC-PCR;Re-inoculation and testing;Recombination
本研究从四川主要栽培水稻品种D优527体内共分离到102株细菌。研究了内生细菌在水稻植株内不同生育期、不同组织部位的动态分布曲线特点，确定编号为SR-15、SR-25、SL-37的三株细菌为优势菌株，并进行了优势菌株的回接、定殖试验，验证其内生性。经生理、生化特性结合形态学的研究，确定了优势内生菌株在微生物学上的分类地位，并进行了与Bt杀虫蛋白基因的重组，以及杀虫蛋白基因的表达及对水稻二化螟防治研究，验证了优势内生细菌作为生防工程载体菌的可行性。此外，对菌株的致病性、促生性等生物学特性进行研究。 取得的主要结果如下： 1 利用改进的细菌分离技术，从健康栽培水稻品种D优527的植株体内共分离到102株细菌，从根系中分离到41株，从叶片分离到37株，从茎分离到24株。其中，革兰氏阳性细菌77株，革兰氏阴性细菌25株。利用ERIC-PCR技术对菌株基因组进行分析，结果显示，菌株基因组间存在多态性。 2 对所获得菌株进行种群、数量的动态分析，研究细菌在根、茎、叶内随水稻生育期变化的动态分布情况，其中，水稻分蘖期和孕穗期是细菌种群数量分布的高峰时期。 3 根据种群分布特点，初步确定SR-15、SR-25、SL-37为优势种群，而SR-15优势性尤为明显，并确定了SR-15、SR-25、SL-37菌株在不同生育期数量动态变化趋势，其中，SR-15在分蘖期和孕穗期数量分布高于其它两菌株。 4 对SR-15、SR-25、SL-37菌株的生物学特性进行研究，结果显示，菌株的生长均符合细菌生长的典型的生长曲线，在不同pH、温度、培养基条件下，菌株生长的动态变化各有差异，且与相关报道的内生细菌生长特性具有一定的相似性。 5 转puc18标记验证内生性试验结果表明，在不同电压下，电击转化细菌的效率不同，在电压为1500V时转化效率最高，而puc18使用量对转化效率影响不明显。转带抗生素标记的puc18于优势细菌，回接水稻再分离，在抗生素培养基上初步筛选和PCR鉴定，与原始菌株一致。并通过电子显微镜超薄切片确定了各菌株在水稻体内的存在部位。验证了SR-15、SR-25、SL-37皆为水稻内生细菌。 6 对SR-15、DR-25、SL-37菌株进行光学显微镜和电子显微镜观察、细胞壁化学成分分析以及生理生化特性分析，确定SR一巧为盐敏芽抱杆菌，SR一25为栖息微球菌，SL一37为浅黄金杆菌。 8应用改进的PEG原生质体转化法和新型高效电击转化法，将含杀虫毒性强的苏云金杆菌6一内毒素基因Cry 1 Ac的质粒导入载体菌SR一15，构建杀虫工程内生细菌，并用电镜观察到基因表达后产生的毒蛋白晶体。 9工程菌株的杀虫试验表明，经超声波破碎后工程菌株发酵液(loscfi对ml一1护cfiUml)浸水稻幼苗，并饲养水稻二化螟幼虫，6天后其校正死亡率达80.1%。
Many bacterial populations were screened from rice plant D-you 527 planted mainly in Sichuan. Three strains were certain to be dominant positions. The endophytic characters of three strains were tested by the nodulation transferring pud 8 plasmid vector. The classification statuses in microbiology of 3 strains were deduced through the research of physiological and biochemical characters. A Delta-endotoxin gene Cry I Ac from B. thuringiensis was transformed to SR-15 strain. The result showed that the transformed gene strain expressed toxin protein, and strong toxicity to Chilo suppressalis.1 One hundred and two bacteria were isolated from roots, stems, leaves and seeds of healthy rice plant D-you 527. Among them, 77 strains were G+, 25 strains were G-. 41 strains were isolated from roots, 37 strains were isolated from leaves and 24 strains were isolated from stems. Bacterial strains were tested different by ERIC-PCR.2 SR-15, SR-25, SL-37 strains were tested to be the dominant position through c distribution curves and characters. Quantity of SR-15 strain was biggest.3 Distribution curves of isolated population in different stages of roots, stems, and leaves were deduced. The result showed that distribution quantity of the dominant bacteria SR-15, SR-25, SL-37 in different stages of roots, stems, and leaves, or different organs were different.4 From the growth curves of three strains, they were consistent with the characters of normal bacteria, those were: lag phase, exponential phase, stationary phase and decline phase. Biological characters of three strains were different in conditions of different pH, temperature and culture media. These characters are similar to the endophytic bacteria reported.5 Efficiency transferring puclS plasmid vector into dominant bacteria in different voltage and plasmid concentration was tested. Transformation frequency in 1500 voltage was highest, but same in different plasmid concentration.6 Bacterial strains transformed puclS plasmid vector were nodulated into the rice plants and screened again through antibiotic plates. The strains screened were tested to be same to original strains through ERIC-PCR. It showed that three strains colonizatied in rice by elect-microscope observation, and were tested that they were all rice endophytic bacteria.7 SR-15, SR-25, SL-37 were identified as Bacillus halmapalus, Micrococcus sedentarius and Aureobacterium flavesnes respectively through chemical components of cell wall, physiological and biochemical characters observed by tests -, microscope and elect-microscope.8 A transformed of SR-15 strain carrying a delta-endotoxin gene from B. thuringiensis subsp. Probe into the express of crystal preliminarly.9 The result showed that SR-15 strain transformed a delta-endotoxin gene Cry I AC from B. thuringiensis subsp could produce toxic protein. Experiment of insect bioassay was that the fermentation production was toxic to the Chilo suppressalis.
|
