【作者】 王占斌；

【导师】 王志英；

【作者基本信息】 东北林业大学 ， 森林保护学， 2007， 博士

【摘要】 木霉菌作为生物防治资源是近年来有害生物防治研究的热点。美国、西欧等国家均已实现工业化生产。随着现代生物技术的不断发展，科学家们已经从生化和分子水平上对拮抗木霉的生防机制进行研究，并有较大的成功。关于木霉菌生防机制的假说很多，本文主要从四个方面对木霉的生防机制进行论述：1、寄生作用与抗生作用；2、竞争作用和产酶机制；3、诱导植物产生抗性：4、辅助机制。通过以上生防机制的阐明，以期更合理地把木霉用于生防领域中。几丁质酶是广泛存在于微生物和植物体内的一类蛋白质，它能催化真菌细胞壁的重要成分—几丁质的水解，从而抑制真菌的生长增殖，提高植物的抗真菌能力。近年来，随着对其作用机理、生化性质、表达调控的深入研究，用几丁质酶基因转化植株正日益成为植物防御真菌病害的有效途径。本试验提取木霉DNA，根据GENBANK上登录的木霉几丁质酶基因序列设计一对引物Tri-1／Tri-2，用PCR方法扩增出几丁质酶基因片段，将PCR产物连接到pMD18-T克隆载体上转化大肠杆菌JM109，克隆并测序。试验中共得到两个相关基因片段，测序后经生物信息学分析确定为几丁质酶基因，两个木霉菌的几丁质酶基因片段与已发表的序列相比其同源性达到99％。杨树做为广泛栽培的重要造林树种之一，因其速生丰产、实用性强、分布广、无性繁殖能力强、基因组比较小，因此杨树成为利用基因工程方法进行遗传改良的理想木本模式植物。杨树作为转基因的木本模式植物，主要是因为它具有以下优点：①分布广泛，经济价值高；②容易进行离体实验和培养，微繁和再生体系已经十分成熟：③易被土壤农杆菌侵染，转化率高。近年来，杨树转基因的研究在抗病虫害育种、抗除草剂、材性改良、生殖发育调整以及转基因树的安全性等方面的进展较快。本研究以小黑杨作为转基因的受体材料，研究了不同预培养时间、工程菌液浓度、浸染时间、共培养时间以及筛选方法对其遗传转化效率的影响。通过不同浓度头孢对小黑杨叶片产愈伤组织及分化的影响实验，筛选培养中加入700mg／L-800 mg／L的头孢唑林钠浓度不会影响小黑杨叶片产生愈伤组织和分化。加入700mg／L的头孢唑林钠抑菌浓度不会影响小黑杨的诱导生根。通过卡那霉素对小黑杨叶片分化和诱导生根所能忍受的临界浓度实验，确定40mg／L的卡那霉素作为叶片转化筛选的临界浓度。以20mg／L的卡那霉素作为生根筛选的临界浓度。在进行小黑杨遗传转化时，预培养的时间以3～4d为最好。预培养时间少于3d，抗性芽率较低，3d时的抗性芽率为1.92％，增加到4d时达到2.08％，5d以后抗性芽率下降。小黑杨转化率并没随着分化率的增加而增加。菌液浓度直接影响转化效果，适宜于小黑杨转化的菌液液度为OD₆₀₀=0.3～0.4。对于小黑杨叶片的遗传转化，共培养时间在2～4d时比较合适，以3d最好。侵染时间在6～15min比较适合小黑杨叶片的遗传转化。实验过程中经分子检测共获得的13株转基因的阳性植株，占到经抗菌素筛选所得到的植株数的4.9％。RT-PCR检测表明有5株未正常表达，有可能产生基因沉默现象有待于今后更进一步的实验验证。对转基因植株在分子检测的同时进行了几丁质酶活力的测定，绝大多数转基因植株叶片几丁质酶活力明显高于对照植株，其中11号植株酶活力和对照比高达3.093倍。但在检测中发现有的转基因植株几丁质酶活力比较低，有的甚至低于对照。可能是外源基因在受体发生了基因沉默（失活）现象。更多还原

【Abstract】 As biological control resource, Trichoderm has become the focus of pest biocontrolresearch in recent years. Trichoderm have important economic value. Trichoderm has been tobe provided for industrial production in American and Western Europe. Along with modernbiological techniques have developed, scientists had researched bio-control mechanism aboutantagonism Trichoderm in biochemistry and molecule level, and had much progress. Aboutbio-control mechanism of Trichoderm, there are many hypothesises. According to the researchprogress, four aspects of discussion are given about the biological control mechanism ofTrichoderm in this paper: 1. Function of parasitism and antibiosis; 2. Competition function andenzyme producing mechanism; 3. Plant resistance to be induced; 4. Assistant mechanism.Trichoderm can be used better in biocontrol areas through the biocontrol mechanism clarified.Chitinase is a group of extent proteins which existed in the plant and microorganism,which is expressed when plants are endangered by fungi. It can hydrolyze chitin which is mainand important component of the cell wall of fungi except for mycete in order to prohibitgrowing of fungi. As its operation mechanism, biological property and expiration regulationwere studied deeply, what chitinase gene was introduced into the plant genome is an efficientway to control fungi disease.Trichoderma viride Pers.ex Fr. was choosed from all female parent of TrichodermaPers.ex Fr. epiphyte preserved in the laboratory. DNA extraction of Trichoderma viride Pers.exFr. was done in the experiment, and then we designed a pair of primers based on the geneencoding of Trichoderma viride chitinase in the GENBANK database. The chitinase gene fromTrichoderma viride Pers.ex Fr. was cloned in JM109 with pMD-18T cloning vector by the PCRamplification method. The sequence was analyzed with the software of Blast on net.There are two correlation gene segments gained from the experiment, which areTrichoderma viride Pers. ex Fr. chitinase gene confirmed by bioinformatics analysis. In order toprovide creditable evidence for correlated investigation in the future, the function of chitinasegene was analyzed based on the above research.Poplar trees is one of widely planted tree species, it is ideal wooden model plants forgenetic modification by the way of gene engineering as its growing fast, best practicability,wide distributing and good asexual production. Poplar trees is transgenic model plants becauseof following advantages:①Poplar trees is grown commercially and fast for its economicimportance as a wood;②Its culture methods is easy and to be known by the researchers, itsmicro-propagation and regeneration system are best know by the researchers;③It has hightransformation rate and is easily to be infected by agrobacterium. Populus transgenic research develop fast in the areas of breeding of disease and insect resistance, herbicide resistance,timber modification, regulation of production and developing, transgenic tree security, and soon.Populus simonii×P. nigra is one of the main strains popularized for its high values, suchas growing fast, good quality, high adaptability etc. So we select the Populus simonii×P, nigraas the research material. Several factors, such as pre-cultivated time, Agrobacteriaconcentration, infection time, co-cultivated time and selecting methods which impactpercentage of transformation were investigated in the research.Experiment of different concentration of Cefazolin Sodium as antibiotic to inhibit thegrowth of Agrobacterium for Populus simonii×p nigra was no effect to leaf callus producingand differentiation. 700mg/L concentration didn’t have bad impacts on bud inducing androoting for Populus simonii×p. nigra during the experiment.Through sensitivity experiment of leaves to Kanamycia, the Kanamycin concentration of40 mg/L was determined when leaves differentiation, rooting by inducing bud concentration ofKanamycin was 20mg/L in rooting culture medium.Successfully established the transformation procedure Agrobacterium-mediated ofPopulus simonii×P. nigra, pre-culture time was 3～4days; resistance bud rate was low when pre-culture time was less than 3 days, it is 1.92％at three days. The resistance bud rate was 2.08％at 4 days, but it was begun to down after 5 days. Centrifuge Agrobacterium when beingdevelopped to the logarithm growth date(OD600=0.5), then dilute Agrobacterium to theconcentration of OD600=0.3～0.4 with MS liquid culture medium or asepsis distilled water forinfection.Selection of transformation time is very important to increase the transformation rate. Forco-culture 2-4 days is suitable, 3 days is best. After being co-cultured 3 days, inoculate on theselecting medium to be filtrated in succession during inducing unsure buds and rooting withKanamycin(containing Kanamycin 40mg/L and Cefazolin Sodium 700mg/L). 6～15 minuteinfection time was suitable for transformation.Some transformants were identified by using molecule detections and thirteen positivetransformants were achieved. It is about 4.9％in the number of the plant to be filtrated byantibiotics. The positive transformants emerges differential amplification strips while negativecomparison didn’t. 5 plants are expressed normally by RT-PCR detection. Others may be genesilenced so they should to be analysed carefully in the future. Chitinase activation of thetransgenic plants was mensurated at the same time, for most transgenic plants its activation ishigher than the normal ones. The chitinase activation of No. 11 is 3.093 times higher than thecontrol. Chitinase activation of some transgenic plants are lower after detection, even lowerthan the control, it may be the reason that foreign gene in the receptors happened gene silence (lost), it need to be detected carefully in the future.更多还原