Study On Transgene Of Fungal Disease Resistance Of Populus Simonii×P.nigra.

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.