【作者】 许继飞；

【导师】 邹莉； 王志英；

【作者基本信息】 东北林业大学 ， 森林保护学， 2006， 硕士

【摘要】 欧美杨系列是中国林科院引进的杨树新品种。因其品质优良,已通过国家级技术鉴定,被国家林业局列为我国西部及华北地区重点推广树种。但是,随着欧美杨的广泛栽培,病虫害的发生越来越严重,因此其抗性育种显得尤为迫切。采用基因工程技术开展转基因杨树的研究,是培育抗逆、抗虫和抗病杨树新品种的有效途径。 本研究以欧美杨108无菌苗为材料,通过对基本培养基的筛选、激素种类和浓度的调整,培养条件的选择等优化了欧美杨108再生体系的培养基和培养条件。在此基础上,采用根癌农杆菌介导法,进行抗病虫天花粉蛋白（Trichosanthin,TCS）基因的遗传转化研究。探讨了影响转化效率的若干因子,首次建立了以潮霉素磷酸转移酶（hpt）为选择标记基因的欧美杨108遗传转化系统,并获得了抗性愈伤和抗性芽。经PCR和PCR—Southern检测,证明目的基因已经整合到欧美杨108基因组中,为进一步开展欧美杨108遗传转化打下了理论基础。主要研究结果如下: 通过筛选得到了优化的叶片分化再生培养基、茎段愈伤组织诱导培养基、不定芽生根培养基及培养条件。叶片分化时,采用MS培养基附加0.6mg·L^-16—BA和0.2mg·L^-1NAA,在光照条件下培养;茎段愈伤组织诱导时,采用WPM培养基附加1.5mg·L^-12,4—D和0.75mg·L^-1KT,在光照条件下培养:不定芽生根和继代生根时,均采用WPM培养基附加一定量的IBA,在光照条件下培养。在这些培养基上外植体的分化率、诱导愈伤率以及生根率均达到了100%,而且生长状况优良。 确定了筛选欧美杨108转化体及选择生根培养时的抗生素浓度。潮霉素抑制叶片分化的浓度为2mg·L^-1;生根培养时的选择浓度为0～2mg·L^-1。比较了头孢唑啉钠和羧卞青霉素抑菌效果及对叶片分化和苗木生根的影响,结果表明头孢唑啉钠和羧卞青霉素浓度分别为600mg·L^-1和400mg·L^-1时,可完全抑制农杆菌LBA4404的生长:在此浓度下,头孢唑啉钠对叶片的分化影响较羧卞青霉素大,而对苗木的生根影响较羧卞青霉素小。因此,在叶片分化时,采用400mg·L^-1的羧卞青霉素抑菌;在生根时,采用600mg·L^-1的头孢唑啉钠抑菌。 最后,探讨了影响遗传转化的丰要因素,首次建立了以潮霉素磷酸转移酶（hpt）为选择标记基因的欧美杨108的遗传转化系统。采用根癌农杆菌介导法转化欧美杨108叶片外植体,共得到18个潮霉素抗性愈伤和抗性芽,转化效率为4.3%。抗性体经PCR和PCR—Southern检测,证实TCS基因已经整合到欧美杨108的基因组中。更多还原

【Abstract】 Poplar is one of the important sylvicultural and commercial tree;it is very popular throughout the world, especially in china. Populus euramericana 108 is a kind of hybrid strain from populus nigra and popolus deltoids. It is the main strain which has been popularized for its high values such as growing fast, high resistance, long fibre etc.by the State Forestry Administration. With the most widely cultivation of Populus euramericana 108, the Arising of Plant Diseases and Pests were more and more frequent. The research of gene transformation on poplar by gene engineering technique is a effective way for the selection of new species of poplar with tolerance, insect-resistant and disease-resistant.In this study, leaf and stem of Populus euramericana 108 were used as experiment material, through the screening to the basic culture medium, the adjustment of bormone kind and proportion, choice of the culture condition, the medium and the culture condition of Populus euramericana 108 regeneration system were optimized.On the basis of the establishment of high frequency regeneration system, adapting to Agrobacterium-mediated to conduct transformation of insect-resistance and disease-resistance genes of TCS gene. Through exploring the factors affecting the genetic transformation, the transgenic system, which selects hpt as The Selectable Marker Genes, was established and the resistant shoot were attained for the first time. The resistant shoots were identified by PCR and PCR-Southern analysis. The results showed that TCS gene had been integrated into Populus euramericana 108 genome.The main conclusion of this experiment was as following:Through the screening to through the screening to the basic culture medium, the adjustment of hormone kind and proportion and choice of the culture condition, the optimal medium and the culture condition were attained.the optimal medium for differentiation from leaf derived from in vitro cultured was MS+6 — BA 0.6 mg·L^-1+ NAA 0.2 mg·L^-1 the optimal medium for Callus induction from stem and Leafstalk was WPM+2, 4 -D 1.5 mg·L^-1 + KT 0.75 mg·L^-1.The optimal medium for Root Induction was WPM+0.4 mg/L IBA. They were cultured under thecontrolled condition of 25±1℃ with 2000 lx of successive light. Differentiation rate, Callus induction rate and and Root Induction rate individual was up to 100%.The appropriate concentration of Hyg for selection culture was determined. The differentiation of leaf explants was inhabited on differentiation medium containing 2mg/L Hyg;all shoots that were transferred to rooting medium containing 0-2mg/L Hyg were inhibited completely to root.The effect of cefotaxime and Carbenicillin in different concentration was evaluated oninhibiting Agrobacterium LBA4404 growth, the leaf differentiation and root. It showed that Agrobacterium LBA4404 cultured on solid differentiation medium containing 600mg/L cefotaxime or 400 mg-L^’ Carbenicillin was killed;in the concentration the effect of cefotaxime to leaf explants was observed than higher that of Carbenicillin and the effect of cefotaxime to shoot rooting was observed than less that of Carbenicillin. So the proper inhibit Agrobacterium LBA4404 concentration was 400mg/L Carbenicillin in leaf differentiation and 600mg/L cefotaxime in shoot rooting.At last, through exploring the factors affecting the genetic transformation, the transgenic system, which selects hpt as The Selectable Marker Genes, was established. Adapting to Agrobacterium-mediated to transform the leaf explants, 18 resistant shoots were obtained and transformation frequency was 4.3%. The resistant shoots were identified by PCR and PCR-Southern analysis. The results showed that TCS gene had been integrated into Popuhis euramericana 108 genome.更多还原