【作者】 张福丽；

【导师】 王志英； 王占斌；

【作者基本信息】 东北林业大学 ， 森林保护学， 2006， 硕士

【摘要】 杨树属杨柳科（Salicaceae）,杨属（Populus）,全世界杨树有100多种,而我国就有57种之多,因此无论对于生态还是对国民经济的促进作用,杨树都起着其它树种所无法比拟的作用。但一直以来,杨树受到真菌病害如杨叶锈病、溃疡病等的威胁,每年给国民经济造成巨大的经济损失,而给生态环境造成的巨大破坏更是无法弥补的。小黑杨（Populus simonii×P.nigra）具有生长快、材质好、适应性强等优点,是林业生产上推广应用的优良品种之一。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。 几丁质酶是植物在受到病原菌侵染时所表达出的一组蛋白质,可以水解构成真菌（除卵菌外）细胞壁的主要组成成分几丁质,从而抑制真菌的生长。 本研究以MS+0.5mg/L 6-BA+0.1mg/L NAA为分化培养基、WPM+0.4mg/L IBA为生根培养基,以外源的几丁质酶基因为目的基因,在此基础上进行了小黑杨的遗传转化研究,通过叶片及组培苗茎段的敏感性试验,最终确定叶片分化时卡那霉素的临界浓度为40mg/L,芽诱导生根时卡那霉素的临界浓度为20mg/L,抑菌抗生素头孢唑林钠700mg/L的浓度不会对小黑杨叶片分化及生根培养产生不利影响。成功摸索出农杆菌介导的小黑杨遗传转化体系:选取生长20d左右的健壮的小黑杨组培苗叶片,通过主叶脉剪2～3下,预培养3～4d;将培养至对数生长期的农杆菌(OD₆₀₀=0.5)离心后用液体MS培养基或无菌蒸馏水稀释成OD₆₀₀=0.3～0.4浓度,进行浸染;侵染时间为6～15min;共培养3d后,转入到筛选培养基中进行诱导不定芽及生根阶段的卡那霉素连续筛选（含有40mg/L卡那霉素和700mg/L的头孢唑林钠）。通过农杆菌介导法将外源几丁质酶基因导入到小黑杨中,为小黑杨的遗传转化及抗性育种提供了理论基础。 其中一部分转化苗通过PCR分子检测,有13株出现了特异性扩增条带,而阴性对照没有特异性条带产生;通过PCR-Southern的进一步分子检测,出现了特异性的杂交条带,阳性率为100%,说明外源基因已经整合到小黑杨基因组中。小黑杨叶片的遗传转化中最高转化率达到4.12%。更多还原

【Abstract】 Poplar belongs to Salicaceae, Populus. There are more than one hundard kinds in the world, while more than fivty-seven in China. So, poplar is more important than other trees for not only protecting ecology but also advancing country economy development. But, poplar is damaged by fungal diaeases all the time, such as Melampsora spp., Botryosphaeria ribis, and so on, which lead to huge loss for the country economy every year. For ecology, the unremedi-ed loss is much bigger. Populus simonii×P nigra is one of the main strains popularized for its high values, such as growing fast, good quality, high adaptability etc.. So we select the Populus simonii×P. nigra as the research material. Several factors, such as pre-cultivated time, Agroba-cteria concentration, infection time, co-cultivated time and selecting methods which impact percentage of transformation were investigated in the research.Chitinase is a group of proteen which is expressed when plants are endanged by fungi. It can hydrolyze chitin which is main and important component of the cell wall of fungi except for Oomycetes in order to prohibit growing of fungi.In this study, the culture medium for leaf regeneration was MS+0.5mg/L 6-BA+0.1mg/L NAA and for rooting was WPM+0.4mg/L IBA. The target gene was foreign chitinase. Then the transformation of Populus simonii ×P. nigra was studied basing on the above preconditions. Through sensitivity experiment of leaves to Kanamycia, the Kanamycin concentration of 40 mg/L was determined when leaves differentiation, rooting by inducing bud concentration of Kanamycin was 20mg/L in rooting culture medium. Concentration of Cefazolin Sodium as antibiotic to inhibit the growth of Agrobacterium was 700mg/L and this concentration didn’t have bad impacts on bud inducing and rooting for Populus simonii ×P. nigra during the experiment. Successfully established the transformation procedure Agrobacterium-mediated of Populus simonii×P nigra: cut vigorous leaves to 2-3 patches through the main vein after growing about 20 days, pre-culture time was 3⁴days;centrifuge Agrobacterium when being developped to the logarithm growth time(OD₆₀₀=0.5), then dilute Agrobacterium to the concentration of OD₆₀₀=0.3-0.4 with MS liquid culture medium or asepsis distilled water for infection;infection time was 6-15 minute;after being co-cultured 3 days, inoculate on the selecting medium to be filtrated in succession during inducing unsureness buds and rooting with Kanamycin（containing Kanamycin 40mg/L and Cefazolin Sodium 700mg/L）. The target gene-foreign chitinase was transformed into Populus simonii×P nigra by Agrobacterium mediated transformation. The theory base was given for Populus simonii ×P. nigra transformation and fastness breeding.The molecule detections were done. Some transformants were identified by using PCRamplification and thirteen positive transformants were achieved. The positive transformants emergenced differential amplification strips while negative comparison didn’t. Then by PCR-Southern analysis, the differential hybridizing strips appeared and the positive frequentcy was 100 percent. The results showed that the foreign gene has been integrated into genome of Populus simoniixR nigra. The highest percentage of transformation for leaves Populus simoniixR nigra was 4.12%.更多还原