【作者】 熊瑾；

【导师】 陈晓阳；

【作者基本信息】 北京林业大学 ， 林木遗传育种， 2006， 硕士

【摘要】 毛白杨(Populus tomentosa)是我国特有的珍贵乡土树种,具有生长快、寿命长、树干通直、材质好等特点,是优良的工业用材树种和防护林树种。但毛白杨的插穗生根困难,扦插成活率低,成为毛白杨新品种不能大规模进入产业化,不易发展毛白杨无性系林业的重要原因。为提高毛白杨的扦插生根能力,本课题组将rolB基因导入毛白杨,以获得具有较强生根能力的转基因植株。本文以转入rolB基因的毛白杨为材料,通过组培苗生根试验,硬枝扦插试验,植株内源激素测定和生长指标测定来研究转rolB基因毛白杨植株的生根能力以及生长变异情况。主要研究结果如下:1.对转rolB基因毛白杨植株进行扩繁,并进行PCR检测,筛选出大量的阳性克隆。2.在无外源激素培养基上,转基因植株的生根率达到80%~100%,而未转化植株的生根率只有13%~20%。转化植株对生长素表现出高度的敏感性,当IBA浓度达1.0mg/L时,转化植株的生根受到抑制。rolB的表达,使转基因植株的生根能力增强,同时对外源激素表现出高度的敏感性。3.硬枝扦插试验中,转基因植株的扦插在无外源激素处理的情况下,生根率显著高于对照,其上半段枝条的扦插生根率达到75.8%,下半段枝条达到87.5%。而未转基因对照植株的上半段枝条的扦插生根率为56.7%,下半段为36.7%。同时转基因植株的下半段枝条的生根能力大于上半段枝条,而对照正好相反。证明rolB基因的表达是近根的部位强于远离根的部位。同时,外源激素处理插条的试验表明,转基因植株的插条对外源激素表现出高度的敏感性,在外源激素达到2000 mg/kg时,转基因植株插条的生根受到抑制。4.转rolB基因毛白杨植株的内源激素测定结果表明,转基因植株在扦插不同时期,插条的激素变化规律与对照相同。转基因植株与对照激素含量的差异出现在扦插后期,此时转基因植株插条的IAA含量显著高于对照,而ABA含量显著低于对照,IAA/ABA的比值显著高于对照。初步推断,转rolB基因毛白杨植株插条生根能力的增加,可能与rolB基因表达调控植物内源激素水平有关系。而转基因植株的组培苗的IAA含量和ABA含量与对照都没有显著差异。5.在对转rolB基因毛白杨植株的大田生长观测中发现,移栽大田当年的转基因毛白杨植株的高生长明显低于对照,表现出了明显的矮化现象。另外转基因植株表现更多还原

【Abstract】 Populus tomentosa is an important tree species in China, with the excellent characters of fast-growing, long-life and nice quality of wood. It is widely planted in the north of China for wood industry. However, it is difficult to root from wood cuttings which reduce its value in industry. In order to improve the rooting ability of the Populus tomentosa, the rolB gene was transformed into the plant and the transgenic clones were obtained. In this work, the transgenic plants were studied through rooting experiments, hardwood cutting experiments, determination of endogenous hormone and variation in growth. The main research work and the results are as follows:1. The clones of the transgenic plants were identified by PCR, and the positive clones were obtained.2. The in vitro rooting results showed that the transgenic clones rooted from 80% to 100% without auxin, while only 13%~20% for the control plants. Transgenic plants showed the high sensitivity to the auxin. When the concentration of IBA reached 1.0 mg/L, the rooting of the transgenic clones were restrained.3. In the experiment of hardwood cutting without any auxin treatments, both the first and the second cuttings of the rolB transformed plants had a significantly higher rooting ability than the cuttings from the untransformed plants. The rooting percentage of the first cutting from transgenic plants was 75.8%, and the second cutting was 75.8%, while only 56.7% and 36.7% for the controls. The second cutting of the rolB plants had significantly higher rooting ability than the first cuttings, while it is contrary to the control. It is proved that the rolB gene had a higher expression in the region close to the root. In the experiment of hardwood cutting treated by auxin, the cuttings of the rolB transformed plants increased sensitivity to auxin. When the concentration of IBA reached 2000mg/kg, the rooting of the transgenic clones was restrained.4. The endogenous hormone of the transgenic plants was determined by GC-MS-SIM. The change of hormone in the rolB plants is similar to the controls in the different period of cutting experiment. In the time of rooting, the cutting of rolB plant showed higher endogenous IAA contents and lower ABA contents than the controls. The expression of the rolB gene was correlated with endogenous hormone and the rooting capacity.5. Growth analysis showed that the stem height of the transgenic clones was lower than the controls in the first year of transplants. The dwarfism appeared in the transgenic plants. The transgenic clones showed a similar physiological index to the untransformed control plants. In the second year of transplant, the dwarfism of transgenic plants was reduced, and grew as well as the controls.更多还原