【作者】 孟丙南；

【导师】 李云；

【作者基本信息】 北京林业大学 ， 林木遗传育种， 2010， 博士

【摘要】 刺槐(Robinia pseudoacacia L.)原产美国,是多年生豆科树种,是适合于用材林、能源林、水土保持林、饲料林和蜜源林等的多用途树种。四倍体刺槐(Tetraploid Robinia pseudoacacia L.)引自韩国,属难生根树种,其扦插生根困难,成活率低。本研究以四倍体刺槐K4无性系等为试验材料,对其硬枝扦插和嫩枝扦插技术进行优化试验,尤其采用越冬沙藏催根法和埋枝黄化催芽法进行扦插研究。重点对埋枝黄化催芽嫩枝扦插生根过程进行形态结构、氧化酶活性和内源激素水平研究,并对其利用双向电泳、质谱分析和生物信息学技术进行蛋白质组学研究,探讨蛋白质水平的扦插不定根形成的调控机制,为四倍体刺槐无性系扦插方法的改进提供了理论基础,为林木无性系的扦插生根机理蛋白质组学研究提供了有益的尝试和借鉴。主要研究成果如下：1.四倍体刺槐K2、K3、K4、YD无性系和当地实生刺槐的硬枝扦插试验表明,插穗部位、生长调节剂种类和生长调节剂处理浓度对硬枝扦插成活率存在显著影响,不同品系间生根能力也存在显著差异,其中YD无性系成活率为87.78%,而四倍体刺槐K4无性系的成活率仅23.33%。2.越冬沙藏催根扦插法以沙藏前和扦插时的两次生长调节剂处理插穗可以提高四倍体刺槐K4无性系的硬枝扦插成活率；配合沙藏时插穗基部垂直向上放置的“倒催根”方法,K4无性系硬枝扦插成活率达71.11%。3.在组织培养幼化苗木建立的采穗圃中采取嫩枝插穗,从插穗部位、生长调节剂种类、处理浓度、处理时间、扦插时期和扦插基质6个因素优化刺槐无性系嫩枝扦插技术,确定了最佳生根效果的组合。其中,K4无性系以6月初,半木质化带顶芽的上部插穗,蛭石作扦插基质,植物生长调节剂ABT 2000 mg/L速蘸处理30 s为最佳处理组合,生根率达77.78%。4.埋枝黄化催芽法是在春季对硬枝插穗进行覆沙催芽处理,利用基部黄化的催芽嫩枝进行扦插的方法。本方法提高四倍体刺槐K4无性系的硬枝扦插成活率达90.6%,而且比常规嫩枝扦插缩短了生根时间。5.针对K4无性系埋枝黄化催芽的黄化嫩枝和未黄化嫩枝扦插生根过程,从形态结构上研究确定了3个不定根形成阶段,即愈伤组织诱导期、不定根原基形成期和不定根伸长期；发现生根过程的氧化酶POD、IAAO和PPO的活性、以及内源激素IAA、ABA、GA3、ZR的水平在不定根形成过程中随着时间会发生相应的变化,与不定根的形成有密切关系。6.建立了稳定性好、分辨率高适合四倍体刺槐埋枝黄化催芽嫩枝插穗韧皮部的蛋白质分离的双向电泳体系。首次对埋枝黄化催芽的黄化嫩枝和未黄化嫩枝的扦插生根过程进行了蛋白质组学研究,确立了特异蛋白质点148个,并对其中蛋白表达丰度较高的40个点进行了蛋白质肽质量指纹谱(PMF)分析。通过MASCOT数据库搜索,明确鉴定了13个蛋白,其中包括核糖蛋白、代谢蛋白、信号蛋白、木质素合成蛋白和叶绿素蛋白等。通过对这些蛋白质在生根过程中功能的分析,从蛋白质表达水平探讨了扦插生根的分子调控机理。更多还原

【Abstract】 Black locust (Robinia pseudoacacia L.) is a leguminous, deciduous, fast growing tree species native to the southeastern USA. It is a multi-purpose tree species suitable for production of timber, fuel wood, land reclamation, beekeeping and forage.Tetraploid Robinia pseudoacacia which was colchicine-induced from black locust in South Korea is considered to be a more promising forage tree than diploid R. pseudoacacia. But it is difficult to root,especially in cutting propagation. The objectives of the present study were to optimize techniques of hardwood cutting and softwood cutting for tetraploid R. pseudoacacia clones. Hardwood cutting after overwintering sand storage root promoting and wood-burying etiolation root promoting were used. Morphological structure, oxidase activity and endogenous hormone levels of wood-burying etiolation root promoting in softwood cutting process were researched. Functional proteomics was also studied by using two-dimensional electrophoresis, mass spectrometry and bioinformatics techniques to investigate mechanism of adventitious root formation on a protein level. These provided not only a theory to the improvement of black locust clone cuttage, but aslo references to proteomics studies on rooting mechanism of forest trees clones. The results were as follows:1. There were significant different survival rate between tetraploid R. pseudoacacia clones K1, K3, K4, YD and local seedlings in cutting positions, plant growth regulators and growth regulator concentrations in hardwood cutting. The difference of rooting ability between clones was also significant. The survival rate of clone YD was 87.8% whereas tetraploid R. pseudoacacia clone K4 was only 23.33%.2. Survival rate of hardwood cutting could be enhanced by combine of growth regulator treatment before sand storage and during cutting after overwintering sand storage. Combining with inverted storage root promoting the survival rate of tetraploid R. pseudoacacia clone K4 was increased to 71.11%.3. Soft wood cuttages of R. pseudoacacia picked from cutting orchard where planted with tissue cultured young seedlings were investigated on cutting positions, plant growth regulators treatment concentrations, treatment time, cutting time and cutting medium. Then the best combination was selected. The rooting rate of clone K4 was up to 77.78% from the best combination that using semi-lignified top cuttings with terminal buds in early June, and using vermiculite as medium and quick dipping for 30 seconds with ABT 2000 mg/L.4. Wood-burying etiolation root promoting was the process of covering hardwood cuttings with sand in spring and cutting with the bottom etiolated softwood. The survival rate of hardwood cutting of tetraploid R. pseudoacacia clone K4 was increased to 89.4% and its rooting time was shorter than the normal softwood cutting by using this method.5. The process of cutting rooting of etiolated softwood and non-etiolated softwood in wood-burying etiolation root promoting of clone K4 was investigated and 3 phases of adventitious root formation in morphological structure were found. These three phases were callus inducement stage, adventitious root primordium formation stage and adventitious root elongating stage. The results also showed that the activity of POD、IAAO and PPO and endogenous hormones IAA、ABA、GA3 and ZR were fluctuating with time during the period of adventitious root formation, and they were highly related to adventitious root formation.6. A system of 2-DE involving the preparation of protein sample, the electrophoresis and staining for the proteomic extracted from tetraploid R. pseudoacacia phloem was developed. And the proteomics of cutting rooting process of etiolated softwood and non-etiolated softwood in wood-burying etiolation root promoting were investigated. Then 148 differential protein spots were found, and 40 of them with high protein expression abundance were analyzed in PMF. Through MASCOT database searching, the identified proteins were involved in RNA proteins, signal transduction, metabolism proteins, lignin proteins and phyllo-proteins.Molecular regulation mechanism of cutting rooting on a protein expression level was discussed by analyzing the function of these proteins during the process of rooting.更多还原