【作者】 宛文鹏；

【导师】 詹亚光；

【作者基本信息】 东北林业大学 ， 遗传学， 2007， 硕士

【摘要】 本论文利用高压静电场处理白蜡属树种的花粉，通过提高花粉生命力促进白蜡属种间杂交亲和性和杂种生活力。为了揭示其分子机理，以经高压静电场处理和对照的水曲柳花粉为研究对象，利用mRNA差异显示（DDRT-PCR）和Reverse Northern杂交技术获得差异表达片段，并对基因片段进行测序和序列分析。所得结果如下：1、白蜡属树种花粉萌发的比较适合培养基为：蔗糖10g·L^-1+琼脂1g·L^-1+尿素0.1g·L^-1+硼酸1g·L（-1）+硝酸钾0.1g·L^-1+硫酸镁0.2g·L^-1+硝酸钙0.1g·L^-1+硫酸锌0.1mg·L^-1+钼酸钠20 mg·L^-1+NAA10mg·L^-1+6-BA1mg·L^-1，pH6.5。2、本试验测定株间花粉生活力最高相差30.9％，说明白蜡属株间花粉生命力差异明显。3、将花粉4℃密封保存有利于保持花粉的生命力。4、经适宜的高压静电场处理，水曲柳的花粉萌发率可比对照高出19.7％。5、经适宜场强处理的白蜡属花粉与水曲柳雌株杂交，座果率明显提高，最多提高13.5％。6、大叶白蜡、小叶白蜡和绒毛白蜡花粉经高压静电场处理后杂交得到的种子在种长、种宽、千粒重三方面几乎都高于未处理得到的杂交种子。种长差别最大的品种前者比后者高出0.25cm（9.40％）；种宽差别最大的品种前者比后者高出0.06cm（9.84％）；千粒重差别最大的品种前者比后者高出13.71g（25.90％）。7、经高压静电场处理花粉后得到的杂交苗在苗高、地茎、光和速率、叶绿素含量方面都明显高于对照苗。8、改良的CTAB法是提取水曲柳花粉总RNA的有效方法。9、水曲柳花粉经高压静电场处理后有特异基因表达。通过Reverse Northern斑点杂交的检测鉴定，共获得6个差异基因片段。通过序列测定及分析，发现6个基因片段分别与拟南芥的核苷控制的离子通道基因、番茄叶绿体的铜锌超氧化物歧化酶（SOD）基因、番茄叶绿体的铜锌超氧化物歧化酶（SOD）基因、拟南芥的核酸调控的离子通道蛋白、与番茄的激素诱导的蛋白酶抑制剂基因、苜蓿科细胞色素P450单氧酶有着较高同源性。氨基酸序列同源性分别高达98％、95％、96％、99％、93％、93％。更多还原

【Abstract】 In this paper the pollens of the species of Fraxinus Linn were treated with high voltage static, which promoted the cross-compatibility by enhancing the vitality of their pollen. In order to study the molecule mechanism of it, both of the control and pollens dealt with high voltage static field were taken for RNA extract.The DNA fragments of differential expressed genes were gotten by DDRT-PCR and were confirmed by Reverse Northern dot blotting, and then they were sequenced and analyzed. The main results were as follows:1, The suitable culture medium for the pollens germination of Fraxinus Linn is : sugar 10 g.L^-1+agar 1 g·L^-1+urea 0.1 g·L^-1+boric acid 0.5 g·L^-1+nitre 0.1 g·L^-1+bitter salt 0.2 g·L^-1 +calcium nitrate 0.1 g·L^-1+zinc salfate 0.1 mg·L^-1+sodium molybdate 20 mg·L^-1+NAA10 mg·L^-1+6-BA 1mg·L^-1,pH 6.5.2、The differences of pollens vitality between individual plants were significant, which up to 30.9 percent at the most among the trees experimented in this paper.3、To keep higher vitality ,pollens should be sealed after dry and stored below 4℃in refrigerator.4、The germination rate of pollen of Fraxinus mandshurica treated with the proper high voltage static field was 19.7 percent higher than that of control.5、The pollens treated with proper high voltage static field could promote hybrid between the species of Fraxinus Linn and get a higher fructification rate, 13.5 percents higher than control at the most.6、The hybrid seeds of Fraxinus sogdiana Bunge, Fraxinus velutina Torr. and Fraxinus rhynchophylla obtained by pollen processed by proper high voltage static shared higher data in seed length, seed width and kilo granule weight than control, and the higher values of these were0.25centimeters（9.40percents）,0.06centimeters（9.84percents）and13.71gram （25.9percents） at the most.7、F1 seedlings from pollen treated by high voltage static were notably different from control（not treatment）in seedling height, ground stem width, photosynthesis rate and chlorophyll content.8、Improved CTAB method is effective in extracting the RNA in the pollen of Fraxinus mandshurica.9、Genes in the pollen of Fraxinus mandshurica expressed differential after processed by high voltage static field. Identified by reverse northern dot blotting, 6 differential expressed gene fragments were gotten. By sequencing and analyzing, we found that the fragments have great identity with the gene for cyclic nucleotide-gated cation channel in Arabidopsis thaliana, with the gene for calmodulin binding / cyclic nucleotide binding / ion channel in Arabidopsis thaliana, with the gene for chloroplast Cu-Zn superoxide dismutase in tomato,with the gene for putative cyclic nucleotide-regulated ion channel protein in Arabidopsis thaIiana, with the gene for auxin-induced proteinase inhibitor （ARPI） gene in Lycopersicon esculentum, and with the cytochrome P450 monooxygenase CYP76E1 （CYP76E1） gene in Medicago truncatula respectively.There nucleotide homologous were 98percents, 95percents, 96percents, 99percents, 93percents and 93percents.更多还原