【作者】 胡雅琴；

【导师】 肖娅萍；

【作者基本信息】 陕西师范大学 ， 植物学， 2004， 硕士

【摘要】 太白红杉(Larix chinensis Beissn)属秦岭特有植物，是森林上限树种，被列为国家二级保护植物。根据文献记载以及我们的野外调查：太白红杉的个体数目少，局限分布于秦岭的少数地区。一般认为，太白红杉是第四纪冰川的孑遗植物，历经冰川的覆盖后，地面上首先恢复的木本植物是松科植物，因此我们有必要探讨落叶松属的系统演化及太白红杉在松科的系统位置。 rbcL基因和matK基因均为叶绿体基因。rbcL基因编码核酮糖-1，5-二磷酸羧化／加氧酶(Rubisco，Rbc)的大亚基，matK基因位于trnK基因的内含子中，编码的成熟酶参与RNA转录体中Ⅱ型内含子的剪切。目前这两个基因已广泛用于植物系统发育分析。 基于以上考虑，我们首先扩增了太白红杉、华北落叶松、大果红杉的rbcL基因和matK基因序列，结合网上共享的松科其它植物的序列对太白红杉的系统位置作了探索。在结果分析中应用了Chromas、SeqVerter、DNASIS2.5、ClustalX2.0、Mega2.0和PAUP4.0~*等整理和分析软件，得到如下结果： 1．rbcL和matK基因在碱基组成上不同，rbcL密码子的pos#1GC含量较高，而matK密码子的pos#3GC含量较高；rbcL3个位点受到的突变作用相同，matK pos#1受到的突变作用较2、3位点大；rbcL的碱基替换大多发生在pos#3，而matK的碱基替换平均发生在3个位点上。rbcL碱基替换TV呈饱和状态，而matK的TS，TV与p-distance都呈线性关系，matK的替换未饱和。 2．在用rbcL和matK序列构建的系统树上，冷杉属、油杉属和金钱松属始终聚在一起；rbcL序列构建的系统树上，银杉属与松属关系较近，而在matK构建的系统树上，银杉属与云杉属聚在一起。 3．在rbcL+matK序列构建的系统树上，红杉组和落叶松组先分成两支，落叶松组的日本落叶松和美加落叶松亲缘关系较近。由于rbcL和matK这两个序列都位于叶绿体上，进化途径有相似性，更限于时间、经费、数据等原因，未得出太白红杉的确切系统位置，但是结合核基因序列或线粒体基因序列，可以得到理想的结果。 4．在建树分析中，用了距离法(邻接法)和性状数据分析法(最大简约 法)2种方法，得到的结果有相似之处，如冷杉属、油杉属和金钱松 属始终优先聚在一起。用最大简约法得出的系统树，尽管没有解决系 统发生问题，但是与距离法得出的结果相比，树状图显示的关系更容 易让人接受。 其次，探讨了不同松科植物材料DNA的提取条件以及rbcL基因和matK基因的PCR一SSCP影响条件。所谓SSCP即单链构象多型性。双链DNA高温解链后即快速冷却，使单链DNA在低温下由于碱基的部分互补发生自身折叠，形成二级结构，通常DNA一级结构的一个碱基发生改变就会导致构型的变异，这种变异经中性聚丙烯酞胺凝胶电泳通过带型差异表现出来。PCR一SSCP就是将PCR产物变性后作的中性聚丙烯酸胺凝胶电泳分析。结果表明:1.同一植物不同部位提取的效果不同，不同的组织器官适用不同的提取 方法:针叶、未木质化新梢及新发的幼苗适合CTAB法，而种子需要 采用蛋白酶K法。我们尝试用一20℃冷藏1个月的针叶和新梢作材料 提取总DNA，也获得了较好的结果，说明常规冷冻可以短期保存材 料。2.在CTAB法提取DNA样品过程中，没有使用RNA酶，粗提样品也没 有进行进一步的纯化处理，对粗提的DNA样品进行rbcL和matK基 因扩增，结果表明DNA样品中存在RNA对扩增结果没有影响。3.PCR一SSCP分析时，凝胶的交联度对带型的清晰程度有很大的影响， 分析时，首先应对凝胶的交联度进行预实验。4.不同的凝胶浓度对带型影响很大，相同大小DNA分子在5%的凝胶 上有清晰的带型，而在3.5%的凝胶上带型不稳定。5.电泳环境温度对电泳带型有影响，4℃电泳sh与20℃电泳sh距离基 本相同，但是后者带型不佳，而前者带型清晰。6.我们分别用lxTBE和O.SXTBE对3.5%和5%的凝胶进行电泳，发 现两种离子浓度下，5%的胶得到清晰的带型，说明离子浓度对电泳结 果没有影响。 第三，多数学者认为，在提取植物总DNA时，多糖是影响提取效果的一个很重要因素。但是在我们的试验中，对太白红杉的种子和幼苗分别进行了多糖含量的测定，结果显示:可溶性多糖并不是影响总DNA提取效果的决定因素。更多还原

【Abstract】 Larix chinensis Beissn is the endemic plant that distributed on hightest-altitude top in Qinling Mountain and has been listed one of rare and endangered plants on China Red Data Book. Based on the data and our field investigation, it is showed that the individual number of L. chinensis Beissn is very small and these plants only distribute on a few mountains. Generally, L. chinensis Beissn is the glacial relict of the Quateranry. After the earth has been covered by galciation for long time, the Pinaceae is the first occurred wood tree. So it is necessary for us to study the phylogeny of Larix and the phylogenic position of L. chinensis.rbcL and matK gene are chloroplastic genes. rbcL gene encodes the large subunit of Rubisco. matK lied in the trnK intron and the maturase encoded by this gene participates the RNA II introns splicing. Now these tow genes have been widely used in the phylogenic analysis of plant.Firstly, we amplifed the rbcL and matK gene of L. chinensis Beissn, L.potaninii var. macrocarpa Law, L. principis-rupprechtii Mayr and thenresearched the phylogenic position of L. chinensis Beissn combining thePinaceae sequences shared in the Internet. During the anglicizing process,we applied many softwares such as Chromas, SeqVerter, DNASI 2.5,ClustalX 2.0, Mega 2.0 and POUP 4.0 *. The results were as follows:1. rbcL and matK gene were different from the base constitution. In rbcL,position #1 included more GC than matK as well as position #3 of matKincluded more GC. Three positions of rbcL were under the samemutation effect and the position #1 of matK was undergoing moremutation effect than the other positions. The base substitution of rbcLtook place more easily on position #3 while the substitution of matKtook place on 3 positions equally. The base transversion (TV) of rbcLtook on saturation state and the transversion (TV), transition (TS) ofmatK were lined with p-distance.2. On the phylogenic tree based on rbcL and matK, Abies, Keteleeria and Pseudolarix were always clustered together. On rbcL tree, Cathaya had tight relation with Pinus and on matK tree, Cathaya clustered with Picea.3. On the phylogenic tree based on rbcL + matK, the Larix took part into tow clades: the Sect. Larix clade and Sect. Multiseriales clade. L.kaempfer and L. laricina had tight relations. Because rbcL and matK all lied on chloroplastic genome and they had similar evolution mode we did not gain the phylogenic position clearly. This was also because we had limited time, little expense and deficient data. But we could get ideal result if we combined nuclear gene and mitochondrial gene sequences.4. During the analyzing process, we applied distance method (neighbor joining method) and characteristic data analysis method (maximum parsimony method). And we got similar results such as Abies, Keteleeria and Pseudolarix were always clustered together firstly. The phylogenic tree by maximum parsimony method showed the more acceptable relationship than that of by distance method though it also had not resolved the phylogenic problem.Secondly, we studied the DNA extracting conditions of different plant materials and the PCR-SSCP conditions of rbcL, matK. Single-strand conformation polymorphism (SSCP) is a method for detecting mutations by sensing conformational differences between mutant and normal DNAs in the single-stranded state using non-denaturing polyacrylamide gel electrophoresis. So SSCP also could detect whether or not DNA fragments are identical in sequence. PCR-SSCP is the analysis method for denatured PCR produce in non-denaturing polyacrylamide gel electrophoresis. The results were followed:1. There was different extracting effect for different plant parts and different tissues or organs should use different extracting method: conifer, non-lignified treetop and seedling should adopt CTAB method while seed should adopt Proteinase K method. We attempted to extract D更多还原