【作者】 周涵韬；

【导师】 林鹏；

【作者基本信息】 厦门大学 ， 植物学， 2001， 博士

【摘要】 红树林（Mangroves）是生长于热带、亚热带陆海交汇的海湾河口潮间带的盐生木本植物群落，它蕴藏着丰富的动植物、微生物基因库，是一种珍贵的生物资源。红树林在维护海岸生态平衡；防风减灾、护堤保岸；环境污染监测、净化与防治等上发挥重要的作用。目前国内外对红树植物开展了大量的生物学研究工作，成果多集中在生态学，生理学，生物化学等领域，而分子生物学领域的研究工作还处于起步阶段。本文运用RAPD分子标记技术，对不同种属红树植物的遗传变异与生态分化进行了研究；首次运用改进的mRNA差别显示技术，从抗盐能力强的泌盐红树植物—白骨壤基因组中分离克隆了抗盐相关基因。同时运用蛋白质双向电泳技术，从白骨壤叶片中分离了抗盐相关蛋白质，并初步分析了该蛋白质的生化特性。具体结果如下： 1．采用改良的CTAB抽提方法，成功地从不同种属红树植物的叶片中提取和纯化了基因组DNA。获得的DNA纯度较好(A₂₆₀／A₂₈₀。在1.6-1.8之间)，得率高（平均得率约为120μgDNA／g F.W.），片段完整（片段大小约为23Kb左右）。适宜于进行PCR扩增及后续分子生物学研究。说明本文首次在前人工作基础上加以改进的CTAB法，广泛适用于富含单宁、多糖、脂类、胶质类物质等的红树植物基因组DNA的提取与纯化。 RAPD扩增反应的稳定性、重复性及扩增带型的丰富性是进行红树植物遗传多样性分析的基础。使用德国Biometra公司T3型PCR仪，规范实验操作，采用如下反应体系：10mmol／L Tris-HCl，pH8.0，50mmol／L KCl，0.01％明胶，80ng模板DNA，2mmol／L MgCl₂，0.2mmol／L dNTPs，0.2μmol／L 10核苷酸随机引物，0.5U TaqDNA聚合酶，总体积25μL。扩增程序为：94℃变性1min，36℃退火1min，72℃延伸2min，共进行40个循环，最后72℃延伸7min。获得的RAPD扩增结果稳定性高，重复性好，扩增带型丰富平均为6—7条。 2．以福建九龙江口龙海红树林自然保护区浮宫种苗园内白骨壤（Avicenniamarina）、桐花树（Aegiceras corniculatum）、无瓣海桑（Sonneratia apetala）、秋茄（Kandelia candel）、木榄（Bruguiera gymnorrhiza）、海莲（Bruguierasexangula）、尖瓣海莲（Bruguiera sexangula var．rhynchopetala）等7种红树植物为材料，运用RAPD技术对这7种红树植物进行了遗传多样性分析。从30个10核苷酸随机引物中筛选出15个有效引物。利用这15个有效引物共扩增出630条DNA带，其中多态性条带535，占总扩增条带的84.92％。利用Nei指数法得出7种红树植物间的遗传一致度和遗传距离，并运用UPGMA统计分析法对红树植物的7个种间的亲缘关系进行聚类分析，DNA分子分类系统图。7个种分为A、B两个大组，A组中包含H个种，B组中包含四个种。白骨壤（马鞭草科）、桐花树（紫金牛科）、无瓣海桑（海桑科）同属于A组。白骨壤与桐花树的遗传距离为0．58而与无瓣海桑的遗传距离为0．60，无瓣海桑与桐花树的遗传距离达0．67。可见这三种红树植物虽然聚类在同一组，但由于彼此之间的遗传距离较远（平均0．63），符合传统形态分类，三种红树植物属于不同科：而同属红树科的秋茄、木榄、海莲、尖瓣海莲经分子聚类一起出现在8 组，它们的平均遗传距离为0．35，分子分类也表明它们聚类为同一科，属于科内属种间关系。相对于秋茄而言，木榄、海莲和尖瓣海莲这三者的亲缘关系更近，平均遗传距离为0．28，属于属内种间关系。这和传统的分类学相吻合。由此表明RAPD分于标记技术能较为客观地反应出红树植物的遗传多样性及其相互间的亲缘关系。 3．进一步以海南东寨港红树林自然保护区中，红树（hjzonhora anjcula ta）红海榄（Rhjzopho。stflosa）、秋茄（K8ndelia candel）、角果木（Cerjopstag81）、木榄（BrugUje。goorrhjza）、海莲（BrugU。e。sex。ngula）、尖瓣海莲（BrugU。e。sexa。gula var．rbopchopetala）等红树科7种红树植物为材料，通过已筛选出的15个有效引物进行RAPD分析，获得了红树科7种红树植物的遗传分类关系。！5个有效引物共扩增出 617 条 DNA带，其中多态性条带 415，占总扩增条带的67．26％。利用咖 指数法得出7种红树植物间的遗传一致度和遗传距离，并运用UPG亚A统计分析法对红树植物的7个种间的亲缘关系进行聚类分析，DNA分子分类系统图。7个种分为A、B两个大组，平均遗传距离为0．41，遗传距离最大的为角果木和尖瓣海莲之间0．56（＜0．6），表明7种红树植物在分于水平上处于同一科。 红树、红海榄及角果木处于A组。红树、红海榄遗传距离为0．23。表明红树与红海榄在分于水平上处于同一属。这与它们在宏观分类上属于红树属相一致。角果木与红树的遗传距离为0．38，与红海榄的遗传距离为0．46，平均为0．42（＞0．4），表明角果木与红树、红海榄为科内属间关系，这与宏观分类结果一致。 秋茄、木榄、海莲、尖瓣海莲同处于B组，并且它们种间的遗传距离和分类关系与在福建九龙江口采样分析结果一致，从而进一步证明了RAPD分子标记技术进行红树植物遗传多样性研究的准确性。B组分为2个亚组。秋茄处于一个亚组，它与更多还原

【Abstract】 Molecular Ecological Studies on Genetic Variation and Salt-Resistant Characteristics of Several Species of MangrovesZhou Han-tao(School of Life Sciences, Xiamen University 361005)AbstractMangroves, the intertidal ecosystems occurring primarily in the tropical regions around the world, are valuable natural resources with high productivity and special wetland habitat. The plant species that are quite diversity to form a part of the ecosystem are constantly under environmental stress due to high saline conditions and extreme temperatures and have adapted themselves to these frequent and fluctuating changes at the special place or in the introduction. These plants, which are either bushy shrubs or small-to-large trees, are of great significance because of their potential in protecting coastlines by preventing sea water inundation; inspecting & purging the environmental pollution: and maintaining coastal ecological balance. However, detailed studies of these plants are lacking in major areas. Especially, the genetic polymorphism of the genomes of the plant species in the mangrove ecosystem is poorly understood, and the substantial accumulation of mucilage, latex, phenolics, tannins and other secondary metabolites in these plants make it a difficult system for the studies of molecular biology. In the study, the techniques of RAPD molecular marker .were established to analysis the genetic polymorphism and genetic relationship in mangroves. The technical systems of mRNA differential display were established to isolate the salt-resistant genes from Avicennia marina. The techniques of two-dimensional gel electrophoresis ~vere established to isolate the salt-resistant proteins from Avicennia marina, and the characteristics of these proteins were also analysed. The concrete results as followed:1.CTAB method used in the experiment of DNA extraction is an effective way to wipe off the substantial accumulation of mucilage, latex, phenolics, tannins and other secondary metabolites in mangrove, The obtainment of the DNA extraction has a high ratio I 2Oug DNA per-gram-fresh-weight leaves. The DNA extraction with value of A2~/A,80 between 1.6-1.8 indicates that it is purified enough for RAPD analysis. DNA bands are integral fragments with the size about 23Kb in 0.8% agarose gel. The DNA extraction can be used as the DNA template for RAPD analysis.To ensure that the results of RAPD will be reproductive and true in mangroves, themajor affecting factors in RAPD were studied. These factors included the concentrations of Taq polymerase enzyme, MgCl2, template, dNTPs, primer and thermal cycles, et aI. The final optimized RAPD conditions in mangoves were: each 25 11 I reaction solution was consisted of l0XBuffer (100mM Tris-HCI, pH8.O, 500mM KC1, 0.1%gelatin), SOng template DNA, 2mmol MgCl2, 0.2ismol/L dNTPs of each, 0.2~.t mol/L primer and O.5U Taq DNA polymerase. The amplification procedure conditions were : denature at 94~C I mm, annealing 36~Cfor I mm, extension 72’C for 2 mm, cycling 40 times, last extension at 72~C for 7 mm. The average amplified bands were 6-7. and the results can be repeated finely.2.DNAs of seven species of mangroves in Jiulongiiang Estuary in Fujian, which are Avicennia marina, Aegiceras corn iculatum, Sonneratia apelala, Kandelia candel, Bruguiera gymnorrhiza, Brziguiera sexangula and Bruguiera sexangula var. rhynchopetala , were extracted as the templates. RAPD analysises on genetic diversity of mangroves were conducted. 15 effective primers were screened from 30 10-oligonucleotide arbitrary primers, and a total of 630 DNA bands were amplified, among which 535 (84.92%) were polymorphic. Based on UPGA cluster analysis on DNA bands amplified by the 15 primers, a DNA molecular dendrogram was established. A standard delimit of genetic distance in mangroves was also established, and compared to the conventional classification, the molecular classification of mangroves was accurate.3.DNAs from 7 species in Rhizophpraceae of Dongzhai Harbour Mangrove Reserve in Hainan, which are Rhiz更多还原