【作者】 李际红；

【导师】 邢世岩；

【作者基本信息】 山东农业大学 ， 森林培育学， 2011， 博士

【摘要】 银杏(Ginkgo biloba L)有许多原始和进化特征。银杏属实际上可能是现存的种子植物中最古老的一个属。国内外大量研究表明银杏是比较形态学、解剖学、胚胎发育及系统发育研究的最理想的试材之一。叶籽银杏(Ginkgo biloba var. epiphylla Mak.)作为银杏家族中的一个特异种质,具有重要的理论和观赏价值。其最大特点在于叶生雌性生殖器官(epiphyllous female reproductive organ,EFRO)的个体发生和结构。它们对银杏的系统发育、起源、演化及亲缘关系的研究具有重要意义。本研究通过对叶籽银杏叶生雌性生殖器官(EFRO)发育与再生过程中DNA甲基化分子机制的研究,以期揭示叶籽银杏雌性生殖器官的形态学本质及其系统学意义。其实验结果和主要结论如下:本研究采用甲基化敏感扩增多态性技术(MSAP),以叶籽银杏、银杏及攀枝花苏铁、铁线蕨两个外类群为材料,从54对MSAP选扩增引物中,选出扩增清晰、可辨且可重复的16-24对MSAP引物组合,扩增获得这四个物种的DNA胞嘧啶甲基化修饰水平及MSAP扩增图谱,结果表明叶籽银杏基因组中至少有48.7%(全甲基化34%、半甲基化14.7%)、银杏44.0%(全甲基化32.8%、半甲基化11.2%)、苏铁39.6%(全甲基化29.2%、半甲基化9.8%)、铁线蕨44.6%(全甲基化29.4%、半甲基化15.2%)的CCGG/GGCC位点发生胞嘧啶甲基化。与相关物种比较,这四个物种的DNA胞嘧啶甲基化水平整体较高。植物基因组DNA胞嘧啶甲基化修饰检出率不同,反映了在基因组水平上的物种的差异性。本研究通过对叶籽银杏、银杏在萌动期和展叶期的CCGG/GGCC位点胞嘧啶甲基化进行的总体分析,结果发现萌动期叶籽银杏甲基化水平47.6%、银杏42.4%,展叶期叶籽银杏着生叶生胚珠的叶籽银杏(YZ)甲基化水平48.3%、没有着生叶生胚珠的叶籽银杏(YC)40.5%、银杏(CK)41.5%,体现为叶籽银杏的甲基化高于YC及CK;同时叶籽银杏、银杏在甲基化模式上存在着丰富的差异,萌动期叶籽银杏相对于银杏去甲基化比例为13.2%,超甲基化比例为14.6%,超甲基化的比例略高于去甲基化;展叶期YZ相对于YC去甲基化比例为8.2%,超甲基化比例为16.0%,YZ相对于CK去甲基化比例为4.8%,超甲基化比例为28%,总体表现为展叶期的YZ相对于YC及CK的超甲基化水平高于萌动期的超甲基化水平。显示萌动期与展叶期叶籽银杏甲基化修饰的水平不同。说明叶籽银杏EFRO发育过程中胞嘧啶甲基化具有时空特异性。本研究对叶籽银杏不同组织DNA胞嘧啶甲基化的水平、模式及模式上的差异类型进行分析,结果发现叶籽银杏不同组织DNA胞嘧啶甲基化的水平不同,其发端期47.7%、雌配子发育前期(前期)46.0%、雌配子发育中期(中期)45.2%、雌配子发育后期(后期)41.8%、叶籽银杏正常胚珠胚乳初期(CK)35.3%。不同组织在甲基化模式上也存在着广泛的差异,表现为发端期相应于前期去甲基化比例为19.7%、超甲基化比例为14.6%,中期去甲基化比例20.0%、超甲基化比例为13.30%,后期去甲基化比例为20.3%、超甲基化比例为14.6%,CK去甲基化比例为20.3%,超甲基化比例为18.8%。不同组织甲基化模式上的差异体现为发端期组织相对于雌配子发育的三个时期的组织(前期、中期、后期)及叶籽银杏正常胚珠胚乳初期组织(CK)的去甲基化水平高于超甲基化水平,发生了去甲基化,这个时期有更多的基因表达,是叶籽银杏EFRO形成的关键时期。叶籽银杏在不同时期、不同组织这种去甲基化/超甲基化的变化式样,对揭示叶籽银杏EFRO形成的可能机制及系统学意义奠定了基础。本研究从54对MSAP选扩引物中,选出16对MSAP选扩引物组合,扩增产生叶籽银杏不同单株清晰可辨的MSAP扩增图谱,检测其甲基化水平,发现不同单株甲基化水平不同,其中,沂源油坊(I油坊)为47.2%、沂源织女洞南(I织南) 44.09%、沂源织女洞北(I织北) 43.8%、沂源白裕(I白裕) 43.3%、沂源中河(I中河) 43.5%、泰安老君堂正常银杏(CK)39.7%。不同单株在甲基化模式上也存在着广泛的差异, I油坊相对于I织南、I织北、CK的去甲基化比例分别为8.7%、13.3%和8.42%,超甲基化比例分别为27.1%、11.22%、13.68%。在同一时期,I油坊较之其他几株表现为较高的超甲基化水平,或许与I油坊树龄大(树龄为1300年,其余在700年左右)有关。即植物形态的构成与年龄有一定的关联。本研究采用分子生物学技术,对部分叶籽银杏、银杏修饰位点及叶籽银杏多态性位点进行克隆和序列分析,通过匹配的同源序列功能注释,发现叶籽银杏、银杏基因组中包括大量的重复序列、转座子序列、转录调控因子、反转录转座子序列、F-box蛋白、叶绿体通道蛋白、钠离子通道蛋白、泛素蛋白、假设蛋白及乙酸脱氢酶在内的多种类型DNA序列中均存在DNA甲基化修饰现象。这些基因在叶籽银杏和银杏中的甲基化状态被修饰的程度不同,表明它们可能有助于叶籽银杏和银杏间的表型差异的形成,对于解读叶籽银杏的形成机制具有一定的意义。更多还原

【Abstract】 Ginkgo biloba L. had many original and evolutional features. In fact, ginkgo genera maybe was the oldest genera. Numerous researches at home and abroad showed that ginkgo was one of the ideal materials for comparative morphology, anatomy, embryonic development, and phylogenies.As a peculiar germplasm of ginkgo, Ginkgo biloba var. epiphylla Mak, had important value in theory and view. Its distinguishing feature was the ontogeny and structure of epiphyllous female reproductive organ (EFRO), which had important significance in phylogenies, origin, development and research on genetic relationship of ginkgo. Studies on DNA methylation mechanism in EFRO development of Ginkgo biloba var. epiphylla Mak had important significance in revealing morphology essence and systematics. The results and main conclusions as follows:MSAP was applied on ginkgo biloba L., ginkgo biloba var. epiphylla Mak., Cycas, and Adiantum edgeworthii Hook. 16-24 pairs of clear and reproducible selective primers were selected from 54 pairs of MSAP. Cytosine methylation modified level and MSAP amplification patterns of this four species obtained from amplification. The results showed that: about 48.7% (34% full methylation, and 14.7% half- methylation) of the CCGG sites in epiphylla ginkgo genome were methylated, 44.0% (32.8% methylation, and 11.2% half- methylation) in ginkgo, 39.6% (29.2% methylation, and 9.8% half- methylation) in Cycas, and 44.6% (29.4% methylation, and 15.2% half-methylation) in Adiantum edgeworthii. Compared with related species, the methylate level of these species was high. The difference of DNA methylate modified detection rate can reflect the differences in genome species.Cytosine methylation of the CCGG sites at budding stage and leaf-expansion stage of ginkgo and epiphylla ginkgo was general analysis. The results obtained were: at budding stage, methylate level was 47.6% of epiphylla ginkgo, and 42.4% of ginkgo; at leaf-expansion stage, methylate level was 48.3% of malformation-leaf (YZ), 40.5% of normal leaf (YC) of epiphylla ginkgo, and 41.5% of ginkgo leaf (CK), which showed that methylate level of YZ was higher than YC and CK. At the same time, Epiphylla ginkgo and ginkgo had rich variation in methylate model. The ratio of demethylate epiphylla ginkgo to ginkgo at budding sta ge was 13.2%, supermethylation rate was 14.6%, which was little higher than demethylate. The ratio of demethylate YZ to YC at leaf-expansion stage was 8.2%, supermethylation rate was 28%. On the whole,it was obvious that YZ showed supermethylation was highest at leaf-expansion stage,meanwhile, which was also higher than budding stage. Methylate modified level was different at budding stage and leaf-expansion stage of epiphylla ginkgo. It was showed that Cytosine methylation of EFRO of epiphylla ginkgo had specificity with time and space.The cytosine methylate level, mode, and variation type of DNA in different tissue of epiphylla ginkgo was analyzed. It showed that cytosine methylate level DNA in different tissue was different, which was 47.7% at budding stage, at early, middle, late stage of female gametophyte development was 46.0%, 45.2%, and 41.8%, respectively, 35.3% at endosperm stage of CK. Methylate mode was different in different tissue, which showed that demethylate ratio was 19.7%, supermethylation ratio was 14.6% at previous stage, demethylate ratio was 20.0%, supermethylation ratio was 13.3% at middle stage, and demethylate ratio was 20.3%, supermethylation ratio was 14.6% at later stage. The demethylate ratio of CK was 20.3%, and supermethylation ratio was 18.8%. The methylate model difference among different tissue showed that demethylate level was higher than supermethylation level of tissue at budding stage to tissue of three stage of gametophyte development (previous stage, middle stage, and later stage), and early tissue of endosperm of CK. Much gene express at this stage, which was the critical stage of epiphylla ovule formation. This demethylate/supermethylation mode of different stage and different tissue of epiphylla ginkgo laid a foundation for EFRO formation mechanism of epiphylla ginkgo and its phylogenies.16 pairs of selective primers were selected from 54 pairs of MSAP. MSAP amplification pattern produced from different individual of epiphylla ginkgo. Methylate level was different in different individual: youfang (I youfang) was 47.2%, south of zhinv cave (I zhinan) was 44.09%, north of zhinv cave (I zhibei) was 43.8%, baiyu(biayu) was 43.3%, zhonghe (I zhonghe) 43.5%, normal ginkgo of laojuntang (CK) was 39.7%. The methylate mode was also different in individual. Demethylate ratio of I youfang to I zhinan, I zhibei, and CK was 8.7%, 13.3%, and 8.42%, supermethylation ratio of which was 27.1%, 11.22%, and 13.68%, respectively. I youfang appeared higher supermethylation level than other individual contemporaneity, perhaps, which was related with old age (I youfang was 1300a, and others were about 700a). There was relationship between plant morphosis and age. Mode sites of epiphylla ginkgo and ginkgo, and polymorphic site of epiphylla ginkgo were cloned and sequence analyzed by protocols in molecular biology. DNA methylate modified was existed many DNA sequence types, such as: repetitive sequence, transposon sequence, transcription regulator, retrotransposons sequence, F-box protein, channel protein of chloroplast, channel protein of Na+, ubiquitin-protein, hypothetical protein and ethyl-dehydrogenase in epiphylla ginkgo and ginkgo genome by matching homologous sequence. The different in methylate modified level of such genes in epiphylla ginkgo and ginkgo showed that it was helpful to epiphylla ginkgo and ginkgo phenotypic differences formation, and had significance in understanding epiphylla ginkgo formation.更多还原