【作者】 曾汉元；

【导师】 刘选明；

【作者基本信息】 湖南农业大学 ， 植物学， 2013， 博士

【摘要】 纤维素类能源草(简称“能源草”)是指以植物体内的纤维素为主要能量来源的草本植物。笔者通过对我国热带和亚热带地区能源草的三轮筛选(文献工作筛选、野外观察筛选和田间试验筛选)和综合评价,确定芦竹为最佳者,然后对芦竹的形态结构、药用成分和ISSR、ITS及纤维素合成酶基因等分子水平的特征进行了研究。主要研究结果如下：1、芦竹等纤维素类能源草资源调查与引种栽培按照“高大,多年生,丛生性好,适应性较强,属于草本植物”的自拟标准,通过查阅工具书、植物标本和中国植物志在线数据库,初步筛选出64种纤维素类能源草,隶属于8科31属,其中6种为我国温带分布种,58种为热带与亚热带分布种。在对我国热带和亚热带地区的能源草的野外调查中,观察到拥有一千平方米以上自然居群且植株高度超过1.5m的只有芒、五节芒、斑茅、南荻、河八王、象草、甜根子草、芦竹、芦苇、光高梁、龙须草和拟高粱等12种。对它们进行了引种栽培。2、芦竹作为能源草的分析与评价通过对芒、五节芒、斑茅、河八王、象草、芦竹、芦苇、光高梁和拟高粱等9种能源草的物候期、抗逆性、农艺性状、年干物质产量、热值、灰分含量、光合特性、纤维素和木质素含量等方面的比较研究,构建了能源草的评价体系,采用指数和法,得出9种能源草的评价总得分为：芦竹>芒>象草>斑茅>五节芒>河八王>光高粱>拟高粱>芦苇,因此,在这9种能源草中,以芦竹最佳,其次是芒和象草。3、芦竹形态结构与几种重要成分的研究观察发现,不同产地的芦竹在茎、叶、花等器官的形态上有较大的差异,表现出丰富的多样性。结合生物碱的组织化学定位研究,观察了芦竹根状茎和茎的解剖结构,生物碱在芦竹中的分布部位主要是维管组织。分别采用酸碱洗涤法和Klason法测定了8个居群芦竹成熟期的纤维素和木质素含量,并测定了芦竹2个居群在不同生长期的纤维素和木质素含量。结果表明：(1)芦竹同一居群不同部位的纤维素与木质素含量由高到低均为：秆>枝>叶；(2)8个居群芦竹纤维素含量由高到低依次为：浙江嵊州芦竹>南京市郊西解芦竹>湖南衡阳芦竹>浙江慈溪芦竹>江苏阜宁芦竹>湖南凤凰芦竹>贵州铜仁芦竹>云南洱源芦竹；纤维素含量最高的是采自浙江嵊州的样品,为49.2%；(3)8个居群芦竹木质素含量由高到低依次是：湖南凤凰芦竹>贵州铜仁芦竹>云南洱源芦竹>浙江嵊州芦竹>南京西解芦竹>湖南衡阳芦竹>江苏阜宁芦竹>浙江慈溪芦竹,木质素含量最高的是采自湖南凤凰县的样品,为27.7%。(4)随着生长期的延长,芦竹的纤维素和木质素含量都有不同程度的提高,在生长期2-6个月时增长快,此后6个月增长缓慢。采用HPLC法测定了5个不同芦竹居群的芦竹碱和芦竹胺的含量,结果表明：(1)同一居群不同器官的芦竹碱和芦竹胺含量由高到低依次为：根状茎>茎>叶。因此,芦竹的药用部位以根状茎最佳。(2)在5个居群中,以江苏南京芦竹样品的芦竹碱和芦竹胺含量最高。4、不同居群芦竹遗传多样性的ISSR分析从UBC801～900中筛选出7个有效引物对芦竹11个居群89个样品进行了ISSR扩增,结果表明：11个居群的芦竹平均遗传多样度Ht=0.3376,居群间遗传分化系数Gst为0.8815,平均基因流为0.0672。芦竹居群内的遗传多样性极低,但物种水平的遗传多样性较高。UPGMA法聚类结果显示：不同居群芦竹的亲缘关系与它们的地理位置有一定的关系,但不完全一致。5、不同居群芦竹及其近缘种的ITS序列分析对芦竹、象草、五节芒、斑茅、河八王、光高粱和拟高粱等7种高大禾草的11个个体的rDNA ITS区进行了克隆和测序,并登录于GenBank数据库。采用DNASTAR、CLUSTAL X和MEGA等软件分析其遗传关系聚类图。结果表明：ITS区序列长度为597～601bp,GC含量为58.6～67.8%,5.8S只有5个变异位点；同一物种的不同种群ITS序列只有0～10个碱基的差异。聚类分析结果与经典分类基本一致。ITS分子标记可作为禾本科的亚科以下类群的分类佐证。6、芦竹纤维素合成酶基因克隆与表达分析根据已报道的纤维素合成酶基因的特点设计上、下游引物,对芦竹的纤维素合成酶基因进行了PCR扩增、克隆和测序,得到1379个碱基组成的纤维素合成酶基因片段。通过在GenBank做Blast比对分析,与已报道的所有物种的纤维素合成酶基因的序列高度同源,可以认定此序列为纤维素合成酶基因片段。分析表明,此序列中有四个内含子区。更多还原

【Abstract】 Cellulosic bioenergy grasses (abbreviated form "bioenergy grass") refers to the grass with cellulose as the main source of energy. Through three times of screening (screening by literature search, field observation and field experiment) and comprehensive evaluation to the cellulosic energy grasses in tropical and subtropical regions of China, we conclude that Arundo donax is the best. And then, we studied its morphology, structure, medicinal components and molecular features such as ISSR, ITS and cellulose synthase genes. The main results are as follows:1. the investigation and introduction and cultivation of energy grass resourceIn accordance with the standard of" tall, perennial, tufted and good, strong adaptability and belonging to grass", by consulting the reference books, plants specimen and flora of China online database,64kinds of cellulosic energy grass belonging to31genus and8families were initial screened out, of which6species are kinds of temperate species,58are kinds of tropical and subtropical species. In the field investigation of energy grass to the tropical and subtropical regions of our country, we found that only12species had one or more natural populations with distribution areas over one thousand square meters and the height of plants were more than1.5m, i.e., Miscanthus sinensis, Miscanthus floridulus, Saccharum arundinaceum, Triarrhena lutarioriparia, Narenga porphyrocoma, Pennisetum purpureum, Saccharum spontaneum, Arundo donax, Phragmites communi, Sorghum nitidum, Eulaliopsis binata, and Sorghum propinquum. All of them were introduced and cultivated.2. analyse and comprehensive evaluation among Arundo donax and other energy grassesBy comparative study of phenophase, resistance, agronomic traits, dry matter yield, calorific value, ash content, photosynthetic characteristics, cellulose and lignin content and other aspects of9kinds of energy grasses Miscanthus sinensis, Miscanthus floridulus, Saccharum arundinaceum, Narenga porphyrocoma, Pennisetum purpureum, Arundo donax, Phragmites communi, Sorghum nitidum and Sorghum propinquum, and their evaluation systems was constructed. Using the index method, the total evaluation score of9energy grasses were obtained. Their scores from the hightest to the lowest were Arundo donax, Miscanthus sinensis, Pennisetum purpureum, Saccharum arundinaceum, Miscanthus floridulus,Narenga porphyrocoma, Sorghum nitidum, Sorghum propinquum, Phragmite communis, respectively. Therefore, among these9energy grasses, Arundo donax was the best, followed by Miscanthus sinensis and Pennisetum purpureum. 3. the study of morphology, structure and some important components of Arundo donaxThe different populations of Arundo donax had great differences in the morphology of stem, leaf and flower, and exhibit great diversity. Combined with histochemical localization of alkaloids, the anatomical structure of rhizomes and stem of Arundo donax were observed. It showed that the distribution of gramine in Arundo donax was mainly in vascular tissue.The methods of acid-base washing procedure and Klason procedure were used to measure the contents of cellulose and lignin of eight different habitats of Arundo donax respectively, and the contents of cellulose and lignin of two populations of Arundo donax at different stages of growth were measured. The results showed that:(1) in the same population, the contents of cellulose and lignin from the highest to the lowest were all stem, branch and leave.(2) As for the stems of all samples, the contents of cellulose from the highest to the lowest were the habitats collected from Shengzhou of Zhejiang Province, Xijie of Nanjing, Hengyang of Hunan, Cixi of Zhejiang, Funing of Jiangsu, Tongren of Guizhou and Eryuan of Yunnan Province, respectively. The sample of Shengzhou had the highest cellulose content which was49.2%.(3) From the highest content of lignin to the lowest, they were habitats of Hengyang in Hunan Province, Eryuan of Yunnan, Xijie of Nanjing, Tongren of Guizhou, Shengzhou of Zhejiang, Funing of Jiangsu, Hengyang of Hunan and Cixi of Zhejiang Province, respectively. Stems in Fenghuang had the highest lignin content, which was27.7%.(4) The contents of cellulose and lignin from eight populations increased at different degrees with the growth of time, and they increased very fast in2-6months, and then increased very slowly.Using the HPLC method, the content of gramine and amine from5different populations of Arundo donax were determined. The results showed that:(1) the content of gramine and amine from the same population but different organs are different, from the highest to the lowest is rhizome, stem and leaf. Thus, the best medicinal part of Arundo donax is its rhizome.(2) in5populations, the sample from Nanjing Jiangsu province had the highest content of gramine and amine.4. ISSR analysis of genetic diversity of Arundo donaxFrom UBC801-900,7efficient ISSR primers were screened, by which89samples from11populations of Arundo donax were analyzed. The results showed that:the average genetic diversity of11populations of Arundo donax is Ht=0.3376.The coefficient of genetic differentiation between populations is Gst=0.8815. The average gene flow was0.0672. The genetic distance within different individuals of Arundo donax is little, whereas it is high between different populations. The clustering results by UPGMA method showed that the phylogenetic among different populations of Arundo donax had some relationships with their geographical location, but not completely consistent.5. ITS sequence analysis of different populations of Arundo donax and related speciesThe internal transcribed spacer (ITS) regions of nuclear ribosomal DNA from11individuals of7species Arundo donax, Pennisetum purpureum, Miscanthus floridulus, Saccharum arundinaceum, Narenga porphyrocoma, Sorghum nitidum and Sorghum propinquum were cloned and sequenced, and the sequences were submitted to the Genbank database. The phylogenetic tree of the11species was studied by DNASTAR, CLUSTALX and MEGA software. Results showed that the length of the ITS regions among7species ranged from597to601bp and the contents of G+C in ITS(ITS1+5.8S+ITS2) regions ranged from58.6to67.8%;5.8S was very conservative with only5variable sites. There were a total of184variable sites and160informative sites in ITS regions among7species, but there were only zero to ten different bases among populations of the same species. The cluster analysis result was consistent with classical taxonomy on the whole. Therefore, ITS molecular marker can be used as an evidence of classification to the taxons below subfamilies of Gramineae.6. the analysis of cloned cellulose synthase gene sequence of Arundo donaxBased on the reported characteristics of cellulose synthase gene, the upstream and downstream primers for cloning cellulose synthase gene of Arundo donax were designed, and then, the cellulose synthase gene of Arundo donax was amplified, cloned and sequenced. We gained a fragment of cellulose synthase gene of1379bases. Through blast analysis on the line of GenBank, we found that it had very high homologous with the sequences of cellulose synthase genes of all reported species.更多还原