| 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. |
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