應(yīng)用DNA復(fù)合條形碼技術(shù)研究太白山中小型土壤動物多樣性
發(fā)布時間：2018-08-22 07:40
【摘要】：物種的正確鑒定是所有生物學(xué)研究的基礎(chǔ),基于形態(tài)學(xué)的物種鑒定需要具有豐富經(jīng)驗的分類學(xué)家,且通常需要大量的時間,并且表型可塑性和隱存種的存在以及一些物種不同時期的形態(tài)學(xué)差異會造成錯誤的鑒定。分類學(xué)家的不斷減少和基于形態(tài)學(xué)的物種鑒定的局限性表明我們需要新的方法來完成分類鑒定。2003年,Hebert等提出了DNA條形碼的概念。DNA條形碼技術(shù)的出現(xiàn)彌補(bǔ)了傳統(tǒng)分類學(xué)存在的一些不足,為分類學(xué)的研究提供了重要的幫助。但是,DNA條形碼技術(shù)只能鑒定單個的生物體,面對混合樣本和環(huán)境樣本時就顯現(xiàn)出了其乏力的一面。混合樣本和環(huán)境樣本通常包含了大量的、分類范圍廣泛的生物個體,分離這些生物個體并一一測定其條形碼序列是一項極大的挑戰(zhàn)。新一代測序技術(shù)的出現(xiàn)與持續(xù)發(fā)展解決了DNA條形碼技術(shù)只能鑒定單個生物體的問題。通過新一代測序技術(shù)可以獲得DNA樣品中每一條DNA分子的序列,因此,我們可以提取混合的生物個體的總DNA,選擇合適的分子標(biāo)記進(jìn)行PCR擴(kuò)增,然后使用新一代測序技術(shù)對PCR產(chǎn)物進(jìn)行測序,將測得的序列與合適的數(shù)據(jù)庫進(jìn)行比對,從而獲得混合樣本或環(huán)境樣本中包含的分類學(xué)信息。這種結(jié)合了DNA條形碼技術(shù)和新一代測序技術(shù)的方法就稱為DNA復(fù)合條形碼(DNAmetabarcoding)技術(shù)。目前,對于我國中西部地區(qū)土壤動物的研究較少。本研究于2014年6月上旬在太白山自然保護(hù)區(qū)及周圍選取高山草甸、針葉林、落葉小葉林、落葉闊葉林和農(nóng)田5種生境進(jìn)行了取樣,使用Tullgren漏斗分離土樣中的中小型土壤動物,以18S rRNA基因和COI基因作為分子標(biāo)記,應(yīng)用DNA復(fù)合條形碼技術(shù)對分離得到的中小型土壤動物進(jìn)行了研究。旨在為進(jìn)一步探討秦嶺土壤動物多樣性格局及其影響因素提供基礎(chǔ)資料,為秦嶺生物多樣性的監(jiān)測與保護(hù)提供科學(xué)依據(jù)。本研究的主要結(jié)果如下：(1)共獲得土壤動物424種,隸屬于3門10綱34目201科322屬,其中高山草甸321種,針葉林315種,落葉小葉林314種,落葉闊葉林339種,農(nóng)田327種。并對分類到種屬水平的類群進(jìn)行了列表。(2)生境的變化會對中小型土壤動物的群落組成造成一定的影響。不同生境的中小型土壤動物群落組成不同,但各生境的共有物種較多,達(dá)217種。而各生境的特有物種很少,分別為：高山草甸10種,針葉林7種,落葉小葉林7種,落葉闊葉林13種,農(nóng)田8種。
[Abstract]:The correct identification of species is the basis of all biological research. Morphological identification of species requires experienced taxonomists and usually takes a lot of time. Moreover, the existence of phenotypic plasticity and cryptic species, as well as the morphological differences of some species at different stages may lead to false identification. The decreasing number of taxonomists and the limitation of species identification based on morphology indicate that we need new methods to complete classification. In 2003, Hebert et al proposed the concept of DNA barcode. Some of the shortcomings of classicology, It provides important help for the study of taxonomy. However, DNA barcode technology can only identify individual organisms, but it shows a weak side in the face of mixed samples and environmental samples. Mixed samples and environmental samples usually contain a large number of biological individuals with a wide range of classification. It is a great challenge to isolate these organisms and determine their barcode sequences one by one. The emergence and sustainable development of new generation sequencing technology solve the problem that DNA barcode can only identify single organism. The sequence of each DNA molecule in a DNA sample can be obtained by a new generation of sequencing techniques. Therefore, we can extract the total DNA of a mixed organism and select the appropriate molecular markers for PCR amplification. Then the PCR products are sequenced by a new generation of sequencing techniques, and the measured sequences are compared with the appropriate database to obtain the taxonomic information contained in the mixed samples or environmental samples. This method, which combines DNA barcode technology and new generation sequencing technology, is called DNA compound barcode (DNAmetabarcoding) technology. At present, there is little research on soil fauna in the central and western regions of China. In this study, five habitats, alpine meadow, coniferous forest, deciduous leaflet forest, deciduous broad-leaved forest and farmland, were sampled in and around Taibai Mountain Nature Reserve in early June 2014. Small and medium-sized soil animals were isolated from soil samples by Tullgren funnel. Using 18s rRNA gene and COI gene as molecular markers, the isolation of small and medium-sized soil animals was studied by using DNA complex barcode technique. The aim of this study was to provide basic data for further study on soil animal diversity pattern and its influencing factors in Qinling Mountains, and to provide scientific basis for monitoring and protection of biodiversity in Qinling Mountains. The main results are as follows: (1) A total of 424 species of soil animals were obtained, belonging to 322 genera of 201 families, 34 orders, 3 phyla, 10 classes, including 321 species of alpine meadow, 315 species of coniferous forest, 314 species of deciduous leaflet forest, 339 species of deciduous broad-leaved forest and 327 species of farmland. At the same time, the taxonomy to the species level was listed. (2) the community composition of small and medium-sized soil fauna would be influenced by the change of habitat. The composition of small and medium soil fauna communities in different habitats is different, but there are 217 species of common species in different habitats. There are 10 species in alpine meadow, 7 species in coniferous forest, 7 species in deciduous leaflet forest, 13 species in deciduous broad-leaved forest and 8 species in farmland.
【學(xué)位授予單位】：陜西師范大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2015
【分類號】：S154.5
本文編號：2196436
[Abstract]:The correct identification of species is the basis of all biological research. Morphological identification of species requires experienced taxonomists and usually takes a lot of time. Moreover, the existence of phenotypic plasticity and cryptic species, as well as the morphological differences of some species at different stages may lead to false identification. The decreasing number of taxonomists and the limitation of species identification based on morphology indicate that we need new methods to complete classification. In 2003, Hebert et al proposed the concept of DNA barcode. Some of the shortcomings of classicology, It provides important help for the study of taxonomy. However, DNA barcode technology can only identify individual organisms, but it shows a weak side in the face of mixed samples and environmental samples. Mixed samples and environmental samples usually contain a large number of biological individuals with a wide range of classification. It is a great challenge to isolate these organisms and determine their barcode sequences one by one. The emergence and sustainable development of new generation sequencing technology solve the problem that DNA barcode can only identify single organism. The sequence of each DNA molecule in a DNA sample can be obtained by a new generation of sequencing techniques. Therefore, we can extract the total DNA of a mixed organism and select the appropriate molecular markers for PCR amplification. Then the PCR products are sequenced by a new generation of sequencing techniques, and the measured sequences are compared with the appropriate database to obtain the taxonomic information contained in the mixed samples or environmental samples. This method, which combines DNA barcode technology and new generation sequencing technology, is called DNA compound barcode (DNAmetabarcoding) technology. At present, there is little research on soil fauna in the central and western regions of China. In this study, five habitats, alpine meadow, coniferous forest, deciduous leaflet forest, deciduous broad-leaved forest and farmland, were sampled in and around Taibai Mountain Nature Reserve in early June 2014. Small and medium-sized soil animals were isolated from soil samples by Tullgren funnel. Using 18s rRNA gene and COI gene as molecular markers, the isolation of small and medium-sized soil animals was studied by using DNA complex barcode technique. The aim of this study was to provide basic data for further study on soil animal diversity pattern and its influencing factors in Qinling Mountains, and to provide scientific basis for monitoring and protection of biodiversity in Qinling Mountains. The main results are as follows: (1) A total of 424 species of soil animals were obtained, belonging to 322 genera of 201 families, 34 orders, 3 phyla, 10 classes, including 321 species of alpine meadow, 315 species of coniferous forest, 314 species of deciduous leaflet forest, 339 species of deciduous broad-leaved forest and 327 species of farmland. At the same time, the taxonomy to the species level was listed. (2) the community composition of small and medium-sized soil fauna would be influenced by the change of habitat. The composition of small and medium soil fauna communities in different habitats is different, but there are 217 species of common species in different habitats. There are 10 species in alpine meadow, 7 species in coniferous forest, 7 species in deciduous leaflet forest, 13 species in deciduous broad-leaved forest and 8 species in farmland.
【學(xué)位授予單位】：陜西師范大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2015
【分類號】：S154.5
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