發(fā)布時(shí)間：2018-04-22 14:29

  本文選題：廬山 + 牯嶺��； 參考：《南昌大學(xué)》2017年碩士論文

【摘要】：DNA條形碼技術(shù)可以快速準(zhǔn)確地鑒定物種,這使其成為傳統(tǒng)植物鑒定體系中的重要補(bǔ)充,同時(shí)也能夠作為新型工具來(lái)很好的保障植物準(zhǔn)確地進(jìn)行分類鑒定。本此實(shí)驗(yàn)主要以廬山或牯嶺命名的8種植物為對(duì)象來(lái)探討DNA條形碼技術(shù)是否可用于該地區(qū)的物種鑒定。目的:利用DNA條形碼技術(shù)來(lái)快速、準(zhǔn)確地對(duì)廬山地區(qū)以廬山或牯嶺命名的8種鄉(xiāng)土植物物種及其密切相關(guān)種進(jìn)行鑒定研究,為后續(xù)進(jìn)行有效的對(duì)以廬山或牯嶺命名的江西鄉(xiāng)土植物分類系統(tǒng)管理打好基礎(chǔ)。方法:本研究選用廬山上采摘的以廬山或牯嶺命名的8種鄉(xiāng)土植物樣本的DNA序列來(lái)進(jìn)行DNA提取,逆轉(zhuǎn)錄-聚合酶鏈?zhǔn)椒磻?yīng)(RT-PCR)擴(kuò)增和測(cè)序。為了擴(kuò)大研究范圍,本次實(shí)驗(yàn)從GenBank中下載了多個(gè)近緣物種的DNA序列,全部序列經(jīng)CodonCode Aligner V軟件拼接、Clustalw 4.0軟件比對(duì)分析、MEGA7.0計(jì)算相關(guān)數(shù)據(jù)和K2P模型構(gòu)建NJ和ML聚類樹等進(jìn)行鑒定分析。結(jié)論:一.在以廬山或牯嶺命名的8種鄉(xiāng)土植物中ITS與psbA-trnH序列進(jìn)行PCR擴(kuò)增并且測(cè)序的成功率為100%。ITS序列所獲得的5種植物序列長(zhǎng)度均在440至724bp之間,比對(duì)長(zhǎng)度為316~661bp,其中變異位點(diǎn)率為31.9%到61.3%,簡(jiǎn)約位點(diǎn)率也達(dá)到了49.1%~54.5%;psbA-trnH序列所獲得的3種植物序列長(zhǎng)度均在475~581bp之間,比對(duì)長(zhǎng)度在351~481bp之間,其中變異位點(diǎn)率為35.5%~52.9%,簡(jiǎn)約位點(diǎn)率也達(dá)到了27.8%~51.6%。此結(jié)果表明兩類序列不但有較大的變異性,而且還存在較多的信息位點(diǎn)來(lái)用于重建可靠的系統(tǒng)進(jìn)化關(guān)系。二.以廬山或牯嶺命名的8種鄉(xiāng)土植物與近緣種其ITS+PsbA-trnH序列中平均GC含量為29.8%~62.5%。以廬山或牯嶺命名的8種鄉(xiāng)土植物種內(nèi)的K2P遺傳距離均為0.000,本研究發(fā)現(xiàn)以廬山或牯嶺命名的8種鄉(xiāng)土植物的ITS+PsbA-trnH序列與其近緣種的差異較大、變異位點(diǎn)較多,8種鄉(xiāng)土植物種間的K2P平均距離為0.0744~0.3117,因此該8種鄉(xiāng)土植物和相關(guān)種間的K2P遺傳距離均大于種內(nèi)的;三.研究時(shí)采用NJ和ML法構(gòu)建系統(tǒng)發(fā)育樹發(fā)現(xiàn)以廬山或牯嶺命名的8種鄉(xiāng)土植物其物種的分支組成均具有很高相似性,這一結(jié)果印證了構(gòu)建的NJ和ML系統(tǒng)樹所用的拓?fù)浣Y(jié)構(gòu)其可信度高。四.研究結(jié)果顯示,ITS+psbA-trnH序列可為物種種間系統(tǒng)的發(fā)育研究提供分子依據(jù):(1)本研究結(jié)果發(fā)現(xiàn)廬山木槿與木槿的近緣關(guān)系最近,所以支持《中國(guó)植物志》將廬山木槿歸屬于木槿屬這一觀點(diǎn);(2)本研究中牯嶺山梅花與絹毛山梅花個(gè)體聚在一起,與其他近緣種明顯分開,其與《中國(guó)植物志》中說(shuō)明的牯嶺山梅花是絹毛山梅花的一變種,親緣關(guān)系最近符合;(3)廬山蠟瓣花(Corylopsis sinensis Hemsl var.calvescens Rend):中國(guó)植物志及一些地方植物志已改為禿蠟瓣花(Corylopsis willottiae Rend)。本研究將廬山蠟瓣花、蠟瓣花和腺蠟瓣花歸為一支,并支持《中國(guó)植物志》第三十五卷把廬山蠟瓣花歸為蠟瓣花屬中的穗花系;(4)本研究發(fā)現(xiàn),ITS序列在景天屬物種種間和種內(nèi)變異均存在顯著的差異,其水平鑒定成功率達(dá)到100%。因此本研究可以準(zhǔn)確地將廬山景天、土佐景天和日本景天區(qū)分開,所以ITS序列可作為景天屬植物的DNA條形碼通用序列;(5)psbA-trnH序列可以準(zhǔn)確的將石韋及其近緣屬進(jìn)行區(qū)分。psbA-trnH序列可以準(zhǔn)確鑒定石韋及其親緣屬這一屬性使其可用于蕨類藥材DNA條形碼的鑒定研究;(6)ITS序列在忍冬屬中具有很高的擴(kuò)增效率和鑒定成功率。我們?cè)趯?shí)驗(yàn)中通過(guò)雙向測(cè)序兩端拼接,有效的解決了測(cè)序過(guò)程中可能會(huì)存在polyA/T結(jié)構(gòu)這一問(wèn)題,從而可得到物種識(shí)別所需要的信息。該序列贊同《中國(guó)植物志》(第七十二卷)將廬山忍冬作為下江忍冬的一變種,歸為紅花亞組;(7)本研究發(fā)現(xiàn),ITS序列可以準(zhǔn)確的區(qū)分廬山懸鉤子、北懸鉤子、絨毛懸鉤子和石生懸鉤子,因此ITS序列作為鑒定懸鉤子及其親緣物種的研究是具有可行性的;(8)本研究發(fā)現(xiàn),ITS序列在東俄芹屬物種的種間和種內(nèi)變異均存在顯著的差異,因此可以準(zhǔn)確地將傘形科中東俄芹屬、茴芹屬、小芹屬和舟瓣芹屬區(qū)分開,所以ITS序列可被視為傘形科植物DNA條形碼中的通用序列。本研究的創(chuàng)新性:首次嘗試在DNA分子層面上對(duì)廬山地區(qū)的鄉(xiāng)土植物進(jìn)行研究。研究依據(jù)分子系統(tǒng)學(xué)理論、基于DNA條形碼在植物鑒定中的原則、利用鑒別植物其種屬的最適DNA條形碼來(lái)構(gòu)建系統(tǒng)進(jìn)化樹,以此確定所要鑒定的植物物種在整個(gè)屬中的系統(tǒng)位置。
[Abstract]:DNA barcode technology can quickly and accurately identify species, which makes it an important supplement in the traditional plant identification system, and can also be used as a new tool to ensure the accurate classification and identification of plants. This experiment is mainly based on the 8 plants named Mount Lu or kushing to explore the availability of DNA barcode technology. Species identification in this area. Objective: to quickly and accurately identify 8 species of native plant species and their close related species named by Mount Lu or kushing in Mount Lu area by using DNA bar code technology, and to make a good basis for the follow-up of the management of the Jiangxi native plant classification system named by Mount Lu or kushing. The DNA sequences of 8 native plant samples taken on the Mount Lu were selected for DNA extraction, and reverse transcription polymerase chain reaction (RT-PCR) was amplified and sequenced. In order to expand the scope of the study, this experiment downloaded DNA sequences of several related species from GenBank, and all the sequences were spliced by CodonCode Aligner V software. Clustalw 4 software comparison analysis, MEGA7.0 calculation related data and K2P model construction NJ and ML cluster tree to identify. Conclusion: 1. The success rate of PCR amplification and sequencing of the ITS and psbA-trnH sequences of the 8 native plants named in Mount Lu or kushing is 440 to 724b of the 5 plant sequences obtained from the 100%.ITS sequence. Between P, the comparison length is 316~661bp, of which the ectopic point rate is 31.9% to 61.3%, and the simple loci rate also reaches 49.1%~54.5%; the length of the 3 plant sequences obtained by the psbA-trnH sequence is between 475~581bp and the length of 351~481bp, and the ectopic point rate is 35.5%~ 52.9%, and the simple loci rate also reaches the 27.8%~51.6%. result table. The two class of Ming two sequences not only have large variability, but also have more information loci to reconstruct reliable phylogenetic relationships. Two. The average GC content in the ITS+PsbA-trnH sequence of 8 native plants and close species named by Mount Lu or Kuling is the K2P genetic distance in the 8 native plant species named by Mount Lu or Kuling. The difference was 0. The study found that the ITS+PsbA-trnH sequence of 8 native plants named in Mount Lu or kushing was different from its close species, with more variation loci and the average distance of K2P between 8 native species of native plants was 0.0744~0.3117, so the K2P genetic distance between the 8 native plants and related species was greater than that in the species; three. N was used in the study. The phylogenetic tree constructed by the J and ML methods found that the branches of 8 native plants named in Mount Lu or kguling were highly similar. The results showed that the topological structure of the constructed NJ and ML system trees had high reliability. Four. The results showed that the ITS+psbA-trnH sequence could provide the development of interspecific system for species. Molecular basis: (1) the results of this study found the closest relationship between Hibiscus hibiscus and hibiscus hibiscus. Therefore, it supports the view of "Chinese flora" to belong to the hibiscus Hibiscus of Mount Lu. (2) in this study, the plum flower of kushing mountain and the spun mountain plum blossom are gathered together, and the other close species are distinctly separated from the Mount Lu. Plum flower is a variety of Sericite plum blossom, and the relationship has recently met; (3) Mount Lu wax petal flower (Corylopsis sinensis Hemsl var.calvescens Rend): Chinese flora and some local plant records have been changed to bald wax petal flower (Corylopsis willottiae Rend). This study has classified the wax petal flower, wax flower and adenosine petal flower of Mount Lu as one, and supported < China. The thirty-fifth volumes of plant Zhi > thirty-fifth rolls of Mount Lu wax flower belong to the Honoka system in the genus paraffin; (4) this study found that there were significant differences in the interspecific and intraspecific variations of the ITS sequence, and the success rate of its level identification reached 100%. so that this study could accurately separate the Mount Lu, Sedum, and Japanese Sedum, so the ITS sequence The column can be used as a general sequence of DNA barcode for Sedum plants; (5) psbA-trnH sequence can accurately distinguish between Shi Wei and its proximal genus in the.PsbA-trnH sequence, which can accurately identify the property of Shi Wei and its relatives so that it can be used for the identification of DNA barcode for ferns; (6) the ITS sequence has a high increasing effect in the genus Lonicera. In the experiment, we can effectively solve the problem that the polyA/T structure may exist in the process of sequencing, and we can get the information needed for the identification of species in the process of sequencing. This sequence agrees with China's plant Chronicles (seventy-second volumes) to make the honeysuckle of Mount Lu as a variety of Lonicera japonica and return to red flower. (7) (7) this study found that the ITS sequence can accurately distinguish the raspberry, the North Rubus, the villus Rubus and the stone Rubus, so the ITS sequence is feasible to identify the raspberry and its related species. (8) this study found that there is a significant difference between the interspecific and intraspecific variations of the ITS sequence in the species of the genus apigenin. Therefore, the parsley family, anise, parsley and parsley, can be separated accurately, so the ITS sequence can be regarded as a general sequence in the DNA barcode of paratoin family. The innovation of this study is the first attempt to study the native plants in the Mount Lu area at the DNA molecular level. On the basis of the principle of DNA barcode in plant identification, the phylogenetic tree is constructed by using the optimum DNA barcode identifying the species of plant species to determine the system location of the plant species identified in the whole genus.

【學(xué)位授予單位】：南昌大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類號(hào)】：Q943.2

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