以庐山或牯岭命名的8种江西乡土植物的DNA分子鉴定研究

发布时间：2018-04-22 14:29

本文选题：庐山 + 牯岭　；参考：《南昌大学》2017年硕士论文

【摘要】：DNA条形码技术可以快速准确地鉴定物种,这使其成为传统植物鉴定体系中的重要补充,同时也能够作为新型工具来很好的保障植物准确地进行分类鉴定。本此实验主要以庐山或牯岭命名的8种植物为对象来探讨DNA条形码技术是否可用于该地区的物种鉴定。目的:利用DNA条形码技术来快速、准确地对庐山地区以庐山或牯岭命名的8种乡土植物物种及其密切相关种进行鉴定研究,为后续进行有效的对以庐山或牯岭命名的江西乡土植物分类系统管理打好基础。方法:本研究选用庐山上采摘的以庐山或牯岭命名的8种乡土植物样本的DNA序列来进行DNA提取,逆转录-聚合酶链式反应(RT-PCR)扩增和测序。为了扩大研究范围,本次实验从GenBank中下载了多个近缘物种的DNA序列,全部序列经CodonCode Aligner V软件拼接、Clustalw 4.0软件比对分析、MEGA7.0计算相关数据和K2P模型构建NJ和ML聚类树等进行鉴定分析。结论:一.在以庐山或牯岭命名的8种乡土植物中ITS与psbA-trnH序列进行PCR扩增并且测序的成功率为100%。ITS序列所获得的5种植物序列长度均在440至724bp之间,比对长度为316~661bp,其中变异位点率为31.9%到61.3%,简约位点率也达到了49.1%~54.5%;psbA-trnH序列所获得的3种植物序列长度均在475~581bp之间,比对长度在351~481bp之间,其中变异位点率为35.5%~52.9%,简约位点率也达到了27.8%~51.6%。此结果表明两类序列不但有较大的变异性,而且还存在较多的信息位点来用于重建可靠的系统进化关系。二.以庐山或牯岭命名的8种乡土植物与近缘种其ITS+PsbA-trnH序列中平均GC含量为29.8%~62.5%。以庐山或牯岭命名的8种乡土植物种内的K2P遗传距离均为0.000,本研究发现以庐山或牯岭命名的8种乡土植物的ITS+PsbA-trnH序列与其近缘种的差异较大、变异位点较多,8种乡土植物种间的K2P平均距离为0.0744~0.3117,因此该8种乡土植物和相关种间的K2P遗传距离均大于种内的;三.研究时采用NJ和ML法构建系统发育树发现以庐山或牯岭命名的8种乡土植物其物种的分支组成均具有很高相似性,这一结果印证了构建的NJ和ML系统树所用的拓扑结构其可信度高。四.研究结果显示,ITS+psbA-trnH序列可为物种种间系统的发育研究提供分子依据:(1)本研究结果发现庐山木槿与木槿的近缘关系最近,所以支持《中国植物志》将庐山木槿归属于木槿属这一观点;(2)本研究中牯岭山梅花与绢毛山梅花个体聚在一起,与其他近缘种明显分开,其与《中国植物志》中说明的牯岭山梅花是绢毛山梅花的一变种,亲缘关系最近符合;(3)庐山蜡瓣花(Corylopsis sinensis Hemsl var.calvescens Rend):中国植物志及一些地方植物志已改为秃蜡瓣花(Corylopsis willottiae Rend)。本研究将庐山蜡瓣花、蜡瓣花和腺蜡瓣花归为一支,并支持《中国植物志》第三十五卷把庐山蜡瓣花归为蜡瓣花属中的穗花系;(4)本研究发现,ITS序列在景天属物种种间和种内变异均存在显著的差异,其水平鉴定成功率达到100%。因此本研究可以准确地将庐山景天、土佐景天和日本景天区分开,所以ITS序列可作为景天属植物的DNA条形码通用序列;(5)psbA-trnH序列可以准确的将石韦及其近缘属进行区分。psbA-trnH序列可以准确鉴定石韦及其亲缘属这一属性使其可用于蕨类药材DNA条形码的鉴定研究;(6)ITS序列在忍冬属中具有很高的扩增效率和鉴定成功率。我们在实验中通过双向测序两端拼接,有效的解决了测序过程中可能会存在polyA/T结构这一问题,从而可得到物种识别所需要的信息。该序列赞同《中国植物志》(第七十二卷)将庐山忍冬作为下江忍冬的一变种,归为红花亚组;(7)本研究发现,ITS序列可以准确的区分庐山悬钩子、北悬钩子、绒毛悬钩子和石生悬钩子,因此ITS序列作为鉴定悬钩子及其亲缘物种的研究是具有可行性的;(8)本研究发现,ITS序列在东俄芹属物种的种间和种内变异均存在显著的差异,因此可以准确地将伞形科中东俄芹属、茴芹属、小芹属和舟瓣芹属区分开,所以ITS序列可被视为伞形科植物DNA条形码中的通用序列。本研究的创新性:首次尝试在DNA分子层面上对庐山地区的乡土植物进行研究。研究依据分子系统学理论、基于DNA条形码在植物鉴定中的原则、利用鉴别植物其种属的最适DNA条形码来构建系统进化树,以此确定所要鉴定的植物物种在整个属中的系统位置。
[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.

【学位授予单位】：南昌大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：Q943.2

【参考文献】