ISSR标记技术在植物物证鉴定中应用的初步研究
发布时间：2018-08-19 10:56
【摘要】： 前言 植物广泛存在于自然界,与人们生活密切相关。在一些刑事案件中,检验案犯无意间带走或留在现场的植物叶片、果实、斑汁、花粉等植物物证,对确定嫌疑人与犯罪现场的关系,认定或排除嫌疑人常起到关键作用。在实际案件中,嫌疑人或受害人身上或尸体上附着的植物样品条件很差,凭借传统的形态学和组织学的方法可能无法确认、区分。随着分子生物学技术的发展,可以应用DNA技术对这些样品进行分析、检验,确定它们的种类与周围环境的相似性。 简单序列重复区间(inter-simple sequence repeat,ISSR)分子标记是在PCR基础上发展起来的一种DNA多态性检测技术。其基本原理是在简单序列重复(simplesequence repeat,SSR)的3′或5′端锚定1～4个核苷酸,然后对反向排列SSR间的一段DNA进行PCR扩增,检测其长度多态性。SSR也称微卫星DNA,是一类由几个(多为1～5个)碱基组成的基序串联重复而成的DNA序列,其中最常见的是二核苷酸重复,即(CA)_n和(TG)_n。SSR在真核生物中的分布是非常普遍的,并且进化变异速度非常快,因而锚定引物的ISSR-PCR可以检测基因组许多位点的差异。与SSR-PCR相比,用于ISSR-PCR的引物不需要预先的DNA测序。ISSR分子标记通常为显性标记,呈孟德尔式遗传,具有简便、快速、稳定、DNA多态性高等优点。本研究采用植物分子生物学领域具有广阔应用价值的ISSR标记技术,旨在建立一种在DNA分子水平对植物物证进行种属鉴定的方法,通过筛选ISSR通用引物,对植物种和品种进行鉴定,从而解决相关法律问题。 材料与方法 采用传统CTAB法结合PVP、β-巯基乙醇和RNase A用于提取植物DNA,参考UBC(University of British Columbia)公布和文献中报道的引物,筛选出扩增条带清晰、稳定性好、多态性高的通用引物,应用PCR技术和聚丙烯酰胺凝胶电泳结合银染显谱的方法,对生活中常见的7类植物(葱、玉米、辣椒、大豆、茄子、水稻和槐树)和沈农265、辽粳294、辽星1、R9311和日本晴共5种水稻进行分型。对不同种属、同一种属不同品系和同一植株不同部位的扩增结果进行比较;同时对将室温干燥、阳光曝晒、潮湿环境和农药处理的检材与未处理检材的扩增结果进行比较;以及确定满足ISSR分析的最低植物叶片取材量。最后采用NTSYS软件进行统计学分析,计算Dice相似系数,对同一种属不同品种间进行UPGMA聚类分析。 结果 使用引物UBC824对7类植物进行PCR扩增,扩增产物主要分布在300～3000bp之间,不同种类具有不同的谱带,可以区分被检7类植物。应用引物UBC835对5种水稻品种扩增,扩增产物的长度位于300bp～3000bp,共检出了28条泳动度不同的电泳谱带,根据检出的扩增产物数目和/或片段长度不同,可以区分水稻的5个不同品种。5份供试水稻的Dice相似系数为0.48～0.71,平均为0.60。本研究通过对不同大小植物叶片和不同含量模板DNA的ISSR扩增产物电泳图谱分析,能够获得可满足ISSR分析的最低需要植物叶片取材量和模板DNA量分别为0.1cm~2和390pg,远不如普通PCR-STR的灵敏度高。应用引物UBC824对分别保存于不同环境中(室温干燥、潮湿环境、阳光曝晒和农药喷洒)的植物检材和保存不同时间的植物检材的ISSR扩增产物电泳谱带的分析结果表明,在相同环境条件下放置时间越长,植物DNA模板改变越明显;在相同作用时间下,阳光曝晒组可以得到更多的谱带,潮湿环境组的条带较少,而室温放置组得到的条带最少。喷洒农药一周内各时段提取检材与未喷洒农药检材扩增产物的电泳谱带数目、泳动位置及产物量均无明显差异。 结论 1、采用传统CTAB法结合PVP、β-巯基乙醇和RNase A提取植物DNA能够满足ISSR-PCR分析的最小检材需要面积为0.1cm~2。 2、应用ISSR-PCR技术,根据引物UBC824的扩增结果可区分葱、玉米、辣椒、大豆、茄子、水稻和槐树共7种植物;根据引物UBC835的扩增结果可对沈农265、辽粳294、辽星1、R9311和日本晴5种水稻检材进行区分,为法医学种属鉴定和农业生产中的品种真实性鉴定提供了一种简便经济有效的方法。 3、应用ISSR-PCR技术对阳光曝晒、潮湿环境、干燥和农药因素作用下的检材分析认为:环境湿度是影响DNA降解的重要因素,而农药因素对于DNA影响较小,提示ISSR标记在对陈旧、变质植物检材的种类鉴定受到一定限制。
[Abstract]:Preface
Plants exist widely in nature and are closely related to people's lives. In some criminal cases, the examination of plant evidences, such as leaves, fruits, juices, pollen, etc., taken or left by the criminal unintentionally plays a key role in determining the relationship between the suspect and the crime scene and in identifying or eliminating the suspect. With the development of molecular biology technology, DNA technology can be used to analyze and test these samples to determine their species similarity with the surrounding environment.
Inter-simple sequence repeat (ISSR) molecular marker is a DNA polymorphism detection technique developed on the basis of PCR. Its basic principle is anchoring 1-4 nucleotides at the 3'or 5' ends of simple sequence repeat (SSR), and then amplifying a segment of DNA between SSRs in reverse sequence by PCR. SSR, also known as microsatellite DNA, is a kind of DNA sequence composed of several (up to 1-5) bases. The most common sequence is dinucleotide duplication, i.e. (CA)_n and (TG)_n. Compared with SSR-PCR, primers used for ISSR-PCR do not require pre-sequencing of DNA. ISSR molecular markers are usually dominant markers, which are Mendelian inheritance, simple, rapid, stable, and have high DNA polymorphism. ISSR molecular biology has wide application value in plant molecular biology. The purpose of SR marker technique is to establish a method for species identification of plant evidences at DNA molecular level, and to identify plant species and varieties by screening ISSR universal primers, so as to solve related legal problems.
Materials and methods
The traditional CTAB method combined with PVP, beta-mercaptoethanol and RNase A was used to extract plant DNA. Referring to the primers published by UBC (University of British Columbia) and reported in the literature, universal primers with clear amplified bands, good stability and high polymorphism were screened. PCR and polyacrylamide gel electrophoresis combined with silver staining were used to antibiotic. Seven common plants (onion, maize, pepper, soybean, eggplant, rice and sophora) and five rice varieties (Shennong 265, Liaojing 294, Liaoxing 1, R9311 and Nipponqing) were typed. The results of amplification were compared between the treated and untreated samples, and the lowest amount of leaf samples satisfying ISSR analysis was determined. Finally, NTSYS software was used for statistical analysis, Dice similarity coefficient was calculated, and UPGMA cluster analysis was carried out among different varieties of the same genus.
Result
Primer UBC824 was used to amplify 7 kinds of plants. The amplified products were mainly distributed between 300 BP and 3000 bp. Different kinds of plants had different bands, which could distinguish 7 kinds of plants. Primer UBC835 was used to amplify 5 rice varieties. The length of amplified products ranged from 300 BP to 3000 bp. 28 electrophoretic bands with different swimming degrees were detected. The similarity coefficients of five tested rice varieties ranged from 0.48 to 0.71, with an average of 0.60. The electrophoretic profiles of ISSR amplified products from leaves of different sizes and templates with different contents of DNA were analyzed to meet the minimum requirements of ISSR analysis. The sensitivity of PCR-STR was much lower than that of conventional PCR-STR when the amount of template DNA and leaf material were 0.1 cm~2 and 390 pg, respectively. The results showed that the longer the exposure time was, the more obvious the change of DNA template was. Under the same exposure time, more bands could be obtained in sunlight exposure group, less bands in humid environment group, and the least bands could be obtained in room temperature exposure group. There was no significant difference in the number of electrophoresis bands, the location of swimming and the quantity of products.
conclusion
1. Using traditional CTAB method combined with PVP, beta-mercaptoethanol and RNase A to extract plant DNA can meet the minimum required area of 0.1 cm~2 for ISSR-PCR analysis.
2. ISSR-PCR was used to distinguish 7 species of onion, maize, pepper, soybean, eggplant, rice and locust according to the amplification results of primer UBC824. According to the amplification results of primer UBC83 5, Shennong 265, Liaojing 294, Liaoxing 1, R9311 and Japan Qing were identified as forensic species and authenticity in agricultural production. Identification provides a simple, economical and effective method.
3. ISSR-PCR analysis of samples exposed to sunlight, humid environment, drying and pesticides showed that environmental humidity was an important factor affecting DNA degradation, while pesticides had little effect on DNA, suggesting that ISSR markers were limited in the identification of obsolete and deteriorated plant samples.
【学位授予单位】：中国医科大学
【学位级别】：硕士
【学位授予年份】：2009
【分类号】：D919
本文编号：2191456
[Abstract]:Preface
Plants exist widely in nature and are closely related to people's lives. In some criminal cases, the examination of plant evidences, such as leaves, fruits, juices, pollen, etc., taken or left by the criminal unintentionally plays a key role in determining the relationship between the suspect and the crime scene and in identifying or eliminating the suspect. With the development of molecular biology technology, DNA technology can be used to analyze and test these samples to determine their species similarity with the surrounding environment.
Inter-simple sequence repeat (ISSR) molecular marker is a DNA polymorphism detection technique developed on the basis of PCR. Its basic principle is anchoring 1-4 nucleotides at the 3'or 5' ends of simple sequence repeat (SSR), and then amplifying a segment of DNA between SSRs in reverse sequence by PCR. SSR, also known as microsatellite DNA, is a kind of DNA sequence composed of several (up to 1-5) bases. The most common sequence is dinucleotide duplication, i.e. (CA)_n and (TG)_n. Compared with SSR-PCR, primers used for ISSR-PCR do not require pre-sequencing of DNA. ISSR molecular markers are usually dominant markers, which are Mendelian inheritance, simple, rapid, stable, and have high DNA polymorphism. ISSR molecular biology has wide application value in plant molecular biology. The purpose of SR marker technique is to establish a method for species identification of plant evidences at DNA molecular level, and to identify plant species and varieties by screening ISSR universal primers, so as to solve related legal problems.
Materials and methods
The traditional CTAB method combined with PVP, beta-mercaptoethanol and RNase A was used to extract plant DNA. Referring to the primers published by UBC (University of British Columbia) and reported in the literature, universal primers with clear amplified bands, good stability and high polymorphism were screened. PCR and polyacrylamide gel electrophoresis combined with silver staining were used to antibiotic. Seven common plants (onion, maize, pepper, soybean, eggplant, rice and sophora) and five rice varieties (Shennong 265, Liaojing 294, Liaoxing 1, R9311 and Nipponqing) were typed. The results of amplification were compared between the treated and untreated samples, and the lowest amount of leaf samples satisfying ISSR analysis was determined. Finally, NTSYS software was used for statistical analysis, Dice similarity coefficient was calculated, and UPGMA cluster analysis was carried out among different varieties of the same genus.
Result
Primer UBC824 was used to amplify 7 kinds of plants. The amplified products were mainly distributed between 300 BP and 3000 bp. Different kinds of plants had different bands, which could distinguish 7 kinds of plants. Primer UBC835 was used to amplify 5 rice varieties. The length of amplified products ranged from 300 BP to 3000 bp. 28 electrophoretic bands with different swimming degrees were detected. The similarity coefficients of five tested rice varieties ranged from 0.48 to 0.71, with an average of 0.60. The electrophoretic profiles of ISSR amplified products from leaves of different sizes and templates with different contents of DNA were analyzed to meet the minimum requirements of ISSR analysis. The sensitivity of PCR-STR was much lower than that of conventional PCR-STR when the amount of template DNA and leaf material were 0.1 cm~2 and 390 pg, respectively. The results showed that the longer the exposure time was, the more obvious the change of DNA template was. Under the same exposure time, more bands could be obtained in sunlight exposure group, less bands in humid environment group, and the least bands could be obtained in room temperature exposure group. There was no significant difference in the number of electrophoresis bands, the location of swimming and the quantity of products.
conclusion
1. Using traditional CTAB method combined with PVP, beta-mercaptoethanol and RNase A to extract plant DNA can meet the minimum required area of 0.1 cm~2 for ISSR-PCR analysis.
2. ISSR-PCR was used to distinguish 7 species of onion, maize, pepper, soybean, eggplant, rice and locust according to the amplification results of primer UBC824. According to the amplification results of primer UBC83 5, Shennong 265, Liaojing 294, Liaoxing 1, R9311 and Japan Qing were identified as forensic species and authenticity in agricultural production. Identification provides a simple, economical and effective method.
3. ISSR-PCR analysis of samples exposed to sunlight, humid environment, drying and pesticides showed that environmental humidity was an important factor affecting DNA degradation, while pesticides had little effect on DNA, suggesting that ISSR markers were limited in the identification of obsolete and deteriorated plant samples.
【学位授予单位】：中国医科大学
【学位级别】：硕士
【学位授予年份】：2009
【分类号】：D919
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