ISSR標記技術在植物物證鑒定中應用的初步研究
發(fā)布時間：2018-08-19 10:56
【摘要】： 前言 植物廣泛存在于自然界,與人們生活密切相關。在一些刑事案件中,檢驗案犯無意間帶走或留在現場的植物葉片、果實、斑汁、花粉等植物物證,對確定嫌疑人與犯罪現場的關系,認定或排除嫌疑人常起到關鍵作用。在實際案件中,嫌疑人或受害人身上或尸體上附著的植物樣品條件很差,憑借傳統(tǒng)的形態(tài)學和組織學的方法可能無法確認、區(qū)分。隨著分子生物學技術的發(fā)展,可以應用DNA技術對這些樣品進行分析、檢驗,確定它們的種類與周圍環(huán)境的相似性。 簡單序列重復區(qū)間(inter-simple sequence repeat,ISSR)分子標記是在PCR基礎上發(fā)展起來的一種DNA多態(tài)性檢測技術。其基本原理是在簡單序列重復(simplesequence repeat,SSR)的3′或5′端錨定1～4個核苷酸,然后對反向排列SSR間的一段DNA進行PCR擴增,檢測其長度多態(tài)性。SSR也稱微衛(wèi)星DNA,是一類由幾個(多為1～5個)堿基組成的基序串聯(lián)重復而成的DNA序列,其中最常見的是二核苷酸重復,即(CA)_n和(TG)_n。SSR在真核生物中的分布是非常普遍的,并且進化變異速度非常快,因而錨定引物的ISSR-PCR可以檢測基因組許多位點的差異。與SSR-PCR相比,用于ISSR-PCR的引物不需要預先的DNA測序。ISSR分子標記通常為顯性標記,呈孟德爾式遺傳,具有簡便、快速、穩(wěn)定、DNA多態(tài)性高等優(yōu)點。本研究采用植物分子生物學領域具有廣闊應用價值的ISSR標記技術,旨在建立一種在DNA分子水平對植物物證進行種屬鑒定的方法,通過篩選ISSR通用引物,對植物種和品種進行鑒定,從而解決相關法律問題。 材料與方法 采用傳統(tǒng)CTAB法結合PVP、β-巰基乙醇和RNase A用于提取植物DNA,參考UBC(University of British Columbia)公布和文獻中報道的引物,篩選出擴增條帶清晰、穩(wěn)定性好、多態(tài)性高的通用引物,應用PCR技術和聚丙烯酰胺凝膠電泳結合銀染顯譜的方法,對生活中常見的7類植物(蔥、玉米、辣椒、大豆、茄子、水稻和槐樹)和沈農265、遼粳294、遼星1、R9311和日本晴共5種水稻進行分型。對不同種屬、同一種屬不同品系和同一植株不同部位的擴增結果進行比較;同時對將室溫干燥、陽光曝曬、潮濕環(huán)境和農藥處理的檢材與未處理檢材的擴增結果進行比較;以及確定滿足ISSR分析的最低植物葉片取材量。最后采用NTSYS軟件進行統(tǒng)計學分析,計算Dice相似系數,對同一種屬不同品種間進行UPGMA聚類分析。 結果 使用引物UBC824對7類植物進行PCR擴增,擴增產物主要分布在300～3000bp之間,不同種類具有不同的譜帶,可以區(qū)分被檢7類植物。應用引物UBC835對5種水稻品種擴增,擴增產物的長度位于300bp～3000bp,共檢出了28條泳動度不同的電泳譜帶,根據檢出的擴增產物數目和/或片段長度不同,可以區(qū)分水稻的5個不同品種。5份供試水稻的Dice相似系數為0.48～0.71,平均為0.60。本研究通過對不同大小植物葉片和不同含量模板DNA的ISSR擴增產物電泳圖譜分析,能夠獲得可滿足ISSR分析的最低需要植物葉片取材量和模板DNA量分別為0.1cm~2和390pg,遠不如普通PCR-STR的靈敏度高。應用引物UBC824對分別保存于不同環(huán)境中(室溫干燥、潮濕環(huán)境、陽光曝曬和農藥噴灑)的植物檢材和保存不同時間的植物檢材的ISSR擴增產物電泳譜帶的分析結果表明,在相同環(huán)境條件下放置時間越長,植物DNA模板改變越明顯;在相同作用時間下,陽光曝曬組可以得到更多的譜帶,潮濕環(huán)境組的條帶較少,而室溫放置組得到的條帶最少。噴灑農藥一周內各時段提取檢材與未噴灑農藥檢材擴增產物的電泳譜帶數目、泳動位置及產物量均無明顯差異。 結論 1、采用傳統(tǒng)CTAB法結合PVP、β-巰基乙醇和RNase A提取植物DNA能夠滿足ISSR-PCR分析的最小檢材需要面積為0.1cm~2。 2、應用ISSR-PCR技術,根據引物UBC824的擴增結果可區(qū)分蔥、玉米、辣椒、大豆、茄子、水稻和槐樹共7種植物;根據引物UBC835的擴增結果可對沈農265、遼粳294、遼星1、R9311和日本晴5種水稻檢材進行區(qū)分,為法醫(yī)學種屬鑒定和農業(yè)生產中的品種真實性鑒定提供了一種簡便經濟有效的方法。 3、應用ISSR-PCR技術對陽光曝曬、潮濕環(huán)境、干燥和農藥因素作用下的檢材分析認為:環(huán)境濕度是影響DNA降解的重要因素,而農藥因素對于DNA影響較小,提示ISSR標記在對陳舊、變質植物檢材的種類鑒定受到一定限制。
[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.
【學位授予單位】：中國醫(yī)科大學
【學位級別】：碩士
【學位授予年份】：2009
【分類號】：D919
本文編號：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.
【學位授予單位】：中國醫(yī)科大學
【學位級別】：碩士
【學位授予年份】：2009
【分類號】：D919
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