ISSR標(biāo)記技術(shù)在植物物證鑒定中應(yīng)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(guān)。在一些刑事案件中,檢驗(yàn)案犯無意間帶走或留在現(xiàn)場的植物葉片、果實(shí)、斑汁、花粉等植物物證,對確定嫌疑人與犯罪現(xiàn)場的關(guān)系,認(rèn)定或排除嫌疑人常起到關(guān)鍵作用。在實(shí)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(xué)和組織學(xué)的方法可能無法確認(rèn)、區(qū)分。隨著分子生物學(xué)技術(shù)的發(fā)展,可以應(yīng)用DNA技術(shù)對這些樣品進(jìn)行分析、檢驗(yàn),確定它們的種類與周圍環(huán)境的相似性。 簡單序列重復(fù)區(qū)間(inter-simple sequence repeat,ISSR)分子標(biāo)記是在PCR基礎(chǔ)上發(fā)展起來的一種DNA多態(tài)性檢測技術(shù)。其基本原理是在簡單序列重復(fù)(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(jìn)行PCR擴(kuò)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(fù)而成的DNA序列,其中最常見的是二核苷酸重復(fù),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(jìn)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(diǎn)的差異。與SSR-PCR相比,用于ISSR-PCR的引物不需要預(yù)先的DNA測序。ISSR分子標(biāo)記通常為顯性標(biāo)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(diǎn)。本研究采用植物分子生物學(xué)領(lǐng)域具有廣闊應(yīng)用價值的ISSR標(biāo)記技術(shù),旨在建立一種在DNA分子水平對植物物證進(jìn)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(jìn)行鑒定,從而解決相關(guān)法律問題。 材料與方法 采用傳統(tǒng)CTAB法結(jié)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(xiàn)中報(bào)道的引物,篩選出擴(kuò)增條帶清晰、穩(wěn)定性好、多態(tài)性高的通用引物,應(yīng)用PCR技術(shù)和聚丙烯酰胺凝膠電泳結(jié)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(nóng)265、遼粳294、遼星1、R9311和日本晴共5種水稻進(jìn)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(kuò)增結(jié)果進(jìn)行比較;同時對將室溫干燥、陽光曝曬、潮濕環(huán)境和農(nóng)藥處理的檢材與未處理檢材的擴(kuò)增結(jié)果進(jìn)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(jìn)行統(tǒng)計(jì)學(xué)分析,計(jì)算Dice相似系數(shù),對同一種屬不同品種間進(jìn)行UPGMA聚類分析。 結(jié)果 使用引物UBC824對7類植物進(jìn)行PCR擴(kuò)增,擴(kuò)增產(chǎn)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(yīng)用引物UBC835對5種水稻品種擴(kuò)增,擴(kuò)增產(chǎn)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(jù)檢出的擴(kuò)增產(chǎn)物數(shù)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(shù)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(kuò)增產(chǎn)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(yuǎn)不如普通PCR-STR的靈敏度高。應(yīng)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(nóng)藥噴灑)的植物檢材和保存不同時間的植物檢材的ISSR擴(kuò)增產(chǎn)物電泳譜帶的分析結(jié)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(nóng)藥一周內(nèi)各時段提取檢材與未噴灑農(nóng)藥檢材擴(kuò)增產(chǎn)物的電泳譜帶數(shù)目、泳動位置及產(chǎn)物量均無明顯差異。 結(jié)論 1、采用傳統(tǒng)CTAB法結(jié)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(yīng)用ISSR-PCR技術(shù),根據(jù)引物UBC824的擴(kuò)增結(jié)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(jù)引物UBC835的擴(kuò)增結(jié)果可對沈農(nóng)265、遼粳294、遼星1、R9311和日本晴5種水稻檢材進(jìn)行區(qū)分,為法醫(yī)學(xué)種屬鑒定和農(nóng)業(yè)生產(chǎn)中的品種真實(shí)性鑒定提供了一種簡便經(jīng)濟(jì)有效的方法。 3、應(yīng)用ISSR-PCR技術(shù)對陽光曝曬、潮濕環(huán)境、干燥和農(nóng)藥因素作用下的檢材分析認(rèn)為:環(huán)境濕度是影響DNA降解的重要因素,而農(nóng)藥因素對于DNA影響較小,提示ISSR標(biāo)記在對陳舊、變質(zhì)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(xué)位授予單位】：中國醫(yī)科大學(xué)
【學(xué)位級別】：碩士
【學(xué)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(xué)位授予單位】：中國醫(yī)科大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2009
【分類號】：D919
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