本文選題：轉(zhuǎn)基因作物 + 環(huán)介導(dǎo)等溫?cái)U(kuò)增��； 參考：《吉林大學(xué)》2017年博士論文

【摘要】：本研究根據(jù)我國轉(zhuǎn)基因生物安全監(jiān)管對快速、精準(zhǔn)、高通量檢測技術(shù)的需求,以轉(zhuǎn)基因作物中常見外源抗蟲基因及新研發(fā)的轉(zhuǎn)基因玉米轉(zhuǎn)化體為靶標(biāo),基于普通PCR、定量PCR、數(shù)字PCR、多重PCR、LAMP等技術(shù),旨在建立相應(yīng)轉(zhuǎn)基因成分的核酸分子檢測方法。本研究還利用基因工程手段將前期從嗜鹽曲霉中克隆、并已在擬南芥中證實(shí)具有耐鹽潛力的AgGlp F基因轉(zhuǎn)入大豆栽培品種,以創(chuàng)制耐鹽轉(zhuǎn)基因大豆新材料,為耐鹽大豆新品種培育提供種質(zhì)資源。主要研究結(jié)果如下:1.為建立轉(zhuǎn)基因作物中抗蟲基因的篩選檢測方法,我們系統(tǒng)分析了國內(nèi)外轉(zhuǎn)基因作物中常用的外源抗蟲基因種類及其核苷酸序列,并根據(jù)序列一致性程度,將cry1Ab、cry1Ac、cry1Ab/Ac、mcry1Ac、cry1A.105、cry2Ab、cry3A、cry3Bb等8種Bt基因分為Cry1A、Cry2A和Cry3A等3個(gè)類別,分別建立了這3類Bt基因的單一PCR檢測方法及通過一次擴(kuò)增同時(shí)檢測這3類基因的三重PCR方法,其中Cry1A類基因檢測方法采用簡并PCR策略。上述方法具有特異性強(qiáng)、靈敏度高等特點(diǎn),檢測靈敏度均可達(dá)0.1%,非常適用于轉(zhuǎn)基因作物中cry1Ab、cry1Ac、cry1Ab/Ac等常見Bt基因的快速高效檢測。2.除了常規(guī)PCR方法,本研究還開展了轉(zhuǎn)基因作物中cry2Ab和cry3A基因LAMP快速檢測方法研究。結(jié)果表明,應(yīng)用這兩種LAMP方法可特異性地將含有相應(yīng)靶標(biāo)基因的轉(zhuǎn)基因作物與其他轉(zhuǎn)基因作物和非轉(zhuǎn)基因作物鑒別開,方法的檢出限均可達(dá)5個(gè)靶標(biāo)DNA序列拷貝,比常規(guī)PCR方法的靈敏度提高了4倍。而且,應(yīng)用LAMP方法可在1個(gè)小時(shí)內(nèi)完成檢測,比常規(guī)PCR方法用時(shí)縮短一半以上。通過在LAMP反應(yīng)體系中加入熒光染料,還能夠?qū)崿F(xiàn)肉眼可視化檢測,為轉(zhuǎn)基因成分的現(xiàn)場快速檢測提供了一種可靠的技術(shù)手段。3.為加強(qiáng)我國自主研發(fā)的轉(zhuǎn)基因玉米IE034和Bt506的安全評價(jià)、安全監(jiān)管及知識產(chǎn)權(quán)保護(hù),本研究開展了這兩種轉(zhuǎn)基因玉米的轉(zhuǎn)化體特異性定性及定量PCR精準(zhǔn)檢測方法研究,包括特異性、靈敏度、檢出限、定量限、準(zhǔn)確度和精確度測試等。結(jié)果表明,IE034和Bt506玉米的轉(zhuǎn)化體特異性PCR方法對相應(yīng)轉(zhuǎn)化體具有嚴(yán)格的專一性,這兩種轉(zhuǎn)基因玉米的定性PCR方法檢測靈敏度均可達(dá)0.05%,相當(dāng)于約20個(gè)基因組DNA拷貝。IE034玉米定量PCR方法的檢測限及定量限分別為8個(gè)和40個(gè)拷貝,對IE034玉米的轉(zhuǎn)化體質(zhì)量分?jǐn)?shù)分別為5%、1%、0.5%和0.23%的4個(gè)實(shí)際樣品的定量測定值與預(yù)期值之間的偏差分別為12.2%、3.0%、8.0%和8.7%。Bt506玉米定量PCR方法的檢測限及定量限分別為4個(gè)和35個(gè)拷貝,對Bt506玉米的轉(zhuǎn)化體質(zhì)量分?jǐn)?shù)分別為5%、2%和0.5%的3個(gè)實(shí)際樣品的定量測定值與預(yù)期值之間的偏差分別為10.96%、18.08%和12.64%,均符合ENGL發(fā)布并被國內(nèi)外同行廣泛認(rèn)可的評價(jià)標(biāo)準(zhǔn)。綜上所述,本研究建立的檢測方法可對IE034及Bt506轉(zhuǎn)化體成分進(jìn)行精確的定性鑒定和定量分析。4.建立了我國自主研發(fā)的轉(zhuǎn)基因玉米Bt38的熒光定量PCR方法(q PCR)。應(yīng)用該方法對2個(gè)實(shí)際樣品的鑒定結(jié)果表明,無論是單重還是雙重定量PCR體系,均能獲得準(zhǔn)確可靠的定值結(jié)果。利用單重q PCR方法獲得的測定值與預(yù)期值之間的偏差分別為7.44%和14.28%,利用雙重q PCR方法定值的偏差分別為2.50%和17.34%。此外,我們還成功將Bt38玉米的q PCR方法轉(zhuǎn)化為微滴式數(shù)字PCR方法(dd PCR)。應(yīng)用Bt38玉米的單重和雙重dd PCR方法,對上述2個(gè)實(shí)際樣品的定量結(jié)果均與q PCR結(jié)果一致,表明建立的Bt38玉米q PCR和dd PCR方法均適合于Bt38轉(zhuǎn)化體成分的身份鑒定及精準(zhǔn)定量分析。5.利用農(nóng)桿菌介導(dǎo)的大豆子葉節(jié)轉(zhuǎn)化法,將AgGlp F基因轉(zhuǎn)入大豆受體材料Willams82中,經(jīng)除草劑篩選及PCR、Southern雜交鑒定,獲得29株轉(zhuǎn)AgGlp F基因大豆陽性植株,并從T1代植株中篩選出AgGlp F基因以單拷貝形式整合到大豆基因組中的3個(gè)轉(zhuǎn)基因大豆品系。反轉(zhuǎn)錄熒光定量PCR結(jié)果表明,AgGlp F基因在轉(zhuǎn)基因材料E8A7027-816和E8A7027-817的葉片、根、莖等不同組織器官中均能穩(wěn)定轉(zhuǎn)錄表達(dá),其中葉片的表達(dá)量最高,其次為莖、根。轉(zhuǎn)基因大豆材料E8A7027-817在200mmol/L的Na Cl鹽脅迫下能夠正常生長,而非轉(zhuǎn)基因大豆對照材料在鹽脅迫處理3周后枯萎死亡,表明過表達(dá)AgGlp F基因能顯著提高大豆的耐鹽性,這為獲得耐鹽轉(zhuǎn)基因大豆提供了一種有效的途徑。此外,建立了AgGlp F基因特異性的熒光定量PCR方法,為轉(zhuǎn)AgGlp F基因大豆后代材料鑒定及基因表達(dá)量分析提供方法支持。
[Abstract]:On the basis of GMO Safety Supervision in China on the fast, accurate, high-throughput detection technology needs to genetically modified crops in common exogenous insect resistant genes and transgenic maize new transformants as the target, based on common PCR, quantitative PCR, digital PCR, multiplex PCR, LAMP technology, to establish the corresponding detection method of nucleic acid molecules genetically modified ingredients. This study also used genetic engineering means to pre from halophilic Aspergillus clones, and confirmed with salt tolerance potential of AgGlp F gene into soybean cultivars in Arabidopsis, to create salt tolerant transgenic soybean germplasm resources to provide new materials, new varieties of salt tolerant soybean cultivation. The main results are as follows 1.: to establish a method of detecting for the screening of transgenic crop gene, we analyzed the domestic and foreign GM crops commonly used in insect resistant gene types and exogenous nucleotide sequences, According to the sequence and the degree of consistency, cry1Ab, cry1Ac, cry1Ab/Ac, mcry1Ac, cry1A.105, cry2Ab, cry3A, cry3Bb and other 8 kinds of Bt gene into Cry1A, Cry2A and Cry3A 3 categories, a single PCR detection method of the 3 kinds of Bt gene were established and amplified through a three PCR method at the same time. Measurement of these 3 genes, including Cry1A gene detection method using degenerate PCR strategy. The method has strong specificity, high sensitivity, the detection sensitivity can reach 0.1%, so it is very suitable for cry1Ab, cry1Ac and cry1Ab/Ac in transgenic crops, because of the common Bt based fast and efficient detection of.2. PCR in addition to conventional methods, this study also carried out cry2Ab and cry3A genes in transgenic crops LAMP method for rapid detection of genetically modified crops. The results showed that the application of the two kinds of LAMP methods specifically will contain the target gene with other transgenic and non transgenic crops Crop identification, the detection limit can reach 5 copies of target DNA sequence, sensitivity than the conventional PCR method is increased by 4 times. Moreover, the application of LAMP detection method can be completed in 1 hours, compared to the conventional PCR method for reduction of more than half. The fluorescent dye is added in the LAMP reaction system, but also can the naked eye visual detection, scene for the rapid detection of genetically modified components provides a reliable technical means to strengthen the safety evaluation.3. China's independent research and development of transgenic maize IE034 and Bt506, safety supervision and protection of intellectual property rights, this paper carried out qualitative and quantitative conversion of specific PCR these two kinds of genetically modified corn precision detection research methods, including specificity, sensitivity, detection limit, limit of quantification, accuracy and precision of testing. The results showed that the transformant specific PCR method of IE034 and Bt506 in maize transformation of corresponding body. There are strict specificity, qualitative PCR method of these two kinds of genetically modified corn detection sensitivity can reach 0.05%, the detection limit and the limit of quantification is equivalent to approximately 20 genome copies of DNA quantitative PCR method.IE034 maize were 8 and 40 copies, the mass fraction of IE034 maize transformants respectively 5%, 1%. Quantitative 4 actual samples 0.5% and 0.23% of the measured value and the expected difference between the value were 12.2%, 3%, 8% and the LOD and LOQ of 8.7%.Bt506 corn quantitative PCR method were 4 and 35 copies, the mass fraction of transformants of Bt506 corn were 5%, 3 and 2% quantitative samples and 0.5% of the measured value and the expected difference between the value were 10.96%, 18.08% and 12.64%, are in line with ENGL issued by the domestic and foreign colleagues and widely accepted evaluation criteria. In summary, this study established the detection method of composition of IE034 and Bt506 transformed body Qualitative identification and quantitative analysis of the precise.4. method was established for fluorescence quantitative PCR transgenic maize Bt38 developed in China (Q PCR). The identification results by this method in 2 real samples showed that both single and double quantitative PCR system can obtain accurate and reliable results of fixed value. Using the single Q method for the determination of PCR value and the expected value of the deviation between 7.44% and 14.28% respectively, the deviation value by the double Q PCR method were 2.50% and 17.34%. respectively. In addition, we also successfully Q PCR method of Bt38 corn into the droplet digital PCR (DD PCR). The application of Bt38 in Maize Single and double DD PCR method, the quantitative of the 2 sample results and Q PCR results showed that Bt38, PCR and DD PCR of maize Q methods were suitable for conversion of Bt38 body composition identification and accurate quantitative analysis of.5. using Agroinoculation Cotyledonary node transformation mediated by bacteria, F AgGlp gene into soybean receptor in Willams82 by herbicide selection and PCR Southern hybridization, 29 strains of transgenic AgGlp soybean F gene positive plants, and from the T1 generation plants screened F gene AgGlp in the form of single copy integration to 3 GM soybean lines in the soybean genome. Reverse transcription fluorescence quantitative PCR results showed that the leaf, the F gene AgGlp in transgenic materials E8A7027-816 and E8A7027-817 of the root, stem in different organs are stable expression, which leaves the expression level was the highest, followed by stems and roots. The transgenic soybean material E8A7027-817 can grow normally in 200mmol/L Na Cl under salt stress, while the non transgenic soybean control materials in salt treated die after 3 weeks, showed that over expression of F gene of AgGlp can significantly improve the salt tolerance of soybean, which in order to obtain transgenic salt tolerance Soybean provides an effective way. In addition, a fluorescence quantitative PCR method for AgGlp F gene is established, which provides support for material identification and gene expression analysis of AgGlp F transgenic soybean progeny.

【學(xué)位授予單位】：吉林大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2017
【分類號】：S565.1
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本文編號：1742722