本文選題：轉(zhuǎn)基因楊樹 + 環(huán)介導(dǎo)等溫?cái)U(kuò)增技術(shù)��； 參考：《分子植物育種》2017年11期

【摘要】：隨著轉(zhuǎn)基因技術(shù)的成熟,轉(zhuǎn)基因生物越來越多,關(guān)于轉(zhuǎn)基因生物的爭(zhēng)議也越來越受到人們的關(guān)注。建立一套能夠簡(jiǎn)便、快速、準(zhǔn)確的檢測(cè)轉(zhuǎn)基因林木的外源基因技術(shù),對(duì)轉(zhuǎn)基因林木的研究有重要意義。采用靈敏、快速的環(huán)介導(dǎo)等溫?cái)U(kuò)增(loop-mediated isothermal amplification,LAMP)技術(shù),建立一套以Bt Cry1Ac或Npt II基因?yàn)榘袠?biāo)的轉(zhuǎn)基因林木的檢測(cè)方法。針對(duì)靶標(biāo)基因的6個(gè)區(qū)域設(shè)計(jì)4種特異引物,優(yōu)化LAMP-PCR反應(yīng)體系中的各個(gè)成分,根據(jù)直接觀察產(chǎn)物中焦磷酸鎂的沉淀情況、Gold View核酸染色劑對(duì)其產(chǎn)物染色情況來判斷反應(yīng)結(jié)果并與將產(chǎn)物經(jīng)過1%瓊脂糖電泳的情況進(jìn)行對(duì)比。比較LAMP-PCR和常規(guī)PCR兩種不同檢測(cè)方法的靈敏度。對(duì)轉(zhuǎn)基因楊樹不同無性系中多種外源基因進(jìn)行檢測(cè)。篩選出了最佳的引物,確定出了最優(yōu)的反應(yīng)體系:3 mmol/L Mg2+、1.4 mmol/L Bst、1.4 mol/L Betaine、7:1的10μmol/L內(nèi)外引物比、2μL10 mmol/L d NTP、以及2μL濃度為200 ng/μL的DNA模板。在PCR儀65℃等溫條件下保溫90 min,可直接觀察到焦磷酸鎂的白色沉淀;經(jīng)過Gold View核酸染色劑染色,可在紫外燈下觀察到熒光綠色;經(jīng)過1%瓊脂糖電泳的檢測(cè),可觀察到清晰的彌散狀條帶。通過對(duì)比試驗(yàn)得出LAMP-PCR的靈敏度比常規(guī)PCR要高。對(duì)實(shí)驗(yàn)室多個(gè)轉(zhuǎn)基因楊樹無性系目的基因的檢測(cè),證明了LAMP-PCR在轉(zhuǎn)基因林木外源基因檢測(cè)中的較高可靠性。本研究在國(guó)內(nèi)外首次采用環(huán)介導(dǎo)等溫?cái)U(kuò)增技術(shù)檢測(cè)轉(zhuǎn)基因林木,建立了一套更簡(jiǎn)便、靈敏、快速、可靠的轉(zhuǎn)基因林木的LAMP-PCR檢測(cè)方法。該技術(shù)在恒溫65℃的條件下保溫90 min即可完成轉(zhuǎn)基因林木的檢測(cè),既節(jié)省了時(shí)間,又降低了成本,為轉(zhuǎn)基因林木的檢測(cè)研究提供了一種新的技術(shù)。
[Abstract]:With the maturity of transgenic technology, more and more genetically modified organisms (GMOs) are becoming more and more controversial. It is of great significance to establish a set of foreign gene technology which can be used to detect the transgenic trees easily, quickly and accurately. A sensitive and rapid loop-mediated isothermal amplification technique was used to detect transgenic trees with BT Cry1Ac or NPT II as target. Four specific primers were designed for the six regions of the target gene to optimize the components of the LAMP-PCR reaction system. According to the precipitation of magnesium pyrophosphate in the product, the reaction result was judged by Gold View nucleic acid stain and compared with that of 1% agarose electrophoresis. The sensitivity of LAMP-PCR and conventional PCR were compared. The foreign genes in different clones of transgenic poplar were detected. The best primers were screened out, and the optimal reaction system: 1. 3 mmol / L Mg2 / 1.4 mmol / L Bst1 1.4 mol / L Bst1 10 渭 mol / L inner and outer primer ratio of 2 渭 L 10 mmol / L d NTP and 2 渭 L concentration of 200 ng/ 渭 L DNA template were obtained. The white precipitate of magnesium pyrophosphate can be observed directly in the isothermal condition of 65 鈩，

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