激發(fā)光源LUYOR-3415RG檢測(cè)煙草瞬轉(zhuǎn)GFP熒光

在我國農(nóng)作物體系中，煙草是重要的經(jīng)濟(jì)作物之一。從經(jīng)濟(jì)貢獻(xiàn)來看，煙草產(chǎn)業(yè)是部分省份的“支柱性產(chǎn)業(yè)"，尤其在云南、貴州、河南、湖南等主產(chǎn)區(qū)，煙草種植覆蓋近千萬農(nóng)戶，直接關(guān)聯(lián)煙農(nóng)收入、地方財(cái)政與就業(yè)穩(wěn)定。煙草是典型的“喜溫、喜光、喜肥"作物，對(duì)生長環(huán)境要求十分苛刻。在現(xiàn)代科研領(lǐng)域中，本氏煙草（Nicotiana benthamiana）是實(shí)驗(yàn)室中重要的模式植物。本氏煙草源于澳洲，容易培養(yǎng)和轉(zhuǎn)化，目前已在各個(gè)國家廣泛用于植物生物學(xué)、基因工程和分子生物學(xué)等領(lǐng)域的研究，例如植物與病原菌互作、代謝工程、功能基因組、合成生物學(xué)和基因編輯等。

近期，中科院華南植物園在《Plant Biotechnology Journal》期刊（IF=10.5，中科院一區(qū)top期刊）發(fā)表文獻(xiàn)《A novel geminivirus-derived 3' flanking sequence of terminator mediates the gene expression enhancement》，文獻(xiàn)中使用熒光蛋白激發(fā)光源LUYOR-3415RG檢測(cè)煙草葉片瞬轉(zhuǎn)GFP熒光，并通過專用濾鏡拍攝煙草GFP熒光表達(dá)照片。LUYOR-3415RG使用便捷、檢測(cè)GFP熒光速度快，大大提高了煙草熒光檢測(cè)效率，為雙生病毒衍生元件的功能驗(yàn)證提供了直觀可靠的技術(shù)支撐。

熒光蛋白激發(fā)光源LUYOR-3415RG可快速檢測(cè)植物基因瞬時(shí)表達(dá)與穩(wěn)定表達(dá)，不用采摘葉片，可直接檢測(cè)植物活體。研究團(tuán)隊(duì)構(gòu)建SBG51系列表達(dá)載體后，通過農(nóng)桿菌介導(dǎo)的本氏煙葉片浸潤實(shí)驗(yàn)，利用熒光蛋白激發(fā)光源LUYOR-3415RG在2-8天的不同時(shí)間點(diǎn)進(jìn)行熒光觀測(cè)。熒光蛋白激發(fā)光源LUYOR-3415RG配備495nm專用濾光片，能高效過濾背景雜光，清晰捕捉到SBG51序列介導(dǎo)的GFP熒光增強(qiáng)效應(yīng)，直觀呈現(xiàn)出它與普通載體在熒光強(qiáng)度和持續(xù)時(shí)間上的差異 ——SBG51-GFP樣本的熒光信號(hào)在接種后第8天仍清晰可見，而對(duì)照組在第6天已因基因沉默消失。

原文摘要

Exploring the new elements to re-design the expression cassette is crucial in synthetic biology. Viruses are one of the most important sources for exploring gene expression elements. In this study, we found that the DNA sequence of the SBG51 deltasatellite from the Sweet potato leaf curl virus (SPLCV) greatly enhanced the gene expression when flanked downstream of the terminator. The SBG51 sequence increased transient GFP gene expression in Nicotiana benthamiana leaves by up to ~6 times and ~10 times compared to the gene expression controlled by the UBQ10 promoter and 35S promoter alone, respectively. The increased GFP gene expression level contributed to the continuous accumulation of GFP protein and GFP fluorescence until 8 days post-inoculation (dpi). The SBG51 sequence also enhanced the gene expression in the transgenic Arabidopsis plants and maintained the spatio-temporal pattern of the FLOWERING LOCUS T (FT) and TOO MANY MOUTHS (TMM) promoters. We identified a123 bp of AT-rich sequence containing seven “ATAAA" or “TTAAA" elements from the SBG51DNA, which had the gene expression enhancement effect. Furthermore, the artificial synthetic sequences containing tandem repeated “ATAAA" or “TTAAA" elements were

sufficient to increase the gene expression but did not alter the polyadenylation of mRNA, similar to the function of matrix attachment regions (MAR). Additionally, the compact artificial synthetic sequence also had an effect on yeast when the expression cassette was integrated into the genome. We conclude that the geminivirus deltasatellite-derived sequence and the “ATAAA"/“TTAAA" elements are powerful tools for enhancing gene expression.

GFP fluorescence photograph and confocal microscopy . The N. benthamiana leaves infiltrated by the GFP expression vector were photographed under (hand-held) light (LUYOR-3415RG) using the 495 nm filter (LUV-495A). The GFP fluorescence of the transgenic Arabidopsis leaves, the

N.       benthamiana leaves 48 h after in filtration, or the protoplasts 24 h after transfection were detected using the LEICA SP8 STED 3X fluorescence microscope confocal system.

原文圖片

該研究證實(shí)，源自雙生病毒δ衛(wèi)星的SBG51序列可使基因表達(dá)量提升10倍。熒光蛋白激發(fā)光源LUYOR-3415RG助力中科院華南植物園基因研究，明確了 "ATAAA"/"TTAAA" 元件的增強(qiáng)功能，為合成生物學(xué)提供了高效便捷的檢測(cè)工具。除煙草外，熒光蛋白激發(fā)光源LUYOR-3415RG還可以檢測(cè)擬南芥、棉花、大豆、草莓等各類植物和作物。

熒光蛋白激發(fā)光源LUYOR-3415RG是一款便攜式、充電電池供電的雙波長熒光激發(fā)光源，客戶可以根據(jù)需要選配兩種光源，單波長6×3W LED發(fā)光，照射面積大。LUYOR-3415RG熒光蛋白激發(fā)光源可有效觀察GFP、eGFP、DsRed、mCherry、tdTomato等綠色熒光蛋白和紅色熒光蛋白的表達(dá)。LUYOR-3415RG在國內(nèi)外高校和科研院所得到廣泛好評(píng)，在全球已有近千篇引用文獻(xiàn)。

文獻(xiàn)DOI：10.1111/pbi.14561