Reichel C, Mathur J, Eckes P, Langenkemper K, Koncz C, Schell J, Reiss B, Maas C
Max-Planck-Institut für Züchtungsforschung, Abteilung Genetische Grundlagen der Pflanzenzüchtung, Köln, Germany.
Proc Natl Acad Sci U S A. 1996 Jun 11;93(12):5888-93. doi: 10.1073/pnas.93.12.5888.
The expression of the jellyfish green fluorescent protein (GFP) in plants was analyzed by transient expression in protoplasts from Nicotiana tabacum, Arabidopsis thaliana, Hordeum vulgare, and Zea mays. Expression of GFP was only observed with a mutated cDNA, from which a recently described cryptic splice site had been removed. However, detectable levels of green fluorescence were only emitted from a small number of protoplasts. Therefore, other mutations in the GFP cDNA leading to single-amino acid exchanges in the chromophore region, which had been previously studied in Escherichia coli, were tested in order to improve the sensitivity of this marker protein. Of the mutations tested so far, the exchange of GFP amino acid tyrosine 66 to histidine (Y66H) led to detection of blue fluorescence in plant protoplasts, while the exchange of amino acid serine 65 to cysteine (S65C) and threonine (S65T) increased the intensity of green fluorescence drastically, thereby significantly raising the detection level for GFP. For GFP S65C, the detectable number of green fluorescing tobacco (BY-2) protoplasts was raised up to 19-fold, while the fluorimetricly determined fluorescence was raised by at least 2 orders of magnitude.
通过在烟草、拟南芥、大麦和玉米的原生质体中瞬时表达,分析了水母绿色荧光蛋白(GFP)在植物中的表达情况。仅在一个去除了最近发现的隐蔽剪接位点的突变cDNA中观察到了GFP的表达。然而,只有少数原生质体发出了可检测水平的绿色荧光。因此,为了提高这种标记蛋白的灵敏度,对之前在大肠杆菌中研究过的导致发色团区域单氨基酸交换的GFP cDNA中的其他突变进行了测试。在目前测试的突变中,GFP氨基酸酪氨酸66突变为组氨酸(Y66H)导致在植物原生质体中检测到蓝色荧光,而氨基酸丝氨酸65突变为半胱氨酸(S65C)和苏氨酸(S65T)则显著提高了绿色荧光的强度,从而显著提高了GFP的检测水平。对于GFP S65C,可检测到的发出绿色荧光的烟草(BY-2)原生质体数量增加到了19倍,而荧光测定的荧光强度至少提高了2个数量级。