Carter Robert W, Schmale Michael C, Gibbs Patrick D L
Marine Biology and Fisheries Division, Rosenstiel School for Marine and Atmospheric Science, University of Miami, 4600 Rickenbacker Cswy, Miami, FL 33149, USA.
Comp Biochem Physiol C Toxicol Pharmacol. 2004 Jul;138(3):259-70. doi: 10.1016/j.cca.2004.07.002.
Many cnidarians display vivid fluorescence under proper lighting conditions. In general, these colors are due to the presence of fluorescent proteins similar to the green fluorescent protein (GFP) originally isolated from the hydrozoan medusa Aequorea victoria (Cnidaria: Hydrozoa). To optimize the search for new fluorescent proteins (FPs), a technique was developed that allows for the rapid cloning and screening of FP genes without the need for a prior knowledge of gene sequence. Using this method, four new FP genes were cloned, a green from Montastraea cavernosa (Anthozoa: Scleractinia: Faviidae), a cyan from Pocillopora damicornis (Anthozoa: Scleractinia: Pocilloporidae), a cyan from Discosoma striata (Anthozoa: Corallimorpharia), and a red from a second Discosoma species. Two additional green FPs were cloned, one from M. cavernosa and one from its congener Montastraea faveolata, from purified cDNA using PCR primers designed for the first M. cavernosa green FP. Each FP has recognizable amino acid sequence motifs that place them conclusively in the GFP protein family. Mutation of these products using a low-stringency PCR protocol followed by screening of large numbers of bacterial colonies allowed rapid creation of mutants with a variety of characteristics, including changes in color, maturation time, and brightness. An enhanced version of the new red FP, DspR1+, matures faster at 30 degrees C than the commercially available DsRed but matures slower than DsRed at 37 degrees C. One of the M. cavernosa green FPs, McaG2, is highly resistant to photobleaching and has a fluorescence quantum yield approximately twice that of EGFP-1.
许多刺胞动物在适当的光照条件下会呈现出鲜艳的荧光。一般来说，这些颜色是由于存在与最初从水螅水母维多利亚多管水母（刺胞动物门：水螅纲）中分离出的绿色荧光蛋白（GFP）相似的荧光蛋白。为了优化对新荧光蛋白（FPs）的搜索，开发了一种技术，该技术无需事先了解基因序列就能快速克隆和筛选FP基因。使用这种方法，克隆了四个新的FP基因，一个来自多孔鹿角珊瑚（珊瑚虫纲：石珊瑚目：鹿角珊瑚科）的绿色荧光蛋白，一个来自鹿角杯形珊瑚（珊瑚虫纲：石珊瑚目：杯形珊瑚科）的青色荧光蛋白，一个来自条纹碟珊瑚（珊瑚虫纲：珊瑚藻目）的青色荧光蛋白，以及来自另一种碟珊瑚的红色荧光蛋白。另外两个绿色荧光蛋白也被克隆出来，一个来自多孔鹿角珊瑚，另一个来自其同属的蜂巢鹿角珊瑚，是使用针对第一个多孔鹿角珊瑚绿色荧光蛋白设计的PCR引物从纯化的cDNA中克隆得到的。每个荧光蛋白都有可识别的氨基酸序列基序，这使它们明确地归属于GFP蛋白家族。使用低严谨度PCR方案对这些产物进行突变，然后筛选大量细菌菌落，能够快速创建具有各种特性的突变体，包括颜色、成熟时间和亮度的变化。新红色荧光蛋白DspR1 +的增强版本在30℃下比市售的DsRed成熟得更快，但在37℃下比DsRed成熟得慢。多孔鹿角珊瑚的一种绿色荧光蛋白McaG2对光漂白具有高度抗性，其荧光量子产率约为EGFP - 1的两倍。
