作 者：
王海英;叶星;白俊杰;夏仕玲;劳海华;简清;王琳
单 位：
上海水产大学生命科学技术学院;中国水产科学研究院珠江水产研究所
关键词：
唐鱼;β-肌动蛋白启动子;红色荧光蛋白;转基因;显微注射;表达
摘 要：
利用PCR技术克隆唐鱼(Tanichthy salbonubes)β-actin基因。所克隆的β-actin基因片段为1464bp,包含长为1374bp的启动调控区和90bp的部分开放阅读框。启动调控区包括105bp的β-actin基因上游调控序列、第一个外显子和第一个内含子。上游调控序列中含有对转录起重要作用的CAATBox、TATABox、CArGBox等元件。将唐鱼β-actin启动调控区克隆到红色荧光表达载体pDsRed2-1上,并显微注射到唐鱼受精卵中,荧光显微镜观察红色荧光蛋白(RFP)的表达。结果表明,RFP在转基因唐鱼中的表达阳性率较高,最高可达51.8%,且RFP的表达水平较高。PCR检测转基因唐鱼的部分器官组织,在被检组织器官中均能检测到外源RFP基因;而RT-PCR以及Southernblot验证显示RFPmRNA的表达有所不同;Southern blot检测肌肉组织基因组DNA,可见比阳性载体大的杂交条带。说明所检测的组织中已发生外源基因RFP的整合,但有些组织存在表达水平较低或者不表达的现象。本实验分离到的β-actin基因启动子序列具有有效的驱动功能,可启动外源基因在唐鱼体内的高效表达,从而为下一步进行功能基因的转化研究奠定基础。
译 名：
Isolation of white cloud mountain minnow β-actin promoter and assay of its transcription activity
作 者：
WANG Hai-ying 1,2,YE Xing1,BAI Jun-jie1,XIA Shi-ling1,LAO Hai-hua1,JIAN Qing1,WANG Lin1 (1. Pearl River Fisheries Institute,Chinese Academy of Fishery Sciences,Guangzhou 510380,China;2. Shanghai Fisheries University,Shanghai 200090,China)
关键词：
Tanichthys albonubes;β-actin gene promoter;red fluorescent protein (RFP);transgene;microinjected;expression detection
摘 要：
β-actin gene is a house-keeper gene and it’s promoter has been reported to be an efficient ubiquitous regulator. The β-actin 5′ flanking region and the initiation intron demonstrate a high level of constitutive promoter activity,and its activity is comparable to,or greater than,that of the SV40 early promoter. White cloud mountain minnow (Tanichthys albonubes) is a small cyprinid fish and also a popular ornamental fish which was originally found only in Guangzhou,China. Through PCR amplification,5′-flanking regions and partial open reading frames of the β-actin gene of white cloud mountain minnow were obtained. The 1 464 bp-long sequence includes 90 bp of partial ORF which encodes a 30 amino acids peptide,and 1 374 bp regulatory sequence which contains the 5′proximal promoter,the first untranslated exon and the initiation intron of β-actin gene. The partial ORF of the β-actin gene shares a high degree of sequence conservation. The similarity of the deduced amino acids was 100% between white cloud mountain minnow and zebrafish,and 96.7% between white cloud mountain minnow and nile tilapia. The proximal promoter region contains elements that were critical for transcription activity,including the CCAAT Box,TATA Box,CArG Box. The regulatory sequence was inserted into the promoterless pDsRed2-1 vector. The linearized recombinant plasmids (pTA-DsRed) were microinjected into the fertilized eggs of white cloud mountain minnow. RFP expression of the transgenic fish was observed by micro fluoroscope. High–frequency generation of transgenic fish which expresses RFP was obtained with the highest positive rate of 51.8%. RFP was expressed at high level in transgenic fish and red fluorescence could even be observed by externals in some fishes. RFP gene could be detected by amplification of the genomic DNA of different organs and tissues of the 4-month-old transgenic fish. The RFP gene transcripts were also analyzed by RT-PCR and Southern blot. Hybridized molecule larger than positive vector was displayed by Southern blot of the transgenic fish genomic DNA. The results showed that the β-actin gene promoter possess effective transcription activity and could promote high level expression of foreign gene. The present study lays foundation for further function gene transfer researches.
