Wang Mao-yuan ,Yang HongZhong Quan-fu,Lai Ming-yong,and Fan Hai-ping

1 Freshwater Fisheries Research Center,Chinese Academy of Fishery Sciences,Wuxi 214081,Jiangsu,China

2 Fujian Institute of Aquctic Product in Freshwater,Fuzhou 350002,China

Introduction

The isolation and identification of promoters is a significant link of constructing expression vector,and the quality of expression vector construction largely depends on the activity of promoters;therefore,it is crucial to look for potent promoters when constructs transgenic recombinant expression vector.In the early study of transgenic fish,the employment of promoters mostly stems from human or other mammals (Zhu et al.,1986;Iyengar et al.,1996).In 1992,after Zhu et al.proposed the concept "all fish genes",looking for effective expression promoters of fish itself had become one of the research hotspots of transgenic fish.At present,most promoters employed in fish genetic engineering mainly derive from promoters of Cyprinus carpio β-actin gene (Liu et al.,1990),Macrozoarces americanus antifreeze protein gene (Du et al.,1992),Danio rerio myosin heavy chain gene (Muller et al.,1997),Oncorhyncus mykiss metal sulfur protein A and B genes (Olsson et al.,1995).Since fish β-actin gene is a kind of housekeeping gene,its promoter possesses equal or even stronger transcriptional activity compared with early SV40 promoters (Robert et al.,1989),thus,it becomes one of the promoters most widely applied in the study of transgenic fish.Although β-actin gene promoters of C.carpio (Liu et al.,1990),Ctenopharyngodon idella (Liu et al.,1990),Mylopharyngodon piceus (Feng et al.,2006),Brachydanio rerio (Higashijima et al.,1997) and Oreochromis niloticus (Hwang et al.,2003) has been isolated and identified,studies of their activities mostly focus on prokaryotic cells rather than on eukaryotic cells.

Oreochromis niloticus belonging to Perciformes Cichlidae Tilapia and native to Lake Tanganyika in Jordan,Africa,has become one of the important economic freshwater aquaculture species,but also a main variety for earn foreign exchange through export of China's aquatic products.The experiment successfully cloned partial promoter sequences of Oreochromis niloticus β-actin gene and inserted the promoter sequences into the promoter deletion plasmid pN1-EGFP containing EGFP reporter gene,resulting in a successful con-struction of eukaryotic expression vector pEGFP-β-actin.Moreover,transferred it into human embryonic kidney epithelial cell HEK 293T by liposome transfection,and for the first time,the activity of Oreochromis niloticus β-actin gene promoters to drive gene expres-sion in eukaryotic cells was verified,which laid a foundation for study on the construction of "all fishes" transgenic Oreochromis niloticus.

Materials and Methods

Animal materials

Oreochromis niloticus was genetically bred by Freshwater Fish,Ministry of Agriculture and preserved by Laboratory of Aquaculture Biology;HEK 293T cell was gifted by Jiangsu Institute of Nuclear Medicine.

Strains and plasmids

Escherichia coli (E.coli) DH5α was preserved by this laboratory;pEGFP-N1 was gifted by Professor Li Wensheng of Zhongshan University;pMD19-T was purchased from TaKaRa Company.

Reagent

Taq DNA polymerase,dNTP,all kinds of DNA restriction endonuclease,T4DNA ligase,a small amount of DNA gel extraction kit and plasmid extraction kits were purchased from TaKaRa Company;Lipofectamine 2000 was purchased from Invitrogen Company.DMEM cell culture medium and fetal bovine serum were purchased from Gibco Company.

Extraction of genomic DNA

Randomly selected four 2-year-old Oreochromis niloticuses,put 30 mL blood in the centrifugal tube through fishtail venous blood,added ACD anticoagulants,extracted by isovolumetric phenol-chloroform referring to Geng et al.(2006) method,injected TE buffer into genomic DNA and kept them at the temperature of 4℃.

Sequence amplification of Oreochromis niloticus β-actin gene promoter

Designed one pair of primers (Pu and Pd) according to Oreochromis niloticus β-actin gene promoter sequences registered in GenBank (accession number: AY116536),meanwhile,the primers were separately introduced into VspⅠand EcoRⅠrestriction sites (underlined mark),synthesized by Shanghai Biocolor BioScience ＆Technology Company (BBST).The primer sequence was as the followings:

Pu: 5'-CCGATTAATGTGAGTGACGCCGGACC AATC-3'

Pd: 5'-GAATTCCCATGTCATCCCAGTTGGTCA CAAT-3'

Regarded the extracted Oreochromis niloticus genomic DNA as the template to amplify PCR.The amplification reaction system was 50 μL,containing 10× buffer 5 μL and dNTP 3 μL with the concentration of 10 μmol • L-1and LA-Taq polymerase 2 U and the template DNA 0.5 μg.Reaction conditions: 10 min of 94℃ initial denaturation;10 s of at 94℃ denaturation;10 min of 68℃ annealing;35 cycles followed by 10 min of 72℃ extension.In addition,the reactor was Eppendorf Mastercycler gradint PCR instrument.Used 1% agarose gel electrophoresis to test PCR products,cut objective belt and recycled plastic recycling reagent box.Recycled products and pMD19-T recombinant vector and transformed E.coli DH5α competent cells,identified and selected some positive clones named pMD19-β-actin by colony PCR,plasmid and enzyme digestion,sent them to Shanghai Biocolor BioScience ＆Technology Company for sequencing.Employed the software of Cluster X (Thompson et al.,1997) and MEGA 3.1 (Kumar et al.,2004) to make a comparison and analysis of Oreochromis niloticus β-actin gene promoter sequences and Oreochromis niloticus (EF026001),Oreochromis niloticus (AY116536),Medaka (S74868),Mylopharyngodon piceus (AY289135),Ctenopharyngodon idella (M25013),Mud carp (DQ241809),Brachydanio rerio (EF026002),C.carpio (M24113) and Human (E06566) β-actin gene promoter sequences.

PEGFP-β-actin eukaryotic cell expression plasmid construction

Mixed pMD19-β-actin and pEGFP-N1 with double digestion VspⅠand EcoRⅠ,respectively at the temperature of 37℃ overnight and connected them by T4ligase followed by plastic recycling fragments.Transformed the connecting products into E.coli DH5α competent cells,identified and selected some positive clones named pEGFP-β-actin through plasmid and enzyme digestion,and sent them to Shanghai Biocolor BioScience ＆Technology Company for sequencing.At the same time,CMV promoter deletion pEGFP-N1 plasmid was used as a negative control;while pEGFP-N1 containing CMV promoter as a positive control.

Cell transfection

Vaccinated HEK 293T cells in a 6-hole plate with 0.5×105per hole before transfection and cultivated it in a culture box under the condition of 37℃and 5% CO2.When the cells were cultivated to about 90%-95%,adopted liposome Lipofectamine 2000 (referring to the manual) to transfect plasmid pEGFP-β-actin,pEGFP-N1 (positive control) and CMV promoter deletion P N1-EGFP plasmid (negative control),respectively.4 h later,replaced the complete medium and continued to develop EGFP;48 h later,observed EGFP expression under the inverted fluorescence microscope.

Results

Analysis of Oreochromis niloticus β-actin promoter sequence

Took the genomic DNA (Fig.1) as the template to obtain a specific fragment of 1 600 bp through PCR amplification (Fig.2).Then,cloned the fragment into pMD19-T vector as well as identified and selected positive recombinants with enzyme digestion for sequencing.Via comparing the sequencing results with registered Oreochromis niloticus β-actin gene sequence,we found that the homology was 99.1% and made sure that the segments obtained were Oreochromis niloticus β-actin gene promoters.

Fig.1 Genomic DNA of Nile tilapia

Fig.2 Amplification of β-actin of Nile tilapia

Oreochromis niloticus β-actin gene promoter region featured the typical vertebrate promoter structure,so –25-–28 was TATA Box;–87-–91 was CAAT Box;while CArG homeopathic action element was –51-–61 between TATA Box and CAAT Box.By comparing of sequence of Oreochromis niloticus and other fishes as well as human β-actin gene promoter region (Fig.3),we could see the homologies were 99.1%-72.5% and 63.9%,respectively.The result showed that β-actin gene promoter regulatory elements of known fishes and higher vertebrates were similar,and the closer the genetic relationship was,the higher the homology was;in addition,they were highly conserved with CCAAT Box,CArG motif and TATA Box homeopathic regulatory elements,and had the similar locations.

Fig.3 Sequence alignment analysis of β-actin promoter

Construction of pEGFP-β-actin eukaryotic cell expression vector

Made use of VspⅠand EcoRⅠdouble digestion pMD19-β-actin recombinant plasmid to get 1 675 bp fragments and connected the fragments with the same restriction enzyme pEGFP-N1 after recycled and purified by T4ligase (Fig.4).Transformed connecting products into E.coli DH5α competent cells,identified and selected some positive clones named pEGFP-β-actin,through plasmid and enzyme digestion (Fig.5).The sequencing result showed that Oreochromis niloticus β-actin promoter was inserted correctly.

Fig.4 pMD19-β-actin of Vsp I and EcoR I enzymes

Fig.5 Results of Vsp I and EcoR I enzymes

pEGFP-β-actin expression in HEK 293T cells

Transferred plasmids pEGFP-β-actin and pEGFP-N1 (positive control) as well as deletion CMV promoter P N1-EGFP (negative control) into HEK 293T cells through liposomal Lipofectamine 2000.24 h later after transfection,partial cells were observed by microscopic examination to emit green fluorescence,but at this moment,the fluorescence was weak.While 48 h after transfection,cellular expression of green fluorescence increased significantly and the fluorescence intensity became strong.

However,72 h later,the fluorescence began to decline.Microscopic examination showed that pEGFPB-actin after transfection had basically no effects on cell growth and cell morphology,instead,EGFP uniformly distributed throughout the cell.Made a contrast of pEGFP-β-actin and pEGFPN1 group cells,we found that in pEGFP-N1 group,the expression green fluorescent cell was about 90%,the fluorescence intensity of the strongest;group pEGFP-β-actin,the cellular expression of green fluorescence was about 50%-60%,but the fluorescence intensity was slightly lower than that of the control group;while pEGFPN1 (negative control) showed no green fluorescence (Fig.6).

Fig.6 Results of EGFP protein transient expression in 293T cells

Discussion

According to the report,the primary structure of fish β-actin gene promoter was similar to that of other vertebrates,featuring of a wide range of promoter expression,including at least three transcriptional regulatory regions: TATA Box,CArG and CCAAT Box (Hwang et al.,2003;Wang et al.,2008).CArG,located between TATA box and CCAAT box,was the binding site of CArG transcription factor,and was also a must for β-actin gene transcription (Wang et al.,2008);in addition,CCAAT Box played a key role in activating exogenous gene expression for β-actin gene promoter (Liliana et al.,1998).At present,many research results showed that these elements were closely related to promoter activity and high-level transcriptional activity,and the absence of them would lead to transcriptional activity significantly reducing,even make β-actin gene promoter activity completely lost (Ponte et al.,1984;Noh et al.,2003;Feng et al.,2006).In this experiment,we had successfully cloned partial promoter sequences of 1 675 bp Oreochromis niloticus β-actin gene,and the sequence analyses showed that its upstream regulatory region also contained these three important homeopathic effect elements as well as similar location to that of other fishes and higher vertebrates.At the same time,compared with the registered Oreochromis niloticus β-actin gene sequence (AY116536;EF026001),we observed that they both had the homology of 99.1% and these differences were present outside TATA Box,CArG,CCAAT Box and conserved sequence,so we presumed that it did not affect the transcriptional activity of promoters.

Domestic and international scholars mostly studied fish promoter activity by directly microinjecting embryo (Hansen et al.,1991;Muller et al.,1996;Muller et al.,1997;Maclean et al.,2002;Rahman et al.,1992).For example,the linearized Danio rerio Mylz2 promoter could obtain a Danio rerio with enhanced green fluorescent protein gene by microinjecting Danio rerio zygote (Jian et al.,2004);microinjecting rare Minnow embryo for Oncorhyncus mykiss histone H3promoter could effectively drive the expression of the reporter gene (Mao et al.,2004);microinjecting Tanichthys albonubes β-actin gene promoter into Tanichthys albonubes zygote could prove the effective driving function (Wang et al.,2008).The advantage of microinjection was that the integration rate of imported exogenous gene was high.However,everything has two sides,and it also has some disadvantages,including precious and expensive equipments,operation techniques requiring long time practice and only limited cells injected.Meanwhile,this method directly took the exogenous substances into the embryo or living tissue to validate its activity from the superficial,but did not rule out the possibility of animal tissue itself initiating the expression.This experiment took advantages of combining liposomes transiently transfecting eukaryotic cells with green fluorescent reporter gene to validate the activity of Oreochromis niloticus β-actin gene promoters.However,transient transfection could not integrate the transfected nucleic acid onto the chromosome,resulting in temporary high-level expression,so we could detect the effect of expression within 24-96 h;since expression and location were independent,it would not be affected by the surrounding chromosome elements.Manpower and time required for transient expression analysis were obviously less than microinjection.Therefore,in the study of human and other vertebrates,combined transient transfection with reporter genes to validate whether promoter activity exists or not as well as its intensity.

There were relatively less reports of fish gene expression in eukaryotic cells,but more reports of Mylopharyngodon piceus β-actin gene promoter microinjected into the zygotes of loach and transfected into Hela cells,both have a stronger driving activity (Feng et al.,2006;Noh et al.,2003).At present,Oreochromis niloticus β-actin gene promoter activity expression was almost researched in embryonic tissues,so research of expression in eukaryotic cells was still a blank.For instance,in 2003,Hwang et al.(2003) employed microinjection to compare the transcription activity of 1.6 kb Oreochromis niloticus β-actin promoter and 1.5 kb C.carpio β-actin promoter,and the result showed that the former transcription activity was stronger than that of the latter in the transgenic Oreochromis niloticus's embryos,while in Brachydanio rerio's,the result was reversed;Yu et al.(2008) microinjected Oreochromis niloticus's 1.6 kb β-actin promoter into Tanichthys albonubes zygote,having an effective driving function.The experiment of cell transfection showed that if negative control had no fluorescence emitted,namely,the possibilities of EGFP itself expression would be excluded;if fluorescence intensity of positive control was maximum,i.e.the driving activity of CMV promoter was stronger than β-actin,which was in line with the research result of Mao et al.(2004),and further validating the promoters cloned by us can effectively drive EGFP exogenous gene expression in HEK 293T cells in eukaryotic cell level;at the same time,the experiment would also attempt to transfect the constructed vector into different cell lines,such as Africa green monkey kidney Vero cells,and we could still see fluorescence,but the intensity was weaker than that of HEK 293T cells.The reason was likely to be that liposomes had different transfection efficiencies in different cells.In addition,the fragment adopted in this experiment was similar to that used in Oreochromis niloticus β-actin gene promoter activity research made by Hwang et al.(2003) and Yu et al.(2008),both controlled in 1 600 bp.However,whether there are more important initiated regulatory elements or enhancers in the upstream of Oreochromis niloticus β-actin gene promoter 5'side wing area still needs further research.

Conclusions

In this experiment,we had successfully cloned partial promoter sequences of 1 675 bp Oreochromis niloticus β-actin gene,and the sequence analysis showed that its upstream regulatory region also contained these three important homeopathic effect elements (TATA Box,CArG and CCAAT Box) as well as similar location to that of other fishes and higher vertebrates.The experiment of cell transfection showed that if negative control had no fluorescence emitted,namely,the possibilities of EGFP itself expression would be excluded;if the fluorescence intensity of positive control was maximum,i.e.the driving activity of CMV promoter was stronger than that of β-actin,and further validateing the promoters cloned by us could effectively drive EGFP exogenous gene expression in HEK 293T cells in eukaryotic cell level.In short,this experiment had successfully constructed β-actin gene promoter-driven recombinant expression vector,and further verified its driving objective gene expression activity in eukaryotic cells,which laid a significant theoretical foundation for fish gene engineering researches.

Du S J,Gong Z,Hew C L,et al.1992.Development of an all-fish gene cassette for gene transfer in aquacultur.Mol Mar BiolBiotechnol,1: 290-300.

Feng H,Ceng J,Luo J,et al.2006.Cloning of black carp β-actin gene and primarily detecting the function of its promoter region.Acta Genetica Sinica,33(2): 133-140.

Geng B,Sun X W,Liang L Q,et al.2006.Analysis the genetic diversity of Aristichthys nobilis in China with 17 microsatellite markers.Hereditas (Beijing),28: 683-688.

Hansen E,Fernandes K,Goldspink G,et al.1991.Strong expression of foreign genes following direct injection into fish muscle.FEBS Letters,290: 73-76.

Higashijima S,Okamoto H,Ueno N,et al.1997.High frequency generation of transgenic zebrafish which reliably express GFP in whole muscles or the whole body by using promoters of zebra fish origin.Developmental Biology,192: 289-299.

Hwang G L,Rahman M A,Razak S A,et al.2003.Isolation and characterization of β-actin tilapia promoter and comparison of its activity with carp β-actin promoter.Biochimica et Biophysica Acta,1625: 11-18.

Iyengar A,Muller F,Maclean N,et al.1996.Regulation and expression of transgenes in fish.Transgen Res,5: 147.

Jian Q,Bai J J,Ye X,et al.2004.Cloning of Mylz2 promoter and generation of green fluorescence transgenic zebrafish.Journal of Fishery Sciences of China,11(5): 391-395.

Kumar S,Tamura K,Nei M.2004.MEGA 3.1: Integrated software for molecular evolutionary genetics analysis andsequence alignment.Briefings in Bioinformatics,5: 150-163.

Liliana D Z,Anne S T,Bruce M P.l998.A 40-base-pairsequence in the 3'end of the β-actin gene regulates β-actin mRNA transcription during myogenesis.Proc Natl Acad Sci USA,85: l389-l393.

Liu Z J,Moav B,Faras A J,et al.1990.Functional analysisof elements affecting expression of the β-actin gene of carp.Mol Cell Biol,10: 3432-3440.

Lju Z J,Zhu Z Y,Roberg K,et al.1990.Isolation and characterization of beta-actin gene of carp (Cyprinus carpio).DNA Seq,1: 125-136.

Maclean N,Hwang G L,Farahmand T.2002.Exploiting transgenic tilapia and the tilapia genome.In: Shimizu N,Aoki T,Hirono I.Aquatic Genomics.Springer-Verlag,Tokyo.

Mao W F,Wang Y P,Sun Y H,et al.2004.Molecular cloning of rainbow trout (Oncorhynchus mykiss) histon H3 promter and activity analysis in rare gudgeon (Gobiocypris rarus).Progress in Natural Science,14(1): 46-50.

Muller F,Gauvry L,Williams D W,et al.1996.The use of transient lacZ expression in fish embryos for comparative analysis of cloned regulatory elements.In: Ennion S J,Goldspink G.Gene expression and manipulation in aquatic organisms.Society for Experimental Biology Seminar Series.Cambridge Univ Press,Cambridge.pp.175-179.

Muller F,Williams D W,Kobolak J,et al.1997.Activator effect of coinjected enhancers on the muscle-specific expression of promoters in zebrafish embryos.Mol Reprod Dev,47: 404-412.

Noh J K,Cho K N,Han E H,et al.2003.Genomic cloning of mud loach Misgurnus mizolepis (Cypriniformes,Cobitidae) β-actin gene and usefulness of its promoter region for fish transgenesis.Marine Biotechnology,5: 244-252.

Olsson P E,Kling P,Erkell L J,et al.1995.Structrual and functional analysis of the rainbow trout (Oncorhyncus mykiss) metallothionein-A gene.Eur J Biochem,230: 344-349.

Ponte P,Ng S,Engel J,et al.l984.Evolutionary conservation in the untranslated regions of actin mRNAs: DNA sequence of a human beta-actinc DNA.Nucl Acids Res,l2: l687-l696.

Rahman A,Maclean N.1992.Fish transgene expression by direct injection into fish muscle.Mol Mar Biol Biotechnol,1: 286-289.

Robert M,Frederickson,Monette R,et al.1989.5'flanking and first intron sequences of the human β-actin gene required for efficient promoter activity.Nucl Acids Res,17(1): 254-270.

Thompson J D,Gibson T J,Plewniak F,et al.1997.The CLUSTAL_X window interface: flexible strategies for multiple sequence alignment aided by quality analysis tools.Nucl Acids Res,25: 4876-4882.

Wang H Y,Ye X,Lao H H,et al.2008.Isolation of white cloud mountain minnow β-actin promoter and assay of its transcription activity.Chinese Journal of Biotechnology,25;24(10): 1768-1775.

Yu E M,Ye X,Wang H Y,et al.2008.Isolation of Nile tilapia (Oreochromis niloticus) β-actin promoter and assay of its transcription activity.Journal of Agricultural Biotechnology,16(2): 242-247.

Zhu Z,Xu K,Li G,et al.1986.Biological effects of human growth hormone gene microinjected into the fertilized gene of loach Misgurnus anguillicaudatus (Cantor).Chinese Science Bulletin,31(14): 988.

Zhu Z,Chen S P,Hu W,et al.1992.Growth hormone gene and the transgenic fish.In: You C B,Chen Z L.Agricultural biotechnology.China Science and Technology Press,Beijing.pp,106-116.

Fig.3 Morphology of Sphaerotheca fuliginea observed after staining with Coomassie brilliant blue

Wang Xue-zheng et al.Identification of Powdery Mildew Pathogen and Ribosomal DNA-ITS Sequence Analysis on Melon.2013,20(4): 10-18.

Fig.2 Histopathology of a male rats with decursin at a dose of 250 mg • kg-1 for 4 weeks

Jiang Cheng-zhe et al.Decursin Safety from Root of Angelica gigas in Rats.2013,20(4): 46-51.

Fig.6 Results of EGFP protein transient expression in 293T cells

Wang Mao-yuan et al.Isolation of Nile Tilapia (Oreochromis niloticus) β-actin Promoter and Assay of Its Transcription Activity.2013,20(4): 64-71.