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UDC 577.21
cDNA, GENOMIC SEQUENCE CLONING AND OVEREXPRESSION OF GLYCERALDEHYDE-3-PHOSPHATE DEHYDROGENASE GENE (GAPDH)
FROM THE GIANT PANDA
© 2010 wan-ru Hou1*, Yi-ling Hou1, Yu-jie Du2, Tian Zhang1, Yan-zhe Hao1
1College of Life Science, China West Normal University, 637002, Nanchong, China 2Biochemical Department, Zhanjiang Education College, 524048, Zhanjiang, China
Received 20.04.2009 Accepted for publication 13.08.2009
GAPDH (glyceraldehyde-3-phosphate dehydrogenase) is a key enzyme of the glycolytic pathway and it is related to the occurrence of some diseases. The cDNA and the genomic sequence of GAPDHwere cloned successfully from the Giant Panda (Ailuropoda melanoleuca) using the RT-PCR technology and Touchdown-PCR, respectively. Both sequences were analyzed preliminarily. The cDNA of GAPDH cloned from the Giant Panda is 1191 bp in size, contains an open reading frame of 1002 bp encoding 333 amino acids. The genomic sequence is 3941 bp in length and was found to possess 10 exons and 9 introns. Alignment analysis indicates that the nucleotide sequence and the deduced amino acid sequence are highly conserved in some mammalian species, including Homo sapiens, Mus musculus, Rat-tus norvegicus, Canis lupus familiaris and Bos taurus. The homologies for the nucleotide sequences of the Giant Panda GAPDH to that of these species are 90.67, 90.92, 90.62, 95.01 and 92.32% respectively, while the homologies for the amino acid sequences are 94.93, 95.5, 95.8, 98.8 and 97.0%. Primary structure analysis revealed that the molecular weight of the putative GAPDH protein is 35.7899 kDa with a theoretical pI of 8.21. Topology prediction showed that there is one Glyceraldehyde 3-phosphate dehydrogenase active site, two N-glycosylation sites, four Casein kinase II phosphorylation sites, seven Protein kinase C phosphorylation sites and eight N-myristoylation sites in the GAPDH protein of the Giant Panda. The GAPDH gene was overexpressed in E. coli BL21. The results indicated that the fusion of GAPDH with the N-terminally His-tagged form gave rise to the accumulation of an expected 43 kDa polypeptide. The SDS-PAGE analysis also showed that the recombinant GAPDH was soluble and thus could be used for further functional studies.
Key words: Cloning, sequence analysis, GAPDH, Giant Panda.
INTRODUCTION
The Giant Panda (Ailuropoda melanoleuca) is a declining species currently found only in China. Studies of the rare wild animal are ofincreasing concern to the world community. In the past, studies of the Giant Panda have been mainly concentrated on fields such as breeding and propagation, ecology, morphology, toxicology, physiology and pathological biochemistry. At present, research on genetic diversity, parentage, phylogenesis et al. has been developed, while reports on functional genetics are scarce
[1-9].
Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is a key enzyme of the glycolytic pathway. Besides its metabolic function as a glycolytic enzyme within the cytoplasm, recent evidence indicates at new roles for the glyceraldehyde-3-phosphate dehydrogenase in essential mammalian cell processes such as apoptosis and proliferation [10-17]. The present experiment also suggests that the protein is involved in the Alzheimer's disease [18, 19], Huntington's disease [20], prostate cancer [21] and Ma-
* E-mail: hwr168@yahoo.com.cn
ternal diabetes [22]. Therefore, it is significant to clone and analyze the GAPDH gene of the Giant Panda and its significance lies not only in the protection of the Giant Panda, but also in the therapy for several kinds of human hereditary diseases.
This study was conducted using the RT-PCR technique to amplify the cDNA sequence of the GAPDHgene from the total RNA extracted from the skeletal muscle of the Giant Panda. Then the sequence characteristics ofthe protein encoded by the cDNA were analyzed and compared with those of humans and of other reported mammalian species. Thus the study will provide scientific data for inquiring into the hereditary traits of the gene from the Giant Panda.
EXPERIMENTAL
Materials and RNA isolation. The skeletal muscle was collected from a dead giant panda at the Wolong Conservation Center of the Giant Panda, Sichuan, China. The collected skeletal muscle was frozen in liquid nitrogen and then used for RNA isolation.
The total RNA was isolated from about 400 mg of muscle tissue using the Total Tissue/Cell RNA Extraction Kits (Waton Inc., Shanghai, China) according to the manufacturer's instructions. All ofthe extracted RNA was dissolved in the DEPC (diethylpyrocarbonate) water and kept at —70°C.
Primers design, RT-PCR, cloning of RT-PCR products and sequencing. The PCR primers were designed by Primer Premier 5.0 based on the mRNA sequence of the GAPDH gene from Homo sapiens (AB062273), Mus mus-culus (AK081405), Rattus norvegicus (BC059110), Canis lupus familiaris (NM_001003142) and Bos taurus (NM_001034034). The specific primers for the GAPDH were as follows:
GAPDH-1F: 5'-GTGCAGTGCCAGCC[T/G]CGTCC-3';
GAPDH-1R:
5'-AGGGCTCCC[T/C]A[A/G]GCCCCTCC -3'.
The first-strand cDNA was synthesized from the total RNA extract using a reverse transcription kit with Oligo dTs as primers according to the manufacturer's instructions (Promega, Shanghai China). 20 |L of the firststrand cDNA synthesis reaction system contained 1 |g of the total RNA extract, 5 mM ofMgCl2, 1mM of dNTPs, 0.5 |g of Oligo dT15, 10 U/|L of an RNase inhibitor, and 15 U of the AMV reverse transcriptase. The mixture was incubated at 42°C for 60 minutes.
The synthesized single-strand cDNA was used as a template. The total reaction volume for DNA amplification was 25 |L. The reaction mixture contained 1.5 mM of MgCl2, 200 |M of each of dATP, dGTP, dCTP and dTTP (Omega, China), 0.3 |M of each primer, 5.0 units ofthe Taq plus DNA polymerase (Sangon Co., Shanghai, China). DNA amplification was performed using a MJ Research thermocycler, Model PTC-200 (Watertown, MA) with a program of 4 minutes at 94.0°C, followed by 30 cycles of 1 minute at 94.0°C, 0.5 minute at 45°C and 1.5 minutes at 72.0°C, and then ended with the final extension for 10 minutes at 72.0°C. After the amplification, PCR products were separated by electrophoresis in a 1.5% agarose gel with the 1x TAE (Tris-acetate-EDTA) buffer, stained with ethidium bromide and visualized under UV light. Expected fragments of PCR products were harvested and purified from the gel using a DNA harvesting kit (Omega, China), and then ligated into the pMD18T vector (Takara, Japan) at 22°C for 12 hours. The recombinant molecules were transformed into E. coli competent cells (JM109), which were then spread on a LB-plate containing 50 |g/mL ampicillin, 200 mg/mL IPTG (iso-propyl-p-D-thiogalactopyranoside) and 20 mg/mL X-gal. 5 colonies were chosen with a vaccination needle for plasmid extraction by the alkali decomposition method. Plasmid DNA was isolated and digested by PstI and ScaII to verify the insert size. The plasmid DNA was se-
quenced by Huada Zhongsheng Scientific Corporation (Beijing, China).
Cloning the genomic sequence of GAPDH. The PCR
primers were designed based on the cDNA sequence of the GAPDH from the Giant Panda obtained above. The specific primers for the genomic sequence were as follows:
GAPDH-2F:
5-GTGCAGTGCCAGCCGCGTCCCCGAG-3';
GAPDH-2R:
5'-AGGGCTCCCCAAGCCCCTCCCCTTC-3'.
The genomic sequence ofthe GAPDHgene was amplified using Touchdown-PCR with the following conditions 94°C for 30 s, 62°C for 45 s, 72°C for 4 minutes in the first cycle and then the annealing temperature was decreased by 0.5°C per cycle; after 20 cycles the conditions were changed to 94°C for 30 s, 52°C for 45 s, 72°C for 5 minutes for another 20 cycles. The amplified fragment was then also purified, ligated into the clone vector and transformed into competent cells of E. coli. Finally, the recombinant fragment was sequenced by Sangon (Shanghai, China).
Data analysis. The sequences were analyzed using the GenScan software (http://genes.mit.edu/GENSCAN.ht ml). Homology research on the GAPDHgene sequence of the Giant Panda in comparison to those of other species was performed using Blast 2.1 (http://www.nc-bi.nlm.nih.gov/blast/). ORF of the DNA sequence was determined using the ORF finder software (http: //www.ncbi.nlm.nih.gov/gorf/gorf.html). Preference of certain nucleotide bases was analyzed by MEGA3.1. The protein structure of the cloned GAPDH sequence and the functional sites of the corresponding GAPDH protein were determined using the PredictProtein software (http://cubic.bioc.columbia.edu/predictprotein/).
Gene expression in E. coli. Based on the known GAP-DH coding sequences, the primers CCCGAGAAT-TCATGGTGAAGG (EcoRI) and TTACTCCTTG-GATCCCATGTGG (BamHI) were designed to amplify the cDNA. The PCR was performed at 94°C for 3 minutes; 35 cycles of 30 s at 94°C, 45 s at 53°C and 1 minute at 72°C; and finally 10 minutes at 72°C. The amplified PCR product was cut and then ligated into the corresponding site of the pET28a vector (Stratagene, USA). The resulting construct was transformed into the E. coli BL21(DE3) strain (Novagen, Darmstadt, Germany) and used for induction by adding 0.2 mmol/L of IPTG (iso-propyl-p-D-thiogalactopyranoside) at an OD600 of 0.6 and then cultured for 4 hours at 37°C, with the BL21(DE3) cells transformed with only the empty vector being the control. The recombinant protein samples were induced for 0.5, 1, 2, 3 and 4 hours, separated by SDS-PAGE and stained with Commassie blue R 250.
The cell suspension after 4 hours of the IPTG induction was sonicated in ice-water for a total of15 min with a
M 12
2000
1000 750
500 250 100
Fig. 1. Reverse transcription polymerase Chain reaction products of GAPDH from the Giant Panda. M — Molecular maker; 1, 2 — the amplified GAPDH; 3 — checked.
microsonicator (Sonics Inc., CA), then the cells were centrifuged at 10000 g for 30
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