• BMB Reports
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• 제40권2호
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• pp.270-276
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• 2007

We have cloned a novel mouse zinc finger protein gene Znf313 by rapid amplification of cDNA ends (RACE) according to the homologue of human ZNF313 gene. The cDNA is 2,163 base pairs (bp) in length and encodes a 229 amino acids (aa) protein with a $C_3HC_4$ ring finger domain and three $C_2H_2$ domains. 89% and 93% nucleotide (nt) and aa sequence identity is observed with its human homologue. Revealed by Northern blot and RT-PCR, full mRNA consists of 2.16 kb and widely expresses in tissues as a single transcript, most abundantly in heart, liver, kidney and testis. The expression of Znf313 in testis is detected in all development stages. Western blot analysis also reveals that Znf313 is expressed in the tissues. Immunohistochemical staining and subcellular localization demonstrate that Znf313 is expressed both in the cytoplasm and nucleus whereas predominantly localized in the nucleus. Present data suggests that Znf313 gene might play a fundamental role in gene transcription and regulation in organism and relates to spermatogenesis.

• Asian Pacific Journal of Cancer Prevention
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• 제15권12호
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• pp.5055-5061
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• 2014

In this study, an anti-oxidant and anti-tumor protein Latcripin-3 of Lentinula edodes C91-3 was expressed in Escherichia coli. for the first time. According to the cDNA library, the full-length gene of Latcripin-3 was cloned by the methods of 3'-full rapid amplification of cDNA Ends (RACE) and 5'-full RACE. The structural domain gene of Latcripin-3 was inserted into the pET32 a(+). The functional protein of Latcripin-3 was expressed in Rosetta-gami (DE3) E. coli, evaluated by Western blotting and mass spectrometry. DPPH testing showed that the protein Latcripin-3 can scavenge free radicals remarkably well. The activity of functional protein Latcripin-3 on A549 cells was studied with flow cytometry and the MTT method. The MTT assay results showed that there was a decreases in cell viability in a dose-dependent and time-dependent manner in protein Latcripin-3 treated groups. Flow cytometry demonstrated that Latcripin-3 can induce apoptosis and block S phase dramatically in human A549 lung cancer cells as compared to the control group. At the same time, the cell ultrastructure observed by transmission electron microscopy supported the results of flow cytometry. This research offers new insights and advantages for identifying anti-oxidant and anti-tumor proteins.

• 원예과학기술지
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• 제29권2호
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• pp.135-143
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• 2011

In the Doritaenopsis hybrid, like most of the orchid species and hybrids, temperature is crucial for the vegetative-to-reproductive transition, and low temperature is required for bud differentiation. To understand the molecular mechanism of this process, an orchid GIGANTEA (GI) gene, DhGI1, was isolated and characterized by using the rapid amplification of cDNA ends (RACE) PCR technique. Sequence analysis showed that the full-length cDNA is 4,022 bp with a major open reading frame of 3,483 bp, and the amino acid sequence showed high similarity to GI proteins in Zea mays, Oryza sativa, Arabidopsis thaliana and other plants. Semi-quantitative RT-PCR revealed that DhGI1 was expressed throughout development and could be detected in roots, stems, leaves, peduncles and flower buds. The expression level of DhGI1 was higher when the plants were flowering at low temperature (22/$18^{\circ}C$ day/night) than the other growth stages. Further analysis indicated that the accumulation of DhGI1 transcripts was significantly increased at low temperature, and concomitantly, initiation of the peduncle was observed. However, DhGI1 levels were low under high temperature (30/$25^{\circ}C$) conditions, and flower initiation was inhibited. These results indicate that the expression of DhGI1 is regulated by low temperature and that DhGI1 may play an important role in inflorescence initiation in this Doritaenopsis hybrid at low temperatures.

• Asian-Australasian Journal of Animal Sciences
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• 제30권5호
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• pp.736-742
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• 2017

Objective: Experiments were conducted to clone the sequence of Wild Argali short palate, lung and nasal epithelium clone 1 (SPLUNC1) cDNA, and to lay the foundation for further study the biological function of Wild Argali SPLUNC1. Methods: The complete sequence of Wild Argali SPLUNC1 cDNA was generated by rapid amplification of cDNA ends. The entire coding sequence was inserted into the pPIC9K vector and expressed in Pichia pastoris (P. pastoris) GS115. The recombinant SPLUNC1 protein was detected by Western blot and purified by $Ni^{2+}$ chelate affinity chromatography. The test of effect of the protein on Mycoplasma ovipneumoniae (MO) was performed with real-time polymerase chain reaction. Results: The Wild Argali SPLUNC1 cDNA was 1,076 bp with an open reading frame of 768 bp, which encoded a 26.49 kDa protein composed of 255 amino acids. Its amino acid sequence shared 98.4%, 96.9%, 94.5%, 90.2%, 80.8%, 78.4%, 78.3%, 72.5%, 72.3%, 68.8% identity with those of SPLUNC1 cDNA from Ovis aries (accession no. NP_001288334.1), Capra hircus (accession no. XP_005688516.1), Pantholops hodgsonii (accession no. XP_005979709.1), Bos taurus (accession no. NP_776851.1), Felis catus (accession no. XP_006929910.1), Homo sapiens (accession no. NP_001230122.1), Sus scrofa (accession no. NP_001005727.1), Chinchilla lanigera (accession no. NP_001269294.1), Mus musculus (accession no. NP_035256.2), and Rattus norvegicus (accession no. NP_742028.1), respectively. The recombinant protein corresponded to the expected molecular mass of 25.47 kDa as judged by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and it was detected in the supernatant of P. pastoris, and it could be purified. The results from the test of inhibition effect of argali recombinant SPLUNC1 protein on MO showed that the product could inhibit MO very well (p<0.01). Conclusion: The amino acid sequence of Wild Argali SPLUNC1 was different from other organisms. The recombinant SPLUNC1 protein has good biological activity.

• 농업생명과학연구
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• 제46권4호
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• pp.9-20
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• 2012

Eugenol은 많은 식물에서 eugenol synthase에 의해 생합성되는 phenylpropene 계통의 휘발성 화합물이다. 그러나, 토마토 과실에서의 특징은 밝혀져 있지 않다. 이에 따라 토마토 'Micro-Tom'으로부터 RACE 기법을 이용하여 완전장 cDNA를 클로닝 하여, SlEGS라 명명하였다. SlEGS의 open reading frame은 921bp로, 307개의 아미노산 서열을 갖는 단백질로 번역되었다. BLAST 결과에 따라 SlEGS는 PhEGS1 및 CbEGS2와 각 67.1, 69.4%의 높은 상동성을 갖는 것으로 나타났다. CLC genomics workbench 프로그램을 이용하여 SlEGS의 아미노산 구성을 분석하였고, Swiss-PDB viewer 프로그램에서 homology modeling 기법으로 SlEGS의 3차원 단백질 구조를 구축한 후 ProSA-web 툴로 3차원 구조의 안정성을 확인 하였다. 또한 ExPASy의 ProtParam 툴을 이용하여 SlEGS의 생리화학적 특성을 분석 하였다. SlEGS의 추정 분자량은 33.93kDA이고 등전점(pI)은 5.85로 산성인 것으로 나타났다. 이와 더불어 SlEGS의 흡광 계수(EC), 불안정성 지수(II), alipathic 지수(AI), GRAVY값 등의 생리화학적 특성에 대한 분석을 실시 하였다.

• Parasites, Hosts and Diseases
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• 제51권3호
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• pp.327-334
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• 2013

Vaccination is considered a promising alternative for controlling tick infestations. Haemaphysalis longicornis midgut proteins separated by SDS-PAGE and transferred to polyvinylidene difluoride (PVDF) membrane were screened for protective value against bites. The western blot demonstrated the immunogenicity of 92 kDa protein (P92). The analysis of the P92 amino acid sequence by LC-MS/MS indicated that it was a H. longicornis paramyosin (Hl-Pmy). The full lenghth cDNA of Hl-Pmy was obtained by rapid amplification of cDNA ends (RACE) which consisted of 2,783 bp with a 161 bp 3' untranslated region. Sequence alignment of tick paramyosin (Pmy) showed that Hl-Pmy shared a high level of conservation among ticks. Comparison with the protective epitope sequence of other invertebrate Pmy, it was calculated that the protective epitope of Hl-Pmy was a peptide (LEEAEGSSETVVEMNKKRDTE) named LEE, which was close to the N-terminal of Hl-Pmy protein. The secondary structure analysis suggested that LEE had non-helical segments within an ${\alpha}$-helical structure. These results provide the basis for developing a vaccine against biting H. longicornis ticks.

• Asian-Australasian Journal of Animal Sciences
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• 제18권9호
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• pp.1237-1241
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• 2005

Mannose-P-dolichol utilization defect 1 (MPDU1) gene is required for utilization of the mannose donor MPD in synthesis of both lipid-linked oligosaccharides (LLOs) and glycosylphosphatidylinositols (GPI) which are important for functions such as protein folding and membrane anchoring. The full length cDNA of the porcine MPDU1 was determined by in silico cloning and rapid amplification of cDNA ends (RACE). The deduced amino acid showed 91% identity to the corresponding human sequence with five predicted transmembrane regions. RT-PCR was performed to detect its expression pattern in five tissues and results showed that it is expressed ubiquitously among the tissues checked. A single nucleotide substitution resulting in the amino acid change (137 Tyr-137 His) was detected within exon 5. Allele frequencies in six pig breeds showed distinctive differences between those Chinese indigenous pigs breeds and European pigs. Using the pig/rodent somatic cell hybrid panel (SCHP), we mapped the porcine MPDU1 gene to SSC12, which is consistent with the comparative mapping result as conservative syntenic groups presented between human chromosome 17 and pig chromosome 12.

• Asian-Australasian Journal of Animal Sciences
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• 제19권7호
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• pp.931-937
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• 2006

The full-length cDNA of the porcine SMPX gene was obtained by the rapid amplification of cDNA ends (RACE). The nucleotide sequences and the predicted protein sequences share high sequence identity with both human and mouse. The promoter of SMPX was sequenced and then analyzed to find the promoter binding sites. The reverse transcriptase-polymerase chain reaction (RT-PCR) revealed that SMPX has a high level of expression in heart and skeletal muscle, a very low expression in lung and spleen and no expression in liver, kidney, fat and brain. Moreover, SMPX has a differential expression level in skeletal muscle, the expression in 65-day embryos being higher than other stages. The porcine SMPX was mapped to SSCXp24 by using a somatic cell hybrid panel (SCHP) and was found closely linked to SW1903 using the radiation hybrid panel IMpRH. An A/G single nucleotide polymorphism (PCR-RFLP) in the 3'-untranslated region (3'-UTR) was detected in eight breeds. The analysis of allele frequency distribution showed that introduced pig breeds (Duroc and Large White) have a higher frequency of allele A while in the Chinese indigenous pig breeds (Qingping pig, Lantang pig, YushanBlack pig, Large Black-White pig, Small Meishan) have a higher frequencies of allele G. The association analysis using an experimental population (188 pigs), which included two cross-bred groups and three pure-blood groups, suggested that the SNP genotype was associated with intramuscular fat content.

• 원예과학기술지
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• 제30권3호
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• pp.243-249
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• 2012

Hexokinase is the first enzyme in the hexose assimilation pathway; it acts as a sensor for plant sugar responses, and it is also important in determining the fruit sugar levels. The full-length cDNA of a hexokinase gene was isolated from loquat through reverse transcription polymerase chain reaction (RT-PCR) and rapid amplification of cDNA ends, which was designated as EjHXK1. EjHXK1 is 1,839 bp long and contains an entire open reading frame encoding 497 amino acids. The predicted protein of EjHXK1 shares 72%-81% similarity with other plant hexokinases. Phylogeny analysis indicated that EjHXK1 is closely related to maize and rice hexokinases. Transient expression of the 35S: EjHXK1-GFP fusion protein was observed on the cell membrane and cytoplasm. Real-time RT-PCR indicated that EjHXK1 is expressed in loquat leaves, stems, flowers, and fruits. EjHXK1 transcripts were higher during early fruit development, but decreases before maturation, which is consistent with hexokinase enzyme activity during fruit development and conducive for hexose accumulation in mature fruits. These results imply that EjHXK1 may play important roles in the regulation of sugar flux during fruit ripening.

• Journal of Plant Biotechnology
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• 제36권4호
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• pp.407-414
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• 2009

A cDNA clone encoding phytocystatin was isolated from Brassica rapa seedlings, through rapid amplification of cDNA ends (RACE). This gene (name as Brcpi1; GenBank accession no.: EF079953) had a total length of 881 bp with an open reading frame of 609 bp, and encoded predicted polypeptide of 203 amino acid (aa) residues including a putative N-terminal signal peptide. Other relevant regions found its sequence included the G and PW conserved aa motifs, and the consensus LARFAV sequence for phytocystatins and the reactive site QVVAG. The BrCPI1 protein shared 95, 94, 81, 80 and 78% identity with other CPI proterins isolated from Brassica oleracea (BoCPI-1), Arabidopsis thaliana (AtCY SB), Glycine max (GmCPI), Oryza sativa (OsCYS-2) and Zea may (ZmCPI) at amino acid level, respectively. Southern blot analysis showed that Brcpi1 was a low copy gene. Expression pattern analysis revealed that Brcpi1 was a tissue-specific expressing gene during reproductive growth and strongly expressed at mature seedling stages. Furthermore, overexpression of Brcpi1 in transgenic Arabidopsis was enhanced tolerance to salt and cold stresses. Meanwhile the juvenile seedling of Brcpi1 transgenic plants was not affected by various concentrations ABA in MS medium. Taken together, the results showed that Brcpi1 functioned as a cysteine protease inhibitor and it exhibited a protective agent against diverse types of abiotic stress, which induced this gene in a tissue- and stress-specific manner.