Molecular Identification and Expression Analysis of Carbonic Anhydrase VII in Pufferfish (Takifugu rubripes)

Kanij Rukshana Sumi,김수철,Jewel Howlader,Md Rajib Sharker,최갑성,최상기,박종인,노일섭,고강희 한국해양과학기술원 2019 Ocean science journal Vol.54 No.3

A carbonic anhydrase VII gene, encoding 277 amino acids, was identified in the intestinal tissue of pufferfish (Takifugu rubripes). The translated protein with an 833-bp complete coding sequence derived from the 1378-bp cloned sequence showed 83% identity with swamp eel CA VII, 76% with zebrafish CA VII, and 77% with coho salmon CA VII-like protein. The cloned protein also showed 68–69% identity with mammalian CA VII. The predicted molecular weight and iso-electric point of the protein were 30.84 kDa and 6.07, respectively. Active site analysis of the pufferfish CA VII indicated that most of the important residues involved in catalytic activity were highly conserved, whereas four cysteine residues at positions 55, 103, 184, and 275 differed from those in human CA II, and were related to cell-defense mechanisms against oxidative damage. Phylogenetic analysis showed that the cloned sequence was clustered within the fish CA VII clade and close to the swamp eel CA VII. Structural modeling of the pufferfish CA VII protein revealed the conservation of zinc binding histidine residues (zinc ion and histidine residues). Differential expression patterns of the pufferfish CA VII were determined with semiquantitative reverse transcription (RT)-PCR and quantitative PCR (q-PCR) as well. The results of q-PCR revealed that the pufferfish CA VII was highly expressed in intestinal tissue. The pufferfish CA VII was also detected within intestinal tissue sections using an in situ hybridization assay.

Molecular Cloning and Characterization of Carbonic Anhydrase XII from Pufferfish ( <i>Takifugu rubripes</i> )

Sumi, Kanij Rukshana,Kim, Soo Cheol,Howlader, Jewel,Lee, Won Kyo,Choi, Kap Seong,Kim, Hoy-Taek,Park, Jong-In,Nou, Ill-Sup,Kho, Kang Hee MDPI 2018 INTERNATIONAL JOURNAL OF MOLECULAR SCIENCES Vol.19 No.3

<P>In this study, an 1888-bp carbonic anhydrase XII (CA XII) sequence was cloned from the brain of the pufferfish, <I>Takifugu rubripes</I>. The cloned sequence contained a coding region of 1470-bp, which was predicted to translate into a protein of 490 amino acid residues. The predicted protein showed between 68–56% identity with the large yellow croaker (<I>Larimichthys crocea</I>), tilapia (<I>Oreochromis niloticus</I>), and Asian arowana (<I>Scleropages formosus</I>) CA XII proteins. It also exhibited 36% and 53% identity with human CA II and CA XII, respectively. The cloned sequence contained a 22 amino acid NH<SUB>2</SUB>-terminal signal sequence and three Asn-Xaa-Ser/Thr sequons, among which one was potentially glycosylated. Four cysteine residues were also identified (Cys-21, Cys-201, Cys-355, and Cys-358), two of which (Cys-21 and Cys-201) could potentially form a disulfide bond. A 22-amino acid COOH-terminal cytoplasmic tail containing a potential site for phosphorylation by protein kinase A was also found. The cloned sequence might be a transmembrane protein, as predicted from in silico and phylogenetic analyses. The active site analysis of the predicted protein showed that its active site residues were highly conserved with tilapia CA XII protein. Homology modeling of the pufferfish CA XII was done using the crystal structure of the extracellular domain of human carbonic anhydrase XII at 1.55 Å resolution as a template. Semi-quantitative reverse transcription (RT)-PCR, quantitative PCR (q-PCR), and in situ hybridization confirmed that pufferfish <I>CA XII</I> is highly expressed in the brain.</P>

Carbonic Anhydrase I in a Cartilaginous Fish, the Shortspine Spurdog (Squalus mitsukurii)

김수철,Kanij Rukshana Sumi,김정우,최명락,민병화,고강희 한국해양과학기술원 2016 Ocean science journal Vol.51 No.3

Carbonic anhydrase (CA), a ubiquitous enzyme found in many species, including fishes, is involved in physiological functions such as pH homeostasis, calcification, photosynthesis, and ionic regulation. CA I, a member of the α-CA family, is a cytoplasmic isozyme involved in carbon dioxide transport, ion exchange, and acid–base balance. Approximately half of the extant shark species occur only in deep waters; however, few published studies on sharks include these taxa. As fisheries worldwide enter deeper waters, the provision of biological data for these little-known taxa is critical to their management and conservation. To address this limitation, we aimed to detect CA I in various tissues of the shortspine spurdog (Squalus mitsukurii) and characterize its physicochemical properties by using sodium dodecyl-sulfate polyacrylamide gel electrophoresis and isoelectric focusing, together with immunohistochemistry. CA I was detected on SDS-PAGE and western blot analysis as a specific band at 29 kDa in various tissues of the shortspine spurdog, and as a specific band at pI 6.5 in various tissues of the shortspine spurdog by IEF and western blot analysis. CA I immunoreactivity in various tissues of the shortspine spurdog was detected in intracellular locations. To our knowledge, this is the first report of the localization of CA isozymes in various tissues of S. mitsukurii.

Identification and Characterization of Peroxidase and Glucan Synthase Like Genes under Podosphaera xanthii Infection and Phytohormone Treatments in Cucumis melo L.

Jewel Howlader,Kanij Rukshana Sumi,Jong-In Park,Hoytaek Kim,Jong-Gu Kang,Ill-Sup Nou 한국원예학회 2016 한국원예학회 학술발표요지 Vol.2016 No.5

Characterization and Expression Analysis of Peroxidases and Glucan Synthase Like Genes in Cucumis melo L.

( Jewel Howlader ),( Kanij Rukshana Sumi ),( Hoy Taek Kim ),( Arif Hasan Khan Robin ),( Jong In Park ),( Mi Young Chung ),( Ill Sup Nou ) 한국육종학회 2016 Plant Breeding and Biotechnology Vol.4 No.2

Powdery mildew (PM) is a severe fungal disease for melon cultivation worldwide. Stress resistance related genes could be important tools to address this problem. In this study, we retrieved defense related peroxidase and glucan synthase genes from Melon Genome Database ``Melonomics``. Thereafter, we analyzed the genes in silico. We conducted protein blast in the NCBI database and found a high degree of homology among them. Based on the highest protein homology we named two isoforms of Cucumis melo peroxidase 2-like genes (CmPrx2-1 and CmPrx2-2) and one glucan synthase1-like gene (CmGLS1). In reverse transcriptionpolymerase chain reaction (PCR), all 3 genes showed organ specific expression in a C. melo line, SCNU1154. Real-time quantitative PCR expression of these 3 genes was conducted in the infected leaf samples by PM fungus Podosphaera xanthii and also treated leaf samples by exogenous phytohormones (salicylic acid and methyl jasmonate). The CmPrx2-2 gene was up-regulated in response to all seven races of PM fungus whereas up-regulation or down-regulation of CmPrx2-1 gene was race-specific. The CmGLS1 gene was down-regulated in response to all races except one race. The CmPrx2-1, CmPrx2-2, and CmGLS1 genes were up-regulated under both salicylic acid and methyl jasmonate treatments but their level of expression was higher in salicylic acid treated plants compared to methyl jasmonate. Therefore, we speculate that defense response of the three tested genes is largely mediated by the salicylic acid signaling pathway under PM infection. Taken together, the data presented herein may be useful resources in the development of PM stress resistant in C. melo L.

Identification and Expression of Retroviral Envelope Polyprotein in the Dogfish Squalus mitsukurii

Kim, Soo Cheol,Sumi, Kanij Rukshana,Choe, Myeong Rak,Kho, Kang Hee The Korean Society of Marine Life Science 2016 한국해양생명과학회지 Vol.1 No.2

Determining the infection history of living organisms is essential for understanding the evolution of infection agents with their host, particularly for key aspects such as immunity. Viruses, which can spread between individuals and often cause disease, have been widely examined. The increasing availability of fish genome sequences has provided specific insights into the diversity and host distribution of retroviruses in fish. The shortspine spurdog (Squalus mitsukurii) is an important elasmobranch species; this medium-sized dogfish typically lives at depths of 100~500 m. However, the retroviral envelope polyprotein in dogfish has not been examined. Thus, the aim of the present study was to identify and analyze the retroviral envelope polyprotein in various tissues of dogfish. The 1334-base pair full-length novel cDNA of dogfish envelope polyprotein (dEnv) was obtained by 3' and 5'-rapid amplification of cDNA end analysis from S. mitsukurii. The open reading frame showed a complete coding sequence of 815 base pairs with a deduced peptide sequence of 183 amino acids that exhibited 34~50% identity with other fish and bird species. It was also expressed according to reverse transcription and real-time polymerase chain reaction in the kidney, liver, intestine, and lung, but not in the gill. This distribution can be assessed by identifying and analyzing endogenous retroviruses in fish, which consists of three main genes: gag, pol and env. Dogfish envelope polyprotein sequence is likely important in evolution and induces rearrangements, altering the regulatory and coding sequences. This is the first report of the identification and molecular characterization of retroviral envelope polyprotein in various tissues of S. mitsukurii.

Carbonic Anhydrase I in a Cartilaginous Fish, the Shortspine Spurdog (Squalus mitsukurii)

Kim, Soo Cheol,Sumi, Kanij Rukshana,Kim, Jung Woo,Choi, Myeong Rak,Min, Byung Hwa,Kho, Kang Hee Korean Ocean Research & Development Institute and 2016 OCEAN SCIENCE JOURNAL Vol.51 No.3

Carbonic anhydrase (CA), a ubiquitous enzyme found in many species, including fishes, is involved in physiological functions such as pH homeostasis, calcification, photosynthesis, and ionic regulation. CA I, a member of the <TEX>${\alpha}$</TEX>-CA family, is a cytoplasmic isozyme involved in carbon dioxide transport, ion exchange, and acid-base balance. Approximately half of the extant shark species occur only in deep waters; however, few published studies on sharks include these taxa. As fisheries worldwide enter deeper waters, the provision of biological data for these little-known taxa is critical to their management and conservation. To address this limitation, we aimed to detect CA I in various tissues of the shortspine spurdog (Squalus mitsukurii) and characterize its physicochemical properties by using sodium dodecyl-sulfate polyacrylamide gel electrophoresis and isoelectric focusing, together with immunohistochemistry. CA I was detected on SDS-PAGE and western blot analysis as a specific band at 29 kDa in various tissues of the shortspine spurdog, and as a specific band at pI 6.5 in various tissues of the shortspine spurdog by IEF and western blot analysis. CA I immunoreactivity in various tissues of the shortspine spurdog was detected in intracellular locations. To our knowledge, this is the first report of the localization of CA isozymes in various tissues of S. mitsukurii.

Molecular Identification and Expression of Myosin Light Chain in Shortspine Spurdog (Squalus mitsukurii)

Kim, Soo Cheol,Sumi, Kanij Rukshana,Sharker, Md Rajib,Kho, Kang Hee The Korean Society of Marine Life Science 2018 한국해양생명과학회지 Vol.3 No.1

Myosin is considered as the vital motor protein in vertebrates and invertebrates. Our present study was conducted to decipher the occurrence of myosin in dog fish (Squalus mitsukurii). We isolated one clone containing 979 bp cDNA sequence, which consisted of a complete coding sequence of 453 bp and a deduced amino acid sequence of 150 amino acids from the open reading frame with molecular weight, isoelectric point and aliphatic index are 16.72 Kda, 4.49 and 78.00, respectively. It contained 428 bp long 3' UTR with single potential polyadenylation signals (AATAAA). The predicted EF CA²⁺ binding domains were identified in residue 6-41, 83-118 and 133-150. A BLAST search indicates this protein exhibits a strong similarity to whale shark (Rhincodon typus) MLC3 (91% identical) and also house mouse (Mus musculus) MLC isoform 3f (81% identical). Phylogenetic analysis revealed that this protein is a MLC 3 isoform like protein. This protein also demonstrates highly conserved region with other myosin proteins. Homology modeling of S. mitsukuri was performed using crystal structure of Gallus gallus skeletal muscle myosin II based on high similarity. Reverse transcription-polymerase chain reaction (PCR), quantitative PCR results exhibits dogfish myosin protein is highly expressed in muscle tissue.

Isolation and molecular identification of carbonic anhydrase VII in shortspine spurdog (Squalus mitsukurii)

Soo Cheol Kim,Kanij Rukshana Sumi,MD Rajib Sharker,Kang Hee Kho 한국수산과학회 양식분과 2019 한국수산과학회 양식분과 학술대회 Vol.2019 No.5

Development of powdery mildew race 5-specifi c SNP markers in Cucumis melo L. using whole-genome resequencing

Jewel Howlader,Yeji Hong,Sathishkumar Natarajan,Kanij Rukshana Sumi,Hoy-Taek Kim,Jong-In Park,Ill-Sup Nou 한국원예학회 2020 Horticulture, Environment, and Biotechnology Vol.61 No.2

Melon ( Cucumis melo L.), belonging to the Cucurbitaceae family, is cultivated worldwide and is highly valued for its fruitquality. However, this important crop is negatively aff ected by several races of powdery mildew (PM) fungus, Podosphaeraxanthii . Hence, exploration of PM race-specifi c resistant markers would be an eff ective strategy to develop race-specifi cmelon cultivars. Young leaves from four melon genotypes, including susceptible SCNU1154 and race-specifi c-resistantEdisto47, PMR5, and MR1 lines, were sequenced by next-generation sequencing, specifi cally whole-genome resequencing(WGR), to detect race-specifi c single nucleotide polymorphism (SNP) markers. Including putative resistance gene ( R -gene)SNPs, 168, 83, and 122 race 5-specifi c SNPs with the exact variation were identifi ed on PM-related chromosome 2, 5, and12, respectively, for distinguishing race-specifi c lines by comparing the WGR data in the C. melo genome. Based on proximityto the PM resistance quantitative trait loci (QTLs) in physical maps, 43, 37, and 48 SNPs were screened on chromosome2, 5, and 12, respectively. Using a derived cleaved amplifi ed polymorphic sequence (dCAPS) method, polymorphismswere only found in three SNPs (SNPR5_119, SNPR5_120, and SNPR5_121) out of 48 against race 5 on chromosome 12. Among these three, a putative R -gene, an NBS-LRR-type SNP, SNPR5_119, displayed similar genotypic and phenotypicvariation to race 5-specifi c susceptible (SCNU1154, PMR45, WMR29, and Edisto47) and resistant (PI414723, PMR5, andMR1) lines in C . melo except for PI124112. Importantly, two other intergenic SNPs, SNPR5_120 and SNPR5_121, showedgenotypic and phenotypic variation between susceptible and resistant lines tested. High-resolution melting (HRM) analysiswas used to further validate the same expression patterns of SNPR5_120 as a dCAPS method in all lines tested. Therefore,the identifi ed PM race 5-specifi c candidate markers described here might be useful for a marker-assisted selection breedingprogram in C. melo .