Cloning and sequence analysis of Ndufb2 full-length cDNA derived from Oncorhynchus mykiss
Rainbow trout belongs to Salmonidae aerobic fish, and it is necessary for high dissolved oxygen content of living water environment. If the dissolved oxygen content of living water is less than 5 mg/L, it will cause the increase of respiratory rate, which is the so-called “aquaculture floating head”...
Saved in:
| Main Authors: | , , , , , |
|---|---|
| Format: | Article |
| Language: | English |
| Published: |
Zhejiang University Press
2013-11-01
|
| Series: | 浙江大学学报. 农业与生命科学版 |
| Subjects: | |
| Online Access: | https://www.academax.com/doi/10.3785/j.issn.1008-9209.2013.05.121 |
| Tags: |
Add Tag
No Tags, Be the first to tag this record!
|
| _version_ | 1849413375634702336 |
|---|---|
| author | WANG Jiaqing BIAN Jia LI Daizong MA Shuang WANG Liang NA Guangning |
| author_facet | WANG Jiaqing BIAN Jia LI Daizong MA Shuang WANG Liang NA Guangning |
| author_sort | WANG Jiaqing |
| collection | DOAJ |
| description | Rainbow trout belongs to Salmonidae aerobic fish, and it is necessary for high dissolved oxygen content of living water environment. If the dissolved oxygen content of living water is less than 5 mg/L, it will cause the increase of respiratory rate, which is the so-called “aquaculture floating head” phenomenon. Because the fish lives in hypoxia environment and the 90% oxygen consumption is in the mitochondria, the transmission mechanism in composition and electronic respiratory chain may be different from the terrestrial animal. At the mitochondrial inner membrane, electrons from NADH and succinate pass through the electron transport chain to oxygen, which is reduced to water. Complex I is one of the main sites at which premature electron leakage to oxygen occurs, thus being one of the main sites of production of harmful superoxide. The first isolation of mitochondrial complex I since 1961, its composition and structure have had a primary understanding, but the specific mechanism of its participation in respiration, especially the function of each subunit is not clear. The protein encoded by Ndufb2 gene is a subunit of the multisubunit NADH: ubiquinone oxidoreductase (complex I). Mammalian complex I is composed of 45 different subunits. This protein has NADH dehydrogenase activity and oxidoreductase activity. It plays an important role in transferring electrons from NADH to the respiratory chain.Reverse transcription PCR (RT-PCR) and rapid amplification of cDNA ends (RACE) methods were used for the isolation of the whole cDNA of Ndufb2 gene from brain of Oncorhynchus mykiss. The assembly taskes of 3' and 5'-RACE sequence were completed by DNAman program. A pair of gene specific primers were designed to amplify the full-length cDNA sequence. ClustalX 1.81 and MEGA 3.0 software were used to calculate the amino acid sequence differences, and then the phylogenetic relationships of rainbow trout Ndufb2 gene sequence with other species were analyzed. Protein phosphorylation sites and protein kinase phosphorylation sites were predicted using online software NetPhosK 1 and NetPhos 2.Sequence analysis results showed that the rainbow trout Ndufb2 cDNA was 899 bp in length, 5'untranslated region (5'UTR) 152 bp, 3'untranslated region (3'UTR) 441 bp, open reading frame (ORF) 306 bp, encoding 101 amino acids. The protein molecular mass was 11.4 ku and isoelectric point was 5.31. This gene sequence had been submitted to the GenBank database (accession number: FJ534641). Mitochondrial target sequence of 50 amino acid motif appeared from the 1st to 50th amino acid site. Two antigenic sites (65-ILWHCWHDPD-74 and 23-QKIVIRK-29) of the Ndufb2 protein were found, and one serine phosphorylation site (aa49), two threonine phosphorylation sites (aa15, aa47) and seven kinase phosphorylation sites were also found. Sequence alignment exhibited 98% identity of amino acids between O. mykiss and Salmo salar, and more than 55% identity of amino acids between O. mykiss and mammal. Phylogenetic tree showed that the Ndufb2 of rainbow trout had the closest relationship with those of Atlantic salmon and zebrafish, then the amphibians, birds, marsupials and mammals. The phylogenetic analysis showed that the constructed Ndufb2 protein phylogenetic tree was consistent with the traditional species classification tree.The above results indicate that the Ndufb2 gene is relatively conservative in the progress of evolution and play an important role in electron transport process of mitochondrial respiratory chain. The cloned full-length cDNA sequence of mitochondrial respiratory chain gene Ndufb2 of rainbow trout lays a theoretical foundation for the structure and function of clarifying fish mitochondrial complex I. |
| format | Article |
| id | doaj-art-70d7bbef6c51428484501578efc09f23 |
| institution | Kabale University |
| issn | 1008-9209 2097-5155 |
| language | English |
| publishDate | 2013-11-01 |
| publisher | Zhejiang University Press |
| record_format | Article |
| series | 浙江大学学报. 农业与生命科学版 |
| spelling | doaj-art-70d7bbef6c51428484501578efc09f232025-08-20T03:34:08ZengZhejiang University Press浙江大学学报. 农业与生命科学版1008-92092097-51552013-11-013960060610.3785/j.issn.1008-9209.2013.05.12110089209Cloning and sequence analysis of Ndufb2 full-length cDNA derived from Oncorhynchus mykissWANG JiaqingBIAN JiaLI DaizongMA ShuangWANG LiangNA GuangningRainbow trout belongs to Salmonidae aerobic fish, and it is necessary for high dissolved oxygen content of living water environment. If the dissolved oxygen content of living water is less than 5 mg/L, it will cause the increase of respiratory rate, which is the so-called “aquaculture floating head” phenomenon. Because the fish lives in hypoxia environment and the 90% oxygen consumption is in the mitochondria, the transmission mechanism in composition and electronic respiratory chain may be different from the terrestrial animal. At the mitochondrial inner membrane, electrons from NADH and succinate pass through the electron transport chain to oxygen, which is reduced to water. Complex I is one of the main sites at which premature electron leakage to oxygen occurs, thus being one of the main sites of production of harmful superoxide. The first isolation of mitochondrial complex I since 1961, its composition and structure have had a primary understanding, but the specific mechanism of its participation in respiration, especially the function of each subunit is not clear. The protein encoded by Ndufb2 gene is a subunit of the multisubunit NADH: ubiquinone oxidoreductase (complex I). Mammalian complex I is composed of 45 different subunits. This protein has NADH dehydrogenase activity and oxidoreductase activity. It plays an important role in transferring electrons from NADH to the respiratory chain.Reverse transcription PCR (RT-PCR) and rapid amplification of cDNA ends (RACE) methods were used for the isolation of the whole cDNA of Ndufb2 gene from brain of Oncorhynchus mykiss. The assembly taskes of 3' and 5'-RACE sequence were completed by DNAman program. A pair of gene specific primers were designed to amplify the full-length cDNA sequence. ClustalX 1.81 and MEGA 3.0 software were used to calculate the amino acid sequence differences, and then the phylogenetic relationships of rainbow trout Ndufb2 gene sequence with other species were analyzed. Protein phosphorylation sites and protein kinase phosphorylation sites were predicted using online software NetPhosK 1 and NetPhos 2.Sequence analysis results showed that the rainbow trout Ndufb2 cDNA was 899 bp in length, 5'untranslated region (5'UTR) 152 bp, 3'untranslated region (3'UTR) 441 bp, open reading frame (ORF) 306 bp, encoding 101 amino acids. The protein molecular mass was 11.4 ku and isoelectric point was 5.31. This gene sequence had been submitted to the GenBank database (accession number: FJ534641). Mitochondrial target sequence of 50 amino acid motif appeared from the 1st to 50th amino acid site. Two antigenic sites (65-ILWHCWHDPD-74 and 23-QKIVIRK-29) of the Ndufb2 protein were found, and one serine phosphorylation site (aa49), two threonine phosphorylation sites (aa15, aa47) and seven kinase phosphorylation sites were also found. Sequence alignment exhibited 98% identity of amino acids between O. mykiss and Salmo salar, and more than 55% identity of amino acids between O. mykiss and mammal. Phylogenetic tree showed that the Ndufb2 of rainbow trout had the closest relationship with those of Atlantic salmon and zebrafish, then the amphibians, birds, marsupials and mammals. The phylogenetic analysis showed that the constructed Ndufb2 protein phylogenetic tree was consistent with the traditional species classification tree.The above results indicate that the Ndufb2 gene is relatively conservative in the progress of evolution and play an important role in electron transport process of mitochondrial respiratory chain. The cloned full-length cDNA sequence of mitochondrial respiratory chain gene Ndufb2 of rainbow trout lays a theoretical foundation for the structure and function of clarifying fish mitochondrial complex I.https://www.academax.com/doi/10.3785/j.issn.1008-9209.2013.05.121rainbow troutOncorhynchus mykiss<italic>Ndufb2</italic> genereverse transcription-polymerase chain reactionrapid amplification of cDNA ends |
| spellingShingle | WANG Jiaqing BIAN Jia LI Daizong MA Shuang WANG Liang NA Guangning Cloning and sequence analysis of Ndufb2 full-length cDNA derived from Oncorhynchus mykiss 浙江大学学报. 农业与生命科学版 rainbow trout Oncorhynchus mykiss <italic>Ndufb2</italic> gene reverse transcription-polymerase chain reaction rapid amplification of cDNA ends |
| title | Cloning and sequence analysis of Ndufb2 full-length cDNA derived from Oncorhynchus mykiss |
| title_full | Cloning and sequence analysis of Ndufb2 full-length cDNA derived from Oncorhynchus mykiss |
| title_fullStr | Cloning and sequence analysis of Ndufb2 full-length cDNA derived from Oncorhynchus mykiss |
| title_full_unstemmed | Cloning and sequence analysis of Ndufb2 full-length cDNA derived from Oncorhynchus mykiss |
| title_short | Cloning and sequence analysis of Ndufb2 full-length cDNA derived from Oncorhynchus mykiss |
| title_sort | cloning and sequence analysis of ndufb2 full length cdna derived from oncorhynchus mykiss |
| topic | rainbow trout Oncorhynchus mykiss <italic>Ndufb2</italic> gene reverse transcription-polymerase chain reaction rapid amplification of cDNA ends |
| url | https://www.academax.com/doi/10.3785/j.issn.1008-9209.2013.05.121 |
| work_keys_str_mv | AT wangjiaqing cloningandsequenceanalysisofndufb2fulllengthcdnaderivedfromoncorhynchusmykiss AT bianjia cloningandsequenceanalysisofndufb2fulllengthcdnaderivedfromoncorhynchusmykiss AT lidaizong cloningandsequenceanalysisofndufb2fulllengthcdnaderivedfromoncorhynchusmykiss AT mashuang cloningandsequenceanalysisofndufb2fulllengthcdnaderivedfromoncorhynchusmykiss AT wangliang cloningandsequenceanalysisofndufb2fulllengthcdnaderivedfromoncorhynchusmykiss AT naguangning cloningandsequenceanalysisofndufb2fulllengthcdnaderivedfromoncorhynchusmykiss |