An Efficient Method for Rapid Amplification ofArisaema heterophyllumAgglutinin Gene Using a Genomic Walking Technique

Juan Lin; Jun Liu; Xiaofen Sun; Xuanwei Zhou; Jiong Fei; Kexuan Tang

doi:10.1081/pb-200054738

Isolation of Alcohol Dehydrogenase cDNA and Basal Regulatory Region from Metroxylon sagu

ISRN Molecular Biology ◽

10.5402/2012/839427 ◽

2012 ◽

Vol 2012 ◽

pp. 1-9

Author(s):

Ching Ching Wee ◽

Hairul Azman Roslan

Keyword(s):

Amino Acids ◽

Alcohol Dehydrogenase ◽

Elaeis Guineensis ◽

Regulatory Region ◽

Putative Promoter Region ◽

Sago Palm ◽

Binding Domains ◽

Genomic Walking ◽

Rapid Amplification ◽

Metroxylon Sagu

Alcohol dehydrogenase (Adh) is a versatile enzyme involved in many biochemical pathways in plants such as in germination and stress tolerance. Sago palm is plant with much importance to the state of Sarawak as one of the most important crops that bring revenue with the advantage of being able to withstand various biotic and abiotic stresses such as heat, pathogens, and water logging. Here we report the isolation of sago palm Adh cDNA and its putative promoter region via the use of rapid amplification of cDNA ends (RACE) and genomic walking. The isolated cDNA was characterized and determined to be 1464 bp long encoding for 380 amino acids. BLAST analysis showed that the Adh is similar to the Adh1 group with 91% and 85% homology with Elaeis guineensis and Washingtonia robusta, respectively. The putative basal msAdh1 regulatory region was further determined to contain promoter signals of TATA and AGGA boxes and predicted amino acids analyses showed several Adh-specific motifs such as the two zinc-binding domains that bind to the adenosine ribose of the coenzyme and binding to alcohol substrate. A phylogenetic tree was also constructed using the predicted amino acid showed clear separation of Adh from bacteria and clustered within the plant Adh group.

Download Full-text

Isolation and Characterization of the First Putative Peroxidase Gene from Oilseed Rape (Brassica napus) which is Highly Homologous to HRPC

Bioscience Reports ◽

10.1007/s10540-006-9021-0 ◽

2006 ◽

Vol 26 (3) ◽

pp. 263-280 ◽

Cited By ~ 3

Author(s):

Weisheng Wu ◽

Jie Lu ◽

Yamin Wei ◽

Jin Wang ◽

Juan Lin ◽

...

Keyword(s):

Brassica Napus ◽

Gene Family ◽

Oilseed Rape ◽

Blot Analysis ◽

Reading Frame ◽

Peroxidase Gene ◽

Genomic Walking ◽

Isolation And Characterization ◽

New Gene ◽

Rapid Amplification

A new gene, designated as BnPrx (GenBank Accession No. DQ078754), was isolated from oilseed rape (Brassica napus) by SMART Rapid Amplification of cDNA Ends (RACE). The full-length cDNA is 1307 bp long and contains a 1062 bp open reading frame (ORF), which encodes a 354 amino acid peroxidase precursor, with a 31 aa N-terminal signal peptide and a 15 aa C-terminal propeptide. The putative protein has a molecular weight of 38.86 kDa and a calculated pI of 5.85. BnPrx shares high identity with HRPC (89%). BnPrx possesses all active residues and two Ca2+ sites present in Horseradish peroxidase isoenzymes C (HRPC) as well as six N-glycosylation sites. The predicted 3-D structure of BnPrx is very similar to that of HRPC. Assisted by genomic walking technology, the genomic DNA of BnPrx was also cloned, consisting of 3 introns and 4 exons. Thirty-two TATA boxes, 18 CAAT boxes and many cis-elements, such as WUN, MeJR, were found in its promoter region. Southern blot analysis indicated that BnPrx belonged to a small gene family. Northern blot analysis revealed that BnPrx was constitutively expressed in all tested tissues, including roots, stems and leaves, with the high expression in leaves and stems. The expression of BnPrx could be induced by methyl jasmonate (MeJA), salicylic acid (SA), cold and H2O2. The cloning and characterizing of BnPrx might not only help us understand the physiological function and molecular evolution of the large peroxidase gene family more comprehensively, but also provide an alternative way of seeking a more effective and economical substitute for HRPC.

Download Full-text

Functional and molecular identification of a TASK-1 potassium channel regulating chloride secretion through CFTR channels in the shark rectal gland: implications for cystic fibrosis

AJP Cell Physiology ◽

10.1152/ajpcell.00030.2016 ◽

2016 ◽

Vol 311 (6) ◽

pp. C884-C894 ◽

Cited By ~ 6

Author(s):

Connor J. Telles ◽

Sarah E. Decker ◽

William W. Motley ◽

Alexander W. Peters ◽

Ali Poyan Mehr ◽

...

Keyword(s):

Potassium Channel ◽

Reversal Potential ◽

Chloride Secretion ◽

Rectal Gland ◽

Degenerate Primers ◽

Reading Frame ◽

Genomic Walking ◽

Shark Rectal Gland ◽

Mammalian Lung ◽

Rapid Amplification

In the shark rectal gland (SRG), apical chloride secretion through CFTR channels is electrically coupled to a basolateral K+ conductance whose type and molecular identity are unknown. We performed studies in the perfused SRG with 17 K+ channel inhibitors to begin this search. Maximal chloride secretion was markedly inhibited by low-perfusate pH, bupivicaine, anandamide, zinc, quinidine, and quinine, consistent with the properties of an acid-sensitive, four-transmembrane, two-pore-domain K+ channel (4TM-K2P). Using PCR with degenerate primers to this family, we identified a TASK-1 fragment in shark rectal gland, brain, gill, and kidney. Using 5′ and 3′ rapid amplification of cDNA ends PCR and genomic walking, we cloned the full-length shark gene (1,282 bp), whose open reading frame encodes a protein of 375 amino acids that was 80% identical to the human TASK-1 protein. We expressed shark and human TASK-1 cRNA in Xenopus oocytes and characterized these channels using two-electrode voltage clamping. Both channels had identical current-voltage relationships (outward rectifying) and a reversal potential of −90 mV. Both were inhibited by quinine, bupivicaine, and acidic pH. The pKa for current inhibition was 7.75 for shark TASK-1 vs. 7.37 for human TASK-1, values similar to the arterial pH for each species. We identified this protein in SRG by Western blot and confocal immunofluorescent microscopy and detected the protein in SRG and human airway cells. Shark TASK-1 is the major K+ channel coupled to chloride secretion in the SRG, is the oldest 4TM 2P family member identified, and is the first TASK-1 channel identified to play a role in setting the driving force for chloride secretion in epithelia. The detection of this potassium channel in mammalian lung tissue has implications for human biology and disease.

Download Full-text