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Molecular Cloning and Characterization of Snf1 Related Protein Kinases in Tomato (Lycopersicon Esculentum)

Molecular Cloning and Characterization of Snf1 Related Protein Kinases in Tomato (Lycopersicon Esculentum)


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This dissertation, "Molecular Cloning and Characterization of SNF1 Related Protein Kinases in Tomato (lycopersicon Esculentum)" by Pui-Yi, Lam, 林佩儀, was obtained from The University of Hong Kong (Pokfulam, Hong Kong) and is being sold pursuant to Creative Commons: Attribution 3.0 Hong Kong License. The content of this dissertation has not been altered in any way. We have altered the formatting in order to facilitate the ease of printing and reading of the dissertation. All rights not granted by the above license are retained by the author. Abstract: Abstract of thesis entitled MOLECULAR CLONING AND CHARACTERIZATION OF SNF1 RELATED PROTEIN KINASES IN TOMATO (Lycopersicon esculentum) Submitted by Lam Pui Yi for the Degree of Master of Philosophy at The University of Hong Kong in April 2003 Two cDNAs encoding SNF1 related protein kinase (SnRK) were cloned from pink tomato fruit. From the amino acid sequences and phylogenetic analysis, they both fell into the SnRK1 family, and were designated as TKIN1 and TKIN2. The complete cDNA sequence of TKIN1 contains 2045 bp, with the entire coding sequence of 1542 bp that encodes a 514 amino acid protein with a calculated molecular mass of 58824 Da. TKIN1 encodes the protein which has the identical amino acid sequence with tomato SNF1 expressed in seeds imbibed in gibberellin for 24 hours. It also showed high similarity to potato StubSNF1, tobacco NPK5 and cucumber SnRK1. Phylogenetic analysis showed that TKIN1 fell into the SnRK1a sub-family. In contrast, the complete cDNA sequence of TKIN2 contains 1974 bp, with the entire coding sequence of 1512 bp encoding a 504 amino acid protein with a predicted molecular mass of 57762 Da. TKIN2 encodes a protein closely related to potato PKIN1. The phylogenetic tree showed that TKIN2 clusters with potato PKIN1 and lies beyond the SnRK1a and SnRK1b sub-families. Both TKIN1 and TKIN2 contain the activation segment required for activation by the upstream kinase. A comparison of the TKIN1 and TKIN2 amino acid sequences showed that they share overall 67.7% identity, with 87.8% similarity within the kinase domain and 50.0% similarity within the C-terminal regulatory domain. This suggests that these two isoforms are enzymatically similar but are differentially regulated. TKIN1 and TKIN2 cDNAs were heterlogously expressed in E. coli as 6xHis- tagged fusion and thioredoxin fusion proteins to confirm that the cDNAs encode active forms of SnRK. They were successfully expressed as soluble fusion proteins in both systems and the fusion proteins were purified by affinity chromatography. The recombinant proteins however showed no activity in the SAMS peptide phosphorylation assay, an assay specific for the SNF1/AMPK family. It seems that a eukaryotic expression system is required for the expression of biologically active TKIN1 and TKIN2. Attempts were made to determine the relative levels of the TKIN1 and TKIN2 mRNAs by northern blot analysis. However, no distinct signal was detected for either gene. This suggests that the mRNAs encoding TKIN1 and TKIN2 may be expressed at levels too low for detection by northern blots. Polyclonal antibodies were raised against the synthetic peptide VSEESLRRPFRKEKT, corresponding to residues 369-383 of TKIN2. This sequence of amino acids is specific for TKIN2 and fulfills the requirements for raising antibodies. The crude antiserum was pre-adsorbed against tomato tissue proteins and this pre-adsorbed antiserum recognizes a polypeptide with an approximate molecular mass of 57.8 kD in crude extracts of green, breaking, turning, pink, orange and red fruits, on western blot. It showed that TKIN2 protein accumulated in green, breaking and turning fruits and its abundance decreased from pink to red fruits, suggesting that TKIN2 likely plays a role in the tomato fruit ripening process. DOI: 10.5353/th_b2773668 Subjects: Protei


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Product Details
  • ISBN-13: 9781374714441
  • Publisher: Open Dissertation Press
  • Publisher Imprint: Open Dissertation Press
  • Height: 279 mm
  • No of Pages: 162
  • Weight: 390 gr
  • ISBN-10: 1374714445
  • Publisher Date: 27 Jan 2017
  • Binding: Paperback
  • Language: English
  • Spine Width: 9 mm
  • Width: 216 mm


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Molecular Cloning and Characterization of Snf1 Related Protein Kinases in Tomato (Lycopersicon Esculentum)
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