Molecular cloning and characterization of SNF1 related protein kinases in tomato (lycopersicon esculentum)
MOLECULAR CLONING AND CHARACTERIZATION OF SNFI RELATED PROTEIN KINASES IN TOMATO (Lycopersicon esculentum)
for the Degree of Master of Philosophy at The University of Hong Kong
in April 2003
Two cDNAs encoding SNFI related protein kinase (SnRK) were cloned from pink tomato fruit. From the amino acid sequences and phylogenetic analysis, they
both fell into the SnRKl family, and were designated as TKINl and TKIN2. The
complete cDNA sequence of TKINl 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. TKINl encodes the protein which has the identical amino acid sequence with tomato SNFI expressed in seeds imbibed in gibberellin for
24 hours. It also showed high similarity to potato StubSNF1, tobacco NPK5 and cucumber SnRKl. Phylogenetic analysis showed that TKINI fell into the SnRKla 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 PKINI. The phylogenetic tree showed that TKIN2 clusters with potato PKINI and lies beyond the SnRKla and SnRKlb sub-families. Both TKINI and TKIN2 contain the activation segment required for activation by the upstream kinase. A comparison of the TKINI 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.
TKINl and TKIN2 cDNAs were heterlogously expressed in E. coli as 6xHistagged 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 SNFl/AMPK family. It seems that a eukaryotic expression system is required for the expression of biologically active TKINI and TKIN2.
Attempts were made to determine the relative levels of the TKINl and TKIN2 mRNAs by northern blot analysis. However, no distinct signal was detected for either gene. This suggests that the mRNAs encoding TKINI 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 ofTKIN2. 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.
School:The University of Hong Kong
School Location:China - Hong Kong SAR
Source Type:Master's Thesis
Keywords:protein kinases molecular cloning tomatoes genetics
Date of Publication:01/01/2003