Molecular biological and biochemical studies of isopentenyltransferase in bamboo (Bambusa oldhamii)
Date Issued
2011
Date
2011
Author(s)
Yeh, Chuan-Shan
Abstract
Cytokinins (CKs) are a class of plant hormones that play a pivotal role in plant development. They induce cell division in the presence of auxins, and induce shoot formation on calli. They also release axillary buds from apical dominance, increase sink strength, and delay senescence. In the CKs synthesis pathway, the isopentenyl group is transferred from DMAPP to the N6of AMP, resulting in the production of isopentenyladenosine-5’-monophosphate (iPMP). This reaction is thought to be catalyzed by isopentenyltransferases (IPT). IPT is a key enzyme in CKs biosynthesis pathway.
A DNA fragment encoding isopentenyltransferase (IPT) was cloned and sequenced from genomic library of Bamboo (Bambusa oldhamii). Library screening by IPT specific probe from degenerate PCR using degenerate oligonucleotide primers based on the conserved sequences of Arabidopsis thaliana AtIPT and Oryza sativa OsIPT isozymes. The 4.7 kb genomic DNA fragment contains a 1,035 bp open reading frame encoding a molecular mass of 37,599 dalton protein with 344 amino acid named BoIPT1 revealed absence of intron in the frame. BoAIPT1 deduced amino acid sequence shares 47-71% identity to OsIPTs. Prediction of biochemical properties of BoAIPT1 amino acid sequence using ExPAsy (Expert Protein Analysis System) proteomics server revealed phosphorylation and glycosylation sites in Ser、Thr and Tyr residues. Isoelectric point of BoIPT1 is 8.47. Subcellular localization prediction of BoIPT1 is a cytosolic protein. Analysis BoAIPT1 promoter by PLACE promoter scanning tool revealed cis-acting regulatory DNA elements involved auxin、gibberellin and light response.
Using northern blot analysis on BoAIPT1 at various growth stages, demonstrated the expression level of BoAIPT1 is higher in bamboo shoot as compared to the etiolated shoot. Using Real-Time RT-PCR to compare the BoAIPT1expresion level in various parts of bamboo shoot and etiolated shoot, results revealed that the expression level was highest in the shoot. Results from GFP revealed that BoAIPT1 was located in the plastid of onion epidermal cell. Expression of BoAIPT1 in E.coli was used to perform enzyme assay, this revealed to ve similar with other reports regarding IPTs of different plants. This suggests that BoAIPT1 uses ATP and DMAPP as substarte to initiate the iP sysnthesis pathway.
Subjects
bamboo
AIPT
cytokinin
Type
thesis
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