Characterization of inflorescence-predominant chitinase gene in Metroxylon sagu via differential display

Abstract

Chitinase is an enzyme that catalyzes the degradation of chitin, commonly induced upon the attack of pathogens and other stresses. A cDNA (MsChi1) was isolated from Metroxylon sagu and expressed predominantly in the inflorescence tissue of M. sagu, suggesting its role in developmental processes. The chitinase cDNA was detected and isolated via differential display and rapid amplification of cDNA ends (RACE). Primers specific to M. saguchitinase were used as probes to amplify the 3'-end and 5'-end regions of chitinase cDNA. Transcript analysis showed that chitinase is expressed in inflorescence and meristem tissues but was not detected in the leaf tissue. Sequence analysis of amplified cDNA fragments of 3'-end and 5'-end regions indicated that the chitinase cDNA was successfully amplified. The M. saguchitinase cDNA isolated was approximately 1,143 bp long and corresponds to 312 predicted amino acids. Alignments of nucleotide and amino acid have grouped this chitinase to family 19 class I chitinase.

Fig. 1

The leaves, meristem and inflorescence samples of M. sagu. a The leaf samples. b The vegetative growth of the palm. c The meristem samples. d The inflorescence sample. e The inflorescence developing palm (photo copyright CRAUN Research Sdn. Bhd, taken from Tie 2004)

Fig. 2

A 2.5% (w/v) agarose gel-electrophoresis of fragments amplified using (a) combination of oligo(dt)₁₅ACP/AP2 primers. The fragments labelled as a, b and c represent fragments msAP21, msAP22 and msAP23, respectively. b Combination of oligo(dt)₁₅ACP/AP3 primers. The arrow represents fragment msAP33. The M, I and L represent meristem, inflorescence and leaf cDNA samples, respectively. S represents a 100 bp Forever Ladder (Seegene)

Fig. 3

A 2.0% (x/v) agarose gel-electrophoresis of amplified 3′-end region of chitinase in M. sagu meristem, inflorescence and leaf tissues. LaneS represents a 100 bp Forever Ladder (Seegene). LanesM, I and L represent amplified product from meristem, inflorescence and leaf tissue, respectively. Lane C represents the negative control. Arrow indicates the 400 bp fragment and dashed arrow shows the 300 bp fragment

Fig. 4

Nucleotide sequence of chitinase gene in M. sagu showing the open reading frame (ORF), stop codon dan polyadenylation site. Asterisk represents the stop codon and the polyadenylation signal is presented as bold underlined nucleotides. The ORF is located between the arrowheads

Fig. 5

Multiple sequence alignment of the deduced amino acids of MsChi1 and class I chitinase from Festuca arundinacea, Oryza sativa, Phaseolusvulgaris, Pyruspyrifolia and Triticumaestivum. Identical amino acid residues are indicated by asterisks, the catalytic residues are denoted as closed triangles, amino acid for chitin binding domain are underlined and chitinase consensus sequence are indicated inbox.Dashed lines are gaps introduced to maximize the identity