Cloning and characterization of pectate lyase from Hevea brasiliensis.
Latex from the commercial Hevea brasiliensis contains 30-50% (w/w) of natural rubber (cis-1,4-polyisoprene), the raw material for the many products of the rubber industry. We have constructed a cDNA library from the latex of H. brasiliensis to investigate the expressed genes and molecular events in the latex. We have isolated two cDNAs from this library, Hb-PEL-1 and Hb-PEL-2 that could encode for pectate lyase enzymes (EC4.2.2.2). From their sequence analysis Hb-PEL-1 and Hb-PEL-2 encode for proteins of 393 and 323 amino acids, respectively. Comparison of these deduced amino acid sequences with other pectate lyase enzymes showed they contained the conserved NADPH, Ca(2+) and substrate binding sites and had a 74% identity to Arabidopsis thaliana pectate lyase. Only the Hb-PEL-1 recombinant protein expressed from Escherichia coli had enzymic activity which was Ca(2+) dependent. Interestingly, Hb-PEL-1 contained an extra internal peptide between amino acid residue 38-108 when compared to Hb-PEL-2 and this peptide was also present in other pectate lyase enzymes. The transcript of pectate lyase (Hb-PEL) in the latex of rubber tree at various times after the first tapping was quantified by real-time PCR using 18s genes as internal standard. Most transcripts were detected on the first day after tapping and then decreased with time. This indicates that the pectate lyase may be involved in either the release of latex by breaking down the laticifer wall or in the development of laticifers.
Chotigeat
Wilaiwan
W
Center for Genomics and Bioinformatics Research, Faculty of Science, 15 Karnjanawanich Road, Prince of Songkla University, Hat Yai, Songkhla, 90110, Thailand. wilaiwan.c@psu.ac.th
Duangchu
Sarapee
S
Wititsuwannakun
Rapepun
R
Phongdara
Amornrat
A
D000595:Amino Acid Sequence; D003001:Cloning, Molecular; D018076:DNA, Complementary / Q000235:genetics; D028482:Hevea / Q000201:enzymology* / Q000235:genetics*; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction; D011133:Polysaccharide-Lyases / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D017386:Sequence Homology, Amino Acid
D018076:DNA, Complementary; D011133:Polysaccharide-Lyases; C023911:pectate lyase