Cloning, sequence analysis, and prokaryotic expression of cDNA encoding a putative non-specific lipid-transfer protein from the bracts of dovetree (Davidia involucrata Baill)

Abstract

The 967-bp cloneP1A5 was isolated from a suppression subtractive hybridization cDNA library of dovetree bracts (Davidia involucrata Baill.). A complete cDNA of 1047 bp was obtained via 5-RACE (5′Rapid Amplification of cDNA End) techniques, using the gene-specific primer P1A5-1. Northern blot analyses showed that the gene was predominantly expressed in the bracts, while the blotting signal from the leaves was weak. Its deduced amino acid sequence was most highly homologous to the lipid-transfer protein 3 precursor isolated from upland cotton, the lipid-transfer protein SDi-9 from the common sunflower, and the non-specific lipid-transfer protein precursor allergen from sweet cherry. It also had features in common with plant nsLTPs (non-specific lipid-transfer proteins), including eight conserved cysteine residues, a high isoelectric point (8.9), and a lack of tryptophans. The deduced amino acid sequence had two transmembrane helices -- the first from Position 5 (Gly) to Position 35 (Val); the second, from Position 28 (Ala) to Position 46 (Leu). A cleavage site for the putative signal peptide was predicted to occur between Positions 28 (Ala) and 29 (Ala). Therefore, the putative mature form of the protein would comprise 92 amino acids, with a molecular weight of 9.2 kD. All these results provide compelling evidence that theP1A5 clone belongs to the nsLTP1 gene family, thus being named theP1A5 putative nsLTPI gene. This is the first nsLTP gene reported from Davidiaceae.