Abstract:

By using rapid amplification of cDNA end ( RACE ) technigue, a cDNA was isolated from the mutation with short staminate inflorescence of Castanea mollissima and was designated as lipid transfer protein (LTP) gene. This cDNA, 660 bp, encoded a 118-amino acid polypeptide with 8 cysteine residues conserved, and a 26-amino acid signal peptide. The deduced polypeptide sequence has submitted to GenBank, the accession No. of gene and protein sequence are AY574218 and AAS79106, respectively. It has 63% similarity to the LTP in cotton and strawberry. Quantitative fluorescence analysis showed the expression level of LTP in short male inflorescence of chestnut was higher than that in normal sample. The gene encoding chestnut LTP was inserted into the ThioFusion^TM expression vector pET32a (+) and constructed the prokaryotic expression vector of pET-LTP. After induced by IPTG for 5 hours, an approximately 30 ku fusion protein was expressed abundantly in the host strain of Rosetta-gami^TM2(DE3). The recombinant LTP protein purified with Ni²⁺-chelating Sepharose Fast Flow column, revealed its microbial inhibition effect on the spore germination of chestnut pathogen Fusarium.

Key words: Castanea mollissima, lipid transfer protein (LTP), real-time quantitative RT-PCR, resistance function