Molecular cloning of a thaumatin-like protein gene from Pyrus pyrifolia and overexpression of this gene in tobacco increased resistance to pathogenic fungi

摘要

Thaumatin-like proteins (TLPs) belong to the pathogenesis-related proteins 5 family, and members of TLP gene family accumulate in plants in response to pathogen infection or environmental stress. The biological functions of plant TLPs are believed to be diverse, and they are involved in host defense against both the biotic and abiotic stresses as well as the other physiological processes. Lots of TLPs were well studied as antifungal proteins in vivo or in vitro. To isolate TLP genes involved in self-defense during fruit development, a novel gene PpTLP was isolated from the fruit of Pyrus pyrifolia Nakai cv Huobali which is a kind of local sand pear with high resistance to biotic and abiotic stresses in Yunnan province of China. The nucleotide sequence of PpTLP was highly homologous with TLPs from other plant species, and the protein encoded by PpTLP has 16 conserved cysteine residues and 5 amino acid residues related with antifungal activity of plant TLPs, an arginine residue, a glutamic acid residue, and 3 aspartic acid residues. What is more, the putative protein PpTLP has the highly conserved main-chain conformation and the folding patterns as other plant TLPs with antifungal activity. PpTLP was abundantly expressed in pericarp and leaf of P. pyrifolia Nakai cv Huobali. Furthermore, in order to verify the function of PpTLP, the constitutive plant expression vector of PpTLP was constructed and transferred into tobacco (Nicotiana tabacum L. cv Xanthi). The Southern blot and quantitative reverse transcription-polymerase chain reaction (QRT-PCR) analyses demonstrated that the PpTLP gene was integrated into the genome of the tobacco transformants and highly expressed in the transgenic lines. The antifungal activity of PpTLP was detected in vitro plates, and the crude protein extract of transgenic tobacco plants inhibited the hyphal growth of Sclerotinia sclerotiorum, Phomopsis sp., Phytophthora parasitica var. nicotianae, and Alternaria sp. in different degrees.