Abstract:

Objective: This paper aims to study the alpha-pinene synthase gene and (-)-limonene synthase gene of Pinus armandi and their expression characteristics, and so as to provide theoretical support for revealing the terpene synthase gene in P. armandi to defense against Dendroctonus armandi and its blue-stain fungus Leptographium qinlingensis. Method: The cDNA sequences of alpha-pinene synthase gene and (-)-limonene synthase gene of P. armandi were cloned by PCR and race techniques, and the nucleotide sequence and amino acid sequence of the encoded protein were analyzed by bioinformatics method, and the phylogenetic tree was constructed. The expression differences of alpha-pinene synthase gene and (-)-limonene synthase gene were analyzed by real-time fluorescent quantitative PCR after being treated with MeJA and L. qinlingensis. Result: The full length of the coding region of alpha-pinene synthase gene was 1 887 bp. The protein was composed of 628 amino acids with a molecular weight of 71.59 kDa and the theoretical isoelectric point of 5.86. The full-length coding region of (-)-limonene synthase gene was 1 905 bp. The encoded protein was composed of 634 amino acids with a molecular weight of 73.01 kDa and the theoretical isoelectric point of 5.97. Real-time fluorescence quantitative PCR analysis showed that the expression of alpha-pinene synthase gene and (-)-limonene synthase gene was up-regulated at different time after MeJA treatment. The expression level of alpha-pinene synthase gene reached the peak on the second day, and that of (-)-limonene synthase gene reached the peak on the fourth day. Alpha-pinene synthase and (-)-limonene synthase gene was induced to express by L.qinlingensis, but the expression levels were significantly different at different time points after treatment with L. qinlingensis. The result showed that alpha-pinene synthase gene and (-)-limonene synthase gene were crucial genes in response to L. qinlingensis and MeJA treatments. Conclusion: The alpha-pinene synthase gene and (-)-limonene synthase gene of P. armandi are able to respond to the treatment of L. qinlingensis and MeJA, and play an important regulatory role in the defense of D. armandi and blue-stain fungus. This study provides a theoretical basis for further control of D. armandi.

Key words: Pinus armandi, terpene synthases, gene expression, MeJA, Leptographium qinlingensis