Research On Defense Functions Of Cytochrome P450 Gene CYP720B19 And Terpenoids In Chinses White Pine(Pinus Armandi Fr.)

The Chinese white pine (Pinus armandi Fr.) is native coniferous species in China, and distributed in the Qinling and Bashan mid-mountains zone. P. armandi not only rapid growth, quality timber, strong natural regeneration ability but also plays an important ecological role in the Qingling and Bashan forest area. A symbiotic blue-stain fungus (Leptographium qinlingensis Tang and Chen) carried by the Chinese white pine beetle (Dendroctonus armandi Tsai et Li) are the causal organism for the colossal death of the healthy P. armandi over the age of 30 years. Although, the biology and ecology of P. armandi has been widely investigated, but Chinese white pine beetle and symbiotic harm effect on P450 gene (CYP720B19) and terpenoid metabolism in the stem phloem and xylem in P. armandi saplings expression are no related research at all Chinese at home and abroad. Therefore, in this study, we have the close relation between terpenoid synthesis and metabolism and resist diseases and insect pest damage. Cytochrome P450 enzymes are the basis of the key factors on conifers chemical resistance mechanism. We investigated transcript levels of one P450 gene (CYP720B19) and terpenoid metabolism in the stem phloem and xylem in P. armandi saplings after treatment with inoculated-pathogen or MeJa, to reveal Chinese white pine bark beetle and symbiotic fungi overcome the host P. armandi of resistance mechanisms. The main results were as follows:(1) We have isolated one P450 gene from P. armandi and determined that it belongs to the CYP720B sub-family. The sequence was submitted to the P450 nomenclature committee and names assigned as CYP720B19. The full-length cDNA of CYP720B19 contains 1,467 bp and encoding 488 amino acids which has high similarities with Abietadienol/abietadienal oxidase. Differential expression of the CYP720B19 gene was significantly different between inoculation with L. qinlingensis, mechanical wounding, and MeJa treatment and Tween treatment. The CYP720B19 gene of was over-expressed significantly in the stem after the treatment with inoculated-pathogen or MeJa compared with the control saplings. Increased expression of the CYP720B19 gene after treatment suggested that it was involved in P. armandi chemical defence against inoculation of L. qinlingensis, wounding by MeJa and mechanical wounding.(2) We determined the effects of the blue-stain fungus L. qinlingensis on monoterpenes and sesquiterpenes in the phloem and xylem of the stem of P. armandi saplings. We found that the total monoterpene concentrations in the phloem and xylem and the total sesquiterpene concentrations in the xylem were significantly higher in L. qinlingensis-inoculated saplings than in control (mechanically wounded) saplings or untreated saplings. Additionally, the proportions of β-pinene in the xylem and limonene+β-phellandrene in the phloem and xylem were significantly higher in L. qinlingensis-inoculated saplings than in both control and untreated saplings. The proportions of individual sesquiterpenes in the phloem and xylem of the stem were significantly only greater in L. qinlingensis-inoculated saplings than in untreated saplings. Based on the results, we suggest that increases in total monoterpene and sesquiterpene concentrations, as well as increases in the concentrations of P-pinene and limonene+β-phellandrene, may play an important defensive role against pathogenic fungi such as blue-stain fungus L. qinlingensis inoculation.(3) We investigated the change of monoterpenes in the stem phloem and xylem in P. armandi saplings after treatment using treatment (MeJa+Tween-20 and Tween-20 as control) and untreated. GC-MS analysis found that the total monoterpene concentrations in the pholem and xylem in MeJa-treated saplings increased significantly compared with control or untreated saplings. Additionally, the proportions of P-pinene in the phloem, β-myrcene in the xylem, and limonene+β-phellandrene in both phloem and xylem of MeJa-treated saplings were increased significantly compared with control or untreated saplings. Based on our results, we suggest that by increasing the total concentration of monoterpene and the concentrations of β-myrcene, P-pinene and limonene+β-phellandrene maybe play an important defensive role against bark beetle harm in P. armandi defense.