关键词: 舞毒蛾, 取食, 小黑杨, 肉桂酸-4-羟化酶, 4-香豆酸CoA连接酶, 机械损伤, 基因表达

Abstract:

Objective: Phenylalanine is a precursor of many plant compounds that are essential for plant reproduction, growth, development, and defense against different types of stress. Phenylalanine biosynthesis pathway is one of the most important secondary metabolic pathways in plants. In the phenylalanine biosynthesis pathway, the cinnamate 4-hydroxylase (C4H) and 4-coumarate CoA ligase (4CL) are involved in the core reaction of phenylpropane metabolism. Lymantria dispar is a major forest pest in the world. In this study, our objective is to explore the effects of Lymantria dispar herbivore on key enzymes in phenylalanine biosynthesis pathway of Populus simonii×P. nigra for inducing defense, and to provide genetic materials for promoting host resistance by genetic engineering method. Method: The full length of C4H and 4CL was cloned by RT-PCR technology and its characteristics were analyzed in this study. The phylogenetic trees of C4H and 4CL were constructed by Neighbor-joining method, respectively. The activities of C4H and 4CL were compared by using spectrophotometry under the treatment of Lymantria dispar herbivore and mechanical wounding. The expression of C4H and 4CL genes under the two wounding treatments was determined by real-time RT-PCR technology. Result: Two C4H genes (namely C4H-1 and C4H-2) belonging to p450 family and one 4CL from multi-domain protein superfamily (AFD_class_I) were cloned and identified from the transcriptome of Populus simonii×P. nigra. The open reading frames of the full-length genes were 1 518-1 740 bp, encoding 505-579 amino acid residues with the molecular mass of 58.00-63.03 kD and theoretical pI of 5.63-9.09. Phylogenetic analysis showed that the C4H-1 of Populus simonii×P. nigra was clustered into a group with that of P. alba. The C4H-2 of Populus simonii×P. nigra was clustered into a group with that of P. tomentosa, P. tremuloides, P. trichocarpa×P. deltoides and P. trichocarpa. The 4CL of Populus simonii×P. nigra is closely related to that of P. trichocarpa×P. deltoids and P. trichocarpa. The results showed that the L. dispar herbivore induced the increase of C4H-1, C4H-2 and 4CL gene expressions by 6.86-, 1.15- and 1.50- fold of the control, respectively. Moreover, mechanical wounding induced up-regulation of C4H-1 gene, however inhibited down-regulation of C4H-2 and 4CL genes with 1.80-, 0.71- and 0.60- fold of the control, respectively. Both L. dispar herbivore and mechanical wounding could induce activities of C4H and 4CL enzymes, and the induced activity of C4H and 4CL enzymes by herbivore was significantly higher than that of mechanical wounding. Conclusion: L. dispar herbivore can significantly induce the increase of C4H and 4CL activity in Populus simonii×P. nigra, which may be due to the increased expression of C4H-1, C4H-2 and 4CL genes, respectively. The above results indicate that L. dispar herbivore is involved in the induced defensive response of phenylalanine biosynthesis pathway in Populus simonii×P. nigra. The results provides a basic theory for further understanding the resistance mechanism and lignin biosynthesis to defend L. dispar herbivore in Populus simonii×P. nigra.

Key words: Lymantria dispar, herbivore, Populus simonii×P. nigra, cinnamate 4-hydroxylase, 4-coumarate CoA ligase, mechanical wounding, gene expression