关键词: 紫苏, 茉莉酸羟基甲基转移酶, 茉莉酸甲酯, 种子发育, 荧光定量PCR

Abstract:

【Objective】Jasmonic acid carboxyl methyltransferase (JMT) is a key enzyme in the biosynthesis of methyl jasmonate. By cloning the Perilla frutescens JMT and studying its expression patterns under different stresses and different developmental stages of seeds, it provides a theoretical basis for studying the role of JMT in plant defense and seed development.【Method】Primers were designed based on the transcriptome sequencing results of Perilla seeds, and the DNA and cDNA sequences of the JMT were cloned from Perilla, named PfJMT. Analysis of the structure, stability, hydrophilicity, subcellular localization and conserved domain of PfJMT by bioinformatics methods. Analysis of the evolutionary relationship between PfJMT and other species JMT proteins using phylogenetic tree. Tissues such as perilla roots, stems, leaves, and flowers in flowering stage were used for tissue-specific expression analysis. Seeds at 5, 10, 15, 20, 25, and 30 days after flowering were used for the study of JMT expression patterns in different developmental stages of seeds. The perilla seedlings with 4 true leaves were sprayed with 25 μmol·L ^-1 methyl jasmonate (MeJA) and 1 mmol·L ^-1 salicylic acid (SA) and the roots were watered, perilla root and leaf tissues from different individuals were taken after 0, 2, 4, 8, 16, 24, and 48 hours of treatment to study the expression pattern of JMT under different stresses.【Result】PfJMT has an ORF of 1 050 bp and encodes 349 amino acids. Bioinformatics analysis showed that PfJMT is an unstable hydrophilic protein, which is located in the cytoplasm and contains a Methyltransf-7 conserved domain. Compared with the JMT protein sequence of other species, the sequence identity of Perilla JMT and Salvia miltiorrhiza JMT was the highest, which was 80.5%, and the sequence with Oryza sativa Japonica Group was the lowest, which was 36.8%. In the analysis of phylogenetic tree based on JMT protein construction system in many different plants, it was found that the dicotyledonous plants such as Perilla and Arabidopsis thaliana and Salvia miltiorrhiza were closely related, but the relationship with the monocotyledons such as Cymbidium ensifolium and Oryza sativa Japonica Group was far. This indicates that JMT may have large differences in the evolution of monocotyledonous and dicotyledonous plants. The results of real-time PCR showed that the relative expression of PfJMT in roots and stems of Perilla was the lowest, leaves and flowers was slightly higher, 5 days after flowering was the highest, and it was gradually down-regulated with the development of seeds. This indicates that JMT plays an important role in seed development. PfJMT expression was significantly down-regulated in Perilla root and leaf tissues treated with MeJA and SA. This result supports the theory that JMT may not directly participate in defense responses, but indirectly participate in plant defense by regulating JA levels.【Conclusion】PfJMT was successfully cloned, and the expression of PfJMT was gradually down-regulated with the development of seeds, and the expression level was significantly down-regulated under exogenous MeJA and SA stress.

Key words: Perilla frutescens, JMT, MeJA, seed development, real-time PCR