紫苏茉莉酸羟基甲基转移酶基因PfJMT的克隆及表达分析

Cloning and Expression Analysis of Jasmonic Acid Carboxyl Methyltransferase Gene from Perilla frutescens

【目的】茉莉酸羟基甲基转移酶(jasmonic acid carboxyl methyltransferase,JMT)是植物茉莉酸甲酯生物合成的关键酶,通过克隆紫苏JMT,并研究其在不同胁迫下和种子不同发育时期的表达模式,为研究JMT在植物防御和种子发育中的作用提供理论依据。【方法】根据紫苏种子转录组测序结果设计引物,从紫苏中克隆得到紫苏茉莉酸羟基甲基转移酶基因的DNA和cDNA序列,命名为PfJMT,通过生物信息学分析该基因的结构、稳定性、亲水性、亚细胞定位及保守结构域等,利用系统进化树分析PfJMT和其他物种JMT蛋白的进化关系。取开花期的紫苏根、茎、叶、花等组织用于组织特异性表达分析。取开花后5、10、15、20、25和30 d的种子用于JMT在种子不同发育时期表达模式的研究。用25μmol·L^(-1)茉莉酸甲酯(methyl jasmonate,MeJA)和1 mmol·L^(-1)水杨酸(salicylic acid,SA)喷洒处理具有4片真叶的紫苏幼苗并浇灌根部,分别于处理0、2、4、8、16、24和48 h后取样,研究JMT在不同胁迫下的表达模式。【结果】PfJMT的ORF长1 050 bp,编码349个氨基酸。生物信息学分析表明,PfJMT为不稳定的亲水蛋白,亚细胞定位于细胞质,含有一个甲基转移酶-7(Methyltransf-7)保守结构域。通过与其他物种的JMT蛋白多序列比对发现,紫苏JMT和丹参JMT的序列一致度最高,为80.5%,和水稻的序列一致度最低,为36.8%。在对多种不同植物基于JMT蛋白构建系统进化树的分析中发现紫苏和拟南芥、丹参等双子叶植物亲缘关系较近,而与建兰、水稻等单子叶植物亲缘关系较远,说明JMT在单子叶和双子叶植物的进化过程中可能存在较大的差异。荧光定量PCR结果表明,PfJMT在紫苏根和茎中的相对表达量最低,叶和花中的相对表达量比根和茎中略高,在开花后5d的种子中的表达量最高,并且随着种子的发育表达逐渐下调,说明JMT在种子发育中起着重要作用。在使用外源MeJA和SA处理的紫苏根、叶组织中,PfJMT表达均显著下调,这一结果支持JMT可能不直接参与防御反应,而是通过调节JA水平间接参与植物防御的理论。【结论】成功克隆获得PfJMT,随着种子的发育PfJMT表达量逐渐下调,并在外源MeJA、SA胁迫下表达量显著下调。

【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 was80.5%, 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.