Identification of microRNAs from Medicinal Plant Murraya koenigii by High-Throughput Sequencing and Their Functional Implications in Secondary Metabolite Biosynthesis

Abstract

MicroRNAs (miRNAs) are small noncoding RNA molecules that play crucial post-transcriptional regulatory roles in plants, including development and stress-response signaling. However, information about their involvement in secondary metabolism is still limited. Murraya koenigii is a popular medicinal plant, better known as curry leaves, that possesses pharmaceutically active secondary metabolites. The present study utilized high-throughput sequencing technology to investigate the miRNA profile of M. koenigii and their association with secondary metabolite biosynthesis. A total of 343,505 unique reads with lengths ranging from 16 to 40 nt were obtained from the sequencing data, among which 142 miRNAs were identified as conserved and 7 as novel miRNAs. Moreover, 6078 corresponding potential target genes of M. koenigii miRNAs were recognized in this study. Interestingly, several conserved and novel miRNAs of M. koenigii were found to target key enzymes of the terpenoid backbone and the flavonoid biosynthesis pathways. Furthermore, to validate the sequencing results, the relative expression of eight randomly selected miRNAs was determined by qPCR. To the best of our knowledge, this is the first report of the M. koenigii miRNA profile that may provide useful information for further elucidation of the involvement of miRNAs in secondary metabolism. These findings might be crucial in the future to generate artificial-miRNA-based, genetically engineered M. koenigii plants for the overproduction of medicinally highly valuable secondary metabolites.

Keywords: Illumina sequencing; Murraya koenigii; gene regulation; medicinal plant; microRNA (miRNA); secondary metabolites.

Figure 1

Length distribution and abundance of the small RNA sequences identified in M. koenigii leaves through Illumina sequencing.

Figure 2

Family members and their respective reads within each miRNA family of M. koenigii.

Figure 3

Secondary structures (stem-loops) of M. koenigii novel miRNA precursors. Mature miRNAs are highlighted in red font.

Figure 4

Biological Process categories of the GO analysis of the potential target genes of both conserved (a) and novel (b) miRNAs from M. koenigii.

Figure 5

MFE-based network interaction of M. koenigii miRNAs and their corresponding potential targets. Coregulation of multiple targets was observed for several of the identified miRNAs (weaker energy interactions were marked as dotted lines).

Figure 6

Target enzymes of conserved miRNAs of M. koenigii in the terpenoid backbone biosynthesis pathway. EC:2.2.1.7—1-deoxy-D-xylulose-5-phosphate synthase; EC:2.3.1.9—acetyl-CoA C-acetyltransferase; EC:4.1.1.33—diphosphomevalonate decarboxylase; EC:2.1.1.100—protein-S-isoprenylcysteine O-methyltransferase; EC:2.5.1.1—geranyl diphosphate synthase; EC:2.3.3.10—hydroxymethylglutaryl-CoA synthase; EC:2.7.1.36—mevalonate kinase. The blue boxes represent the targeted enzymes of the corresponding known miRNAs.

Figure 7

Target enzymes of conserved miRNAs of M. koenigii in the flavonoid biosynthesis pathway. EC:2.3.1.74—chalcone synthase; EC:5.5.1.6—chalcone isomerase; EC:2.1.1.104—caffeoyl-CoA O-methyltransferase; EC:2.3.1.133—shikimate O-hydroxycinnamoyltransferase; EC:2.4.1.357—phlorizin synthase. The blue boxes represent the targeted enzymes of the corresponding known miRNAs.

Figure 8

Target enzymes of the novel miRNAs of M. koenigii in the terpenoid backbone biosynthesis pathway. EC:4.2.3.27—isoprene synthase; EC:2.7.1.148—4-diphosphocytidyl-2-C-methyl-D-erythritol kinase; EC:2.3.3.10—hydroxymethylglutaryl-CoA synthase. The red boxes represent the targeted enzymes of the corresponding novel miRNAs.

Figure 9

Quantitative PCR analysis of conserved and novel miRNAs of M. koenigii. The relative expression (Log10) of 4 conserved and 4 novel miRNAs was detected by qPCR. U6 was used as an endogenous control. The expression level of miR156a-5p was set as control and taken as 1, and the expression level in all other miRNAs was quantified relative to it. The analysis was performed as triplicates, and the error bars indicate standard deviations. Significant differences between miRNAs at p < 0.05 by ANOVA and Tukey’s test are indicated with different letters.