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Title:	Functional analysis of selected micro RNAS targeting terpenoid biosynthesis in Arabidopsis Thaliana
Authors:	Muhammad Sajad (P80698)
Supervisor:	Ismanizan Ismail, Prof. Dr.
Keywords:	Small interfering RNA Terpenes Arabidopsis thaliana Universiti Kebangsaan Malaysia -- Dissertations Dissertations, Academic -- Malaysia
Issue Date:	19-Jun-2019
Description:	MicroRNAs (miRNAs) are small non-coding RNAs and regulate gene expression at the posttranscriptional level by suppressing the target transcripts (mRNA) through cleavage or translational inhibition. Terpenoids are secondary metabolites (SMs) with high value for agriculture, pharmaceutical and food industry. A little is known about miRNAs in regulating the synthesis of terpenoids. In-silico approaches were used to predict miRNAs and their target transcripts involved in the regulation of terpenoids biosynthesis. To achieve this, 427 mature miRNAs were retrieved from miRBase (Version 21), and target transcripts involved in terpenoids biosynthesis were mined from various research articles, books and databases. The transcript sequences were retrieved from The Arabidopsis Information Resource (TAIR) and The National Centre for Biotechnology Information (NCBI). Plant small RNA target (psRNATarget) analysis server was used and 16 different miRNAs were predicted with their target transcripts possibly involved in the regulation of terpenoids biosynthesis. The selection was further narrowed down, and finally, three miRNAs; miR826b, miR2937 and miR854e, were selected based on the importance of their target transcripts, DXS (1-Deoxy-d-xylulose 5-Phosphate Synthase 1; DXPS1), GGPS2 (Geranylgeranyl pyrophosphate synthase 2) and TPS13 (Terpenoid Synthase 13), respectively. Three transgenic Arabidopsis lines, overexpressing above-selected miRNAs, were developed using miRNA overexpression vector, pMDC32BAtMIR390a-B/c. Around ten homozygous lines (T3 generation) of each construct were screened using hygromycin (50 μg/ml) antibiotic selection, followed by genomic PCR, with amplicon size of 597 bps and nucleotide sequence analysis. Relative expression analysis of overexpressed miRNAs and their target transcripts were carried out using RT-qPCR. In transgenic plants as compared with the WT plants, there observed 1.52, 1.51 and1.35 fold up regulations for miR826b, miR2937 and miR854e, respectively. Whereas, 1.42, 2.63 and 1.25 folds down-regulation were observed in the target transcripts; DXS, GGPS2 and TPS13, respectively. GC-MS chromatography analysis was carried out to check the change in abundance for isoprenoids in the transgenic plants as compared to WT plants. In transgenic plants overexpressing miR826b, there observed a decrease in abundance of phytol and trans-beta-lonone by 1.12 and 0.31 folds, respectively. In transgenic plants overexpressing miR2937, a 8.6 folds decrease in abundance phytol whereas 0.28 folds decrease in abundance of heneicosane as compared with WT plants. The results for plants over-expressing miR854e showed that an increased in abundance of phytol, heneicosane and caryophyllene by 4.6, 0.23 and 1.31 folds, respectively, in transgenic as compared to WT plants. Interestingly, in all three transgenic lines; over-expressing miR826b,miR2937 and miR854e, respectively, a change in abundance of phytol was observed. Since, phytol is an essential component of chlorophyll, this study suggested that that all the studied miRNAs may contribute in plant normal growth and development.,Ph.D.
Pages:	135
Call Number:	QP623.5.S63M837 2019 tesis
Publisher:	UKM, Bangi
Appears in Collections:	Institute of Systems Biology / Institut Biologi Sistem (INBIOSIS)

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