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https://ptsldigital.ukm.my/jspui/handle/123456789/499439| Title: | Regulatory sequences involved in lignin biosynthetic pathway in acacia mangium |
| Authors: | Ong Seong Siang (P48446) |
| Supervisor: | Wickneswari Ratnam, Prof. Dr. |
| Keywords: | Regulatory Pathway Acacia mangium Biosynthesis |
| Issue Date: | 11-Jan-2012 |
| Description: | Several studies in model species have suggested that non-coding RNAs play a pivotal role in regulating the entire monolignol biosynthetic pathway. Small RNA profiling is an important tool for the identification of a complete set of differentially expressed small RNAs in secondary xylem. From two small RNA libraries generated for Acacia mangium, about 10 million sequence reads were obtained in secondary xylem of Am48 with high lignin content (41%) and a corresponding 14 million sequence reads were obtained in secondary xylem of Am54 with low lignin content (21%) using Illumina GAII sequencing platform. Our results suggested that A. mangium small RNAs are composed of a set of 12 highly conserved miRNA families found in the plant miRNA database, 82 novel miRNAs and a large proportion of non-conserved small RNAs with low expression levels. The predicted target genes of these conserved and non-conserved miRNAs include transcription factors associated with regulation of the lignin biosynthetic pathway genes. Two to nine fold difference in small RNA counts between secondary xylem tissues with contrasting lignin content suggested that a cascade of miRNAs play an independent and additive role in regulating the lignin biosynthetic pathway in Acacia species. Semi-qPCR in leaf, phloem, xylem, xylem stress compression wood and xylem stress tension wood showed that only nine amgmiRNA families are detected across three different genotypes of four year old A. mangium. Subsequent analysis using RT-qPCR across six different genotypes of two and four year olds strongly suggested that six highly conserved amg-miRNA families (amg-miR166, amg-miR172, amg-miR168, amg-miR394, amg-miR156 and amgmiR159) were important regulators in monolignol biosynthetic pathway as revealed from their expression levels in xylem stress compression wood and xylem stress tension wood. Nine different targets of amg-miR166 were isolated using Rapid Amplification of cDNA Ends (RACE). Blast analysis of the assembled sequences showed that two of our HD-ZIP III genes belong to ATHB15, one belongs to each ATHB8 and REVOLUTA groups while the remaining are novel sequences. Among all these highly conserved amg-miRNA families, only amg-miR166 was 1000 fold more expressed in leaf and phloem tissues compared to xylem tissue. Amg-miR166 exhibited negative correlation with the nine different characterized HD-ZIP ΙΙΙ mRNA verifying their role in lignin regulation. However, only two HD-ZIP III isoforms showed the presence of the TAATNATTA-like sequences which act as binding site for regulation of the monolignol pathway genes. RT-qPCR analysis showed that C4H, CCoAOMT and CAD genes were upregulated three fold in compression wood as compared to tension wood. In silico analysis using psRNATarget Analysis Server revealed that a single miRNA can regulate the stability of several hundreds of transcripts. Subsequent experimental validation using nucleotide substitution assay indicated that transcription factor silencing are controlled not only by the 5' but also 3' end of the miRNA. Nucleotides within position 11 to 18 were critical for pairing with the target mRNA. We found that precursor structure of amg-miR166 undergoes exhaustive sequence variation even within members of the same family. Our findings have revealed the potential role of miRNAs in regulating the monolignol biosynthetic pathway which can be utilised in the development of gene constructs for silencing of specific genes with minimal effect on plant fitness and viability.,Ph.D |
| Pages: | 165 |
| Call Number: | QP517.B57.O569 2012 |
| Publisher: | UKM, Bangi |
| Appears in Collections: | Faculty of Science and Technology / Fakulti Sains dan Teknologi |
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| File | Description | Size | Format | |
|---|---|---|---|---|
| ukmvital_74631+Source01+Source010.PDF Restricted Access | 3.31 MB | Adobe PDF |  View/Open |
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