MicroRNA in Developing and Germinating Arabidopsis thaliana Seeds Jing Sun Mentor: Dr. Hiro Nonogaki.

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Presentation transcript:

MicroRNA in Developing and Germinating Arabidopsis thaliana Seeds Jing Sun Mentor: Dr. Hiro Nonogaki

Importance of Seeds Environment Protection of the embryo Dispersal of offspring Agriculture and food

Project Goal To examine the involvement of microRNA in gene expression in Arabidopsis thaliana seeds and siliques

What are MicroRNAs? Discovered in 1993 by Dr. Victor Ambros and colleagues Originally for viral studies Involved in posttranscriptional gene regulation Photo by Amanda Weatherman

How are MiRNAs Produced? Single Stranded RNA Double Stranded Hairpin Loop Pri-microRNA MicroRNA Pre-microRNA

mRNA cleavage MiRNA Function in Gene Regulation DNARNAProtein TranslationTranscription

mRNA cleavage MiRNA Function in Gene Regulation DNARNAProtein TranslationTranscription

Biological Function of MiRNA Developmental control Environmental response Homeostasis

Methodology Overview Screening Extraction, Purification, Separation Detection & Identification GUS Expression Functional Analysis

RNA Extraction Phenol-SDS Extraction Method Isopropanol Fractionation Low Molecular Weight RNA (5S RNA, tRNA, siRNA, microRNA) High Molecular Weight RNA (18S RNA, 28S RNA, rRNA, mRNA)

RNA Purification & Separation ppt1 ppt (kb) 28SrRNA 18SrRNA SmallRNAs 0% 20-50% ppt1 ppt (kb) 28SrRNA 18SrRNA SmallRNAs 0% 20-50%

RNA Probe Detection TargetmiRNA Antisenseprobe Digoxigeninlabel C A G C C A A G G A U G A C U U G C C G A GUC G GU C UA CUG A A C G G CU D D *** * Extra bases Anti – DIG Ab AlkalinePhosphatase AP

Microarrays But not yet commercially available for microRNA Examines the whole genome Seed/SeedArray.htm

Membrane Probe solution (170µl) miR162 miR161 miR172 miR171miR170 miR168 Screening of Expressed miRNAs

MicroRNAs Expressed Developing siliques Micro RNA

Relative Expression (%) Germination Relative Expression (%) Germination microRNA MicroRNAs Expressed

Target MicroRNAs Developing Silique MicroRNA Relative Intensity miR156: SPL 13 miR157: SPL 5 miR160: ARF miR393: TIR/F-BOX miR396: GRF

When Target Gene Expressed Reverse Transcription Polymerase Chain Reaction (RT-PCR) 6 hr 12 hr 18 hr 24 hr Northern Blotting 6 hr12 hr18 hr24 hr

Where Gene Expressed Selection Marker Antibiotic resistance Kanamycin Reporter Gene GUS Target Gene Promoter

Localization GUS Expression – vascular bundles GUS Expression – stem, flower, silique

Functional Analysis Mutate microRNA binding site Observe phenotype (Palatnik et al., 2003; Nature 425: )

MicroRNA Resistant Target Wild Type Control

MicroRNA Resistant Target Wild Type

MicroRNA Resistant Target

Summary Screened for microRNAs in developing and germinating seeds GUS expression target genes Characterizing mutations target genes 100 Target gene expression

Acknowledgements Dr. Hiro Nonogaki Howard Hughes Medical Institute Dr. Kevin Ahern Dr. Chris Mathews Ernest and Pauline Jaworski Summer Research Grant Dr. Jim Carrington Tanja Homrichhausen Po-Pu (Eric) Liu Jessica Hewitt Nobuya Koizuka Ruth Martin

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