Application of laser capture microdissection to analyze specific cell type gene expression in Norway spruce

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Title: Application of laser capture microdissection to analyze specific cell type gene expression in Norway spruce
Author: Zhao, Lei
Contributor: University of Helsinki, Faculty of Agriculture and Forestry, Department of Agricultural Sciences
Publisher: Helsingfors universitet
Date: 2016
Language: eng
Thesis level: master's thesis
Discipline: Bioteknik (MAAT)
Biotechnology (MAAT)
Biotekniikka (MAAT)
Abstract: In Norway spruce (Picea abies L. Karst.) lignin forms a major part of the xylem cell walls and renders the tracheids water resistant while giving support to the cell walls and the whole tree trunk. In Norway spruce lignin is polymerized mainly from coniferyl alcohol but the origins of this monolignol are not known. In our study, we employed laser capture microdissection (LCMD) system to isolate ray parenchyma cells and xylem tracheids from thin (30-40 μm), tangential cryomicrotome sections of developing xylem of the spruce trunk. We wanted to analyse the monolignol biosynthesis pathway gene expression separately in these cell types. Our aim was to examine the possibility that coniferyl alcohol is produced in the tracheid cells, or whether also the neighboring cells (ray cells) contribute to the biosynthesis of monolignols during the lignification as has been confirmed in angiosperms before. Total RNA extracted from the collected material was used to perform low mRNA input sequencing on the Illumina HiSeq platform to identify transcripts potentially involved in monolignol biosynthesis and secretion. As a control material we used whole cryomicrotome sections containing both xylem tracheids and rays. Deep sequencing was performed to generate quantitative expression data within a particular cell type. Differential gene expression was conducted with a Chipster analysis software by using DESeq2. Altogether, 936 genes were differentially expressed between whole sections and ray cells, and 424 transcripts were more abundant in ray cells, while 512 transcripts were less abundant. Comparison between whole sections and xylem tracheids illustrated that 18 transcripts were more abundant in xylem tracheids and 275 transcripts were less abundant. Our study demonstrated the usefulness of LCMD combined with RNA-Seq to characterize gene expression in specific cell types.
Subject: differential gene expression
laser capture microdissection (LCMD)
monolignol biosynthesis pathway
Norway spruce
ray cells
xylem tracheids

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