Trichome isolation with and without fixation using laser microdissection and pressure catapulting followed by RNA amplification: Expression of genes of terpene metabolism in apical and sub-apical trichome cells of Artemisia annua L.

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• 作者：Linda Olofsson ; Anneli Lundgren ; Peter E. Brodelius ; ^{peter.brodelius@lnu.se}
• 关键词：ADS ; amorpha-4 ; 11-diene synthase ; ALDH1 ; aldehyde dehydrogenase 1 ; CPR ; cytochrome P450 reductase ; CPS ; caryophyllene synthase ; CYP71AV1 ; amorphadiene-12-hydroxylase ; DBR2 ; artemisinic aldehyde ¦¤11(13) reductase ; DXR ; 1-deoxy-d-xylulose 5-phosphate reduc
• 刊名：Plant Science
• 出版年：2012
• 出版时间：February 2012
• 年：2012
• 卷：183
• 期：Complete
• 页码：9-13
• 全文大小：178 K

文摘

The aim of this project was to evaluate the effect of fixation on plant material prior to Laser Microdissection and Pressure Catapulting (LMPC) and to identify an appropriate method for preserving good RNA quality after cell isolation. Therefore, flower buds from Artemisia annua L. were exposed to either the fixative formaldehyde or a non-fixative buffer prior to cell isolation by LMPC. Proteinase K was used after cell isolation from fixed plant tissue, in an attempt to improve the RNA yield. The ability to detect gene expression using real-time quantitative PCR with or without previous amplification of RNA from cells isolated by LMPC was also evaluated. Conclusively, we describe a new technique, without fixation, enabling complete isolation of intact glandular secretory trichomes and specific single trichome cells of A. annua. This method is based on LMPC and preserves good RNA quality for subsequent RNA expression studies of both whole trichomes, apical and sub-apical cells from trichomes of A. annua. Using this method, expression of genes of terpene metabolism was studied by real-time quantitative PCR. Expression of genes involved in artemisinin biosynthesis was observed in both apical and sub-apical cells.