Effective methods for practical application of gene expression analysis in field-grown rice roots
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Background and aims
Gene expression analysis under field conditions improves our understanding of crop responses to fluctuating environmental conditions. We evaluated RNA extraction methods and reference genes for a quantitative real-time PCR study of rice roots grown under field conditions.
Roots were collected from paddy (Andosol) at different growth stages, from tillering to ripening. RNA was extracted by three methods using commercially available RNA-extraction kits, and RNA yield and quality were compared.
Our RNA extraction method enabled us to obtain adequate concentration and yield of RNA from the roots during a broad period of growth. Adequate RNA concentration was successfully extracted from the field-grown rice roots using large quantities of tissue samples and by purifying the samples prior to the extraction. Among the seven candidate reference genes, TATA-binding protein coding gene (TBP2) was observed to be highly stable across all the root samples and growth stages, in real-time PCR assays. Root gene expression analysis of water channel- and nitrogen uptake-related proteins clearly indicated dynamic changes (with respect to diurnal and growth stages) in their expression levels and their associations with meteorological factors. Some aquaporin genes such as OsPIP1;3, OsPIP2;4, and OsPIP2;5, showed clear diurnal changes. Among them, the expression pattern of OsPIP2;5 paralleled the potential evaporation (Ep) and leaf stomatal conductance on a sunny day. The gene expression analysis clearly showed diverse expression patterns; the gene expression levels of many aquaporins decreased after panicle formation stage, whereas the abundance of some genes such as OsNIP2;1 and OsAMT1;3 increased at the later growth stage.
Our method will be useful for evaluating the variations of gene expression in the roots of field-grown rice under fluctuating environmental condition.
KeywordsGene expression analysis Rice (Oryza sativa L.) RNA extraction Reference gene Root
Cyclin domain containing protein
Days after transplanting
Eukaryotic elongation factor 1-alpha
Combination of commercially available RNA extraction kits, Fruit-mate for RNA Purification, RNAiso Plus, and RNeasy Plant Mini Kit
NOD26-like intrinsic protein
Plasma membrane intrinsic protein
RNeasy Plant Mini Kit
Tonoplast intrinsic protein
Ubiquitin-conjugating enzyme E2
This research was financially supported in part by a Grant-in-Aid from a Japanese Society for the Promotion of Science (JSPS) fellowship awarded to M.M, and by JSPS KAKENHI Grant to M.MH (No. JP16H04865) and T. K. (Nos. JP25292152, JP17H03896). We are grateful to Toshikazu Kawakata for providing meteorological data and Eisaku Kumagai, Fumihiko Saito, Nozomi Yokota, and Shin Fukui for their technical assistance. We also thank Kensaku Suzuki, Kaoru Zenbayashi, and Atsushi Nagasaka for their valuable comments on this study.
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