Accurate and high resolution in situ hybridization analysis of gene expression in secondary stem tissues.
Identifieur interne : 004904 ( Main/Corpus ); précédent : 004903; suivant : 004905Accurate and high resolution in situ hybridization analysis of gene expression in secondary stem tissues.
Auteurs : S. Regan ; V. Bourquin ; H. Tuominen ; B. SundbergSource :
- The Plant journal : for cell and molecular biology [ 0960-7412 ] ; 1999.
English descriptors
- KwdEn :
- Gene Expression (MeSH), Genes, Plant (MeSH), Glucuronidase (genetics), In Situ Hybridization (methods), Molecular Sequence Data (MeSH), Plant Stems (metabolism), Plants (genetics), Plants (metabolism), Plants, Genetically Modified (MeSH), RNA, Messenger (genetics), RNA, Messenger (metabolism), RNA, Plant (genetics), RNA, Plant (metabolism), Rhizobium (genetics), Transformation, Genetic (MeSH), beta-Glucosidase (genetics).
- MESH :
- chemical , genetics : Glucuronidase, RNA, Messenger, RNA, Plant, beta-Glucosidase.
- genetics : Plants, Rhizobium.
- metabolism : Plant Stems, Plants, RNA, Messenger, RNA, Plant.
- methods : In Situ Hybridization.
- Gene Expression, Genes, Plant, Molecular Sequence Data, Plants, Genetically Modified, Transformation, Genetic.
Abstract
Accurate in situ hybridization analysis in secondary stem tissues of plants has been hindered by specific characteristics of these tissues. First, secondary cell walls non-specifically bind probes used for in situ hybridization thus preventing gene expression analysis in the lignified regions of the stem, such as the xylem. Second, the mRNA in the cambial meristem and its recent derivatives are prone to inadequate fixation when conventional techniques are used. Here we describe an in situ hybridization technique which uses fast freezing and freeze substitution to cryoimmobilize the mRNA followed by embedding in a methacrylate resin for high-resolution analysis of gene expression. By using a transgenic poplar line harbouring rolC:uidA, rolC:iaaM, the gene expression pattern could be compared with histochemical GUS staining. This in situ hybridization technique results in superior preservation of cellular contents, retention of mRNA in all cell types in the poplar stem, a significant reduction of non-specific binding to secondary cell walls and a resolution not previously possible in secondary tissues. This technique will be particularly valuable for the expression analysis of genes involved in xylogenesis and wood formation.
DOI: 10.1046/j.1365-313x.1999.00536.x
PubMed: 10476083
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pubmed:10476083Le document en format XML
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<author><name sortKey="Regan, S" sort="Regan, S" uniqKey="Regan S" first="S" last="Regan">S. Regan</name>
<affiliation><nlm:affiliation>Department of Forest Genetics and Plant Physiology, Swedish University of Agricultural Sciences, Umeå.</nlm:affiliation>
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<author><name sortKey="Bourquin, V" sort="Bourquin, V" uniqKey="Bourquin V" first="V" last="Bourquin">V. Bourquin</name>
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<author><name sortKey="Tuominen, H" sort="Tuominen, H" uniqKey="Tuominen H" first="H" last="Tuominen">H. Tuominen</name>
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<author><name sortKey="Sundberg, B" sort="Sundberg, B" uniqKey="Sundberg B" first="B" last="Sundberg">B. Sundberg</name>
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<author><name sortKey="Regan, S" sort="Regan, S" uniqKey="Regan S" first="S" last="Regan">S. Regan</name>
<affiliation><nlm:affiliation>Department of Forest Genetics and Plant Physiology, Swedish University of Agricultural Sciences, Umeå.</nlm:affiliation>
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<profileDesc><textClass><keywords scheme="KwdEn" xml:lang="en"><term>Gene Expression (MeSH)</term>
<term>Genes, Plant (MeSH)</term>
<term>Glucuronidase (genetics)</term>
<term>In Situ Hybridization (methods)</term>
<term>Molecular Sequence Data (MeSH)</term>
<term>Plant Stems (metabolism)</term>
<term>Plants (genetics)</term>
<term>Plants (metabolism)</term>
<term>Plants, Genetically Modified (MeSH)</term>
<term>RNA, Messenger (genetics)</term>
<term>RNA, Messenger (metabolism)</term>
<term>RNA, Plant (genetics)</term>
<term>RNA, Plant (metabolism)</term>
<term>Rhizobium (genetics)</term>
<term>Transformation, Genetic (MeSH)</term>
<term>beta-Glucosidase (genetics)</term>
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<term>RNA, Messenger</term>
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<term>beta-Glucosidase</term>
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<term>Rhizobium</term>
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<term>Plants</term>
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<keywords scheme="MESH" xml:lang="en"><term>Gene Expression</term>
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<front><div type="abstract" xml:lang="en">Accurate in situ hybridization analysis in secondary stem tissues of plants has been hindered by specific characteristics of these tissues. First, secondary cell walls non-specifically bind probes used for in situ hybridization thus preventing gene expression analysis in the lignified regions of the stem, such as the xylem. Second, the mRNA in the cambial meristem and its recent derivatives are prone to inadequate fixation when conventional techniques are used. Here we describe an in situ hybridization technique which uses fast freezing and freeze substitution to cryoimmobilize the mRNA followed by embedding in a methacrylate resin for high-resolution analysis of gene expression. By using a transgenic poplar line harbouring rolC:uidA, rolC:iaaM, the gene expression pattern could be compared with histochemical GUS staining. This in situ hybridization technique results in superior preservation of cellular contents, retention of mRNA in all cell types in the poplar stem, a significant reduction of non-specific binding to secondary cell walls and a resolution not previously possible in secondary tissues. This technique will be particularly valuable for the expression analysis of genes involved in xylogenesis and wood formation.</div>
</front>
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<Abstract><AbstractText>Accurate in situ hybridization analysis in secondary stem tissues of plants has been hindered by specific characteristics of these tissues. First, secondary cell walls non-specifically bind probes used for in situ hybridization thus preventing gene expression analysis in the lignified regions of the stem, such as the xylem. Second, the mRNA in the cambial meristem and its recent derivatives are prone to inadequate fixation when conventional techniques are used. Here we describe an in situ hybridization technique which uses fast freezing and freeze substitution to cryoimmobilize the mRNA followed by embedding in a methacrylate resin for high-resolution analysis of gene expression. By using a transgenic poplar line harbouring rolC:uidA, rolC:iaaM, the gene expression pattern could be compared with histochemical GUS staining. This in situ hybridization technique results in superior preservation of cellular contents, retention of mRNA in all cell types in the poplar stem, a significant reduction of non-specific binding to secondary cell walls and a resolution not previously possible in secondary tissues. This technique will be particularly valuable for the expression analysis of genes involved in xylogenesis and wood formation.</AbstractText>
</Abstract>
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