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Localisation of abundant and organ-specific genes expressed in Rosa hybrida leaves and flower buds by direct in situ RT-PCR.

ABSTRACT: In situ PCR is a technique that allows specific nucleic acid sequences to be detected in individual cells and tissues. In situ PCR and IS-RT-PCR are elegant techniques that can increase both sensitivity and throughput, but they are, at best, only semiquantitative; therefore, it is desirable first to ascertain the expression pattern by conventional means to establish the suitable conditions for each probe. In plants, in situ RT-PCR is widely used in the expression localisation of specific genes, including MADS-box and other function-specific genes or housekeeping genes in floral buds and other organs. This method is especially useful in small organs or during early developmental stages when the separation of particular parts is impossible. In this paper, we compared three different labelling and immunodetection methods by using in situ RT-PCR in Rosa hybrida flower buds and leaves. As target genes, we used the abundant ?-actin and RhFUL gene, which is expressed only in the leaves and petals/sepals of flower buds. We used digoxygenin-11-dUTP, biotin-11-dUTP, and fluorescein-12-dUTP-labelled nucleotides and antidig-AP/ streptavidin-fluorescein-labelled antibodies. All of the used methods gave strong, specific signal and all of them may be used in localization of gene expression on tissue level in rose organs.

SUBMITTER: Jedrzejuk A

PROVIDER: S-EPMC3354552 | biostudies-literature | 2012

REPOSITORIES: biostudies-literature

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Localisation of abundant and organ-specific genes expressed in Rosa hybrida leaves and flower buds by direct in situ RT-PCR.

Jedrzejuk Agata A Mibus Heiko H Serek Margrethe M

TheScientificWorldJournal 20120501

In situ PCR is a technique that allows specific nucleic acid sequences to be detected in individual cells and tissues. In situ PCR and IS-RT-PCR are elegant techniques that can increase both sensitivity and throughput, but they are, at best, only semiquantitative; therefore, it is desirable first to ascertain the expression pattern by conventional means to establish the suitable conditions for each probe. In plants, in situ RT-PCR is widely used in the expression localisation of specific genes, ...[more]

PMID: 22629162

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Project description:BackgroundThere has been increased consumption of blueberries in recent years fueled in part because of their many recognized health benefits. Blueberry fruit is very high in anthocyanins, which have been linked to improved night vision, prevention of macular degeneration, anti-cancer activity, and reduced risk of heart disease. Very few genomic resources have been available for blueberry, however. Further development of genomic resources like expressed sequence tags (ESTs), molecular markers, and genetic linkage maps could lead to more rapid genetic improvement. Marker-assisted selection could be used to combine traits for climatic adaptation with fruit and nutritional quality traits.ResultsEfforts to sequence the transcriptome of the commercial highbush blueberry (Vaccinium corymbosum) cultivar Bluecrop and use the sequences to identify genes associated with cold acclimation and fruit development and develop SSR markers for mapping studies are presented here. Transcriptome sequences were generated from blueberry fruit at different stages of development, flower buds at different stages of cold acclimation, and leaves by next-generation Roche 454 sequencing. Over 600,000 reads were assembled into approximately 15,000 contigs and 124,000 singletons. The assembled sequences were annotated and functionally mapped to Gene Ontology (GO) terms. Frequency of the most abundant sequences in each of the libraries was compared across all libraries to identify genes that are potentially differentially expressed during cold acclimation and fruit development. Real-time PCR was performed to confirm their differential expression patterns. Overall, 14 out of 17 of the genes examined had differential expression patterns similar to what was predicted from their reads alone. The assembled sequences were also mined for SSRs. From these sequences, 15,886 blueberry EST-SSR loci were identified. Primers were designed from 7,705 of the SSR-containing sequences with adequate flanking sequence. One hundred primer pairs were tested for amplification and polymorphism among parents of two blueberry populations currently being used for genetic linkage map construction. The tetraploid mapping population was based on a cross between the highbush cultivars Draper and Jewel (V. darrowii is also in the background of 'Jewel'). The diploid mapping population was based on a cross between an F1 hybrid of V. darrowii and diploid V. corymbosum and another diploid V. corymbosum. The overall amplification rate of the SSR primers was 68% and the polymorphism rate was 43%.ConclusionsThese results indicate that this large collection of 454 ESTs will be a valuable resource for identifying genes that are potentially differentially expressed and play important roles in flower bud development, cold acclimation, chilling unit accumulation, and fruit development in blueberry and related species. In addition, the ESTs have already proved useful for the development of SSR and EST-PCR markers, and are currently being used for construction of genetic linkage maps in blueberry.

Project description:Chestnut (Castanea mollisima) is an important woody food crop, but its yield has been low in cultivation, mainly due to the problems of fewer female flowers and more male flowers. Therefore, regulating the transition of chestnut flowers and effectively balancing the proportion of male and female to improve the yield are key factor to be solved in production. In this study, the chestnut floral buds in pre- and post-winter were used as materials. The data of metabolites, hormones, and gene expression during flower bud differentiation of chestnut were analyzed by transcriptomics and metabolomics to preliminarily reveal the possible reason of male and female flower bud transformation in pre- and post-winter. The analysis of Differentially Expressed Genes (DEGs) showed that there were 6323 DEGs in the Complete mixed flower bud (CMF) group in pre- and post-winter, of which 3448 genes were up-regulated and 2875 genes were down-regulated. There were 8037 DEGs in the Incomplete mixed flower bud (IMF) in pre- and post-winter, of which 4546 genes were up-regulated and 3491 genes were down-regulated. A total of 726 genes from the two flower buds were enriched into 251 Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways in post winter, of which plant hormone signal transduction accounted for 4.13%. The analysis results of differential metabolites showed that the differential metabolites of the two flower buds were mainly concentrated in the secondary metabolic synthesis pathway. The difference of hormone content showed that the content of Gibberellin 9 (GA9) and GA19 in CMF was higher than that in IMF in pre-winter, but the opposite in post-winter. Methyl jasmonate (MeJA) content was only very high in CMF in pre-winter, while Jasmonoyl-(l)-Isoleucine (JA-ILE) showed high content in CMF in post-winter. In post-winter, higher concentration of JA-ILE was positively correlated with the expression of Flowering Locus T (CmFT), and CmFT gene was significantly positively correlated with the expression levels of MYC2-1, MYC2-2 and LFY 3 (LEAFY 3). The higher concentration of JA-ILE was negatively correlated with the transcription level of JAZ1-3. In vitro experiments further verified that Jasmonate-Zim 1–3 (JAZ 1–3) combined with MYC2-1 inhibited the transcription of CmFT gene, while MYC2-1 alone promoted the expression of FT. The results suggested that a higher concentration of GA is conducive to breaking the dormancy of flower buds and promoting the development of male flower buds, while a lower concentration of GA and a higher concentration of JA-ILE are conducive to the differentiation and formation of female flower buds in post-winter, in which JAZ1-3 and MYC2-1 play a key role in the differentiation of female flower buds of chestnut.

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Localisation of abundant and organ-specific genes expressed in Rosa hybrida leaves and flower buds by direct in situ RT-PCR.

Publications

Localisation of abundant and organ-specific genes expressed in Rosa hybrida leaves and flower buds by direct in situ RT-PCR.

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