A method for examining expression of homologous genes in plant polyploids

Song, Keming; Osborn, Thomas C.

doi:10.1007/bf00040689

Cited by 14 publications

(4 citation statements)

References 25 publications

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“…For northern blot analyses, the RNAs (10 pg) were separated by formaldehyde agarose gel electrophoresis (Maniatis et al, 1982) and blotted onto Hybond-N+ membranes (Amersham). For the expression analysis of the two homologous genes, RT-PCR with RFLP was carried out as described (Song and Osborn, 1994). Primer sequences are 5'-GATATGATGGCGGAGGCAAAG-3' and 5 ' -AGAGTTAATTAAGCACAGGGCTTC-3'.…”

Section: Differential Gene Expression Analysismentioning

confidence: 99%

Fruit-Specific Expression of a Defensin-Type Gene Family in Bell Pepper (Upregulation during Ripening and upon Wounding)

et al. 1996

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We have isolated a 454-bp cDNA that encodes a nove1 fruit-specific defensin from bell pepper (Capsicum annuum). The encoded 75-amino-acid polypeptide contains an N-terminal domain characteristic of a signal peptide and a 48-amino-acid mature domain named 11. The mature protein, from which the N-terminal amino acid sequence was determined, contains eight cysteines that form four intramolecular disulfide bridges, suggesting a monomeric form for 11. In healthy fruits J1 is undetectable at the green stage but high levels accumulate during ripening. In wound areas of the green fruit the accumulation of 11 dramatically increased, suggesting a role for Jl in the plant's defense response. Moreover, we have demonstrated that 11 possesses an antifungal activity. We have isolated and characterized the corresponding two homologous genes (jl-1 and jl-2) that exist in the hell pepper genome. Both genes are interrupted hy the insertion, at the same position, of one intron of 853 bp for j7-7 and 4900 bp for j7-2. Northern blot and reverse transcriptase-polymerase chain reaction and restriction fragment length polymorphism analyses revealed that jl-7 transcripts are present only in fruits, only in trace amounts in mature green fruits, and that they accumulate to high levels in fully ripe fruits, whereas no jl-2 transcripts were detected in the samples monitored.

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Section: Differential Gene Expression Analysismentioning

confidence: 99%

Fruit-Specific Expression of a Defensin-Type Gene Family in Bell Pepper (Upregulation during Ripening and upon Wounding)

et al. 1996

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“…This technique has been used to gain remarkable insight into the global frequency of silencing in synthetic (9,10) and natural (11) polyploids; nevertheless, the method is not easily adapted to evaluate the expression status of a specific gene pair. Finally, the presence/ absence of specific homoeologous pairs has been evaluated using RFLP analysis of RT-PCR products [RT-PCR/RFLP (12)]; however, this approach is only semi-quantitative (13), and it reveals nonidentical transcripts only when they differ by a restriction site.…”

Section: Introductionmentioning

confidence: 99%

Quantitative Analysis of Transcript Accumulation from Genes Duplicated by Polyploidy Using cDNASSCP

Cronn

Adams

2003

BioTechniques

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Repeated rounds of polyploidy have been commonplace in the lineages leading to modern eukaryotic genomes, giving rise to widespread gene duplication. Genes duplicated by polyploidy, or homoeologs, may continue to be expressed at equal levels following polyploidization or their expression may be dramatically altered. In this report, we describe how SSCP analysis of RT-PCR products can be used to evaluate the expression status (presence and relative quantity) of highly similar homoeologous gene pairs from an allotetraploid genome. This cDNA-SSCP approach was used to evaluate transcript abundance in synthetic tetraploid mRNA pools (i.e., mixtures of diploid products) and three natural homoeologous gene pairs expressed in tetraploid cotton (Gossypium hirsutum) ovules. Results from replicated tests show that cDNA-SSCP reliably separates duplicated transcripts with 99% sequence identity. Most significantly, the method yields quantitative estimates of transcript ratios in template pools that range from equimolar to approximately 100:1.

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“…In Brassica , synthetic amphidiploids have been synthesized and characterized at the morphological, cytological and molecular levels and comparisons to the natural counterparts have suggested their role in the evolution of amphidiploidy (Song et al, 1993). This may be due to changes either in expression patterns of homologous genes or at the whole genome level caused by interactions between the cytoplasmic and nuclear genomes (Song et al, 1993;Song and Osborn, 1994). Chromosomal rearrangements also cause rapid divergence of the genomes of artificial polyploids from their progenitors (Song et al, 1995).…”

Section: Polyploidymentioning

confidence: 99%