Effect of ploidy increase on transgene expression: example from Citrus diploid cybrid and allotetraploid somatic hybrid expressing the EGFP gene.
Identifieur interne : 000C18 ( Ncbi/Checkpoint ); précédent : 000C17; suivant : 000C19Effect of ploidy increase on transgene expression: example from Citrus diploid cybrid and allotetraploid somatic hybrid expressing the EGFP gene.
Auteurs : Shi-Xiao Xu [République populaire de Chine] ; Xiao-Dong Cai ; Bin Tan ; Ding-Li Li ; Wen-Wu GuoSource :
- Protoplasma [ 1615-6102 ] ; 2011.
English descriptors
- KwdEn :
- MESH :
- chemical , biosynthesis : Green Fluorescent Proteins.
- genetics : Citrus, Citrus sinensis, Green Fluorescent Proteins, Plants, Genetically Modified.
- Diploidy, Gene Expression, Hybridization, Genetic, Tetraploidy, Transgenes.
Abstract
Polyploidization is an important speciation mechanism for all eukaryotes, and it has profound impacts on biodiversity dynamics and ecosystem functioning. Green fluorescent protein (GFP) has been used as an effective marker to visually screen somatic hybrids at an early stage in protoplast fusion. We have previously reported that the intensity of GFP fluorescence of regenerated embryoids was also an early indicator of ploidy level. However, little is known concerning the effects of ploidy increase on the GFP expression in citrus somatic hybrids at the plant level. Herein, allotetraploid and diploid cybrid plants with enhanced GFP (EGFP) expression were regenerated from the fusion of embryogenic callus protoplasts from 'Murcott' tangor (Citrus reticulata Blanco × Citrus sinensis (L.) Osbeck) and mesophyll protoplasts from transgenic 'Valencia' orange (C. sinensis (L.) Osbeck) expressing the EGFP gene, via electrofusion. Subsequent simple sequence repeat (SSR), chloroplast simple sequence repeat and cleaved amplified polymorphic sequence analysis revealed that the two regenerated tetraploid plants were true allotetraploid somatic hybrids possessing nuclear genomic DNA of both parents and cytoplasmic DNA from the callus parent, while the five regenerated diploid plants were cybrids containing nuclear DNA of the leaf parent and with complex segregation of cytoplasmic DNA. Furthermore, EGFP expression was compared in cells and protoplasts from mature leaves of these diploid cybrids and allotetraploid somatic hybrids. Results showed that the intensity of GFP fluorescence per cell or protoplast in diploid was generally brighter than in allotetraploid. Moreover, same hybridization signal was detected on allotetraploid and diploid plants by Southern blot analysis. By real-time RT-PCR and Western blot analysis, GFP expression level of the diploid cybrid was revealed significantly higher than that of the allotetraploid somatic hybrid. These results suggest that ploidy level conversion can affect transgene expression and citrus diploid cybrid and allotetraploid somatic hybrid represents another example of gene regulation coupled to ploidy.
DOI: 10.1007/s00709-010-0200-2
PubMed: 20734092
Affiliations:
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<term>Green Fluorescent Proteins (biosynthesis)</term>
<term>Green Fluorescent Proteins (genetics)</term>
<term>Hybridization, Genetic</term>
<term>Plants, Genetically Modified (genetics)</term>
<term>Tetraploidy</term>
<term>Transgenes</term>
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<term>Citrus sinensis</term>
<term>Green Fluorescent Proteins</term>
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<keywords scheme="MESH" xml:lang="en"><term>Diploidy</term>
<term>Gene Expression</term>
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<front><div type="abstract" xml:lang="en">Polyploidization is an important speciation mechanism for all eukaryotes, and it has profound impacts on biodiversity dynamics and ecosystem functioning. Green fluorescent protein (GFP) has been used as an effective marker to visually screen somatic hybrids at an early stage in protoplast fusion. We have previously reported that the intensity of GFP fluorescence of regenerated embryoids was also an early indicator of ploidy level. However, little is known concerning the effects of ploidy increase on the GFP expression in citrus somatic hybrids at the plant level. Herein, allotetraploid and diploid cybrid plants with enhanced GFP (EGFP) expression were regenerated from the fusion of embryogenic callus protoplasts from 'Murcott' tangor (Citrus reticulata Blanco × Citrus sinensis (L.) Osbeck) and mesophyll protoplasts from transgenic 'Valencia' orange (C. sinensis (L.) Osbeck) expressing the EGFP gene, via electrofusion. Subsequent simple sequence repeat (SSR), chloroplast simple sequence repeat and cleaved amplified polymorphic sequence analysis revealed that the two regenerated tetraploid plants were true allotetraploid somatic hybrids possessing nuclear genomic DNA of both parents and cytoplasmic DNA from the callus parent, while the five regenerated diploid plants were cybrids containing nuclear DNA of the leaf parent and with complex segregation of cytoplasmic DNA. Furthermore, EGFP expression was compared in cells and protoplasts from mature leaves of these diploid cybrids and allotetraploid somatic hybrids. Results showed that the intensity of GFP fluorescence per cell or protoplast in diploid was generally brighter than in allotetraploid. Moreover, same hybridization signal was detected on allotetraploid and diploid plants by Southern blot analysis. By real-time RT-PCR and Western blot analysis, GFP expression level of the diploid cybrid was revealed significantly higher than that of the allotetraploid somatic hybrid. These results suggest that ploidy level conversion can affect transgene expression and citrus diploid cybrid and allotetraploid somatic hybrid represents another example of gene regulation coupled to ploidy.</div>
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<name sortKey="Guo, Wen Wu" sort="Guo, Wen Wu" uniqKey="Guo W" first="Wen-Wu" last="Guo">Wen-Wu Guo</name>
<name sortKey="Li, Ding Li" sort="Li, Ding Li" uniqKey="Li D" first="Ding-Li" last="Li">Ding-Li Li</name>
<name sortKey="Tan, Bin" sort="Tan, Bin" uniqKey="Tan B" first="Bin" last="Tan">Bin Tan</name>
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<country name="République populaire de Chine"><noRegion><name sortKey="Xu, Shi Xiao" sort="Xu, Shi Xiao" uniqKey="Xu S" first="Shi-Xiao" last="Xu">Shi-Xiao Xu</name>
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