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Identification and molecular characterization of tissue-preferred rice genes and their upstream regularly sequences on a genome-wide level.

Jiang SY, Vanitha J, Bai Y, Ramachandran S - BMC Plant Biol. (2014)

Bottom Line: Interestingly, the LOC_Os10g34360 URS was found to be vascular bundle preferred and its activities were restricted only to vascular bundles of leaves, roots and florets.By genome-wide analysis, we have identified tissue-preferred URSs, five of which were further characterized using transgenic plants harboring URS::GUS constructs.These data might provide some evidence for possible functions of the genes and be a valuable resource for tissue-preferred candidate URSs for plant transgenesis.

View Article: PubMed Central - PubMed

ABSTRACT

Background: Gene upstream regularly sequences (URSs) can be used as one of the tools to annotate the biological functions of corresponding genes. In addition, tissue-preferred URSs are frequently used to drive the transgene expression exclusively in targeted tissues during plant transgenesis. Although many rice URSs have been molecularly characterized, it is still necessary and valuable to identify URSs that will benefit plant transformation and aid in analyzing gene function.

Results: In this study, we identified and characterized root-, seed-, leaf-, and panicle-preferred genes on a genome-wide level in rice. Subsequently, their expression patterns were confirmed through quantitative real-time RT-PCR (qRT-PCR) by randomly selecting 9candidate tissue-preferred genes. In addition, 5 tissue-preferred URSs were characterized by investigating the URS::GUS transgenic plants. Of these URS::GUS analyses, the transgenic plants harboring LOC_Os03g11350 URS::GUS construct showed the GUS activity only in young pollen. In contrast, when LOC_Os10g22450 URS was used to drive the reporter GUS gene, the GUS activity was detected only in mature pollen. Interestingly, the LOC_Os10g34360 URS was found to be vascular bundle preferred and its activities were restricted only to vascular bundles of leaves, roots and florets. In addition, we have also identified two URSs from genes LOC_Os02G15090 and LOC_Os06g31070 expressed in a seed-preferred manner showing the highest expression levels of GUS activities in mature seeds.

Conclusion: By genome-wide analysis, we have identified tissue-preferred URSs, five of which were further characterized using transgenic plants harboring URS::GUS constructs. These data might provide some evidence for possible functions of the genes and be a valuable resource for tissue-preferred candidate URSs for plant transgenesis.

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GUS activities in the URS::GUStransgenic plants for the gene with locus nameLOC_Os03g11350. (A) Different stages of rice florets/seeds. (B) Enlarged rice florets. (C) Enlarged rice young anthers. (D) Pollen at the uni-nucleate stage. In (A) to (D), left and right images were from WT and the transgenic plants, respectively. Bars: 1 mm in (A) to (C) and 50 μm in (D).
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Fig3: GUS activities in the URS::GUStransgenic plants for the gene with locus nameLOC_Os03g11350. (A) Different stages of rice florets/seeds. (B) Enlarged rice florets. (C) Enlarged rice young anthers. (D) Pollen at the uni-nucleate stage. In (A) to (D), left and right images were from WT and the transgenic plants, respectively. Bars: 1 mm in (A) to (C) and 50 μm in (D).

Mentions: Our data from qRT-PCR analysis showed that the gene LOC_Os03g11350 was mainly expressed at the early stage of panicle development (Figure 2D). To further investigate the expression patterns at the cellular level, we generated the URS::GUS (encoding β-glucuronidase) transgenic plants. For each gene, around 2 Kb URS region upstream of start codon of the gene was used for URS motif searches and primer selection. For the gene LOC_Os03g11350, the 1,805 bp URS fragment was amplified from the rice genome using the primers listed in the Additional file 6. The fragment was subsequently cloned upstream of the reporter GUS gene. Following the cloning, this construct was transformed into the rice genome by Agrobacterium-mediated transformation. The investigation on the URS::GUS plants showed that no GUS activity was observed in leaves or roots or any other non-reproductive tissues. The GUS activity was detected only at the early stage of panicle development (Figure 3A). Further investigation showed that the GUS activity was limited only to the anthers but not in the floret husks (Figure 3B and C). The GUS-stained anthers were then squeezed with a forceps and pollen was subjected to further observation under microscope. The result showed that the activity of the URS was restricted to young pollen at the uninucleate stage (Figure 3D, data not shown in the other stages). The qRT-PCR was carried out to analyze expression abundance of the GUS reporter gene and the result confirmed that the gene LOC_Os03g11350 was mainly expressed in 0–5 cm long immature panicles (Additional file 7A).Figure 3


Identification and molecular characterization of tissue-preferred rice genes and their upstream regularly sequences on a genome-wide level.

Jiang SY, Vanitha J, Bai Y, Ramachandran S - BMC Plant Biol. (2014)

GUS activities in the URS::GUStransgenic plants for the gene with locus nameLOC_Os03g11350. (A) Different stages of rice florets/seeds. (B) Enlarged rice florets. (C) Enlarged rice young anthers. (D) Pollen at the uni-nucleate stage. In (A) to (D), left and right images were from WT and the transgenic plants, respectively. Bars: 1 mm in (A) to (C) and 50 μm in (D).
© Copyright Policy - open-access
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC4248441&req=5

Fig3: GUS activities in the URS::GUStransgenic plants for the gene with locus nameLOC_Os03g11350. (A) Different stages of rice florets/seeds. (B) Enlarged rice florets. (C) Enlarged rice young anthers. (D) Pollen at the uni-nucleate stage. In (A) to (D), left and right images were from WT and the transgenic plants, respectively. Bars: 1 mm in (A) to (C) and 50 μm in (D).
Mentions: Our data from qRT-PCR analysis showed that the gene LOC_Os03g11350 was mainly expressed at the early stage of panicle development (Figure 2D). To further investigate the expression patterns at the cellular level, we generated the URS::GUS (encoding β-glucuronidase) transgenic plants. For each gene, around 2 Kb URS region upstream of start codon of the gene was used for URS motif searches and primer selection. For the gene LOC_Os03g11350, the 1,805 bp URS fragment was amplified from the rice genome using the primers listed in the Additional file 6. The fragment was subsequently cloned upstream of the reporter GUS gene. Following the cloning, this construct was transformed into the rice genome by Agrobacterium-mediated transformation. The investigation on the URS::GUS plants showed that no GUS activity was observed in leaves or roots or any other non-reproductive tissues. The GUS activity was detected only at the early stage of panicle development (Figure 3A). Further investigation showed that the GUS activity was limited only to the anthers but not in the floret husks (Figure 3B and C). The GUS-stained anthers were then squeezed with a forceps and pollen was subjected to further observation under microscope. The result showed that the activity of the URS was restricted to young pollen at the uninucleate stage (Figure 3D, data not shown in the other stages). The qRT-PCR was carried out to analyze expression abundance of the GUS reporter gene and the result confirmed that the gene LOC_Os03g11350 was mainly expressed in 0–5 cm long immature panicles (Additional file 7A).Figure 3

Bottom Line: Interestingly, the LOC_Os10g34360 URS was found to be vascular bundle preferred and its activities were restricted only to vascular bundles of leaves, roots and florets.By genome-wide analysis, we have identified tissue-preferred URSs, five of which were further characterized using transgenic plants harboring URS::GUS constructs.These data might provide some evidence for possible functions of the genes and be a valuable resource for tissue-preferred candidate URSs for plant transgenesis.

View Article: PubMed Central - PubMed

ABSTRACT

Background: Gene upstream regularly sequences (URSs) can be used as one of the tools to annotate the biological functions of corresponding genes. In addition, tissue-preferred URSs are frequently used to drive the transgene expression exclusively in targeted tissues during plant transgenesis. Although many rice URSs have been molecularly characterized, it is still necessary and valuable to identify URSs that will benefit plant transformation and aid in analyzing gene function.

Results: In this study, we identified and characterized root-, seed-, leaf-, and panicle-preferred genes on a genome-wide level in rice. Subsequently, their expression patterns were confirmed through quantitative real-time RT-PCR (qRT-PCR) by randomly selecting 9candidate tissue-preferred genes. In addition, 5 tissue-preferred URSs were characterized by investigating the URS::GUS transgenic plants. Of these URS::GUS analyses, the transgenic plants harboring LOC_Os03g11350 URS::GUS construct showed the GUS activity only in young pollen. In contrast, when LOC_Os10g22450 URS was used to drive the reporter GUS gene, the GUS activity was detected only in mature pollen. Interestingly, the LOC_Os10g34360 URS was found to be vascular bundle preferred and its activities were restricted only to vascular bundles of leaves, roots and florets. In addition, we have also identified two URSs from genes LOC_Os02G15090 and LOC_Os06g31070 expressed in a seed-preferred manner showing the highest expression levels of GUS activities in mature seeds.

Conclusion: By genome-wide analysis, we have identified tissue-preferred URSs, five of which were further characterized using transgenic plants harboring URS::GUS constructs. These data might provide some evidence for possible functions of the genes and be a valuable resource for tissue-preferred candidate URSs for plant transgenesis.

Show MeSH
Related in: MedlinePlus