Figure 1.
Procedure for construction of the novel T-vector.
(A) Two bridge fragments were amplified from the rice genome and were then cloned into pGEM-T Easy Vector (Promega). The two fragments, ligated together by SpeI, contain HindIII/AhdI in 5′-region and AhdI/NcoI sites in 3′ region. The resulted HindIII-NcoI fragment (AhdI fragment) was subcloned into the GFP-contaiing pCAMBIA 1300. (B) AhdI fragment and GFP containing pCAMBIA 1300. (C) The novel T-overhang vector was produced by digesting the vector in (B) with AhdI. The partial sequences with T-overhang (green letters) as cloned insert were shown above the digested vector. The start codon “ATG” of the reporter GFP gene was also highlighted with green color.
Figure 2.
Direct PCR cloning to generate a promoter::GFP cassette.
The figure shows the steps how to generate a promoter::GFP cassette for plant transgenesis. The pDsTGFP was digested by AdhI to produce T-overhang vector. The vector was then ligated with a PCR fragment purified from agarose gel. The ligation product was used for Ecoli transformation to generate the desired plasmid DNA for Agrobacterium-mediated transformation.
Figure 3.
Expression patterns of pollen-specific genes by two sets of microarray data.
The normalized microarray expression values in callus cells for the dataset GSE279881 and in seedlings for the dataset GSE17002 were set as controls, respectively. Their values were set as “1” and all other values were calculated by comparing with the control values. Then, all the values are log transformed (base 2 for simplicity) and the resulted log2 values were used for the heat mapping. Samples were labelled as below: 1, callus cells; 2, leaves; 3, roots; 4, uninucleate microspores; 5, bicellular pollens; 6, tricellular pollens; 7, mature pollen grains; 8, germinated pollen grains; I, seedlings; II, pollens at anthesis; III, sperms at anthesis. Red, black, and green colors indicated that transformed expression values were >0, = 0, and <0, respectively, in the matrix. The red stars indicate the genes with inconsistent expression patterns when compared with the values from the MPSS dataset.
Figure 4.
Expression heat map of pollen-specific genes by the RNA_Seq data.
In the RNA_Seq dataset, a total of 10 different developmental stages of tissues were collected for the expression analysis. These tissues were labelled as below: 1, shoots; 2, leaves-20 days; 3, pre-emergence inflorescence; 4, post-emergence inflorescence; 5, anther; 6, pistil; 7, seed-5 days after pollination (DAP); 8, seed-10DAP; 9, embryo-25DAP; 10, endosperm-25 DAP. The normalized expression values were directly used to generate heat map. The values “0” indicated that no expression signal was detected for these genes in the corresponding tissues. The larger the expression values are, the stronger the genes show their expression. The prefix “LOC_” in locus names is omitted for convenience in this figure.
Figure 5.
Detail analysis in the expression patterns of the gene LOC_Os10g35930.
(A) The expression profile of the gene shown by the MPSS dataset. Samples were labeled as below: 1, 3-day old germinating seeds; 2, 10 days of germinating seedlings; 3, 14-day old young leaves; 4, 14day old young roots; 5, 60-day old mature leaves; 6, 60-day old mature roots; 7, 60-day old stems; 8, immature panicles; 9, mature pollens; 10, merismatic tissues; 11, ovary and mature stigmas; 12, rice developing seeds. (B) The gene showed mature inflorescence specific expression according to the microarray dataset with the GEO accession number GSE6893. Samples were labelled as below: 1, 7-day old roots; 2, mature leaves; 3, young leaves; 4, shoot apical meristem; 5, 0–3 cm inflorescence; 6, 3–5 cm inflorescence; 7, 5–10 inflorescence; 8, 10–15 cm inflorescence; 9, 15–22 cm inflorescence; 10, 22–30 cm inflorescence; 11, 0–2 day old seeds, 12, 3–4 day old seeds; 13, 5–10 day old seeds; 14, 11–20 day old seeds; 15, 21–29 day old seeds. (C) The gene showed the highest expression at mature pollens by the microarray dataset with the GEO accession number GSE17002. Samples were labelled as below: 1, seedling; 2, pollens at anthesis; 3, sperms at anthesis. (D) The gene showed the highest expression level at mature and germinated pollen grains. Samples were labelled as below: 1, callus cells; 2, leaves; 3, roots; 4, uninucleate microspores; 5, bicellular pollens; 6, tricellular pollens; 7, mature pollen grains; 8, germinated pollen grains. (E) The gene showed the anther-specific expression by the RNA_Seq data. The samples were labelled as below: 1, shoots; 2, 20 day old leaves; 3, pre-emergence inflorescence; 4, post-emergence inflorescence; 5, anthers; 6, pistil; 7, 5 day old seeds; 8, 10 day old seeds; 9, 25 day old seeds; 10, , 25 day old endosperms. (F) The gene showed the highest expression at the booting and flowering panicles by qRT-PCR. Samples were labelled as below: 1, two-week old leaves; 2, two-month old leaves; 3, two-week old roots; 4, two-month old roots; 5, 0–5 cm long panicles; 6, 5–10 cm long panicles; 7, more than 10 cm long panicles; 8, booting panicles; 9, flowering panicles; 10, milky seeds; and 11, mature seeds.
Figure 6.
The GFP expression activities in various tissues shown by transgenic rice carrying the promoter::GFP construct.
The 1,492 bp of promoter from the upstream of start codon of the gene LOC_Os10g35930 was used to drive the expression of the reporter GFP gene. (A) No GFP signal in WT (top) and transgenic (bottom) roots. (B) No GFP expression in WT (top) and transgenic (bottom) leaves. (C) No GFP signal in WT (left) and transgenic (right) stems. (D) No GFP expression in WT (top) and transgenic (bottom) young panicles with less than 3 cm length. (E) No GFP expression in WT (top) and transgenic (bottom) young florets. (F) No GFP expression in WT (left) and transgenic (right) young anthers at the uninucleate microspore stage. (G) No GFP expression in WT (left) and transgenic (right) young anthers at the bicellular pollen stage. (H) No GFP expression in WT (left) and strong GFP signal in transgenic anthers (right) at the mature pollen stage. Bars = 1 mm.
Figure 7.
The GFP expression activites at various stages of pollens shown by transgenic rice carrying the promoter::GFP construct.
(A) No GFP expression in WT (left) and transgenic (right) pollens at the uninucleate microspore stage. (B) No GFP expression in WT (left) and strong GFP expression in transgenic (right) tricellular pollens. (C) Enlarged images showing the transgenic mature pollens under visible light (left) and GFP signals under epifluorescence (right). (D) The left image shows mature pollen carrying the promoter::GFP cassette under visible light. The middle image shows GFP signal in the same pollen under epifluorescence and the right image shows DAPI staining at 3 nuclei in the pollen under UV light. (E) Germinated pollens under visible light (left) and their GFP expression level under epifluorescence (right). GFP signal was only observed in the mature pollen and no GFP signal was detected in a germinated pollen and its tube. Arrows indicate the germinated pollen tubes. Bars = 50 µm.