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. 2020 Dec 11:11:602680.
doi: 10.3389/fpls.2020.602680. eCollection 2020.

Targeted Transgene Expression in Rice Using a Callus Strong Promoter for Selectable Marker Gene Control

Jie Zhou  1 , Dongyue Li  1 , Chao Zheng  2 , Rumeng Xu  3 , Ersong Zheng  2 , Yong Yang  1 , Yang Chen  1 , Chulang Yu  4 , Chengqi Yan  5 , Jianping Chen  4 , Xuming Wang  1
Affiliations

Targeted Transgene Expression in Rice Using a Callus Strong Promoter for Selectable Marker Gene Control

Jie Zhou et al. Front Plant Sci. .

Abstract

Precise expression of a transgene in the desired manner is important for plant genetic engineering and gene function deciphering, but it is a challenge to obtain specific transgene expression free from the interference of the constitutive promoters used to express the selectable marker gene, such as the Cauliflower mosaic virus (CaMV) 35S promoter. So, the solutions to avoid these inappropriate regulations are largely demanded. In this study, we report the characterization of a callus strong promoter (CSP1) in rice and its application for accurate transgene expression. Our results indicate that the high expression of the CSP1 promoter in the callus enables efficient selection of hygromycin equivalent to that provided by the CaMV 35S promoter, whereas its expression in other tissues is low. To evaluate possible leaky effects, the expression of a β-glucuronidase reporter driven by six specific promoters involving hormone signaling, pathogen response, cell fate determination, and proliferation was observed in transgenic rice plants generated by CSP1-mediated selection. Distinct β-glucuronidase expression was found consistently in most of the transgenic lines obtained for each promoter. In addition, we applied these specific marker lines to investigate the root cellular responses to exogenous cytokinin and auxin treatment. The results reveal that the root growth inhibition by cytokinin was differently regulated at high and low concentrations. In summary, we have established the feasibility of using callus-specific promoter-dependent selection to mitigate the transgene misexpression in rice. By enabling efficient transformation, rice plants with reliable transgene expression will be easily acquired for broad applications.

Keywords: callus strong promoter; selectable marker; transgene expression; transgenic rice; β-glucuronidase.

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Conflict of interest statement

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Figures

FIGURE 1
FIGURE 1
Tissue expression pattern of CSP1 in rice. (A) Tissue expression atlas of CSP1 gene. Gene expression profile among 39 specific tissues was retrieved from transcriptomes by the Genevestigator program (www.genevestigator.com). (B) Real-time RT-PCR analysis of CSP1 gene expression in different rice tissues. Roots and leaves were collected from 14-day-old solution cultured seedlings. Flag leaf, young spikelets were collected from soil-grown plants at maximum tillering stage. Embryogenic calli were collected from mature seeds after 30 days of inoculation on callus induction medium. ACT1 was used as the internal reference gene, and relative expression level in each sample is shown as a fold difference compared with roots. Data are means ± SD of three technical replicates as representative of three independent experiments. (C–E) Expression pattern of CSP1:GUS in roots of 9-day-old seedlings. Three parts of the primary root are shown from distal to proximal end: primary root tip (C), elongation zone (D), differentiation zone with developed lateral roots (E). Scale bars, 100 mm. (F–J) Expression of CSP1:GUS in leaf tip of 9-day-old seedling (F), tip of lemma and palea (G), anther and ovary (H) of mature flower, geminated embryo (I), and resistant calli (J). Scale bars, 1 mm. Images are representatives of three independent GUS-positive transgenic lines.
FIGURE 2
FIGURE 2
CSP1 promoter has high transcriptional activity in callus comparable with that of the CaMV 35S promoter. (A) Schematic diagram showing the structure of the HPT-GUS vectors with CaMV 35S and CSP1 promoters near the left border (LB) and controlling the expression of HPT. A promoterless GUS cassette with multiple cloning sites (MCS) was designed for histochemical analysis. (B) Growth of transformed calli 21 days after the onset of first-round selection on 50 mg/L Hyg (S1) and 14 days after the second-round selection on fresh medium with the same Hyg concentration (S2). Left panel, transformed with 35S controlled HPT-GUS vector; right panel, transformed with CSP1 controlled HPT-GUS vector. A representative dish of calli from two independent transformations with each vector is shown. (C) Hyg-tolerant growth of the calli transformed by the HPT-GUS vectors with HPT controlled by 35S and CSP1, respectively. Weight of fresh calli per dish was measured at the beginning of the first (S1) and second (S2) rounds of selection. Data are mean ± SD (n ≥ 5). Experiments were performed twice in two independent transformations (indicated as –1 and –2) with each vector. No significant difference was found at P < 0.1 in a Student’s t-test. (D,E) Relative HPT and GUS expression levels in secondary resistant calli transformed by 35S and CSP1-controlled HPT-GUS vectors. Data are mean ± SD of three technical replicates in each of two independent experiments (indicated as Rep 1 and Rep 2). * and *** indicate a significant difference at P < 0.05 and 0.001, respectively, in a Student’s t-test.
FIGURE 3
FIGURE 3
Comparison of copy numbers among transgenic T0 plants transformed with HPT-GUS vectors controlled by the CaMV 35S and CSP1 promoters. (A) Schematic diagram showing the structure of the HPT-GUS vectors with CaMV 35S and CSP1 promoters in reverse and upstream of the promoterless GUS gene and controlling the expression of HPT. Probe for Southern blot hybridization was designed from the 3′ end of HPT gene after NdeI. (B,C) Southern blot analysis of integrated T-DNA copy number in 16 independent T0 transgenic plants transformed with HPT-GUS vectors controlled by 35S (B) or CSP1 (C) promoters using the HPT probe shown in (A).
FIGURE 4
FIGURE 4
GUS leaky expression in calli transformed by HPT-GUS vectors with HPT controlled by the CaMV 35S and CSP1 promoters in two stacking configurations. (A) GUS leaky expression in calli transformed by the 35S-HPT-GUS vector with the CaMV 35S promoter near the LB. (B) GUS leaky expression in calli transformed by the CSP1-HPT-GUS vector with the CSP1 promoter near the LB. (C) GUS leaky expression in calli transformed by the HPT-35S-GUS vector with the 35S promoter in reverse and near the GUS gene. (D) GUS leaky expression in calli transformed by the HPT-CSP1-GUS vector with the CSP1 promoter in reverse and near the GUS gene. Figures are representative calli from two independent transformations by each vector. Bars: 1 mm. (E–G), Histograms showing the numbers of spots of GUS leaky expression in calli transformed by 35S-HPT-GUS (E), CSP1-HPT-GUS (F), and HPT-CSP1-GUS (G) vectors in two independent experiments, and one of them was shown. A total of 30 transformed calli per vector were selected for GUS staining overnight at 37°C after 2 weeks of selection. X-axis indicates the range of numbers of GUS spots per callus from 0 to 50 and divided into 11 intervals: 0, 1–5, 6–10, 11–15, 16–20, 21–25, 26–30, 31–35, 36–40, 41–45, and 46–50. Y-axis shows the number of calli that corresponds to each range. Average number of GUS spots per callus derived from each vector is shown on each histogram.
FIGURE 5
FIGURE 5
Specific GUS staining patterns in the roots of QHB/CYCB1;1/DR5/TCSn/RR6/PR1b:GUS transgenic lines selected by vectors based on CSP1-HPT-MCS-GUS. Primary roots of 8-day-old seedlings were subjected to GUS staining at 37°C for 22 h (QHB, TCSn, and PR1b) or 30 min (CYCB1;1, DR5, and RR6). GUS staining images of the root tips (lower panels), root segments with initiated lateral root primordia (middle panels), and root segments with elongated lateral roots (upper panels) were shown. Bars, 100 μm. Images are representative GUS expression patterns in T1 generation of at least three lines.
FIGURE 6
FIGURE 6
Cytokinin and auxin responsiveness in QHB/CYCB1;1/TCSn/DR5:GUS transgenic lines selected by vectors based on CSP1-HPT-MCS-GUS. (A) Six-day-old QHB:GUS transgenic seedlings (T2) were subjected to 100-μM KT treatment for 0, 6, 10, and 24 h. Primary root tips were collected at each time point and subjected to GUS staining for 6 h to show the progressively reduced GUS expression in response to prolonged KT treatment. (B) Six-day-old CYCB1;1:GUS transgenic seedlings (T2) were treated with 100-μM KT for the same periods as (A). Primary root tips were collected at each time point and subjected to GUS staining for 1 h. (C) Six-day-old TCSn:GUS transgenic seedlings (T2) were treated with 100-μM KT in fresh culture solution for 24 h or without KT as mock treatment. Primary root tips (left two panels) and a mature root zone with lateral roots (right two panels) were dissected for GUS staining. Strongly induced GUS expressions in the entire root are clearly shown after 7-h staining. (D) Six-day-old DR5:GUS transgenic seedlings (T2) were treated with 1-μM NAA in fresh culture solution for 24 h or without NAA as mock treatment. Primary root tips (left two panels) and a mature root zone with lateral roots (right two panels) were dissected for GUS staining. Increased GUS expression is clearly shown after 30-min staining. Bars, 100 μm. Images are representative GUS expression patterns of at least three lines.
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