Next Article in Journal / Special Issue
Over-Expression of Larch DAL1 Accelerates Life-Cycle Progression in Arabidopsis
Previous Article in Journal
Magnetic Treatment Improves the Seedling Growth, Nitrogen Metabolism, and Mineral Nutrient Contents in Populus × euramericana ‘Neva’ under Cadmium Stress
Previous Article in Special Issue
Genome-Wide Investigation of the MiR166 Family Provides New Insights into Its Involvement in the Drought Stress Responses of Tea Plants (Camellia sinensis (L.) O. Kuntze)
 
 
Search for Articles:
Title / Keyword
Author / Affiliation / Email
Journal
Article Type
 
 
Advanced Search
 
Section
Special Issue
Volume
Issue
Number
Page
 
Journals
Forests
Volume 13
Issue 6
10.3390/f13060952
forests-logo
Submit to this Journal Review for this Journal Propose a Special Issue
► Article Menu

Article Menu

share Share announcement Help format_quote Cite question_answer Discuss in SciProfiles thumb_up
...
Endorse textsms
...
Comment
first_page
settings
Order Article Reprints
Font Type:
Arial Georgia Verdana
Font Size:
Aa Aa Aa
Line Spacing:
Column Width:
Background:
Article

Identification and Functional Evaluation of Three Polyubiquitin Promoters from Hevea brasiliensis

by
Shichao Xin
1,2,
Jinu Udayabhanu
1,2,
Xuemei Dai
1,2,
Yuwei Hua
1,2,
Yueting Fan
1,2,
Huasun Huang
1,2 and
Tiandai Huang
1,2,*
1
Key Laboratory of Biology and Genetic Resources of Rubber Tree, Ministry of Agriculture and Rural Affairs, State Key Laboratory Incubation Base for Cultivation & Physiology of Tropical Crops, Rubber Research Institute, Chinese Academy of Tropical Agricultural Sciences, Haikou 571101, China
2
Haikou Key Laboratory of Innovation of Seedlings of Tropical Plants, Haikou 571101, China
*
Author to whom correspondence should be addressed.
Forests 2022, 13(6), 952; https://0-doi-org.brum.beds.ac.uk/10.3390/f13060952
Submission received: 11 April 2022 / Revised: 24 May 2022 / Accepted: 15 June 2022 / Published: 17 June 2022
(This article belongs to the Special Issue Tree Genetics: Molecular and Functional Characterization of Genes)
Browse Figures
Versions Notes

Abstract

:
Hevea brasiliensis is an economically important tree species that provides the only commercial source of natural rubber. The replacement of the CaMV35S promoter by endogenous polyubiquitin promoters may be a viable way to improve the genetic transformation of this species. However, no endogenous polyubiquitin promoters in Hevea have been reported yet. Here, we identified three Hevea polyubiquitin genes HbUBI10.1, HbUBI10.2 and HbUBI10.3, which encode ubiquitin monomers having nearly identical amino acid sequences to that of AtUBQ10. The genomic fragments upstream of these HbUBI genes, including the signature leading introns, were amplified as putative HbUBI promoters. In silico analysis showed that a number of cis-acting elements which are conserved within strong constitutive polyubiquitin promoters were presented in these HbUBI promoters. Transcriptomic data revealed that HbUBI10.1 and HbUBI10.2 had a constitutive expression in Hevea plants. Semi-quantitative RT-PCR showed that these three HbUBI genes were expressed higher than the GUS gene driven by CaMV35S in transgenic Hevea leaves. All three HbUBI promoters exhibited the capability to direct GFP expression in both transient and stable transformation assays, although they produced lower protoplast transformation efficiencies than the CaMV35S promoter. These HbUBI promoters will expand the availability of promoters for driving the transgene expression in Hevea genetic engineering.

1. Introduction

Hevea brasiliensis, also known as the rubber tree, is an economically important crop that provides the only commercial source of natural rubber. As a cross-pollinated perennial tree species with a long juvenile phase, the genetic improvement of Hevea through conventional breeding is troublesome and time-consuming [1]. Genetic engineering is a useful technique to accelerate the trait improvement of this species. Previously, multiple transformation methods have been developed to introduce genes of interest into Hevea, thereby improving agronomic traits related to latex yield, disease and stress response, latex quality, etc. [1,2,3,4,5].
In plant transformation systems, the promoter is one of the most important components that determine the expression levels of selectable markers, antibiotic resistance genes and genes of interest, thus affecting the screening and regeneration efficiency of the transgenic system and the performance of transgenic lines. The CaMV35S promoter derived from cauliflower mosaic virus (CaMV) is capable of directing the constitutive expression of adjacent genes in transgenic plants, which makes it the most commonly used promoter both in the stable and transient transformation of Hevea [3,6,7,8]. However, the use of the CaMV35S promoter still presents some shortcomings that may limit the efficiency and efficacy of Hevea transformation. Constitutive promoters isolated from plant pathogens can lead to abnormal conditions in transgenic plants [9]. It has also been extensively reported that, in multiple plant species, the transgenic hosts can recognize and inactivate intrusive DNA by DNA methylation and histone modification within the CaMV35S promoter region, which represses the expression of transgenes, leading to lethality in the selection process [10,11,12,13]. Additionally, when repeatedly used in multi-gene constructs, for expressing multiple transgenes of interest, the multicopy events of 35S promoter regions greatly increase the incidence of methylation-associated transgene silencing [14,15], thereby making transgenic products less efficacious. Moreover, the insertion of the viral DNA sequence into the host plant genome may cause regulatory concerns. Therefore, promoters of plant origin are drawing increasing interests in generating transgenic plants.
Ubiquitin is a highly conserved eukaryotic protein that is expressed at considerable levels in diverse plant tissues at different development stages, under both normal and stress conditions [16,17,18]. The ubiquitin gene promoters are good candidates for driving the expression of transgenes in plants [19]. A soybean polyubiquitin gene promoter Gmubi showed high levels of constitutive expression and represents an alternative to viral promoters for driving gene expression in soybeans [20]. The promoters of two polyubiquitin genes, PvUbi1 and PvUbi2, were previously isolated from switchgrass [21]. More recently, PvUbi and maize Zmubi promoters were respectively used to constitutively express two transgenes, xplA and xplB, in transgenic switchgrass, conferring the ability to remove toxic hexahydro-1,3,5-trinitro-1,3,5-triazine in soil [22]. The use of the native combinations of four polyubiquitin gene promoters and corresponding terminators resulted in an up to >3-fold increase in transgene expression in maize [19]. GUS expression levels from constructs containing RUBQ1 or RUB2 rice ubiquitin promoters were 8- to 35-fold higher in transgenic rice plants, respectively, as compared with that of the CaMV35S promoter [23]. When used in regulating selectable marker genes in maize, the plant-derived constitutive promoter ZmUbi1 produced a transformation efficiency of 43.8%, which is significantly higher than those of two viral promoters, CaMV35T (25%) and SCBV (8%) [24]. In multiplexed genome editing system in alfalfa, the replacement of the CaMV35S promoter by the Arabidopsis ubiquitin promoter to drive Cas9 expression led to a significant improvement in genome editing efficiency [25]. Ren, et al. [26] identified a novel ubiquitin promoter in grapes, which could improve the expression of Cas9, and thereby significantly increase the editing efficiency.
It is also worth noting that some polyubiquitin promoters have shown contrasting expression levels in different plant species [27]. The ubiquitin promoter of Gladiolus was shown to give low levels of transgene expression in freesia, Easter lily, tobacco, rice and rose, but high levels when reintroduced in Gladiolus [28], which is probably due to the lack of corresponding factors in heterologous systems [20]. Hernandez-Garcia and Finer [27] suggested that promoters intended for biotechnological applications should be functionally characterized in the same plant species of interest. It is therefore of great value to identify efficient endogenous polyubiquitin promoters in Hevea, and use them to improve the genetic engineering of Hevea.
In this study, three polyubiquitin promoters were isolated from Hevea. Analyses of sequences and expression profiles were performed, and their capabilities of driving transgene expression were evaluated both in transient and stable transformation studies. Our study provides potential tools for improving the genetic modification of Hevea via transgenic techniques.

2. Materials and Methods

2.1. Plant Materials

Hevea plant materials used in present study were collected from the “Hainan Innovation Base for production of Natural Rubber New Planting Material” (Danzhou, China). Hevea genomic DNA was extracted from the young leaves of Hevea clone Reyan7-33-97, and used for the amplification of promoter fragments. Two- to three-month-old Reyan7-33-97 trees were subjected to dark incubation for the generation of etiolated leaves, which were harvested for the isolation of mesophyll protoplasts. Anther-derived somatic embryos used for Hevea transformation were produced following Hua et al. [29].

2.2. Isolation and Bioinformatic Analysis of Polyubiquitin Genes and Their Promoters in Hevea

Polyubiquitin genes are highly conserved across species, and thus the sequence of the Arabidopsis polyubiquitin gene AtUBQ10 (accession No.: AT4G05320), whose promoter has been extensively used for driving transgene expressions [30,31,32], was used as the query to search for polyubiquitin genes in the reference genome of Hevea brasiliensis (http://hevea.catas.cn (accessed on 1 November 2019)) [33]. Three Hevea polyubiquitin genes having high sequence similarities with that of AtUBQ10 were obtained and designated as HbUBI10.1, HbUBI10.2, and HbUBI10.3, respectively. Multiple sequence alignment was performed using DNAMAN 6.0 (Lynnon Biosoft, San Ramon, CA, USA). These polyubiquitin genes and their upstream fragments of approximately 1000–1500 bp in length were amplified by PCR using the primers listed in Table S1 with the Hevea genomic DNA as a template, and then were sequenced. The isolated promoter sequences upstream of three HbUBI genes were analyzed using the PlantCARE database (https://bioinformatics.psb.ugent.be/webtools/plantcare/html/ (accessed on 21 October 2020)) for the identification of putative cis-acting elements.

2.3. Analysis of the Expressions of Three HbUBI Genes in Hevea Plants

Expression profiles of HbUBI10.1 and HbUBI10.2 polyubiquitin genes in various tissues of multiple Hevea clones under different conditions were obtained from the HeveaDB website (http://hevea.catas.cn/tool/v1/toExpression (accessed on 1 June 2021)) [34]. The normalized expression levels for each gene were calculated as fragments per kilobase of transcript per million fragments mapped (FPKM). A heatmap was constructed using TBtools to display the expressions of these HbUBI genes [35].
Semi-quantitative RT-PCR was performed using the primers listed in Table S1. Three individual 1-year-old transgenic Hevea plants, T1, T2, and T3 were analyzed. First-strand cDNA was synthesized from one microgram of total RNA in a 20 µL reaction mixture using the EasyScript® First-Strand cDNA Synthesis SuperMix Kit (TransGen Biotech, Beijing, China). To achieve proper results, a pre-experiment was performed to determine the appropriate cycle number and the optimum annealing temperature range for each primer pair. The cycle number of 30 and annealing temperature of 57 °C were shown to be applicable for all primer pairs. The semi-quantitative RT-PCR was performed using 100 ng cDNA templates, 0.2 μM of each primer, and Premix Taq DNA Polymerase (TakaraBio, Shiga, Japan) in a reaction volume of 20 µL. The reactions were performed in triplicate for each sample using the following program: initial denaturation at 94 °C for 2 min, followed by 30 cycles consisting of denaturation at 98 °C for 10 s, annealing at 57 °C for 30 s, and extension at 72 °C for 30 s. The final extension step was a 72 °C hold for 10 min. The resulting PCR products were then sequenced to confirm the specificity. The Hevea DEAD-box RNA helicase 8 gene (HbRH8, LOC110669478) was used as the internal control [36]. Agarose gels (1%, w/v) were used to analyze the PCR products, and the nucleic acids were stained with ethidium bromide. Gel images were taken and the band intensities were determined by Image Lab 6.0 software (Bio-Rad, Hercules, CA, USA). Relative expressions of three HbUBI genes and GUS gene were calculated by normalizing their band intensities to the band intensity of the HbRH8 reference gene.

2.4. Vector Construction

The promoters of three HbUBI genes were amplified by PCR from the Hevea genomic DNA using the primers listed in Table S1. For the construction of the transient expression vector, each PCR fragment was constructed into the pJIT163hGFP vector [37] using the restriction enzymes SacI (NEB, MA, USA) and NcoI (NEB, MA, USA) to replace the 2× CaMV35S promoter (Figure 1a). The resulting proHbUBI-pJIT163hGFP plasmids were used for transient transformation of Hevea protoplasts. Subsequently, a PCR reaction was carried out with proHbUBI-pJIT163hGFP plasmids as the template to create proHbUBI-hGFP fragments flanked by SacI/PstI restriction sites using the primers listed in Table S1. These PCR products were then introduced into the linearized pCAMBIA3300 vector (Cambia, Australia) to create the stable transformation vector proHbUBI-hGFP-pCAMBIA3300 (Figure 1b). The resulting plasmids were then transformed into Agrobacterium tumefaciens strain EHA105 by the freeze–thaw method [38] and used for the transformation of Hevea embryos.

2.5. Hevea Protoplasts Isolation and Polyethylene Glycol (PEG)-Mediated Transformation

The isolation and PEG-mediated transformation of Hevea mesophyll protoplasts were performed as previously described [6]. Briefly, about 2 g of 5–7-day-old etiolated leaves in the color-changing stage were collected for protoplasts’ isolation. Two hundred microliters of protoplast solution (6 × 106 /mL) was transformed with 50 μL of transient expression vector DNA (1 μg/μL), and then incubated in the dark at 26 °C for 48 h. The pJIT163hGFP plasmid carrying a green fluorescent protein (GFP) gene driven by the 2× CaMV35S promoter was transformed as the control. Ten microscope fields were surveyed for each sample under a Leica AF6000 fluorescence microscope (Leica, Wetzlar, Germany). Three biological replicates were performed for statistical analysis to determine the transformation efficiency.

2.6. Agrobacterium-Mediated Transformation of Hevea Somatic Embryos

Hevea embryos about 1.5 cm in size were collected and transformed by Agrobacterium tumefaciens EHA105 bearing the proHbUBI-hGFP-pCAMBIA3300 construct, following the methods previously described [8]. After transformation, 0.75 mg/L basta was used for the selection of positive transformants. Basta-resistant embryos were then surveyed for GFP expression under a Leica AF6000 fluorescence microscope (Leica, Wetzlar, Germany).

3. Results

3.1. Isolation of Three Polyubiquitin Promoters in Hevea

In search for endogenous polyubiquitin promoters in Hevea that could potentially be used as alternatives to the CaMV35S promoter, we performed BLAST searches of the reference genome of Hevea using AtUBQ10 as a query. Three polyubiquitin genes that share high sequence similarities to the query were identified and designated as HbUBI10.1, HbUBI10.2 and HbUBI10.3 (Figure S1). The HbUBI10.1 and HbUBI10.2 genes encode polyubiquitins consisting of six ubiquitin repeats in a head-to tail, spacerless arrangement, which is consistent with that of AtUBQ10. In contrast, the HbUBI10.3 has a shorter coding sequence encoding five ubiquitin monomers. Additionally, the ubiquitin monomers encoded by these three HbUBI genes have an amino acid sequence identical to that of AtUBQ10, except for a single amino acid difference in the first ubiquitin monomer in HbUBI10.1 and HbUBI10.2 (Figure S2). All of these HbUBI genes consist of a long leading intron, ranging from 760 to 1175 bp in length (Figure 2a), which is required in the polyubiquitin promoters to induce strong gene expression [32,39,40]. Therefore, in this study, DNA fragments of 1237, 1146 and 1380 bp in length upstream of the ATG start codon of the HbUBI10.1, HbUBI10.2 and HbUBI10.3 genes, including the leading introns, were respectively amplified as putative HbUBI promoters for subsequent experiments (Figure 2a). The sequences for HbUBI10.1, HbUBI10.2 and HbUBI10.3 promoters were deposited at the National Center for Biotechnology Information (NCBI) under the accession numbers ON110822, ON110823 and ON110824, respectively. PlantCARE analysis showed that these three HbUBI promoters consist of a large number of TATA-box and CAAT-box motifs at numerous positions, which are core elements required for transcription initiation (Figure 2b, Table S2). Two light responsive elements, G-box and the GATA-motif that were commonly present in other constitutive promoters were also found in these three HbUBI promoters. The promoters of HbUBI10.1 and HbUBI10.3 respectively harbor two and one G-box motifs. The GATA-motif appeared one and two times in the HbUBI10.2 and HbUBI10.3 promoter sequences, respectively. Additionally, multiple putative cis-acting elements that are associated with hormones and abiotic stresses response are present on these HbUBI promoters (Figure 2b, Table S2).

3.2. Expression Analysis of Three HbUBI Genes

We obtained the expression profiles of HbUBI10.1 and HbUBI10.2 in Hevea plants by mining the transcriptome data downloaded from HeveaDB [34]. The results show that these two genes are constitutively expressed in diverse Hevea tissues at different development stages (Figure 3a). Notably, HbUBI10.1 is highly expressed in laticifers and latex, where natural rubber is biosynthesized. The expression of HbUBI10.3 was not yet available in HeveaDB. Further study is required to investigate the expression profiles of HbUBI10.3.
Semi-quantitative RT-PCR was performed to compare the endogenous expression levels of three HbUBI genes with that of the GUS gene driven by the CaMV35S promoter in the leaves of 1-year-old transgenic Hevea plants (Figure 3b–d). The results show that the GUS gene driven by CaMV35S promoter is comparably expressed among three transgenic Hevea plants, while these three HbUBI genes show slightly higher endogenous expression levels than that of the GUS gene (Figure 3c,d). These results suggest that the use of promoters from these HbUBI genes may give higher levels of transgene expression than that of the CaMV35S promoter in Hevea transformation.

3.3. Functional Evaluation of Three HbUBI Promoters in Hevea Tissues

To determine whether these HbUBI promoters could be used as alternatives to CaMV35S for Hevea transformation, both transient and stable transformation assays were performed to evaluate the capabilities of three HbUBI promoters in directing transgene expression in Hevea tissues.
These three HbUBI promoters were fused to the GFP reporter gene, and transformed into Hevea protoplasts. GFP fluorescence was observed in Hevea protoplasts transformed by plasmids containing each of these three HbUBI promoters (Figure 4), confirming their transcriptional activities. Transformation efficiencies were calculated according to the numbers of protoplasts with GFP fluorescence. The control plasmid incorporating the 2× CaMV35S promoter gave a transformation efficiency of 48%. However, when the GFP gene was associated with three HbUBI promoters, the transformation efficiencies were around 35% (Figure 4), which were lower than that of the 2× CaMV35S promoter control.
Agrobacterium-transformed Hevea embryos could be used as a preliminary indicator of the transcription activities of promoters in directing stable transgene expression. As shown in Figure 5, all three HbUBI promoters successfully promoted the expression of GFP in Hevea embryos. These results indicate that these three HbUBI promoters faithfully drive the expression of transgenes, and are therefore suitable alternatives to the CaMV35S promoter in Hevea transformation.

4. Discussion

In previously reported Hevea transformation systems, the CaMV35S promoter was extensively used to direct the constitutive expressions of selectable markers, reporter genes and genes of interest [3,5,7,8]. However, the use of the CaMV35S promoter for driving transgene expression in plant transformation is not ideal, either because of the incidence of promoter methylation-induced transgene repression during the development of transgenic plants [12,13], or the biosafety concerns raised by the pathogen—the-origin of the CaMV35S promoter. With the aim to provide alternative promoters to the CaMV35S for Hevea genetic transformation, we settled on the polyubiquitin promoters that confer a higher transformation efficiency, transgene expression levels and expression stability in various plant species when compared with those of the CaMV35S promoter [23,24,31].
The polyubiquitin genes encode the polyubiquitin precursor protein containing tandemly repeated ubiquitin monomers [18]. This study identified three polyubiquitin genes HbUBI10.1, HbUBI10.2 and HbUBI10.3 in Hevea, which respectively encode six, six and five repeats of ubiquitin monomers, having an identical amino acid sequence to AtUBQ10 except for one amino acid difference in the first monomer of HbUBI10.1 and HbUBI10.2 (Figures S1 and S2). These findings support the previous reports that the ubiquitins are highly conserved among different plant species [18,21].
Previous studies showed that polyubiquitin promoters of around 1000 bp in length are generally used to drive transgene expression [20]. A longer sequence upstream of tomato UBIQUITIN10 directs higher adjacent gene expression [32]. However, small promoters enable the elaboration of compact vectors, and therefore may produce higher transformation efficiencies [41]. All three Hevea polyubiquitin genes identified in the present study possess a long leading intron upstream of the ATG start codon (Figure 2a), which is a common feature of polyubiquitin genes. This intron is considered part of the promoter, and contributes to very high levels of adjacent gene expression in transiently and stably transformed plant tissues [27,30,32,39,40]. Removal of the intron from the promoter region either reduces the strength of the promoter or results in a complete loss of promoter activity [23,27]. Taking into consideration the above, the fragments upstream of three HbUBI genes including the signature leading intron, ranging from 1146–1380 bp in length, were used for further analysis.
The promoters of these three HbUBI genes were shown to contain numerous copies of TATA-box and CAAT-box motifs (Figure 2b, Table S2), which act as major determinants of promoter efficiency during gene transcription [42]. In addition to these core promoter elements, two light-responsive elements, the GATA-motif and G-box motif, were identified, which are well conserved motifs in other strong constitutive promoters [27,42]. The G-box motif, which has been proposed to contribute considerably to the strong expression mediated by polyubiquitin promoters [27,43], was present two and one times in the promoters of HbUBI10.1 and HbUBI10.3, respectively. One and two copies of GATA-motifs were identified in the promoters of HbUBI10.2 and HbUBI10.3, respectively. These results suggest that these three HbUBI promoters might be involved in effective transcription. Additionally, the presence of a large number of environment-responsive cis-acting elements in these HbUBI promoters may also contribute to the total transcription activities [27].
Transcriptomic data provide a better understanding of the global regulation of gene expression, and therefore facilitate the identification of endogenous promoters in economically important crops [26,41]. Several transcriptomes for a number of Hevea clones are now available [34,44,45]. According to the transcriptome database, HbUBI10.1 and HbUBI10.2 genes were highly expressed in various tissues and development stages (Figure 3a), indicating that their promoters should have constitutive activities. Although the activities of these three HbUBI promoters in directing transgenes in whole Hevea plants were not accomplished in this study, the higher endogenous expression levels of three HbUBI genes, as compared with the expression driven by the CaMV35S promoter in transgenic Hevea leaves, suggest that these HbUBI promoters may be capable of directing the strong expression of transgenes (Figure 3c,d).
Both the constitutive promoters of apple MdUBQ10 and Arabidopsis AtUBQ10 drove the expression of target genes in apple protoplast cells significantly better than the CaMV35S promoter [46]. However, in our present study, the vector incorporating the 2× CaMV35S promoter produced higher protoplast transformation efficiency than the HbUBI promoter-based transient vectors (Figure 4), which may be attributed to its smaller size (737 bp in length), independent of the transcriptional activities of those incorporated promoters. These results demonstrate the capabilities of HbUBI promoters in driving transient expression in Hevea protoplasts. However, further promoter truncation analysis is required to reduce the length of these HbUBI promoters while maintaining their transcriptional activities, thus reducing the construct size, and therefore facilitating genetic transformation [27].
Transient expression is not subject to chromatin-based gene regulation and, thus, the transient assay does not reflect the full functionality of promoters [27]. Given that the recovery of whole transgenic Hevea plants takes at least several months, Agrobacterium-transformed Hevea embryos were therefore used for the rapid validation of the transcription activities of these HbUBI promoters in directing stable transgene expression. In basta-resistant embryos, all three HbUBI promoters produced GFP fluorescence (Figure 5), demonstrating their capabilities in driving the stable expression of reporter genes or selection markers in Hevea transformation. Further long-term investigations are required to document the comprehensive expression profiles of these HbUBI promoters in whole transgenic Hevea plants at different development stages. The maize ubiquitin promoter ZmUbi1 produced significant higher maize transformation efficiency than those of two viral promoters, the CaMV35S promoter and sugarcane bacilliform virus promoter, when used in driving the expression of selectable marker genes [24]. Therefore, the future use of these HbUBI promoters as replacements of the CaMV35S promoter for expressing selectable markers may also produce higher transformation efficiencies in Hevea.
Additionally, polyubiquitin promoters show great potential in CRISPR-mediated genome editing systems. In allotetraploid tobacco plants, UBIQUITIN10 promoters from several plant species directed the higher expression of selection markers than that of the CaMV35S promoter, and thereby significantly improved the efficiencies of Agrobacterium-mediated transformation and CRISPR/Cas9-mediated mutagenesis [32]. The use of a grape endogenous polyubiquitin promoter UBQ2 can significantly improve the editing efficiencies in grape cells and stable transgenic plants by increasing the expression of Cas9 [26]. In a multiplexed genome editing system in alfalfa, the replacement of the CaMV35S promoter by the Arabidopsis ubiquitin promoter to drive Cas9 expression led to a significant improvement in genome editing efficiency [25]. Targeted genome editing in Hevea was recently achieved using the CRISPR/Cas9 system, wherein the expression of Cas9 was driven by the CaMV35S promoter [47]. Future applications of these HbUBI promoters in Hevea CRISPR systems may also improve the efficiencies of targeted mutagenesis.

5. Conclusions

This study identified three Hevea polyubiquitin genes by BLASTing the Hevea genome. Expression analysis showed that HbUBI10.1 and HbUBI10.2 were constitutively expressed in Hevea plants, and all three HbUBI genes exhibited higher endogenous expression than the GUS gene driven by the CaMV35S promoter in transgenic Hevea leaves. We isolated the promoter fragments of these three HbUBI genes, and confirmed that these promoters could direct GFP expression in both transient and stable assays. These results suggest that these HbUBI promoters could be potentially used as alternatives to the CaMV35S promoter for driving the constitutive expression of transgenes in Hevea genetic modification programs.

Supplementary Materials

The following supporting information can be downloaded at: https://0-www-mdpi-com.brum.beds.ac.uk/article/10.3390/f13060952/s1, Figure S1: Sequence alignment of coding regions of three Hevea polyubiquitin genes with that of AtUBQ10; Figure S2: Alignment of the amino acid sequences of three HbUBI polyubiquitins and AtUBQ10; Table S1: Primers used in present study; Table S2: Putative cis-acting elements identified in three HbUBI promoters.

Author Contributions

Conceptualization, H.H. and T.H.; Data curation, S.X.; Funding acquisition, Y.H.; Investigation, S.X., J.U. and X.D.; Methodology, S.X.; Project administration, T.H.; Resources, X.D.; Supervision, H.H.; Validation, S.X., J.U. and Y.F.; Writing—original draft, S.X.; Writing—review & editing, S.X. S.X. and J.U. contributed equally to this study. All authors have read and agreed to the published version of the manuscript.

Funding

This research was funded by the National Natural Science Foundation of China (grant number 31872708), the Hainan Provincial Natural Science Foundation of China (grant number 322MS137), and the National Key R&D Program of China (grant number 2019YFD1001102).

Institutional Review Board Statement

Not applicable.

Informed Consent Statement

Not applicable.

Data Availability Statement

The sequences for the three Hevea polyubiquitin promoters have been deposited at National Center for Biotechnology Information (NCBI) under the following accession numbers: ON110822 (HbUBI10.1), ON110823 (HbUBI10.2) and ON110824 (HbUBI10.3). The materials used in this study are available from the authors upon request.

Conflicts of Interest

The authors declare no conflict of interest.

References

  1. Thulaseedharan, A.; Jayashree, R.; Kala, R.G.; Rekha, K.; Sobha, S.; Jayasree, P.K.; Kumari, S.S.; Saleena, A.; Venkatachalam, P. Rubber Tree. In Compendium of Transgenic Crop Plants; Kole, C., Hall, T.C., Eds.; Blackwell Publishing Ltd.: Malden, MA, USA, 2009; pp. 117–140. [Google Scholar]
  2. Arokiaraj, P.; Florian, R.; Obermayr, E.; Samsul, B.A.; Yeang, H.Y. Expression of human serum albumin in transgenic Hevea brasiliensis. J. Rubber Res. 2002, 5, 157–166. [Google Scholar]
  3. Lestari, R.; Rio, M.; Martin, F.; Leclercq, J.; Woraathasin, N.; Roques, S.; Dessailly, F.; Clément-Vidal, A.; Sanier, C.; Fabre, D.; et al. Overexpression of Hevea brasiliensis ethylene response factor HbERF-IXc5 enhances growth and tolerance to abiotic stress and affects laticifer differentiation. Plant Biotechnol. J. 2018, 16, 322–336. [Google Scholar] [CrossRef] [Green Version]
  4. Huang, T.D.; Li, J.; Li, Y.T.; Huang, H.S.; Hua, Y.W. Somatic Embryo, an Alternative Target Tissue for Agrobacterium-Mediated Transformation in Hevea brasiliensis. J. Rubber Res. 2015, 18, 171–188. [Google Scholar]
  5. Martin, F.; Abati, V.; Burel, A.; Clement-Vidal, A.; Sanier, C.; Fabre, D.; Woraathasin, N.; Rio, M.; Besret, P.; Farinas, B.; et al. Overexpression of EcGSH1 induces glutathione production and alters somatic embryogenesis and plant development in Hevea brasiliensis. Ind. Crops Prod. 2018, 112, 803–814. [Google Scholar] [CrossRef]
  6. Fan, Y.; Xin, S.; Dai, X.; Yang, X.; Huang, H.; Hua, Y. Efficient genome editing of rubber tree (Hevea brasiliensis) protoplasts using CRISPR/Cas9 ribonucleoproteins. Ind. Crops Prod. 2020, 146, 112146. [Google Scholar] [CrossRef]
  7. Leclercq, J.; Lardet, L.; Martin, F.; Chapuset, T.; Oliver, G.; Montoro, P. The green fluorescent protein as an efficient selection marker for Agrobacterium tumefaciens-mediated transformation in Hevea brasiliensis (Müll. Arg). Plant Cell Rep. 2010, 29, 513–522. [Google Scholar] [CrossRef]
  8. Huang, T.; Xin, S.; Fang, Y.; Chen, T.; Chang, J.; Ko, N.C.K.; Huang, H.; Hua, Y. Use of a novel R2R3-MYB transcriptional activator of anthocyanin biosynthesis as visual selection marker for rubber tree (Hevea brasiliensis) transformation. Ind. Crops Prod. 2021, 174, 114225. [Google Scholar] [CrossRef]
  9. Ali, S.; Kim, W.-C. A Fruitful Decade Using Synthetic Promoters in the Improvement of Transgenic Plants. Front. Plant Sci. 2019, 10, 1433. [Google Scholar] [CrossRef]
  10. Mishiba, K.; Nishihara, M.; Nakatsuka, T.; Abe, Y.; Hirano, H.; Yokoi, T.; Kikuchi, A.; Yamamura, S. Consistent transcriptional silencing of 35S-driven transgenes in gentian. Plant J. 2005, 44, 541–556. [Google Scholar] [CrossRef]
  11. Kanazawa, A.; O’Dell, M.; Hellens, R.P. The Binding of Nuclear Factors to the as-1 Element in the CaMV 35S Promoter is Affected by Cytosine Methylation in vitro. Plant Biol. 2007, 9, 435–441. [Google Scholar] [CrossRef]
  12. Okumura, A.; Shimada, A.; Yamasaki, S.; Horino, T.; Iwata, Y.; Koizumi, N.; Nishihara, M.; Mishiba, K. CaMV-35S promoter sequence-specific DNA methylation in lettuce. Plant Cell Rep. 2016, 35, 43–51. [Google Scholar] [CrossRef] [PubMed]
  13. Weinhold, A.; Kallenbach, M.; Baldwin, I.T. Progressive 35S promoter methylation increases rapidly during vegetative development in transgenic Nicotiana attenuata plants. BMC Plant Biol. 2013, 13, 99. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  14. Muskens, M.W.M.; Vissers, A.P.A.; Mol, J.N.M.; Kooter, J.M. Role of inverted DNA repeats in transcriptional and post-transcriptional gene silencing. Plant Mol. Biol. 2000, 43, 243–260. [Google Scholar] [CrossRef]
  15. Peremarti, A.; Twyman, R.M.; Gómez-Galera, S.; Naqvi, S.; Farré, G.; Sabalza, M.; Miralpeix, B.; Dashevskaya, S.; Yuan, D.; Ramessar, K.; et al. Promoter diversity in multigene transformation. Plant Mol. Biol. 2010, 73, 363–378. [Google Scholar] [CrossRef] [PubMed]
  16. Sullivan, J.A.; Shirasu, K.; Deng, X.W. The diverse roles of ubiquitin and the 26S proteasome in the life of plants. Nat. Rev. Genet. 2003, 4, 948–958. [Google Scholar] [CrossRef]
  17. Han, S.-W.; Jung, B.-K.; Park, S.-H.; Ryu, K.-Y. Reversible Regulation of Polyubiquitin Gene UBC via Modified Inducible CRISPR/Cas9 System. Int. J. Mol. Sci. 2019, 20, 3168. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  18. Stone, S.L. Chapter 26—Ubiquitination of Plant Transcription Factors. In Plant Transcription Factors; Gonzalez, D.H., Ed.; Academic Press: Boston, MA, USA, 2016; pp. 395–409. [Google Scholar]
  19. Wang, P.-H.; Kumar, S.; Zeng, J.; McEwan, R.; Wright, T.; Gupta, M. Transcription Terminator-Mediated Enhancement in Transgene Expression in Maize: Preponderance of the AUGAAU Motif Overlapping With Poly(A) Signals. Front. Plant Sci. 2020, 11, 1498. [Google Scholar] [CrossRef] [PubMed]
  20. Hernandez-Garcia, C.M.; Martinelli, A.P.; Bouchard, R.A.; Finer, J.J. A soybean (Glycine max) polyubiquitin promoter gives strong constitutive expression in transgenic soybean. Plant Cell Rep. 2009, 28, 837–849. [Google Scholar] [CrossRef]
  21. Mann, D.G.J.; King, Z.R.; Liu, W.; Joyce, B.L.; Percifield, R.J.; Hawkins, J.S.; LaFayette, P.R.; Artelt, B.J.; Burris, J.N.; Mazarei, M.; et al. Switchgrass (Panicum virgatum L.) polyubiquitin gene (PvUbi1 and PvUbi2) promoters for use in plant transformation. BMC Biotechnol. 2011, 11, 74. [Google Scholar] [CrossRef] [Green Version]
  22. Cary, T.J.; Rylott, E.L.; Zhang, L.; Routsong, R.M.; Palazzo, A.J.; Strand, S.E.; Bruce, N.C. Field trial demonstrating phytoremediation of the military explosive RDX by XplA/XplB-expressing switchgrass. Nat. Biotechnol. 2021, 39, 1216–1219. [Google Scholar] [CrossRef]
  23. Wang, J.; Oard, J.H. Rice ubiquitin promoters: Deletion analysis and potential usefulness in plant transformation systems. Plant Cell Rep. 2003, 22, 129–134. [Google Scholar] [CrossRef] [PubMed]
  24. Beringer, J.; Chen, W.; Garton, R.; Sardesai, N.; Wang, P.-H.; Zhou, N.; Gupta, M.; Wu, H. Comparison of the impact of viral and plant-derived promoters regulating selectable marker gene on maize transformation and transgene expression. Plant Cell Rep. 2017, 36, 519–528. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  25. Wolabu, T.W.; Cong, L.; Park, J.-J.; Bao, Q.; Chen, M.; Sun, J.; Xu, B.; Ge, Y.; Chai, M.; Liu, Z.; et al. Development of a Highly Efficient Multiplex Genome Editing System in Outcrossing Tetraploid Alfalfa (Medicago sativa). Front. Plant. Sci. 2020, 11, 1063. [Google Scholar] [CrossRef] [PubMed]
  26. Ren, C.; Liu, Y.; Guo, Y.; Duan, W.; Fan, P.; Li, S.; Liang, Z. Optimizing the CRISPR/Cas9 system for genome editing in grape by using grape promoters. Hortic. Res. 2021, 8, 52. [Google Scholar] [CrossRef] [PubMed]
  27. Hernandez-Garcia, C.M.; Finer, J.J. Identification and validation of promoters and cis-acting regulatory elements. Plant Sci. 2014, 217–218, 109–119. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  28. Joung, Y.H.; Kamo, K. Expression of a polyubiquitin promoter isolated from Gladiolus. Plant Cell Rep. 2006, 25, 1081–1088. [Google Scholar] [CrossRef]
  29. Hua, Y.W.; Huang, T.D.; Huang, H.S. Micropropagation of self-rooting juvenile clones by secondary somatic embryogenesis in Hevea brasiliensis. Plant Breed. 2010, 129, 202–207. [Google Scholar] [CrossRef]
  30. Grefen, C.; Donald, N.; Hashimoto, K.; Kudla, J.; Schumacher, K.; Blatt, M.R. A ubiquitin-10 promoter-based vector set for fluorescent protein tagging facilitates temporal stability and native protein distribution in transient and stable expression studies. Plant J. 2010, 64, 355–365. [Google Scholar] [CrossRef]
  31. Wolabu, T.W.; Park, J.-J.; Chen, M.; Cong, L.; Ge, Y.; Jiang, Q.; Debnath, S.; Li, G.; Wen, J.; Wang, Z. Improving the genome editing efficiency of CRISPR/Cas9 in Arabidopsis and Medicago truncatula. Planta 2020, 252, 15. [Google Scholar] [CrossRef]
  32. Kumar, M.; Ayzenshtat, D.; Marko, A.; Bocobza, S. Optimization of T-DNA configuration with UBIQUITIN10 promoters and tRNA–sgRNA complexes promotes highly efficient genome editing in allotetraploid tobacco. Plant Cell Rep. 2022, 41, 175–194. [Google Scholar] [CrossRef]
  33. Tang, C.; Yang, M.; Fang, Y.; Luo, Y.; Gao, S.; Xiao, X.; An, Z.; Zhou, B.; Zhang, B.; Tan, X.; et al. The rubber tree genome reveals new insights into rubber production and species adaptation. Nat. Plants 2016, 2, 16073. [Google Scholar] [CrossRef] [Green Version]
  34. Cheng, H. HeveaDB: A Hub for Rubber Tree Genetic and Genomic Resources. In The Rubber Tree Genome; Matsui, M., Chow, K.-S., Eds.; Springer International Publishing: Cham, Switzerland, 2020; pp. 137–152. [Google Scholar]
  35. Chen, C.; Chen, H.; Zhang, Y.; Thomas, H.R.; Frank, M.H.; He, Y.; Xia, R. TBtools: An Integrative Toolkit Developed for Interactive Analyses of Big Biological Data. Mol. Plant 2020, 13, 1194–1202. [Google Scholar] [CrossRef] [PubMed]
  36. Li, H.; Qin, Y.; Xiao, X.; Tang, C. Screening of valid reference genes for real-time RT-PCR data normalization in Hevea brasiliensis and expression validation of a sucrose transporter gene HbSUT3. Plant Sci. 2011, 181, 132–139. [Google Scholar] [CrossRef] [PubMed]
  37. Wang, K.; Tang, D.; Hong, L.; Xu, W.; Huang, J.; Li, M.; Gu, M.; Xue, Y.; Cheng, Z. DEP and AFO Regulate Reproductive Habit in Rice. PLoS Genet. 2010, 6, e1000818. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  38. Horsch, R.B.; Rogers, S.G.; Fraley, R.T. Transgenic plants. Cold Spring Harb. Symp. Quant. Biol. 1985, 50, 433–437. [Google Scholar] [CrossRef]
  39. Hernandez-Garcia, C.M.; Bouchard, R.A.; Rushton, P.J.; Jones, M.L.; Chen, X.; Timko, M.P.; Finer, J.J. High level transgenic expression of soybean (Glycine max) GmERF and Gmubi gene promoters isolated by a novel promoter analysis pipeline. BMC Plant Biol. 2010, 10, 237. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  40. Torre, C.M.D.L.; Finer, J.J. The intron and 5′ distal region of the soybean Gmubi promoter contribute to very high levels of gene expression in transiently and stably transformed tissues. Plant Cell Rep. 2015, 34, 111–120. [Google Scholar] [CrossRef] [PubMed]
  41. Hernandez-Garcia, C.M.; Finer, J.J. A novel cis-acting element in the GmERF3 promoter contributes to inducible gene expression in soybean and tobacco after wounding. Plant Cell Rep. 2016, 35, 303–316. [Google Scholar] [CrossRef]
  42. Erpen, L.; Tavano, E.C.R.; Harakava, R.; Dutt, M.; Grosser, J.W.; Piedade, S.M.S.; Mendes, B.M.J.; Mourão Filho, F.A.A. Isolation, characterization, and evaluation of three Citrus sinensis-derived constitutive gene promoters. Plant Cell Rep. 2018, 37, 1113–1125. [Google Scholar] [CrossRef]
  43. Subhi, S.M.; Ghulam, K.A.P.; Omar, A.R. Isolation of an oil palm constitutive promoter derived from ubiquitin extension protein (uep2) gene. J. Oil Palm Res. 2019, 31, 28–41. [Google Scholar] [CrossRef] [Green Version]
  44. Yang, H.; Wang, X.; Wei, Y.; Deng, Z.; Liu, H.; Chen, J.; Dai, L.; Xia, Z.; He, G.; Li, D. Transcriptomic analyses reveal molecular mechanisms underlying growth heterosis and weakness of rubber tree seedlings. BMC Plant Biol. 2018, 18, 10. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  45. Xin, S.; Hua, Y.; Li, J.; Dai, X.; Yang, X.; Udayabhanu, J.; Huang, H.; Huang, T. Comparative analysis of latex transcriptomes reveals the potential mechanisms underlying rubber molecular weight variations between the Hevea brasiliensis clones RRIM600 and Reyan7-33–97. BMC Plant Biol. 2021, 21, 244. [Google Scholar] [CrossRef] [PubMed]
  46. Wang, X.; Xu, L.; Liu, X.; Xin, L.; Wu, S.; Chen, X. Development of potent promoters that drive the efficient expression of genes in apple protoplasts. Hortic. Res. 2021, 8, 211. [Google Scholar] [CrossRef] [PubMed]
  47. Dai, X.; Yang, X.; Wang, C.; Fan, Y.; Xin, S.; Hua, Y.; Wang, K.; Huang, H. CRISPR/Cas9-mediated genome editing in Hevea brasiliensis. Ind. Crops Prod. 2021, 164, 113418. [Google Scholar] [CrossRef]
Forests 13 00952 g001 550
Figure 1. Schematic representation of the vectors used for Hevea transformation. (a) Transient expression vectors used for protoplasts transformation contain an GFP reporter gene driven by three Hevea polyubiquitin promoters, respectively. (b) T-DNA region of the binary vector used for Agrobacterium-mediated transformation of Hevea embryos.
Figure 1. Schematic representation of the vectors used for Hevea transformation. (a) Transient expression vectors used for protoplasts transformation contain an GFP reporter gene driven by three Hevea polyubiquitin promoters, respectively. (b) T-DNA region of the binary vector used for Agrobacterium-mediated transformation of Hevea embryos.
Forests 13 00952 g001
Forests 13 00952 g002 550
Figure 2. Analysis of three HbUBI genes and their promoters. (a) Schematic representation of the genomic structures of three polyubiquitin genes. (b) Putative cis-elements identified in these three HbUBI promoter regions. Abbreviations: CDS, coding sequence; UTR, untranslated region; DRE, dehydration-responsive element; MeJA, methyl jasmonic acid; STRE, stress response element; ABA, abscisic acid.
Figure 2. Analysis of three HbUBI genes and their promoters. (a) Schematic representation of the genomic structures of three polyubiquitin genes. (b) Putative cis-elements identified in these three HbUBI promoter regions. Abbreviations: CDS, coding sequence; UTR, untranslated region; DRE, dehydration-responsive element; MeJA, methyl jasmonic acid; STRE, stress response element; ABA, abscisic acid.
Forests 13 00952 g002
Forests 13 00952 g003 550
Figure 3. Expressions of three HbUBI genes in Hevea brasiliensis. (a) Expression profiles of HbUBI10.1 and HbUBI10.2 genes in various tissues of multiple Hevea clones at different developmental stages. Normalized transcript levels are displayed as FPKM values in the heatmap. Row names denote the Hevea clone, type of tissue, age and treatment. Abbreviations: JA, jasmonic acid; TPD, tapping panel dryness; stageB, bronze stage; stageBC, color-changing stage; stageC, light green stage; stageD, stable stage. (b) GUS staining of the leaves from 1-year-old transgenic Hevea plants harboring a GUS gene driven by the CaMV35S promoter. (c) Semi-quantitative RT-PCR analysis of the endogenous expression levels of three HbUBI genes in transgenic Hevea plants bearing a GUS gene driven by the CaMV35S promoter. WT represents the wild-type control, while T1, T2 and T3 represent three individual transgenic Hevea plants. (d) Relative expressions of three HbUBI genes and GUS in the leaves of 1-year-old transgenic Hevea plants. The mean relative expressions with standard deviation were calculated from three replicates of semi-quantitative RT-PCR reactions.
Figure 3. Expressions of three HbUBI genes in Hevea brasiliensis. (a) Expression profiles of HbUBI10.1 and HbUBI10.2 genes in various tissues of multiple Hevea clones at different developmental stages. Normalized transcript levels are displayed as FPKM values in the heatmap. Row names denote the Hevea clone, type of tissue, age and treatment. Abbreviations: JA, jasmonic acid; TPD, tapping panel dryness; stageB, bronze stage; stageBC, color-changing stage; stageC, light green stage; stageD, stable stage. (b) GUS staining of the leaves from 1-year-old transgenic Hevea plants harboring a GUS gene driven by the CaMV35S promoter. (c) Semi-quantitative RT-PCR analysis of the endogenous expression levels of three HbUBI genes in transgenic Hevea plants bearing a GUS gene driven by the CaMV35S promoter. WT represents the wild-type control, while T1, T2 and T3 represent three individual transgenic Hevea plants. (d) Relative expressions of three HbUBI genes and GUS in the leaves of 1-year-old transgenic Hevea plants. The mean relative expressions with standard deviation were calculated from three replicates of semi-quantitative RT-PCR reactions.
Forests 13 00952 g003
Forests 13 00952 g004 550
Figure 4. Transient expression assays in Hevea mesophyll protoplasts. Transformation efficiencies were calculated from three replicates. Size bars indicate 50 μm.
Figure 4. Transient expression assays in Hevea mesophyll protoplasts. Transformation efficiencies were calculated from three replicates. Size bars indicate 50 μm.
Forests 13 00952 g004
Forests 13 00952 g005 550
Figure 5. Transgenic Hevea embryos bearing a GFP reporter gene that was respectively driven by HbUBI10.1, HbUBI10.2 and HbUBI10.3 promoters. Non-transformed embryos were used as negative controls.
Figure 5. Transgenic Hevea embryos bearing a GFP reporter gene that was respectively driven by HbUBI10.1, HbUBI10.2 and HbUBI10.3 promoters. Non-transformed embryos were used as negative controls.
Forests 13 00952 g005
Publisher’s Note: MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Share and Cite

MDPI and ACS Style

Xin, S.; Udayabhanu, J.; Dai, X.; Hua, Y.; Fan, Y.; Huang, H.; Huang, T. Identification and Functional Evaluation of Three Polyubiquitin Promoters from Hevea brasiliensis. Forests 2022, 13, 952. https://0-doi-org.brum.beds.ac.uk/10.3390/f13060952

AMA Style

Xin S, Udayabhanu J, Dai X, Hua Y, Fan Y, Huang H, Huang T. Identification and Functional Evaluation of Three Polyubiquitin Promoters from Hevea brasiliensis. Forests. 2022; 13(6):952. https://0-doi-org.brum.beds.ac.uk/10.3390/f13060952

Chicago/Turabian Style

Xin, Shichao, Jinu Udayabhanu, Xuemei Dai, Yuwei Hua, Yueting Fan, Huasun Huang, and Tiandai Huang. 2022. "Identification and Functional Evaluation of Three Polyubiquitin Promoters from Hevea brasiliensis" Forests 13, no. 6: 952. https://0-doi-org.brum.beds.ac.uk/10.3390/f13060952

Note that from the first issue of 2016, this journal uses article numbers instead of page numbers. See further details here.

Article Metrics

Back to TopTop