Molecular cloning, structural and expression profiling of DlRan genes during somatic embryogenesis in Dimocarpus longan Lour.
© Fang et al. 2016
Received: 17 November 2015
Accepted: 16 February 2016
Published: 25 February 2016
To clone and examine expression profiles of DlRan genes during somatic embryogenesis in Dimocarpus longan Lour. Thirty cDNA sequences and two genomic sequences encoding DlRan proteins were isolated from longan embryogenic cultures. Structural analysis of DlRan genes revealed that the longan Ran gene family is more expanded than that of Arabidopsis. Expression analysis of DlRan genes during somatic embryogenesis uncovered a high abundance of DlRan genes in early embryogenic cultures and heart- and torpedo-shaped embryos. The expression of DlRan genes in embryogenic calli was affected by exogenous 2,4-dichlorophenoxyacetic acid treatment. DlRan is involved in 2,4-D induced somatic embryogenesis and development of somatic embryos in longan.
KeywordsCloning Dimocarpus longan Gene expression Ras-related nuclear protein Somatic embryogenesis
Ras-related nuclear protein (Ran) is a highly conserved, small GTPase family that is essential to multiple cellular processes in eukaryotes (Clarke and Zhang 2008). The roles of Ran have been extensively researched and well documented in animals. In contrast, little is known about Ran in plants.
Plant Ran proteins share high homology and perform similar functions in the regulation of mitotic progress with their counterparts in yeast and animals (Lü et al. 2011; Lee et al. 2008; Wang et al. 2006; Zang et al. 2010). Furthermore, Ran is involved in mediating responses to external stimuli, such as heat, salt and drought stresses (Ferreira et al. 2006; Jiang et al. 2007; Xu and Huang 2008, 2010; Yoshimura et al. 2008; Zang et al. 2010). Inhibition expression of OsRan2 in rice leads to pleiotropic developmental abnormalities (Chen et al. 2011; Zang et al. 2010). These results suggest that Ran is crucial to plant growth and development.
Longan (Dimocarpus longan Lour.), an evergreen fruit tree of great commercial value, is distributed in subtropical and tropical countries (Matsumoto 2006; Zheng et al. 2009). Longan embryo development is of great scientific interest because of its role in fruit quality and yield. The developmental regulation of Ran during the middle stage of longan somatic embryogenesis (SE) implies a role for Ran in this process (Fang et al. 2011). Furthermore, Ran has been proposed as a target for breeding and production improvement in longan (Fang et al. 2014) because of its role in delaying flowering and enhancing cold tolerance in other plants (Chen et al. 2011; Wang et al. 2006). Nevertheless, cloning and characterization of longan Ran has not yet been reported.
In this study, 30 cDNA sequences and two genomic sequences encoding DlRan proteins were isolated. We analyzed the structures of DlRan genes, and investigated their expression profiles during SE and under exogenous 2,4-dichlorophenoxyacetic acid (2,4-D) treatment. On the basis of our results, we propose that DlRan is involved in cell division during longan SE and participates in 2,4-D-induced SE through signal transduction.
The establishment and maintenance of our longan embryogenic callus line “Honghezi” was described in Lai et al. (2000). The synchronization of embryogenic cultures at different developmental stages was carried out as described previously (Fang et al. 2014). All cultures were kept in dark conditions at 25 ± 1 °C.
Total RNA was extracted from embryogenic cultures using TriPure Isolation Reagent (Roche Molecular Biochemicals, Basel, Switzerland) and then treated with DNase I (Takara, China) to remove genomic DNA.
5′ and 3′ rapid amplification of cDNA ends (RACE)
Specific primers used for 3′ and 5′ RACE and corresponding products
Outer primer: RanF2
Nested primer: RanF3
Outer primer: RanF4
Nested primer: RanF5
Ran3′-3, Ran3′-4, Ran3′-5, Ran3′-6, Ran3′-7, Ran3′-8, Ran3′-9, Ran3′-10, Ran3′-11, Ran3′-12
Outer primer: RanF8
Nested primer: RanF9
Ran3′-13, Ran3′-14, Ran3′-15
Outer primer: RanR3
Nested primer: RanR4
Ran5′-1, Ran5′-2, Ran5′-3, Ran5′-4, Ran5′-5
Outer primer: RanR5
Nested primer: RanR6
Ran5′-6, Ran5′-7, Ran5′-8, Ran5′-9, Ran5′-10, Ran5′-11
Outer primer: RanR7
Nested primer: RanR8
Outer primer: RanR9
Nested primer: RanR10
Ran5′-13, Ran5′-14, Ran5′-15
Outer primer: RanR12
Nested primer: RanR13
Outer primer: RanR11
Nested primer: RanR13
Primers used in this study
Primer sequences (5′–3′)
Primer sequences (5′–3′)
CCCCTTGAAA ACCAGATAAA ATG
DNA extraction and isolation of genomic DNA encoding DlRan
Total genomic DNA was isolated from longan embryogenic calli with a Plant Genomic DNA kit (Tiangen, China). A 2389-bp DlRan DNA sequence was obtained using specific primers (RanF18 and RanR29; Table 2) and Takara LA Taq (Takara) and was designated as DlRan3A (GenBank accession no. JQ775539). The genomic sequence of DlRan3B (JQ279697) has been characterized previously (Fang et al. 2013).
Quantitative real-time PCR analysis
Primers used for qRT-PCR analysis
Primer sequences (5′–3′)
N type DlRans
D type DlRans
Treatment of embryogenic calli with 2,4-D
Embryogenic calli cultured on M0 medium (Murashige-Skoog basal salts, 2% sucrose and 6 g/L agar, pH 5.8) supplemented with 1 mg 2,4-D/l were transferred and maintained for 24 h on M0 medium or M0 medium supplemented with either 0.5, 1.5 or 2.0 mg/l of 2,4-D. All samples were frozen in liquid nitrogen after harvesting and stored at −80 °C.
Predicted protein sequences were analyzed and theoretical isoelectric points (pIs) and mass values of mature peptides were calculated using the PeptideMass program (http://us.expasy.org/tools/peptidemass.html). Amino acid sequence alignment was performed using DNAMAN software. A phylogenetic tree of Ran proteins was constructed using MEGA5 software.
Cloning of DlRan cDNAs from torpedo-shaped somatic embryos of longan
GenBank accession numbers of Ran cDNAs and primer pairs used for their amplifications
Primer pairs (forward/reverse)
First PCR: RanF10/3P
Nested PCR: RanF11/3NP
First PCR: RanF10/3P
Nested PCR: RanF11/3NP
First PCR: RanF10/3P
Nested PCR: RanF11/3NP
First PCR: RanF10/3P
Nested PCR: RanF11/3NP
First PCR: RanF10/3P
Nested PCR: RanF11/3NP
First PCR: 5P/RanR25
Nested PCR: 5NP/RanR26
Sequence analyses and molecular characterization of DlRan genes
Calculated molecular mass and predicted pI of DlRan proteins
Molecular weight (Da)
Expression analysis of DlRan genes during SE in longan
The effect of 2,4-D on expression of DlRan genes in longan embryogenic calli
Characterization of an expanded Ran gene family in longan
The Ran gene family comprises a small number of genes found in different organisms, namely one member in humans and Schizosaccharomyces pombe and four in Arabidopsis (Ma 2007; Takai et al. 2001). In this study, 30 DlRan cDNAs were cloned from torpedo-shaped embryos in longan. Alignments between DlRan cDNA sequences and genomic DNA sequences suggested the existence of more Ran genes in the longan genome. Phylogenetic analysis revealed that seven deduced DlRan proteins are closely related to Ran3 from other species. Our results suggest that the longan Ran gene family is expanded compared with Arabidopsis (Ma 2007). The estimated size of the longan genome is 444 Mb (VanBuren et al. 2011), about threefold larger than that of Arabidopsis. Nevertheless, the exact number of Ran genes in longan cannot be determined until whole genome sequencing is completed. Sequence features of the longan Ran gene family that may be unique to this species and cannot be determined until all Ran genes have been isolated from the longan genome.
Regulation of DlRan gene expression
In the present study, DlRan genes were significantly upregulated at the heart-shaped embryo stage. At the torpedo-shaped embryo stage, DlRan genes were downregulated whereas the Ran protein was rapidly upregulated. Our results indicate that the expression patterns of DlRan genes were different from that of the Ran protein identified in our previous study (Fang et al. 2011; Lai et al. 2012). Discordance between protein and mRNA expression is a common phenomenon in eukaryotic cells (Skrzycki et al. 2010; Wang et al. 2010). We speculate that unidentified post-transcriptional mechanisms participate in regulation of DlRan gene expression.
We found that changes in synonymous codon usage gave rise to mRNA secondary structure alterations among DlRan3C-1, DlRan3C-2 and DlRan3C-3. Although synonymous mutations have no effect on the resulting protein sequence, the selection of synonymous codons affects the modulation of gene expression and cellular functions (Plotkin and Kudla 2011). The differential usage of synonymous codons among these transcripts may be functional, but further tests are required to confirm this hypothesis.
Potential functions of DlRan genes during SE in longan
The involvement of Ran in longan SE has been demonstrated previously (Fang et al. 2011). Our results indicated that reduction of 2,4-D concentration in the medium, which promotes initiation of somatic embryo development, enhanced DlRan gene expression. This result further supports the involvement of DlRan in longan SE. Plant Ran is involved in cell proliferation (Lü et al. 2011; Wang et al. 2006). The sequence alignment in the present study indicates that DlRan proteins are highly conserved with respect to Ran proteins from other plants, suggesting similar functionality. Our expression analysis showed that DlRan gene transcripts are more abundant during SE stages associated with active cell division. The high expression of DlRan genes observed at heart- and torpedo-shaped stages may be related to the cell proliferation that gives rise to the cotyledons and radicle. We believe that DlRan proteins may regulate mitotic progress in a manner similar to their homologs in other plants.
2,4-D was shown to alter Ran expression when applied at different concentrations. Auxin plays pivotal roles in SE. 2,4-D, the most commonly used synthetic auxin for induction of SE (Karami and Saidi 2010), affects the indole acetic acid (IAA) synthetic pathway and promotes IAA accumulation (Michalczuk et al. 1992a, b). Ectopic postembryonic expression of LEC2 has been shown to induce somatic embryo formation (Stone et al. 2001). LEC2 has been proposed to induce SE by promoting auxin activity, and 2,4-D exerts effects similar to those of ectopic LEC2 expression (Stone et al. 2008). Su et al. (2009) have suggested that exogenous auxin levels play an important role in determining expression patterns of WUS, a correct expression of which is essential for somatic embryo induction. 2,4-D can induce SE, but also inhibits somatic embryo development (Aiqing et al. 2011). Pan et al. (2010) found that treatment with high concentrations of 2,4-D changed the proteome of Valencia embryogenic callus. Although the mechanisms involved in induction of SE by 2,4-D and the inhibitory effect of this auxin on somatic embryo development remain to be uncovered, 2,4-D functions by altering gene expression in plant cells through signal transduction. Ran is a vital regulator of nucleocytoplasmic trafficking in plants (Meier and Somers 2011; Merkle 2011). Numerous studies have detailed the involvement of Ran in plant responses to hormonal and environmental signaling (Ferreira et al. 2006; Jiang et al. 2007; Kriegs et al. 2006; Lee et al. 2008; Mahong et al. 2012; Wang et al. 2006; Xu and Huang 2010; Yoshimura et al. 2008). Ran is involved in auxin signaling (Wang et al. 2006) and it is unsurprising to find that Ran expression is influenced by 2,4-D. 1 mg 2,4-D/l is necessary to maintain longan calli at embryogenic state, remove or reduce the concentration of 2,4-D initiates the development of somatic embryos. Nucleocytoplasmic transport and cell division are essential during the formation of somatic embryos. It is reasonable that the expression of Ran was enhanced by reducing the concentration of 2,4-D. Properly increasing the concentration of 2,4-D promote the proliferation of longan calli and improve the expression of Ran. However, 2 mg 2,4-D/l inhibit the growth of longan calli and cause browning, which can explain the repression effect of 2 mg 2,4-D/l on Ran level. Our results further support the involvement of Ran in auxin signal transduction. Zang et al. (2010) have suggested that Ran participates in abiotic response signaling by modulating the nuclear transportation of proteins and RNA. Taking the results of these studies and ours into consideration, we speculate that DlRan may participate in 2,4-D-induced SE by transmitting 2,4-D signals and may regulate the expression of embryogenesis-related genes by controlling nuclear trafficking.
In this study, 30 cDNA and two genomic DNA sequences of DlRan genes were isolated. We also revealed the expression profiles of DlRan genes during SE and under exogenous 2,4-D treatment. Our results suggest the importance of DlRan genes in longan embryo development. Future research should focus on the elucidation of mechanisms involved in regulation of DlRan gene expression and the functions of different DlRan genes during SE in longan.
ZF and YL conceived and designed the experiments. ZF, CL and YZ performed the experiments. ZF, CL and ZL analyzed the data. ZF and ZL wrote the paper. All authors read and approved the final manuscript.
This work was funded by the National Natural Science Foundation of China (31272149 and 31572088) and Fujian provincial Major Special Project of Agricultural science and technology (2015NZ0002-1).
The authors declare that they have no competing interests.
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