Advances in Applied Agricultural Sciences 2 (2014); 09: 54-63
Alignment and phylogeny studies of rolC gene of Agrobacterium rhizogenes and their cellular homologues in plants
Joshua P.V. 1, SalomiSuneetha D.R. 2* and Arundhati A. 3
1 Deprtment. of Biotechnology, Dr. SVKP & Dr.KS Raju Arts & Science College, Penugonda, India. 2 Horticultural College and Research Institute, Dr. YSR Horticultural University, Venkataramannagudem, India. 3 Deprtment. of Botany, Andhra University, Visakhapatnam, India.
The focus of the present study is the comparative analysis of rolC gene of Agrobacterium rhizogenes and its cellular homologues present in plants. The occurrence of rolC gene was identified in different species and varieties of Nicotiana, Kalanchoe and Klenia. Initially, the presence of rol genes in plants was identified from the preliminary studies on severe morphological variations, tumorigenesis, rhizogenesis and phytohormone independent growth under culture conditions. The molecular study for the presence or absence of rolC gene homologues in plants was carried out by the PCR amplification of their genomic DNA using rolC gene of A. rhizogenes as primer. The amplified fragments were sequenced and deposited in GenBank. In the present analysis, the rolC gene present in different strains of A. rhizogenes, the novel Genbank accepted rolC gene sequences and their homologous sequences extracted from Genbank were pooled. Multiple sequence alignment and molecular phylogeny were carried out for these nineteen rolC sequences. Results were discussed in terms of the presence of bacterial oncogenes in plants, the donor strain for these genes, the homology, variations and their molecular phylogeny.
Agrobacterium rhizogenes is one of the important bacterial genera present in plants. The presence of cellular T-DNA genes in plants represents an ancient horizontal transfer of genes between plants and an ancestor of A. rhizogenes (Furner et al., 1986; Christey and Braun, 2005). Nicotiana is one of the common genera containing the bacterial oncogenes of Agrobacterium rhizogenes (Joshua et al., 2009). In tobacco genome integration of T-DNA is through insertion at double stranded breaks by nonhomologous end joining (Chilton and Que, 2003).
In general, the manifestation of Ri plasmid (rol) genes are dwarfed phenotype, reduced apical dominance, smaller wrinkled leaves, increased rooting, altered flowering and reduced fertility (Casanova et al., 2005). The presence of rol genes in N. glauca x N. langsdorffii hybrid plants showed tumorigenesis, severe morphological variations and phytohormone independent growth (Suneetha et al., 2006) i.e the ability of cells to grow on MS basal medium (Murashige and Skoog, 1962) without exogenous supplementation of phytohormones.
rol gene homologous sequences are known to be present in several members across different families of the plant kingdom; some of the families include Solanaceae, Crassulaceae, Asteraceace and the species of the genera Nicotiana, Petunia and Daucus (Frundt et al., 1998). The members of Nicotiana include N. glauca x N. langsdorffii hybrid (Suneetha et al., 2006), N. tabacum var. Samsun, N. undulata (Joshua et al., 2009), N. rustica, N. plumbaginifolia, N. rustica x N. plumbaginifolia hybrid, Kleinia amaniensis, Kalanchoe daigremontiana and Kalanchoe blossfeldiana show the presence of rol genes but they differ in the combination of genes present. The rol gene oncogenes of A. rhizogenes not only act as the modulators of plant growth and cell differentiation but are also potential activators of secondary metabolism in transformed cells via uncommon signal transduction pathways as studied in the members of Solanaceae, Araliaceae, Rubiaceae, Vitaceae and Rosaceae families (Bulgakov, 2008).
Expression of rolC gene was employed in floriculture in improving the ornamental and horticultural traits (Casanova et al., 2005) specifically, rose (Souq et al., 1996), lily (Mercuri et al., 2003) and carnation plants. rolC gene expression resulted in 15 fold increase of anthraquinones and increase in the content of arabinogalactan (AG) in the transformed calli of Rubia cordifolia (Shkryl et al., 2008; Giunter et al., 2013), production of 25 tropane alkaloids in transgenic Datura innoxia (Jousse et al., 2010) and in molecular breeding towards compact growth as studied in Kalanchoe blossfeldiana (Christensen et al., 2008). They played a very important role in modulation of stress resistance of transformed cells, changed sucrose metabolism, and the inhibition of programmed cell death (Bulgakovet al., 2013).
The present work is an effort towards the sequencing of cellular rolC gene sequences in different members of Nicotiana, Kalanchoe and Klenia. Comparative analysis between rolC gene sequences of prokaryotic origin and their eukaryotic homologues present across different families and genera and to study their molecular phylogeny.
Materials and Methods
DNA isolation, amplification and sequencing
The work was carried out in the Department of Biochemistry, Andhra University, Visakhapatnam. Genomic DNA was extracted from fresh tender leaves of Nicotiana tabacum var.Samsun, Nicotiana rustica, Nicotiana plumbaginifolia, Nicotiana rustica x Nicotiana plumbaginifolia, Nicotiana undulata, Kalanchoe blossfeldiana, Kalanchoe daigramontiana, Klenia amanuensis using Dellaporta method of plant DNA extraction (Dellaporta et al., 1983). The extraction procedure includes the reaction with extraction buffer 1 M Tris HCl (pH 8.0), 0.5 M EDTA (pH 8.0), 5M NaCl, 10 µM β – mercaptoethanol, 1% Polyvinyl pyrrolidone), 20% SDS at 65ºC, precipitation of protein with 5 M potassium acetate, precipitation of DNA with iso-propanol and suspension of crude DNA in resuspension buffer1 (100 mM EDTA and 250 mM Tris HCl). The DNA was purified using RNase, phenol:chloroform:isoamylalcohol (25:24:1), precipitated with 3 M sodium acetate, chilled ethanol and resuspended in resuspension buffer2 (50 mM EDTA and 100 mM Tris HCl). The concentration and purity of DNA were estimated using UV-VIS spectrophotometer (Systronics, Model-117).
DNA amplification reaction was carried out in Appendorf’s master cycler using the genomic DNA of Nicotiana tabacum var. Samsun, Nicotiana rustica, Nicotiana plumbaginifolia, Nicotiana rustica x Nicotiana plumbaginifolia, Nicotiana undulata, Kalanchoe blossfeldiana, Kalanchoe daigramontiana, Klenia amaniensisas templates and 18mer sequences of rolC genes of A. rhizogenesas primers (Biotech Desk Pvt. Ltd),Taq DNA polymerase and its buffer with MgCl2 and dNTP mix.
Table 1: List of rolC sequences considered for Multiple Sequence Alignment
Source of the rolC gene/cellular homologue
Nicotiana tabacum var.Samsun
Nicotiana rustica x Nicotiana plumbaginifolia
Agrobacterium rhizogenes pRi1724 DNA
Agrobacterium rhizogenes strain A4
Agrobacterium rhizogenes strain pRiA4
Panax ginsengrolC 4- Pq
Panax ginsengrolC2- Pq
Agrobacterium rhizogenes pRi1724 DNA
Agrobacterium rhizogenes pRi1724 DNA
The rolC primer sequences are as follows
Thermal profile was as follows: Initial denaturation at 94ºC for 4 min, Denaturation at 94ºC for 1 min, Annealing at 59ºC for 1 min and Extension at 72ºC for 1.5 min – this reaction was repeated for 30 cycles; Final extension at 72ºC for 10 min. Amplified fragments of DNA were separated on gel electrophoresis using 1.2% agarose gel and the size of the fragments were measured with reference to 100 bp ladder.
Specific PCR amplified fragments of the size of rolC genes were gel eluted from low melting agarose gel (1%) using elution buffer(1 M Tris-Cl, 0.5 M EDTA; pH 8.0), precipitated using 10 M ammonium acetate, dissolved in 0.1 M Tris-Cl (pH 8.0) and were sequenced utilizing the services of MWG Biotech Pvt. Ltd., Bangalore, India. The sequences were extracted in Fasta format using Chromas software downloaded from http://www.technelysium.com.au/chromas.html
The sequences of cellular rolC genes present in Nicotiana tabacum var. Samsun, Nicotiana rustica, Nicotiana plumbaginifolia, Nicotiana rustica x Nicotiana plumbaginifolia, Nicotiana undulata, Kalanchoe blossfeldiana, Kalanchoe daigramontiana, Klenia amanuensis were submitted to GenBank of NCBI (National Centre for Biotechnology Information) using BankIt tool (www.ncbi.nlm.nih.gov/websub/tool) for further evaluation of authenticity, novelty of the sequences and for accession numbers.
Multiple Sequence alignment (MSA)
The rolC genes of Agrobacterium rhizogenes and their cellular homologues were extracted from Genbank database (www.ncbi.nlm.nih.gov) 194.0 release of February 2013. The sequences were categorized as (i) sequences from different strains of A. rhizogenes, (ii) sequences isolated, sequenced in the laboratory and deposited to GenBank and (iii) the sequences of cellular homologues already present in the database. Altogether, 19 sequences for rolC were considered for the present study (Table 1).
The rolC gene sequences were labeled in Fasta format. The abbreviated notation of the rolC gene sequence, the source and the accession number were given in table 1. MSA was carried out using default Gonnet Matrix of Clustalw2 tool maintained at EBI (http://www.ebi.ac.uk/). The alignment between the rolC genes of A. rhizogenes and the cellular rol gene homologues, conservation pattern and the variations were studied.
The phylogenetic relationship was studied for the nineteen above said rolC gene sequences using the cladogram constructed by following Neighbor Joining method as implemented in ClustalW2.
Results and Discussion
Plants have unique behavior of coexisting with microorganisms, especially with Agrobacterium rhizogenes. Several members of the plant kingdom like Kalanchoe, Petunia, Daucus and 15 out of 42 species of Nicotiana so far studied were found to contain the TL-DNA genes (Intrieri and Buiatti, 2001). In the present study, the cellular rolC genes were identified amplified and sequenced for the first time in different members of Nicotiana and the homology with bacterial rolC gene was compared.
PCR amplification, sequencing and Genbank accession numbers
The DNA isolated from the leaf samples of Nicotiana tabacum var.Samsun, Nicotiana rustica, Nicotiana plumbaginifolia, Nicotiana rustica x Nicotiana plumbaginifolia, Nicotiana undulata, Kalanchoe blossfeldiana, Kalanchoe daigramontiana, Klenia amaniensis, it’s purity was checked and quantified. Presenceof rolC gene in plant genome was confirmed by the amplification of 260 bp which was equal to the size of bacterial rolC gene. The product of rolC gene is a β-glucosidase which hydrolyzes cytokinin N- and O-glucosides and releases the active form of cytokinin from inactive conjugates, which alter the endogenous auxin/cytokinin balance (Estruch et al., 1991). The amplified fragments were sequenced and were found to be highly homologous with the rolC gene of Agrobacterium rhizogenes. The sequences deposited to NCBI-Genbank received accession numbers (GU182970; GU182971; GU182972; GU182973; GU182974; GU182975; GU265856; GU182976) as listed in Table 1.
Fig. 1. Multiple sequence alignment of rolC gene of A. rhizogenes and its cellular plant homologues
Multiple Sequence alignment
The alignment included a total of nineteen sequences – five sequences from different strains of Agrobacterium rhizogenes (Ar_rolC1 to Ar_rolC5) and fourteen cellular rolC gene sequences from plants (Fig. 1). The cellular rolC gene homologues were members from different families viz., Solanaceae, Crussulaceae, Brassicaceae, Asteraceae (Furner et al., 1986; Frundt et al., 1998). Among them, the majority of rolC gene homologues were from the species and varieties of the genus Nicotiana. In plants, eight sequences are from different members of Nicotiana (Nd_rolC, Nc_rolC, Nts_rolB, Nr_rolB, Nu_rolC, Np_rolC, Nrp_rolC and Ng_rolB), two sequences from Panax (Pq_rolC1, Pq_rolC), four sequences one each from different plants Ka_rolC, Kb_rolC, Kd_rolC and Cr_rolB. The length of nucleotide sequence considered for study was ranging from 417 bp – 460 bp. Total number of stars include 108, which represent the conserved base pairs throughout the alignment.
Fig.2. Cladogram of rolC gene of A. rhizogenes and its cellular plant homologues
The rolC gene sequences showed very high scores (80-100) irrespective of their occurrence in prokaryotic or eukaryotic cells, with different conservation patterns.
Basing on the alignments 19 rolC gene sequences were divided into three groups. First group included the top five sequences – two (Nd_rolC and Nc_rolC) sequences were homologous to Ar_rolC3, 4 and 5 with the scores in the range of 82-99%. The scores among the Ar_rolC 3, 4 and 5 was 100% representing that they were completely homologous. These sequences were from the rolC of Agrobacterium rhizogenes pRi1724. Nd_rolC and Nc_rolC (Nicotiana debneyi and Nicotiana cordifolia) were similar in their alignment except few substitutions (Transitions and Transversions -G to A, C with T, A to T) with rolC of Agrobacterium rhizogenes pRi1724.
Second group included the middle twelve sequences from Nts_rolC to Nrp_rolC, which were aligned with Ar_rolC1 (A. rhizogenes strain A4) and with scores in the range of 99-100%. This group involves all the novel genes sequenced in this study. They showed maximum conservation among the three groups except few mutations occurred in Nicotaina tabacum var. Samsun, N. rustica, N. rustica x N. plumbaginifolia and Kalanchoe blossfeldiana. Single nucleotide addition and deletion mutations were commonly observed, but an insertion of three nucleotides (AAT) and four nucleotides (CGGC) was also recorded in N. rustica x N. plumbaginifolia. The 1st and 2nd groups were very similar at several sites of the alignment. The 3rd group involved only two sequences which were formed between Ng_rolC and Ar_rolC2 (N. glauca and (pRiA4). N. glauca showed several substitution mutations when compared with the other groups.
The phylogenetic relationship was studied among 19 sequences of rolC (Fig. 2). The construction was based on the distances between the sequences, the sequences with minimum distance were considered as nearest neighbors and the results were viewed as cladogarm with Java supporting software. The cladogram of rolC showed two distinct nodes – a small node with three rolC sequences Kalanchoe daigremontaina and Nicotiana undulata were homologous and Nicotiana plumbaginifoliawas diverged from them. A. rhizogenes rolC1 (Strain A4) was their bacterial nearest neighbor.
The 2nd was a big node consisting of ten sequences with four arms. Ar_rolC3, Ar_rolC4 and Ar_rolC5 (pRi1724) formed an arm representing that they were identical and showing their common origin. Nicotiana debneyi and Nicotiana cordifolia were diverged from them and were the nearest neighbors for them. N. glauca shared homology with Ar_rolC2 (pRiA4). N. rustica x N. plumbaginifolia diverged from these sequences. Nicotaina tabacum var. Samsun was homologous to Kalanchoe blossfeldiana which formed an out group from Ar_rolC1 (strain A4). The rolC sequences of Kalanchoe daigremontaina and Nicotiana undulata were conserved forming an arm and N. plumbaginifolia was diverged from them. No divergence was observed between Ar_rolC1 (Strain A4) and Nicotaina rustica. The cellular homologues which contain the rol genes through artificial transformation (Catharanthus roseus, Pinus guiseng, synthetic construct) did not show any divergence from the donor strain, hence showed no distance in the molecular phylogeny.
The present study facilitated to identify the presence of rolC gene in N. tabacum var. Samsun, N. undulata, N. rustica, N. plumbaginifolia, N. rustica x N. plumbaginifolia, Kalanchoe daigremontiana, Kalanchoe blossfeldiana and Kleinia amaniensis. Eight novel bacterial rolC genes present in plants were PCR amplified and the fragments were sequenced, their unique accession numbers were received from GenBank database. Their multiple sequence alignment and molecular phylogeny assisted to study the similarities, differences and divergence patterns among the prokaryotic rolC gene sequences and their eukaryotic cellular homologues. A. rhizogenes Agropine strain A4 was commonly observed with its rolC gene sequences showing a close similarity with the cellular rol genes. Hence, A. rhizogenes Agropine strain A4 could be the donor strain for the entry of T-DNA into the genome of different species and varieties of Nicotiana and the members of succulents.
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