MOXEKymPHÁa EHomma, 2010, moM 44, № 5, c. 786-791
= TEHOMHKA, TPAHCKPHnTOMHKA =
UDC 577.21
ISOLATION AND CHARACTERIZATION OF STRESS INDUCED Ty 1-copia LIKE RETROTRANSPOSABLE ELEMENTS IN Chickpea (Cicer arietinum L.)
© 2010 M. K. Rajput*, K. C. Upadhyaya
School of Life Sciences, Jawaharlal Nehru University, New Delhi 110067, India Received December 21, 2009 Accepted for publication February 16, 2010
Almost all active plant retroelements are known to be induced by various biotic and abiotic stresses. Here, we show the presence of transcriptionally active Tyl-copia like retroelements in chickpea and their induction in response to abiotic stress. Eight Tyl-copia retrotransposon like mRNA sequences were reverse transcribed, amplified, cloned and characterized from stressed plants. These mRNA sequences were not detected in chickpea plants grown under normal conditions. Basing on the similarity analysis, these RT transcript sequences were classified into three families. It is proposed that all sequences except CARE7 might be transcript sequences of functional retrotransposons. The mRNA sequence CARE3 shows 99% nucleotide identity to a genomic Tyl-co-pia like sequence present in the Genbank with accession no. AJ535883.
Key words: Cicer arietinum, retrotransposon, reverse transcriptase, stress, Tyl-copia.
Retrotransposons dominate not only plant but almost all eukaryotic genomes. These genetic elements replicate via RNA intermediates and increase the genome size by their amplification and insertion into new genomic sites. They are classified into LTR and non-LTR retrotransposons depending on the presence or absence of long terminal repeats (LTRs) at their termini. Internally retrotransposons contain gag (group antigen) and Pol (polymerase) genes. The polyprotein encoded by the pol gene can be processed into reverse transcriptase, protease, RNaseH and integrase or en-donuclease proteins [1]. The LTR-retrotransposons are divided into Tyl-copia and Ty3-gypsy groups. The integrase domain is upstream of the reverse tran-scriptase domain in the copia group and down stream of it in the gypsy group [2]. Retrotransposons are quiescent during normal plant development but some of them get activated in response to various biotic and abiotic stresses. The stress induced transcriptional activation of well-characterized plant retroelements is tightly linked to the molecular pathways activated by stress, and their activation is under the control of the cis-regulatory sequences similar to those of plant defense genes [3]. Stress induced activation of plant retrotransposons may be a contributory factor in soma-clonal variations, and it may be utilized to isolate new active elements [4]. In this way these elements are possibly associated with stress alleviation phenomena in plants [4, 5]. Since these elements transpose by the
copy-and-paste mechanism they have been implicated in genomic expansion during evolution [6, 7].
The presence of retroelements in high copy numbers in heterogeneous populations, their dispersion throughout the genome and their insertion into new genomic sites without losing the parental copies are some of the properties of the retroelements which make them particularly suitable candidates for generating molecular markers for DNA finger printing, genetic linkage mapping and phylogenetic analysis for a variety of crop plants. Besides most retrotransposon insertions are irreversible, therefore, changes remain relatively fixed, which is good attribute for analyzing phylogenetic relationships [1, 7, 8].
With respect to retrotransposon research chickpea has received little attention. Only a complete Ty3-gyp-sy-like retrotransposon, two Ty3-gypsy-like sequences and a few Ty1-copia retrotransposon-like sequences have been reported [9—12]. In this study we report partial transcripts of Ty.1 -copia--like retrotransposons from chickpea, which are induced in response to desiccation.
EXPERIMENTAL
Seeds of the chickpea variety Pusa 362 were grown in the soil in November, which is the most favorable season for it in New Delhi, India. Ten days old seedlings were subjected to abiotic stress (desiccation
Abbreviations: cAF — copia active family; CARE — Cicer arietinum retroelement; LTR — long terminal repeats; ORF — open reading frame; RT — reverse transcriptase.
* E-mail: mkrajput@hotmail.com
stress) by keeping them at 25°C for 5 days without water to get a state between temporary and permanent wilting. Total RNA from the stressed and normal plants was extracted using RNeasy Plant mini kit ("Qiagen", Germany). To eliminate the possibility of genomic DNA contamination, the RNA samples were treated with the RNase free DNase I as according to the manufacturer's instructions. The concentration of RNA samples was determined by the following formula: Absorption at 260 nm x 40 (^g/mL) x dilution factor. The integrity of RNA in the samples was confirmed by 1.2% (w/v) MOPS-agarose gel electrophoresis [13].
For reverse transcription and amplification the conserved regions DVKTAF and YVDDMDP of the reverse transcriptase of copia-like retrotransposons were selected as forward and reverse primers [14, 15]. RT-PCR was carried out using "Promega's" Access RT-PCR system with modifications as follows: 1 ^g RNA-template, 1.5 mM MgCl2, 200 nM of each of the primers (5'-GGGATC-CAYRTCRTCNACRTANARNA-3' and 5'-ATTC-GAYGTNAARCANGCNTTYYT-3'). The reverse transcription and amplification were performed in a MJ Research thermal cycler with the following parameters: 40°C for 40 min, 94°C for 5 min, followed by 35 cycles of 94°C for 1 min, 48°C for 1 min and 72°C for 1 min, followed by a final extension step at 72°C for 10 min. The amplified products were eluted from the 1.5% (w/v) TAE-Agarose gel [12] and cloned into a pGEM-Teasy vector ("Prome-ga"). The nucleotide sequence was determined by using an automated DNA sequencer ("Applied" Biosystems).
Pair-wise and multiple sequence alignments were carried out using ClustalW (1.83) (http://www.ebi.ac.uk/ clustalw) tools [16]. The sequences were compared using the Feng and Doolittle [17] method and the ORFs were determined by using the NCBI ORF finder tool at the following URL — http://www.ncbi.nlm.nih.gov/gorf/ gorf.html.
RESULTS AND DISCUSSION
Several Ty1-copia retrotransposon-like genomic sequences have been reported in chickpea [10, 11] but this is the first report on the isolation and characterization of stress induced Ty1-copia retrotransposon-like mRNA sequences in chickpea. The transcript sequences of reverse transcriptase conserved domains of the Ty1-copia group of retrotransposons from desiccated chickpea plants were isolated by RT-PCR. The expected amplicon of ~280 bp was purified from the agarose gel and cloned into a pGEM-Teasy vector (Fig. 1). After sequencing, eight reverse transcriptase sequence clones were detected, named and submitted to Genbank to obtain accession numbers (Table 1). In addition to a ~280-bp amplicon, two bands of higher
M S
Fig. 1. Reverse transcription and amplification of stress induced Ty1-copia retrotransposon-like sequences from chickpea. Lane S contains RT-PCR products. Arrow indicates Ty1-copia reverse transcriptase specific cDNA band (~280 bp). Other high molecular weight bands are nonspecific amplifications. Lane M contains 100-bp DNA ladder.
molecular weights (~450-bp and ~600-bp) were also observed in the gel. These high molecular weight products were also cloned and sequenced. However, the sequences of these amplicons showed homology to the protein kinase-like sequences. Such amplification might be due to the degeneration of the priming oligo-nucleotides. No reverse transcription-amplification was detected from the RNA isolated from the plants grown under normal conditions.
Stress induced activation
Detection of RT specific RNA sequences in desiccated plants and their absence in normally grown plants show that the Ty1-copia-like retrotransposons in chickpea can be activated in response to abiotic stress. This is not surprising because abiotic stresses have been reported to activate some plant retrotrans-posons [3, 18—20]. The first active plant retrotranspo-son Tnt1 was isolated from tobacco by screening millions of protoplasts [21]. Interestingly, the fungal extract which was used to prepare the protoplast was responsible for the induction of Tnt1. Hirochika [4] designed a protocol to isolate copia-like active retro-elements. This procedure involves using the reverse transcription polymerase chain reaction (RT-PCR)
MO^EKyraPHAtf EHOnoraa TOM 44 № 5 2010
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Table 1. Classification of the Tyl-copia group retrotransposon reverse transcriptase mRNA sequences on the basis of their identities. cAF — copia active family
Name of family Members No. of members Intra-family identity (a.a), % Inter-family identity (a.a), % Intra-family identity (ntd), % Inter-family identity (ntd), % Accession no.
cAFl CARE2 CARE4 CARE5 CARE8 CARE9 5 76-93 18-49 95-97 53-61 AJ544276 AJ544278 AJ544279 AJ888305 AJ888306
cAF2 CARE3 CARE6 2 63 31-49 88 52-89 AJ544277 AM182261
cAF3 CARE7 1 - 18-39 - 53-89 AJ544280
CARE — Cicer arietinum retroelement.
Table 2. Reverse transcriptase sequences of Tyl-copia group retrotransposons used for phylogenetic analysis
Accession no./sequence Organism
AAT90482 Vigna radiata
AAF37865 Ipomoea batatas
AF231939 Ipomoea batatas
BAA11674 Nicotiana tabacum
CAJ09744 Camellia sinensis
DQ100159 Camellia sinensis
CAA13065 Solanum tuberosum
ABD19095 Phelipanche tunetana
X13777 Nicotiana tabacum
AAG44306 Aegiceras corniculatum
AAC34608 Lycopersicon esculentum
CAD11851 Brassica oleracea
AJ414059 Brassica rapa
ABD19043 Phelipanche bungeana
AAT73707 Populus ciliate
AAG44354 Spiranthes sinensis
AF416818 Musa acuminate
AY654426 Citrus sinensis
AC087545 Oryza sativa
DQ105074 Malus x domestica
AJ535857 /CART37 Cicer arietinum
AJ535859 /CART50 Cicer arietinum
AJ535883 /CART157 Cicer arietinum
AJ535894 /CART366 Cicer arietinum
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