научная статья по теме CHARACTERIZATION OF HETEROGENEITY IN TY1-COPIA GROUP RETROTRANSPOSONS IN CHICKPEA (CICER ARIETINUM L.) Биология

Текст научной статьи на тему «CHARACTERIZATION OF HETEROGENEITY IN TY1-COPIA GROUP RETROTRANSPOSONS IN CHICKPEA (CICER ARIETINUM L.)»

MOXEKymPHÁa EHomma, 2010, moM 44, № 4, c. 601-607

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UDC 577.21

CHARACTERIZATION OF HETEROGENEITY IN Tyl-copia GROUP RETROTRANSPOSONS IN Chickpea (Cicer arietinum L.)

© 2010 M. K. Rajput*, K. C. Upadhyaya

School of Life Sciences, Jawaharlal Nehru University, New Delhi 110067, India Received September 01, 2009 Accepted for publication October 21, 2009

Retrotransposons represent a major fraction of the plant genome and play a significant role in the molecular evolution through sequence re-organization. In order to access the diversity in Tyl-copia group of retrotransposons in chickpea, reverse transcriptase (RT) conserved domain specific primers were selected to amplify RT conserved sequences. Thirty-six amplified fragments were cloned and characterized. On the basis of deduced amino acid homology among them, these sequences were grouped into five families. These sequence families showed from 34 to 8l% inter-family homology at the amino acid level. Although these sequences belong to a highly conserved region no two sequences were identical. The results show that there is a high degree of heterogeneity among the Tyl-copia group of retroelements in chickpea. The genomic Southern hybridization with one of the reverse transcriptase sequences as a probe shows the presence of a large population of the Tyl-copia group of retrotransposons in chickpea.

Key words: retrotransposon, Tyl-copia, reverse transcriptase, chickpea, Cicer arietinum.

Retrotransposons make up a large portion of the plant genome ranging from 6% in Arabidopsis to over 70% in maize. On the basis of the presence or absence of long terminal repeats (LTRs) at their termini, retrotransposons are classified into LTR and non-LTR retrotransposons. Retrotransposons generally contain gag (group antigen) and pol genes. The pol gene harbours many domains viz. protease, integrase, reverse transcriptase (RT) and RNaseH. Depending on the arrangement of the reverse transcriptase and integrase domains, the LTR-retrotransposons are divided into Tyl-copia and Ty3-gypsy groups [1]. In copia-like retrotransposons, the integrase or the endonuclease domain is positioned upstream of the reverse tran-scriptase domain, while in the gypsy type it is downstream of the reverse transcriptase domain, but functionally they are both similar. All known active plant retrotransposons are quiescent during normal development but some of them can get activated tran-scriptionally as well as transpositionally in response to various biotic and abiotic stresses [2]. These elements are reported to play an important role in stress alleviated phenomenon in plants [3].

Retroelements have also been implicated in ge-nomic expansion during evolution [4]. The "C-value-paradox", i.e. the non-correspondence of the structural complexity to the functional complexity in or-

ganisms, could be explained by the amount of retro-elements in the genome [5]. The heterogeneous populations of retrotransposons have been reported in various plant species [6—9]. Due to their heterogeneous populations with a high copy number and dispersal throughout the genome, retrotransposons are now being utilized as molecular markers in DNA fingerprinting, genetic linkage mapping and phylogenet-ic analysis [10].

Chickpea (Cicer arietinum L.) is one of the most important legume crops in the Indian subcontinent and ranks third in the world for pulse production. This self-pollinating annual crop with a somatic chromosome number of 2n = 16 has a genome size of ~931 Mb [11]. Such a large genome is expected to have a significant fraction of retroelements. In order to characterize the heterogeneous population of the copia group of retrotransposons in chickpea, we have isolated genomic reverse transcriptase (RT) sequences by PCR using primers designed from the conserved domains of the RT region.

EXPERIMENTAL

DNA from chickpea variety Pusa 362 was isolated using standard procedures of Ausubel et al. [12]. Two reverse transcriptase conserved domains (DVKTAF

Abbreviations: RT — reverse transcriptase; CART — Cicer arietinum retrotransposon; cF — copia family; LTR — Long Terminal Repeat; ORF — open reading frame.

* e-mail: mkrajput@hotmail.com

-pol-

5'LTR gag protease Integrase RT RNaseH 3'LTR

DVKTAF YVDDMDP

Fig. 1. Typical structure of a Tyl-copia retrotransposon. The arrows indicate the positions of the forward and reverse primers used to amplify conserved reverse transcriptase domains.

and YVDDMDP) were selected for forward and reverse primers for PCR amplification [6, 13]. PCR was performed in 50 pi, with 500 ng of the genomic DNA, 100 pM of each of the dNTPs, 1.5 mM MgCl2, 50 pmol of each of the primers (5'-GGGATC-CAYRTCRTCNACRTANARNA-3' and 5'-ATTC-GAYGTNAARCANGCNTTYYT-3') and 2 U of the Taq DNA polymerase ("Promega"). The thermal cycling was performed in a MJ Research thermal cycler with the following parameters: 94°C for 5 min, followed by 35 cycles of 94°C for 1 min, 47°C for 1 min and 72°C for 1.5 min, then followed by a final extension step at 72°C for 10 min. The expected amplified product was eluted from the 1.5% (w/v) TAE-Agarose gel and cloned into a pGEM-Teasy vector ("Promega"). The nucleotide

sequence was determined by using an automated DNA sequencer ("Applied Biosystems").

Chickpea genomic DNA was digested with appropriate enzymes (EcoRI or Hindlll), fractionated on a 0.7% (w/v) TAE-Agarose gel and blotted onto a Hy-bond N+ nylon membrane ("Amersham"). The hybridization was performed under conditions ofhigh stringency using the clone CART77 sequence as a probe and following the methods of Sambrook et al. [14].

Pair-wise and multiple sequence alignments were carried out using clustalW (1.83) (http://www.ebi.ac.uk/clustalw) tools [15]. The sequences were classified according to the method of Feng and Doolittle [16] 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.

M A

Fig. 2. Amplification of Tyl-copia retrotransposon-like sequences from chickpea genome. Lane A contains Tyl-copia RT specific amplification products. Arrow indicates the expected ~280 bp band. Lane M contains 100-bp DNA ladder.

RESULTS AND DISCUSSION

Structurally the Ty1-copia group of retrotrans-posons contains 5'- and 3'-LTRs flanking an internal gag-pol region. The pol gene contains many conserved domains but the reverse transcriptase (RT) domain of the pol gene is highly conserved in various plant species. This conserved domain has been selected to design degenerate oligonucleotide primers in order to amplify the conserved RT sequences from a large spectrum of the copia group of elements [13]. Further, these reverse transcriptase conserved domains have been used to characterize the heterogeneity and phylogeny among the copia group of retrotransposons in many plant species. In this study, the degenerate oligonucleotide primers corresponding to the DVKTAF and YVDDMDP regions were used to amplify the conserved RT domains (Fig. 1). An amplicon of ~280 bp was obtained from the chickpea genome using polymerase chain reaction (Fig. 2). Considering the examples of other angiosperms it was hypothesized that the am-plicon would contain heterogeneous populations of RT sequences from a wide spectrum of copia-like elements [4, 5, 17]. Therefore, these amplified sequences were purified from the gel and cloned into a pGEM-Teasy vector. Sequences of 36 independent clones were determined, named and submitted to the

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Fig. 3. Alignment ofthe deduced amino acid sequences corresponding to the reverse transcriptase domain (RT) of the Ty1-copia group re-trotransposons in chickpea. Dashes (-) show gaps, stars (*) denote identical, colons (:) denote conserved and semicolons (;) denote semi-conserved amino acid residues in all sequences. Numerals on the right are the number of amino acid residues in the sequences.

GenBank to obtain accession numbers (Table 1). Two copia reverse transcriptase-like sequences from chickpea have been reported by Sant et al. [18], but

both of these elements do not show any significant homology to the elements reported in the present study because the priming regions in the two studies

Table l. Accession numbers of the Ty1-copia group reverse transcriptase sequences of chickpea reported in this study

S. no. Name of Clone Accession no. S. no. Name of Clone Accession no.

1 CART 9 AJ535750 19 CART 120 AJ535874

2 CART 14 AJ535875 20 CART 123 AJ535877

3 CART 33 AJ535856 21 CART 126 AJ535878

4 CART 37 AJ535857 22 CART 135 AJ535879

5 CART 40 AJ535858 23 CART 141 AJ535881

6 CART 50 AJ53

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