Isolation of Retroelement from Plant Genomic DNA (PCR法从植物寻找逆转录元件)

Retroelements and their derivatives are a ubiquitous and abundant component of plant genomes. From the 1990s, PCR based techniques have been developed to isolate the elements from genomic DNA of different plants, and the methods and primers used are presented here.

Major classes of retroelements include the Pseudoviridae (Ty1-copia ), the Metaviridae (Ty3 -gypsy) and the Retroposineae LINE (non-LTR) groups. All reverse transcribing elements can be included in a universal classification which is presented on a linked page . Mixed PCR products representing the full heterogeneous pool of retrotransposons from each group are obtained by PCR with degenerate primers ( degenerate nucleotide symbols link), as listed below. Some of the species tested are also listed, but experience of us and many others is that the primers succeed in most or all cases. Non-degenerate primers for amplifying individual transposons are obtained from sequences in the databases, and of course can be used at lower annealing temperatures to isolate elements. PCR protocols and programmes are given at the bottom of the page.

Revision 20-05-2005: Added New Paper Hill, Flavell et al. and emphasized Pseudo/MetaViridae (not Pseudovirideae or Metavirideae which would refer to another level of classification of retroelements or retrotransposons)12/7/07: reduced amount of Taq polymerase recommended (we use enzyme from YorkBio, BioLine or Promega - the source of the enzyme does make a difference to the results).

Pseudoviridae ( Ty1-copia) Degenerate Primers

These work for virtually every higher plant species (tested in over 75 species, failed in one). Upstream Primer: 5'ACNGCNTTYYTNCAYGG encoding TAFLHG Downstream Primer: 5'ARCATRTCRTCNACRTA encoding YVDDML (in reverse). These two primers, from Flavell et al. (1992 a and b below), amplify a band of approximately 270bp. The primer pair 5'CARATGGARGTNAARAC encoding QMDVKT and 5'CATRTCRTCNACRTA encoding YVDDM (missing the L at the end of the Flavell primers) is used by Hirochika and Hirochika (1993 below).

Original papers are by three groups at about the same time: Flavell AJ, Smith DB, Kumar A. 1992 Extreme heterogeneity of Ty1-copia group retrotransposons in plants. Mol Gene Genet 231: 233-242 Flavell AJ, Dunbar E, Anderson R, Pearce SR, Hartley R, Kumar A. 1992. Ty1-copia group retrotransposons are ubiquitous and heterogeneous in higher plants. Nucleic Acids Res. 20: 3639-3644.

Voytas DF, Cummings MP, Konieczny A, Ausubel FM, Rodermel SR. 1992. copia-like retrotransposons are ubiquitous among plants. Proc.Natl.Acad.Sci.USA 89: 7124-7128

Hirochika H, Hirochika R. 1993. Tyl-copia group retrotransposons as ubiquitous components of plant genomes. Jpn J Genet 68: 35-46; Hirochika H, Fukuchi A, Kikuchi F. 1992. Retrotransposon families in rice. Mol.Gen.Genet. 233: 209-216.

Metaviridae (Ty3-gypsy) Element Degenerate Primers

We (Heslop-Harrison and colleagues) have used the following in Hordeum (Vershinin et al., 2002), sugar beet (Kubis et al. 1998) and a variety of gymnosperms (Friesen et al. 2001, the original reference for these primers; see references page of this website for the citations ).

Forward primers: GyRT1 : 5' MRNA TGTGYGTNGAYTAYMG encoding RMCVDYR GyRT3 : 5' YKNWSNGGNTAYCAYCARAT encoding LSGYHQI

Reverse primers GyRT4 : 5' RCAYTTNSWNARYTTNGCR encoding YAKLSKC

GyRT1/GyRT4 will give a 420bp product. GyRT3/ GyRT4 will give an approx 300bp product.

Jump to nucleotide degeneracy symbols

These were published in Friesen et al. 2001, Kubis et al. 1998

Alan Schulman11 Forward primer: 5' GTITAWYKTIGAYGAYRTIYTIRT Reverse primer: 5' ICKYTCISWYTGICCRTCISTYTGIGG

Andy Flavell Gypsy Primers were in Molecular Biology and Evolution 2000 or MGG 2005. They work well in 4 out of 4 species tested to date (beans'n nematodes). The Ty3-gypsy-group retrotransposon sequences were amplified using primer 8717 (5-TAYCCNHTNCCNCGNATHGA-3) or 8718 (5-TAYCCNHTNCCNAGRATHGA-3), both encoding YPLPRID, together with 5-ARCATRTCRTCNACRTA-3, encoding YVDDML (Flavell et al. 1992a). The up-stream Ty3-gypsy primers were based on an alignment of the plant Ty3-gypsy elements CIN-full, Huck, Leviathan, Reina, Tekay (Jeff Bennetzen, personal communication), Del 1 (Accession No. X13886) and Ifg7 (AJ004945) -quoted from Hill et al. (2005 MGG - see below for citation)

LINE Element Degenerate Primers

Pat Heslop-Harrison These work well in Hordeum, Allium, Oryza, Secale, Nicotiana and Antirrhinum. 5' RVNRANTTYCGNCCNATHTC 3' (named BEL1) encoding [E/D/K/N/S][E/D/N] FRPIS 3' TCYGTCCCCCTRGGRRACAG 5' (BEL2) encoding RQGDPLS (very similar to Andy's downstream primer) Bel1/Bel2 PCR products should be approximately 410bp.

Modified with changed specificity at the 3' end by Sybille Kubis (Kubis SE, Castilho AMMF, Vershinin AV, Heslop-Harrison JS. 2003. Retroelements, transposons and methylation status in the genome of oil palm (E laeis guineensis ) and the relationship to somaclonal variation. Plant Molecular Biology 52 : 69-79), work well in oil palm and Brassica (Alix K, Heslop-Harrison JS. 2004 . The diversity of retroelements in diploid and allotetraploid Brassica species. Plant Molecular Biology 54 : 895-909) and mosses, ferns and liverwords (Elsebeth Kolmos)

BEL1MF: 5'-RVNRANTTYCGNCCNATHAG-3' and

BEL2MR: 5'-GACARRGGRTCCCCCTGNCK-3'.

Andy Flavell These work well in Vicia . Upstream Primer: 5' CCNGGNCCNGAYGGNWT encoding PGPDG[IMF] Downstream Primer: 5' SWNARNGGRTCNCCYTG encoding QGDPLSP or: 5' SWNARNGGRCANCCYTG encoding QGCPLSP These primers together amplify a band of approximately 650bp.

Hill P, Burford D, Martin DMA and Flavell AJ Retrotransposon populations of Vicia species with varying genome size Molecular Genetics and Genomics Published online: 13 May 2005 DOI: 10.1007/s00438-005-1141-x Have published more primers based on Wright et al (1996): Reverse transcriptase (rt) gene fragments were amplified from LINE retrotransposons in Vicia DNA s by degenerate PCR using primers DVO144 (5-GGGATCCNGGNCCNGAYGGNWT-3) and 10712 (5-SWNARNGGRTCNCCYTG-3), which were derived from those described by Wright et al.

(Wright DA, Ke N, Smalle J, Hauge BM, Goodman HM, Voytas DF (1996) Multiple non-LTR retrotransposons in the genome of Arabidopsis thaliana. Genetics 142:569�578): probably for protein amino acids DPGPDG and QGDPLSP

PCR Programmes

Typical Mix for PCR amplification: We use 50 μl  for both running on a gel and cloning, 15 μl for analysis of amplification alone. We use a Biometra T-gradient PCR machine.