- Email: [email protected]
Use of reverse transcription-polymerase chain reaction for cloning of coat protein-encoding genes of cymbidium mosaic virus
GENE AN INTERNATIONAL GENES
ELSEVIER
, J O U R N A L ON
AND GENOME5
Gene 179 (1996) 105-107
Use of reverse transcription-polymerase chain reaction for cloning of coat protein-encoding genes of cymbidium mosaic virus 1 Pattana Srifah a,,, Suvit Loprasert a, Nanyawan Rungroj b a Biotechnology Laboratory, Chulabhorn Research Institute, Laksi, Bangkok 10210, Thailand b Department of Plant Pathology, Kasetsart University, Bangkok 10900, Thailand Received 3 February 1996; revised 7 April 1996; accepted 8 April 1996
Abstract
A reverse transcription-polymerase chain reaction (RT-PCR) has been developed for the cloning of coat protein-encoding genes (CP) of cymbidium mosaic virus (CyMV) isolated from three different infected species of Thai orchid: Cattleya, Mokara and Oncidium. The analysis of the compiled sequences of the cDNA clones shows a single open reading frame which encoded a CyMV CP gene. The gene is 669 nucleotides (nt) long and codes for a 23 761-Da protein. The nt sequences of CyMV CP from the Thai isolates showed that some of them differ at a single nt and share 97% homology, but all of them share only 88% homology to the Singapore Oncidium isolate. In addition, the CyMV CP, unlike that from other potexviruses, is distinctive and differs greatly from the amino acid composition deduced from the nt sequence of the CP. Keywords: Orchid; Plant virus; Potexvirus; Gene sequencing; Homology
Cymbidium mosaic virus (CyMV) is widely distributed and known to reduce plant vigor and lower flower quality in orchids, which affects their economic value (Zetter et al., 1990). CyMV, a member of the potexvirus group, contains about 6 - 7 - k b positive-sense genomic RNA that is capped and polyadenylated (Francki et al., 1985). Its R N A genome contained a 660-nt CP gene, presumably expressed from subgenomic R N A (Bendena et al., 1987), located in the 3'-terminal portion of the C y M V genome (Chia et al., 1992). Infected Thai orchids (Cattleya, Mokara and Oncidium) were examined and total R N A extracted by the hot phenol/LiC1 method as described by Verwoerd et al. * Corresponding author. Tel. +66 2 5740622, ext. 1402; Fax +66 2 5740616; e-mail: [email protected] ~Presented at the Chulabhorn Research Institute International Conference on 'Biotechnology Research and Applications for Sustainable Development (BRASD)', Central Plaza Hotel, Bangkok, Thailand, 7 10 August 1995. Abbreviations: aa, amino acid(s); AMV, avian myeloblastosisvirus; bp, base pair(s); cDNA, DNA complementaryto RNA; CP, coat protein(s); CP, gene(s) encoding CP; CyMV, cymbidium mosaic virus; dNTP, deoxynucleotide triphosphate; kb, kilobase(s) or 1000 bp; nt, nucleotide(s); ORF, open reading frame; PAUP, see text; PCR, polymerase chain reaction; PMV, papaya mosaic virus; PVX, potato virus X; RT, reverse transcription(tase) ; u, unit(s); WC1MV, white clover mosaic virus. 0378-1119/96/$15.00 © 1996 Elsevier Science B.V. All rights reserved PH S0378-1119(96)00419-2
(1989). The RNAs were reverse transcribed into c D N A of C y M V CP genes by the R T - P C R method using sense primer 5'-TAG GAT C C T G G C G A G G G T TAA G and antisense primer 5'-TTG GAT CCT T T T T T T T T T TT. AMV RT was added to the P C R mixture containing 0.5 ~tg of extracted RNA/100 ng of each primer/0.5 m M all four dNTPs/1 u Taq D N A polymerase/10x P C R buffer (Perkin-Elmer kit). Temperature cycles were 15 min at 42°C, 5 min at 99°C and 5 min at 5°C for firststrand c D N A synthesis, followed by 30 cycles of 30 s at 94°C for denaturing, 1 min at 55°C for annealing and 2 min at 72°C for primer extension. Each of these c D N A fragments was cloned into the BamHI site of pKSBluescript plasmid for nt sequencing. Each c D N A clone from each isolation contains a single 669-nt O R F almost identical to that encoded by the C y M V gene CP (Mr of 23 761). Comparison of coding regions revealed that Mokara C y M V CP gene shared 97% homology to Cattleya and Oncidium C y M V CP genes. These Thai isolates differed at a single nt, whereas their homology to Singapore Oncidium isolation previously reported by Chia et al. (1992) was only 88%; their deduced proteins shared about 94, 96 and 72% aa homology, respectively, to the Singapore isolate. The strong homologies were found mainly in the N-terminal portions of the CP genes (Fig. 1). The alignment of C y M V CP aa sequences with those of other potexviruses revealed that the CP O R F
P. Srifah et al./Gene 179 (1996) 105-107
106
CyMV-Cat CyMV-Mok CyMV_-OncT CyMV-OncS
10 MGEPTPTPAA ********** ****~***** **********
20 TYSAADPTSS ********** *********A *********A
30 40 PKLADLTAIK YSPVTSSIVT ********** t*******A* ******A*** ****"***A* ******A*** ***I****A*
50 PEEIKAITQL ********** ********** **********
CyMV-Cat CyMV-Mok CyMV-OncT CyMV-OncS
60 70 80 WVNNLGLPAD TVGAADIDLA RAYADVGDSK * * * * * * * * * * * * * * * A * * * * * * * * * * * A ** * * " * * * ~ * * * * * V P * A ***~ * * * * * * * A * * * * * * * * * * * * * * * T * A ***~ * * * * * * * A * *
CyMV-Cat CyMV-Mok CyMV-OncT CyMV-OncS
110 120 130 140 150 AQIFVANVTP RQFCAYYAKV VKNQMIATND PPANWAKAGF QEDTRFAAFD *K******** ********** *W*L*L**** ********** ********** *K******** *********" *W*L*L**** **G******* ********** ********** ********** *W*L*L**** ********** **********
CyMV-Cat CyMV-MoK CyMV-OncT CyMV-OncS
160 170 180 190 200 GFDTVDSTAT LKPTEWQRRP TDRERAASI GKYGALARQR IQNGNLITNI F**A*****A *E'A****** ********** ********** ********** F**A*****A *E-A****** ********** ********** ********** F**A****GA *E*A*CSAA* LTATC*LDRE VRRPCPSAYP ERQPHHQHCR
CyMV-Cat CyMV-Mok CyMV-OncT CyMV-OncS
210 220 AEVTKGHLGS TNTLYALPAP PTE * * * * * * * * * * * * * * * * * * * * *** * * * * * * * * * * * * * * * * * * * * *** GHQGPSW*HQ HSLRS*CTPY
90 i00 SATLLGVCPT KPDVRRASLA ********** **t******* ******F*** *******T** ******F*** **-****A**
Fig. 1. Alignment of aa sequences for CyMV CP isolated from various kinds of orchid varieties, including Cattleya (Cat), Mokara (Mok), and Oncidium (OncT, Thai isolate; OncS, Singapore isolate), with a length of 223, 223, 223 and 220 aa, respectively. Identical aa residues are indicated by asterisks.
WClMV PMV MV-MOk WCIMV PMV
MGEPTPTPAAT ........ :YSAADPTSSPKLADLTAIKYSPVTSSIATP MSAPASTTQATGSTTSTTTKTAGATPATASGLFTIPDGDFFSTARAWAS MAT .................... TTATTPPSLTDIRALKYTSSTVSVASP MSKSSMSTPNTAFPAITQEQMSSIKVDPTSNL
41 50
-EEIKAITQLWVNNLGLPADTVGAAAIDLARAYADVGASKSATLL DAVATNEDLSEIE~IWKN-MKVPTDTMTQAAWKLVRHCADVGSSAQTEMI A ...... EIEAITKTWAETFKIPMDVLPLACWDLARAFADVGASSKSELP --LPSQEQLKSVSTL-MVAAKVPAASVTTVALELVNFCYDNGSSAYTTVT
85 99 74 79
,.
MV-Mok
.
°*
,
*
*
30 32
*.*
WCIMV PMV
GVCPTKPDVRRASLAAKIFVANVTPRQFCAYYVVWLMLATNDPPANW DTGPYANGISRARLAAAI-KEVCTLRQFCMKYAPVVWNWMLTNNSPPANW GDSAALAGVSRKQLAQAI-KIHCTIRQFCMYFANIVWNIMLDTKTPPASW GPSSI-PEISLAQLASIVKASGTSLRKFCRYFAPIIWNLR-TDKMAPANW
134 149 123 127
CyMV-Mok PVX WCIMV
AKAGFQEDTRFAAFDFFDAVDSTAALEPAE-WQRRPTDRERAAHSIGKYG QAQGFKPEHKFAAFDFFNGVTNPAAIMPKEGLIRPPSEAEMNAA(}TAAFV SKLGYKEESKFAGFDFFDGVNHPAALMPADGLIRGPSDAEILA~AKQV
184 198 173
PMV
EASGYKPSAKFAAFD~FDGVENPAAMQPPSGLIRSPTQEERIAN~TNKQV
177
. .
MV-Mok WCIMV PMV
.*~.**
*,°*
*~°
*
*
*..
*
*
ALARQRIQNGNLITNIAEVTKGHLGSTNTLYALPAPPTE KITK/%RA~SNDFASLDAAVTRGRITGTTTAEAVVTLPPP ALHR ,DAKPTWHKRCQLC . . . . . . . . . . . HLFQAAAQDNNFTSNSAFITKGQISGSTPTIQFL-PPPE
223 237 190 215
Fig. 2. Alignment of aa sequences for CP of CyMV, Mokara (Mok) isolate, and those of other potexviruses. PMV, papaya mosaic virus; PVX, potato virus X; and WC1MV, white clover mosaic virus. Identical aa residues are indicated by asterisks and similar aa residues by dots below the aa sequence. Dashes represent the conceptually deleted aa, as a result of the alignment.
CyMV PVX
PMV WCIMV Fig. 3. Dendrogram calculated by the PAUP program showing the relationships between the aligned CP of four potexviruses.
P. Srifah et al./Gene 179 (1996) 105-107
contained short sequence motif consensus (Fig. 2). The alignment revealed rather strong homologies mainly in the mid-region of the CP but the N- and C-terminal portions showed poor homology. The relationships of potexvirus CP, whose sequence has already been reported, were assessed by determining the percentage of non-identical aa residues in each pair of aligned sequences. A dendrogram was computed for these differences by P A U P (Phylogenetics Analysis Using Parsimony Version 3.1.1) program. As shown in Fig. 3, the potexvirus CP, potato virus X (PVX), is closely related to papaya mosaic virus (PMV). CyMV was the most distant from all potexviruses.
107
References Bendena, W.G., Bancroft,J.B. and Mackie, G.A.(1987) Molecularcloning of clover yellowmosaic virus RNA:identification of coat protein coding sequences in vivo and in vitro. Virology 157, 276-284. Chia, T.F., Chan, Y.S. and Chua, N.H. (1992) Characterization of cymbidium mosaic virus coat protein gene and its expression in transgenic tobacco plants. Plant Mol. Biol. 18, 1091 1099. Francki, R.I.B., Milne, R.G. and Hatta, T. (1985) Atlas of Plant Viruses, Vol. 2. CRC Press, Boca Raton, FL, pp. 159-172. Verwoerd, T.C., Dekker, B.M.M. and Hoekema,A. (1989) A small scale procedure for rapid isolation of plant virus. Nucleic Acids Res. 17, 2362. Zetter, F.W., Ko, N.J., Wisler, G.C., Elliott, M.S. and Wong, S.M. (1990) Viruses of orchids and their control. Plant Dis. 74, 621-626.
ELSEVIER
, J O U R N A L ON
AND GENOME5
Gene 179 (1996) 105-107
Use of reverse transcription-polymerase chain reaction for cloning of coat protein-encoding genes of cymbidium mosaic virus 1 Pattana Srifah a,,, Suvit Loprasert a, Nanyawan Rungroj b a Biotechnology Laboratory, Chulabhorn Research Institute, Laksi, Bangkok 10210, Thailand b Department of Plant Pathology, Kasetsart University, Bangkok 10900, Thailand Received 3 February 1996; revised 7 April 1996; accepted 8 April 1996
Abstract
A reverse transcription-polymerase chain reaction (RT-PCR) has been developed for the cloning of coat protein-encoding genes (CP) of cymbidium mosaic virus (CyMV) isolated from three different infected species of Thai orchid: Cattleya, Mokara and Oncidium. The analysis of the compiled sequences of the cDNA clones shows a single open reading frame which encoded a CyMV CP gene. The gene is 669 nucleotides (nt) long and codes for a 23 761-Da protein. The nt sequences of CyMV CP from the Thai isolates showed that some of them differ at a single nt and share 97% homology, but all of them share only 88% homology to the Singapore Oncidium isolate. In addition, the CyMV CP, unlike that from other potexviruses, is distinctive and differs greatly from the amino acid composition deduced from the nt sequence of the CP. Keywords: Orchid; Plant virus; Potexvirus; Gene sequencing; Homology
Cymbidium mosaic virus (CyMV) is widely distributed and known to reduce plant vigor and lower flower quality in orchids, which affects their economic value (Zetter et al., 1990). CyMV, a member of the potexvirus group, contains about 6 - 7 - k b positive-sense genomic RNA that is capped and polyadenylated (Francki et al., 1985). Its R N A genome contained a 660-nt CP gene, presumably expressed from subgenomic R N A (Bendena et al., 1987), located in the 3'-terminal portion of the C y M V genome (Chia et al., 1992). Infected Thai orchids (Cattleya, Mokara and Oncidium) were examined and total R N A extracted by the hot phenol/LiC1 method as described by Verwoerd et al. * Corresponding author. Tel. +66 2 5740622, ext. 1402; Fax +66 2 5740616; e-mail: [email protected] ~Presented at the Chulabhorn Research Institute International Conference on 'Biotechnology Research and Applications for Sustainable Development (BRASD)', Central Plaza Hotel, Bangkok, Thailand, 7 10 August 1995. Abbreviations: aa, amino acid(s); AMV, avian myeloblastosisvirus; bp, base pair(s); cDNA, DNA complementaryto RNA; CP, coat protein(s); CP, gene(s) encoding CP; CyMV, cymbidium mosaic virus; dNTP, deoxynucleotide triphosphate; kb, kilobase(s) or 1000 bp; nt, nucleotide(s); ORF, open reading frame; PAUP, see text; PCR, polymerase chain reaction; PMV, papaya mosaic virus; PVX, potato virus X; RT, reverse transcription(tase) ; u, unit(s); WC1MV, white clover mosaic virus. 0378-1119/96/$15.00 © 1996 Elsevier Science B.V. All rights reserved PH S0378-1119(96)00419-2
(1989). The RNAs were reverse transcribed into c D N A of C y M V CP genes by the R T - P C R method using sense primer 5'-TAG GAT C C T G G C G A G G G T TAA G and antisense primer 5'-TTG GAT CCT T T T T T T T T T TT. AMV RT was added to the P C R mixture containing 0.5 ~tg of extracted RNA/100 ng of each primer/0.5 m M all four dNTPs/1 u Taq D N A polymerase/10x P C R buffer (Perkin-Elmer kit). Temperature cycles were 15 min at 42°C, 5 min at 99°C and 5 min at 5°C for firststrand c D N A synthesis, followed by 30 cycles of 30 s at 94°C for denaturing, 1 min at 55°C for annealing and 2 min at 72°C for primer extension. Each of these c D N A fragments was cloned into the BamHI site of pKSBluescript plasmid for nt sequencing. Each c D N A clone from each isolation contains a single 669-nt O R F almost identical to that encoded by the C y M V gene CP (Mr of 23 761). Comparison of coding regions revealed that Mokara C y M V CP gene shared 97% homology to Cattleya and Oncidium C y M V CP genes. These Thai isolates differed at a single nt, whereas their homology to Singapore Oncidium isolation previously reported by Chia et al. (1992) was only 88%; their deduced proteins shared about 94, 96 and 72% aa homology, respectively, to the Singapore isolate. The strong homologies were found mainly in the N-terminal portions of the CP genes (Fig. 1). The alignment of C y M V CP aa sequences with those of other potexviruses revealed that the CP O R F
P. Srifah et al./Gene 179 (1996) 105-107
106
CyMV-Cat CyMV-Mok CyMV_-OncT CyMV-OncS
10 MGEPTPTPAA ********** ****~***** **********
20 TYSAADPTSS ********** *********A *********A
30 40 PKLADLTAIK YSPVTSSIVT ********** t*******A* ******A*** ****"***A* ******A*** ***I****A*
50 PEEIKAITQL ********** ********** **********
CyMV-Cat CyMV-Mok CyMV-OncT CyMV-OncS
60 70 80 WVNNLGLPAD TVGAADIDLA RAYADVGDSK * * * * * * * * * * * * * * * A * * * * * * * * * * * A ** * * " * * * ~ * * * * * V P * A ***~ * * * * * * * A * * * * * * * * * * * * * * * T * A ***~ * * * * * * * A * *
CyMV-Cat CyMV-Mok CyMV-OncT CyMV-OncS
110 120 130 140 150 AQIFVANVTP RQFCAYYAKV VKNQMIATND PPANWAKAGF QEDTRFAAFD *K******** ********** *W*L*L**** ********** ********** *K******** *********" *W*L*L**** **G******* ********** ********** ********** *W*L*L**** ********** **********
CyMV-Cat CyMV-MoK CyMV-OncT CyMV-OncS
160 170 180 190 200 GFDTVDSTAT LKPTEWQRRP TDRERAASI GKYGALARQR IQNGNLITNI F**A*****A *E'A****** ********** ********** ********** F**A*****A *E-A****** ********** ********** ********** F**A****GA *E*A*CSAA* LTATC*LDRE VRRPCPSAYP ERQPHHQHCR
CyMV-Cat CyMV-Mok CyMV-OncT CyMV-OncS
210 220 AEVTKGHLGS TNTLYALPAP PTE * * * * * * * * * * * * * * * * * * * * *** * * * * * * * * * * * * * * * * * * * * *** GHQGPSW*HQ HSLRS*CTPY
90 i00 SATLLGVCPT KPDVRRASLA ********** **t******* ******F*** *******T** ******F*** **-****A**
Fig. 1. Alignment of aa sequences for CyMV CP isolated from various kinds of orchid varieties, including Cattleya (Cat), Mokara (Mok), and Oncidium (OncT, Thai isolate; OncS, Singapore isolate), with a length of 223, 223, 223 and 220 aa, respectively. Identical aa residues are indicated by asterisks.
WClMV PMV MV-MOk WCIMV PMV
MGEPTPTPAAT ........ :YSAADPTSSPKLADLTAIKYSPVTSSIATP MSAPASTTQATGSTTSTTTKTAGATPATASGLFTIPDGDFFSTARAWAS MAT .................... TTATTPPSLTDIRALKYTSSTVSVASP MSKSSMSTPNTAFPAITQEQMSSIKVDPTSNL
41 50
-EEIKAITQLWVNNLGLPADTVGAAAIDLARAYADVGASKSATLL DAVATNEDLSEIE~IWKN-MKVPTDTMTQAAWKLVRHCADVGSSAQTEMI A ...... EIEAITKTWAETFKIPMDVLPLACWDLARAFADVGASSKSELP --LPSQEQLKSVSTL-MVAAKVPAASVTTVALELVNFCYDNGSSAYTTVT
85 99 74 79
,.
MV-Mok
.
°*
,
*
*
30 32
*.*
WCIMV PMV
GVCPTKPDVRRASLAAKIFVANVTPRQFCAYYVVWLMLATNDPPANW DTGPYANGISRARLAAAI-KEVCTLRQFCMKYAPVVWNWMLTNNSPPANW GDSAALAGVSRKQLAQAI-KIHCTIRQFCMYFANIVWNIMLDTKTPPASW GPSSI-PEISLAQLASIVKASGTSLRKFCRYFAPIIWNLR-TDKMAPANW
134 149 123 127
CyMV-Mok PVX WCIMV
AKAGFQEDTRFAAFDFFDAVDSTAALEPAE-WQRRPTDRERAAHSIGKYG QAQGFKPEHKFAAFDFFNGVTNPAAIMPKEGLIRPPSEAEMNAA(}TAAFV SKLGYKEESKFAGFDFFDGVNHPAALMPADGLIRGPSDAEILA~AKQV
184 198 173
PMV
EASGYKPSAKFAAFD~FDGVENPAAMQPPSGLIRSPTQEERIAN~TNKQV
177
. .
MV-Mok WCIMV PMV
.*~.**
*,°*
*~°
*
*
*..
*
*
ALARQRIQNGNLITNIAEVTKGHLGSTNTLYALPAPPTE KITK/%RA~SNDFASLDAAVTRGRITGTTTAEAVVTLPPP ALHR ,DAKPTWHKRCQLC . . . . . . . . . . . HLFQAAAQDNNFTSNSAFITKGQISGSTPTIQFL-PPPE
223 237 190 215
Fig. 2. Alignment of aa sequences for CP of CyMV, Mokara (Mok) isolate, and those of other potexviruses. PMV, papaya mosaic virus; PVX, potato virus X; and WC1MV, white clover mosaic virus. Identical aa residues are indicated by asterisks and similar aa residues by dots below the aa sequence. Dashes represent the conceptually deleted aa, as a result of the alignment.
CyMV PVX
PMV WCIMV Fig. 3. Dendrogram calculated by the PAUP program showing the relationships between the aligned CP of four potexviruses.
P. Srifah et al./Gene 179 (1996) 105-107
contained short sequence motif consensus (Fig. 2). The alignment revealed rather strong homologies mainly in the mid-region of the CP but the N- and C-terminal portions showed poor homology. The relationships of potexvirus CP, whose sequence has already been reported, were assessed by determining the percentage of non-identical aa residues in each pair of aligned sequences. A dendrogram was computed for these differences by P A U P (Phylogenetics Analysis Using Parsimony Version 3.1.1) program. As shown in Fig. 3, the potexvirus CP, potato virus X (PVX), is closely related to papaya mosaic virus (PMV). CyMV was the most distant from all potexviruses.
107
References Bendena, W.G., Bancroft,J.B. and Mackie, G.A.(1987) Molecularcloning of clover yellowmosaic virus RNA:identification of coat protein coding sequences in vivo and in vitro. Virology 157, 276-284. Chia, T.F., Chan, Y.S. and Chua, N.H. (1992) Characterization of cymbidium mosaic virus coat protein gene and its expression in transgenic tobacco plants. Plant Mol. Biol. 18, 1091 1099. Francki, R.I.B., Milne, R.G. and Hatta, T. (1985) Atlas of Plant Viruses, Vol. 2. CRC Press, Boca Raton, FL, pp. 159-172. Verwoerd, T.C., Dekker, B.M.M. and Hoekema,A. (1989) A small scale procedure for rapid isolation of plant virus. Nucleic Acids Res. 17, 2362. Zetter, F.W., Ko, N.J., Wisler, G.C., Elliott, M.S. and Wong, S.M. (1990) Viruses of orchids and their control. Plant Dis. 74, 621-626.