Kloning Gen Coat Protein (CP) Carnation Mottle Virus (CarMV) pada Vektor Ekspresi [Cloning of Carnation Mottle Virus (CarMV) Coat Protein Gene into Expression Vector]

<p>Carnation mottle virus (CarMV) termasuk anggota genus Carmovirus dalam famili Tombusviridae. Virus ini banyak ditemukan menginfeksi tanaman anyelir di Jawa Barat dan menyebabkan gejala mottle. Sebagai langkah awal untuk memproduksi antiserum melalui teknik ekspresi gen CP perlu diklon pada vektor yang sesuai. Penelitian ini bertujuan mendapatkan klon CarMV yang berfungsi melalui kloning dan subkloning gen CP CarMV ke dalam vektor ekspresi yang sesuai. Penelitian dilakukan dalam beberapa tahap, yaitu ekstraksi RNA total dan amplifikasi cDNA CarMV dengan RT-PCR, menggunakan primer spesifik CarMVF dan CarMVR yang mengandung situs enzim restriksi XhoI dan BamHI, kloning dan subkloning DNA sisipan, serta konfirmasi transforman. Rekombinan gen sisipan CP CarMV dalam bakteri dikonfirmasi dengan koloni PCR. Gen CP CarMV berhasil dikloning ke dalam TA vektor pTZ57R/T dan disubkloning ke vektor ekspresi pET28a. Sekuen rekombinan CP CarMV berhasil dikonfirmasi melalui perunutan DNA. Penelitian lebih lanjut diperlukan untuk mendapatkan produksi antigen rekombinan yang melimpah pada bakteri ekspresi dan kondisi yang sesuai.</p><p><strong>Keywords</strong></p><p>Dianthus caryophillus L.; Carmovirus; Kloning; Subkloning; Bakteri ekspresi</p><p><strong>Abstract</strong></p><p>Carnation mottle virus (CarMV) is a type member of Carmovirus genus in family of Tombusvirus. The virus infects carnation plants in the centre area production of West Java and it cause mottle symptoms. The research aimed to obtain functional clone(s) of CarMV CP gene in suitable expression kloning vector. The research was carried out through several steps, namely total RNA extraction and amplification of cDNA of CP CarMV by RT-PCR using specific primer pairs CarMVF and CarMVR containing restriction enzyme sites XhoI and BamHI, respectively, TA cloning, and subcloning into expression vector pET28a and confirmation of recombinant plasmids by colony PCR. CarMV CP gen was successfully cloned into TA cloning vector pTZ57R/T and subcloned into vector pET28a, alsowere confirmed by DNA sequencing. Future experiment is necessary to be conducted to obtain abundance recombinant antigen production of CarMV CP in suitable expression condition and bacterial host.</p>

Deteksi Cepat Carnation mottle virus pada Tanaman Anyelir (Dianthus caryophyllus L.)

<em>Carnation mottle virus </em>(CarMV) merupakan virus penting pada tanaman anyelir di Indonesia maupun di dunia. Deteksi virus yang mudah dan cepat, diperlukan untuk memantau sumber induk anyelir bebas virus. Tujuan penelitian adalah mengevaluasi tiga metode preparasi RNA total yang mudah dan cepat dari tanaman anyelir sebagai templat <em>one step </em>RT-PCR. Sumber RNA total adalah dari daun dan batang anyelir terinfeksi CarMV. Metode yang dievaluasi yaitu SDT, SEM, dan kit komersial sebagai pembanding. Optimasi dilakukan terhadap konsentrasi akhir primer (0.4 – 1.0 µM) dan MgCl₂(1.5 dan 2.0 mM). Metode SDT dan SEM berhasil mendapat RNA total dari tanaman anyelir baik dari sampel daun maupun batang. Keberhasilan yang didapat dengan metode SDT dan SEM sebanding dengan kit komersial. <em>One step</em> RT-PCR RNA total yang digabungkan dengan metode SDT dan SEM menghasilkan intensitas DNA yang sebanding dengan kit komersial. RNA total dari daun sebagai sumber templat <em>one step</em> RT-PCR terbaik dibandingkan batang. Preparasi RNA total dengan metode SDT dan SEM adalah metode cepat, mudah, dan murah dalam menyediakan templat <em>one step</em> RT-PCR. Konsentrasi primer 0.4 µM dan MgCl₂2 mM merupakan konsentrasi optimum dan menghasilkan hasil amplifikasi terbaik

Eliminasi Carnation Mottle Virus Menggunakan Senyawa Antiviral pada Kultur Jaringan Anyelir (Dianthus caryophyllus L.)

Carnation mottle virus (CarMV) merupakan salah satu virus penting pada tanaman anyelir dan semua kultivar anyelir yang ditanam di Jawa Barat terinfeksi oleh virus ini. Penelitian bertujuan mendapatkan metode eliminasi CarMV yang efektif untuk membebaskan planlet anyelir dari virus. Inisiasi eksplant terinfeksi dilakukan pada media MSO dan perbanyakan planlet dilakukan pada media perbanyakan MS yang mengandung 1,0 ppm BA dan 0,5 ppm kinetin (MSZ). Metode eliminasi CarMV yang diuji terdiri atas perlakuan 2-thiourasil dan amantadin dengan konsentrasi masing-masing 0, 5, 10, 15, 20, 25, dan 30 ppm, dan ribavirin 5 ppm sebagai pembanding. Tunas apikal planlet ditanam pada media perlakuan (MSZA). Setelah tunas tumbuh, meristem terminal diambil 0,5 mm untuk ditanam pada media MSZ. Kultur meristem terminal dari planlet pada perlakuan 2- thiourasil menghasilkan planlet bebas virus sebesar 0 – 57%. Konsentrasi 2-thiourasil 25 ppm menghasilkan persentase planlet bebas virus tertinggi, namun perlakuan tersebut toksik pada tanaman. Perlakuan amantadin menghasilkan 25,0 – 54,55% planlet bebas virus. Di antara perlakuan yang diuji, perlakuan antiviral amantadin dengan konsentrasi 5 – 30 ppm lebih optimal menghasilkan planlet anyelir bebas CarMV dan tidak toksik terhadap tanaman. Perlakuan amantadin 5 – 20 ppm mampu menghambat virus lebih tinggi dibandingkan perlakuan 2-thiourasil pada konsentrasi yang sama. Amantadin 5 – 30 ppm menghasilkan tingkat penghambatan virus sebesar 42,94 – 59,57%, sedangkan 2-thiourasil sebesar -8,18 – 63,03%. Senyawa 2-thiourasil dan amantadin berpotensi sebagai agen antiviral untuk mendapatkan tanaman anyelir bebas CarMV.

Identification and molecular characterization of Carnation mottle virus Brazilian isolates from carnation

Carnation mottle virus (CarMV), associated with carnation plants showing or not symptoms, was identified by host range, serology and molecular analysis of the coat protein gene. Seven samples were assayed by biological and serological tests. Two of them, one from São Paulo and another from Minas Gerais states, Brazil, which presented higher absorbance values in DAS-ELISA, were selected for molecular studies. Foliar samples were submitted to total RNA extraction, RT-PCR with specific primers, and amplicons obtained were sequenced. Phylogenetic analyses were carried out using the PAUP program after determining the nucleotide substitution model. The identity percentages between Brazilian sequences were 99%. When sequences of CP carnation isolates from other countries were compared, the identity ranged from 96-99%. CarMV isolates from São Paulo and Minas Gerais states are the first sequences obtained in Brazil, and analysis showed that they belong to the PK group and showed only two amino acids changes at positions 61 and 260. The virus presents a high genetic stability and it is readily mechanically transmitted from infected to healthy plants. This is the first report of CarMV in Minas Gerais state, of CP nucleotide sequences from Brazilian CarMV isolates, as well as molecular phylogenetic analysis in Brazil.

Identification of Carnation mottle virus from Lisianthus Plants in Taiwan

Lisianthus (Eustoma exaltatum (L.) Salisb. ex G. Don subsp. russellianum (Hook.) Kartesz) is an economically important ornamental crop in Taiwan. Over the past decade, nine viruses have been identified or detected in lisianthus including: Bean yellow mosaic virus (BYMV), Lisianthus necrosis virus (LNV) (2), Cucumber mosaic virus (CMV) (1), Turnip mosaic virus (TuMV), Tomato spotted wilt virus (TSWV), Broad bean wilt virus (BBWV), Tomato mosaic virus (ToMV), Pepper veinal mottle virus (PVMV), and Ageratum yellow vein virus (AYVV) (4). In May 2007 (late period of growing season) in central Taiwan, systemic necrotic spots, which are similar to that caused by LNV (2), were found on approximately 20% of the lisianthus plants. Spherical virus particles, approximately 32 nm in diameter, were found in the crude sap of infected lisianthus collected from the fields. However, the diseased samples did not react with antisera against domestic lisianthus-infecting spherical viruses, LNV (2) and CMV (1). A virus culture was isolated via mechanical inoculation on Chenopodium quinoa and serologically identified as Carnation mottle virus (CarMV) by ELISA, western blotting, and immunoelectron microscopy using antiserum against the CarMV zantedeschia strain (3). The virus induced necrotic local lesions on the inoculated leaves of C. quinoa, C. amaranticolor, Gomphrena globosa, Cucurbita moschata, Phaseolus angularis, P. vulgaris, and Vigna unguiculata. Lisianthus was previously reported as a local lesion host for CarMV (3). In current studies with 8 of 10 lisianthus plants, the newly isolated virus induced necrotic local lesions on inoculated leaves 20 days post inoculation (dpi). However, systemic necrotic lesions on noninoculated upper leaves, as were observed in the fields, appeared 120 dpi on inoculated plants, indicating that CarMV induces systemic infection in lisianthus during late growth stages. Noninoculated plants did not develop symptoms. Complementary DNA fragments of viral genomic RNA were amplified with a specific primer of the coat protein gene (3) and sets of degenerate primer for CarMV. The amplified cDNA fragments were cloned and sequenced. The full-length sequence was submitted as GenBank Accession No. FJ843021. The genomic RNA consists of 4,003 nucleotides and has an identical genome organization to that reported for members of the genus Carmovirus. The nucleotide sequence of the full-length genome shares more than 95% identity to isolates of CarMV (GenBank Accession Nos. AF192772, AJ304989, AJ811998, NC_001265, and X02986), and the nucleotide and deduced amino acid sequence of coat protein shares more than 98% identity with that of CarMV-TW (AY383566) (3), CarMV-FO25 (EF622206), CarMV-Italy-Ca1 (EF622207), and CarMV-Netherland Ca2 (EF622210). To our knowledge, this is the first report of natural infection of CarMV in lisianthus in Taiwan. References: (1) C. C. Chen and C. C. Hu, Plant Prot. Bull. 41:179, 1999. (2) C. C. Chen et al. Plant Dis. 84:506, 2000. (3) C. C. Chen et al. Plant Dis. 87:1539, 2003. (4) Y. H. Cheng et al. J. Taiwan Agric. Res. 58:196, 2009.

First Report of Carnation mottle virus in Phalaenopsis Orchids

In November 2003, two Phalaenopsis orchids from two different nurseries with symptoms of chlorotic rings on leaves were observed in Changhua County of central Taiwan. Symptomatic plants were collected and examined for the presence of viruses. Electron microscopic examination of ultrathin sections of leaf tissues from the symptomatic orchids found isometric virions of 32 nm in diameter. Subsequently, an isolate (herein designated as ‘92-orchid-1’) with particles of similar size were isolated from one symptomatic orchid and established in Chenopodium quinoa (3). After indirect ELISA tests using antisera against Carnation mottle virus (CarMV), Cucumber mosaic virus, Cymbidium ringspot virus, Tomato bushy stunt virus, Capsicum chlorosis virus, Impatiens necrotic spot virus, Tomato spotted wilt virus, Tomato ringspot virus, and Lisianthus necrosis virus, this isolate reacted positively with the antiserum produced against CarMV (1). CarMV-TW-infected and healthy C. quinoa were used as positive and negative controls, respectively. To further characterize this virus, the conserved region of the polymerase gene (ORF1RT) of Carmoviruses was amplified with degenerate primer pairs, FJJ2003-17 (5′-TATATCTCGAGCAA(A/C)TAGGGG(G/T)GCCT) and FJJ2003-18 (5′-TATAGGATCCCC(C/T)A(A/T)(A/G)GC(A/T)GTGTTCA), by reverse transcription (RT)-PCR using the total RNA isolated from the leaves of 92-orchid-1-, CarMV-TW-infected, and healthy C. quinoa (3). The 894-nt ORF1RT conserved region of isolate 92-orchid-1 (GenBank Accession No. HQ117873) shared 97.1, 65.6, 61.7, and 63.5% nucleotide identities and 98.3, 70.2, 66.1, and 64.7% amino acid identities with those of CarMV (X02986), Pelargonium flower break virus (NC_005286), Saguaro cactus virus (NC_001780), and Angelonia flower break virus (NC_007733), respectively. The sequence comparison of the ORF1RT conserved region indicated that 92-orchid-1 was a carmovirus related to CarMV. Sequence analyses of the coat protein (CP) gene (GenBank Accession No. HQ117872) amplified with the specific CP primer pairs of CarMV (FJJ2004-53: 5′-ACTGCGCTCGAGCTACTCTGTTGACAGTTCTA, and 2004-54: 5′-ATATATGGATCCCGTCCCGCCGTGTGTGTCTA) showed the isolate shared 95.8 to 98.8% nucleotide identities and 96.8 to 98.9% amino acid identities with those of 40 CarMV isolates. Furthermore, the CP gene shared 96.9, 97.0, and 98.8% nucleotide identities and 98.0, 95.7, and 98.3% amino acid identities with isolates from carnation (GenBank Accession No. AY383566) (1), calla lily (GenBank Accession No. HQ117870) (2), and lisianthus (GenBank Accession No. FJ843021), respectively, in Taiwan. These results suggested that this isolate was CarMV but distinct from the above-mentioned three isolates and designated CarMV-Ph. From 2004 to 2007, a further survey of 280 symptomatic Phalaenopsis plants by ELISA using CarMV polyclonal antibodies (1) found that approximately 4% of those tested were infected. To our knowledge, this is the first report of CarMV in Phalaenopsis orchids and the occurrence has substantial implications for the important nursery and floral industry in Taiwan. References: (1) C. C. Chen et al. Plant Pathol. Bull. 12:199, 2003. (2) C. C. Chen et al. Plant Dis. 87:1539, 2003. (3) Y. X. Zheng et al. Eur. J. Plant Pathol. 121:87, 2008.