Transformation of overlapping, movement protein and coat protein gene into Nicotiana tabacum

Research output: Contribution to journalArticle

Abstract

A study was carried out to transform overlapping (OVG) gene, movement protein (MP) gene and the coat protein (CP) gene into Nicotiana tabacum. The cDNAs of all the three genes mentioned were successfully obtained. The 200 bp cDNA of the OVG gene was amplified via RT-PCR and subsequently used in the random mutagenesis process, to generate three mutants, ovg1, ovg4 and ovg5. cDNA of MP with an estimated size of 870 bp and its site-directed mutants, mpA and mpD were also generated in addition to the 760 bp wildtype CP gene obtained from a previous research. All the cDNAs were individually cloned into pPCR-Script, and subsequently sub-cloned into the plant transformation vector, pCAMBIA3301. Each of the recombinant pCAMBIA3301 plasmids, harbouring one of the above mentioned genes and mutant alleles of interest, was then transformed into Agrobacterium tumefaciens LBA4404. Transformation experiments into tobacco plants (Nicotiana tabacum cv. White Burley] were carried out by the leaf-disc co-cultivation method. Primary screening of transformants revealed an average of 39.33% putative transformants (positives) for all constructs used (59% pCAMBIA-CP and 56% pCAMBIA-OVGori and mutant ovg1, ovg4, ovg5 and ovg7 and 3% pCAMBIA-MP, mutants mpA and mpC). Further screening of the surviving transfomants showed that 72% (70% pCAMBIA-CP, 90% pCAMBIA-OVGori and mutants, 56% pCAMBIA-MP and mutants) showed GUS (β-glucuronidase) activity when assayed. Integration of the transgene into the genome was confirmed via PCR and Southern-PCR methods. First generation screening of randomly selected putatively transformed plants carrying constructs of pCAMBIA3301-CP, pCAMBIA3301-OVGori and pCAMBIA3301-ovg4 were 100% positive in the PCR and Southern PCR analysis producing ∼760 bp, ∼200 bp and ∼800 bp bands for CP, OVG and gus genes respectively. Further testing was carried out via Southern-DNA method using the same transformants. The results showed positive integration of transgene of pCAMBIA3301-CP and pCAMBIA3301-OVGori into tobacco plantlets.

Original languageEnglish
Pages (from-to)11-20
Number of pages10
JournalJournal of Pure and Applied Microbiology
Volume3
Issue number1
Publication statusPublished - Apr 2009

Fingerprint

Capsid Proteins
Tobacco
Overlapping Genes
Genes
Polymerase Chain Reaction
Complementary DNA
Proteins
Mutant Proteins
Transgenes
Agrobacterium tumefaciens
Glucuronidase
Mutagenesis
Plasmids
Alleles
Genome
DNA
Research

Keywords

  • Coat protein
  • Genome integration
  • Movement protein
  • Overlapping gene
  • Transformation

ASJC Scopus subject areas

  • Applied Microbiology and Biotechnology
  • Biotechnology
  • Microbiology

Cite this

Transformation of overlapping, movement protein and coat protein gene into Nicotiana tabacum. / K. Nadarajah, Kalaivani; Leng, Tan Sze.

In: Journal of Pure and Applied Microbiology, Vol. 3, No. 1, 04.2009, p. 11-20.

Research output: Contribution to journalArticle

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abstract = "A study was carried out to transform overlapping (OVG) gene, movement protein (MP) gene and the coat protein (CP) gene into Nicotiana tabacum. The cDNAs of all the three genes mentioned were successfully obtained. The 200 bp cDNA of the OVG gene was amplified via RT-PCR and subsequently used in the random mutagenesis process, to generate three mutants, ovg1, ovg4 and ovg5. cDNA of MP with an estimated size of 870 bp and its site-directed mutants, mpA and mpD were also generated in addition to the 760 bp wildtype CP gene obtained from a previous research. All the cDNAs were individually cloned into pPCR-Script, and subsequently sub-cloned into the plant transformation vector, pCAMBIA3301. Each of the recombinant pCAMBIA3301 plasmids, harbouring one of the above mentioned genes and mutant alleles of interest, was then transformed into Agrobacterium tumefaciens LBA4404. Transformation experiments into tobacco plants (Nicotiana tabacum cv. White Burley] were carried out by the leaf-disc co-cultivation method. Primary screening of transformants revealed an average of 39.33{\%} putative transformants (positives) for all constructs used (59{\%} pCAMBIA-CP and 56{\%} pCAMBIA-OVGori and mutant ovg1, ovg4, ovg5 and ovg7 and 3{\%} pCAMBIA-MP, mutants mpA and mpC). Further screening of the surviving transfomants showed that 72{\%} (70{\%} pCAMBIA-CP, 90{\%} pCAMBIA-OVGori and mutants, 56{\%} pCAMBIA-MP and mutants) showed GUS (β-glucuronidase) activity when assayed. Integration of the transgene into the genome was confirmed via PCR and Southern-PCR methods. First generation screening of randomly selected putatively transformed plants carrying constructs of pCAMBIA3301-CP, pCAMBIA3301-OVGori and pCAMBIA3301-ovg4 were 100{\%} positive in the PCR and Southern PCR analysis producing ∼760 bp, ∼200 bp and ∼800 bp bands for CP, OVG and gus genes respectively. Further testing was carried out via Southern-DNA method using the same transformants. The results showed positive integration of transgene of pCAMBIA3301-CP and pCAMBIA3301-OVGori into tobacco plantlets.",
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