Cloning of a chitinase-like cDNA (hs2), its transfer to creeping bentgrass (Agrostis palustris Huds.) and development of brown patch (Rhizoctonia solani) disease resistant transgenic lines

Benli Chai, Shahina B. Maqbool, Ravindra K. Hajela, David Green, Joseph M. Vargas, Donald Warkentin, Robab Sabzikar, Mariam B. Sticklen

Research output: Contribution to journalArticle

22 Citations (Scopus)

Abstract

We isolated a cDNA clone (hs2) encoding a chitinase-like protein from a triploid Dutch elm disease resistant American elm (Ulmus americana NPS-3-487). Amino acid sequence of this chitinase-like protein showed 65-78% homology to other plant chitinases. A plasmid KYLX71-pHS2 was constructed using the hs2 sequence under the control of cauliflower mosaic virus 35S promoter and nos terminator. This construct was used to genetically engineer creeping bentgrass (Agrostis palustris Huds.) via Biolistic® PDS-100/He system. Plasmid JS101 containing the bar herbicide resistance selectable marker gene regulated by the rice actin 1 (Act1) promoter was used as a selectable co-transformation marker. Transgenic plants were produced with a 21.1% transformation frequency on the basis of bialophos selection. The co -transformation frequency for hs2 was 5.68%. Linked transgenes (bar and mtld) were co-integrated with a frequency of 100% as expected and 21.3% of the transgenic plants showed integration of all three genes. Southern blot analysis of transformants showed that the hs2 transgene copy numbers ranged from 1 to 5. Northern blot hybridization confirmed transcription of the transgenes, and western blot determined the HS2 protein expression. No correlation was found between gene copy number and the level of gene expression. Disease resistance profiles of five independent transgenic lines in greenhouse trials revealed that two lines (711 and 9603) were significantly (P < 0.05) resistant to Rhizoctonia solani, the causative agent of brown patch disease.

Original languageEnglish (US)
Pages (from-to)183-193
Number of pages11
JournalPlant Science
Volume163
Issue number2
DOIs
StatePublished - Aug 2002
Externally publishedYes

Fingerprint

Agrostis
Agrostis stolonifera var. palustris
Rhizoctonia
Chitinases
Agrostis stolonifera
Cloning
Thanatephorus cucumeris
chitinase
Transgenes
Ulmus americana
transgenes
Organism Cloning
molecular cloning
Complementary DNA
Genes
Genetically Modified Plants
genetically modified organisms
transgenic plants
plasmids
Plasmids

Keywords

  • American elm (Ulmus americana)
  • Brown patch disease (Rhizoctonia solani)
  • CDNA (hs2)
  • Chitinase-like
  • Creeping bentgrass (Agrostis palustris Huds)

ASJC Scopus subject areas

  • Plant Science
  • Biochemistry
  • Biotechnology

Cite this

Cloning of a chitinase-like cDNA (hs2), its transfer to creeping bentgrass (Agrostis palustris Huds.) and development of brown patch (Rhizoctonia solani) disease resistant transgenic lines. / Chai, Benli; Maqbool, Shahina B.; Hajela, Ravindra K.; Green, David; Vargas, Joseph M.; Warkentin, Donald; Sabzikar, Robab; Sticklen, Mariam B.

In: Plant Science, Vol. 163, No. 2, 08.2002, p. 183-193.

Research output: Contribution to journalArticle

Chai, Benli ; Maqbool, Shahina B. ; Hajela, Ravindra K. ; Green, David ; Vargas, Joseph M. ; Warkentin, Donald ; Sabzikar, Robab ; Sticklen, Mariam B. / Cloning of a chitinase-like cDNA (hs2), its transfer to creeping bentgrass (Agrostis palustris Huds.) and development of brown patch (Rhizoctonia solani) disease resistant transgenic lines. In: Plant Science. 2002 ; Vol. 163, No. 2. pp. 183-193.
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AU - Chai, Benli

AU - Maqbool, Shahina B.

AU - Hajela, Ravindra K.

AU - Green, David

AU - Vargas, Joseph M.

AU - Warkentin, Donald

AU - Sabzikar, Robab

AU - Sticklen, Mariam B.

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N2 - We isolated a cDNA clone (hs2) encoding a chitinase-like protein from a triploid Dutch elm disease resistant American elm (Ulmus americana NPS-3-487). Amino acid sequence of this chitinase-like protein showed 65-78% homology to other plant chitinases. A plasmid KYLX71-pHS2 was constructed using the hs2 sequence under the control of cauliflower mosaic virus 35S promoter and nos terminator. This construct was used to genetically engineer creeping bentgrass (Agrostis palustris Huds.) via Biolistic® PDS-100/He system. Plasmid JS101 containing the bar herbicide resistance selectable marker gene regulated by the rice actin 1 (Act1) promoter was used as a selectable co-transformation marker. Transgenic plants were produced with a 21.1% transformation frequency on the basis of bialophos selection. The co -transformation frequency for hs2 was 5.68%. Linked transgenes (bar and mtld) were co-integrated with a frequency of 100% as expected and 21.3% of the transgenic plants showed integration of all three genes. Southern blot analysis of transformants showed that the hs2 transgene copy numbers ranged from 1 to 5. Northern blot hybridization confirmed transcription of the transgenes, and western blot determined the HS2 protein expression. No correlation was found between gene copy number and the level of gene expression. Disease resistance profiles of five independent transgenic lines in greenhouse trials revealed that two lines (711 and 9603) were significantly (P < 0.05) resistant to Rhizoctonia solani, the causative agent of brown patch disease.

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