Theoretical and Applied Genetics

, Volume 98, Issue 3, pp 371-378

Inheritance, expression, and silencing of a chitinase transgene in rice

  • S. ChareonpornwattanaAffiliated withDepartment of Biochemistry, Kansas State University, Manhattan, KS 66506, USA, and Department of Biotechnology, Mahidol University, Bangkok, Thailand
  • , K. V. TharaAffiliated withDepartment of Biochemistry, Willard Hall, Kansas State University, Manhattan, KS 66506-3702, USA Fax: +1 785-532-7278 E-mail: smk@ksu.edu
  • , L. WangAffiliated withDepartment of Biochemistry, Willard Hall, Kansas State University, Manhattan, KS 66506-3702, USA Fax: +1 785-532-7278 E-mail: smk@ksu.edu
  • , S. K. DattaAffiliated withPlant Tissue Culture Laboratory, International Rice Research Institute, P.O. Box 933, 1099 Manila, The Philippines
  • , W. PanbangredAffiliated withDepartment of Biotechnology, Mahidol University, Bangkok, Thailand
  • , S. MuthukrishnanAffiliated withDepartment of Biochemistry, Willard Hall, Kansas State University, Manhattan, KS 66506-3702, USA Fax: +1 785-532-7278 E-mail: smk@ksu.edu

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Abstract

 The inheritance and expression of a transgene locus consisting of multiple copies of a rice chitinase gene under the control of the CaMV 35S promoter was studied in the T3 and T4 generations of a transformed line that expressed the chitinase at a high level. All T3 progeny of a homozygous T2 parent expressed the chitinase constitutively at 3 weeks after germination, but a proportion of the progeny had undetectable levels of chitinase 8 weeks after germination, indicating silencing of the transgene. Transgene silencing was also observed among progeny of a hemizygous parent. However, we did not observe chitinase gene silencing among progeny of another homozygous line that expressed the transgenic chitinase at a five- to tenfold lower level. Thus, expression level, rather than copy number, of the transgene appears to be critical for silencing. Silencing was observed in the leaf, sheath, and root tissues of the plant, indicating that it is not restricted to specific tissues. Silencing was first observed in the youngest leaves and only later in the oldest leaves of the same plant. There was co-silencing of the selectable marker gene, hpt, which is also driven by the CaMV 35S promoter. Unlike the two transgenes (chitinase and marker), the resident homologous chitinase gene with seed-specific expression and two nonhomologous chitinase genes induced in the leaves upon pathogen infection were not silenced. The silent phenotype was inherited in the T4 generation plants, while progeny of expressing plants exhibited silencing. The chitinase transgene appeared intact, and no evidence for gross alterations or methylation of CCGG sites was found. The silent phenotype could not be reversed by treatment with 5-azacytidine. Northern blot analysis and nuclear run-on transcription studies indicated that silencing occurred at the transcriptional level. The implications of transgene silencing in genetic engineering of monocot plants for disease resistance are discussed.

Key words Chitinase Gene-silencing hpt-gene-silencing Rice Transcriptional silencing