Production and analysis of cor47 Gene-Containing Transgenic Rice Plants

Section of Biochemistry. Molecular and Cell Biology, Cornell University. Ithaca, NY 14853, USA

Introduction

Important abiotic stresses that result in lowering rice yield include drought, high soil salinity and low temperature. Plant tolerance of these stresses is thought to be controlled by a number of genes and involves several component traits which are likely to be quantitative in nature. More than ten individual genes have been identified and cloned, which are expressed specifically in response to different abiotic stresses (for reviews, see Ingram and Bartels 1996; Bray 1997). Potentially, any one of these genes can confer a certain degree of stress tolerance. However, the potential contribution of each of these genes needs to be tested, one at a time, by generating and analyzing transgenic plants.

Several genes have been reported to be stimulated by high salinity, drought and low temperature, and the corresponding eDNA sequences have been cloned in Arabidopsis (Gilmour et a!. 1992). One of the cDNAs is known as cor47.

We constructed a plasmid, pCOR47 (Figure 1), containing a chimenc gene with a

1.1-kb cDNA sequence of the Arabidopsis cor47 cDNA (Gilmour eta!. 1992) fused to the

Acti promoter from rice and the potato PinIl 3� terminator sequence (Actl-cor47-PinIl

3�). The same plasmid includes a selectable marker cassette (35S promoter-bar-nos

3�). We introduced the plasmid into rice suspension cells via the biolistic method (Cao et al. 1992). Suspension cell cultures were established from embryogenic calli induced from mature embryos of rice (Oryza sativa L. cv. Kenfeng) after one month of subculture in liquid AA2 medium, with changes of medium every other day. Selection and regeneration of
transformed cells were performed on the basis of resistance to ammonium glufosinate. The presence and integration status of the transgene were conducted in 56 independent transgenic lines by PCR and Southern blot analysis based on the procedure of Duan eta!. (1996). The result of Southern blot analysis (Figure 2) showed that several of the R1 progeny of heterozygous herbicide-resistant plant lines harbored between one and three copies of the transgene. These plants appeared phenotypically normal and were selected for further analysis.

We used three different transgenic lines (#2, #25 and #27) and one non-transgenic control plant line to determine the cor47 mRNA levels in different tissues by Northern slot-blot analysis. Both cor47 transgenic lines in the R0 and R1 generations showed increased expression levels of the cor47 gene in all three major tissues tested (leaf, stem and root), as compared to the control plant (NT) (Figure 3). The slot-blot results are semi- quantitative because the purity of the RNA samples was not known and may not have been the same in different samples.

We analyzed the response of cor47-transgenic rice plants grown under drought conditions. To start water stress, water was withheld from the trays for 5 days. The stressed plants were then supplied with water for 2 days to allow the plants to recover. A total of four such cycles (28 days) was applied. Our preliminary result showed that R1 generation cor47-transgenic rice plants are more tolerant to water deficiency under greenhouse conditions than control plants (Table 1). More specifically, both cor2- and cor25-transgenic lines showed a much faster growth rate as indicated by plant height/root length and dry weight of shoot and root, as compared to the control plant (NT).

In summary, we have produced a number of transgenic rice lines that harbor a cor47 cDNA-containing plasmid. Our preliminary data demonstrated that cor47 gene-encoded protein is correlated with a faster growth rate under drought conditions.

Table 1. Growth performances of cor47-transgenic and control rice plants under water deficit conditions

The plants used in this experiment were 40-day-old seedlings of R, generation plants. H = height; DW = dry weight; L = length.

References Bray, E.A., 1997. Plant responses to water deficit. Trends in Plant Science 2: 48-54. Cao, I., X. Duan, D. McElroy and R. Wu, 1992. Regeneration of herbicide-resistant transgenic rice plants following microprojectile-mediated transformation of suspension culture cells. Plant Cell Reports 11: 586-591. Duan, X.L., X. Li, Q. Xue, M. Abo-E1-Saad, D. Xu and R. Wu, 1996. Transgenic rice plants harboring an introduced potato proteinase inhibitor II gene are insect resistant. Nature Biotech. 14: 494-498. Gilmour, Si., N.N. Artus and M.F. Thomashow, 1992. cDNA sequence analysis and expression of two cold regulated genes of Arabidopsis thaliana. Plant Mo!. Biol. 18: 13-21. Ingram, J. and D. Bartels, 1996. The molecular basis of dehydration tolerance in plants. Annu. Rev. Plant Physiol. Plant Mol. Biol. 47: 377-403.

References
Bray, E.A., 1997. Plant responses to water deficit. Trends in Plant Science 2: 48-54.

Cao, I., X. Duan, D. McElroy and R. Wu, 1992. Regeneration of herbicide-resistant transgenic rice plants following microprojectile-mediated transformation of suspension culture cells. Plant Cell Reports 11:

Duan, X.L., X. Li, Q. Xue, M. Abo-E1-Saad, D. Xu and R. Wu, 1996. Transgenic rice plants harboring an introduced potato proteinase inhibitor II gene are insect resistant. Nature Biotech. 14: 494-498.

Gilmour, Si., N.N. Artus and M.F. Thomashow, 1992. cDNA sequence analysis and expression of two cold regulated genes of Arabidopsis thaliana. Plant Mo!. Biol. 18: 13-21.

Ingram, J. and D. Bartels, 1996. The molecular basis of dehydration tolerance in plants. Annu. Rev. Plant Physiol. Plant Mol. Biol. 47: 377-403.