Gene Construct and Transformation of Rice Plants

A full length glutelin cDNA and its promoter region were isolated from a Japanese cultivar, Sasanishiki. A partial fragment at the 5' end of the cDNA was cut out and assembled to make eight tandem repeats of the fragment. This was done because a preliminary study with in vitro translation implied that antisense RNA transcribed from the repeats can inhibit the translation of the glutelin gene more effectively than single, full length, antisense RNA. The antisense construct was assembled from the glutelin promoter region, the first intron of the castor bean catalase gene, the eight tandem repeats in antisense orientation, and the NOS terminator, in that order.

The assembled construct was transferred to a super-binary vector of A. tumefaciens carrying two T-DNA regions, one of which incorporates a hygromycin resistance gene, hpt.14 Calluses derived from mature embryos of japonica rice (cv. Tsukinohikari) were trans formed, and hygromycin resistant plants were obtained as described by Hiei et al.2

Out of 106 hygromycin resistant regenerates, 14 plants were selected for low copy number of the antisense gene, normal plant shape, and high fertility. The selfed seeds of these plants were harvested in a greenhouse experiment, and their glutelin content was determined by density measurements of glutelin bands made visible with CBB in SDS-PAGE analyses oftotal seed proteins. Two plants that showed considerable reduction in the glutelin content were selected for further examination, and their progeny formed lines H39 and H75.

H39 showed two locus segregation for the antisense gene, one of which was not linked with the hpt gene. Plants possessing the antisense gene in a homozygous state and lacking the hpt gene were selected. H75 showed one locus segregation of the two linked transgenes, and homozygous plants were selected.

The progeny of H39 and H75 were grown in an isolated field experiment. Northern blot analysis revealed that these two lines showed considerable decrease in glutelin mRNA in immature grains. Mature seed proteins of these lines were also analyzed. H39 and H75 showed thinner glutelin bands than those of wild type plants, but thicker bands of prolamin, another storage protein group. The total nitrogen of the glutelin fraction of H39 was lower than that of the wild type plants, although the total nitrogen in its whole grains was almost the same as that of the wild type plants. These results imply that glutelin synthesis and accumulation were inhibited by the antisense gene but that prolamin possibly increased, resulting in little change overall in total protein content in H39. It is possible that inhibition of synthesis of one protein results in a compensatory increase in other proteins.

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