Isolation of an anther-specific gene from rice (Oryza sativa L.)

Jang-Yong Lee, Purdue University

Abstract

In order to develop a system by which male sterility and fertility of rice can be controlled, it is necessary to have regulating elements (promoters) that regulate the expression of genes expressed only in the male organ during anther development. A rice anther (tapetum)-specific cDNA and corresponding genomic DNA have been cloned from IR54 rice variety. A panicle cDNA library was constructed in Uni-Zap XR vector, and differentially screened first for panicle specificity and second for anther specificity. Two cDNA clones which were cross hybridizable with each other were selected and a 0.52kb EcoRI and XhoI fragment of a cDNA clone (RTS1) was used as the probe for screening of an IR54 genomic library constructed in Lambda-Gem 11 vector. A positive genomic clone (RTS2) was isolated and sequenced. Northern blot and in situ hybridization results showed that the gene was predominantly expressed in the tapetum of the anther. The mRNA of the gene began to accumulate in the spikelet at a time normally associated with the early stage of meiosis, reached a maximum at the stage of vigorous meiosis, and disappeared prior to anthesis. There was no expression observed in leaves or seeds. Sequence comparison between the cDNA clone (RTS1) and the corresponding genomic clone (RTS2) and a primer extension assay showed that this gene has no introns and a continuous coding region of 285 base pairs coding for a polypeptide of 94 amino acids. The coding region of the gene has a high GC content of 75.8% (216/285). A comparison of the nucleotide and deduced amino acid sequences with those in data banks has not shown any significant homology to known sequences. However, there is a sequence, GAATTTGTTA, in the promoter region, which differs only by one or two nucleotides from one of the conserved sequence motifs in the promoter region of two of the pollen-specific genes of tomato. In order to confirm the anther-specificity and to determine the regulatory sequences of the anther-specific gene (RTS2) various chimeric plasmid constructions containing different lengths of the 5$\sp\prime$ controlling region of the rice anther-specific promoter driving gusA gene were made. The various constructs were used to test promoter activity transiently in rice tissues using particle bombardment and in stably transformed tobacco plants using Agrobacterium as the transformation vector. Although the rice tissues bombarded with these constructs showed some tissue specificity, GUS staining in bombarded anthers was weak as compared to bombarded spiklets. The transformed tobacco plants with these constructs, which were confirmed by Southern blot analysis to have gusA gene, did not show any GUS activity. The promoter function and regulation of RTS2 for anther-specificity may be limited to rice, and it will be necessary to obtain rice plants transformed with these anther-specific promoter constructs and to compare the expression of the gusA gene in these transgenic rice plants to determine the DNA sequences responsible for controlling the anther-specific expression.

Degree

Ph.D.

Advisors

Hodges, Purdue University.

Subject Area

Molecular biology|Agronomy

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