A maize α-zein promoter drives an endosperm-specific expression of transgene in rice.

An alpha-zein promoter isolated from maize containing P-box, E motif sequence TGTAAAGT, opaque-2 box and TATA box was studied for its tissue-specific expression in rice. A 1,098 bp promoter region of alpha-zein gene, fused to the upstream of gusA reporter gene was used for transforming rice immature embryos (ASD 16 or IR 64) via the particle bombardment-mediated method. PCR analysis of putative transformants demonstrated the presence of transgenes (the zein promoter, gusA and hpt). Nineteen out of 37 and two out of five events generated from ASD 16 and IR 64 were found to be GUS-positive. A histological staining analysis performed on sections of mature T1 seeds revealed that the GUS expression was limited to the endosperm and not to the pericarp or the endothelial region. GUS expression was observed only in the following seed development stages : milky (14-15 DAF), soft dough (17-18 DAF), hard dough (20-23 DAF), and mature stages (28-30 DAF) of zein-gusA transformed (T0) plants. On the contrary a constitutive expression of GUS was evident in CaMV35S-gusA plants. PCR and Southern blotting analyses on T1 plants demonstrated a stable integration and inheritance of transgene in the subsequent T1 generation. GUS assay on T2 seeds revealed that the expression of gusA gene driven by alpha-zein promoter was stable and tissue-specific over two generations. Results suggest that this alpha-zein promoter could serve as an alternative promoter to drive endosperm-specific expression of transgenes in rice and other cereal transformation experiments.

Genetic transformation of tropical maize (Zea mays L.) inbred line with a phytase gene from Aspergillus niger.

A full-length cDNA of phyA gene of Aspergillus niger, encoding phytase enzyme, was cloned and expressed in E. coli BL21 cells and assayed for its activity. The phyA cDNA consisted of 1404 bp, which encoded 467 amino acid residues. The phytase activity of purified phytase was 826.33 U/mL. The phyA gene under the control of endosperm-specific promoters was transformed into an Indian maize inbred line, UMI29, using particle bombardment-mediated transformation method to generate transgenic maize plants over-expressing phytase in seeds. PCR and GUS analyses demonstrated the presence of transgenes in T0 transgenic plants and their stable inheritance in the T1 progenies. Three transgenic events expressing detectable level of A. niger phytase were characterized by western blot analysis. Phytase activity of 463.158 U/kg of seed was observed in one of the events, JB-UMI29-Z17/2. The phytase activity of transgenic maize seeds was 5.5- to 7-fold higher than the wild-type UMI29 seeds and, consequently, the seeds had 0.6- to 5-fold higher inorganic phosphorus content.

Pyramiding transgenic resistance in elite indica rice cultivars against the sheath blight and bacterial blight.

Elite indica rice cultivars were cotransformed with genes expressing a rice chitinase (chi11) and a thaumatin-like protein (tlp) conferring resistance to fungal pathogens and a serine-threonine kinase (Xa21) conferring bacterial blight resistance, through particle bombardment, with a view to pyramiding sheath blight and bacterial blight resistance. Molecular analyses of putative transgenic lines by polymerase chain reaction, Southern Blot hybridization, and Western Blotting revealed stable integration and expression of the transgenes in a few independent transgenic lines. Progeny analyses showed the stable inheritance of transgenes to their progeny. Coexpression of chitinase and thaumatin-like protein in the progenies of a transgenic Pusa Basmati1 line revealed an enhanced resistance to the sheath blight pathogen, Rhizoctonia solani, as compared to that in the lines expressing the individual genes. A transgenic Pusa Basmati1 line pyramided with chi11, tlp, and Xa21 showed an enhanced resistance to both sheath blight and bacterial blight.