A SNP in OsMCA1 responding for a plant architecture defect by deactivation of bioactive GA in rice.

Plant architecture directly affects biomass in higher plants, especially grain yields in agricultural crops. In this study, we characterized a recessive mutant, plant architecture determinant (pad), derived from the Oryza sativa ssp. indica cultivar MH86. The mutant exhibited severe dwarf phenotypes, including shorter and stunted leaves, fewer secondary branches during both the vegetative and reproductive growth stages. Cytological studies revealed that pad mutant growth defects are primarily due to the inhibition of cell expansion. The PAD gene was isolated using a map-based cloning strategy. It encodes a plasma membrane protein OsMCA1 and a SNP responsible for a single amino acid change was found in the mutant. PAD was universally expressed in rice tissues from the vegetative to reproductive growth stages, especially in seedlings, nodes and rachillae. Quantitative real-time PCR analysis revealed that the most of the genes responding to gibberellin (GA) metabolism were up-regulated in pad mutant internodes. The endogenous GA content measurement revealed that the levels of GA1 were significantly decreased in the third internode of pad mutants. Moreover, a GA response assay suggested that OsMCA1/PAD might be involved in the regulation of GA metabolism and signal transduction. Our results revealed the pad is a loss-of-function mutant of the OsMCA1/PAD, leading to upregulation of genes related to GA deactivation, which decreased bioactive GA levels.

Quantitation of the residual DNA from rice-derived recombinant human serum albumin.

Residual DNA in recombinant protein pharmaceuticals can potentially cause safety issues in clinical applications; thus, maximum residual limit has been established by drug safety authorities. Assays for residual DNA in Escherichia coli, yeast, and Chinese hamster ovary (CHO) cell expression systems have been established, but no rice residual DNA assay for rice expression systems has been designed. To develop an assay for the quantification of residual DNA that is produced from rice seed, we established a sensitive assay using quantitative real-time polymerase chain reaction (qPCR) based on the 5S ribosomal RNA (rRNA) genes. We found that a 40-cycle qPCR exhibited a linear response when the template concentration was in the range of 2×10(4) to 0.2pg of DNA per reaction in TaqMan and SYBR Green I assays. The amplification efficiency was 103 to 104%, and the amount of residual DNA from recombinant human serum albumin from Oryza sativa (OsrHSA) was less than 3.8ng per dosage, which was lower than that recommended by the World Health Organization (WHO). Our results indicate that the current purification protocol could efficiently remove residual DNA during manufacturing and processing. Furthermore, this protocol could be viable in other cereal crop endosperm expression systems for developing a residual DNA quantitation assay using the highly conserved 5S rRNA gene of the crops.