Cloning And Functional Analysis Of ZmDWF1, A Maize Gene Involved In Brassinosteroids Biosynthetic Pathway

Brassinosteroids (BR) are natural plant growth regulators required for plant growth and development. Brassinolide (BL) is the most bioactive form found to date. BR-deficient or BR-insensitive mutants display dwarfism. Molecular genetic and physiological studies on BR-related mutants of dicot plants play a critical role in normal plant growth and development. However, little is known about the function of BR in monocots (grasses), except for the phenotypic analysis of three rice mutants. The Arabidopsis DWF1/DIM is a bifunctional protein which catalyses both the A24 isomeration, and subsequently, the â–³24 (25) reduction in BR biosynthesis pathway.To better understand the role of DWF1 of monocot plants in BR biosynthesis, we firstly cloned a maize gene, ZmDWFl, which has extensive sequence similarity to Arabidopsis DWF1. ZmDWFl is predicted to encode a 562-amino-acid protein that has 95% and 86% similarity with rice OsDWFl and Arabidopsis DWF1, respectively. Southern blot result showed that one copy of ZmDWFl is present in maize genome. Northern blot analyses revealed that expression of ZmDWFl is temporally and spatially regulated during development, and especially in young root tip. Immunofluorescence localization experiment in maize root tip showed that there are large amount of bright red granule-like fluorescence in the cells of root tip (calyptrogen), and weak or no such fluorescence in the other cells. So these results maybe imply the presence of complete BR biosynthetic pathway in maize root, especially in root tip (calyptrogen).Secondly, although protein sequence hydropathy prediction result showed ZmDWFl is an integrated membrane protein, we still successfully expressed the full-length protein fused into the plasmid pET30a (+), then purified the 70 KD fusion proteins by Ni-NTA agarose affinity chromatography column. After immunning rabbit four times, the rabbit polyclonal antibodies were obtained. We also localized the putative membrane protein in the endoplasmic reticulum membrane using immunogold-labeling method. This is the first reported directly localized experiment of BR-related protein.Thirdly, transgenic maize plants with double-stranded ZmDWFl RNA were generated mediated by Agrobacterium. PCR and Southern blot analysis demonstrated the integration of this RNAi construct into maize genome. Western blot result in the early and late stage maize leaves also demonstrated the expected cosuppression of ZmDWFl gene in transgenic dwarfed plants, and the suppressed degree is consistently with dwarfed degree. Transgenic plants had dwarfed phenotype varying in severity, showed a range of abnormalities in organ growth and development, the most striking of which were defects in the elongation of the stem and leaves, and in the differentiation of the vascular bundles. Light microscopic observations revealed that these abnormalities were primarily owing to a failure in the organization and polar elongation of the leaf and stem cells. Electron microscopic observationsfurther showed the abnormalities in stoma and chloroplast. Based on this, we discuss the biological function of BR in maize plants.Finally, transgenic Arabidopsis plants with the same construct showed dwarfed phenotype like dwfl mutant. PCR-Southern blot analysis demonstrated that the integration of this RNAi construct into Arabidopsis genome. Western blot results also demonstrated the expected cosuppression of ZmDWFl gene in transgenic dwarfed plants. This fact demonstrated the heterologous gene silencing is possible based on the high sequence similarity between the maize and Arabidopsis DWF1 homologues. So maybe this construct can be used in dwarfed gene engineering of other crops.