Using Virus-inducing gene silencing (VIGS) technique to study the function of PhLA in Petunia flowers
Date Issued
2011
Date
2011
Author(s)
Chen, Hsiao-wei
Abstract
The plant develops various shapes of flowers in nature in order to attract pollinators for increasing the progenies of next generation. Asymmetry is one special trait for distinguishing different species of flowers. In previous research, CYC was discovered to be a major gene deciding the dorsal-ventral asymmetry of the flower in Antirrhinum. CYC is a TCP transcription factor and the BLAST result from petunia EST database identified two CYC-like DNA segments and applied to virus-induced gene silencing (VIGS) for petunia cultivar ‘Primtime Blue’. In the VIGS treated plants with reduced target gene expression based on the CYC-like segments, comparing to the control plant instead of showing different morphology of flower asymmetry, greater curvature of the petal lobe was observed. The full cDNA sequence of the silenced gene was obtained by RACE PCR and its derived amino acid sequence was used for alignment with other genes. The alignment result classified this gene into TCP gene family and the CIN subgroup, is closely related to its homologs CINCINNATA in snapdragon (Antirrihnum majas), TCP4 in Arabidopsis (Arabidopsis thaliana L. ) and an ortholog of LA in tomato (Solanum lycopersicon L.), thus named the gene as PhLA. Previous research about AmCIN, AtTCP4 and SlLA in either mutants or transformants showed defects in both leaves and flowers, however in petunia only exhibits abnormal growth in the flower when PhLA expression is down regulated. In this research, based on the phenotype observed in the VIGS treated plants, a number of techniques such as RC-PCR semi-quantification, in situ hybridization and scanning electronic microscopy (Hennig et al.) were used to analyze the role of PhLA in the development of petunia flowers. So far, it is indicated cells around the central vein area of petal lobes turned from conical shape to flatter and greater type which is parallel to the phenotype of petal lobes measured of greater maximum width, shorter minimum width and greater heights. Moreover, a miR319 recognition site near the 3’-UTR of PhLA was identified; previous research of miR319 in Arabidopsis and tomato verified that its expression affects the growth of both flowers and leaves by down regulating target genes. It is suspected that PhLA is regulated by miR319 and may be a deciding factor of flower morphology. In summary, it is expected to understand the evolutionary role of PhLA and the influence of regulatory mechanism of miR319 and PhLA to the flower curvature with the use of VIGS technique.
Subjects
TCP transcription factor
PhLA
miR319
virus-induced gene silencing (VIGS)
flower curvature
Type
thesis
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