Isolation And Expression Analysis Of Auxin And Cytokinin-response Genes In Hyacinthus Orientalis In Vitro Floral-organ Regeneration System

Explants of Hyacinthus orientalis cultured on MS medium with 2,4-D (0.1 mg/L) and 6-BA (2.0 mg/L) can produce floral meristem directly. This in vitro system suggests the floral meristem can be induced by exogenous hormones. By using this system, we try to isolate the hormone-responsive genes which may related to floral regeneration.A cDNA labrary was constructed by using the floral meristem materials from the explants cultured on the medium containing hormones for 5 days and 10 days.Screenings of cDNA library was carried out by a microarray technique. Two probes were used. Positive probe was made from the floral meristem materials cultured for 5 days and 10 days with hormones by RNA reverse transcription and labelling with radioactive dCTP; two negative probes from uncultured explants and it cultured for 5 days and 10 days. Then, we did calculating signal arrays, sequencing, sequence analysis and alignments on GeneBank etc. Finally 229 different ESTs were got from the cDNA library. By combining the information from the cDNA sequences with functions of their homologs, we suggest that two kinds of hormones may play important roles during flower regeneration in a few ways: (i) proteolytic system of ubiqutin/26S proteosome complex, (ii) actin-dependent vesicular cycling of auxinefflux carrier, (iii) regulating transcription of genes, (iv) phosphorylation and dephosphorylation.The four genes were selected for northern analysis. The results are as follows:Exogenous hormones enhance HFT gene expression. Sequence alignment suggests HFT (Hyacinthus forkhead transcription factor) is a novel gene. Its expressing level was enhanced by the hormones, and lower level were maintained in explants uncultured or cultured without hormones. HFT expresses in roots, leaves, tepals, stamens and ovaries in the plant.Hormones promote expression ofHZFl, & Hyacinthus zinc finger gene, during 24 hours cultured. However, the hormones repress its expression after day 5. HZF1 has no or low level mRNA accumulation in the eplants cultured without hormones, and its expression were found in all organs in the plant. So it is response to the exogeneous hoemones at an early stage.Hormones are essential for maintaining the expression of HRZ, a ring zinc finger gene. HRZ expresses in the explants cultured with hormones for 10 days and the level of expression is declined at day 15. It expresses in all organs in the plant. But no its mRNA was detected without hormones. So it may functions in the early developmental events of regenerated flower.A homolog of GA Sbeta-hydroxylase gene expression pattern was examined. Hybridization signals of this gene could be detected at day 5 and however, they appear again after day 10 when explants cultured with hormones. It has the similar pattern when the explants cultured without hormones or uncultured. This gene may encode the enzyme which may be involved in GA biosynthesis.