The roles of homeodomain proteins during the clamp cell formation in a bipolar mushroom, Pholiota nameko

서지정보

ㆍ발행기관 : 한국버섯학회 ㆍ수록지정보 : 한국버섯학회지 / 9권 / 1호
ㆍ저자명 : Ruirong Yi, Hiroyuki Mukaiyama, Takashi Tachikawa, Norihiro Shimomura, Tadanori Aimi

목차

ABSTRACT
Introduction
Materials and methods
Fungal strains
Mycelium preparation, DNA and RNA extraction
Amplification of A3-hox1 and A3-hox2 genes
Co-transformation method
DAPI and Fluorescent Brightener 28 staining andmicroscopic observation
Construction of two plasmid vectors for overexpressionof the A3-hox1 and A3-hox2 genes
Southern hybridization
Real-time PCR assay
Results and discussions
A single introduced hox gene is insufficient to inducetrue clamps in high frequency
Two separated, introduced hox gene increases thefrequency of clamps
Two introduced combined hox gene also increasethe frequency of fused hook cell
Greater expression of the hox genes drive the realclamp formation
Different growth condition was observed in differentkinds of transformants
Different expression amount of four hox gene indifferent kinds of transformants
REFERENCES

영어 초록

In the bipolar basidiomycete Pholiota nameko, a pair of homeodomain protein genes located at the A mating-type locus regulates mating compatibility. In the present study, we used a DNA-mediated transformation system in P. nameko to investigate the homeodomain proteins that control the clamp formation. When a single homeodomain protein gene (A3- hox1 or A3-hox2) from the A3 monokaryon strain was introduced into the A4 monokaryon strain, the transformants produced many pseudo-clamps but very few clamps. When two homeodomain protein genes (A3-hox1 and A3-hox2) were transformed either separately or together into the A4 monokaryon, the ratio of clamps to the clamp-like cells in the transformants was significantly increased to approximately 50%. We, therefore, concluded that the gene dosage of homeodomain protein genes is important for clamp formation. When the sip promoter was connected to the coding region of A3-hox1 and A3-hox2 and the fused fragments were introduced into NGW19-6 (A4), the transformants achieved more than 85% clamp formation and exhibited two nuclei per cell, similar to the dikaryon (NGW12 -163 × NGW19-6). The results of real-time RT-PCR confirmed that sip promoter activity is greater than that of the native promoter of homeodomain protein genes in P. nameko. So, we concluded that nearly 100% clamp formation requires high expression levels of homeodomain protein genes and that altered expression of the A mating-type genes alone is sufficient to drive true clamp formation.

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