| Summary: | 碩士 === 國立中興大學 === 分子生物研究所 === 82 === According to the sequence information for the promoter region
of TA29, a pair of primers AS29A and AS28B that could
specifically amplify this promoter region was synthesized and a
DNA fragment of 366 bp containing the promoter region of TA29
was then amplified from tobacco genomic DNA by polymerase chain
reaction ( PCR ). To test the tissue specific function of this
PCR amplified promoter fragment, a chimeric gene construct
pBIA129 that contains this 366 bps promoter region fused to GUS
reporter gene was obtained. Through agrobacteria transformation
system, five kanamycin resistant tobacco plants were screened
and the existence of this chimeric gene for each kanamycin
resistant tobacco plant was demonstrated by PCR and Southern
analysis. Histochemical analysis demonstrated that the GUS
enzyme activity was expressed only in the tapetal cells of the
third stage of tobacco anther. The results indicated that the
PCR amplified promoter fragment could program the temporal and
spatial expression of GUS gene in transgenic tobaccos. A cDNA
clone BA112 that derived from the mRNA of Brassic anther was
previously demonstrated to be tapetal specific by Northern blot
and in situ hybridization analysis. To search for this tapetal
specific gene, a genomic library of Brassic napus was screened
with BA112 cDNA probe. After screening for about 2.5×105
plaques, there were 36 clones showed positively hybridized to
BA112 cDNA but only one clone gBA05 showed strongly hybridized
to an oligonucleotide probe BA19, an oligonucleotide probe
locates at the 5' end of BA112.
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