| Summary: | 碩士 === 國立中興大學 === 分子生物研究所 === 81 === To identify if the chloroplast promoter contains its own
specific sequence, many more promoters from various chloroplast
genes will be required. A plasmid pUTT containing two E. coli
thr terminators locate at two ends of the multiple cloning
sites was used as the vector for shotgun screening of spinach
chloro plast promoters. The clones that expressed promoter
activity after in vitro transcription were isolated and
characterized. The promoter-containing clones were first
sequenced and verfied by comparing to the known spinach and
tobacco chloroplast DNA sequences from Gene Bank. Twenty-two
clones, including the 5' regions of atpB, psaA, psaB, psbD,
rbcL, rpoC, rps4, rps14, trnC1, trnF1, trnS1, trnS2, trnT1 and
trnV1 were identified. A clone that contains the light-induced
promoter of psbD gene was further characterized. The psbD gene
is a light regulated gene and encodes a photosystem II reaction
center D2 protein. The essential promoter sequences for the
transcription of psbD gene were analyzed by 5' end deletion
with exonuclease III. The in vitro transcription results of
deleted mutants revealed that three transcription initiation
sites locate within -641 to -1619 nt upstream from the
translation start site. The three trans- cription initiation
sites for the psbD gene were measured to be located at "-1180",
"-1040" and "-990" respectively according to the size of
transcripts. The results of primer extension analysis for psbD
RNA revealed a set of mltiple transcription initaition sites
within the -850 to -950 region of psbD genes.
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