| Summary: | 碩士 === 國立臺灣大學 === 園藝學系 === 81 === The first and key enzyme in the branch pathway of
phenylpropanoid biosynthesis is chalcone synthase (CHS).
It catalizes the condensation of the acyl residues from one
molecule of 4-coumaroyl-CoA and three molecules of malonyl-
CoA. In order to isolate the complementary DNA
sequences for CHS mRNA degenerate oligonucleotide
primers corresponding to conserved region of chalcone
synthase from various plant species were synthesized.
These primers were used for polymerase chain reaction
to amplify DNA fragments from first strand cDNA
synthesized from poly(A)+ RNA isolated from Dtps. Queen
Scarlet `A77-30' petals. The synthesized fragments ranged
between 100 and 600bp. The sequence of these DNA fragments
have been determined and their deduced amino acid sequences
showed that they were derived from CHS genes. Approximately
260,000 plaque-forming units were screened from the
Phalaenopsis (Lucky Lady x Pinglong Cardinal) `B79-19' cDNA
library constructed in λ ZAP II vector by plaque
hybridization with 32P-labelled CHS fragments and we got 11
positive putative clones. Purified indivifually,
Phalaenopsis chalcone synthase cDNA were digested to draw
restriction maps. One of the cDNA (pOCHS01) was chosen for
sequence analysis. The coding region encodes 390 amino acids
and was determinated to be 42.5 kDa with an isoelectric point
of 6.0. The identity of Phalaenopsis CHS to other CHS
polypeptide from different plant species varies between 59.5
and 64.9%. According to Southern analysis of genomic DNA
digested with BamHI and EcoRI, we show that both Phalaenopsis
and Doritaenopsis chalcone synthase belong to multigene
family. Genomic Southern analysis of F1 progeny and their
white and pink flower patterns showed that DNA patterns of
progeny was similar to the maternal parent.
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