| Summary: | 博士 === 國立中興大學 === 植物學系 === 86 === Thirty-eight cDNA clones representing genes expressed in the
rice embryo by differential screening of a cDNA library made
with poly(A) RNA from embryos at the stage of 10-days after
pollination have been isolated. Eighteen cDNA clones displayed
either a significant nucleic acid or amino acid sequences
similarity to sequences kno?充n the GenBank, i.e., two rice
oleosins, four globulin or glutelin-like proteins, two ABA
response proteins, two LEA-like proteins, one trypsin inhibitor,
one metallothionein-like protein, one protein kinase, one
leucine-zipper containing protein, one proline-rich protein and
three genes with unknown function. Experimental results
indicated the presence of redundant cDNA clones for genes that
produced abundant amount of mRNAs in the embryo. RNA slot blot
hybridization demonstrated the embryo-specific expression for
thirty-seven cDNA clones. The majority of the identified cDNA
had not been known in rice. Results in this study contribute
toward the on-going attempt to isolate embryo-specific and novel
genes in rice. The oleosin, LEA-like protein, trypsin inhibitor,
one unknown gene and metallothionein-like protein were analyzed
and characterized.Plant seeds store triacylglycerols (TAGs) as
energy sources for germination and postgerminative growth of
seedlings. The TAGs are preserved in small discrete
intracellular organelles called oil bodies. An oil body contains
a TAG matrix surrounded by a layer of phospholipid and proteins
termed oleosin. We have isolated and sequenced two rice embryo-
specific cDNA clones Ose701 and Ose721, which encode the 16 kD
and 18 kD oleosin isoforms respectively. The cDNA nucleotide
sequences of Ose701 shares 72% similarity to maize ole16 , and
the cDNA nucleotide sequences of Ose721 shares 72% and 74%
similarity to that of maize ole18 and barley ole1, respectively.
Several families of proteins that accumulate to high levels in
plant embryonic tissues during the maturation phase of seed
development were termed late embryogenesis abundant (LEA)
proteins. We have isolated and sequenced a rice embryo-specific
cDNA clone Ose730. The cDNA fragment of Ose730 is 953
nucleotides in length, the deduced translation product consist
of 200 amino acids with eight conserved 11-amino acid tandem
repeated structure, a typical structure motif of the group 3 LEA
proteins. The amino acid sequences of cDNA clone Ose730 shows
70% similarity to maize LEA protein. Southern blot analysis
showed that Ose730 is a single copy nuclear gene in rice genome.
By Northern blot analysis Ose730 expression was shown to be
detected in embryos of 5DAP to embryos from mature, harvested
after drying seed, and mRNA was not detected in de-embryo
grains, seedling, young leaves, roots and anthers. Theses
results revealed the embryo-specific expression for Ose730
clone.The proteinase inhibitors are widespread in both
monocotyledons and dicotyledons and appear to function as part
of the defense system against insect attack. We report the
nucleotide sequences of a rice embryo-specific cDNA clone Ose727
A encoding amino acid sequences almost identical to that of rice
bran trypsin inhibitor (RBTI). The amino acids of RBTI showed a
high degree of sequence homology to wheat germ trypsin inhibitor
and soybean Bowman-Birk proteinase inhibitor. These proteinase
inhibitors all belong to the serine proteinase inhibitors
family. The cDNA fragment of Ose727A is 1050 nucleotides in
length, the deduced translation product consist of 195 amino
acid residues and the amino acid sequence of 60 to 192 shows
100% identity to the 133 amino acid of RBTI. The additional 59
amino acid residues on the N-terminal of cDNA Ose727A may serve
as a signal peptide of the RBTI. In addition to the 59 amino
acid residues on the N-terminus, three extra amino acids Ala-
Glu-Asp on the C-terminus of the deduced protein product are not
found in RBTI.We have isolated and sequenced a rice embryo-
specific clone Ose705, which is one of the three cDNA clones
were suggested to be novel rice embryo-specific genes. The
Ose705 had the most abundant mRNA levels in the embryo, and this
abundant was consistent with the high redundancy of cDNA clones
selected by this approach, thirty-nine clones belonged to the
group of Ose705 (31% of the 126 clones). The cDNA fragment of
Ose705 is 1709 nucleotides in length, and the 3'' untranslated
region including three putative polyadenylation signals . The
deduced translation product consist of 393 amino acid residues.
Southern blot analysis showed that Ose705 is a single copy
nuclear gene in rice genome. Northern blot analysis demonstrated
that Ose705 has the highest level mRNA expression in 9 to 20 DAP
embryos, low level in 5 to 7 DAP embryos and 25 to 30 DAP
embryos, and mRNA was not detected in callus, de-embryo grains,
roots, coleoptides, young leaves, mature leaves, and anthers.
Theses results revealed the embryo-specific expression for
Ose705 clone. A genomic clone containing Os705 was isolated and
sequenced. The 5''-flanking DNA sequence revealed several
putative regulatory elements, including a G-box (CACGTG), a G/A
hybrid motif (CACGTA), a T/G hybrid motif(AACGTG) and two ABA
responsive element (CACGTGGCA). The transcriptional start site
was determined by primer extension and CapFinder method. By
amino acid sequences analysis Ose705 deduced translation product
was shown a hydrophilic protein, which the 1-26 amino acid
residues on the N-terminus may serve as a signal peptide, and
secreted to outside.A cDNA clone encoding a putative
metallothionein-like protein gene, OsMTe, was isolated from rice
embryo (Oryza sativa L. cv Lomello). The deduced protein product
is 84 amino acids with an arrangement of cysteine residues
characteristic of type 2 plant metallothioneins. Northern blot
analysis showed that transcript of OsMTe gene were expression
specifically in the rice embryo with the highest level of mRNA
detected in the very early stage (0-4 DAP) of immature embryo.
In situ hybridization analysis demonstrated OsMTe expression in
the meristem region of embryo. The metal-binding characteristics
of the OsMTe was determined by atomic absorption
spectrophotometry. The results revealed that OsMTe has high
affinity for Zn2+. The result of Southern blot analysis
suggested that there is a single copy in the rice genome. A
genomic clone containing OsMTe gene was isolated and sequenced.
The 5''-flanking DNA sequence revealed several putative
regulatory elements, including a C-box (GACGTC), a A/G hybrid
motif (TACGTG) and an element (TGCTCTCC) resembling the metal
responsive element. A cap site for the encoded mRNA was
determined by primer extension and CapFinder method. The deduced
amino acid sequence was compared to other type 2
metallothioneins and the characteristics of the cysteine-rich
domains and its possible role in rice embryo development are
discussed.
|
|---|
