Please use this identifier to cite or link to this item: http://hdl.handle.net/11455/20689
標題: 水稻胚發育時期特有表現基因之分離與分析
Isolation and characterization of rice genes that are specifically expressed during embryo development
作者: 陳鵬文
Chen, Peng-Wen
關鍵字: rice embryo;水稻胚;development;發育
出版社: 植物學系
摘要: 
利用水稻授粉後 10 天胚的 poly(A) RNA 為材料,構築了一個 cDNA 基
因庫。經差異性篩選法得 38 組具有胚發育時期特有表現的 cDNA 選殖體
。其中 18 組 cDNA 選殖體經核酸及胺基酸序列分析比對之後,發現有
二個屬於油體膜蛋白基因,四個屬於儲藏性蛋白基因,二個為受 ABA 誘
導之基因,二個似 LEA 的基因,及蛋白抑制基因、金屬硫蛋白基因、
蛋白激基因、含 leucine-zipper 之蛋白基因、 proline-rich 之蛋白
基因各一個及三個尚不知功能之基因。在篩選 cDNA 選殖體之過程中,發
現高表現量之基因,被?G充z選之機會增加。分析不同組織基因表現之結
果,證實篩得之 cDNA 選殖體均為胚發育時期特有表現之基因。這些選殖
體大部分在水稻中仍未被研究,因此本研究對篩選水稻新基因具有重要貢
獻。接著從 38 組具有胚發育時期特有表現的 cDNA 選殖體中,選取油體
膜蛋白基因、LEA 基因、蛋白抑制基因、一個尚不知功能之基因及金屬
硫蛋白基因,做進一步的特性分析與研究。植物種子內富含三醯基甘油,
作為種子萌芽及萌芽後幼苗生長之能量來源。三醯基甘油儲存在細胞內一
個小而不連續之胞器,謂之油脂體。油脂體內含有大量的三醯基甘油基質
,整個油脂體表面由一層磷脂及蛋白構成,其中含量最豐富的蛋白稱為油
體膜蛋白。我們所分離的二個水稻胚發育時期特有表現的 cDNA 選殖體
Ose701 及 Ose721,即屬於水稻 16 kD 及 18 kD 同型異構之油體膜蛋白
,並且分別與玉米的油體膜蛋白基因 ole16 及 ole18 核酸序列有 72
% 的相似度。植物種子發育過程中,在接近成熟時期有許多蛋白會大量表
現,並累積在種子胚體組織內。這類蛋白被稱為胚發育晚期大量表現蛋白
,簡稱 LEA 蛋白。我們所分離的水稻胚發育時期特有表現的 cDNA 選殖
體 Ose730,該基因包括了 200 個胺基酸,並且具有 8 個 LEA group 3
蛋白之 11-mer 胺基酸重覆序列。因此,認為 Ose730 這組選殖體屬於
LEA group 3 蛋白。經胺基酸序列比對,發現與玉米的 LEA 蛋白有 70
﹪ 之相似度。已知許多 LEA 蛋白為多基因家族,但是從南方墨點分析結
果看來,Ose730 在水稻基因組中是單一基因。此外,也進一步分析
Ose730 mRNA 的表現情形。發現在授粉後 5 天的胚體便有 Ose730 mRNA
的表現,而且一直持續表現至成熟並已乾燥之種子胚體。並且除了水稻胚
體有表現外,水稻的其他組織則沒有任何的表現,此說明了 Ose730 為水
稻胚特有表現基因。一般的榖類作物富含蛋白抑制基因,並且許多這類
基因蛋白也從許多禾本科作物中被分離與純化。我們所分離的水稻胚發育
時期特有表現的 cDNA 選殖體 Ose727A 即屬於米糠蛋白抑制基因,該
基因與小麥胚芽蛋白抑制基因及大豆 Bowman-Birk 蛋白抑制基因之
蛋白胺基酸序列有極高的相似度,均屬於絲胺酸蛋白抑制基因家族。
Ose727A 具有 195 個胺基酸。其中胺基酸 60 至 192 與水稻米糠蛋白
抑制基因之 133 個胺基酸序列完全一樣。除此之外,胺基端多了 59 個
胺基酸,認為可能是米糠蛋白抑制基因之一段訊號胜。另外,基端
還有三個胺基酸 Ala-Glu-Asp 為米糠蛋白抑制基因所沒有的。我們所
分離的 38 個水稻胚發育時期特有表現的 cDNA 選殖體中,Ose705 為三
個尚不知功能之基因的其中一個。Ose705 不僅比其他選殖組有較大量的
mRNA 表現,而且該 Ose705 更是由 39 個相同或是極相似的選殖系所構
成,佔了全部 126 個選殖系的 31 ﹪。此為所有 38 組選殖體中最大的
一組。Ose705 為 1709 bp 之全長 cDNA 選殖系,3'' 非轉譯區域具有 3
個典型的 polyadenylation 訊號。從南方墨點分析結果看來,Ose705 在
水稻基因組中是單一基因。此外,進一步分析 Ose705 mRNA 的表現情形
,發現 Ose705 在整個水稻胚體發育過程中,發育初期並不表現,或是表
現量很低。授粉後 5 到 7 天表現量開始上升,到授粉後 9 天則有最高
的表現量,並持續表現至 20 天。接著在授粉後 25 天表現量急遽下降
,30 天的表現量已很少,40 天胚體則幾乎不表現。除了水稻胚體有表現
外,水稻的其他組織則沒有任何的表現發生。此說明了 Ose705 為水稻胚
特有表現基因。Ose705 之基因組選殖體 osg705 被進一步的分離與分析
。osg705 之 5''非轉錄區域,存在一些調控因子 ,包括一個 G-box,G/A
hybrid,T/G hybrid 及二個 ABRE 。經 primer extension 及
CapFinder PCR 分析,定出主要之轉錄作用起始點。胺基酸分析推測
Ose705 之蛋白產物為一鹼性親水性蛋白,可能具有一個訊號胜,將該
蛋白送往胞外。除此之外,我們在 Ose705 親水性蛋白區域發現二個
22-mer 重複序列,這些重複序列的功能為何,值得進一步的探討。
Ose712 這組選殖體屬於金屬硫蛋白基因,該基因含有 84 個胺基酸,從
蛋白的分析發現該蛋白胺基端及基端富含半胱胺酸,並且從半胱胺酸的
排列發現,該基因屬於植物 type 2 MT-like 基因,稱為 OsMTe。南方墨
點分析顯示 OsMTe 在水稻基因組中是單一基因。進一步利用北方墨點分
析及原位雜交技術,觀察 OsMTe mRNA 的表現情形。發現在授粉後 0-4
天的水稻胚有最高的表現量,而且表現在胚的分生組織部位。此外,金屬
結合試驗顯示 OsMTe 可以與 Zn2+ 相結合。OsMTe 之基因組選殖體
osg712 被進一步的分離與分析。osg712 之 5'' 非轉錄區域,具有一些調
控因子 ,包括一個 C-box,A/G hybrid,以及一個似 MRE 因子。經
primer extension 及 CapFinder PCR 分析,主要轉錄作用起始點亦被決
定。綜合與其他植物 type 2 MT-like 基因之比較以及特性分析之結果,
進一步說明 OsMTe 基因在水稻胚可能扮演的角色。

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.
URI: http://hdl.handle.net/11455/20689
Appears in Collections:生命科學系所

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