Isolation and characterization of rice genes that are specifically expressed during embryo development

• Main Authors: Chen, Peng-Wen, 陳鵬文
• Other Authors: Tseng Yi-Hsiung, Chen Liang-Jwu
• Format: Others
• Language: zh-TW
• Published: 1997
• Online Access: http://ndltd.ncl.edu.tw/handle/68087214492787712019

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.