Jae Yoon Kim 1) , Jae Han Park, and, Yong Weon Seo 1)

1. C SD/ade-/His-/Leu-/Trp-/X-gal SD/Leu-/Trp.- II II 0.02 III I I III ABA+GA3 HvSAMS 1185bp GA3 Spermidine ABA I Ethylene Barley ****** Wounding 0h 1h 6h 12h 24h 48h NaCl 360bp 18bp 426bp 24bp 357bp 0h 1h 6h 12h 24h 48h 0h 1h 6h 12h 24h 48h 0h 1h 6h 12h 24h 48h II Rice 1000 HvVDAC C Mock 0h 1h 6h 12h 24h 48h H3PO3 HCl HvVDAC HvVDAC HvVDAC 0h 1h 6h 12h 24h 48h III 0h 1/2h 1h 3h 6h 12h Litchi IV 155 HvVDAC VI VI IV HvVDAC HvVDAC IV Papaya V 380 Populus I rRNA rRNA rRNA VI rRNA rRNA Elaeagnaceae II 641 ********* rRNA Positive Control (PC) rRNA Dendrobium III NC 817 V V NC IV Catharanthus2 1000 Negative Control (NC) V Periwinkle PC PC 177 VI Carnation 128 549 Tomato 427 Kidney Phaseolus 1000 128 998 lima Pisum Araidopsis 183 Tabacco 790 Catharanthus3 1000 915 Actinidiaceae EcoRI Xba I Xho I Hind III 9 Kb A B IPTG induction time IPTG induction time 3 Kb A B 0 1/6 1 2 4h 0 1/6 1 2 4h 9766 45 31 21 14 G L S R HvSAMS 1 2 3 4 5 6 7 8 9 10 11 rRNA HvSAMS rRNA GA3 ABA 0h 1h 6h 12h 24h 48h 0h 1h 6h 12h 24h 48h HvSAMS HvSAMS rRNA rRNA ABA+GA3 Spermidine 0h 1h 6h 12h 24h 48h 0h 1h 6h 12h 24h 48h HvSAMS HvSAMS B A rRNA 0.63 rRNA 0.748 0.716 Wounding NaCl 0.156 0.169 0h 1h 6h 12h 24h 48h 0h 1/2h 1h 3h 6h 12h 0.147 HvSAMS HvSAMS 0.643 0.149 rRNA rRNA 0.5 1.0 Ethylene L S G R C Mock 0h 1h 6h 12h 24h 48h H3PO3 HCl HvSAMS HvVDAC rRNA rRNA 1 2 3 4 5 6 7 8 9 10 EcoRI Xho I XbaI Hind III HvVDAC rRNA Isolation and Functional Characterization of genes related to Kernel Development by Differential Hybridization and Yeast Two Hybrid Screening in extra-early barley Jae Yoon Kim1), Jae Han Park, and, Yong Weon Seo1) 1) Devision of Biotechnology and Genetic Engineering, Korea University, Seoul, Korea Abstract A cDNA library was constructed using kernels of early mature barley (possessing eam10). A S-AdenosylMethionine Syntase (HvSAMS, Hordeum vulgare S-AdenosylMethionine Syntase) gene that was differentially expressed in the grain development of 3 days after fertilization was isolated and its tissue/developmental specific expression was analyzed. The cDNA encoding HvSAMS contained a 1185 bp open reading frame (ORF) that encoded 394 amino acids. Southern blot analysis showed at least 2 copies were existed in barley. Transcript levels of HvSAMS mRNA were highest at -6, -3, 0 DAF (Days After Fertilization) and in stem, grain, and root tissues. The expression of HvSAMS was detected in leaves in response to abiotic stresses (salt and wounding) and elicitors (ABA, GA3, ABA+GA3, and spermidine). Coding region of HvSAMS was cloned in the protein expression vector(pET32) and transformed into the host cells(BL21). Translational products of HvSAMS was successfully identified through 1D SDS-PAGE after induction with IPTG. Hybridization with a anti-HIS antibody at the expected size was obtained (52 kDa).  In order to identify proteins that interacted with HvSAMS, a yeast two hybridization library [Transformants : 4.48 X 106 cell/ml (SD/Leu-/Trp-)] was constructed. One clone that showed homology to wheat VDAC1, was selected and was designated as HvVDAC (Hordeum vulgare Voltage Dependent Anion Channels). The cDNA encoding HvVDAC contained a 828 bp open reading frame (ORF) that encoded 275 amino acids. The sequence comparison indicated that HvVDAC was similar to wheat VDAC1 with 93% homology. The N-terminal region of HvSAMS fused to GAL4 DNA-binding domain bound to HvVDAC. Southern analysis of barley DNA using a DIG labled full-length cDNA probe of HvVDAC was conducted. Digestion of each EcoR I, Xba I, Hind III showed one hybridized and two bands were detected with Xho I digestion. Its expression was detected at -3, 0, 3, 7 DAF and predominantly high in grain tissues. The response modes of HvVDAC to elicitors except ethylene were similar to HvSAMS. ◄ Fig. 2 Southern blot analysis ofthegenomic DNA of K800. The DNA was digested with EcoRI, XbaI, XhoI, and HindIII, and the resulting DNA fragments were separated by 0.8% agarose gel electrophoresis, transferred onto nylon membrane and hybridized with DIG labeled full-length HvSAMS probe. Table 1 Differentially expressed clones in grain of K800 (GSHO 1732 GS96, eam10). ▼ ▲Fig. 1 Alignment of the deduced amino acid sequences of SAMS from different plant species. A; Alignment of the HvSAMS sequence. Red letters indicated sequence identity. The two conserved motifs (asterisks) were boxed. B; Phylogenetic dendrogram of SAMS amino acid sequences were compared to other SAMS sequences in a ClustalW. ◄Fig. 7 Alignment of deduced amino acid sequences with HvVDACs from different plant species. Residues that were associated with the eukaryotic porin domain was indicated as blue box. ▲Fig. 3 Northern blot hybridization of HvSAMS gene in grain of the barley during development. The grain materials were harvested during and development of DAF - 6, - 3, 0, 3, 7, 10, 13, 16, 18, 22, and 26. d : days after fertilization. cv. K800 ▲ Fig. 6 Expression of the HvSAMS in a bacterial system. The HvSAMS cDNA, cloned in pET 32c, was expressed by transformed E. coli BL21(DE3). IPTG, at final concentration of 1mM, was served as an inducer for HvSAMS expression. The cell lysate protein was separated by SDS-PAGE (left) and western botting (right) ▲ Fig. 4 Northern blot hybridization of the HvSAMS gene in different tissues. Total RNA (10 ㎍ per sample) of four tissues from the barley was fractionated on a 1% denaturing agarose gel. G: grain, L: leaf, S: stem, and R: Root. ▲Fig. 10 Northern blot hybridization of HvSAMS gene at different stages of grain development. The grain tissues were harvested at DAF - 6, - 3, 0, 3, 7, 10, 13, 16, 18, 22d. DAF : days after fertilization. cv. K800 ◄Fig. 8 A: BaitDiagram of the bait fragment. B: Quantitative β-galactosidase assay of fig. 8A. C: Growth on SD/Leu-/Trp-, SD/Ade-/His-/Leu-/Trp-/x-gal medium of each bait fragments. ▲ Fig. 11 Northern blot hybridization of the HvVDAC gene in different tissues. Total RNA (10 ㎍ per sample) of four tissues from the barley was fractionated on a 1% denaturing agarose gel. G: grain, L: leaf, S: stem, and R: Root. ◄Fig. 12 Northern blot analysis of the HvVDAC gene in leaves of extra‐early mature barley (K800). Leaf tissues were collected at 1h, 6h, 12h, 24h, and 48h after treated with GA3, ABA, GA3+ABA, spermidine, salt, and ethylene. Leaf tissues were collected at 1/2h, 3h, 6h, and 12h after treatment. C : control, Mock: H2O treatment, h : hour. ◄ Fig. 9 Southern blot analysis ofthegenomic DNA of K800. Genomic DNA was digested with EcoRI, XhoI, XbaI, and HindIII, and the resulting DNA fragments were separated by 0.8% agarose gel electrophoresis, transferred onto nylon membrane and hybridized with DIG labeled full-length HvVDAC probe. ▲ Fig. 5 Northern blot analysis of the HvSAMS in leaves of extra‐early mature barley (K800, possess eam 10). The leaf material was harvested from the plant with GA3, ABA, GA3+ABA, spermidine, salt, and ethylene treatment for 1h, 6h, 12h, 24h, and 48h. The wounding stress was treated 1/2h, 3h, 6h, and 12h. C : control, Mock: H2O treatment, h : hour. AcknowledgementsThis work was financially supported grant from the BG21, RDA, Rep. Of Korea † Tel : 02-3290-3464, E-mail : seoag@korea.ac.kr