1. Isolation and Functional Characterization of genes related to Kernel Development by Differential Hybridization and Yeast Two Hybrid Screening in extra-early barley Jae Yoon Kim 1), Jae Han Park, and, Yong Weon Seo 1) 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 10 6 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. † Tel : 02-3290-3464, E-mail : seoag@korea.ac.kr ▲ 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. Table 1 Differentially expressed clones in grain of K800 (GSHO 1732 GS96, eam10). ▼ ▲ 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. 2 Southern blot analysis of the genomic 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. Acknowledgements This work was financially supported grant from the BG21, RDA, Rep. Of Korea EcoRI Xba I Xho I Hind III 9 Kb 3 Kb G L S R HvSAMS rRNA 1 2 3 4 5 6 7 8 9 10 11 HvSAMS rRNA ▲ 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. ****** ********* AB Actinidiaceae Catharanthus3 Tabacco 915 Araidopsis 1000 Pisum 790 lima Phaseolus Kidney 998 Tomato Carnation 1000 549 Periwinkle Catharanthus2 427 1000 183 177 Dendrobium Elaeagnaceae 128 641 Populus Papaya Litchi 380 128 155 Rice 817 Barley HvSAMS 1000 0.02 ▲ 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: H 2 O treatment, h : hour. ▲ 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) 9766 45 31 21 14 9766 45 31 21 14 0 1/6 1 2 4h 0 1/6 1 2 4h IPTG induction time AB 0 1/6 1 2 4h 0 1/6 1 2 4h ◄ 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. 0h 1h 6h 12h 24h 48h GA 3 HvSAMS rRNA 0h 1h 6h 12h 24h 48h ABA HvSAMS rRNA 0h 1h 6h 12h 24h 48h ABA+GA 3 HvSAMS rRNA 0h 1h 6h 12h 24h 48h Spermidine HvSAMS rRNA 0h 1/2h 1h 3h 6h 12h Wounding rRNA 0h 1h 6h 12h 24h 48h NaCl HvSAMS rRNA C Mock 0h 1h 6h 12h 24h 48h H 3 PO 3 HCl Ethylene HvSAMS rRNA HvSAMS 0h 1h 6h 12h 24h 48h GA 3 HvVDAC rRNA 0h 1h 6h 12h 24h 48h ABA HvVDAC rRNA 0h 1h 6h 12h 24h 48h ABA+GA 3 HvVDAC rRNA 0h 1h 6h 12h 24h 48h Spermidine HvVDAC rRNA 0h 1/2h 1h 3h 6h 12h Wounding rRNA 0h 1h 6h 12h 24h 48h NaCl HvVDAC rRNA HvVDAC C Mock 0h 1h 6h 12h 24h 48h H 3 PO 3 HCl Ethylene HvVDAC rRNA L S G R HvVDAC rRNA HvVDAC rRNA 1 2 3 4 5 6 7 8 9 10 ▲ 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. 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. 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 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: H 2 O treatment, h : hour. ◄ 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: H 2 O treatment, h : hour. EcoRI Xho I XbaI Hind III ◄ Fig. 9 Southern blot analysis of the genomic 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. 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. A360bp18bp426bp24bp357bp 1185bp I II III IV V VI II III IV V PC I VI NC SD/Leu-/Trp.- SD/ade-/His-/Leu-/Trp-/X-gal C II III IV V PC I VI NC I II III IV V VI Negative Control (NC) Positive Control (PC) 0.51.0B0.63 0.716 0.156 0.147 0.643 0.149 0.748 0.169