Isolation and Functional Characterization of genes related to Kernel Isolation and Functional Characterization of genes related to Kernel Development by Differential Hybridization and Yeast Two Hybrid Development by Differential Hybridization and Yeast Two Hybrid Screening in extra-early barley 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 t hat was differentially expressed in the grain development of 3 days after fertilization was isolated and its tissue/developmental specific expression was analyzed. The cDNA encodi ng 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 H vSAMS 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 st resses (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 h ost 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 c onstructed. One clone that showed homology to wheat VDAC1, was selected and was designated as HvVDAC (H ordeum v ulgare V oltage D ependent A nion C hannels). The cDNA encoding HvVDAC contain ed 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 regio n 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 th Fig. 4 Northern blot hybridization of th e e HvSAMS HvSAMS gene in different tissues. Total gene in different tissues. Total RNA (10 RNA (10 ㎍ ㎍ per sample) of four tissues fro per sample) of four tissues fro m the barley was fractionated on a 1% dena m the barley was fractionated on a 1% dena turing agarose gel. G: turing agarose gel. G: grain, L: leaf, S: stem, and R: Root. grain, L: leaf, S: stem, and R: Root. Table 1 Differentially expressed clones in grain of Table 1 Differentially expressed clones in grain of K800 (GSHO 1732 GS96, K800 (GSHO 1732 GS96, eam10 eam10 ). ). ▼ ▼ ▲ ▲ Fig. 3 Fig. 3 Northern blot hybridization of Northern blot hybridization of HvSAMS HvSAMS gene in gr gene in gr ain of the barley during development. The grain materials ain of the barley during development. The grain materials were harvested during and development of DAF - 6, - 3, 0, were harvested during and development of DAF - 6, - 3, 0, 3, 7, 10, 13, 16, 18, 22, and 26. d : 3, 7, 10, 13, 16, 18, 22, and 26. d : days after fertilization. cv. K800 days after fertilization. cv. K800 ◄ ◄ Fig. 2 Southern bl Fig. 2 Southern bl ot analysis of ot analysis of the the g g enomic DNA of K800. enomic DNA of K800. The DNA was digested The DNA was digested with EcoRI, XbaI, Xh with EcoRI, XbaI, Xh oI, and HindIII, and oI, and HindIII, and the resulting DNA fr the resulting DNA fr agments were separat agments were separat ed by 0.8% agarose g ed by 0.8% agarose g el electrophoresis, el electrophoresis, transferred onto nyl transferred onto nyl on membrane and hybr on membrane and hybr idized with DIG labe idized with DIG labe led full-length led full-length HvSA HvSA MS MS probe. 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 Fig. 1 Alignment of the deduced amino acid sequences of SAMS from different plant species. A; Alignment of the SAMS from different plant species. A; Alignment of the HvS HvS AMS AMS sequence. Red letters indicated sequence identity. The sequence. Red letters indicated sequence identity. The two conserved motifs (asterisks) were boxed. B; Phylogenet two conserved motifs (asterisks) were boxed. B; Phylogenet ic dendrogram of SAMS amino acid sequences were compared t ic dendrogram of SAMS amino acid sequences were compared t o other SAMS sequences in a ClustalW. o other SAMS sequences in a ClustalW. ****** ******* ** A B 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 Fig. 5 Northern blot analysis of the Northern blot analysis of the HvSAMS HvSAMS in leaves of extra‐early mature barley (K800, in leaves of extra‐early mature barley (K800, possess possess eam 10 eam 10 ). The leaf material was harvest ). The leaf material was harvest ed from the plant with GA3, ABA, GA3+ABA, sper ed from the plant with GA3, ABA, GA3+ABA, sper midine, salt, and ethylene treatment for 1h, 6 midine, salt, and ethylene treatment for 1h, 6 h, 12h, 24h, and 48h. The wounding stress was h, 12h, 24h, and 48h. The wounding stress was treated 1/2h, 3h, 6h, and 12h. C : control, Mo treated 1/2h, 3h, 6h, and 12h. C : control, Mo ck: H ck: H 2 O O treatment, h : hour. treatment, h : hour. ▲ ▲ Fig. 6 Fig. 6 Expression of the Expression of the HvSAMS HvSAMS in a bact in a bact erial system. The erial system. The HvSAMS HvSAMS cDNA, cloned in pE cDNA, cloned in pE T 32c, was expressed by transformed E. coli T 32c, was expressed by transformed E. coli BL21(DE3). IPTG, at final concentration of 1 BL21(DE3). IPTG, at final concentration of 1 mM, was served as an inducer for mM, was served as an inducer for HvSAMS HvSAMS exp exp ression. The cell lysate protein was separat ression. The cell lysate protein was separat ed by SDS-PAGE (left) and western botting (r ed by SDS-PAGE (left) and western botting (r ight) ight) 9766 45 31 9766 45 31 21 14 21 14 0 1/6 1 0 1/6 1 2 4h 2 4h IPTG induction IPTG induction time time IPTG induction IPTG induction time time A A B B 0 1/6 1 0 1/6 1 2 4h 2 4h ◄ ◄ Fig. 7 Alignment of dedu Fig. 7 Alignment of dedu ced amino acid sequences w ced amino acid sequences w ith ith HvVDACs HvVDACs from different from different plant species. Residues th plant species. Residues th at were associated with th at were associated with th e eukaryotic porin domain e eukaryotic porin domain was indicated as was indicated as blue box. blue box. 0h 1h 6h 12h 24h 48h GA 3 HvS AMS rRNA 0h 1h 6h 12h 24h 48h ABA HvS AMS rRNA 0h 1h 6h 12h 24h 48h ABA+GA 3 HvS AMS rRNA 0h 1h 6h 12h 24h 48h Spermid ine HvS AMS rRNA 0h 1/2h 1h 3h 6h 12h Wounding rRNA 0h 1h 6h 12h 24h 48h Na Cl HvS AMS rRNA C Mock 0h 1h 6h 12h 24h 48h H 3 PO 3 HCl Ethylene HvS AMS rRNA HvS AMS 0h 1h 6h 12h 24h 48h GA 3 HvV DAC rRNA 0h 1h 6h 12h 24h 48h ABA HvV DAC rRNA 0h 1h 6h 12h 24h 48h ABA+GA 3 HvV DAC rRNA 0h 1h 6h 12h 24h 48h Spermid ine HvV DAC rRNA 0h 1/2h 1h 3h 6h 12h Wounding rRNA 0h 1h 6h 12h 24h 48h Na Cl HvV DAC rRNA HvV DAC C Mock 0h 1h 6h 12h 24h 48h H 3 PO 3 HCl Ethylene HvV DAC rRNA L S G R HvV DAC rRNA HvV DAC rRNA 1 2 3 4 5 6 7 8 9 10 ▲ ▲ Fig. 11 Northern blot hybridization of the Fig. 11 Northern blot hybridization of the HvVDA HvVDA C C gene in different tissues. Total RNA (10 gene in different tissues. Total RNA (10 ㎍ ㎍ per s per s ample) of four tissues from the barley was fractio ample) of four tissues from the barley was fractio nated on a 1% denaturing agarose nated on a 1% denaturing agarose gel. G: grain, L: leaf, S: stem, and R: Root. gel. G: grain, L: leaf, S: stem, and R: Root. ▲ ▲ Fig. 10 Fig. 10 Northern blot hybridization of Northern blot hybridization of HvSAMS HvSAMS gene at d gene at d ifferent stages of grain development. The grain tissues w ifferent stages of grain development. The grain tissues w ere harvested at DAF - 6, - 3, 0, 3, 7, 10, 13, 16, 18, 2 ere harvested at DAF - 6, - 3, 0, 3, 7, 10, 13, 16, 18, 2 2d. DAF : days after fertilization. cv. K800 2d. DAF : days after fertilization. cv. K800 ◄ ◄ Fig. 12 Fig. 12 Northern Northern blot analysis of th blot analysis of th e e HvVDAC HvVDAC gene in le gene in le aves of extra‐early aves of extra‐early mature barley (K80 mature barley (K80 0). Leaf tissues we 0). Leaf tissues we re collected at re collected at 1h, 6 1h, 6 h, 12h, 24h, and 48h h, 12h, 24h, and 48h after tr tr eated with GA3, AB eated with GA3, AB A, GA3+ABA, spermid A, GA3+ABA, spermid ine, salt, and ethy ine, salt, and ethy lene. Leaf tissues lene. Leaf tissues were collected at were collected at 1/2h, 3h, 6h, and 1 1/2h, 3h, 6h, and 1 2h after treatment. 2h after treatment. C : control, Mock: C : control, Mock: H H 2 O treatment, h : h O treatment, h : h our. our. EcoRI Xho I Xb aI Hind III ◄ ◄ Fig. 9 Southern blot Fig. 9 Southern blot analysis of analysis of the the genomi genomi c DNA of K800. Genomic c DNA of K800. Genomic DNA was digested with DNA was digested with EcoRI, XhoI, XbaI, and EcoRI, XhoI, XbaI, and HindIII, and the resul HindIII, and the resul ting DNA fragments wer ting DNA fragments wer e separated by 0.8% ag e separated by 0.8% ag arose gel electrophore arose gel electrophore sis, transferred onto sis, transferred onto nylon membrane and hyb nylon membrane and hyb ridized with DIG label ridized with DIG label ed full-length ed full-length HvVDAC HvVDAC probe. probe. ◄ ◄ Fig. 8 Fig. 8 A: Bait A: Bait Diagram of the b Diagram of the b ait fragment. B: ait fragment. B: Quantitative Quantitative β β -g -g alactosidase ass alactosidase ass ay of fig. 8A. ay of fig. 8A. C: Growth on SD/ C: Growth on SD/ Leu-/Trp-, SD/Ad Leu-/Trp-, SD/Ad e-/His-/Leu-/Trp e-/His-/Leu-/Trp -/x-gal medium o -/x-gal medium o f each bait frag f each bait frag ments. ments. A A 360bp 360bp 18bp 18bp 426bp 426bp 24bp 24bp 357bp 357bp 1185bp 1185bp I I I I I I II II I IV IV V V V I II II III III IV IV V PC PC I VI VI NC NC SD/Leu-/Trp.- SD/Leu-/Trp.- SD/ade-/His-/Leu-/Trp-/X-gal SD/ade-/His-/Leu-/Trp-/X-gal C C II II III III IV IV V PC PC I VI VI NC NC I I I I I II II I I IV IV V V I Negative Negative Control Control (NC) (NC) Positive Positive Control Control (PC) (PC) 0.5 0.5 1.0 1.0 B B 0.63 0.63 0.71 0.71 6 0.15 0.15 6 0.14 0.14 7 0.64 0.64 3 0.14 0.14 9 9 0.74 0.74 8 8 0.16 0.16 9