Isolation and Functional Characterization of genes related to Kernel Development by Isolation and Functional Characterization of genes related to Kernel Development by Differential Hybridization and Yeast Two Hybrid Screening in extra-early barleyDifferential 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
AbstractA 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 : [email protected]
▲ ▲ Fig. 4 Northern blot hybridization of the Fig. 4 Northern blot hybridization of the HvSAMS HvSAMS gene in different tissues. Total RNA (10 gene in different tissues. Total RNA (10 ㎍ ㎍ per sampper sample) of four tissues from the barley was fractionated ole) of four tissues from the barley was fractionated on a 1% denaturing agarose gel. G: n a 1% denaturing 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 K800 (GSHO 1732 Table 1 Differentially expressed clones in grain of K800 (GSHO 1732
GS96, GS96, eam10eam10). ). ▼▼
▲▲ Fig. 3 Fig. 3 Northern blot hybridization of Northern blot hybridization of HvSAMS HvSAMS gene in grain of the barlgene in grain of the barl
ey during development. The grain materials were harvested during and dey during development. The grain materials were harvested during and d
evelopment of DAF - 6, - 3, 0, 3, 7, 10, 13, 16, 18, 22, and 26. d : evelopment of DAF - 6, - 3, 0, 3, 7, 10, 13, 16, 18, 22, and 26. d :
days after fertilization. cv. K800days after fertilization. cv. K800
◄ ◄ Fig. 2 Southern blot anFig. 2 Southern blot an
alysis ofalysis of thethe genomic DNgenomic DN
A of K800. The DNA was A of K800. The DNA was
digested with EcoRI, Xbadigested with EcoRI, Xba
I, XhoI, and HindIII, and thI, XhoI, and HindIII, and th
e resulting DNA fragmente resulting DNA fragment
s were separated by 0.8% s were separated by 0.8%
agarose gel electrophoreagarose gel electrophore
sis, transferred onto nylosis, transferred onto nylo
n membrane and hybridizn membrane and hybridiz
ed with DIG labeled full-leed with DIG labeled full-le
ngth ngth HvSAMSHvSAMS 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 SAMS from dFig. 1 Alignment of the deduced amino acid sequences of SAMS from d
ifferent plant species. A; Alignment of the ifferent plant species. A; Alignment of the HvSAMSHvSAMS sequence. Red letters sequence. Red letters
indicated sequence identity. The two conserved motifs (asterisks) were bindicated sequence identity. The two conserved motifs (asterisks) were b
oxed. B; Phylogenetic dendrogram of SAMS amino acid sequences were coxed. B; Phylogenetic dendrogram of SAMS amino acid sequences were c
ompared to other SAMS sequences in a ClustalW. ompared to 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. 5Fig. 5 Northern blot analysis of the Northern blot analysis of the HvSAMSHvSAMS in leaves o in leaves of extra‐early mature barley (K800, possess f extra‐early mature barley (K800, possess eam 10eam 10). The le). The leaf material was harvested from the plant with GA3, ABA, af material was harvested from the plant with GA3, ABA, GA3+ABA, spermidine, salt, and ethylene treatment for 1GA3+ABA, spermidine, salt, and ethylene treatment for 1h, 6h, 12h, 24h, and 48h. The wounding stress was treated h, 6h, 12h, 24h, and 48h. The wounding stress was treated 1/2h, 3h, 6h, and 12h. C : control, Mock: H1/2h, 3h, 6h, and 12h. C : control, Mock: H22O O treatment, h : hour.treatment, h : hour.
▲ ▲ Fig. 6 Fig. 6 Expression of the Expression of the HvSAMSHvSAMS in a bacterial syste in a bacterial syste
m. The m. The HvSAMSHvSAMS cDNA, cloned in pET 32c, was express cDNA, cloned in pET 32c, was express
ed by transformed E. coli BL21(DE3). IPTG, at final coned by transformed E. coli BL21(DE3). IPTG, at final con
centration of 1mM, was served as an inducer for centration of 1mM, was served as an inducer for HvSAHvSA
MSMS expression. The cell lysate protein was separated b expression. The cell lysate protein was separated b
y SDS-PAGE (left) and western botting (right)y SDS-PAGE (left) and western botting (right)
97
66
45
31
21
9
76
6 4
5 3
1 2
1
14
14
0 1/6 1 2 0 1/6 1 2 4h4h
IPTG induction IPTG induction timetime
IPTG induction IPTG induction timetime
AA BB 0 1/6 1 2 0 1/6 1 2 4h4h
◄◄ Fig. 7 Alignment of deduced aFig. 7 Alignment of deduced a
mino acid sequences with mino acid sequences with HvVDHvVD
ACsACs from different plant species. from different plant species.
Residues that were associated wResidues that were associated w
ith the eukaryotic porin domain ith the eukaryotic porin domain
was indicated as was indicated as
blue box.blue box.
0h 1h 6h 12h 24h 48h
GA3
HvSAMS
rRNA
0h 1h 6h 12h 24h 48h
ABA
HvSAMS
rRNA
0h 1h 6h 12h 24h 48h
ABA+GA3
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 H3PO3 HCl
Ethylene
HvSAMS
rRNA
HvSAMS
0h 1h 6h 12h 24h 48h
GA3
HvVDAC
rRNA
0h 1h 6h 12h 24h 48h
ABA
HvVDAC
rRNA
0h 1h 6h 12h 24h 48h
ABA+GA3
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 H3PO3 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 Fig. 11 Northern blot hybridization of the HvVDAC HvVDAC gene in digene in different tissues. Total RNA (10 fferent tissues. Total RNA (10 ㎍ ㎍ per sample) of four tissues frper sample) of four tissues from the barley was fractionated on a 1% denaturing agarose om the barley was fractionated 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 different stagegene at different stage
s of grain development. The grain tissues were harvested at DAF - 6, - 3, s 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. K8000, 3, 7, 10, 13, 16, 18, 22d. DAF : days after fertilization. cv. K800
◄◄ Fig. 12Fig. 12 Northern blot Northern blot analysis of the analysis of the HvVDACHvVDAC gene in leaves of extra‐egene in leaves of extra‐early mature barley (K80arly mature barley (K800). Leaf tissues were coll0). Leaf tissues were collected at ected at 1h, 6h, 12h, 24h, and 48h1h, 6h, 12h, 24h, and 48h after treated with GA3, ABtreated with GA3, ABA, GA3+ABA, spermidinA, GA3+ABA, spermidine, salt, and ethylene. Leae, salt, and ethylene. Leaf tissues were collected f tissues were collected at 1/2h, 3h, 6h, and 12h at 1/2h, 3h, 6h, and 12h after treatment. C : contrafter treatment. C : control, Mock: Hol, Mock: H22O treatment, O treatment, h : hour.h : hour.
EcoRI Xho I XbaI Hind III
◄ ◄ Fig. 9 Southern blot analFig. 9 Southern blot anal
ysis ofysis of thethe genomic DNA of genomic DNA of
K800. Genomic DNA was diK800. Genomic DNA was di
gested with EcoRI, XhoI, Xbgested with EcoRI, XhoI, Xb
aI, and HindIII, and the resulaI, and HindIII, and the resul
ting DNA fragments were seting DNA fragments were se
parated by 0.8% agarose geparated by 0.8% agarose ge
l electrophoresis, transferrel electrophoresis, transferre
d onto nylon membrane and onto nylon membrane an
d hybridized with DIG labeled hybridized with DIG labele
d full-length d full-length HvVDACHvVDAC prob prob
e. e.
◄◄ Fig. 8Fig. 8 A: BaitDiag A: BaitDiagram of the bait fragram of the bait fragment. B: Quantitativment. B: Quantitative e ββ-galactosidase a-galactosidase assay of fig. 8A. ssay of fig. 8A. C: GC: Growth on SD/Leu-/Trrowth on SD/Leu-/Trp-, SD/Ade-/His-/Leup-, SD/Ade-/His-/Leu-/Trp-/x-gal medium -/Trp-/x-gal medium of each bait fragmeof each bait fragments. nts.
AA
360bp360bp 18bp18bp 426bp426bp 24bp24bp 357bp357bp
1185bp1185bp
IIIIII
IIIIIIIVIV
VV
VIVI
IIII
IIIIII
IVIV
VV
PCPC
II
VIVI
NCNC
SD/Leu-/Trp.-SD/Leu-/Trp.- SD/ade-/His-/Leu-/Trp-/X-galSD/ade-/His-/Leu-/Trp-/X-galCC
IIII
IIIIII
IVIV
VV
PCPC
II
VIVI
NCNC
IIIIII
IIIIII
IVIV
VVVIVI
Negative Negative Control Control (NC)(NC)
Positive Positive Control Control (PC)(PC)
0.50.5 1.01.0
BB
0.630.63
0.7160.7160.1560.156
0.1470.147
0.6430.643
0.1490.149
0.7480.748
0.1690.169
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