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Phần III: Nghiên cứu vai trò của các gien liên quan đến khả năng chống chịu lạnh ở ngô: Quá trình biểu hiện và trình tự vùng promoter của các gien này
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Bằng kỹ thuật PVR-CDNA Select Subtraction (hay còn có tên gọi khác là SSH- Supperssion Subtractive Hybridization) tác giả đã phân lập được 18 gien có biểu hiện cao trong điều kiện lạnh 6 độ C và 13 độ C. trong số 18 gien này, gien ZmCO16.1 có tần số xuất hiện rất cao (49%) trong thư viện cDNA.
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Nội dung Text: Phần III: Nghiên cứu vai trò của các gien liên quan đến khả năng chống chịu lạnh ở ngô: Quá trình biểu hiện và trình tự vùng promoter của các gien này
31(3): 71-80 Tap chf SINH HOC 9-2009<br />
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<br />
<br />
ARTICLE 3: CHARACTERIZATION OF THE STRESS-INDUCED GENE<br />
ZMCOI6.1 IN MAIZE: EXPRESSION AND PROMOTER SEQUENCES<br />
THUY HA NGUYEN<br />
Institute of Agricultural Genetics, Hanoi,Vietnam<br />
JORGLEIPNER<br />
' Institute of Plant Sciences, Zyiich, Switzerland<br />
ORLENE GUERRA-PERAZA<br />
University of Guelph, Ontario. Canada<br />
PETER STAMP<br />
Institute of Plant Sciences, Zyrlcli, Switzerland<br />
<br />
ABSTRACT: Using cDNA subtraction technique, 18 cold stress responsive-genes were identified, among<br />
them a novel gene, ZmC016.1, whose function is still unknown. Analysis of the ZmC0I6.1 promoter<br />
sequence revealed several conserved stress-responsive cis-acting elements. Further expression characterization<br />
shows that ZmCOId.l is induced, in addition by cold, by other abiotic stresses such as drought and NaCI as<br />
well as by signalling molecules such as ABA and SA. The results indicate that ZmC0I6.1 is a general stress<br />
responsive gene. A possible regulation mechanism is presented where ZmC0I6.1 is alternatively spliced<br />
yielding two transcripts whose levels are changed upon different stress treatments. Furthermore the predicted<br />
ZmC0I6.1 amino acid sequence and its homologue show high similarity with proteins in rice and Arabidopsis<br />
suggesting that it belongs to a conserved protein in plants.<br />
<br />
<br />
Cold-acclimation in plants involves multiple tolerance [8]. These results suggest that this<br />
changes in morphology, metabolism such as transcriptional regulation mechanism is<br />
accumulation of abscisic acid (ABA) and conserved among several plant species. In<br />
salicylic acid (SA), changes in membrane lipid addition, CBF type transcription factors have<br />
composition, formation of compatible osmolytes been found in other plants although the function<br />
and production of antioxidants. These processes remains to be evaluated. However, there are also<br />
are accompanied by notable changes in the level indications of the existence of CBF-<br />
of various gene transcripts and proteins [16]. independent cold acclimaction [5]. Gene<br />
Our understanding of the molecular pathways in expression is regulated not only at the<br />
cold acclimation has changed dramatically with transcriptional level but can also be regulated by<br />
the discovery of the C-repeat post-transcriptional events such as alternative<br />
(CRT)/dehydration-responsive element (DRE) splicing, translational and post-translational<br />
binding transcription factors (CBF) in the model modifications like phosphorylation [2].<br />
organism Arabidopsis thaliana. The CBFs bind Whilst the molecular pathways of<br />
to CRT/DRE elements present in the promoter acclimation to low temperature are well<br />
regions of many cold- and dehydration- understood for the model plant Arabidopsis [1,<br />
responsive genes such as cold-regulated (COR) 16], the knowledge about the molecular basis of<br />
genes [4, 17]. In these hnes, over-expression of cold-acclimation in maize is still rudimentary.<br />
Arabidopsis CBF induces COR gene expression Furthermore, low , temperature stress in<br />
in the chilling-sensitive tomato (Lycoperslcon Arabidopsis occurs at subzero temperatures<br />
esculentum), resulting in protection against while maize growth is challenged already at<br />
chilling stress at 0°C and improved freezing temperatures below 20°C suggesting that<br />
<br />
71<br />
divergent acclimation pathways might be separated by electrophoresis, using 2.0% M<br />
employed. In order to characterize the molecular agarose gel, and monitored using Gel Doc 2000<br />
pathways induced in maize in response to cold (Bio-Rad Company, USA).<br />
stress, a previous study [12-14] identified The cDNA from the PCR amplification was<br />
several nsovel genes, including ZmC016.1, cloned into the pDrive vector (Qiagen AG,<br />
whose transcript level increases after exposure Switzerland) and transformed into E. coli DH5D<br />
to low non-freezing temperature. The aim of this cells. Clones were sequenced by MWG (MWG-<br />
study was to characterize this novel gene for a Biotech AG, Ebersberg, Germany).<br />
better insight into its role during cold response.<br />
We show that ZmC0I6.1 is in addition to cold 3. Abiotic stress and signalling molecule<br />
also highly induced under drought and salt treatments<br />
stress and by signalling molecules like salicylic Abiotic stress or signalling molecules were<br />
acid and abscicic acid suggesting ZmC0I6.l as applied to maize plants when the third leaf was<br />
being a conserved general stress response gene. fully developed. The plants were cold-stressed<br />
Furthermore, the expression of ZmC0I6.1 is by decreasing the temperature to 6°C or 13°C.<br />
modified by alternative splicing in response to For the drought stress, maize plants were<br />
abiotic stress. removed from the hydroponic culture and were<br />
I. MATERIAL AND METHODS left to dry in the growth chamber. The salt<br />
treatment was induced by adding NaCI to the<br />
1. Plant material and growth conditions Hoagland solution to obtain a concentration of<br />
Maize seeds of the genotype ETH-DH7 150 mM. Stress signalhng molecules were<br />
were grown in half Hoagland solution (H2395, applied to the hydroponic culture at a final<br />
Sigma Chemical Co., USA) supplemented with concentration of 100 pM salicylic acid (SA) or<br />
0.5% Fe-sequestrene, 6 mM K"" and 4 mM Ca^"". 100 pM abscisic acid (ABA). All the treatments<br />
Before treatment, plants were grown until the were imposed in the dark. Control plants<br />
thud leaf was fully developed at 25/22°C (unstressed) were collected prior applying the<br />
(day/night) in growth chambers (Conviron selected stress treatments. The middle part of<br />
PGW36, Winnipeg, Canada) at a 12-hour third leaves were harvested, frozen in liquid<br />
photoperiod, a light intensity of 300 pmol m"^ s ' nitrogen and stored at -80°C until assay.<br />
and a relative humidity df 60/70% (day/night). 4. Bioinformatics<br />
2. Reverse transcriptase (RT)-PCR, cloning<br />
A similarity search was performed using the<br />
and analysis of cDNA basic local alignment search tool (BLAST)<br />
Total RNA was extracted from maize leaf (National Centre for Biotechnology Information<br />
samples using Tri Reagent according to Sigma's (NIH, Bethesda, MD, USA) (http://www.ncbi.<br />
protocol for RNA isolation. 1.5 pg total RNA of nlm.nih.gov/BLAST/) and the NCBI BLAST2<br />
each sample was reverse transcribed to first- service maintained by the Swiss Institute of<br />
strand cDNAs using oligo (dT)23 primer in a Bioinformatics (http://au.expasy.org/tools/blast/).<br />
total volume of 20 pi, according to the supplier's PLACE (http://www.dna.affrc.go.jp/PLACE/), a<br />
instructions (Advantage RT-for-PCR Kits, DB database of motifs found in plant cii'-acting<br />
Biosciences, Clontech, USA). Synthesized regulatory DNA elements was used to scan the<br />
cDNAs were diluted in 100 pi H2O and then 4 promoter of the ZmC0I6.1 gene. Splicing<br />
pi diluted cDNAs were used as templates for prediction was realized using the Genscan<br />
PCR amplification in a volume of 20 pi as program (http://genes.mit.edu/GENSCAN.html).<br />
follows: 25 cucles at 95°C for 30s, 57°C for 30 Phylogenetic tree was made using the<br />
s and 72°C for 60 s and finally with an CLUSTAL W program.<br />
extension at 72°C for 5 minutes. The maize<br />
coding genes ubiquitin, ZmUBI (accession II. RESULTS<br />
number S94466), was used as an internal 1. A novel cold induced gene, ZmCOI6.1 is<br />
standard. Amplified PCR products (15 pi) were conserved in plant species<br />
72<br />
A previous study using the chilling tolerant indicates that the novel cold-induced gene<br />
maize genotype ETH-DH7 identified several ZmC016.1 is conserved in plant species.<br />
novel cold-induced genes [12-14]. From this Gene homologues and orthologs share<br />
study, one gene, ZmC0I6.1, represented by four identity on the amino acid level where similarity<br />
different cloned fragments was sorted out for in particular regions might be indicative of<br />
further characterization based on the high level domains or motifs important for function. To<br />
of occurrence in the screening. To determine the identify putative domains, a comparison of the<br />
complete sequence of ZmC 016.1, ten amino acid sequences mentioned above with<br />
oligonucleotides, which covered the the ZmCOId.l predicted protein sequence was<br />
AZM4_69676 sequence from the maize performed and the results obtained revealed<br />
genotype B73 (tgi_maize/) and which showed similar domains specifically at the N- and C-<br />
96 % homology with ZmC0I6.1 detected terminals (data not shown). The most conserved<br />
fragment, were designed to amplify this region was the C-terminus with the putative<br />
sequence, only, but not AZM4_12960 homolog motif L-P-[FY]-[TV]-V-P-F. • Furthermore, a<br />
sequence, which shows 81 % homology with lysine-rich region was identified at the N-<br />
ZmC0I6.1 fragments. Overlapping regions of terminal of all the sequences. The function of<br />
the corresponding gene in the ETH-DH7 these motifs has not been described previously,<br />
genotype were amplified. The overlapping suggesting that they are novel. Analysis of the<br />
fragments were sequenced, assembled and amino acid sequence for transmembrane regions<br />
annotated in the Genbank (accession number by TMpred [6] did not reveal the presence of<br />
DQ060243) [12-14]. transmembrane domains, thus, predicting that<br />
To investigate the possible existence of ZmC0I6.1 is a soluble protein. This result<br />
homologues and/or orthologues of the suggests that ZmCOI6.1 and its maize homolog<br />
ZmC0I6.1 predicted amino acid sequence, a are conserved in plant species sharing high<br />
database search was carried out. The database similarity at least for two domains at the amino<br />
analysis identified nine amino acid sequences, acid level.<br />
similar to the ZmC0I6.1 sequence: one maize a. ZmC016.1 gene Is alternatively spliced<br />
homologue, two from Oriza sativa (rice)<br />
(Os03gl3810 and OslOg03550 in the TIGR rice To better understand the time course of cold<br />
genome annotation database, induction ofthe ZmC 016.1 gene, an experiment<br />
was conducted, in which seedlings were<br />
http://www.tigr.org) and six from Arabidopsis<br />
exposed to 6°C for 24 hours and samples were<br />
thaliana (Atlg20100, Atlg75860, At2gl7787,<br />
collected after one, two, four, six, 12 and 24<br />
At3g07280, At4g35940 and At5g48610) (figure<br />
hours to analyze early and later response. The<br />
1). ZmC0I6.1 also shares nucleotide sequence<br />
expression of ZmC0I6.1 increased with time of<br />
similarity with ESTs from wheat (Tritlciim<br />
exposure to cold confirming its regulation by<br />
aestivum L.), barley (Hordeum vulgare L.),<br />
cold. Upon analysis by RT-PCR, ZmC0I6.1<br />
sugarcane (Saccharum ojfidnarum L.) and<br />
obtained two fragments, referred as sfl and sf2.<br />
sorghum (Sorghum bicolor L.) (data not shown).<br />
To determine whether the fragments sfl and sf2<br />
Using the amino acid sequences, the<br />
were indeed transcripts from the ZmC016.1<br />
phylogenetic relationship between sequences<br />
gene and not the expression product of another<br />
derived from maize, rice and Arabidopsis were<br />
gene(s), both forms were cloned using the<br />
analysed excluding the ESTs coding for an<br />
oligonucleotides 6551-2 and ZmC0I6.1h_R and<br />
incomplete protein (figure 1). This analysis<br />
subsequently sequenced. The sequence analysis<br />
revealed three main groups: one consisted of<br />
revealed that both cDNA forms were identical<br />
ZMC0I6.1 and a maize homologue<br />
with the specific parts of the ZmC016.1 gene<br />
AZM4_12960 sequence together with the rice<br />
(data not shown).At normal growth condition<br />
sequences, the second group accommodated the<br />
(non-stress condition), both fragments sfl and<br />
Arabidopsis sequences At4g35940, At2gl7787,<br />
sf2 have 3 exons and 2 introns. Under stress<br />
At3g07280 and At5g48610 and the third dne<br />
treatments, the intron II is splice out in sfl and<br />
Atlg75860 and Atlg20100. This analysis<br />
the intron 12 in both sfl and sf2 (figure 2).<br />
<br />
73<br />
• ZmC 016.1<br />
^AZM4_12960<br />
•0s03g13810<br />
•0s10g03550<br />
I—AT1G20100<br />
•AT1C75860<br />
r-AT4G35940<br />
•AT2G17787<br />
•At3g07280<br />
^—At5g48G10<br />
<br />
Figure 1. ZmC0I6.1 is conserved in plants as shown by phylogenetic analysis of the deduced<br />
ZmC0I6.1 amino acid sequence, homolog and ortholog sequences. The phylogenetic tree of the<br />
amino acid sequences of ZmC0I6.1, maize homologue and orthologues in rice and Arabidopsis<br />
were constructed using the CLUSTAL W program<br />
<br />
<br />
<br />
<br />
Figure 2. The splicing structure of ZmC0I6.1 to yield sfl and sf2, as predicted from gene analysis.<br />
Thick lines represent exons (El, E2 and E3) and thin lines introns II (nucleotide position from 744<br />
and 1866) and 12 (nucleotide position from 2180 and 2280). Angled lines represent fragments<br />
spliced out to yield sfl and sf2 respectively. Triangles indicate the position of the start ( T ) and stop<br />
(A) codon. The predicted alternative splicing transcripts are sfl containing El, II, E2 and E3 and<br />
sf2 containing E l , E2 and E3<br />
<br />
Interestingly, we found that sfl and sf2 were 13"C. The data shown that, at 13°C treatment,<br />
amplified from the samples taken at 0 hour the sf2 transcript was also present and remained<br />
(control) as well as under cold treatment (figure stable over time, while the levels of sfl<br />
3). A lower level of sfl was found under control increased rapidly (figure 3). These results show<br />
conditions, but the levels increased with the that ZmC016.1 is induced at short exposure to<br />
length of time exposed to cold stress. The cold and increases with time. The RT-PCR<br />
smaller cDNA fragment, sf2, decreased during suggests that the expression is characterized by<br />
exposure to 6°C from 1 to 12 hours but started the appearance of two fragments.<br />
to increase at 24 hours. To test the effect of The presence of two fragments in the<br />
suboptimal temperature 13°C on the expression analysis of ZmC016.1 expression pointed to the<br />
of ZmC016.1 and the expression of the two possibility that alternative splicing is taking<br />
fragments, the similar experimental set-up at place. Analysis of the putative spliced forms of<br />
<br />
74<br />
ZinC016.1, sfl and sf2, revealed that sfl but was less obvious in the second. For further<br />
expanded from nucleotide 640 to nucleotide analysis of the gene, the splicing predictor<br />
3196 with 101 nucleotides missing between the GENSCAN program [3] was used to verify the<br />
positions 2179 and 2281 (12) (Figure 3). In the results described above. This program predicted<br />
sf2 transcript, the regions between 744 and 1867 the donor sites of the first and second introns,<br />
(11) and between 2179 and 2281 were missing. the acceptor site of the second intron as well as<br />
To identify the positions of the introns and the branch point region of the second intron but<br />
exons as well as the splicing points, the not of the first one. Other splicing regions in the<br />
Z111COI6.I sequence was analyzed to determine first intron were predicted by GENSCAN, which<br />
the splicing consensus sequence, AG/GTAAGT, corresponded neither to the two spliced forms<br />
of the introns 5'-splice donor site and TGCAG/G found in this study nor to any of the other<br />
of the 3'-splice acceptor site as well as the expressed sequence tags (EST) in the database<br />
consensus branch point region CUR AY (R, (data not shown). Similar pattern where also<br />
purine; Y, pyrimidine) [9]. Both the first and found in the sequence of rice and Arabidopsis<br />
second introns had a conserved 5'-splice donor (data not shown). These results show that<br />
site. However, the 3'-acceptor site was ZmC 016.1 sequence harbours conserved<br />
conserved in the second intron but less splicing points that would give potential<br />
conserved in the first. The branch point products of sizes that are in agreement with sfl<br />
sequence was well conserved in the first intron and sf2 obtained in the RT-PCR analysis.<br />
<br />
Time at 6°C Oh lb 2h 12 h 24 h<br />
sfl j^is^f^^sm<br />
sf2<br />
<br />
ZmUBI<br />
<br />
Time at 13°C Oh 1h 2h<br />
sfl w^"-''''f^w>^*w^^m<br />
ZmUBI<br />
<br />
<br />
Figure 3. Expression of ZmC0I6.1 gene under abiotic stresses: ZmC0I6.1 is induced by cold,<br />
expression increases with time of exposure and is alternatively spliced. The effect of low (6°C) and<br />
suboptimal (13°C) temperature (in the dark) on the expression and alternative splicing of the<br />
ZmC0I6.1 gene was examined. 0 hour indicates samples taken prior to treatment. RT-PCR was<br />
performed with the primers 6551-2 and ZmC0I6.1b_R to analyse the expression of the ZmC0I6.1<br />
transcripts. ZmUBI was used as the internal control.<br />
<br />
2. ZmC0I6.1 gene alternative splicing stress. Therefore, the induction ofthe ZmC0I6.1<br />
occurred under different abiotic stresses gene was tested for drought and salt stress and<br />
and signalling molecules with signalling molecules known to induce<br />
stress responses, for example to abscisic acid<br />
In a previous study the expression of (ABA) and salicylic acid (SA). The ZmC0I6.1<br />
ZmC0I6.1 was changed in response to different gene transcript accumulated under drought and<br />
abiotic stresses [12]. The question arose whether under salt stress as well as after treatment with<br />
the alternative splicing occurs in the same way SA and ABA. The strongest induction was<br />
under other abiotic stresses or after treatment obtained during drought and during the ABA<br />
with signalling molecules as it did under cold treatment, where the sfl transcript increased but<br />
<br />
75<br />
sf2 remained at low levels (figure 4). Under These results show the conser\^.ion of<br />
sodium chloride and jasmonic acid treatment the alternative splicing of ZmC0I6.1 gene in<br />
expression of the sf2 transcript was suppressed response to abiotic stress other then cold and<br />
(figure 4 and data not shown). induction by signaling molecules.<br />
<br />
Drought NaCI ABA<br />
<br />
<br />
<br />
<br />
ZmUBI<br />
A^<br />
<br />
Figure 4. Expression of fragment sfl of ZmC0I6.1 gene: sfl is increased in maize leaves (ETH-<br />
DH7) in response to various stress treatments (drought, NaC,SA and ABA). RT-PCR was performed<br />
with cDNA produced from RNA extracted from maize seedlings at 0 hour of treatment at 25°C and<br />
after 6 and 24 hours of exposure of maize seedlings to stresses. Ubiquitin (ZmUBI) was used as the<br />
internal control.<br />
<br />
3. The ZmCOI6.I gene promoter contains DRE/CRT-HvCBF2, LTRE-core and LTRE-1.<br />
predicted conserved stress cis-acting Other ds-acimg elements were identified, which<br />
elements are involved in abiotic and biotic stress: MYBl,<br />
ABRE-like G-box, MYB-core and ASFl (Figure<br />
Genes that are induced by stress or other 5; Table 1). Some of these cis-acting elements<br />
treatments usually harbour short sequences, cis- were also present in some of the promoters of the<br />
acting elements, within the promoter that are orthologs of ZmC0I6.1 suggesting that they share<br />
identified by transcription factor, thereby a common feature of pxjssible transcriptional<br />
regulating gene expression. To analyze the regulation (data not shown). This result shows the<br />
ZmC0I6.1 promoter cw-acting elements, the presence of cis-acting element motifs in the<br />
search was performed in a database using the promoter of ZmC0l6.1 and the complexity<br />
PLACE program (PLACE/). Several cis-acting regulation of ZMC0I6.1 gene expression upon<br />
elements were identified in the ZmC0I6.1 induction by different abiotic stresses.<br />
promoter, including the low temperature-<br />
responsive elements MYC, DRE/CRT-core, IIL DISCUSSION<br />
<br />
1 cgctgtgtcgcctagaaatagcgatgtggtacattccgcaccgcacatcgtcacgacggacgcgccttac<br />
ccggcttgcgctggcaacgcgacc|cacgtg[ccggtccgtgattgcgggttg|ccgac|gcttctaggtcggt<br />
71 MYCIG-boxl ABRE-like DRE/CRT-corelLTRE-core<br />
tccgggtcgtgggccctcatacacgttgcgtgcgccccgggaacactcaagtactcaaccccggctccga<br />
141 ACGT<br />
agt^cgacjtgcaagcggggcccacacgctcttaacctagctgcacccgcgacgcgtagttgcagcgcatc<br />
211 LTRE-core<br />
gccattcacagcacccgcatataggt|ctgttg|cactgacatggcgtcccaccacgggcctgtgcc|caact<br />
281 MYB-core MYCIMYB2<br />
gtcagtgaattcgttccggaaacaacgcgtaaccgagactgacgcgctagttgcccgcacgactcggcct<br />
351 DRE1-Rabl7 ASFl<br />
cctcgcccccggctttaaatagtggcgtacccccatcccatagaagagactctttcatttccttctaccg<br />
421 Predicted core Promoter INR<br />
cagcctcagaattcccccctcccccgtagcgaaaccctagccgccacgccaaaaCcaaatdccgccgagc<br />
491 AGC-boxIGCC-core MYBlpREalpha<br />
76<br />
561 ccgaaa|ttttccggcgggttccttgccgcgaatcgattgatttcgagcgattcgactcctttgtgatctc<br />
LTRE-1 IHSE-like<br />
631 tcggcggggtagagcgcggtcgaccgtcggccaigtcgaggtgcttcccctacccgccaccggggt|acgt<br />
DRE/CRT-HvCBF2 KBRE-like|lACGT<br />
701 ycggaacccagtggccgtggccgagccggagtcgaccgctaaggtttgttgaaccttcggatttaca-<br />
DRE/CRT-HvCBF2<br />
Figure 5. ZmCOI6.1 promoter contains motifs of conserved cis-acting elements involved in stress.<br />
The scheme of the ZmCOId.l promoter region and the 5'-end of the transcript showing predicted<br />
position of stress-responsive cis-acting elements motifs (for details see Table 1). The sequence is<br />
numbered according to the sequence (DQ060243). A hyphen denotes the absence of the<br />
corresponding nucleotides residues. The predicted translational start codon is bold and in italics.<br />
Capital letters indicate transcribed regions and lower case letters are non-transcribed regions.<br />
<br />
• : ; • : Table 1<br />
<br />
Stress-responsive cis-acting elements present in the ZmCOI6.1 promoter region<br />
(see Figure 5), the abiotic/biotic stresses, in which they are involved and the conserved<br />
sequences. 'as-1-Iike elements are characterized by two imperfect TGACGTCA motifs,<br />
separated by 4 bp, "ll-bp ethylene-responsive element, TAAGAGCCGCC, •'G-box is 5'-C/A-<br />
ACACGTGGCA-3'with a CACGTG hexanucleotide core.<br />
'K = G or T; N = A, C, G or T; R = A or G; W = A or T; Y = C or T<br />
<br />
''Recognition<br />
cis-acting element Involvement<br />
Sequence<br />
MYC Drought (ABA), cold CANNTG<br />
MYB-core Drought stress CNGTTR<br />
MYBl Dehydration stress (ABA) WAACCA<br />
MYB2 Dehydration stress (ABA) YAACKG<br />
ABRE-hke| Drought (etiolation) ACGTG<br />
DRE/CRT-core Cold- and dehydration-responsive expression TACCGAC<br />
DRE/CRT-HvCBF2 Low temperature GTCGAC<br />
DRE1-Rabl7 Drought (ABA) ACCGAGA<br />
LTRE-core Low temperature CCGAC<br />
LTRE-1 Low temperature CCGAAA<br />
ASFL Auxin and/or salicylic acid; perhaps light regulation TGACG<br />
INR Light-responsive YTCANTYY<br />
AGC-box ,GCC-box Ethylene (=ethylene-inducible defense genes) AGCCGCC<br />
GCC-core Defense, jasmonate GCCGCC<br />
ACGT Drought (etiolation) ACGT<br />
G-box Pathogen, ABA, light CACGTG'<br />
Realpha Etiolation AACCAA<br />
CNNGAANNNTFC<br />
HSE-Uke Heat shock, pathogen<br />
NNG<br />
<br />
<br />
<br />
77<br />
In order to get more insight into the response ZmC0I6.1 are all classical sites, witn the<br />
of maize to low temperature, we have exception of that at the splice acceptor site in<br />
characterized a novel cold-induced gene intron 1 (II). The II of sfl contains several stop<br />
ZmC0l6.1. The sequence analysis reveals codons, making it a non-translated transcript,<br />
ZmC016 A is a conserved gene in plants showing although h cannot be ruled out that translation<br />
high similarity to sequences from rice and takes place by avoiding the II intron code region<br />
Arabidopsis and also to ESTs from wheat by means of an unconventional mechanism as<br />
(Tiiticum aestivum L.), barley (Hordeum vulgare for example ribosomal shunting or internal<br />
L.), sugarcane (Saccharum ojfidnarum L.) and initiation. Assuming that translation starts at the<br />
sorghum (Sorghum bicolor L.). The deduced same position in sfl as in sf2, the deduced<br />
amino acid sequence indicates that these proteins amino acid sequence of sfl would be only 29<br />
are possibly soluble and they share several motifs amino acids long due to a stop codon at the<br />
of high identity whose function still remains to be beginning of exon 1. Start of translation at the<br />
characterized. Although preliminary results show ATG in position 2376 (13) would result in a 285<br />
that ZmC016.1 homolog is induced by cold stiess amino acids long protein which would share the<br />
it remains to be investigated for response to other C-terminus of sf2 (figure 2). This analysis<br />
stresses [12]. suggests sfl as non-functional transcript.<br />
The ZmC0I6.1 gene is induced by several The predicted sites for alternative splicing<br />
abiotic stresses and signaling molecules were also present in the sequence of rice, as<br />
indicating that the ZmC016.1 is probably a shown by the alternative splicing form from the<br />
general stress-regulated gene. This is also locus Os03g 13810 (TIGR, rice genome<br />
supported by the fact that its promoter contains annotation database) suggesting that the<br />
several c/i'-acting elements, suggesting possible orthologs are not only similar on the amino acid<br />
regulation by different transcription factors. level but as well share the same alternative<br />
The presence of regulative modules within splicing modification. As in the ZmC016.1<br />
the promoter is common in stress-induced genes gene, two mRNA forms are produced from<br />
[1, 16]. These elements overlap in function with Os03gl3810, where the first intron is retained in<br />
regard to induction in response to stress, as the larger one and the second intron is spliced<br />
exemplified by the promoter induction of the out in both of them (unpublished).<br />
drought-induced gene RAB17 [7]. However, it The balance between sfl and sf2 of<br />
remains to be determined whether all the ZmC016.1 was affected by the low temperature<br />
predicted cii'-acting elements are important for and being more stable at 13°C than at 6°C; at<br />
the regulation of ZmC0I6.1 gene expression; 6°C there is more sfl than sf2. This difference<br />
the induction by different environmental stress in the transcript level at 6°C and 13°C suggests<br />
point in this direction. The increased expression that alternative splicing might play an important<br />
of ZmC016.1 upon treatment with ABA and SA role in the regulation of ZmC016.1 expression<br />
suggests that ZffiC0I6.1 acts downstream of the depending on the strength of the low<br />
effector pathways of these signaling molecules. temperature stress. It is possible that sf2 is<br />
necessary for normal growth of the plant acting,<br />
The results of this study show that the probably as a negative regulator of the stress<br />
ZmC0I6.1 gene expression is characterized by response. These results are supported by<br />
alternative splicing producing two transcripts, preliminary results showing that a T-DNA<br />
sfl and sf2. Alternative splicing, also known as insertion in the Arabidopsis heterolog of<br />
differential splicing, is a conserved mechanism ZmC0I6.1 gene (At4G35940) is more tolerant<br />
regulating a large part of the expression of many than wilt type plants to cold, drought and salt<br />
genes [8]. The modules in ZmC0I6.1 that are stress (resuhs not shown). Under a strong cold<br />
involved in splicing were identified by stress (6°C) sf2 was down-regulated or<br />
comparing its sequence with conserved splicing remained constant. Therefore, to compensate for<br />
motifs and by means of the GENSCAN induction of the gene through the cw-acting<br />
program, which corroborated the intron elements in the promoter, alternative splicing<br />
retention mechanism. The splicing sites within<br />
78<br />
would be favoured to produce a transcript, sfl, 2. Bade J. et al., 2003: Plant Molecular<br />
which is probably non-functional On the other Biology, 52: 53-68.<br />
hand, at 13°C the function of ZmC016.1 would<br />
3. Burge C , Karlin S., 1997: lournal of<br />
be more important; alternative splicing would be<br />
balanced towards the "functional" transcript sf2 Molecular Biology, 268: 78-94.<br />
as shown by its increase at 13°C in contrast to 4. Chinnusamy V., Zhu J., Zhu J. K., 2007:<br />
6°C. This would be a link to its possible role as Trends in Plant Science, 12(10): 444-451.<br />
a negative regulator. The fact that the sf2<br />
transcript also accumulates in response to 5. Dubouzet J. G. et al., 2003: Plant lournal,<br />
signalling molecules, such as abscisid acid and 33:751-63.<br />
salicylic acid indicates that sfl probably 6. Hofmann K., Stoffel W., 1993: Biol.<br />
regulates the expression of the ZmC016.1 gene Chem. Hoppe-Seyler, 374: 166.<br />
and is not an artifact of the abiotic stresses. It is<br />
important to mention that 12 is spliced out in 7. Kizis D and Pages M., 2002: Plant lournal,<br />
both, sfl and sf2 transcripts; this indicates the 30: 679-689.<br />
specifity of intron retention when plants are 8. Lee J. T. et al., 2003: Plant Cell and<br />
exposed to adverse conditions or to signalling<br />
Environment, 26: 1181-1190.<br />
molecules. The retention of unspliced introns in<br />
a fraction of the transcripts seems to be common 9. Lorkovic Z. J. et al., 2000: Trends Plant<br />
in plants and could either reflect low efficiency Science, 5: 160-167.<br />
of splicing or a regulatory process [9]. In<br />
10. Mastrangelo A. M. et al., 2005: Planta,<br />
support of the later it was found in Arabidopsis<br />
that a high fraction of the alternatively spliced 221:705-715.<br />
forms were retained introns [11]. Interestingly, ll.Ner-Gaon H. et al., 2004: The Plant<br />
the transcripts with retained introns were for the Journal, 39: 877-885.<br />
most part transcripts of stress and<br />
external/internal stimuli-related genes. An 12. Nguyen Thuy Ha et al., 2008: Journal of<br />
intron retention mechanism has been described Biology, 30(2): 77-87.<br />
recently for cold-regulated genes in durum 13. Nguyen H. T. et al., 2009: Plant Physiol.<br />
wheat. In this study, genes coding for a putative Biochem., 47: 116-122.<br />
ribokinase and a C3H2C3 RING-finger protein<br />
were characterized by the stress-induced 14. Orlene Guerra-Peraza, Ha Thuy Nguyen,<br />
retention of a subset of introns in the mature Peter Stamp, Jorg Leipner, 2009: Plant<br />
mRNA [10]. Science, 176: 783-791<br />
<br />
It remains to be characterized how 15. Vanninl C. et al., 2004: Plant Journal, 37:<br />
alternative splicing regulates the activity of 115-127.<br />
ZmC0I6.1 but most importantly how ZmC0I6.1 16. Thomashow M. F., 1999: Annual Review<br />
regulates the stress response in maize. of Plant Physiology & Plant Molecular<br />
Biology, 50: 571-599.<br />
REFERENCE<br />
17. Zhang F. L. et al., 2008: Plant Science,<br />
I. AbeH. etal., 2003: Plant Cell 15: 63-78. 174:510-518.<br />
<br />
<br />
<br />
<br />
79<br />
PHAN HI: NGHIEN CUU VAI TRO CUA CAC GIEN LIEN QUAN DEN<br />
KHA NANG CHONG CHIU LANH 6 NGO: QUA TRINH BIEU HIEN VA<br />
TRINH TU VUNG PROMOTER CUA CAC GIEN NAY<br />
N G U Y E N TIHJY HA, JORG LEIPNER,<br />
<br />
ORLENE GUERRA-PERAZA, PETER STAMP<br />
<br />
TOMTAT<br />
Bang ky thuat PCR-cDNA Select Subtraction (hay con co ten goi khac la SSH- Suppression Subtractive<br />
Hybridization) chung toi da phan lap dugc 18 gien co bieu hien cao trong didu kien lanh 6"C va 13"C. Trong<br />
sd 18 gien nay, gien ZmC0I6.I co tan sd xua't hien rat cao (49%) trong thu vien cDNA. Qua phan tfch san<br />
pham RT- PCR cho thay gien ZniC0I6.I co bieu hien cao khong nhii'ng trong dieu kien nhiet do tha'p ma con<br />
CO phan ung vai cac tac nhan khac nhu kho han. mudi man va cac phan tir truyen tin hieu stress nhu ABA va<br />
SA nhu vay co the khing dinh ZmC0I6.I co vai tro cua gien chju trach nhiem phan ung lai khi gap di^u kien<br />
sdng ba't lgi. Ket qua phan tich cho tha'y, sir bieu hien cua gien ZmC0I6.I do 2 yeu td phien ma quy dinh.<br />
Ngoai ra, qua phan tfch virng promoter ciia gien nay cho tha'y, gien co chira nhidu yeu td chiu trach nhiem<br />
phan Qng lai khi gap di6u kien sdng ba't lgi gidng nhu d gen liia va Arabidopsis.<br />
<br />
Ngdy nhdn hdi: 20-4-2008<br />
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80<br />
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