WO2012110855A1 - Gmwrky49 transcriptional gene and use thereof for enhancing plant tolerance to salt and/or drought - Google Patents
Gmwrky49 transcriptional gene and use thereof for enhancing plant tolerance to salt and/or drought Download PDFInfo
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- WO2012110855A1 WO2012110855A1 PCT/IB2011/050632 IB2011050632W WO2012110855A1 WO 2012110855 A1 WO2012110855 A1 WO 2012110855A1 IB 2011050632 W IB2011050632 W IB 2011050632W WO 2012110855 A1 WO2012110855 A1 WO 2012110855A1
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- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
- C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
- C12N15/8241—Phenotypically and genetically modified plants via recombinant DNA technology
- C12N15/8261—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield
- C12N15/8271—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance
- C12N15/8273—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance for drought, cold, salt resistance
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- This invention relates generally to agro-biotechnology and plant molecular biology.
- it relates to transgenic plants having novel features, methods of producing such plants and polynucleotides and polypeptides, methods of cloning and gene expression to confer salt and/or drought tolerance on plants and other organisms.
- the invention relates to the use of Gm WRKY polynucleotides and transgenic plants expressing these polynucleotides and polypeptides.
- Soybean is one of the most important cash corps.
- the salt and/or drought tolerance is particularly important for soybean.
- no transgenic salt and/or drought tolerant soybean plant has been developed so far.
- it is important to find out transcription factors associated with salt and/or drought tolerance for growing a soybean plant with tolerance to salt and/or drought and thereby increasing its production.
- plants receive extracellular changes of environment and transfer them into cells to induce expressions of some responding genes via many pathways and synthesize some functional proteins, osmoregulation substances as well as transcription factors for signal transmission and gene expression regulation so that plants are able to make corresponding responses to environmental changes and avoid damages caused by high salt, drought, and/or low temperature stresses.
- osmoregulation substances as well as transcription factors for signal transmission and gene expression regulation so that plants are able to make corresponding responses to environmental changes and avoid damages caused by high salt, drought, and/or low temperature stresses.
- Abiotic stress inducible genes are classified into two groups. The products of the first group include effector proteins that help in cell membrane system protection, water holding, controling ion homeostasis etc.
- proteins include osmoprotectants, LEA, aquaporins, chaperones and detoxification enzymes.
- the second group of gene produce regulatory proteins involved in perception of signal, signal transduction and transcriptional regulation of gene expression. These proteins include kinases, phoshoinositide metabolisms' enzymes and transcription factors.
- transcription factor families have been found to be induced by salt and/or drought stresses, such as DREB, ERF, WRKY, MYB, bZIP, and NAC families (Hasegawa et al., 2000; He et al., 2005; Seki et al., 2003; Zhu, 2002; Zhou et al., 2008; Liao et al., 2008a, 2008b ).
- DREB1A and AtMYB2 improved the salt and drought tolerance of transgenic plants when transferred into Arabidopsis (Abe et al., 2003; Kasuga et al., 1999).
- Alfin1 a PHD finger protein, was identified as a salt-induced transcriptional factor and enhanced the stress tolerance by ectopic expression in transgenic plants (Bastola et al., 1998).
- These transcriptional factors ultimately regulate the expression of functional genes in response to environmental stresses. When plants encounter stresses, transcription factor as a controlling gene is able to regulate the expression of a series of downstream genes to enhance the tolerance of plants to the stresses.
- Ge et al (2010) utilized Affymetrix® Soybean GeneChip® to conduct transcriptional profiling on Glycine soja roots subjected to 50 mmol/L NaHCO 3 treatment. In a total of 7088 probe sets, 3307 were up-regulated and 5720 were down-regulated at various time points. The number of significantly stress regulated genes increased dramatically after 3 h stress treatment and peaked at 6 h. GO enrichment test revealed that most of the differentially expressed genes were involved in signal transduction, energy, transcription, secondary metabolism, transport, disease and defence response (Ge et al. 2010).
- One object of the present invention is to provide an isolated polynucleotide capable of giving a plant, preferably soybean, tolerance to salt and/or drought stress, which comprises a nucleotide sequence as shown in SEQ ID NO:1 or a conservative variant or degenerate sequence comprising one or more substitutions, deletions, additions and/or insertions in the said nucleotide sequence, or a sequence hybridizable with the said sequence under moderate stringent condition, or a complementary sequence thereof, or a variant or derivative having at least 90% homology and same or similar biological function to the said nucleotide sequence.
- Another object of the present invention is to provide an expression vector comprising the said polynucleotide sequence.
- Another object of the present invention is to provide a host cell transformed or transfected by the said expression vector.
- Another object of the present invention is to provide a use of the said polynucleotide sequence for increasing salt and/or drought stress tolerance in plants, preferably soybean.
- One aspect of the present invention provides a method for determining whether a test plant, for example a dicot, has been exposed to at least one stress condition, for example an abiotic stress, comprising determining polynucleotide expression in the test plant to produce an expression profile and comparing the expression profile of the test plant to the expression profile of at least one reference plant that has been exposed to at least one stress, for example an abiotic stress.
- the expressed polynucleotides are selected from the group consisting of the polynucleotide sequences contained in the sequence listing.
- the test and reference plants are soybean plants and the expressed polynucleotides are selected from the group consisting of SEQ ID No: 1 or a functional portion thereof.
- the object of the present invention is to isolate a DNA fragment comprising a complete encoding region of transcription factor gene, to clone it, and to use it for improvement of soybean or other plants to salt and/or drought tolerance.
- the present invention is based on the discovery by structure analysis of the obtained gene that belongs to plant-specific transcription factor WRKY family, and thus the said transcription factor is named as GmWRKY49.
- the term 'isolated polynucleotide capable of giving a plant tolerance to salt and/or drought stress' represents the polynucleotide sequence as shown in SEQ ID NO:1 and further comprises all variants or derivatives having at least 90% homology and same or similar biological function to the sequence as shown in SEQ ID NO:1.
- 'isolated' means 'artificially changed from natural status and/or separated/extracted/isolated from natural environment'.
- an 'isolated' component or substance existing in nature is 'isolated', it has been changed or removed from its initial environment or been subject to both.
- a polynucleotide or polypeptide naturally existing in live animal is not 'isolated', but the same polynucleotide or polypeptide separated or extracted from its natural status is 'isolated', which is exactly the term used herein.
- 'polynucleotide(s)' means a single or double stranded polymer of deoxyribonucleotide or ribonucleotide bases and includes DNA both sense and anti-sense strands, and corresponding RNA molecules, including HnRNA and mRNA molecules, and comprehends cDNA, genomic DNA and recombinant DNA, as well as wholly or partially synthesized polynucleotides.
- An HnRNA molecule contains introns and corresponds to a DNA molecule in a generally one-to-one manner.
- An mRNA molecule corresponds to an HnRNA and DNA molecule from which the introns have been excised.
- a polynucleotide may consist of an entire gene, or any portion thereof.
- Operable anti-sense polynucleotides may comprise a fragment of the corresponding polynucleotide, and the definition of 'polynucleotide' therefore includes all such operable anti-sense fragments.
- a nucleotide 'variant' is a sequence that differs from the recited nucleotide sequence in having one or more nucleotide deletions, substitutions or additions. Such modifications may be readily introduced using standard mutagenesis techniques, such as oligonucleotide-directed site-specific mutagenesis, for example, by Adelman et al. (1983). Nucleotide variants may be naturally occurring allelic variants, or non-naturally occurring variants. Variant nucleotide sequences preferably exhibit at least about 70%, more preferably at least about 80% and most preferably at least about 90% homology (determined as described below) to the recited sequence.
- 'homology' when used in relation to nucleic acids refers to a degree of complementarity either partial or complete homology.
- 'Sequence identity' refers to a measure of relatedness between two or more nucleic acids, and is given as a percentage with reference to the total comparison length. The identity calculation takes into account those nucleotide residues that are identical and in the same relative positions in their respective larger sequences. Calculations of identity may be performed by algorithms contained within computer programs such as 'GAP' (Genetics Computer Group, Madison, Wis.) and 'ALIGN' (DNAStar, Madison, Wis.).
- a partially complementary sequence is one that at least partially inhibits (or competes with) a completely complementary sequence from hybridizing to a target nucleic acid is referred to using the functional term 'substantially homologous'.
- the inhibition of hybridization of the completely complementary sequence to the target sequence may be examined using a hybridization assay (Southern or Northern blot and the like) under conditions of low stringency.
- a substantially homologous sequence or probe will compete for and inhibit the binding (in other words, the hybridization) of a sequence which is completely homologous to a target under conditions of low stringency. This is not to say that conditions of low stringency are such that non-specific binding is permitted; low stringency conditions require that the binding of two sequences to one another be a specific (in other words, selective) interaction.
- the absence of non-specific binding may be tested by the use of a second target which lacks even a partial degree of complementarity (for example, less than about 30% identity); in the absence of non-specific binding the probe will not hybridize to the second non-complementary target.
- a partial degree of complementarity for example, less than about 30% identity
- the term 'substantially homologous' refers to any probe which can hybridize to either or both strands of the double-stranded nucleic acid sequence under conditions of low to high stringency as described in the below.
- Low stringency conditions in reference to nucleic acid hybridization comprise conditions equivalent to binding or hybridization of 500 nucleotides long probe at 42 °C in a solution consisting of 5 ⁇ SSPE (43.8 g/l NaCl, 6.9 g/l NaH 2 PO 4 ⁇ H 2 O and 1.85 g /l EDTA, pH adjusted to 7.4 with NaOH), 0.1% SDS, 5 ⁇ Denhardt's reagent [50 ⁇ Denhardt's contains per 500 ml: 5 g Ficoll (Type 400, Pharmacia), 5 g BSA (Fraction V; Sigma)] and 100 ug/ml denatured salmon sperm DNA followed by washing in a solution comprising 5 ⁇ SSPE, 0.1% SDS at 42 °C when a probe of about 500 nucleotides in length is employed.
- 5 ⁇ SSPE 43.8 g/l NaCl, 6.9 g/l NaH 2 PO 4 ⁇ H 2 O and 1.85 g /l EDTA, pH adjusted
- High stringency conditions in reference to nucleic acid hybridization comprise conditions equivalent to binding or hybridization of 500 nucleotides long probe at 42 °C in a solution consisting of 5 ⁇ SSPE (43.8 g/l NaCl, 6.9 g/l NaH 2 PO 4 ⁇ H 2 O and 1.85 g/l EDTA, pH adjusted to 7.4 with NaOH), 0.5% SDS, 5 ⁇ Denhardt's reagent and 100 ug/ml denatured salmon sperm DNA followed by washing in a solution comprising 0.1 ⁇ SSPE, 1.0% SDS at 42 °C.
- low stringency conditions Numerous equivalent conditions may be employed to comprise low stringency conditions; factors such as the length, nature of the probe and target (DNA, RNA, base composition), concentration of the salts or other components (for example, the presence or absence of dextran sulfate formamide, polyethylene glycol etc) and the hybridization solution may be varied to generate conditions of low stringency hybridization different from, but equivalent to, the above listed conditions.
- the conditions are well known in the art that promote hybridization under conditions of high stringency (for example, increasing hybridization temperature and/or wash steps, use of formamide in the hybridization solution, etc.).
- the term 'substantially homologous' refers to any probe that can hybridize the single-stranded nucleic acid sequence (in other words, it is the complement of) under conditions of low to high stringency as described above.
- 'hybridization' refers to the pairing of complementary nucleotides (in other words nucleic acids). Hybridization and its strength (in other words, the strength of association/pairing between the nucleic acids) is impacted by such factors like degree of complementation between the nucleic acids, stringency of the conditions involved, the T m of newly formed hybrid, and the G:C ratio within the sequence of pairing nucleic acids. Pairing of complementary nucleotides within its structure of a single nucleic acid molecule is said to be 'self-hybridized'.
- T m refers to the 'melting temperature' of a nucleic acid.
- the melting temperature is the temperature at which a population of double-stranded nucleic acid molecules becomes half dissociated into single strands.
- 'stringency' refers to the conditions of temperature, ionic strength, and the presence of other compounds such as organic solvents etc, under which nucleic acid hybridizations are conducted. With 'high stringency' conditions, nucleic acid base pairing will occur only between nucleic acid fragments that have a high frequency of complementary base sequences. Thus, conditions of 'low' stringency are often required with nucleic acids derived from genetically divers organisms, as the frequency of complementary sequences is usually less.
- the 'percentage of sequence identity' is determined by comparing two optimally aligned sequences over a comparison window of at least 20 positions, wherein the portion of the polynucleotide sequence in the comparison window may comprise additions or deletions (i.e., gaps) of 20 percent or less, usually 5 to 15 percent, or 10 to 12 percent, as compared to the reference sequences (which does not comprise additions or deletions) for optimal alignment of the two sequences.
- the percentage is calculated by determining the number of positions at which the identical nucleic acid bases or amino acid residue occurs to match in both sequences, dividing by the total number of positions in the reference sequence (i.e. window size) and multiplying by 100.
- nucleotide sequence having at least 95% identity to the reference nucleotide sequence up to 5% nucleotides with reference to the total nucleotides of the reference sequence could be deleted or substituted or inserted or combination of deletion, insertion and substitution by other nucleotides.
- mutations in the reference sequence could occur at any position between and including 5- or 3-terminal position of the reference nucleotide sequence, and they exist in the reference nucleotide sequence either in individual manner or in one or more adjacent groups.
- One aspect of the present invention relates to an isolated polynucleotide capable of giving a plant salt and/or drought stress tolerance, which comprises a nucleotide sequence as shown in SEQ ID NO:1 or a conservative variant or degenerative sequence comprising one or more substitutions, deletions, additions and/or insertions into the said nucleotide sequence, or a hybridizable sequence under moderate stringent condition, or a complementary sequence thereof, or a variant or derivative of the said nucleotide sequence having at least 90% homology having same or similar biological function.
- the said polynucleotide consists of the DNA sequence given as SEQ ID NO:1.
- the gene or homologous gene of the present invention can be screened from cDNA and genomic libraries by using a polynucleotide-specific oligonucleotide primer/probe such as the cloned GmWRKY49 gene.
- the GmWRKY49 gene of the present invention and any DNA fragment of interest or DNA fragment homologous to it can also be obtained/amplified from genome, mRNA and cDNA by using PCR (polymerase chain reaction) technology.
- a sequence comprising GmWRKY49 gene can be isolated/obtained by using the above techniques, and can be transferred into any expression vector capable of carrying the gene of interest into the plant and expression of the gene of interest (an exogenous gene) thereof.
- the transgenic plant, with enhanced salt and/or drought tolerance can be obtained via its transformation with the said sequence and any expression vector capable of inducing the expression of an exogenous gene in the plant.
- PCR can be used for amplifying the sequence from cDNA, wherein the said cDNA is prepared via reverse transcriptase (RT) PCR from the isolated RNA.
- RT reverse transcriptase
- a sequence-specific oligonucleotide primer can be designed or purchased or synthesized for this amplification based on the sequence as shown in SEQ ID NO:1.
- PCR product can be separated by gel electrophoresis and detected by methods well known by those skilled in the art.
- sequence-specific oligonucleotide primer/probe' refers to an oligonucleotide sequence having at least 80%, preferably at least 90%, more preferably at least 95% identity to the said polynucleotide, or to the anti-sense oligonucleotide of the said polynucleotide.
- the very useful oligonucleotide primer and/or probe in the present invention has at least 10-40 nucleotides.
- the oligonucleotide primer includes at least about 10 consecutive nucleotides of the said polynucleotide.
- the oligonucleotide used in the present invention includes at least about 15 consecutive nucleotides of the said polynucleotide.
- Another aspect of the present invention relates to an expression vector comprising the said polynucleotide sequence.
- Any strong or inducible promoter can be added before starting nucleotide of the gene of the present invention to construct/insert into a plant expression vector.
- Enhancers can also be used while constructing the gene of the present invention into a plant expression vector, and these enhancer regions can be ATG initiation codons, adjacent region initiation codons, etc. The insertion of the enhancers must be identical to the reading frame of the encoding sequence in order to ensure the translation of whole sequence.
- the expression vector carrying the GmWRKY49 gene of the present invention can be introduced into plant or other living cells by conventional biological methods such as Ti plasmid, plant virus vector, microinjection, direct DNA transformation, electroporation and the like (Weissbach, 1998; Geiserson and Corey, 1998) .
- the preferred plant of the present invention is soybean.
- the plants of the present invention also include but are not limited to: cotton, soybean, corn, rice, barley, wheat, Brassica, tomato, potato, tobacco, pepper, Arabidopsis , sunflower, etc, also includes non-agronomic species which are useful in developing appropriate expression vectors such as tobacco, rapid cycling Brassica species, and Arabidopsis thaliana .
- these vectors comprise the above mentioned polynucleotide sequence of the invention operably linked to a promoter, other regulatory sequences (for example, enhancers, polyadenylation signals, etc.) required for expression in a plant and some suitable selection marker(s) for screening/identification of the expression vector carrying the said polynucleotide sequence of the present invention.
- 'operably-linked' is intended to mean that the nucleotide sequence of interest is linked to the regulatory sequence in a manner that allows for expression of the nucleotide sequence (e.g., in an in vitro transcription/translation system or in a host cell when the vector is introduced into the host cell).
- the recombinant expression cassette will contain in addition to a GmWRKY49 polynucleotide, a promoter functional in a plant cell, a transcription initiation site (if the coding sequence to be transcribed lacks one), and a transcription termination/polyadenylation sequence(See for example, Odell et al. (1985); Rosenberg et al. (1987); Guerineau et al. (1991).
- the termination/polyadenylation region may be obtained from the same gene as the promoter sequence or may be obtained from different genes.
- Unique restriction enzyme sites at the 5' and 3' ends of the cassette are typically included to allow for easy insertion into a pre-existing vector.
- Promoters used in the present invention include but are not limited to constitutive promoters, tissue-, organ-, and developmentally-specific promoters, and inducible promoters.
- Examples of promoters include but are not limited to: constitutive promoter 35S of cauliflower mosaic virus (Odell, et al., 1985); a wound-inducible promoter from tomato, leucine amino peptidase 'LAP', (Chao et al., 1999); a chemically-inducible promoter from tobacco, Pathogenesis-Related 1 (PR1) (induced by salicylic acid and BTH (benzothiadiazole-7-carbothioic acid S-methyl ester)); a tomato proteinase inhibitor II promoter (PIN2) or LAP promoter (both inducible with methyl jasmonate); a heat shock promoter (U.S.
- Selection markers used routinely in transformation include the nptII gene which confers resistance to kanamycin and related antibiotics (Bevan et al. (1983), the bar gene which confers resistance to the herbicide phosphinothricin (White et al. (1990); Spencer et al. (1990), the hph gene which confers resistance to the antibiotic hygromycin (Blochlinger et al., 1984), etc.
- the vector is adapted for use in an Agrobacterium mediated transfection process (See for example, U.S. Pat. Nos. 5,981,839; 5,981,840; and 6,051,757; all of which are incorporated herein by reference).
- the first system is called the 'cointegrate' system having the shuttle vector which contains the gene of interest inserted by genetic recombination into a non-oncogenic Ti plasmid that contains both the cis- and trans-acting elements required for plant transformation.
- the second system is called the 'binary' system. In this system two plasmids are used; the gene of interest is inserted into a shuttle vector containing the cis-acting elements required for plant transformation while other necessary functions are provided in trans by the non-oncogenic Ti plasmid.
- useful vectors having polynucleotide sequence of the present invention are microinjected directly into plant cells.
- the vector is transferred into the plant cell by using polyethylene glycol (PEG) (Krens et al.,1982; Crossway et al.,1986); protoplasts fusion with other entities, either minicells, cells, lysosomes or other fusible lipid-surfaced bodies (Fraley et al. (1982); protoplast transformation (EP 0 292 435); direct gene transfer (Paszkowski et al., 1984; Hayashimoto et al., 1990).
- PEG polyethylene glycol
- the vector may also be introduced into the plant cells by electroporation.
- Plant protoplasts are electroporated in the presence of plasmids containing the gene construct via electrical impulses of high field strength. This technique reversibly permeabilize biomembranes allowing the introduction of the plasmids. Electroporated plant protoplasts reform the cell wall, divide, and form plant callus.
- the vector is introduced through ballistic particle acceleration devices (for example, available from Agracetus, Inc., Madison, Wis. and Dupont, Inc., Wilmington, Del.) (See for example, U.S. Pat. No. 4,945,050; and McCabe et al., 1988; Christou et al., 1990 (soybean); Sanford et al., 1987 (onion); Klein et al., 1988; Koziel et al., 1993 (maize); Hill et al., 1995 (rice); Vasil et al., 1993 (wheat); Wan et al., 1994 (barley); Umbeck et al., 1987 (cotton); Casas et al., 1993 (sorghum); Somers et al., 1992 (oat).
- ballistic particle acceleration devices for example, available from Agracetus, Inc., Madison, Wis. and Dupont, Inc., Wilmington, Del.
- the vectors comprising a nucleic acid sequence of the present invention are transferred via Agrobacterium-mediated transformation (Hinchee et al., 1988; Ishida et al., 1996).
- Agrobacterium is a gram-negative genus of the Rhizomaceae.
- the species of Agrobacterium are responsible for development of plant tumors such as crown gall and hairy root disease.
- opines amino acid derivatives
- the bacterial genes responsible for expression of opines are a convenient source of control elements for chimeric expression cassettes.
- Heterologous genetic sequences for example, nucleic acid sequences of the present invention operatively linked to a promoter
- the Ti plasmid is transmitted to plant cells on infection by Agrobacterium tumefaciens , and is stably integrated into the plant genome (Schell, 1987).
- Figure. 1 Procedural steps involved in identification, isolation, cloning and expression analysis of GmWRKY49 gene.
- FIG. 2 The GmWRKY49 gene expression levels detected by digital gene expression profiling (DGEP) under normal, salt and drought stress in cultivated and wild soybeans.
- DGEP digital gene expression profiling
- Figure. 3 The expression cassette containing the gene of interest of present invention.
- FIG. 4 The overexpression of GmWRKY49 gene in transgenic soybean plants and the growth of transgenic seedlings (5 week old) after 07 days of high salinity (200 mM NaCl) stress.
- the expression of tags from said GmWRKY49 gene increased in specific environments of 200 mM NaCl and/or 6000 PEG within Glycine max and Glycine soja except in 6000 PEG where its expression was significantly decresed in Glycine max.
- Expression level of said GmWRKY49 transcriptional gene was greater in Glycine soja (believed to be salt tolerant wild soybean accession collected from Chinese coast of Yellow Sea).
- the cDNA clones corresponding to the respective tags are from soybean cultivar Market No. 1 (believed to be salt sensitive).
- the said polynucleotides are the cDNA fragment of GmWRKY49 gene. The inventors of the present invention found that this is new stress-associated regulatory gene.
- the present invention is further demonstrated with examples in combination with figures, and described methods for isolating and cloning the DNA fragment comprising the whole encoding region of the said GmWRKY49 gene and for verifying its function, based on the initial research/experimentation of the present invention (see procedural steps of experimentation in Fig. 1).
- the corresponding full length CDS sequence of Gm WRKY49 was downloaded from www.phytozome.com, and respective sequence specific forward and reverse primers were designed.
- the corresponding polynucleotide sequences were amplified from the cDNA of soybean variety 'Market No. 1', and the amplification product was the sequence No 1 of the present invention.
- the specific steps comprised: extracting the total RNAs from the soybean variety 'Market No. 1' and synthesizing first-strand of cDNA by reverse transcription using reverse transcriptase kit (Fermentas Life Sciences) following fanufacturer's instructions.
- the total RNAs were extracted with TRIzol ® reagent (Invitrogen & Co.) after salt and/or drought-stress treatment following the TRIzol® reagent specifications.
- the cDNA obtained was used as the template for amplification, wherein the reaction conditions were: predenaturation at 94 °C for 3 min; 94°C for 30 s, 55°C for 30 s, 72°C for 1 min 45 s, 35 cycles; and elongation at 72°C for 6 mins.
- the amplified product of PCR was separated on 1.0% agarose gel.
- the product was gel extracted using gel extraction kit (Axygen Biosciences) and linked to pCXSN vector via TA-cloning.
- the linked product was transformed into E. coli strain DH5-alpha; and screening and sequencing of positive clones was done to obtain the desired DNA fragment comprising GmWRKY49 gene.
- the said pCXSN vector carries double tobacco mosaic virus promotor 35S with constitutive and over expression characteristics, and is mediated by Agrobacterium.
- the said polynucleotides were designated as pCXSN-GmWRKY49.
- the plasmid carrying overexpression cassette of GmWRKY49 gene was isolated using plasmid extraction kit (Axygen Biosciences) following manufacturer's instructions.
- the expression cassette was transformed into Agrobactarium following procedures well known in the art.
- soybean genetic transformation system mediated by Agrobacterium, it is introduced into the soybean variety 'Market No. 1'. Specific procedures include: soybean seeds were sown in the pot till emergance of its cotyledonous leaves; Agrobacterium carrying GmWRKY49 gene was grown in LB medium containing kanamycin at 28 ° C shaking @180 rpm, harvested by centrifugation at 5000 rpm for two mins at room temperature, the pellet was suspended gently in 10 mM MgCl 2 solution followed by two washings, the OD 600 of final suspension was adjusted to 0.6; Agrobactarium carrying GmWRKY49 gene was injected at the junction of cotyledonous leaves at cotyledonous leaf stage. The inject point was covered with soil. Water was applied as per requirement. The seedlings were grown in the greenhouse at 25°C, 12 h of photoperiod and around 30% humidity.
- Agrobacterium Culture (1) Agrobacterium was streaked and pre-cultured on plates containing LB culture medium with corresponding resistance at 28°C for 48 h (2 days); (2) The Agrobacterium was transferred to the above-mentioned suspension medium and cultured overnight in a shaking machine at 28°C shaking @180 rpm.
- transgenic seedlings carrying GmWRKY49 gene and empty pCXSN vector as control were treated with high salinity (200 mM) and/or drought (6000 PEG).
- soybean plants injected with overexpression cassette were up-rooted from the pots; seedlings having roots at the inject point were selected; all real roots were removed from the selected transgenic plants keeping only roots emerging at the inject point (juncture of two cotyledons); roots were immersed into 1/2 strength Hoagland (Hoagland and Arnon, 1950) culture solution contained in 100 mL glasstubes; after 03 days, only one root at the inject point was kept and rest were removed; after 01 week growth of seedlings in solution culture NaCl and/or PEG was added in the culture solution to establish a 200 mM NaCl and/or 6000 PEG stress; data were recorded after one week of stress. The culture solutions were changed/refreshed on alternate days. The experiment was repeated for 3 times.
- Morphometric data were recorded using following scale to be used at 3-5 node stage of soybean seedling: 1, Dead (whole plant wilted and no recovery possible); 2, Severe wilting (apical wilting + full leaf wilted and rolled); 3, Moderate wilting (apical normal + half-leaf wilted); 4, Low wilting (lower two leaves wilted only); 5, Normal (no symptom of stress).
- Fig. 4 showed the growth condition of overexpression transgenic plants when the control plants all died under the stress for 07 days. The result showed that GmWRKY49 transgenic plants could improve the tolerance of plants to high-salinity and/or drought stress.
- SEQ ID NO:1 in the sequence listing shows the polynucleotide sequence of DNA fragment which is isolated and cloned in the present invention and comprises an GmWRKY49 gene encoding region.
- SEQ ID NO:2 in the sequence listing shows the polypeptide sequence encoded by polynucleotides in the present invention.
- Pethe VV, Winicov I () 'Alfin1 a novel zinc-finger protein in alfalfa roots that binds to promoter elements in the salt-inducible MsPRP2 gene, ' Plant Mol Biol, 1998, pp. 1123-1135, vol. 38.
- Liao Y, Zou HF, Wei W, Hao YJ, Tian AG, et al. 'Soybean GmbZIP44, GmbZIP62 and GmbZIP78 genes function as negative regulator of ABA signaling and confer salt and freezing tolerance in transgenic Arabidopsis' Planta, 2008b,pp. 225-240 vol. 228.
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Abstract
The invention relates to GmWRKY49 transcriptional polypeptides, polynucleotides that encode them and provides methods of using polypeptides and polynucleotides capable of enhancing tolerance of a plant to salt and/or drought stress. Sequence information of the polynucleotides and polypeptides is also disclosed.
Description
This invention relates generally to
agro-biotechnology and plant molecular biology. In
particular, it relates to transgenic plants having
novel features, methods of producing such plants and
polynucleotides and polypeptides, methods of cloning and
gene expression to confer salt and/or drought
tolerance on plants and other organisms. More
specifically, the invention relates to the use of
GmWRKY polynucleotides and transgenic plants
expressing these polynucleotides and polypeptides.
Although salinity has become an
alarming problem of agriculture throughout the world,
the development of this menace is of greater magnitude
in arid and semi-arid areas. These areas are
characterized by hot and dry climate, and amount of
rainfall fluctuates markedly from year to year. Water
evaporation exceeds than precipitation in these areas of
the world, so these soils are inherently high in salts
and require more irrigation to be productive. Since
irrigation water contains salts and minerals, thus an
application of irrigation is also an application of
salts which further compounds the salinity problem.
Soybean is one of the most important cash
corps. The salt and/or drought tolerance is particularly
important for soybean. However, no transgenic salt and/or
drought tolerant soybean plant has been developed so far.
Thus, it is important to find out transcription factors
associated with salt and/or drought tolerance for growing
a soybean plant with tolerance to salt and/or drought and
thereby increasing its production.
Global climate change impacts high
to environmental stresses, which limit the
productivity of agricultural crops around the world.
Amongst the environmental stresses, the adverse
effects of soil salinity and/or drought on crop
production are more drastic. The salts near the soil
surface develop highly stressful conditions for
plant growth, and ultimately limit yield or
result in total plant death.
To adapt or tolerate stressful
environmental factors, plants receive
extracellular changes of environment and transfer
them into cells to induce expressions of some
responding genes via many pathways and
synthesize some functional proteins, osmoregulation
substances as well as transcription factors for
signal transmission and gene expression regulation
so that plants are able to make corresponding
responses to environmental changes and avoid
damages caused by high salt, drought, and/or low
temperature stresses. (Xiong et al, Cell
signaling during cold, drought and salt stress.
Plant Cell. 14 (suppl), S165-S183, 2002). Abiotic
stress inducible genes are classified into two
groups. The products of the first group include
effector proteins that help in cell membrane
system protection, water holding, controling ion
homeostasis etc. These proteins include
osmoprotectants, LEA, aquaporins, chaperones and
detoxification enzymes. The second group of gene
produce regulatory proteins involved in perception
of signal, signal transduction and
transcriptional regulation of gene expression. These
proteins include kinases, phoshoinositide
metabolisms' enzymes and transcription
factors. Several transcription factor families have
been found to be induced by salt and/or drought
stresses, such as DREB, ERF, WRKY, MYB, bZIP, and
NAC families (Hasegawa et al., 2000; He et al.,
2005; Seki et al., 2003; Zhu, 2002; Zhou et al.,
2008; Liao et al., 2008a, 2008b ). DREB1A and AtMYB2
improved the salt and drought tolerance of
transgenic plants when transferred into
Arabidopsis (Abe et al., 2003; Kasuga et al., 1999).
Alfin1, a PHD finger protein, was identified as
a salt-induced transcriptional factor and enhanced
the stress tolerance by ectopic expression in
transgenic plants (Bastola et al., 1998). These
transcriptional factors ultimately regulate the
expression of functional genes in response to
environmental stresses. When plants encounter
stresses, transcription factor as a controlling gene
is able to regulate the expression of a series
of downstream genes to enhance the tolerance of
plants to the stresses.
Ge et al (2010) utilized Affymetrix®
Soybean GeneChip® to conduct transcriptional
profiling on Glycine soja roots subjected
to 50 mmol/L NaHCO3 treatment. In a total
of 7088 probe sets, 3307 were up-regulated and
5720 were down-regulated at various time points.
The number of significantly stress regulated genes
increased dramatically after 3 h stress
treatment and peaked at 6 h. GO enrichment test
revealed that most of the differentially expressed
genes were involved in signal transduction,
energy, transcription, secondary metabolism,
transport, disease and defence response (Ge et
al. 2010).
For reclamation of salt affected
soils, chemical and engineering approaches were
adopted to leach the salts down in water table
but due to high establishment costs and escalating
energy prices, these measures were found to be
of limited use. The biological/genetic approach has
always been thought to be one of the effective and
cheaper means of tackling the problem of
salinity(Shannon, 1984; Hollington, 2000).
For a long time conventional
breeding approach has been practiced to overcome
the problem of salinity and/or drought but resulted
in release of only few cultivars, which are not
commercially very popular. Traditional or
conventional breeding based on selection and
crossing to introduce or enhance desired traits
has been the avenue of germplasm improvement
prior to the development of methods for genetic
engineering. Although conventional crop breeding
programs have improved yields for crops grown in
stressful environments, it is growing belief that
further increase will mostly be achieved through
targeted manipulation of genes involved in
stress tolerance. Genomics particularly functional
genomics makes important contributions to both
traditional and molecular methods of germplasm
improvement, and thus offer one of the cost
effective measures to tackle the problem of soil
salinity and/or drought.
One object of the present invention
is to provide an isolated polynucleotide capable
of giving a plant, preferably soybean, tolerance
to salt and/or drought stress, which comprises a
nucleotide sequence as shown in SEQ ID NO:1 or a
conservative variant or degenerate sequence
comprising one or more substitutions, deletions,
additions and/or insertions in the said
nucleotide sequence, or a sequence hybridizable with
the said sequence under moderate stringent
condition, or a complementary sequence thereof, or a
variant or derivative having at least 90% homology
and same or similar biological function to the
said nucleotide sequence.
Another object of the present
invention is to provide an expression vector
comprising the said polynucleotide sequence.
Another object of the present
invention is to provide a host cell transformed
or transfected by the said expression vector.
Another object of the present
invention is to provide a use of the said
polynucleotide sequence for increasing salt and/or
drought stress tolerance in plants, preferably soybean.
One aspect of the present
invention provides a method for determining
whether a test plant, for example a dicot, has been
exposed to at least one stress condition, for
example an abiotic stress, comprising
determining polynucleotide expression in the test
plant to produce an expression profile and
comparing the expression profile of the test plant
to the expression profile of at least one
reference plant that has been exposed to at
least one stress, for example an abiotic stress. In
one embodiment the expressed polynucleotides are
selected from the group consisting of the
polynucleotide sequences contained in the sequence
listing. In another embodiment, the test and
reference plants are soybean plants and the
expressed polynucleotides are selected from the
group consisting of SEQ ID No: 1 or a functional
portion thereof.
The object of the present
invention is to isolate a DNA fragment
comprising a complete encoding region of
transcription factor gene, to clone it, and to
use it for improvement of soybean or other plants to
salt and/or drought tolerance. The present invention
is based on the discovery by structure analysis
of the obtained gene that belongs to plant-specific
transcription factor WRKY family, and thus the said
transcription factor is named as GmWRKY49.
In the present invention, the term
'isolated polynucleotide capable of giving
a plant tolerance to salt and/or drought
stress' represents the polynucleotide sequence
as shown in SEQ ID NO:1 and further comprises
all variants or derivatives having at least 90%
homology and same or similar biological function
to the sequence as shown in SEQ ID NO:1.
The term 'isolated' means
'artificially changed from natural status
and/or separated/extracted/isolated from natural
environment'. Thus, if an 'isolated'
component or substance existing in nature is
'isolated', it has been changed or removed
from its initial environment or been subject to
both. For example, a polynucleotide or polypeptide
naturally existing in live animal is not
'isolated', but the same polynucleotide or
polypeptide separated or extracted from its natural
status is 'isolated', which is exactly
the term used herein.
The term
'polynucleotide(s)', as used herein,
means a single or double stranded polymer of
deoxyribonucleotide or ribonucleotide bases and
includes DNA both sense and anti-sense strands, and
corresponding RNA molecules, including HnRNA and
mRNA molecules, and comprehends cDNA, genomic
DNA and recombinant DNA, as well as wholly or
partially synthesized polynucleotides. An HnRNA
molecule contains introns and corresponds to a
DNA molecule in a generally one-to-one manner. An
mRNA molecule corresponds to an HnRNA and DNA
molecule from which the introns have been
excised. A polynucleotide may consist of an entire
gene, or any portion thereof. Operable
anti-sense polynucleotides may comprise a fragment
of the corresponding polynucleotide, and the
definition of 'polynucleotide' therefore
includes all such operable anti-sense fragments.
A nucleotide 'variant'
is a sequence that differs from the recited
nucleotide sequence in having one or more nucleotide
deletions, substitutions or additions. Such
modifications may be readily introduced using
standard mutagenesis techniques, such as
oligonucleotide-directed site-specific mutagenesis,
for example, by Adelman et al. (1983).
Nucleotide variants may be naturally occurring
allelic variants, or non-naturally occurring
variants. Variant nucleotide sequences preferably
exhibit at least about 70%, more preferably at least
about 80% and most preferably at least about 90%
homology (determined as described below) to the
recited sequence.
The term 'homology', in
other words 'identity', when used in
relation to nucleic acids refers to a degree of
complementarity either partial or complete
homology. 'Sequence identity' refers to a
measure of relatedness between two or more
nucleic acids, and is given as a percentage with
reference to the total comparison length. The
identity calculation takes into account those
nucleotide residues that are identical and in the
same relative positions in their respective
larger sequences. Calculations of identity may
be performed by algorithms contained within computer
programs such as 'GAP' (Genetics
Computer Group, Madison, Wis.) and 'ALIGN'
(DNAStar, Madison, Wis.). A partially
complementary sequence is one that at least
partially inhibits (or competes with) a completely
complementary sequence from hybridizing to a
target nucleic acid is referred to using the
functional term 'substantially
homologous'. The inhibition of hybridization of
the completely complementary sequence to the
target sequence may be examined using a
hybridization assay (Southern or Northern blot and
the like) under conditions of low stringency. A
substantially homologous sequence or probe will
compete for and inhibit the binding (in other
words, the hybridization) of a sequence which is
completely homologous to a target under conditions
of low stringency. This is not to say that
conditions of low stringency are such that
non-specific binding is permitted; low
stringency conditions require that the binding of
two sequences to one another be a specific (in
other words, selective) interaction. The absence
of non-specific binding may be tested by the use of
a second target which lacks even a partial
degree of complementarity (for example, less than
about 30% identity); in the absence of non-specific
binding the probe will not hybridize to the
second non-complementary target.
When used in reference to a
double-stranded nucleic acid sequence such as a
cDNA or genomic clone, the term 'substantially
homologous' refers to any probe which can
hybridize to either or both strands of the
double-stranded nucleic acid sequence under
conditions of low to high stringency as
described in the below.
Low stringency conditions in
reference to nucleic acid hybridization comprise
conditions equivalent to binding or hybridization
of 500 nucleotides long probe at 42 °C in a solution
consisting of 5×SSPE (43.8 g/l NaCl, 6.9 g/l
NaH2PO4 × H2O and
1.85 g /l EDTA, pH adjusted to 7.4 with NaOH),
0.1% SDS, 5×Denhardt's reagent
[50×Denhardt's contains per 500 ml: 5 g Ficoll
(Type 400, Pharmacia), 5 g BSA (Fraction V;
Sigma)] and 100 ug/ml denatured salmon sperm DNA
followed by washing in a solution comprising
5×SSPE, 0.1% SDS at 42 °C when a probe of about
500 nucleotides in length is employed.
High stringency conditions in
reference to nucleic acid hybridization comprise
conditions equivalent to binding or
hybridization of 500 nucleotides long probe at 42 °C
in a solution consisting of 5×SSPE (43.8 g/l
NaCl, 6.9 g/l NaH2PO4 ×
H2O and 1.85 g/l EDTA, pH adjusted to 7.4
with NaOH), 0.5% SDS, 5×Denhardt's reagent
and 100 ug/ml denatured salmon sperm DNA followed by
washing in a solution comprising 0.1×SSPE, 1.0% SDS
at 42 °C.
Numerous equivalent conditions may
be employed to comprise low stringency
conditions; factors such as the length, nature of
the probe and target (DNA, RNA, base
composition), concentration of the salts or
other components (for example, the presence or
absence of dextran sulfate formamide,
polyethylene glycol etc) and the hybridization
solution may be varied to generate conditions of
low stringency hybridization different from, but
equivalent to, the above listed conditions. In
addition, the conditions are well known in the
art that promote hybridization under conditions of
high stringency (for example, increasing
hybridization temperature and/or wash steps, use
of formamide in the hybridization solution, etc.).
When used in reference to a
single-stranded nucleic acid sequence, the term
'substantially homologous' refers to any
probe that can hybridize the single-stranded
nucleic acid sequence (in other words, it is the
complement of) under conditions of low to high
stringency as described above.
The term 'hybridization'
refers to the pairing of complementary
nucleotides (in other words nucleic acids).
Hybridization and its strength (in other words,
the strength of association/pairing between the
nucleic acids) is impacted by such factors like
degree of complementation between the nucleic
acids, stringency of the conditions involved, the T m of newly formed hybrid, and the G:C ratio
within the sequence of pairing nucleic acids.
Pairing of complementary nucleotides within its
structure of a single nucleic acid molecule is said
to be 'self-hybridized'.
The term 'T m ' refers to the 'melting
temperature' of a nucleic acid. The melting
temperature is the temperature at which a
population of double-stranded nucleic acid molecules
becomes half dissociated into single strands. The T m of nucleic acids is calculated using an
equation well known in the art. As indicated by
standard references, a simple estimate of the T m value may be calculated by the equation: T m =81.5+0.41 (% G+C), when a nucleic acid is
in aqueous solution at 1M NaCl (See for example,
Anderson and Young (1985). Other references include
more sophisticated computations that take
structural as well as sequence characteristics into
account for the calculation of T m .
As used herein the term
'stringency' refers to the conditions
of temperature, ionic strength, and the presence of
other compounds such as organic solvents etc,
under which nucleic acid hybridizations are
conducted. With 'high stringency'
conditions, nucleic acid base pairing will occur
only between nucleic acid fragments that have a high
frequency of complementary base sequences. Thus,
conditions of 'low' stringency are often
required with nucleic acids derived from genetically
divers organisms, as the frequency of
complementary sequences is usually less.
Preferably, the 'percentage
of sequence identity' is determined by
comparing two optimally aligned sequences over a
comparison window of at least 20 positions,
wherein the portion of the polynucleotide
sequence in the comparison window may comprise
additions or deletions (i.e., gaps) of 20
percent or less, usually 5 to 15 percent, or 10 to
12 percent, as compared to the reference
sequences (which does not comprise additions or
deletions) for optimal alignment of the two
sequences. The percentage is calculated by
determining the number of positions at which the
identical nucleic acid bases or amino acid
residue occurs to match in both sequences,
dividing by the total number of positions in the
reference sequence (i.e. window size) and
multiplying by 100.
In other words, for obtaining a
polynucleotide with a nucleotide sequence having
at least 95% identity to the reference
nucleotide sequence, up to 5% nucleotides with
reference to the total nucleotides of the
reference sequence could be deleted or substituted
or inserted or combination of deletion,
insertion and substitution by other nucleotides.
These mutations in the reference sequence could
occur at any position between and including 5-
or 3-terminal position of the reference
nucleotide sequence, and they exist in the reference
nucleotide sequence either in individual manner
or in one or more adjacent groups.
One aspect of the present
invention relates to an isolated polynucleotide
capable of giving a plant salt and/or drought stress
tolerance, which comprises a nucleotide sequence as
shown in SEQ ID NO:1 or a conservative variant
or degenerative sequence comprising one or more
substitutions, deletions, additions and/or
insertions into the said nucleotide sequence, or
a hybridizable sequence under moderate stringent
condition, or a complementary sequence thereof,
or a variant or derivative of the said
nucleotide sequence having at least 90% homology
having same or similar biological function.
In one embodiment of the present
invention, the said polynucleotide consists of
the DNA sequence given as SEQ ID NO:1.
The gene or homologous gene of the
present invention can be screened from cDNA and
genomic libraries by using a
polynucleotide-specific oligonucleotide primer/probe
such as the cloned GmWRKY49 gene. The GmWRKY49
gene of the present invention and any DNA fragment
of interest or DNA fragment homologous to it can
also be obtained/amplified from genome, mRNA and
cDNA by using PCR (polymerase chain reaction)
technology. A sequence comprising GmWRKY49 gene
can be isolated/obtained by using the above
techniques, and can be transferred into any
expression vector capable of carrying the gene
of interest into the plant and expression of the
gene of interest (an exogenous gene) thereof.
The transgenic plant, with enhanced salt and/or
drought tolerance, can be obtained via its
transformation with the said sequence and any
expression vector capable of inducing the expression
of an exogenous gene in the plant.
For example, PCR can be used for
amplifying the sequence from cDNA, wherein the
said cDNA is prepared via reverse transcriptase
(RT) PCR from the isolated RNA. A sequence-specific
oligonucleotide primer can be designed or
purchased or synthesized for this amplification
based on the sequence as shown in SEQ ID NO:1.
After PCR amplification, PCR product can be
separated by gel electrophoresis and detected by
methods well known by those skilled in the art.
The term 'sequence-specific
oligonucleotide primer/probe' refers to an
oligonucleotide sequence having at least 80%,
preferably at least 90%, more preferably at least
95% identity to the said polynucleotide, or to
the anti-sense oligonucleotide of the said
polynucleotide.
The very useful oligonucleotide
primer and/or probe in the present invention has
at least 10-40 nucleotides. In one preferable
embodiment, the oligonucleotide primer includes at
least about 10 consecutive nucleotides of the
said polynucleotide. Preferably, the
oligonucleotide used in the present invention
includes at least about 15 consecutive
nucleotides of the said polynucleotide. The
technologies based on PCR test and hybridization
in situ test are well known in the art.
Another aspect of the present
invention relates to an expression vector
comprising the said polynucleotide sequence. Any
strong or inducible promoter can be added before
starting nucleotide of the gene of the present
invention to construct/insert into a plant
expression vector. Enhancers can also be used
while constructing the gene of the present invention
into a plant expression vector, and these enhancer
regions can be ATG initiation codons, adjacent
region initiation codons, etc. The insertion of the
enhancers must be identical to the reading frame of
the encoding sequence in order to ensure the
translation of whole sequence.
The expression vector carrying the
GmWRKY49 gene of the present invention can be
introduced into plant or other living cells by
conventional biological methods such as Ti plasmid,
plant virus vector, microinjection, direct DNA
transformation, electroporation and the like
(Weissbach, 1998; Geiserson and Corey, 1998) .
The preferred plant of the present
invention is soybean. The plants of the present
invention also include but are not limited to:
cotton, soybean, corn, rice, barley, wheat,
Brassica, tomato, potato, tobacco, pepper,
Arabidopsis, sunflower, etc, also includes
non-agronomic species which are useful in developing
appropriate expression vectors such as tobacco,
rapid cycling Brassica species, and
Arabidopsis thaliana .
Methods which are well known to
those skilled in the art may be used to
construct expression vectors. These methods include
in vitro recombinant DNA techniques, synthetic
techniques, and in vivo genetic recombination.
Such techniques are widely described in the art (See
for example, Sambrook. et al (1989).
Generally, these vectors comprise
the above mentioned polynucleotide sequence of
the invention operably linked to a promoter,
other regulatory sequences (for example, enhancers,
polyadenylation signals, etc.) required for
expression in a plant and some suitable selection
marker(s) for screening/identification of the
expression vector carrying the said
polynucleotide sequence of the present invention.
Within a recombinant expression vector,
'operably-linked' is intended to mean that
the nucleotide sequence of interest is linked to
the regulatory sequence in a manner that allows
for expression of the nucleotide sequence (e.g., in
an in vitro transcription/translation system or
in a host cell when the vector is introduced
into the host cell).
For expression in plants, the
recombinant expression cassette will contain in
addition to a GmWRKY49 polynucleotide, a
promoter functional in a plant cell, a transcription
initiation site (if the coding sequence to be
transcribed lacks one), and a transcription
termination/polyadenylation sequence(See for
example, Odell et al. (1985); Rosenberg et al.
(1987); Guerineau et al. (1991). The
termination/polyadenylation region may be obtained
from the same gene as the promoter sequence or
may be obtained from different genes. Unique
restriction enzyme sites at the 5' and
3' ends of the cassette are typically included
to allow for easy insertion into a pre-existing
vector. Promoters used in the present invention
include but are not limited to constitutive
promoters, tissue-, organ-, and
developmentally-specific promoters, and inducible
promoters. Examples of promoters include but are not
limited to: constitutive promoter 35S of
cauliflower mosaic virus (Odell, et al., 1985); a
wound-inducible promoter from tomato, leucine amino
peptidase 'LAP', (Chao et al., 1999);
a chemically-inducible promoter from tobacco,
Pathogenesis-Related 1 (PR1) (induced by
salicylic acid and BTH
(benzothiadiazole-7-carbothioic acid S-methyl
ester)); a tomato proteinase inhibitor II promoter
(PIN2) or LAP promoter (both inducible with
methyl jasmonate); a heat shock promoter (U.S.
Pat. No. 5,187,267); a tetracycline-inducible
promoter (U.S. Pat. No. 5,057,422); and
seed-specific promoters, such as those for seed
storage proteins (for example, phaseolin, napin,
oleosin, and a promoter for soybean beta
conglycin (Beachy et al. (1985). All references
cited herein are incorporated in their entirety.
Numerous expression/transformation
vectors are available for plant transformation.
The selection of a suitable vector will depend
upon the transformation technique to be used and the
target species for transformation. For certain
target species, different antibiotic or herbicide
selection markers are preferred. Selection markers
used routinely in transformation include the
nptII gene which confers resistance to kanamycin and
related antibiotics (Bevan et al. (1983), the bar
gene which confers resistance to the herbicide
phosphinothricin (White et al. (1990); Spencer et
al. (1990), the hph gene which confers
resistance to the antibiotic hygromycin (Blochlinger
et al., 1984), etc.
In some preferred embodiments, the
vector is adapted for use in an Agrobacterium
mediated transfection process (See for example,
U.S. Pat. Nos. 5,981,839; 5,981,840; and 6,051,757;
all of which are incorporated herein by
reference).
Two systems of recombinant Ti and
Ri plasmid vector systems are now in use. The
first system is called the 'cointegrate'
system having the shuttle vector which contains the
gene of interest inserted by genetic
recombination into a non-oncogenic Ti plasmid that
contains both the cis- and trans-acting elements
required for plant transformation. For example,
in the pMLJI shuttle vector and the non-oncogenic Ti
plasmid pGV3850. The second system is called the
'binary' system. In this system two
plasmids are used; the gene of interest is
inserted into a shuttle vector containing the
cis-acting elements required for plant
transformation while other necessary functions
are provided in trans by the non-oncogenic Ti
plasmid. For example, the pBIN19 shuttle vector
and the non-oncogenic Ti plasmid PAL4404. Some of
these vectors are commercially available.
In some embodiments, useful
vectors having polynucleotide sequence of the
present invention are microinjected directly
into plant cells. In other embodiments, the vector
is transferred into the plant cell by using
polyethylene glycol (PEG) (Krens et al.,1982;
Crossway et al.,1986); protoplasts fusion with
other entities, either minicells, cells,
lysosomes or other fusible lipid-surfaced bodies
(Fraley et al. (1982); protoplast transformation
(EP 0 292 435); direct gene transfer (Paszkowski et
al., 1984; Hayashimoto et al., 1990).
In further embodiments, the vector
may also be introduced into the plant cells by
electroporation. (Riggs et al. (1986). Plant
protoplasts are electroporated in the presence of
plasmids containing the gene construct via
electrical impulses of high field strength. This
technique reversibly permeabilize biomembranes
allowing the introduction of the plasmids.
Electroporated plant protoplasts reform the cell
wall, divide, and form plant callus.
In still further embodiments, the
vector is introduced through ballistic particle
acceleration devices (for example, available
from Agracetus, Inc., Madison, Wis. and Dupont,
Inc., Wilmington, Del.) (See for example, U.S.
Pat. No. 4,945,050; and McCabe et al., 1988;
Christou et al., 1990 (soybean); Sanford et al.,
1987 (onion); Klein et al., 1988; Koziel et al.,
1993 (maize); Hill et al., 1995 (rice); Vasil et
al., 1993 (wheat); Wan et al., 1994 (barley);
Umbeck et al., 1987 (cotton); Casas et al., 1993
(sorghum); Somers et al., 1992 (oat).
In yet some embodiments, in
addition to direct transformation, the vectors
comprising a nucleic acid sequence of the present
invention are transferred via Agrobacterium-mediated
transformation (Hinchee et al., 1988; Ishida et
al., 1996). Agrobacterium is a gram-negative genus
of the Rhizomaceae. The species of Agrobacterium
are responsible for development of plant tumors
such as crown gall and hairy root disease. In the
dedifferentiated tissue characteristic of the
tumors, opines (amino acid derivatives) are
produced and catabolized. The bacterial genes
responsible for expression of opines are a
convenient source of control elements for chimeric
expression cassettes. Heterologous genetic
sequences (for example, nucleic acid sequences
of the present invention operatively linked to a
promoter), can be introduced into appropriate
plant cells, by means of the Ti plasmid of
Agrobacterium tumefaciens. The Ti
plasmid is transmitted to plant cells on infection
by Agrobacterium tumefaciens, and is
stably integrated into the plant genome (Schell,
1987).
Figure. 1: Procedural steps involved in
identification, isolation, cloning and expression
analysis of GmWRKY49 gene.
Figure. 2: The GmWRKY49 gene expression
levels detected by digital gene expression profiling
(DGEP) under normal, salt and drought stress in
cultivated and wild soybeans.
Figure. 3: The expression cassette
containing the gene of interest of present invention.
Figure. 4: The overexpression of
GmWRKY49 gene in transgenic soybean plants and the
growth of transgenic seedlings (5 week old) after 07
days of high salinity (200 mM NaCl) stress.
During the initial research period of
the present invention, the expression of tags from
said GmWRKY49 gene increased in specific environments
of 200 mM NaCl and/or 6000 PEG within Glycine max
and Glycine soja except in 6000 PEG where its
expression was significantly decresed in Glycine
max. Expression level of said GmWRKY49
transcriptional gene was greater in Glycine soja
(believed to be salt tolerant wild soybean accession
collected from Chinese coast of Yellow Sea). The cDNA
clones corresponding to the respective tags are from
soybean cultivar Market No. 1 (believed to be salt
sensitive). The said polynucleotides are the cDNA
fragment of GmWRKY49 gene. The inventors of the present
invention found that this is new stress-associated
regulatory gene.
Specifically, (1) it was found by
digital gene expression profiling (DGEP) using Taq
sequencing technique that the expression amount of the
said polynucleotides in the soybean variety 'Market
No. 1' (Glycine max) for GmWRKY49
increased 1.4 folds after 200m MNaCl stress while
decreased 4.4 folds after 6000 PEG drought stress
treatment. The expression level of the said
polynucleotides was significantly high (2.1 and 1.0
folds after salt and drought stress respectively) in
accession of Glycine soja collected from Sea
coast (Fig. 2). Due to the significant difference in
magnitude of expression before and after stress
treatment, in sensitive and tolerant accessions, and
the functional characteristics of the polynucleotides,
it is deemed that the said polynucleotides participated
in the regulation of expression of genes under salt
and/or drought stress; and (2) the overexpression of
the intact genes in transgenic plants exhibited a
significant increase in high salt and/or drought
tolerance (Fig. 4).
The above results show that said
GmWRKY49 gene is stress-associated regulatory gene and
participate in the regulation of high salt and/or
drought tolerance.
The present invention is further
demonstrated with examples in combination with
figures, and described methods for isolating and
cloning the DNA fragment comprising the whole encoding
region of the said GmWRKY49 gene and for verifying its
function, based on the initial
research/experimentation of the present invention (see
procedural steps of experimentation in Fig. 1).
A skilled in the art can determine the
basic technical features of the present invention
according to the following description and examples,
and can further make any change and modification to
the present invention without leaving the spirit and
scope of the present invention in order to adapt to
various uses and conditions.
According to the DGEP strongly salt
and/or drought inducible tags were found, and upon
blast nr these tags were found to be the members of
GmWRKY transcriptional gene family.
The corresponding full length CDS
sequence of GmWRKY49 was downloaded from
www.phytozome.com, and respective sequence specific
forward and reverse primers were designed. The
corresponding polynucleotide sequences were amplified
from the cDNA of soybean variety 'Market No.
1', and the amplification product was the sequence
No 1 of the present invention.
The specific steps comprised:
extracting the total RNAs from the soybean variety
'Market No. 1' and synthesizing first-strand
of cDNA by reverse transcription using reverse
transcriptase kit (Fermentas Life Sciences) following
fanufacturer's instructions. The total RNAs were
extracted with TRIzol ® reagent (Invitrogen & Co.)
after salt and/or drought-stress treatment following
the TRIzol® reagent specifications. The cDNA obtained
was used as the template for amplification, wherein
the reaction conditions were: predenaturation at 94 °C
for 3 min; 94°C for 30 s, 55°C for 30 s, 72°C for 1
min 45 s, 35 cycles; and elongation at 72°C for 6 mins.
The amplified product of PCR was separated on 1.0%
agarose gel. The product was gel extracted using gel
extraction kit (Axygen Biosciences) and linked to pCXSN
vector via TA-cloning. The linked product was
transformed into E. coli strain DH5-alpha; and
screening and sequencing of positive clones was done to
obtain the desired DNA fragment comprising GmWRKY49
gene. The said pCXSN vector carries double tobacco
mosaic virus promotor 35S with constitutive and over
expression characteristics, and is mediated by
Agrobacterium. The said polynucleotides were
designated as pCXSN-GmWRKY49.
In order to illustrate the function
of the said gene, it was overexpressed in soybean
seedlings and verified by the phenotype of transgenic
plants.
The plasmid carrying overexpression
cassette of GmWRKY49 gene was isolated using plasmid
extraction kit (Axygen Biosciences) following
manufacturer's instructions. The expression
cassette was transformed into Agrobactarium following
procedures well known in the art.
By using the soybean genetic
transformation system mediated by Agrobacterium, it is
introduced into the soybean variety 'Market No.
1'. Specific procedures include: soybean seeds were
sown in the pot till emergance of its cotyledonous
leaves; Agrobacterium carrying GmWRKY49 gene was grown
in LB medium containing kanamycin at 28 ° C shaking @180
rpm, harvested by centrifugation at 5000 rpm for two
mins at room temperature, the pellet was suspended
gently in 10 mM MgCl2 solution followed by
two washings, the OD600 of final suspension
was adjusted to 0.6; Agrobactarium carrying GmWRKY49
gene was injected at the junction of cotyledonous leaves
at cotyledonous leaf stage. The inject point was
covered with soil. Water was applied as per
requirement. The seedlings were grown in the greenhouse
at 25°C, 12 h of photoperiod and around 30% humidity.
Agrobacterium Culture (1)
Agrobacterium was streaked and pre-cultured on plates
containing LB culture medium with corresponding
resistance at 28°C for 48 h (2 days); (2) The
Agrobacterium was transferred to the above-mentioned
suspension medium and cultured overnight in a shaking
machine at 28°C shaking @180 rpm.
In order to verify whether the salt
and/or drought tolerance of transgenic soybean is
related to GmWRKY49 gene, transgenic seedlings
carrying GmWRKY49 gene and empty pCXSN vector as control
were treated with high salinity (200 mM) and/or
drought (6000 PEG). The specific methods were as
follows: soybean plants injected with overexpression
cassette were up-rooted from the pots; seedlings
having roots at the inject point were selected; all
real roots were removed from the selected transgenic
plants keeping only roots emerging at the inject point
(juncture of two cotyledons); roots were immersed into
1/2 strength Hoagland (Hoagland and Arnon, 1950)
culture solution contained in 100 mL glasstubes; after
03 days, only one root at the inject point was kept
and rest were removed; after 01 week growth of
seedlings in solution culture NaCl and/or PEG was added
in the culture solution to establish a 200 mM NaCl
and/or 6000 PEG stress; data were recorded after one
week of stress. The culture solutions were
changed/refreshed on alternate days. The experiment
was repeated for 3 times.
Morphometric data were recorded using
following scale to be used at 3-5 node stage of
soybean seedling: 1, Dead (whole plant wilted and no
recovery possible); 2, Severe wilting (apical wilting +
full leaf wilted and rolled); 3, Moderate wilting
(apical normal + half-leaf wilted); 4, Low wilting
(lower two leaves wilted only); 5, Normal (no symptom of
stress). Fig. 4 showed the growth condition of
overexpression transgenic plants when the control
plants all died under the stress for 07 days. The result
showed that GmWRKY49 transgenic plants could improve
the tolerance of plants to high-salinity and/or
drought stress.
In order to verify whether the
healthy plants are transgenic and contain the
corresponding GmWRKY49 gene, respective gene's
amplification through PCR was adapted using genomic DNA
as template. Only transgenic plants showed
corresponding gene's amplification compared to
control plants (Fig 4).
SEQ ID NO:1 in the sequence listing
shows the polynucleotide sequence of DNA fragment
which is isolated and cloned in the present invention
and comprises an GmWRKY49 gene encoding region.
SEQ ID NO:2 in the sequence listing
shows the polypeptide sequence encoded by
polynucleotides in the present invention.
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Claims (8)
- An expression vector comprising a promoter operably linked to a polynucleotide consisting of the nucleotide sequence as shown in SEQ ID NO:1 or a variant thereof having at least 90% homology to SEQ ID NO:1, wherein SEQ ID NO:1 encode transcription factor providing salt and/or drought stress tolerance in a plant.
- A host cell transformed or transfected by the expression vector according to claim 1, wherein said host cell is a prokaryotic cell or a eukaryotic cell.
- A method for increasing salt and/or drought tolerance in a plant, the method comprising the steps of introducing into a plant a polynucleotide comprising the nucleotide sequence as shown in SEQ ID NO:1 or a variant thereof having at least 90% homology to SEQ ID NO:1, wherein SEQ ID NO:1 encodes transcription factor providing tolerance to drought and/or salt stress in a plant.
- The method according to claim 4, wherein the plant is selected from the monocot or dicot group.
- An isolated polypeptide comprising amino acid sequences selected from the group consisting of SEQ IDNO: 2 or a variant thereof having at least 95% homology to SEQ ID NO: 2.
- A salt and/or drought tolerant transgenic plant produced by the method of claim 3.
- A transgenic seed produced by the transgenic plant of claim 6, wherein said seed produces a salt and/or drought tolerant plant.
- A method of identifying salt and/or drought tolerant plant, wherein the plant comprising transgenic root(s) having green leaves as compared to a control plant after exposure to salt and/or drought stress.
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CN108424438A (en) * | 2018-05-17 | 2018-08-21 | 青岛大学 | A kind of wheat powdery mildew resistance-associated protein TaWRKY49 and its encoding gene and application |
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Cited By (4)
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CN106967729A (en) * | 2017-04-16 | 2017-07-21 | 陈帅 | Application of the WRKY transcription factors in resistant transgenic sweet orange is prepared |
CN106967729B (en) * | 2017-04-16 | 2020-11-06 | 章驰 | Application of WRKY transcription factor in preparation of stress-resistant transgenic sweet orange |
CN108424438A (en) * | 2018-05-17 | 2018-08-21 | 青岛大学 | A kind of wheat powdery mildew resistance-associated protein TaWRKY49 and its encoding gene and application |
CN108424438B (en) * | 2018-05-17 | 2021-06-25 | 青岛大学 | Wheat powdery mildew resistance-related protein TaWRKY49, and coding gene and application thereof |
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