WO2012168763A1 - Gm bzip110 transcriptional gene and use thereof for enhancing plant tolerance to salt and/or drought - Google Patents

Abstract

GmbZIP 110 transcriptional polypeptides, polynucleotides that encode the polypeptides, and methods for enhancing tolerance of a plant to salt and/or drought stress using said polypeptides and polynucleotides are provided. Sequence information of the polynucleotides and polypeptides is also provided.

Description

Gm bZIP110 TRANSCRIPTIONAL GENE AND USE THEREOF FOR ENHANCING PLANT TOLERANCE TO SALT AND/OR DROUGHT Technical Field

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 GmbZIP polynucleotides and transgenic plants expressing these polynucleotides and polypeptides.

Background Art

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.

Technical Problem

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.

Technical Solution

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).

Advantageous Effects

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.

Summary of the Invention

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.

Detailed Description of the Invention

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 bZIP family, and thus the said transcription factor is named as GmbZIP110.

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 GmbZIP110 gene. The GmbZIP110 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 GmbZIP110 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 GmbZIP110 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 GmbZIP110 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).

Description of Drawings

Figure. 1: Procedural steps involved in identification, isolation, cloning and expression analysis of GmbZIP110 gene.

Figure. 2: The GmbZIP110 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 GmbZIP110 gene in transgenic soybean plants and the growth of transgenic seedlings (5 week old) after 07 days of high salinity (200 mM NaCl) stress.

Best Mode Examples

During the initial research period of the present invention, the expression of tags from said GmbZIP110 gene increased in specific environments of 200 mM NaCl and/or 6000 PEG within Glycine soja and didn't differentially expressed in Glycine max. Expression level of said GmbZIP110 transcriptional gene was greater in Glycine soja (believed to be salt tolerant wild soybean accession collected from Chinese coast of Yellow Sea). The clones corresponding to the respective tags are from soybean cultivar Market No. 1 (believed to be salt sensitive). The said polynucleotides are the DNA/cDNA fragment of GmbZIP110 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 level of the said polynucleotides was significantly high (1.8 and 0.7 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 tolerant accession, 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 GmbZIP110 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 GmbZIP110 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.

Mode for Invention Example 1 Isolation and Cloning GmbZIP110 Gene

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 GmbZIP transcriptional gene family.

The corresponding full length CDS sequence of GmbZIP110 was downloaded from www.phytozome.com, and respective sequence specific forward and reverse primers were designed. The corresponding polynucleotide sequences were amplified from the DNA/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 said polynucleotide was also amplified using genomic DNA extracted using CTAB protcol of DNA extraction, which is well known in the art - The gene GmbZIP110 consists of a single exon. 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 GmbZIP110 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-GmbZIP110.

Example 2 Transformation of GmbZIP110 Genes' Overexpression Cassette

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 GmbZIP110 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 was 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 GmbZIP110 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 GmbZIP110 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.

Example 3 Salt and/or Drought Screening of Transgenic Seedlings of GmbZIP110 Gene

In order to verify whether the salt and/or drought tolerance of transgenic soybean is related to GmbZIP110 gene, transgenic seedlings carrying GmbZIP110 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 GmbZIP110 transgenic plants could improve the tolerance of plants to high-salinity and/or drought stress.

Example 4 Verification of the Transgenic Plants Carrying GmbZIP110 gene

In order to verify whether the healthy plants are transgenic and contain the corresponding GmbZIP110 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).

Sequence List Text

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 GmbZIP110 gene encoding region.

SEQ ID NO:2 in the sequence listing shows the polypeptide sequence encoded by polynucleotides in the present invention.

References

Patents

US Patents
4945050	July 1990	Sanford et al.
5187267	February 1993	Comai et al.
5057422	October 1991	Bol et al.
5981839	November 1999	Knauf et al.
5981840	November 1999	Zhao et al.
6051757	April 2000	Barton et al.
EP Patents
0 292 435	May., 1988

Non-patents

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Claims (8)

1. An expression vector comprising a promoter operably linked to a polynucleotide consisting of the nucleotide sequence as shown in SEQ ID NO:1 or complementary nucleotide sequence of SEQ ID NO:1 or a variant thereof having at least 90% homology to SEQ ID NO:1 or its complementary sequence, wherein SEQ ID NO:1 or its complementary nucleotide sequence encodes transcription factor providing salt and/or drought stress tolerance in a plant.
2. 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.
3. 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 complementary nucleotide sequence of SEQ ID NO:1 or a variant thereof having at least 90% homology to SEQ ID NO:1 or its complementary nucleotide sequence, wherein SEQ ID NO:1 or its complementary nucleotide sequence encodes transcription factor providing tolerance to drought and/or salt stress in a plant.
4. The method according to claim 3, wherein the plant is selected from the monocot or dicot group.
5. 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.
6. A salt and/or drought tolerant transgenic plant produced by the method of claim 3.
7. A transgenic seed produced by the transgenic plant of claim 6, wherein said seed produces a salt and/or drought tolerant plant.
8. 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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