Field of the invention :-

The present invention relates to a hybrid-type histidine kinase gene isolated from indica rice IR64, and clones produced thereby.

In particular, the present invention relates to a hybrid-type histidine kinase gene isolated from indica rice IR64 which has been found to be capable of osmosensing and inducible by multiple stresses, and found to be capable of improving the multiple stress tolerance of the crop plants in such a manner that the crop plants thus produced are capable of coping-up with the more than one environmental abiotic stress conditions, and thereby the crop plants have been found to have increased economic value while maintaining the yield thereof.

The present invention also relates to a method of isolation of hybrid-type histidine kinase gene from indica rice IR64, and its sequence listing and its method of cloning, at least, with yeast expression vector and plant expression vector.

The present invention also relates to clones produced by cloning of hybrid- type histidine kinase gene isolated from indica rice IR64 with yeast expression vector and plant expression vector.

The present invention also relates to a method of improving multiple stress tolerance of crop plants by employing hybrid-type histidine kinase gene isolated from indica rice IR64, and crop plants having improved multiple stress tolerance.

The present invention also relates to use of hybrid-type histidine kinase gene isolated from indica rice IR64 improving multiple stress tolerance of crop plants. The present invention also relates to functional characteristics of hybrid-type histidine kinase gene isolated from indica rice IR64.

Background of the invention:-

Rice is an important staple crop for human consumption at the global level.

The genome size of rice is about 430 Mb, which is comparatively smaller than various other cereal crops, which varies from about 430 Mb to about 17000 Mb, for example the genome size of the bread wheat is about 16,900 Mb, maize is about 2,600 Mb, sorghum is about 735 Mb, which makes rice the ideal model cereal crop.

It is observed that the environmental stresses are caused either due to biotic factors, for example the virus, bacteria, fungus, insects or nematodes; or due to abiotic factors, for example the extremes of water availability, that is, flooding, and/or extremes of saline condition, that is, higher salinity, and/or extreme shortage of water, that is, drought, and/or extremes of temperature, that is, the temperature being either very high or very low. Collectively these factors affect the potential yield of the crop plants. Further, the changing climatic conditions and global warming have been perceived as potential threats to food self-sufficiency. Owing to the impressive progress made in understanding the plant-pathogen interactions, transgenic plants which are able to survive better under insect attack, which is biotic factor, are now a reality. The common examples of such varieties are Bt cotton and Bt brinjal.

However, the crop plants which can withstand the abiotic stresses are yet to be made available, primarily because abiotic stresses are multigenic in nature.

Therefore, there is an urgent need to modify crop plants so that they become capable of surviving and maintaining a high yield under extreme environmental abiotic stress conditions, and thus, resulting in stress tolerant plants, that is, there is an urgent need to improve multiple stress tolerance of crop plants so that these can overcome problems caused by environmental stresses.

One of the possible ways by which the stress tolerance capability of crops can be enhanced is through genetic engineering where one or more genes are engineered to give the desired trait. With these objectives in mind, several plants have been modified in recent past. One such example involves genes which can synthesize osmolytes to maintain the water potential of the cell under osmotic stress conditions. Other categories include the modification of genes, which are capable of selectively pumping the toxic sodium ions into the vacuole, and thus are capable to keep the cytoplasm free of toxicity.

However, neither much is understood nor much has been made available as far as the sensing machinery associated with the osmotic stresses is concerned.

In lower organisms, for example the yeast, osmosensing is far better understood, and therefore, functional complementation of the yeast osmosensitive mutants with the target gene from crop plants would be a possible way to functionally validate novel genes for similar functions. Genetic modification of crop plants with respect to their osmosensing machinery would be highly desirable since it would result in enhanced tolerance towards osmotic stresses.

Several attempts have been made for the transgenic plants where performance has been enhanced under a specific stress, say salinity or drought or any other abiotic stress. These transgenic plants so far have been utilizing genes which are induced under a single specific stress.

However, no transgenic plant so far has been made available which utilizes the gene which is induced under multiple stresses, and hence, such genes are highly desirable and advantageous, and therefore, the need of the present time is to have a gene which can withstand multiple stresses so that the plant utilizing such gene can withstand multiple stresses while maintaining its potential yield.

Need of the Invention:-

Therefore, there is an urgent need to have a gene which is capable of osmosensing and is inducible by multiple stresses, and capable of improving the multiple stress tolerance of the crop plants, particularly of the transgenic plants so as to make the plants capable of coping-up with the more than one environmental abiotic stress conditions, and thereby, increasing their economic value while maintaining the yield thereof, and of a method of isolation thereof, and of sequence listing thereof, and of method of cloning of such gene, at least, with yeast expression vector and plant expression vector, and of clones produced thereby on cloning with yeast expression vector and plant expression vector. There is also an urgent need to have a method of improving multiple stress tolerance of crop plants, and crop plants having improved multiple stress tolerance. Further, there is also an urgent need to have functional characteristics of such gene.

Problems to be solved by the invention:-

The present invention, therefore, aims to solve problems of poor stress tolerance of crop plants by providing a gene which should be capable of osmosensing and inducible by multiple stresses, and capable of improving the multiple stress tolerance of crop plants, particularly of transgenic plants so as to make the plants capable of being coping-up with more than one environmental abiotic stress conditions, and method of isolation of such gene from indica rice IR64, and method of improving multiple stress tolerance of crop plants.

Objects of the invention:-

Accordingly, the main object of the present invention is to provide a hybrid- type histidine kinase gene which is isolated from indica rice IR64, and is capable of osmosensing and inducible by multiple stresses, and therefore, is capable of improving the multiple stress tolerance of the crop plants so as to make the plants capable of coping-up with the more than one environmental abiotic stress conditions, and hence, increasing the economic value of the crop plants while still maintaining the potential yield thereof.

This is also an object of the present invention to provide a method of isolation of hybrid-type histidine kinase gene which is isolated from indica rice IR64.

This is also an object of the present invention to provide a sequence listing of the hybrid-type histidine kinase gene which is isolated from indica rice IR64. This is also an object of the present invention to provide method of cloning the hybrid-type histidine kinase gene which is isolated from indica rice IR64, at least, with yeast expression vector and the plant expression vector.

This is also an object of the present invention to provide clones produced by cloning of hybrid-type histidine kinase gene isolated from indica rice IR64 with yeast expression vector and the plant expression vector.

This is also an object of the present invention to provide functional characteristics of the hybrid-type histidine kinase gene which is isolated from indica rice IR64.

This is also an object of the present invention to provide a method for improving multiple stress tolerance of crop plants by employing hybrid-type histidine kinase gene isolated from indica rice IR64.

This is also an object of the present invention to provide crop plants having improved multiple stress tolerance.

This is also an object of the present invention to provide use of hybrid-type histidine kinase gene isolated from indica rice IR64 to improve multiple stress tolerance of crop plants.

The other objects and advantages will be more apparent from the following description and nature of the invention when it is read in conjunction with the accompanying figures, which are not intended to limit its scope.

Brief Description of the Accompanying Figures of the invention:-

Figure IA illustrates primer sequences used for the isolation of gene from indica rice IR64 in accordance with the most preferred embodiment of the present invention. Figure IB illustrates various conditions for polymerase chain reaction (PCR) for the isolation of the gene from indica rice IR64 and cloning thereof in accordance with the most preferred embodiment of the present invention.

Figure 1C illustrates agarose gel electrophoresis of polymerase chain reaction

(PCR) product showing an amplification product of a size of about 2.6Kb, more precisely of size of about 2598 bp of the gene isolated from indica rice IR64 in accordance with the most preferred embodiment of the present invention.

Figure 2 A illustrates the complete Gene sequence of open reading frame

[ORF] of OsHkSb in accordance with the most preferred embodiment of the present invention.

Figure 2B illustrates the complete deduced amino acid sequence for protein encoded by ORF of OsHk3b in accordance with the most preferred embodiment of the present invention.

Figure 3 A illustrates confirmation of cloning of gene of the present invention into the pYES2 vector in accordance with the most preferred embodiment of the present invention.

Figure 3B illustrates confirmation of cloning of gene of the present invention into the pCAMBIA1304 vector in accordance with the most preferred embodiment of the present invention.

Figure 4 illustrates functional complementation of yeast SLNl -temperature dependent osmosensitive mutant HS 13 by the rice OsHKSb ORF in accordance with the most preferred embodiment of the present invention.

Figure 5 illustrates the histogram showing the inducibility of OsHK3b by various abiotic stresses in rice in accordance with the most preferred embodiment of the present invention. Figure 6 Oryza sativa cv IR64 transformation and regeneration - illustrates seeds of rice IR-64 step - [a]; rice callus formation on callus induction media step - [b]; sub-cultured rice callus kept on callus induction media for about 5-7 days step - [c]; co-infection of rice calli with Agrobacterium strain LBA4404 containing OsHK3b construct step - [d]; co-cultivation of co-infected rice calli step - [e]; washing of overgrown Agrobacterium with antibiotic cefotaxim step - [fj; transfer of washed callus on selection plate step - [g]; transformed callus kept on regeneration medium step - [h]; regenerated plantlets as transferred on fresh regeneration medium step - [i]; completely regenerated plant as transferred into culture tubes for hardening step - [J]; transgenic plant from step - [j] is further transferred into Earthen pot and kept in green house step - [k] in accordance with the most preferred embodiment of the present invention.

Figure 7 A is illustrative of confirmation of multiple stress tolerance of leaves of plant grown in Figure 6 by leaf disc assay showing the bleaching rate of different leaf samples under control and salinity stress conditions in accordance with the most preferred embodiment of the present invention.

Figure 7B illustrates total chlorophyll content of the leaves of plant grown in Figure 6 in accordance with the most preferred embodiment of the present invention.

Figure 8 is illustrative of confirmation of successful integration of OsHK3b gene of present invention into genome of IR64 transgenic plants using plasmid - pCAMBIA1304 in accordance with most preferred embodiment of the present invention.

Figure 9 is illustrative of confirmation of multiple stress tolerance Of T ₁ plants, in terms of seed germination under saline conditions, grown from seeds of plants grown in Figure 6 in accordance with the most preferred embodiment of the present invention.

Figure 10 is illustrative of comparative confirmation of multiple stress tolerance of Ti plants grown for 7 days under normal conditions, and thereafter, grown for another 10 days under saline conditions by measuring its chlorophyll contents.

The detailed legends of the Figures have been provided at the end of this description.

Description and Preferred Embodiments of the Invention:-

It is now understood that prior art neither discloses nor teaches that hybrid- type histidine kinase gene having capability of osmosensing and inducible by multiple stresses, and capability of improving the multiple stress tolerance of crop plants, particularly of transgenic plants can be isolated from indica rice IR64.

Further, the prior art neither discloses nor teaches that hybrid-type histidine kinase gene isolated from indica rice IR64 can be cloned, at least, with yeast expression vector and the plant expression vector.

Further, the prior art neither discloses nor teaches that crop plants can be improved for their multiple stress tolerance by employing such isolated gene so as to make the plants capable of being coping-up with more than one environmental abiotic stress conditions to solve problems of poor stress tolerance of crop plants.

With aim to isolate a hybrid-type histidine kinase gene from indica rice IR64, the inventors have surprisingly found that if cDNA isolated from IR64 seedling is treated by polymerase chain reaction (PCR) with forward primer OsHk3bF and reverse primer OsHk3bR, wherein step of annealing is carried out at about 55 C, then hybrid-type histidine kinase gene gets isolated, and the isolated gene has been surprisingly found to have capability of osmosensing and inducible by multiple stresses, and capability of improving the multiple stress tolerance of crop plants, particularly of transgenic plants .

With aim to clone hybrid-type histidine kinase gene isolated from indica rice IR64, at least, with yeast expression vector and the plant expression vector, the inventors have surprisingly found that isolated gene upon amplifying by treatment with forward primer OsHk3bφe/F and reverse primer OsHk3bSpelR by employing polymerase chain reaction (PCR), the gene of present invention gets cloned into yeast expression vector and also into plant expression vector.

With aim to improve multiple stress tolerance of crop plants, the inventors have surprisingly found that when calli grown from seeds of IR64 are co-infected with a medium containing cloned gene, the IR64 gets transformed and grows to a complete transgenic IR64 plant having overexpression of gene of present invention, and such grown transgenic plants have been surprisingly found to have improved multiple stress tolerance against more than one environmental stresses, meaning thereby such grown plants have been found to be capable of surviving better under the abiotic stress conditions.

It has also been found that seeds of transgenic plants grown by employing clones of gene of present invention also have improved multiple stress tolerance during seed germination, and both, root and shoot, surprisingly, do not demonstrate reduced growth, and the plants grown therefrom have also been found to have improved multiple stress tolerance as compared to germination of seeds of normal crop plants and plants grown therefrom. Therefore, the problems of poor stress tolerance of crop plants have been solved by present invention.

Accordingly, the present invention relates to method for isolation of hybrid- type histidine kinase gene from the indica rice IR64, comprising treatment of cDNA isolated from IR64 seedling with forward primer OsHk3bF and reverse primer OsHk3bR by polymerase chain reaction (PCR), wherein step of annealing is carried out at about 55 ⁰ C, and wherein hybrid-type histidine kinase gene - OsHK3b is isolated from its plasmid pTOPO-OSHOb.

In accordance with present invention, the forward primer OsHk3bF is identified as 5ΑTGACGTTCGCGAGGTACGC3' and reverse primer OsHl ^β bR is identified as 5'CTATTCAACTTGGTCATGATTTTGS' (Figure IA).

In accordance with present invention, the primers were synthesized by:- a) aligning amino acids sequence (protein) of the SLNl (osmosensor) of yeast with amino acid sequences (Protein) of 14 members of histidine kinase [HK] family in rice (Oryza sativa japonica), wherein one member of HK family -

OsHk3b in rice (Oryza sativa japonica) among the 14 members family was found to be more close to SLNl (osmosensor) of yeast; b) - based on amino acid sequences of OsHk3b from rice (Oryza sativa japonica), its corresponding nucleotide sequence is taken; c) analyzing its ORF using commercially available software; d) designing forward primer OsHk3bF identified as 5 'ATGACGTTCGCGAGGTACGC3 ' and reverse primer OsHk3bR identified as 5'CTATTCAACTTGGTCATGATTTTGS' from ORF of OsHk3b using commercially available primer designing software; e) identifying nucleotide sequences of forward primer and reverse primer; and f) preparing forward primer OsHk3bF identified as 5 'ATGACGTTCGCGAGGTACGC3 ' and reverse primer OsHk3bR identified as 5'CTATTCAACTTGGTCATGATTTTGS'.

In accordance with one of the preferred embodiments of the present invention, analysis of ORF was performed by using commercially available online software (www.ncbi.nlm.nih.gov/proj ects/gorf/).

In accordance with one of the preferred embodiments of the present invention, designing of forward primer OsHk3bF identified as

5ΑTGACGTTCGCGAGGTACGC3' and reverse primer OsHk3bR identified as 5 'CTATTC AACTTGGTC ATGATTTTG3 ' from ORF of OsHk3b was carried out by using commercially available online primer designing software (www.idtdna.com/analyzer/applications/oligoanalyzer/).

In accordance with present invention, cDNA is isolated from seedlings of rice IR64, particularly from seedlings of rice cv IR64 comprising following steps:- a) isolating total RNA from salinity stressed leaf tissue of IR64 seedling; b) isolating mRNA from total RNA by employing the streptavidin paramagnetic beads and biotin-labeled oligo d(T) ₂ o primer; c) synthesizing first strand cDNA from mRNA by employing conventionally available first strand cDNA synthesis kit.

In accordance with present invention, the polymerase chain reaction (PCR) comprises steps of:-

i) preparing reaction mixture of cDNA, and forward primer OsHk3bF identified as 5ΑTGACGTTCGCGAGGTACGC3' and reverse primer OsHk3bR identified as 5'CTATTCAACTTGGTCATGATTTTGS'; ii) initial denaturation of reaction mixture from step - i) at about 94°C preferably for about 5 min; iii) denaturation of reaction mixture from step - ii) for about 34 cycles at about 94°C preferably for about 1 min; iv) annealing of reaction mixture from step - iii) at about 55°C preferably for about 1 min; v) extension of reaction mixture from step - iv) at about 72°C preferably for about 3 min; and vi) final extension of reaction mixture from step - v) at about 72°C preferably for about 7 min.

In accordance with present invention, the method for isolation of hybrid-type histidine kinase gene from the indica rice IR64, further comprises following steps :-

1) preparing ρTOPO-OsHK3b recombinant plasmid by cloning amplified fragment of gene into TOPO-T A2.1 vector;

2) isolating the gene - OsHK3b from pTOPO-OsHK3b recombinant plasmid using standard M 13 forward and reverse primers; wherein isolation of gene - OsHK3b is confirmed [identified] by size of DNA which is about 2.6Kb, more precisely about 2598 bp.

In accordance with present invention, after performing polymerase chain reaction [PCR] with selected primer many copies of target DNA are made, which is not seen in solution but when ligated into TOPO-TA cloning vector which stabilizes target DNA, the transformed colonies can be seen, however the amplified product can be visualized upon electrophoresis in agarose coupled to staining with ethidium bromide. The TOPO-TA is only used for stabilization of amplified product that also make easy for sequencing. The gene formed means putative isolation. After formation as in Figure 1C isolation is confirmed by sequencing, band on the agarose gel confirms that it has been isolated.

In one embodiment, the present invention relates to hybrid-type histidine kinase gene isolated from indica rice IR64, and the isolated gene has been found to be capable of acting as osmosensor, and having capability of osmosensing and inducible by multiple stresses, and capability of improving the multiple stress tolerance of crop plants, particularly of transgenic plants so as to make the plants capable of coping-up with more than one environmental abiotic stress conditions, and hence, to increase their economic value while maintaining its potential yield.

In another embodiment, the present invention relates to sequence listing of the hybrid-type histidine kinase gene isolated from indica rice IR64, wherein the gene is sequenced after formation of the pT0P0-0sHK3b recombinant plasmid, and wherein complete Gene sequence of open reading frame [ORF] of gene - OsHk3b is 2598 bp and is as given in accompanying Figure 2A a reference to which is drawn here, and wherein the gene - OsHK3b of present invention has been submitted to National Center for Biological Resources (NCBI) http://www.ncbi.nlm.nih.gov/nuccore/198387187 under GenBank accession number Bankit 1121378 FJ004641 on August 7, 2008. The copy of acknowledgment from the National Center for Biological Resources (NCBI) is enclosed.

In one embodiment, the present invention relates to a hybrid-type histidine kinase gene, the complete Gene sequence of open reading frame [ORF] thereof - OsHk3b is 2598 bp and as given in Figure 2A.

In accordance with one embodiment of present invention, hybrid-type histidine kinase gene - OsHK3b is identifiable by GenBank accession number Bankit 1121378 FJ004641 of National Center for Biological Resources (NCBI). In another embodiment, the present invention relates to a method of cloning the hybrid-type histidine kinase gene isolated from indica rice IR64 into yeast expression vector namely - pYES2, comprising steps of:-

a) amplifying gene - OsHk3b from pTOPO-OsHK3b plasmid by treating pTOPO-OsHK3b plasmid with forward primer OsΗkVoSpelF and reverse primer OsHk3b£>peiR by employing polymerase chain reaction [PCR];

wherein the amplified fragment of OsHk3b gene containing the complete open reading frame [ORF] of the gene along with additional Spel site - OsHk3b gets cloned into Xbal site of pYES2.

In accordance with present invention, the primers employed for cloning the isolated gene are same as employed to isolate the gene, but the primers for cloning the isolated gene contains additional Spel site that facilitates cloning of OsHk3b gene into pYES2.

In accordance with preferred embodiment of the present invention, after polymerase chain reaction [PCR] of OsHk3b with forward primer OsHk3b5peiF and reverse primer OsHk3bSpβ7R, pYES2 is added.

In accordance with present invention, the polymerase chain reaction [PCR] to amplify the gene - OsHkSb from pTOPO-OsHK3b plasmid comprises steps of:-

i) preparing reaction mixture of pTOPO-OsHK3b plasmid, and forward primer OsHk3b5peiF and reverse primer OsHlάbSpelR; ■ ii) initial denaturation of reaction mixture from step - i) at about 94°C preferably for about 5 min; iii) denaturation of reaction mixture from step - ii) for about 34 cycles at about 94°C preferably for about 1 min; iv) annealing of reaction mixture from step - iii) at about 55 ⁰ C preferably for about 1 min; v) extension of reaction mixture from step - iv) at about 72°C preferably for about 3 min; and vi) final extension of reaction mixture from step - v) at about 72°C preferably for about 7 min.

In accordance with preferred embodiment of the present invention, after polymerase chain reaction [PCR] of OsHk3b with forward primer OsHk3bSpeIF and reverse primer OsHk3b5pe7R, pYES2 is added.

In another embodiment, the present invention relates to a clone of hybrid-type histidine kinase gene isolated from indica rice IR64 into yeast expression vector namely - pYES2, which is identified as pYES2-OsHk3b.

In another embodiment, the present invention relates to a method of cloning the hybrid-type histidine kinase gene isolated from indica rice IR64 into plant expression vector, preferably rice expression vector namely - pCAMBIA1304, comprising steps of:-

a) amplifying gene - OsHk3b from pTOPO-OsHK3b plasmid by treating pTOPO-OsHK3b plasmid with forward primer OsHk3h-§?e7F and reverse primer OsHk3b<SζpeiR by employing polymerase chain reaction [PCR];

wherein the amplified fragment of OsHk3b gene containing the complete open reading frame [ORF] of the gene along with additional Spel site - OsHk3b gets cloned into Spel site of pC AMBIAl 304.

In accordance with present invention, the primers employed for cloning the isolated gene are same as employed to isolate the gene, but the primers for cloning the isolated gene contains additional Spel site that facilitates cloning of OsHk3b gene into pCAMBIA1304. In accordance with preferred embodiment of the present invention, after polymerase chain reaction [PCR] of OsHk3b with forward primer OsHkSbS^eiF and reverse primer OsHk3bS/?e/R, pCAMBIA1304 is added.

In accordance with present invention, the polymerase chain reaction [PCR] to amplify the gene - OsHk3b from pTOPO-OsHK3b plasmid comprises steps of:-

i) preparing reaction mixture of pTOPO-OsHK3b plasmid, and forward primer OsHk3bS/7eiF and reverse primer OsΗk3bSpeIR.; ii) initial denaturation of reaction mixture from step - i) at about 94°C preferably for about 5 min; iii) denaturation of reaction mixture from step - ii) for about 34 cycles at about 94°C preferably for about 1 min; iv) annealing of reaction mixture from step - iii) at about 55 ⁰ C preferably for about 1 min; v) extension of reaction mixture from step - iv) at about 72°C preferably for about 3 min; and vi) final extension of reaction mixture from step — v) at about 72°C preferably for about 7 min.

In accordance with preferred embodiment of the present invention, after polymerase chain reaction [PCR] of OsHk3b with forward primer OsHk3b»SζpeiF and reverse primer OsHk3bSpe/R, pCAMBIA1304 is added.

In another embodiment, the present invention relates to a clone of hybrid-type histidine kinase gene isolated from indica rice IR64 into rice expression vector namely - pCAMBIA1304, which is identified as pCAMBIA1304-OsHk3b.

In one embodiment, the present invention relates to a method for improving multiple stress tolerance of crop plants, comprising co-infecting rice calli grown from seeds of IR64 with a medium containing cloned gene and allowing the co-infected calli into complete transgenic IR64 plant, wherein the medium is Agrobacterium strain LBA4404, and cloned gene is pCAMBIA1304-OsHk3b. In accordance with present invention, a method for improving multiple stress tolerance of crop plants, comprises steps of:-

a) co-infecting rice calli grown from seeds of IR64 with a medium containing cloned gene; b) co-cultivating the co-infected rice calli from step — a); c) washing overgrown medium from co-cultivated rice calli from step — b) with antibiotic; d) transferring the washed callus from step - c) on a selection plate for growth of transformed calli; e) treating the transformed calli from step - d) with regeneration media; f) repeating step of treatment of transformed calli from step - e) with fresh regeneration media till it forms a completely regenerated plant; g) hardening the completely regenerated plant from step - f) in a culture tube; h) transferring the hardened plant from step - g) to earthen pot for development into a complete transgenic IR64 plant; wherein the medium is Agrobacterhim strain LBA4404, and cloned gene is pCAMBIA1304-OsHk3b.

In accordance with one of the preferred embodiments of the present invention, step - b) of co-cultivation performed by keeping the co-infected rice calli from step a) in dark for about 48 hours.

In accordance with one of the preferred embodiments of the present invention, the culture tube in step - g) contains regenerating rice plants in regenerating media.

In accordance with present invention, the antibiotic is cefotaxim, and the regeneration media comprises commercially available Moorashige and Skoog media (Sigma, India).

In one embodiment, the present invention relates to crop plants having improved multiple stress tolerance, as produced by employing clone of gene isolated from indica rice IR64 of present invention, wherein the transgenic plants have been found to have overexpression of hybrid-type histidine kinase gene isolated from indica rice IR64, and improved multiple stress tolerance against more than one environmental stresses, meaning thereby transgenic plants produced in accordance with present invention have been found to be capable of surviving better under the abiotic stress conditions, and therefore, overcoming problems of poor stress tolerance.

In one embodiment, the present invention relates to second generation plants produced from seeds of first generation transgenic plants which were grown by employing clones of gene of present invention, wherein subsequently [second generation] grown transgenic plants have also been found to have improved multiple stress tolerance, and even during seed germination of first generation of transgenic plants, root and shoot, surprisingly, were found to demonstrate better growth, at least, were not found to demonstrating reduced growth. On the contrary, when a plant is grown from seedlings which were not infected with clones of gene of present invention were found to have poor tolerance to environmental stresses, and during further germination of seeds from these normal [control] plants, growth of root and shoot were found to be slow.

In one embodiment, the present invention relates to use of hybrid-type histidine kinase gene isolated from indica rice IR64, to improve multiple stress tolerance of crop plants.

The present invention is now described with reference to accompanying figures.

In accordance with the most preferred embodiment of the present invention, it comprises following steps :-

A] Isolation of a Hybrid-type Histidine Kinase Gene from Indica Rice IR64:-

In accordance with the most preferred embodiment of the present invention, the full length gene, that is, a stretch of DNA coding for a protein, of OsHK3b is isolated from rice cv IR64 essentially employing the polymerase chain reaction (PCR) which is carried out by essentially by employing the forward primer OsHk3bF identified as 5ΑTGACGTTCGCGAGGTACGC3' and reverse primer OsHk3bR identified as 5'CTATTCAACTTGGTCATGATTTTGS' (Figure IA), which have been judicially selected. It has been surprisingly observed that if an attempt is made to isolate the desired gene from rice IR 64 by employing any other primer, the desired gene does not isolate.

In accordance with this invention, the polymerase chain reaction (PCR) is carried out under the conditions as illustrated in the Figure IB which have also been judicially selected. It has been surprisingly observed that if an attempt is made to carry out the PCR reaction at the conditions different than the conditions illustrated in accompanying Figure IB, the desired gene does not isolate.

In accordance with this invention, the PCR conditions [Figure IB] are - initial denaturation is carried out at about 94 ⁰ C for about 5 min followed by about 34 cycles of denaturation carried out at about 94 ⁰ C for about 1 min, annealing at about 55 ⁰ C for about 1 min, extension at about 72°C for about 3 min and final extension at about

72°C for about 7 min by employing the OsHk3bF and OsHk3bR primers [Figure IB].

In accordance with the present invention, the total RNA is isolated from salinity stressed leaf tissue of IR64 seedling. Followed by isolation of the mRNA from total RNA by employing the streptavidin paramagnetic beads and biotin-labeled oligo d(T) ₂ o primer. The first strand cDNA is synthesized from mRNA by employing the first strand cDNA synthesis kit. The polymerase chain reaction [PCR] is carried out as described herein above. The amplified fragment is cloned into TOPO-TA2.1 vector to get pTOPO-OsHK3b recombinant plasmid. The accompanying Figure 1C illustrates a PCR amplified product from the plasmid pTOPO-OsHK3b using standard

M 13 forward and reverse primers. The size of this DNA as seen from the gel (Figure

1C) is about 2.6Kb, more precisely about 2598 bp of OsHk3b, which confirms that the gene - OsHK3b has been isolated.

B] Sequencing of a Hybrid-type Histidine Kinase Gene isolated from Indica Rice IR64:- In accordance with the present invention, in one embodiment, the gene isolated from indica rice IR 64 is sequenced after formation of the pTOPO-OsHK3b recombinant plasmid. The DNA sequence as obtained after sequencing is illustrated in the accompanying Figure 2A, which illustrates the complete Gene sequence of ORF of OsHkSb which is 2598 bp and has been submitted to National Center for Biological Resources (NCBI) under GenBank accession number Bankit 1121378 FJ004641 on August 7, 2008. The copy of acknowledgment from the National Center for Biological Resources (NCBI) is enclosed.

In accordance with this invention, the DNA sequence as obtained is analyzed using BLAST search, which surprisingly has been found to be homologous to the known gene classified as the hybrid type histidine kinase gene of other organisms with minor variations belonging only to the rice IR64. Further, the isolated cDNA has been confirmed to have about 2598 bp length of ORF which is capable of encoding a polypeptide of about 865 amino acid residues (Figure 2B). The predicted molecular mass of deduced polypeptide is found to be about 95.90 kDa. The predicted protein contains both - a transmitter and - a receiver domain. These are characteristic features of a hybrid type histidine kinase. It also showed a conserved histidine residue at 291 position as illustrated in the accompanying Figure 2B by underlining in line 6 from top in the transmitter domain and a conserved aspartate residue at 772 position as illustrated in the accompanying Figure 2B by underlining in second line from bottom in the receiver domain. The accompanied Figure 2B illustrates the complete deduced amino acid sequence for protein encoded by ORF of OsHk3b which is about 95.9 kDa, wherein the transmitter domain has a conserved Histidine residue at 291 position (H, underlined therein). The receiver domain has a conserved Aspartate residue at position 771 (D, underlined therein).

Accordingly, the present invention provides a gene from indica rice cv IR64 which possess all characteristic feature of the hybrid type histidine kinase gene. The DNA sequence of the isolated OsHk3b gene has also been submitted to National Center for Biological Resources (NCBI) under the accession number bankitl 121378 on August 7, 2008. The copy of acknowledgment from the National Center for Biological Resources (NCBI) is enclosed. C] Cloning of a hybrid-type histidine kinase gene isolated from Indica Rice IR64:-

Cl] Cloning into yeast expression vector - pYES2:-

In accordance with present invention, the gene - OsHk3b of the present invention is cloned into yeast expression vector namely - pYES2 by amplifying the gene - OsHk3b from pTOPO-OsHK3b by employing OsΗk3bSpeR and OsΑkVoSpelR. primers as illustrated in the accompanying Figure IA by employing the polymerase chain reaction [PCR] under the conditions as illustrated in the accompanying Figure IB, wherein the amplified fragment containing the complete open reading frame [ORF] of the gene - OsHkSb is cloned into Xbal site of pYES2 and in-frame cloning is confirmed by restriction digestion using Sphl [Figure 3A], wherein the restriction digestion of pYES2-OsHk3b plasmid with Sphl to confirm the orientation of OsHK3b ORF; M: lkb DNA ladder; 1,2,3,4 and 5 are pYES2-OsHK3b plasmid; 6: pYES2 and 7: undigested pYES2 -OsHk3b plasmid, and the schematic representation of pYES2-OsHk3b has been shown on the top of figure.

Accordingly, the present invention provides a gene - OsHk3b which is capable of being cloned into yeast expression vector pYES2-OsHk3b and produces the clone of gene - OsHkSb into yeast expression vector pYES2-OsHk3b.

C2. Cloning into plant expression vector - pCAMBIA1304

In accordance with present invention, the gene - OsHk3b of the present invention is cloned into plant expression vector namely - pCAMBIA1304 by amplifying the gene - OsHkJb from pTOPO-OsHK3b by employing OsHkSbSpelF and OsHkSbSpeIR primers as illustrated in the accompanying Figure IA by employing the polymerase chain reaction [PCR] under the conditions as illustrated in the accompanying Figure IB, wherein the amplified fragment containing the complete open reading frame of the gene - OsHlύb is cloned into Spel site of pC AMBIAl 304 and in-frame cloning is confirmed by restriction digestion using Sphl [Figure 3B], wherein restriction digestion of pCAMBIA1304-OsHK3b plasmid with Sphl to confirm the orientation of OsHk3b ORF; M: lkb DNA ladder;l,2,3 and 4: pCAMBIA1304-OsHK3b plasmid; 5: pCAMBIA1304 and 6: undigested pCAMBIA1304-OsHK3b plasmid. Schematic representation of pCAMBIA1304- OsHk3b has been shown on the top of figure.

Accordingly, the present invention provides a gene - OsHk3b which is capable of being cloned into plant expression vector pCAMBIA1304-OsHk3b and produces the clone of gene - OsHk3b into plant expression vector pCAMBIA1304-OsHk3b.

D] Functional Characterization [Advantages] of a hybrid-type histidine kinase gene isolated from rice IR 64:-

Dl] To establish that the presently provided gene - OsHk3b is capable of functioning as an osmosensor, i.e. its advantage in rescuing the osmosensitive mutation in yeast, the Yeast (Saccharomyces cerevisiae) strain HS 13 with a temperature-sensitive mutation in SLNl gene is employed for complementation experiments (sln-Ts) in accordance with the preferred embodiment of the present invention. It is observed that the HS 13 mutant grow well at 28 ⁰ C (Figure 4A) but fail to grow at 37 ⁰ C (Figure 4B) on YPD media (Yeast, Peptone and Dextrose media) as well as on YPD containing salt (20OmM NaCl). In accordance with the present invention, the HS 13 mutant is transformed with (i) pYES2-OsHk3b, (ii) vector pYES2 (vector without OsHk3b) and (iii) functional slnl gene (from yeast) to produce a modified HS 13 strain which is tested further. Further, in accordance with the present invention, the HS 13 carrying pYES2 (vector without OsHk3b) works as negative control (VC) while HS 13 carrying functional slnl gene works as positive control (SLNl). When all these strains are streaked on YPD media and incubated either at 28 ⁰ C or at 37 ⁰ C, it is surprisingly observed that after about 72h of incubation, HS 13 transformed with pYES2-OsHk3b and HS 13 carrying functional slnl gene are able to grow even at 37 ⁰ C (Figure 4B), whereas HS 13 alone or HS 13 carrying pYES2 (VC) are not able to grow at 37 ⁰ C, thereby confirms that the complementation of slnl mutant with OsHk3b is achievable, indicating the advantage of gene - OsHk3b in rescuing the osmosensitive mutation in yeast.

After having established the advantage of the gene - OsHk3b as osmosensor which may be used to improve tolerance towards salinity stress, an attempt is made to find out the mode of action of this gene by checking the role of conserved Histidine and/or Aspartate residue of OsHk3b gene, site directed mutagenesis of OsHk3b gene are carried out to mutate the conserved Histidine residue of transmitter domain into Valine (HK3H*, HS13 carrying histidine mutated OSHK3H291V) and conserved Aspartate residue into Glutamate (HK3D*, HS 13 carrying aspartate mutated OSHK3D772E) in the expressed OsHk3b protein. [Site directed mutagenesis of pYES2-OsHk3b is carried out to mutate the conserved Histidine residue of transmitter domain into Valine using forward primer, OSHK3bHisMUTF GGCTACTGTTTCAGTTGAGATCAGAACTC and reverse primer, OSHK3bHisMUTR AGTTCTGATCTCAACTGAAACAGTAGCC; whereas conserved Aspartate residue into Glutamate using forward primer, OSHK3bAspMUTF

GATGCTTGTTTCATGCTCATACAGATGCCAG and reverse primer, OSHK3bAspMUTR -CTGGCATCTGTATGAGCATGAAACAAGCATC using quick change site directed mutagenesis kit].

The above analysis confirms that the mutation in the yeast HS 13, transformed with these mutated OsHk3b conserved residues could not complement the sin 1 -Ts mutant allele containing host HS13 at 37 ⁰ C (Figure 4B), which confirms that OsHk3b is a confirmed hybrid type Histidine kinase and conserved Histidine and Aspartate are involved in the phosphorylation process for the growth of HSl 3 at 37 ⁰ C. Further, the transformed yeast (OsHk3b) is found to be capable to tolerate the salinity stress upto 20OmM NaCl (YPD containing 20OmM NaCl), whereas the vector transformed (VC) or the untransformed HS 13 cells could not survive at 37 ⁰ C (Figure 4D). This analysis clearly establishes that the protein encoding OsHk3b gene is capable of acting as an osmosensor and can be used for improving of tolerance towards salinity stress.

D2] To establish that the presently provided gene - OsHk3b is inducible by different abiotic stresses, a real time PCR analysis (qRT-PCR) is performed to quantify the expression of its mRNA. This analysis is carried out using various cDNA samples made from the mRNA of IR64 seedlings which are subjected to various abiotic stresses, i.e., salinity (S), drought (D), ABA (Abscisic acid), Heat (42 ⁰ C ) and Cold (4 ⁰ C) for about 8 hours and about 24 hours. The real time PCR amplification results as illustrated in the histogram in Figure 5, surprisingly indicate the inducibility of this gene by the different environmental signal in the form of abiotic stresses. Further, differential regulation of OsHk3b transcripts under salinity, drought, ABA, Heat and cold could also be seen surprisingly. The accumulation of OstIK3b transcript increases with time from about 8 hours to about 24 hours in all the stress conditions such as under salinity (Figure 5B, C), drought (Figure 5D, E), ABA (Figure 5F, G), heat (Figure 5H, I) and cold (Figure 5J, K), thereby indicating the role of this gene in multiple abiotic stresses, and hence, its advantage in improving the tolerance of plants for various stresses especially, high temperature, low temperature, salinity as well as drought and so on.

E] Improvement of multiple stress tolerance of crop plants - Use of hybrid- type histidine kinase gene isolated from rice IR 64 in improvement of multiple stress tolerance of crop plants :-

To establish that the presently provided gene - OsHk3b is capable of improving stress, particularly the abiotic stress tolerance in plants [transgenic rice], the IR64 plant is transformed with gene - OsHK3b under the control of judicially selected constitutive promoter using pCAMBIA1304-OsHk3b, and to have a comparison, the IR 64 plant is also transformed with vector control (VC having no OsHk3b gene).

In accordance with the present invention, the transformation of IR64 is achieved by surface sterilizing the seeds of IR-64 (Figure 6a) and transferring on the callus induction media for callus formation, the grown callus (Figure 6b) is further sub-cultured and again kept on the callus induction media for about 5-7 days (Figure 6c), these calli are co-infected with Agrobacterium strain LBA4404, containing pCAMBIA1304-OsHk3b (Figure 6d), the co-infected rice calli is further kept for co- cultivation for about 2-3 days (Figure 6e), the overgrown Agrobacterium (on rice calli) is washed with antibiotic - cefotaxim (Figure 6f), the washed callus is then transferred on a selection plate for growth of the transformed calli (Figure 6g), the transformed calli is further kept on the regeneration media (Figure 6h) and sub- cultured on fresh regeneration media (Figure 6i), the completely regenerated plant is transferred into culture tubes for hardening (Figure 6j), then the hardened plant is further transferred into earthen pots and kept in a green house to develop complete transgenic IR64 plants (Figure 6k). To establish the possible role of gene - OsHk3b in improving stress tolerance in rice, leaf disc assay analysis is performed. In this analysis, the discs of leaf are cut from the various plants and incubated in solution containing high salinity (20OmM NaCl), and also a control is developed where instead of salt, normal water is added. After about 96h of incubation, comparison of the discs is done and their general viability is looked for (Fig 7A), the estimation of chlorophyll for these samples is also carried out and the results are presented in Figure 7B. This analysis indicates that the transgenic IR64 plants (over expressing OsHk3b) shows relatively less bleaching even under control conditions (without salinity stress) in T9 (Fig. 7A5 T29 (Fig. 7A7) as compared to the non-transformed IR64 i.e., WT (Fig. 7Al) and VC (Fig. 7A3). Similarly, more retention of chlorophyll in IR64 transgenic plants T9 (Fig. 7B6) and T23 (Fig. 7B8) is also observed than its non-transgenic counterparts, WT (Fig. 7B2) and VC (Fig. 7B4) under the salinity stress confirming that overexpression of gene - OsHk3b in transgenic rice plants enables them to survive better under abiotic stress especially salinity.

F] Confirmation of OsHk3b Transgenic Rice Plants :-

To confirm the integration of OsHk3b gene into the genome of IR64 overexpression lines, tissue PCR was done using gene -OsHk3b specific forward primer and vector (pCAMBIA1304) specific reverse primer. The amplification show a band of 1.4 kb size in lane number 4, 7, 8, 9, 10, 11, 18, 19, 23, 32 indicates the successful integration of OsHk3b transgene into the genome of these IR64 transgenic plants (Figure 8).

G] Transgenic Lines of Rice (T ₁ ) show Tolerance towards Salinity Stress during Seed Germination:-

Seeds of wild type IR64 (WT) and overexpression OsHk3b were kept in petridish containing cotton saturated with V ₂ Yoshida containing 200 mM NaCl (salinity stress). The inventors have surprisingly found that the germination rate of seeds on 200 mM NaCl is more in case of overexpression OsHk3b lines in comparison to the wild type seeds (Fig. 2A). When length of root and shoot of germinated seedlings were measured after fourth days of growth, inventors surprisingly found that both root and shoot length was more in overexpression lines, but less in the wild type (Fig. 2B). To determine the available K ⁺ and Na ⁺ ions in the transgenic plants, inventors have estimated the amount of available ions by flame photometer and found that overexpressing transgenic plants retain more K ⁺ /Na ⁺ ratio than the wild type plants (Fig. 2C). This analysis indicates that OsHk3b overexpression in rice provide salinity stress tolerance to germinating seeds.

H] Transgenic Seedlings Overexpressing OsHk3b are Better Adapted to Salinity Stress as Compared to WT:

Transgenic rice seedlings (Tl) were further used for testing the tolerance towards salinity. Seven days old seedlings were transferred into 200 niM NaCl containing half Yoshida medium, whereas seedlings grown in 1/2 Yoshida medium without NaCl was used as control. Photographs were taken after ten days of stress. Inventors found that overexpression OsHk3b overexprssing plants were surprisingly able to withstand more salinity stress (Fig. 3B) and retain more chlorophyll (Fig. 3C) than the wild type plant under salinity stress condition. Even under non-stress conditions, the growth of OsHk3b overexpression plants was found to be more than that of wild type lines of OsHK3b (Fig. 3A). This analysis further indicated that OsHk3b overexpression in rice provide salinity stress tolerance to seedlings.

The detailed Legends of the Accompanying Figures:-

Figure IA: Primer sequences used for the isolation of OsHk3b gene

Figure IB: Conditions for Polymerase Chain Reaction (PCR)

Figure 1C: Agarose gel electrophoresis of polymerase chain reaction product showing an amplification product of size 2.6Kb (precisely 2598 bp) of OsHk3b.

Figure 2A: The complete Gene sequence of ORF of OsHk3b which is 2598 bp (submitted to NCBI under GenBank accession number Bankit 1121378 FJ004641). Figure 2B: The complete deduced amino acid sequence for protein encoded by

ORF of OsHkSb which is 95.9 kDa. Transmitter domain has a conserved Histidine residue at 291 position (H, shown in red). Receiver domain has a conserved Aspartate residue at position 771 (D, shown in blue). The sequence has been submitted to NCBI under GenBank accession number Bankit 1121378 FJ004641).

Figure 3 A: Confirmation of cloning of OsHk3b into pYES2 vector: Restriction digestion of pYES2-OsHk3b plasmid with SphlXo confirm the orientation of OsHK3b

ORF; M: lkb DNA ladder; 1,2,3,4 and 5 are pYES2-OsHK3b plasmid; 6: pYES2 and 7: undigested pYES2 -OsHk3b plasmid. Schematic representation of pYES2-OsHk3b has been shown on the top of figure.

Figure 3B: Confirmation of cloning of OsHk3b into pCAMBIA1304 vector: Restriction digestion of pCAMBIA1304-OsHK3b plasmid with Sphl to confirm the orientation of OsHk3b ORF; M: lkb DNA ladder; 1,2,3 and 4: pCAMBIA1304- OsHIOb plasmid; 5: pCAMBIA1304 and 6: undigested pCAMBIA1304-OsHK3b plasmid. Schematic representation of pCAMBIA1304-OsHk3b has been shown on the top of figure.

Figure 4: Functional complementation of yeast SLNl -temperature sensitive osmosensing mutant HS 13 by the rice OsHK3b ORF. Complementation of the mutation by expression of OSHK3B ORF; Sensitivity of yeast strains cultured at 28 ⁰ C, 37 ⁰ C for three days on normal YPD media and YPD with 20OmM NaCl (saline medium). Strains used in this experiment:

WT, wild-type yeast;

KSU, sin 1-ts mutant;

VC, HS 13 carrying vector (ρYES2) only;

OSHK3a and OSHK3b, HS 13 carrying expression pYES2-OsHK3b; SIM , HS 13 carrying wild type expression sin 1 allele;

HK3H*, HS13 carrying histidine mutated pYES2-OsHK3b (OsHK3H291V) and

HK3D*, HS 13 carrying aspartate mutated pYES2-OsHK3b (OsHK3D772E)

4A: Incubation of different yeast strains on YPD medium at 28 ⁰ C 4B: Incubation of different yeast strains on YPD medium at 28 ⁰ C 4C: Incubation of different yeast strains on salinity stress (YPD containing 20OmM NaCl) medium at 28 ⁰ C;

4D: Incubation of different yeast strains on salinity stress (YPD containing 20OmM NaCl) medium at 28 ⁰ C;

4E: Schematic representation of different yeast strains used for streaking in 4A, 4B, 4C and 4D.

Figure 5: The histogram showing the inducibility of OsHK3b by various abiotic stresses in rice. X axis has different samples; Y axis shows relative mRNA expression. qRT-PCR analysis was performed using cDNA synthesized from mRNA of IR64 (IR) under S (20OmM NaCl), D (dehydration), ABA (10OuM Abscisic acid), Heat (42 ⁰ C) and Cold (4 ⁰ C) at 8 (hrs) and 24 (24hrs) of stress along with C (control) samples.

Figure 6: Oryza sativa Cv IR64 transformation and regeneration, a. Seeds of Rice IR-64; b. Rice callus formation on callus induction media; c. sub-cultured rice callus kept on callus induction media for 5-7 days; d. co-infection of rice calli with Agrobacterivm strain LBA4404 containing pCAMBIA1304-OsHK3b construct; e. co- cultivation of co-infected rice calli; f. washing of overgrown Agrobacterium with Antibiotic cefotaxim; g. Transfer of washed callus on selection plate; h. transformed callus was kept on regeneration medium; I. regenerated plantlets was transferred on fresh regeneration medium; j. completely regenerated plant was transferred into culture tubes for hardening; k. plant from step j is further transferred into earthen pot and kept in green house

Figure 7A: Leaf Disc assay. Showing the bleaching rate of different leaf samples under control and salinity stress.

Figure 7B: Total Chlorophyll content. X axis: different samples, Y: axis total chlorophyll content in μg per gram fresh weight of leaves WT, IR64

VC, IR64 transformed with vector without OsHfGb gene; T9, IR64 transformed with vector with OsHK3b gene (plant number 9); T23, IR64 transformed with vector with OsHK3b gene (plant number 23).

Figure 8: illustrates confirmation of IR64-OsHk3b regenerated plants by tissue PCR. (A) PCR amplification was done using the tissue of regenerated plantlets as template and vector specific forward and gene specific reverse primers. M: DNA marker; 4, 7, 8, 9, 10, 11, 12, 18, 19, 23, 32 are the different OsHk3b transgenic plantlets used for tissue PCR, IR: non-transformed IR64 i.e. wild type plants and +ve: pCAMBIA-OsHk3b used as template.

Figure 9: illustrates confirmation of multiple stress tolerance by germination test for IR64-OsHk3b transgenic seeds (Ti) in presence of 200 mM NaCl. (A) Seeds of WT: Wild type; OEl: overexpressing OsHK3b line, were kept in 200 mM NaCl containing 1/2 Yoshida media; (B) Measurement of shoot length; (C) Measurement of root length; (E) Measurement of K ⁺ /Na ⁺ ratio. Data was taken after 96 h of salinity stress.

Figure 10: illustrates growth of IR64-OsHk3b transgenic seedlings (Ti) under control (without salinity) and Salinity stress condition and their chlorophyll measurement. (A) in normal Vi Yoshida media (B) in NaCl (20OmM) supplemented ¹ A Yoshida media. WT: wild type IR64, OEl: overexpressing OsffiGb line. (C) Chlorophyll measurement from control salinity stressed seedlings. 7 days old seedlings grown under control conditions were subjected to salinity stress (200 mM NaCl) for 10 days after the photograph was taken.