WO2008148298A1

WO2008148298A1 - Improvement of cold tolerance in plants by protein kinase gene oscipk03 from rice

Info

Publication number: WO2008148298A1
Application number: PCT/CN2008/001047
Authority: WO
Inventors: Yong Xiang; Lizhong Xiong; Yuemin Huang
Original assignee: Huazhong Agricultural University
Priority date: 2007-06-01
Filing date: 2008-05-29
Publication date: 2008-12-11
Also published as: WO2008148298A8; CN101200724A

Abstract

The clone isolation, function confirmation and use of the OsCIPK03 gene from rice associated with the plant tolerance to cold stress are provided. The gene is (a) the DNA sequence as show in 1-1707 bp of SEQ ID NO : 1, or (b) a DNA sequence that encodes a protein substantially equivalent to the protein encoded by (a).

Description

Improvement of Cold Tolerance in Plants by Protein Kinase Gene OsCIPK03 from Rice

Technical Field

The present invention relates to the field of plant biotechnology, and more particularly, to the isolation, cloning, function identification and use of a DNA fragment (gene) from rice that is associated with cold tolerance in plants. The cold-tolerance of transgenic plants is markedly improved by transferring a complete translation region (Coding sequence) of the gene linked with the constitutive promoter of tobacco mosaic virus (CaMV35S).

Background Art

The growth of plants is naturally subjected to the influence of the environment. For example, drought, salt injury and low temperature always lead to great reduction of crop production, thereby posing a challenge to the development of agriculture in many areas. To cope with or adapt to the adverse effect of environment, plants perceive the changes of extracellular environmental conditions and signal the cells through many pathways. In response, the cells induce expression of some responding genes to generate some functional proteins, osmoregulation substances and transcription factors for signal transmission and gene expression regulation, all of which protect the cells from stress impairment of drought, high salinity, low temperature and the like, so that plants are able to make corresponding responses to environmental changes (Xiong et al., Cell signaling during cold, drought and salt stress. Plant CeIL 14 (suppl), Sl 65-Sl 83, 2002). The expression of these functional genes is essential for the response to the outer environment and thus the survival of the plants. Protein kinase families in plants such as CDPK % CIPK > MAPK and the like are found to play an important role in the transduction of stress signals and tolerance of plants to the stresses. As the expression of genes for these protein kinases can be induced or repressed under different environmental stresses, it is believed that these genes are important in the process of response of plants to the stress. Therefore, the separation and identification of these functional genes that contribute to plant response to environmental stresses are of significance in genetic modification and breeding of crops for their tolerance to the stress. Attempts have been made to improve plant tolerance to stress. Transgenic Arabidopsis plants that overexpress DREBlA and DREB2A showed increased tolerance to low temperature, drought and high salinity as compared to their wild type counterparts (Liu Q et al., "Two transcription factors, DREBl and DREB2, with an EREBP/AP2 DNA domains separate two cellular signal transduction pathways in drought- and low-temperature-responsive gene expression, respectively, in Arabidopsis." Plant Cell. 1998, 10: 1391-1406). The research group of Thomashow at Michigan State University (U.S.A.) also bred plants with enhanced cold tolerance via genetic transformation with Arabidopsis CBFl gene.

Rice is one of the most important cereal crops, and rice with improved cold tolerance is of great significance to human. Therefore, there exists an urgent need to identify the functional genes associated with cold tolerance and breed cold and chill tolerant rice lines in order to increase the yield of rice.

Summary of the Invention

An object of the present invention is to isolate and clone from rice a DNA fragment containing the complete coding region of a gene associated with cold tolerance, and to use the gene to improve the stress tolerance of rice and other plants, and particularly, to increase the cold tolerance of transgenic plants through gene modification. Structural analysis of this stress-associated gene, named OsCIP 'K03, revealed that it belongs to plant CIPK protein kinase family.

The present invention relates to the isolation and use of a DNA fragment containing OsCIPK03 gene, which confers plants with enhanced tolerance to stress such as low temperature and the like. Said DNA fragment is as shown in SEQ ID NO: 1, or is a highly homologous DNA sequence substantially equivalent to SEQ ID NO: 1, or is a subfragment functionally equivalent to SEQ ID NO: 1.

The gene of the present invention or a homologous gene thereof can be obtained by screening a cDNA or genomic library using a cloned OsCIPK03 gene as the probe. Alternatively, the OsCIPK03 gene of the present invention and any DNA segments of interest or homologous DNA segments thereof may also be obtained by amplification from genomic DNA, mRNA and cDNA using PCR (polymerase chain reaction) technology. The sequence containing OsCIPK03 gene can be isolated using the above methods. By transforming plants with said isolated sequence in any expression vector that can direct the expression of an exogenous gene in plant, transgenic plants with enhanced tolerance to low temperature can be produced. According to the present invention, in the process of constructing the gene of the present application into the expression vector of the plant, any strong promoter or inducible promoter can be added to the position preceding the transcription initiation nucleotide, or alternatively, an enhancer may be used. Such an enhancer region can be ATG start code and start code of contiguous regions and the like, provided that the enhancer region is in the same frame as the coding sequence to ensure the translation of a complete sequence. The expression vector bearing the OsCIPKOS gene of the present invention can be introduced into plant cells by conventional biotechnological means such as Ti plasmid, plant viral vector, direct DNA transformation, microinjection, electroporation and the like (Weissbach, 1998, Method for Plant Molecular Biology VIII, Academy Press, New York, pp. 411-463; Geiserson and Corey, 1998, Plant Molecular Biology (2^nd Edition)).

The expression vector comprising the OsCIPKOi gene of the present invention can be used to transform a host that is selected from a wide variety of plants including rice, so as to cultivate cold tolerant plant lines.

The gene of the present invention is expressed by induction of stress, and therefore its promoter is an inducible-type promoter. By inserting both a promoter segment and any gene of interest of the present invention into an appropriate expression vector and transforming a plant host, expression of the gene can be induced under stress conditions, thereby improving the tolerance performance of the plants in response to stress.

The present invention will be further illustrated hereunder by specific examples.

Brief Description of the Drawings

SEQ ID No: 1 in the Sequence Listing shows the sequence of the DNA fragment isolated and cloned in accordance with the present invention, comprising the OsCIPKOi gene encoding region.

Fig. 1 is the flow chart of isolation and identification of the OsClPKOi gene.

Fig. 2 shows the expression levels of the OsCIPKOS gene at different time points under different stresses including drought, high salinity, cold, PEG and ABA, as measured by Northern hybridization.

Fig. 3 shows the expression vector used for genetic transformation of rice. The vector over- expressing OsCIPKOS is obtained by replacing the portion from attRl to attR2 as shown in the figure with the target gene OsCIPKOS through a LR recombination reaction.

Fig. 4 shows that over-expression of the OsCIPKOS gene in rice enhances the cold tolerance of the plant. A. Expression of the OsCIPKOS gene in the transgenic plants. The last lane is the control, and the other lanes are the individual event of transgenic plants; B. Growth of the transgenic plants and the wild type control under normal conditions; C. Growth of the transgenic plants and the wild type control after low temperature stress (4°C stress for 5 days and normal growth for 5 days); D. Survival rate of the transgenic plants and the wild type control after low temperature stress (4°C stress for 4 days and normal growth for 7 days).

Fig.5 shows that the over-expression of the OsCIPKOS gene increases the proline content in the plants. The plants were grown to the 4-leaf stage and were subjected to low temperature stress at 4 ⁰C and samples were taken at day 0, 1, 3, 6 to determine the proline content.

Fig. 6 shows the determination of the expression amounts of genes related to proline synthesis and transportation in the plants over-expressing OsCIPK03.

Examples

At the initial stage of the work leading to the present invention, a cDNA clone designated EI101L12 derived from the rice variety MingHui No. 63 (a rice variety widely cultivated in China) was obtained. This cDNA is the full-length cDNA of the OsCIPK03 gene, a gene associated with stress tolerance. The association of the gene with stress tolerance is evidenced by the following facts. Firstly, it was observed upon an analysis using a cDNA chip technique that the expression amount of the cDNA clone EI 10 ILl 2 in the rice variety "ZhongHan No. 5" (a publicly used rice variety available from Shanghai Academy of Agricultural Sciences, China) was increased by 2.5 times after a drought stress treatment for 15 days. Sequencing of the clone revealed the gene to be OsCIP K03 (Genebank accession number AKl 11929). In view of significant difference in the expression amount of the clone after drought treatment and its functional characteristics, it is considered that the gene as represented by the EIlOlLl 2 clone is involved in regulating and controlling expression of genes under stresses. Secondly,, analysis of the expression profile of the gene under stresses (see Fig.2) revealed that the expression, level of the gene was markedly increased during stress treatments. Thirdly, the transgenic plants with over-expression of the full-length gene showed significantly enhanced tolerance to cold (see Fig. 4). All these results showed that the OsCIPK03 gene is a stress-associated gene involved in regulation and controlling of the plant against stress.

The present invention will become more apparent from the following descriptions of the examples, which illustrate the methods for isolating and cloning the DNA fragment comprising the complete encoding region of the OsCIPK03 gene and for confirming the function of the OsCIPK03 gene on the basis of the above-said initial stage of the work (flow scheme of the present invention is shown in Fig. 1). From the description of the following examples, the basic features of the present invention will become apparent to one skilled in the art, and many changes and modifications can be made to the present invention to adapt it to various uses and conditions without departing from the spirit and scope of the present invention.

Example 1: Isolation and cloning of a DNA fragment containing the OsCIPK03 gene segment Analysis on the expression profiles of drought inducible genes of the rice variety "ZhongHan No. 5" (a publicly used rice variety available from Shanghai Academy of Agricultural Sciences, China) led to the finding of an EST (expression sequence tag) that is strongly induced by drought, the expression amount of which was increased by 2.5 times or above at the later stage of drought stress. Sequence analysis indicated that this gene is a member of the CIPK protein kinase family and is a full-length sequence that corresponds to the cDNA clone 001-015-H02 in the database of KOME (Knowledged-based Oryza Molecular Biological Encyclopedia) of Japan (http://cdna01.dna.affrc.go.jp). According to this cloned sequence, the primers OsCIPK03F

(5'-CACCATGTATAGGGCTAAGAGGGCT-S'. sequence specific primer plus linker CACC site) and OsCIPK03R (5'-TTGAAACTCACAAACTGTCA-S') were designed, and 1-1653 bp of the clone was amplified by reverse transcription from the variety "ZhongHan No. 5". The amplification product is the 1-1653 bp sequence of the present invention (Fig. 3). Particular steps are the following: extracting total RNA from the leaves of the rice variety "ZhongHan No. 5" subjected to drought stress treatment using a TRIZOL reagent (purchased from Invitrogen Inc.) according to the instructions of the manufacture; reverse transcribing with a reverse transcriptase (purchased from Invitrogen Inc.) to synthesize cDNA first chain; amplifying the reverse transcribed product with the nested primer designed according to the sequence of cDNA clone 001-015-H02 (the reaction conditions were: predenaturation at 94°C for 2 min; 30 cycles of 94°C for 30 sec, 55⁰C for 30 sec, 72°C for 2 min; extension at 72°C for 5 min); ligating the PCR products obtained from amplification into a pGEM-T vector (purchased from Promega Inc.); and screening and sequencing the positive clones to obtain the desired full-length gene. Such a clone was named PGEM-OsCIPK03.

Example 2: Detection of induced expression of rice endogenous gene OsCIPK03

The rice variety "Zhonghan No. 5" was used as the material and treated at 3-leaf stage with drought stress, cold stress, high-salinity stress, abscisic acid (ABA) and polyethylene glycol (PEG) respectively. The drought stress treatment was conducted by stopping the supply of water to rice seedlings and sampling at 0 h, 3 h, 6 h, 12 h and 24h. The cold stress treatment was conducted by placing the rice seedlings in a cold chamber at 4°C and sampling at 0 h, 3 h, 6 h, 12 h and 24h. The high-salinity stress treatment was conducted by immersing the seedling root in the 200 mM/L NaCl solution and sampling at 0 h, 5h, 14h and 24h. The ABA treatment was conducted by immersing the seedling root in the 100 μM/L ABA solution and sampling at 0 h, 3 h, 6 h, 12 h and 24h. The PEG treatment was conducted by immersing the seedling root in the 20% PEG6000 solution and sampling at 0 h, 3 h, 5 h and 12 h. The total RNA of the leaves were extracted (Trizol reagent, purchased from Invitrogen), then subjected to RNA membrane transfer and finally to Northern hybridization using OsCIPK03 as the probe according to the experimental methods described in Joseph Sambrook, "Molecular Cloning", Science Press, Peking, 1999. The results showed that expression of the OsCIPK03 gene cloned in the present invention was induced by drought, cold, high-salinity, ABA and PEG (as shown in Fig.2), therefore the gene encodes a protein kinase associated with stress.

Example 3: Construction and transformation of a vector over-expressing OsCIPK03 gene

The above Example 2 showed that expression of the OsCIP K03 gene of the present invention can be induced by drought, cold, high-salinity, abscisic acid (ABA) and polyethylene glycol (PEG). In order to better illustrate the function of this gene, it was over-expressed in rice and the phenotype of transgenic plants was characterized. Steps were the following: amplifying exogenous fragments by PCR using the positive clone pGEM-OsCIPK03 obtained in Example 1 as the template and using the primers and conditions in Example 1; ligating the exogenous fragments into the intermediate vector pENTR/D-TOPO (purchased from Invitrogen, refer to the instruction of the kit for specific steps); ligating the gene of interest into the genetic transformation vector pCB2004H bearing the tobacco mosaic virus promoter (CaM35S) via LR recombination reaction (refer to the instruction of LR recombination reaction kit from Invitrogen); transforming E. coli. strain DHlOβ (purchased from Invitrogen); and screening positive clones to obtain the transformed vector.

The transformed vector was introduced into the rice variety "ZhongHua No. 11" (a publicly used rice variety available from Institute of Crop Science, Chinese Academy of Agricultural Sciences) using a rice genetic transformation system mediated by Agrobacterium. A transgenic plant was obtained through precultivation, infestation, co-cultivation, screen of the callus with hygromycin resistance, differentiation, rooting, seedling establishment and transplanting. One of the obtained transgenic rice plants was designated as T35. The present applicants obtained in total 23 independent transgenic rice plants.

The genetic transformation system of rice (japonica rice subspecies) mediated by Agrobacterium, established by National Key Laboratory of Crop Genetic Improvement where the present applicants work, was used as the genetic transformation system of rice. The genetic transformation was conducted as follows.

(1) Abbreviations of Reagents and Solutions

The abbreviations of phytohormones used in culture media of the present invention were as follows: 6-BA (6-Benzylaminopurine); CN (Carbenicillin); KT (Kinetin); NAA (Napthalene acetic acid); IAA (Indole-3 -acetic acid); 2,4-D (2,4-Dichlorophenoxyacetic acid); AS (Acetosringone); CH (Casein Enzymatic Hydrolysate); HN (Hygromycin B); DMSO (Dimethyl Sulfoxide); N6mac (N6 macroelements solution); N6mic (N6 microelements solution); MSmac (MS macroelements solution); MSmic (MS microelements solution).

(2) Formulation of Primary Solutions

1) Preparation of N6 macroelements mother solution (1OX concentrate):

These compounds were dissolved in succession and then the volume was brought to 1000 ml with distilled water at room temperature.

2) Formulation of N6 microelements mother solution (10OX concentrate): KI 0.08 g

H₃BO₃ 0.16 g

MnSO₄ «4H₂O 0.44 g

ZnSO₄ '7H₂O 0.15 g

These compounds were dissolved and then the volume was brought to 1000 ml with distilled water at room temperature.

3) Formulation of ferric salt (Fe₂EDTA) stock solution (10OX concentrate):

800 ml double distilled water was prepared and heated to 70°C, then 3.73g Na₂EDTA'2H₂O was added and fully dissolved. The resulting solution was kept in 70⁰C water bath for 2 h, then brought to 1000 ml and stored at 4⁰C for use.

4) Formulation of vitamins stock solution (10OX concentrate): Nicotinic acid 0.1 g

VitaminB 1 (Thiamine HCl) 0.1 g

VitaminBό (Pyridoxine HCl) 0.1 g

Glycine 0.2 g

Inositol 10 g

Distilled water was added to dissolve the compounds and the resulting solution was brought to 1000 ml and stored at 4°C for use.

5) Formulation of MS macroelements mother solution (1OX concentrate): NH₄NO₃ 16.5 g

KNO₃ 19.O g

KH₂PO₄ 1.7 g

MgSO₄ '7H₂O 3.7 g

CaCl₂ '2H₂O 4.4 g

6) Formulation of MS microelements mother solution (10Ox concentration): KI 0.083 g

H₃BO₃ 0.62 g

MnSO₄ '4H₂O 0.86 g

Na₂MoO₄ '2H₂O 0.025 g

CuSO₄ '5H₂O 0.0025 g

7) Formulation of 2,4-D stock solution (1 mg/ml):

100 mg 2,4-D was weighed and dissolved in 1 ml 1 N potassium hydroxide for 5 minutes, then 10 ml distilled water was added for complete dissolution. The resulting solution was brought to 100 ml and stored at room temperature.

8) Formulation of 6-BA stock solution (1 mg/ml):

100 mg 6-BA was weighed and dissolved in 1 ml 1 N potassium hydroxide for 5 minutes, then 10 ml distilled water was added for complete dissolution. The resulting solution was brought to 100 ml and stored at room temperature.

9) Formulation of NAA stock solution (1 mg/ml):

100 mg NAA was weighed and dissolved in 1 ml 1 N potassium hydroxide for 5 minutes, then 10 ml distilled water was added for complete dissolution. The resulting solution was brought to 100 ml and stored at 4⁰C for use.

10) Formulation of IAA stock solution (1 mg/ml):

100 mg IAA was weighed and dissolved in 1 ml 1 N potassium hydroxide for 5 minutes, then 10 ml distilled water was added for complete dissolution. The resulting solution was brought to 100 ml and stored at 4°C in a large Erlenmeyer flask added with 300 ml distilled water and 2.78g FeSO₄ •7H₂O. In another large Erlenmeyer flask 300 ml distilled water was added.

11) Formulation of glucose stock solution (0.5 g/ml):

125 g glucose was weighed and dissolved with distilled water. The resulting solution was brought to 250 ml, sterilized and stored at 4°C for use.

12) Formulation of AS stock solution:

0.392g AS was weighed and 10 ml DMSO was measured, then they were transferred to a 1.5ml centrifuge tube and stored at 4°C for use.

13) Formulation of 1 N potassium hydroxide stock solution:

5.6 g potassium hydroxide was weighed and dissolved with distilled water. The resulting solution was brought to 100 ml and stored at room temperature for use.

(3) Culture Media Formulae for Genetic Transformation of Rice

1) Induction Culture Medium:

N6max mother solution ( 1 OX) 100 ml

N6mix mother solution ( 100X) 10 ml

Fe²⁺EDTA stock solution ( 100X) 10 ml

Vitamins stock solution ( 100X) 10 ml

2,4-D stock solution 2.5 ml

Proline 0.3 g

CH 0.6 g

Sucrose 3O g

Phytagel 3 g

Distilled water was added to a volume of 900 ml, and the pH value was adjusted to 5.9 with 1 N potassium hydroxide. The resulting mixture was boiled and brought to 1000 ml. The resulting medium was dispensed into 50 ml Erlenmeyer flasks (25 ml/flask), and the flasks were sealed and sterilized.

2) Secondary Culture Medium:

N6max mother solution ( 1 OX) 100 ml

N6mix mother solution ( 100X) 10 ml

Fe²⁺ EDTA stock solution (100X) 10 ml

Vitamins stock solution ( 100X) 10 ml

2,4-D stock solution 2.0 ml

Proline 0.5 g CH 0.6 g

Sucrose 30 g

Phytagel 3 g

3) Pre-culture Medium:

Nόmax mother solution (10X) 12.5 ml

N6mix mother solution ( 100X) 1.25 ml

Fe²⁺EDTA stock solution (100X) 2.5 ml

Vitamins stock solution ( 100X) 2.5 ml

2,4-D stock solution 0.75 ml

CH 0.15 g

Sucrose 5 g

Agarose 1.75 g

Distilled water was added to a volume of 250 ml, and the pH value was adjusted to 5.6 with 1 N potassium hydroxide. The resulting medium was sealed and sterilized.

Prior to use, the medium was heated to dissolve and 5 ml glucose stock solution and 250 μl AS stock solution were added. The resulting medium was dispensed into the culture dishes (25 ml/dish).

4) Co-culture medium:

Nόmax mother solution (10X) 12.5 ml

N6mix mother solution ( 100X) 1.25 ml

Fe²⁺EDTA stock solution (100X) 2.5 ml

Vitamins stock solution ( 100X) 2.5 ml

2,4-D stock solution 0.75 ml

CH 0.2 g

Sucrose 5 g

Agarose 1.75 g

Prior to use, the medium was heated to dissolve and 5 ml glucose stock solution and 250 μl AS stock solution were added. The resulting medium was dispensed into the culture dishes (25 ml/dish). 5) Suspension Medium:

N6max mother solution (10X) 5 ml

N6mix mother solution (100X) 0.5 ml

Fe²⁺EDTA stock solution ( 100X) 0.5 ml

Vitamins stock solution (100X) 1 ml

2,4-D stock solution 0.2 ml

CH 0.08 g

Sucrose 2 g

Distilled water was added to a volume of 100 ml, and the pH value was adjusted to 5.4. The resulting medium was dispensed into two 100 ml Erlenmeyer flasks and the flasks were sealed and sterilized.

Prior to use, 1 ml glucose stock solution and 100 μl AS stock solution were added.

6) Selective Medium:

N6max mother solution (10X) 25 ml

N6mix mother solution ( 100X) 2.5 ml

Fe²⁺EDTA stock solution ( 100X) 2.5 ml

Vitamins stock solution (100X) 2.5 ml

2,4-D stock solution 0.625 ml

CH 0.15 g

Sucrose 7.5 g

Agarose 1.75 g

Distilled water was added to a volume of 250 ml, and the pH value was adjusted to 6.0. The resulting medium was sealed and sterilized.

Prior to use, the medium was dissolved and 250 μl HN and 400 ppm CN were added. The resulting medium was dispensed into the culture dishes (25 ml/dish).

7) Pre-differentiation Medium:

N6max mother solution (10X) 25 ml

N6mix mother solution (100X) 2.5 ml

Fe²⁺EDTA stock solution ( 100X) 2.5 ml

Vitamins stock solution (100X) 2.5 ml

6-BA stock solution 0.5 ml

KT stock solution 0.5 ml NAA stock solution 50 μl

IAA stock solution 50 μl

CH 0.15 g

Sucrose 7.5 g

Agarose 1.75 g

Distilled water was added to a volume of 250 ml, and the pH value was adjusted to 5.9 with IN potassium hydroxide. The resulting medium was sealed and sterilized.

Prior to use, the medium was dissolved and 250 μl HN and 200 ppm CN were added. The resulting medium was dispensed into the culture dishes (25 ml/dish).

8) Differentiation Medium:

N6max mother solution ( 1 OX) 100 ml

N6mix mother solution ( 100X) 10 ml

Fe²⁺EDTA stock solution ( 100X) 10 ml

Vitamins stock solution (100X) 10 ml

6-BA stock solution 2 ml

KT stock solution 2 ml

NAA stock solution 0.2 ml

IAA stock solution 0.2 ml

CH I g

Sucrose 3Og

Phytagel 3g

Distilled water was added to a volume of 900 ml, and the pH value was adjusted to 6.0 with IN potassium hydroxide. The resulting mixture was boiled and brought to 1000 ml. The resulting medium was dispensed into 50 ml Erlenmeyer flasks (50 ml/flask), and the flasks were sealed and sterilized.

9) Rooting Medium:

MSmax mother solution (10X) 50 ml

MSmix mother solution (100X) 5 ml

Fe²⁺EDTA stock solution ( 100X) 5 ml

Vitamins stock solution (100X) 5 ml

Sucrose 30 g

Phytagel 3 g

Distilled water was added to a volume of 900 ml, and the pH value was adjusted to 5.8 with IN potassium hydroxide. The resulting mixture was boiled and brought to 1000 ml. The resulting medium was dispensed into the rooting tubes (50 ml/tube), and the tubes were sealed and sterilized. (4) Steps of Genetic Transformation Mediated by Agrobacterium

4.1 Callus Induction

(1) Mature rice seeds of "ZHONGHUA No. 11" (Institute of Crop Science, Chinese Academy of Agricultural Sciences) were husked, and then were successively treated with 70% alcohol for 1 minute and surface-disinfected with 0.15% HgCl₂ for 15 minutes;

(2) The seeds were washed with sterilized water for 4-5 times;

(3) The seeds were put onto the induction medium;

(4) The seeded medium was placed in darkness for 4- week culture at 25±1°C.

4.2 Callus Subculture

The bright yellow, compact and relatively dry embryogenic callus was selected, put onto the secondary culture medium, and cultured in darkness for 2 weeks at 25±1°C.

4.3 Pre-culture

The compact and relatively dry embryogenic callus was selected, put onto the pre-culture medium, and cultured in darkness for 2 weeks at 25±1°C.

4.4 Agrobacrium Culture

(1) Agrobacterium EHA 105 (a strain commercially available from Cambia) was pre-cultured on the LA culture medium with corresponding resistance selection at 28°C for 2 days;

(2) The Agrobacterium was transferred to the suspension medium and cultured on the shaking table at 28°C for 2-3 hours.

4.5 Agrobacterium Infestation

(1) The pre-cultured callus was transferred into a sterilized bottle;

(2) The Agrobacterium suspension was adjusted to OD600 0.8-1.0;

(3) The callus was immersed in the Agrobacterium suspension for 30 minute;

(4) The callus was transferred onto a sterilized filter paper and dried, and then put onto the co-culture medium for 3-day culture at 19-2O⁰C.

4.6 Washing and Selective Culture of Callus

(1) The callus was washed with sterilized water until no Agrobacterium was observed;

(2) The callus was immersed in sterilized water containing 400 ppm carbenicillin (CN) for 30 minutes;

(3) The callus was transferred onto a sterilized filter paper and dried; (4) The callus was transferred onto the selective medium and selectively cultured for 2-3 times, 2 weeks for each time. (The screening concentration of hygromycin was 400 mg for the first culture and 250 ppm for the latter cultures).

4.7 Differentiation

(1) The resistant callus obtained was transferred to the pre-differentiation medium, and cultured in darkness for 5-7 weeks;

(2) The callus obtained from the pre-differentiation culture was transferred to the differentiation medium, and cultured in the culture room at 26°C, 14 hours under light (light intensity 1,000-1,500 Ix) and 10 hours in darkness every day.

4.8 Rooting

(1) The roots of the callus generated during differentiation were cut off;

(2) The callus was then transferred to the rooting medium, and cultured at 26°C under light for 2-3 weeks, with the culture conditions same as those of step (2) in 4.7.

4.9 Transplantation

The residual medium on the roots of the callus was washed off, and the seedlings with good roots were transferred into the greenhouse. The greenhouse was maintained moisturized in the first few days.

Example 4: Cold tolerance screening of the OsCIPK03 gene transgenic T2 family at seedling stage

In order to verify whether the cold tolerance of the transgenic rice plants was enhanced and whether such an enhancement was associated with the introduced OsCIPK03 gene, the expression of the OsCIPK03 gene in the transgenic rice plants was detected using Northern hybridization technology (Fig.4A shows the results of Northern hybridization, the hybridization method was the same as used in Example 2), and part of the families of T2 generation plants of the present invention was screened for the cold tolerance. Particular steps were as followings. The seeds of the T2 generation families were germinated in the rooting media containing 50 mg/ml hygromycin for 5 days. Seedlings having substantially the same level of germination were transplanted into small red pails, and wild type control plants were also cultivated. It was found that the transgenic families and the wild type controls showed no difference in development and growth (Fig. 4B). When the plants grew to the 4-leaf stage, they were subjected to low temperature treatment at 4°C for 5 days (24 hours per day). At day 5 no significant difference was observed in the phenotype of either the transgenic plants or the control plants. However, when they were shifted to normal conditions to recover growth for 5 days, it was observed that most of the control plants died while most of the transgenic plants survived (Fig. 4C). In another group of experiments, the plants at the 4-leaf stage were subjected to low temperature treatment at 4⁰C for 5 days (24 hours per day) followed by recovery growth for 7 days. Statistics of the survival rate of the plants showed that the survival rate of the plants over-expressing OsCIPK03 (68.2%-90.4%) was significant different from that of the control plants (18.5%) (Fig. 4D). This proved that the OsCIPK03 gene was without doubt associated with cold tolerance and its over-expression could enhance the cold tolerance of transgenic plants, and that the enhancement of tolerance of transgenic rice plants was surely associated with the introduced OsCIPK03 gene.

Example 5: Determination of proline content in the OsCIPK03 gene transgenic T2 family

Proline content in plants increases significantly under stresses. Content of proline in plants reflects to some extent the stress tolerance of the plant. Increase of proline content in plant tissues under low temperature enhances the cold tolerance of the plant. During the experiments, the transgenic rice plants of the present invention, when grown to the 4-leaf stage, were subjected to low temperature stress at 4 ⁰C, and samples were taken at day 0, 1, 3, 6 to determine the proline content. The experimental results, as shown in Fig. 5, indicated that the proline content in the OsCIPK03 over-expressing family under cold stress was 2-4 times that of the wild type control. These results demonstrated that the over-expression of the cloned OsCIPK03 gene of the present invention led to the increase of proline content, and hence to the significant enhancement of the cold tolerance of the transgenic rice plants.

Determination of proline content is based on the following principle. Extraction of plant samples with sulfosalicylic acid releases the proline into the solution of sulfosalicylic acid. Treatment of the solution by acidic ninhydrin under heat turns the solution red. All the pigments are transferred into the toluene phase by extraction with toluene, with the shade of the pigment being a representation of the content of the proline. The proline content is determined by colorimetry at 520 nm, followed by reference to the standard curve or calculation using the regression equation.

Specifically, the proline content was determined as follows:

5.1 Materials, equipments and reagents

5.1.1 Materials: The materials for determination were the leaves of the transgenic rice plants of the present invention and the leaves of the non-transgenic rice plants.

5.1.2 Equipment: 1. Type 722 spectrophotometer; 2. Mortar; 3. 100 ml small beaker; 4. Volumetric flask; 5. Large test tube; 6. Common test tube; 7. Pipette; 8. Syringe; 9. Water bath; 10 Funnel; 11. Funnel stand; 12. Filter paper; 13. Scissor.

5.1.3 Reagents: 1. Acidic ninhydrin solution: 1.25 g ninhydrin was added into 30 ml of glacial acetic acid and 20 ml of 6mol/L phosphoric acid, and heated (7O⁰C) under stirring to dissolve. The resulting solution was stored in a refrigerator for use; 2. 3% sulfosalicylic acid: 3 g sulfosalicylic acid was dissolved in distilled water and then the volume was brought up to 100 ml; 3. Glacial acetic acid; 4. Toluene.

5.2 Experiment procedures:

5.2.1 Preparation of standard curve: (1) 25 mg proline was accurately weighed on an analytical balance, transferred into a small beaker and dissolved with a small amount of distilled water. The resulting solution was transferred into a 250 ml volumetric flask and brought to the graduation line with distilled water. This standard proline solution contained 100 μg proline per ml. (2) Serial dilutions of the proline solution was prepared by transferring 0.5, 1.5, 2.0, 2.5 and 3.0 ml of the proline stock solution to six 50 ml volumetric flasks respectively, and bringing to the graduation line with distilled water and shaking well. The proline concentrations in the flasks were 1, 2, 3, 4, 5 and 6 μg/ml respectively. (3) Six test tubes were each charged with 2 ml of the respective serial dilutions of the proline solution as well as 2 ml of glacial acetic acid and 2 ml of acidic ninhydrin, and each tube was heated in a boiling water bath for 30 minutes. (4) After cooling, each tube was accurately added with 4 ml toluene and shaken for 30 seconds, then was let stand still for a while for all the pigment to transfer to the toluene phase. (5) The upper proline-toluene solution in each tube was transferred into a cuvette using a syringe, and spectro-photometered at 520 nm using toluene solution as the blank. (6) The standard curve was plotted from the calculated regression equation representing change of absorbance (Y) with proline concentration (X), and it was used for the calculation of the proline content in a 2 ml test sample solution (μg/2ml).

5.2.2 Determination of the sample: (1) Extraction of Proline: 0.5 g of each of the test leaves subjected to different treatments were accurately weighed and placed into large test tubes. 5 ml of 3% sulfosalicylic acid solution was then added into each tube and extraction was conducted in a boiling water bath for 10 minutes (with the tubes being shaken frequently). After cooling, each of the extraction solutions was filtered into a clean test tube, and the each of the filtrates was the proline extract. (2) 2 ml of each of the extracts was transferred into a separate clean test tube with glass plug and 2 ml of glacial acetic acid and 2 ml of acidic ninhydrin were added. The tubes were heated in a boiling water bath for 30 minutes and the solutions turned red. (3) After cooling, 4 ml of toluene was added into each tube, and the tubes were shaken for 30 seconds and then let them stand still for a while. The upper solution from each tube was transferred into a 10 ml centrifuge tube and centrifuged at 3,000 rpm for 5 minutes. (4) The upper red proline-toluene solution in each centrifuge tube was pipetted into a cuvette and spectro-photometered at 520 nm using toluene solution as the blank to obtain the absorbance.

5.2.3 Calculation: The proline content in the 2 ml test sample solution (X μg/ml) was calculated by using the regression equation (or referring to the standard curve), and the percentage of proline in the sample was thereby calculated. The proline content (μg/g sample) was calculated by the following formula:

Proline content (μg/g sample) = [X x 5 / 2] / sample weight (g)

The results of this example are shown in Fig. 5.

Example 6: Determination of the expression amounts of genes related to proline synthesis and transportation in the wild-type and OsCIPK03 gene transgenic family

The increase in proline content may be due to the change in the expression amounts of genes related to proline synthesis and transportation. Therefore we used real-time PCR to determine the expression amounts of two genes related to proline synthesis and two genes related to proline transportation in the wild-type and OsCIPK03 gene transgenic family. Experiment results showed that the expression amounts of these four genes were increased to a different degree (5- to 10-fold) in the OsCIPK03 over-expressing plants. Experiment results as shown in Fig. 6 indicate that the over-expression of OsCIPK03 increased the expression of genes related to proline synthesis and transportation, thereby leading to the increase in proline content and hence to the enhancement of cold tolerance of the transgenic plants. The experiment was performed on an ABI 7500 Realtime PCR apparatus. The reaction system was 10 μl of 2^χ SYBR Green Master Mix Reagent (Applied Biosystems), 1.0 μl of cDNA templates, 200 nM gene-specific primers, with a total volume of 20 μl. The reaction conditions were: the first step, 95 ⁰C 3 min; the second step, 95 ⁰C 30 sec, 60 ⁰C 30 sec and 72 ⁰C 1 min, for 40 cycles. The GeneBank accession numbers for these four genes are: AK102633, AKl 01230, AK067118, AK0666298, respectively. The primers used in the real-time PCR detection were respectively: for AK102633, S'-CTCAAATCAAGGCGTCAACTAAGA-S' and 5'-TTTGTCAATATATACGTGGCATATACCA-S', for AK101230, 5'-CGCCCCTCCCCGTATCT-3' and 5'-AGGAATGCGGCAACAAGTG-S', for AK067118, 5^I-AGGGACGATGGAGTTCTAAAGCT-3^I and 5'-GGGATTCCAAAGGCAAAAAGA-S', and for AK0666298,

S'-GAGGAGGCTACCTGACTGTCAAC-S' and 5'-GCTCATGAAGTCGCCAAGGA-S'. The housekeeper gene in rice, i.e. Actin 1 (GeneBank accession number X 16280) was used as the internal control in PCR, with primers being 5'-TGGCATCTCTCAGCACATTCC-3 and ^I-TGCACAATGGATGGGTCAGA-3^t.

The results of this example are shown in Fig. 6.

Claims

1. A transformed plant comprising a recombinant DNA construct comprising a promoter functional in a plant cell positioned to provide for expression of a polynucleotide having a sequence with at least about 70%, 75%, 80%, 85%, 90%, or 95% identity to SEQ ID NO:1 or to a functionally active fragment thereof.

2. A transformed plant comprising a recombinant DNA construct comprising a promoter functional in a plant cell positioned to provide for expression of a polynucleotide encoding a polypeptide at least about 70%, 75%, 80%, 85%, 90%, or 95% identical to that encoded by the polynucleotide sequence of SEQ ID NO: 1.

3. The transformed plant according to claims 1 or 2, wherein said plant is a crop plant.

4. A method of producing a transformed plant having an improved property, wherein said method comprises transforming a plant with a recombinant construct comprising a promoter functional in a plant cell positioned to provide for expression of a polynucleotide encoding a polypeptide useful for improving plant cold tolerance, drought tolerance, or salt tolerance, wherein said polynucleotide has a sequence with at least about 70%, 75%, 80%, 85%, 90%, or 95% identity to SEQ ID NO:1 or to a functionally active fragment thereof.

5. A method of producing a transformed plant having an improved property, wherein said method comprises transforming a plant with a recombinant construct comprising a promoter functional in a plant cell positioned to provide for expression of a polynucleotide encoding a polypeptide useful for improving plant cold tolerance, drought tolerance, or salt tolerance, wherein said polypeptide is at least about 70%, 75%, 80%, 85%, 90%, or 95% identical to that encoded by the polynucleotide sequence of SEQ ID NO: 1.

6. The method according to claims 4 or 5, wherein said transformed plant is a crop plant.

7. A plant exhibiting an improved property as compared to the control plant, wherein the altered trait is selected from the group consisting of greater cold tolerance, greater tolerance to water deprivation, and greater salt tolerance, or combinations thereof, wherein the plant has greater expression or activity of a polypeptide encoded by a polynucleotide that has at least about 70%, 75%, 80%, 85%, 90%, or 95% identity to SEQ ID NO: 1 or to a functionally active fragment thereof.

8. A plant exhibiting an improved property as compared to the control plant, wherein the altered trait is selected from the group consisting of greater cold tolerance, greater tolerance to water deprivation, and greater salt tolerance, or combinations thereof, wherein the plant has greater expression or activity of a polypeptide at least about 70%, 75%, 80%, 85%, 90%, or 95% identical to that encoded by the polynucleotide sequence of SEQ ID NO: 1.

9. The transformed plant according to claims 8 or 9, wherein said plant is a crop plant.