CN101200724A - Improving Plant Cold Tolerance Using Rice Protein Kinase Gene OsCIPK03 - Google Patents

Abstract

The present invention belongs to the technical field of plant gene engineering and concretely relates to the separation clone of paddy DNA segment OsCIPK03 related to cold tolerance and a function validation and an application thereof. The concrete step is that a complete translated region (Coding sequence) of gene is combined with the combined type promoter (CaM 35S) of tobacco flower and grass virus and then is shifted to the paddy directly to ensure that the cold tolerance capability of a transgenic paddy plant is improved obviously. Cloned OsCIPK03 gene has a DNA sequence which is shown in the positions from 1 to 1707 of the sequence list SEQ ID NO: 1 or the DNA sequence of protein, the coding of which is the same as the protein coded by SEQ ID NO: 1.

Description

Improving Plant Cold Tolerance Using Rice Protein Kinase Gene OsCIPK03

technical field

The invention relates to the field of plant biotechnology. It specifically relates to the isolation and cloning, functional verification and application of a rice DNA fragment (gene). Said gene is related to plant cold tolerance. The complete translation region (Coding sequence) of the gene was combined with the constitutive promoter of tobacco mosaic virus (CaMV35S) and then directly transferred into normal plants, and the cold tolerance of the transgenic plants was significantly improved.

Background technique

Plants are affected by many environmental factors during the growth process. Drought, salt damage and low temperature often lead to large-scale crop yield reduction, which is the bottleneck of agricultural development in many areas. In order to resist or adapt to unfavorable environmental factors, the plant senses changes in the extracellular environmental conditions and transmits them into the cells through various channels, which will induce the expression of some response genes, and produce some genes that can protect the cells from stresses such as drought, high salt, and low temperature. Damaged functional proteins, osmotic regulators, and transcription factors that transmit signals and regulate gene expression, so as to respond to external changes (Xiong et al. Cell signaling during cold, drought and salt stress. Plant Cell.14 (suppl), S165-S183, 2002). The expression of those functional genes plays an important role in the plant's response to the environment and ultimately its survival. At present, it has been found in many plants that protein kinase families such as CDPK, CIPK, and MAPK play an important role in the transmission of plant stress signals and the adaptability to stress. These protein kinase genes can be induced to express or inhibited under different stresses, so it is considered that these protein kinase genes play a very important role in the process of plant response to stress. Therefore, the isolation and identification of such functional genes that can improve the plant's response to stress is of great significance to the genetic improvement of crop stress resistance and breeding. At present, people have tried to improve plant resistance. The transgenic Arabidopsis plants cultivated by DREB1A and DREB2A have stronger low temperature tolerance, drought tolerance and high salt tolerance than wild type (Liu Q et al. Two transcription factors, DREB1 and DREB2 , with an EREBP/AP2 DNA domains separate two cellular signal thansduction pathways in drought-andlow-temperature-responsive gene expression, respectively, in Arabidopsis. Plant Cell. 1998, 10: 1391-1406.). The Thomashow MF research team at Michigan State University in the United States used the CBF1 gene of Arabidopsis thaliana for genetic transformation and also bred plants with enhanced cold tolerance.

Rice is one of the most important food crops. Cold-tolerant rice is of great significance to us. Therefore, finding out the functional genes related to cold resistance and cultivating cold-resistant and cold-resistant varieties is of great significance to improve rice yield.

Contents of the invention

The purpose of the present invention is to isolate and clone a DNA segment containing a complete coding segment of a cold tolerance-related gene from rice, and use this gene to improve the stress resistance of rice or other plants, especially through transgenics, to improve the cold tolerance of transgenic plants. Structural analysis of this gene belongs to the plant CIPK protein kinase family and is related to stress. The cloned gene was named OsCIPK03.

The present invention relates to the isolation and application of a DNA segment containing OsCIPK03 gene, which endows plants with enhanced tolerance under low temperature and other adversity conditions. Wherein, the fragment is as shown in the sequence table SEQ ID NO: 1, or is basically equivalent to the highly homologous DNA sequence shown in SEQ ID NO: 1, or its function is equivalent to a subunit of the sequence shown in SEQ ID NO: 1 fragment.

The cloned OsCIPK03 gene can be used as a probe to obtain the gene or homologous gene of the present invention by screening from cDNA and genome libraries. Similarly, PCR (polymerase chain reaction) technology can also be used to amplify the OsCIPK03 gene of the present invention and any interested section of DNA or a section of DNA homologous thereto from genome, mRNA and cDNA. Using the above techniques, the sequence containing the OsCIPK03 gene can be isolated, and the sequence can be transformed with any expression vector that can guide the expression of foreign genes in plants to transform plants, and transgenic plants with enhanced tolerance to low temperature stress can be obtained. When the gene of the present invention is constructed into a plant expression vector, any strong promoter or inducible promoter is added before its transcription initiation nucleotide. When the gene of the present invention is constructed into a plant expression vector, enhancers can also be used. These enhancer regions can be ATG start codons and adjacent region start codons, etc., but must be the same as the reading frame of the coding sequence, to Guaranteed translation of the entire sequence.

The expression vector carrying the OsCIPK03 gene of the present invention can be imported into plant cells by conventional biotechnological methods such as Ti plasmid, plant virus vector, direct DNA transformation, microinjection, electroporation (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 including the OsCIPK03 gene of the present invention can be used to transform the host into various plants including rice to cultivate cold-resistant plant varieties.

The gene of the present invention is induced by stress, so its promoter is an inducible promoter. The promoter segment of the present invention and any gene of interest are simultaneously connected into a suitable expression vector, and transformed into a plant host. Under the condition of inducible expression gene, improve the plant's tolerance to adversity.

The present invention will be further described below in conjunction with specific embodiments.

Description of drawings

Sequence listing SEQ ID NO: 1 shows the sequence of the DNA fragment containing the coding region of the OsCIPK03 gene isolated and cloned in the present invention.

Figure 1: Flow chart of OsCIPK03 gene isolation and identification.

Figure 2: Northern hybridization was used to detect the expression level of OsCIPK03 gene at different time points of drought, high salt, low temperature, PEG and ABA and other adversity stresses.

Figure 3: Vectors used for genetic transformation of rice. The OsCIPK03 overexpression vector was obtained by replacing the attR1-attR2 part in the figure with the sequence of the target gene OsCIPK03 through LR recombination reaction.

Figure 4: Overexpression of OsCIPK03 gene in rice can improve the ability of plants to tolerate low temperature. A, OsCIPK03 gene expression in transgenic plants, the last one is the control, and the rest are independent transgenic plants. B, the growth of the transgenic family and the wild-type control under normal conditions; C, the growth of the transgenic family and the wild-type control after low temperature stress (4°C stress for 5 days, normal growth for 5 days); D, the transgenic family and the wild-type control Survival rate statistics after low temperature stress (4°C stress for 4 days, normal growth for 7 days).

Figure 5: Overexpression of OsCIPK03 gene increases proline content in plants. When the plants grew to the four-leaf stage, they were subjected to low temperature stress at 4°C, and samples were taken at 0, 1, 3, and 6 days for the determination of proline content.

Figure 6: Detection of the expression levels of proline synthesis and transport-related genes in OsCIPK03 overexpression plants.

Detailed ways

The preliminary work of the present invention obtained the cDNA clone EI101L12 derived from rice variety Minghui 63 (a rice variety widely popularized in China). The cDNA is the full-length cDNA of the OsCIPK03 gene, which is a stress-resistance-related gene. The main basis is as follows: (1) cDNA clone EI101L12 was found to be effective in the drought stress treatment of the rice variety "Zhonghan 5" (a rice variety publicly provided by the Shanghai Academy of Agricultural Sciences, China) by using cDNA chip technology analysis. Day expression increased 2.5 times. It was sequenced and analyzed to find that the gene is OsCIPK03 (Genebank accession number is AK111929). In view of the obvious difference in the expression of this clone after drought treatment and its functional characteristics, it is considered that the gene represented by the EI101L12 clone is involved in the regulation of gene expression under stress. (2) The expression profile analysis was carried out under stress conditions (Fig. 2), and it was found that the expression level was significantly increased during the stress treatment. (3), the full-length gene is overexpressed in the plant, and the cold tolerance ability of the transgenic plant is greatly enhanced ( FIG. 4 ). These results indicated that the OsCIPK03 gene is a stress-related regulatory gene involved in the regulation of plants to stress.

The following examples further define the present invention, and describe the method for isolating and cloning a DNA fragment containing the complete coding segment of the OsCIPK03 gene and verifying the function of the OsCIPK03 gene based on the previous work of the present invention (the flow chart of the invention is shown in FIG. 1 ). From the following descriptions and these examples, those skilled in the art can ascertain the essential characteristics of the present invention, and without departing from the spirit and scope of the present invention, various changes and modifications can be made to the present invention so as to be applicable to various uses and conditions.

Example 1: Isolation and cloning of DNA fragments comprising OsCIPK03 gene segments

Through the analysis of the drought-induced gene expression profile of the rice variety "Zhonghan 5" (a publicly used rice variety provided by the Shanghai Academy of Agricultural Sciences in China), it was found that a drought-induced gene was strongly induced (the expression level increased by 2.5 times in the late stage of drought stress) The EST (Expressed Sequence Signature) of the above) was found through sequence analysis that the gene is a member of the CIPK protein kinase family and is a full-length sequence, which corresponds to the Japanese rice full-length database (http://cdna01.dna.affrc. go.jp) cDNA clone 001-015-H02. According to the cloned sequence, primers OsCIPK03F (5'-CACCATGTATAGGGCTAAGAGGGCT-3', sequence-specific primer plus adapter CACC site) and OsCIPK03R (5'-TTGAAACTCACAAACTGTCA-3') were designed, and the 1-1653bp sequence of the clone was obtained from the No. 5" variety was reverse-transcribed and amplified. The amplified product is the sequence 1-1653bp of the present invention ( FIG. 3 ). The specific steps are: use TRIZOL reagent (purchased from Invitrogen Company) to extract leaf total RNA from the rice variety "Zhonghan No. 5" treated with drought stress (extraction method is according to the above-mentioned TRIZOL reagent instruction manual), use reverse transcriptase (purchased from Invitrogen company) to synthesize the first strand cDNA by reverse transcription. The nested primers designed according to the sequence of the cDNA clone 001-015-H02 were amplified from the reverse transcription product. The reaction conditions were: 94°C for 2 min; 94°C for 30 sec, 55°C for 30 sec, 72°C for 2 min, and 30 cycle; 72°C extension for 5 min. The amplified PGR product was ligated into the pGEM-T vector (purchased from Promega), positive clones were screened and sequenced to obtain the desired full-length gene. This clone was named PGEM-OsCIPK03.

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

The rice variety "Zhonghan 5" was used as the material, and it was subjected to drought, chilling injury, high-salt stress, and abscisic acid (ABA) and polyethylene glycol (PEG) treatments at the 3-leaf stage. In drought treatment, the rice seedlings were cut off from water, and samples were taken after 0h, 3h, 6h, 12h, and 24h. The chilling injury treatment is to put the rice seedlings in a 4°C growth box, and take samples after 0h, 3h, 6h, 12h, and 24h. For high salt stress, the roots of the seedlings were soaked in 200mM/L NaCl solution and samples were taken after 0h, 5h, 14h, and 24h. For ABA treatment, the roots of the seedlings were soaked in 100 μM/L ABA solution and samples were taken after 0h, 3h, 6h, 12h and 24h. PEG treatment is to soak the roots of seedlings in 20% PEG6000 solution and take samples after 0h, 3h, 5h and 12h. After extracting the total RNA (Trizol reagent, purchased from Invitrogen Company) of the leaves, carry out the RNA transfer membrane according to the relevant experimental operation method of "Molecular Cloning" (J. Sambrook, Science Press, Beijing, 1999 edition), and use OsCIPK03 as the probe Needle to do Northern hybridization. The results show that the gene OsCIPK03 cloned in the present invention can be induced and expressed by drought, chilling injury, high salt, ABA and PEG (as shown in FIG. 2 ), and is a protein kinase related to stress.

Embodiment 3, construction of OsCIPK03 gene overexpression vector, transformation

According to the result of embodiment 2, it is known that gene OsCIPK03 of the present invention can be induced by drought, cold damage, high salt, abscisic acid (ABA) and polyethylene glycol (PEG), in order to better clarify the function of this gene, The applicant overexpressed it in rice and verified it from the phenotype of transgenic plants. The method is: first use the positive clone pGEM-OsCIPK03 plasmid obtained in Example 1 as a template, use the primers and conditions in Example 1 to amplify the foreign fragment by PCR, and import the foreign fragment into the intermediate vector pENTR/D-TOPO (The vector was purchased from Invitrogen Company, see the kit instructions for specific steps), and the target gene was further recombined into the genetic gene carrying the tobacco mosaic virus promoter (CaM35S) through LR recombination reaction (refer to Invitrogen Company's LR reaction recombination kit instruction manual) Transformation vector pCB2004H. Transform Escherichia coli DH10β (the strain was purchased from Invitrogen). Positive clones were screened to obtain transformation vectors.

It was introduced into the rice variety Zhonghua 11 (a publicly used rice variety provided by the Institute of Crop Science, Chinese Academy of Agricultural Sciences) through the Agrobacterium-mediated rice genetic transformation system, after pre-cultivation, infection, co-cultivation, and screening The callus with hygrotoxin resistance, differentiated, rooted, and transplanted to obtain transgenic plants. The obtained transgenic rice plants were named T35. The present invention obtains 23 independent transgenic rice plants in total.

The genetic transformation system of rice (subspecies japonica) mediated by Agrobacterium adopts the transformation system established by the State Key Laboratory of Crop Genetic Improvement where the applicant works. The specific steps of the system are as follows:

(1) Abbreviation of reagents and solutions

The abbreviation of the plant hormone used in culture medium among the present invention is as follows: 6-BA (6-BenzylaminoPurine, 6-benzyl adenine); CN (Carbenicillin, carbenicillin); KT (Kinetin, kinetin); NAA ( Napthalene acetic acid, naphthalene acetic acid); IAA (Indole-3-acetic acid, indole acetic acid); 2,4D (2,4-Dichlorophenoxyacetic acid, 2,4-dichlorophenoxyacetic acid); AS (Acetosringone, acetyl clove Ketone); CH (Casein Enzymatic Hydrolysate, hydrolyzed casein); HN (Hygromycin B, hygromycin); DMSO (Dimethyl Sulfoxide, dimethyl sulfoxide); N6max (N6 macro-component solution); N6mix (N6 micro-component solution ); MSmax (MS macrocomponent solution); MSmix (MS microcomponent solution)

(2) Main solution formula

1) Preparation of N6 medium macroelement mother solution [10 times concentrated solution (10X)]:

Potassium nitrate (KNO ₃ ) 28.3g

Potassium dihydrogen phosphate (KH ₂ PO ₄ ) 4.0g

Ammonium sulfate ((NH ₄ ) ₂ SO ₄ ) 4.63g

Magnesium sulfate (MgSO ₄ ·7H ₂ O) 1.85g

Calcium chloride (CaCl ₂ 2H ₂ O) 1.66g

Dissolve one by one, then dilute to 1000ml at room temperature.

2) Preparation of N6 medium trace element mother solution [100 times concentrated solution (100X)]

Potassium iodide (KI) 0.08g

Boric acid (H ₃ BO ₃ ) 0.16g

Manganese sulfate (MnSO ₄ 4H ₂ O) 0.44g

Zinc sulfate (ZnSO ₄ ·7H ₂ O) 0.15g

Dissolve at room temperature and dilute to 1000ml.

3) Preparation of iron salt (Fe ₂ EDTA) stock solution (100X)

Prepare 800ml of double distilled water and heat it to 70°C, add 3.73g of disodium ethylenediammonium tetraacetate (Na ₂ EDTA·2H ₂ O), fully dissolve it and keep it in a water bath at 70°C for 2 hours, dilute to 1000ml, set at 4°C Save for later.

4) Preparation of vitamin stock solution (100X)

Nicotinic acid 0.1g

Vitamin B1 (Thiamine HCl) 0.1g

Vitamin B6 (Pyridoxine HCl) 0.1g

Glycine 0.2g

Inositol 10g

Add water to make up to 1000ml, and store at 4°C for later use.

5) Preparation of MS medium macroelement mother solution (10X)

Ammonium nitrate (NH ₄ NO ₃ ) 16.5g

Potassium nitrate 19.0g

Potassium dihydrogen phosphate 1.7g

Magnesium Sulfate 3.7g

Calcium chloride 4.4g

Dissolve at room temperature and dilute to 1000ml.

6) Preparation of MS medium trace element mother solution (100X)

Potassium iodide 0.083g

Boric acid 0.62g

Manganese sulfate 0.86g

Sodium molybdate (Na ₂ MoO ₄ 2H ₂ O) 0.025g

Copper sulfate (CuSO ₄ 5H ₂ O) 0.0025g

Dissolve at room temperature and dilute to 1000ml.

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

Weigh 100mg of 2,4-D, dissolve in 1ml 1N potassium hydroxide for 5 minutes, then add 10ml of distilled water to dissolve completely, then dilute to 100ml, and store at room temperature.

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

Weigh 100mg of 6-BA, dissolve in 1ml 1N potassium hydroxide for 5 minutes, then add 10ml of distilled water to dissolve completely, then dilute to 100ml and store at room temperature.

9) Preparation of naphthaleneacetic acid (NAA) stock solution (1mg/ml):

Weigh 100mg of NAA, dissolve it with 1ml 1N potassium hydroxide for 5 minutes, then add 10ml of distilled water to dissolve completely, then dilute to 100ml, store at 4°C for later use.

10) Preparation of indole acetic acid (IAA) stock solution (1mg/ml):

Weigh 100mg of IAA, dissolve it in 1ml 1N potassium hydroxide for 5 minutes, then add 10ml of distilled water to dissolve completely, then adjust the volume to 100ml, store at 4°C, add 300ml of distilled water and ferric sulfate (FeSO ₄ 7H ₂ O ) 2.78g. Add 300ml of distilled water to another large triangular flask.

11) Preparation of glucose storage solution (0.5g/ml):

Weigh 125g of glucose, then dissolve it in distilled water to a volume of 250ml, and store it at 4°C after sterilization.

12) Preparation of AS stock solution:

Weigh 0.392g of AS and 10ml of DMSO, dispense into 1.5ml centrifuge tubes, and store at 4°C for later use.

13) 1N potassium hydroxide stock solution

Weigh 5.6g of potassium hydroxide, dissolve it in distilled water to 100ml, and store it at room temperature for later use.

(3) Medium formula for genetic transformation of rice

1) Induction medium

N6max mother solution (10X) 100ml

N6mix mother solution (100X) 10ml

Fe ²⁺ EDTA stock solution (100X) 10ml

Vitamin stock solution (100X) 10ml

2,4-D stock solution 2.5ml

Proline (Proline) 0.3g

CH 0.6g

Sucrose 30g

Phytagel 3g

Add distilled water to 900ml, adjust the pH value to 5.9 with 1N potassium hydroxide, boil and set the volume to 1000ml, dispense into 50ml Erlenmeyer flasks (25ml/bottle), seal and sterilize.

2) subculture medium

N6max mother solution (10X) 100ml

N6mix mother solution (100X) 10ml

Fe ²⁺ EDTA stock solution (100X) 10ml

Vitamin stock solution (100X) 10ml

2,4-D stock solution 2.0ml

Proline 0.5g

CH 0.6g

Sucrose 30g

Phytagel 3g

Add distilled water to 900ml, adjust the pH value to 5.9 with 1N potassium hydroxide, boil and set the volume to 1000ml, dispense into 50ml Erlenmeyer flasks (25ml/bottle), seal and sterilize. 3) Pre-medium

N6max mother solution (10X) 12.5ml

N6mix mother solution (100X) 1.25ml

Fe ²⁺ EDTA stock solution (100X) 2.5ml

Vitamin stock solution (100X) 2.5ml

2,4-D stock solution 0.75ml

CH 0.15g

Sucrose 5g

Agarose 1.75g

Add distilled water to 250ml, adjust the pH value to 5.6 with 1N potassium hydroxide, seal and sterilize.

Heat to dissolve the medium before use, add 5ml of glucose stock solution and 250μl of AS stock solution, and pour them into Petri dishes (25ml/dish).

4) Co-culture medium

N6max mother solution (10X) 12.5ml

N6mix mother solution (100X) 1.25ml

Fe ²⁺ EDTA stock solution (100X) 2.5ml

Vitamin stock solution (100X) 2.5ml

2,4-D stock solution 0.75ml

CH 0.2g

Sucrose 5g

Agar powder 1.75g

Add 250ml of distilled water, adjust the pH value to 5.6 with 1N potassium hydroxide, seal and sterilize.

Heat to dissolve the culture medium before use, add 5ml of glucose stock solution and 250μl of AS stock solution, and pour them into Petri dishes (25ml/dish).

5) Suspension medium

N6max mother solution (10X) 5ml

N6mix mother solution (100X) 0.5ml

Fe ²⁺ EDTA stock solution (100X) 0.5ml

Vitamin stock solution (100X) 1ml

2,4-D stock solution 0.2ml

CH 0.08g

Sucrose 2g

Add distilled water to 100ml, adjust the pH value to 5.4, divide into two 100ml Erlenmeyer flasks, seal and sterilize.

Add 1ml glucose stock solution and 100μl AS stock solution before use.

6) Select medium

N6max mother solution (10X) 25ml

N6mix mother solution (100X) 2.5ml

Fe ²⁺ EDTA stock solution (100X) 2.5ml

Vitamin stock solution (100X) 2.5ml

2,4-D stock solution 0.625ml

CH 0.15g

Sucrose 7.5g

Agar powder 1.75g

Add distilled water to 250ml, adjust the pH value to 6.0, seal and sterilize.

Dissolve the medium before use, add 250μl HN and 400ppm CN, aliquot and pour into Petri dishes (25ml/dish).

7) Pre-differentiation medium

N6max mother solution (10X) 25ml

N6mix mother solution (100X) 2.5ml

Fe ²⁺ EDTA stock solution (100X) 2.5ml

Vitamin stock solution (100X) 2.5ml

6-BA stock solution 0.5ml

KT stock solution 0.5ml

NAA stock solution 50μl

IAA stock solution 50μl

CH 0.15g

Sucrose 7.5g

Agar powder 1.75g

Add distilled water to 250ml, adjust the pH value to 5.9 with 1N potassium hydroxide, seal and sterilize.

Dissolve the medium before use, add 250μl HN and 200ppm CN, and pour into petri dishes (25ml/dish).

8) Differentiation medium

N6max mother solution (10X) 100ml

N6mix mother solution (100X) 10ml

Fe ²⁺ EDTA stock solution (100X) 10ml

Vitamin stock solution (100X) 10ml

6-BA stock solution 2ml

KT stock solution 2ml

NAA stock solution 0.2ml

IAA stock solution 0.2ml

CH 1g

Sucrose 30g

Phytagel 3g

Add distilled water to 900ml, and adjust the pH value to 6.0 with 1N potassium hydroxide.

Boil and set the volume to 1000ml, dispense into 50ml Erlenmeyer flasks (50ml/bottle), seal and sterilize.

9) Rooting medium

MSmax mother solution (10X) 50ml

MSmix mother solution (100X) 5ml

Fe ² EDTA stock solution (100X) 5ml

Vitamin stock solution (100X) 5ml

Sucrose 30g

Phytagel 3g

Add distilled water to 900ml, and adjust the pH value to 5.8 with 1N potassium hydroxide.

Boil and set the volume to 1000ml, dispense into rooting tubes (25ml/tube), seal and sterilize.

(4) Agrobacterium-mediated genetic transformation step

4.1 Callus induction

(1) Ripe rice seeds (Zhonghua 11, Institute of Crop Science, Chinese Academy of Agricultural Sciences) were dehulled, then treated with 70% ethanol for 1 minute, and 0.15% mercuric chloride (HgCl ₂ ) was used to sterilize the surface of the seeds for 15 minutes;

(2) Wash the seeds 4-5 times with sterilized water;

(3) seeds are placed on the induction medium;

(4) Culture the inoculated medium in a dark place for 4 weeks at a temperature of 25±1°C.

4.2 Callus subculture

Select bright yellow, compact and relatively dry embryogenic calli, and place them on the subculture medium for 2 weeks in the dark at a temperature of 25±1°C.

4.3 Pre-cultivation

Select compact and relatively dry embryogenic callus, put it on the pre-medium and culture it in the dark for 2 weeks at a temperature of 25±1°C.

4.4 Agrobacterium culture

(1) Pre-cultivate Agrobacterium EHA105 (derived from CAMBIA, a commercial strain) on LA medium with corresponding resistance selection for two days at 28°C;

(2) Transfer the Agrobacterium to the suspension medium, and culture on a shaker at 28° C. for 2-3 hours.

4.5 Agrobacterium infection

(1) transfer the pre-cultured callus to a sterilized bottle;

(2) adjust the suspension of Agrobacterium to OD ₆₀₀ 0.8-1.0;

(3) Soak the callus in the Agrobacterium suspension for 30 minutes;

(4) Transfer the callus to a sterilized filter paper and blot dry; then place it on a co-culture medium for 3 days at a temperature of 19-20°C.

4.6 Callus washing and selection culture

(1) Wash the callus with sterilized water until the Agrobacterium cannot be seen;

(2) Soak in sterilized water containing 400ppm carbenicillin (CN) for 30 minutes;

(3) transfer the callus to dry on the sterilized filter paper;

(4) Transfer the callus to selection medium for selection 2-3 times, each time for 2 weeks. (The screening concentration of hygromycin is 400ppm for the first time, and 250ppm for the second and subsequent times)

4.7 Differentiation

(1) transfer the resistant callus to the dark place on the pre-differentiation medium and cultivate it for 5-7 weeks;

(2) Transfer the pre-differentiation cultured callus to the differentiation medium, and culture in the light for 14 hours (illumination intensity 1000-1500 lx) every day at a temperature of 26° C. in the culture room, and culture in the dark for 10 hours.

4.8 Rooting

(1) cut off the roots produced during differentiation;

(2) Then transfer it to the rooting medium and cultivate it under the light for 2-3 weeks, and the culture condition is the same as that described in step (2) of 4.7.

4.9 Transplanting

Wash off the residual medium on the roots and transfer the seedlings with a good root system to the greenhouse while keeping them moist for the first few days.

Example 4: Screening of OsCIPK03 gene transgenic T2 family for cold tolerance at seedling stage

In order to verify whether the cold tolerance of transgenic rice plants is enhanced and whether its enhancement is related to the transferred OsCIPK03 gene, the present invention uses Northern hybridization technology to detect the expression of OsCIPK03 gene in transgenic rice plants (Fig. 4A is Northern hybridization (method is the same as in Example 2) result, and the part families of T2 generation plants of the present invention have been carried out cold tolerance screening.Concrete steps are as follows: the seed of T2 generation family germinates after 5 days in the rooting medium that contains 50mg/ml ampicillin, will germinate consistent seedlings Transplanted into small red buckets, and planted wild-type control plants, it was found that the growth and development of the transgenic family and the wild-type control were not significantly different (Fig. 4B). When the plants grew to the 4-leaf stage, they were subjected to 4°C low temperature Treatment (processing 24 hours a day), after 5 days of treatment, the phenotypes of the transgenic and control plants were observed, and there seemed to be no significant difference, but after they were transferred to normal conditions to resume growth for 5 days, it was found that most of the control plants were dead, while the transgenic Most of the lines can survive (Fig. 4C). In another group of experiments, the plants at the 4-leaf stage were treated at 4°C for 5 days at low temperature (24 hours per day) and then resumed growth for 7 days, and the survival rate of the plants was calculated and found , the plant survival rate (68.2%-90.4%) of the overexpression family of OsCIPK03 is extremely significantly different from that of the control (18.5%) (Fig. 4D). This result shows that the OsCIPK03 gene is indeed related to cold tolerance, and its overexpression can improve the tolerance of transgenic plants. The cold tolerance of transgenic rice plants is indeed related to the transgenic OsCIPK03 gene.

Example 5: Determination of Proline Content in OsCIPK03 Gene Transgenic T2 Family

Under stress conditions, the proline content in plants increased significantly. The proline content in plants reflects the stress resistance of plants to a certain extent. Under low temperature conditions, the proline in plant tissues increases, which can improve the cold resistance of plants. During the experiment, the transgenic rice plants of the present invention were subjected to low temperature stress at 4°C when they grew to the four-leaf stage, and samples were taken at 0, 1, 3, and 6 days for proline content determination. The experimental results are shown in Figure 5. It shows that the amount of proline accumulated in the OsCIPK03 overexpression family is 2-4 times that of the wild-type control when subjected to cold stress. The result shows that the overexpression of the cloned OsCIPK03 gene of the present invention causes the increase of the proline content, thereby significantly improving the low temperature tolerance of the transgenic rice plants.

The main principle of proline content determination is: when plant samples are extracted with sulfosalicylic acid, proline will be free in the solution of sulfosalicylic acid, and then the solution will turn red after heat treatment with acidic ninhydrin , and then treated with toluene, the pigment is all transferred to toluene, and the depth of the pigment indicates the level of proline content. Colorimetric at a wavelength of 520nm, find out (or use regression equation to calculate) the content of proline from the standard curve.

The specific steps of proline content determination are as follows:

1. Materials, equipment and reagents

(1) Materials: the plants to be tested are leaves of transgenic rice plants and leaves of non-transgenic rice plants of the present invention.

(2) Equipment: 1.722 type spectrophotometer; 2. Mortar; 3.100ml small beaker; 4. Volumetric flask; 5. Large test tube; 6. Ordinary test tube; 7. Pipette; 8. Syringe; 9. Water bath pot; 10. funnel; 11. funnel holder; 12. filter paper; 13 scissors.

(3) Reagent 1. Acidic ninhydrin solution: dissolve 1.25g ninhydrin in 30ml glacial acetic acid and 20ml6mol/L phosphoric acid, stir and heat (70°C) to dissolve, and store in a refrigerator; 2.3% sulfosalicylic acid : 3g sulfosalicylic acid was dissolved in distilled water and the volume was adjusted to 100ml; 3. Glacial acetic acid; 4. Toluene.

2. Experimental steps

1. Drawing of the standard curve (1) Accurately weigh 25mg of proline on an analytical balance, pour it into a small beaker, dissolve it with a small amount of distilled water, then pour it into a 250ml volumetric flask, add distilled water to make it up to the mark, and the standard solution Contains 100μg of proline per ml. (2) Preparation of serial proline concentrations Take six 50ml volumetric flasks, fill them with proline stock solution 0.5, 1.0, 1.5, 2.0, 2.5 and 3.0ml respectively, dilute to the mark with distilled water, shake well, the Proline concentrations were 1, 2, 3, 4, 5 and 6 μg/ml, respectively. (3) Take 6 test tubes, respectively draw 2ml of proline solution of serial standard concentration, 2ml of glacial acetic acid and 2ml of acidic ninhydrin solution, and heat each tube in a boiling water bath for 30min. (4) After cooling, add 4ml of toluene to each test tube accurately, shake for 30 seconds, and let it stand for a while, so that all the pigments are transferred to the toluene solution. (5) Use a syringe to gently draw the proline toluene solution in the upper layer of each tube into the cuvette, and use the toluene solution as a blank control, and perform colorimetry at a wavelength of 520nm. (6) Drawing of the standard curve: first obtain the regression equation that the absorbance value (Y) changes according to the proline concentration (X), then draw the standard curve according to the regression equation, and calculate the content of proline (μg) in the 2ml assay solution /2ml).

2. Determination of sample (1) extraction of proline: accurately take by weighing each 0.5g of different processed plant leaves to be tested, put respectively in the large tube, then add the sulfosalicylic acid solution of 5ml3% to each tube respectively, Extract in a boiling water bath for 10 minutes (shake frequently during the extraction process), filter in a clean test tube after cooling, and the filtrate is the proline extract. (2) Pipette 2ml of the extract into another clean test tube with a glass stopper, add 2ml of glacial acetic acid and 2ml of acidic ninhydrin reagent, heat in a boiling water bath for 30min, and the solution will turn red. (3) After cooling, add 4ml of toluene, shake for 30S, let stand for a while, take the supernatant into a 10ml centrifuge tube, and centrifuge at 3000rpm for 5min. (4) Use a pipette to gently absorb the upper proline red toluene solution into the cuvette, use toluene as a blank control, and compare the color at a wavelength of 520nm on a spectrophotometer to obtain the absorbance value.

3. Calculation of results Calculate (or find out from the standard curve) the proline content (Xμg/2ml) in the 2ml measuring solution according to the regression equation, and then calculate the percentage of the proline content in the sample. Calculated as follows:

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

The effect of this embodiment is shown in accompanying drawing 5.

Example 6: Detection of expression levels of proline synthesis and transport-related genes in wild-type and OsCIPK03 gene transgenic families

The increase in proline content may be due to changes in the expression of proline synthesis and transport-related genes. We used Realtime-PCR to detect the expression of two proline synthesis and two proline transport genes in wild-type and OsCIPK03 Expression levels in gene transgenic lines. The experimental results showed that the expression levels of these four genes were increased to varying degrees (5-10 times) in OsCIPK03 overexpressed plants. The experimental results ( FIG. 6 ) indicated that the overexpression of OsCIPK03 increased the expression of proline synthesis and transport-related genes, resulting in an increase in proline content, thereby improving the cold tolerance of the transgenic plants. The experiment was carried out on ABI7500 Realtime-PCR instrument. The reaction system was 10μl 2×SYBR Green Master Mix Reagent (Applied Biosystems), 1.0ul cDNA template, 200nM gene-specific primers, and the total volume was 20μl. The reaction conditions are the first step: 95°C, 3 minutes; the second step (40 cycles) 95°C, 30 seconds, 60°C, 30 seconds, 72°C, 1 minute. The GenBank numbers of these four genes are: AK102633, AK101230, AK067118, AK0666298. The primers used for Realtime-PCR detection are: AK102633 (5'-CTCAAATCAAGGCGTCAACTAAGA- and 5'-TTTGTCAATATACGTGGCATATACCA-3'), AK101230 (5'-CGCCCCTCCCCGTATCT-3' and 5'-AGGAATGCGGCAACAAGTG-3'), 1 8 5 0 6 7 '-AGGGACGATGGAGTTTCTAAAGCT-3' and 5'-GGGATTCCAAAGGCAAAAAGA 3'), AK0666298 (5'-GAGGAGGCTACCTGACTGTCAAC-3' and 5'GCTCATGAAGTCGCCAAGGA-3'). The rice housekeeping gene Actinel (GenBank number X16280) was used as an internal control for PCR, and its primers were 5'-TGGCATCTCTCAGCACATTCC-3 and 5'-TGCACAATGGATGGGTCAGA-3'.

The effect of this embodiment is shown in Figure 6 .

sequence listing

<110> Huazhong Agricultural University

<120>Using rice protein kinase gene OsCIPK03 to improve plant cold tolerance

<130>

<141>2007-05-31

<160>1

<170>PatentIn version 3.1

<210>1

<211>1707

<212>DNA

<213> Rice (Oryza sativa)

<220>

<221> gene

<222>(1)..(1707)

<223>

<400>1

atgtataggg ctaagagggc tgcattatct ccaaaggtga agcgccgtgt agggaagtat 60

gagctcgggc gcaccattgg agaaggaacc tttgcaaagg tccggtttgc gaagaacact 120

gaaaatgacg aaccagttgc tatcaaaatc cttgacaagg agaaggttca gaagcacaga 180

ttggttgaac agattaggcg tgaaatttgt actatgaagt tagtaaagca tcctaatgtt 240

gttcggctgt tcgaggtcat gggaagtaaa gcaagaattt tcattgttct ggaatatgtt 300

actggaggag agctctttga aatcattgca actaatggaa ggttgaagga ggaggaagca 360

cgaaaatact ttcaacaact tatcaatgca gttgactact gccacagtag gggtgtgtac 420

cacagagact tgaagttaga aaatttgctg cttgatgctt ctggaaacct gaaagtatct 480

gactttggtt tgagtgcttt aaccgagcaa gtgaaggctg acggtttgct gcacacgaca 540

tgtggaactc ctaattatgt tgctccagag gtgattgagg acagaggcta tgatggggca 600

gctgcagata tctggtcttg tggggtaatc ctttatgttc tgcttgctgg gtttttacca 660

tttgaggatg acaacatcat tgctctttat aaaaagatct ctgaagctca gtttacctgt 720

ccctcttggt tttctactgg agctaagaag ctgatcacca gaattctgga tcccaaccct 780

acaactagga tcaccatttc tcaaatactg gaagatcctt ggttcaaaaa gggttacaaa 840

ccgcctgtat ttgacgagaa atatgaaact agttttgacg atgtcgatgc tgcttttgga 900

gactccgaag accggcatgt caaagaagaa actgaagatc agcctacctc tatgaacgcg 960

tttgaactca tttcactgaa tcaggcactg aatctggaca atttgttcga ggcaaaaaag 1020

gagtataaaa gagagacaag attcacatca caatgtcctc caaaagaaat tatcaccaag 1080

attgaagaag ctgcaaagcc acttggattt gatattcaaa agaaaaatta caagatgcgc 1140

atggagaacc tgaaagcagg tagaaaaggc aatctcaatg ttgcaactga ggttttccaa 1200

gtagctccat ccttacatgt ggttgagctc aagaaggcaa aggggggacac tctggagttc 1260

caaaagttct acagaaccct gtcgacccag ctcaaggacg tggtctggaa gtgcgacggc 1320

gaggtcgaag gcaacggcgc cgcggcgtga acgtggtttt tgccatggct ttcggggcac 1380

cggttcttcg tgtacatagc tgctctgcca tcatcaatgg ggtgttcgcc gtagagtagc 1440

tttttgtaac aagagaaaaa ggaaagaaaa aaagagaggg aaagattcgt tggttcgttg 1500

ataggtaggc tgctccgatg aaacaacggg cgcgatgcct gctgtggatg agcttgtcgc 1560

cgtgttagtt catttattct gtggcctcgg aaggttgtaa cgggacacaa tcagatgccg 1620

caatgcaacg ggctgacagt ttgtgagttt caaacaacat ttgaataagt ctagcctcct 1680

ccgtcaacta taggcggagt ttgattc 1707

Claims (4)

1.OsCIPK03 gene mediated give the dna sequence dna of plant to the low temperature stress tolerance, it is the dna sequence dna shown in the 1-1707 position among (a) SEQ ID NO:1, or (b) coding and the identical protein DNA sequence of (a) encoded protein matter.

2. the described dna sequence dna of claim 1 that suitable promotor connects.

3. the described dna sequence dna of claim 2, it is the dna sequence dna shown in the SEQ ID NO:1.

4. each described dna sequence dna of claim 1-3 is increasing paddy rice to the application in the low temperature stress tolerance.

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