OsCIP1, a protein related to cold tolerance during the booting stage of rice, and its encoding gene and applications.

Abstract

This invention relates to the field of biotechnology, disclosing a rice cold tolerance protein OsCIP1, its encoding gene, and its applications. The OsCIP1 protein is shown in F1, F2, or F3 as follows: F1) The amino acid sequence is that of the protein shown in SEQ ID NO.2; F2) A protein obtained by substituting and / or deleting and / or adding amino acid residues to the protein of F1), possessing more than 75% identity with the protein shown in F1) and exhibiting activity in regulating cold tolerance during the rice seedling stage; F3) A fusion protein obtained by attaching a protein tag to the N-terminus and / or C-terminus of F1) or F2). The OsCIP1 protein provided by this invention can improve the cold tolerance of rice during the booting stage, which is of great significance for new variety breeding, environmental sanitation, and food security, and has significant application and promotion value.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, and in particular to OsCIP1, a protein related to cold tolerance during the booting stage of rice, its encoding gene, and its application in improving cold tolerance during the booting stage of rice. Background Technology

[0002] Rice is one of the world's three major food crops, with more than half of the global population relying on it for sustenance. Originating in tropical and subtropical regions, rice is highly sensitive to temperature throughout its growth cycle. In recent years, the frequency of global climate anomalies has increased, leading to a rise in rice yield reductions caused by low temperatures, making low temperatures one of the adverse factors limiting rice production.

[0003] Chilling injury in rice refers to the phenomenon where rice is exposed to continuous or short-term temperatures below the critical temperature, causing delayed growth and, in severe cases, damage to nutrients or reproductive organs, leading to abnormal development and reduced yield. Based on the timing of the chilling injury, it can be divided into bud stage chilling injury, seedling stage chilling injury, and reproductive growth stage (booting and flowering stage) chilling injury. Reproductive growth stage chilling injury refers to the low-temperature stress experienced by rice from the beginning of reproductive growth until flowering, ultimately resulting in abnormal pollen development, inability of anthers to dehisce and release pollen normally, and inability of pollen scattered on the stigma to germinate and fertilize properly, thus affecting normal flowering and pollination and resulting in empty grains. Because the occurrence of chilling injury during the booting and flowering stages is very close, it is sometimes difficult to strictly distinguish between the two in practice, and they are often collectively referred to as booting-flowering stage chilling injury. Chilling injury encountered during the booting stage is particularly important because it can cause irreversible yield losses.

[0004] Therefore, chilling injury during the booting stage is the period that most severely affects rice yield, making research on cold tolerance during this stage extremely important. However, developing cold-tolerant rice varieties using traditional breeding methods is a long process and progresses slowly. In contrast, genetic engineering technology can directly modify the genetic background of plants at the gene level, allowing for targeted modification of genetic traits. Discovering cold-tolerant genes during the booting stage of rice has significant theoretical and practical implications for breeding new cold-tolerant rice varieties. Summary of the Invention

[0005] In view of this, the present invention provides a cold tolerance-related protein OsCIP1 in rice during the booting stage, its encoding gene, and its application in improving cold tolerance during the booting stage of rice. To address the problem of long cycles and slow progress in breeding cold-tolerant rice varieties using traditional breeding methods in the prior art, the present invention adopts the following technical solution to achieve the above objective:

[0006] One object of the present invention is to provide a method for improving the cold resistance of rice during the booting stage.

[0007] The method provided by the present invention includes the following steps: introducing a nucleic acid molecule encoding the OsCIP1 protein shown in SEQ ID NO:2 into a target rice to obtain transgenic rice, wherein the transgenic rice has higher cold tolerance during the booting stage than the target rice.

[0008] In the above method, the higher cold tolerance of the transgenic rice during the booting stage compared to the target rice is reflected in the higher seed setting rate of the transgenic rice under low temperature stress during the booting stage.

[0009] The low temperature is 16-17 ℃.

[0010] Another object of the present invention is to provide the application of the OsCIP1 protein shown in SEQ ID NO:2, the nucleic acid molecule encoding the OsCIP1 protein shown in SEQ ID NO:2, or the recombinant vector, expression cassette or recombinant bacteria containing said nucleic acid molecule in improving the cold tolerance of rice during the booting stage.

[0011] In the above applications, the improvement of cold tolerance during the booting stage of rice is reflected in the increased seed setting rate of rice under low temperature stress during the booting stage.

[0012] The low temperature is 16-17 ℃.

[0013] Compared with the prior art, the beneficial effects of the present invention are: it improves the cold resistance of rice during the booting stage, shortens the cycle of breeding cold-resistant rice during the booting stage, and provides new genetic resources for breeding new cold-resistant rice varieties.

[0014] This invention provides the application of the OsCIP1 protein and its encoding gene (SEQ ID NO:2) in improving cold tolerance during the booting stage of rice. This invention improves cold tolerance during the booting stage of rice, which is of great significance for new variety breeding, environmental sanitation, and food security, and has significant application and promotion value. Attached Figure Description

[0015] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the accompanying drawings used in the description of the embodiments or the prior art will be briefly introduced below.

[0016] Figure 1 This is a schematic diagram of the OsCIP1 screening process in Embodiment 1 of the present invention;

[0017] Figure 2 This is the expression pattern of OsCIP1 in different tissues of the rice variety Kunming Xiaobaigu in Example 1 of the present invention. The internal reference is the OsActin1 gene. The young spikelets 1, 3, 5, 7, 10, and 15 represent spikelets with lengths of 1cm, 3cm, 5cm, 7cm, 10cm, and 15cm, respectively.

[0018] Figure 3This is a diagram illustrating the expression pattern of OsCIP1 in the panicle of the rice variety Kunming Xiaobai under cold stress in Example 1 of the present invention, with the OsActin1 gene as the internal reference.

[0019] Figure 4 The results of real-time quantitative PCR detection of different transgenic lines of OsCIP1 overexpressing plants are shown, with the OsActin1 gene as the internal control.

[0020] Figure 5 Schematic diagrams of different cooling methods;

[0021] Figure 6 Phenotypic image of panicle after cold treatment during the booting stage of OsCIP1 overexpressing plants;

[0022] Figure 7 A statistical chart showing the seed setting rate of OsCIP1 overexpressing plants and control plants after cold water irrigation during the heading stage;

[0023] Figure 8 A statistical chart showing the seed setting rate of OsCIP1 overexpressing plants and control plants after cold treatment in an artificial climate chamber during the heading stage;

[0024] Figure 9 A statistical chart showing the fruit set rate of OsCIP1 overexpressing plants and control plants grown in high-altitude areas. Detailed Implementation

[0025] This invention discloses a cold tolerance-related protein OsCIP1 in rice during the booting stage, its encoding gene, and its applications.

[0026] It should be understood that the expression “one or more of…” individually includes each of the objects described after the expression, as well as various different combinations of two or more of the described objects, unless otherwise understood from the context and usage. The expression “and / or” combined with three or more described objects should be understood to have the same meaning, unless otherwise understood from the context.

[0027] The terms “including,” “having,” or “containing,” including the use of their grammatical synonyms, should generally be understood as open-ended and non-restrictive, for example, not excluding other unstated elements or steps, unless otherwise specifically stated or understood from the context.

[0028] It should be understood that the order of the steps or the order in which certain actions are performed is not important as long as the invention remains operational. Furthermore, two or more steps or actions can be performed simultaneously.

[0029] The use of any and all instances or exemplary language such as “e.g.” or “including” in this document is merely intended to better illustrate the invention and is not intended to limit the scope of the invention unless the claims are made. No language in this specification should be construed as indicating that any unclaimed element is essential to the practice of the invention.

[0030] Furthermore, the numerical ranges and parameters used to define the present invention are approximate values, and the relevant values ​​in the specific embodiments have been presented as precisely as possible. However, any value inevitably contains standard deviations due to individual test methods. Therefore, unless explicitly stated otherwise, it should be understood that all ranges, quantities, values, and percentages used in this disclosure are modified with the word "approximately". Here, "approximately" generally means that the actual value is within plus or minus 10%, 5%, 1%, or 0.5% of a specific value or range.

[0031] The rice variety Kunming Xiaobaigu (KMXBG) described in the following examples is described in the non-patent literature “Dai Luyuan et al. Genetic analysis of cold resistance index traits of Yunnan rice variety Kunming Xiaobaigu. Chinese Journal of Rice Science, 1999, 13(2):73-76.”, which can be obtained by the public from China Agricultural University (i.e. the applicant) to replicate the experiments of this application.

[0032] The rice variety Towada in the following examples is referred to as WT or wild type below and is described in the non-patent literature “Dai Luyuan et al. Genetic analysis of cold resistance index traits of Yunnan rice variety Kunming Xiaobaigu. Chinese Journal of Rice Science, 1999, 13(2):73-76.” The public can obtain it from China Agricultural University (i.e. the applicant) to repeat the experiments of this application.

[0033] The acquisition and utilization of the aforementioned genetic resources comply with relevant laws and regulations.

[0034] In the quantitative experiments described below, three replicate experiments were conducted, and the average value of the results was taken.

[0035] The vector pCM1307-3FLAG used in the following examples is described in the non-patent literature “J Li, B Tang, Y Li, CLi, Z Li, et al. (2021) Rice SPL10 positively regulates trichome development through expression of HL6 and auxin-related genes . J. Integr. Plant Biol. 63(8): 1521-1537.”, which can be obtained by the public from China Agricultural University (i.e. the applicant) to replicate the experiments of this application, but may not be used for other purposes.

[0036] The vectors pMDC32-CTB4a and pGEX-4T-1-CTB4a in the following examples KD The invention is described in the non-patent literature “ZZhang, J Li, Y Pan, J Li, Z Li, et al. (2017) Natural variation in CTB4aenhances rice adaptation to cold habitats. Nat Commun. 8: 14788.”, which can be obtained by the public from China Agricultural University (i.e. the applicant) to replicate the experiment of this application, but may not be used for other purposes.

[0037] The Agrobacterium tumefaciens strain EHA105 in the following examples is described in the non-patent literature “Gao Shiwu et al. Study on factors affecting the transformation efficiency of Agrobacterium tumefaciens EHA105 competent cells. Journal of Tropical Biology. March 2012, Vol. 3, No. 1”, which can be obtained by the public from China Agricultural University (i.e. the applicant) to replicate the experiments of this application.

[0038] The following examples used Excel statistical software to process the data. The experimental results are expressed as mean ± standard deviation. Duncant's test was used, and different letters indicate significant differences (P < 0.05).

[0039] Alternating light and dark culture, with the following conditions: 28℃; 14 hours of light culture / 10 hours of dark culture; light intensity during light culture: 90 μE / m². 2 / s.

[0040] YEP liquid medium: Dissolve 10g of yeast extract, 5g of NaCl and 10g of tryptone in distilled water, then bring the volume to 1L with distilled water and autoclave at 121℃ for 15min.

[0041] YEP solid medium: Dissolve 10g yeast extract, 5g NaCl and 10g tryptone in distilled water, then bring the volume to 1L with distilled water, and finally add 15g agar powder. Autoclave at 121℃ for 15min.

[0042] AAM liquid culture medium: Dissolve 50 ml of AAM (20×), 5 mL of MS (200×), 10 mL of AAM organic (100×), 10 mL of iron salt (100×), 500 mg of hydrolyzed casein, 68.5 g of sucrose, 36 g of glucose, 75 mg of glycine, 877 mg of glutamine, 266 mg of aspartic acid, and 228 mg of arginine in an appropriate amount of deionized water, then bring the volume to 1 L with deionized water, adjust the pH to 5.5, and autoclave at 115℃ for 15 min; finally, add 1 ml of 20 mg / ml acetylsuccione solution and mix well.

[0043] AAM (20×): Dissolve 6.645 g CaCl2, 58.8 g KCl, 3.4 g KH2PO4 and 7.4 g MgSO4·7H2O in an appropriate amount of distilled water, and then make up to 1 L with distilled water.

[0044] MS micro-dissolve 166 mg KI, 3.38 g MnSO4·H2O, 1.24 g H3BO3, 50 mg Na2MoO4·2H2O, 1.72 g ZnSO4·7H2O, 5 g CoCl2·6H2O and 5 g CuSO4·5H2O in an appropriate amount of distilled water, and then bring the volume to 1 L with distilled water.

[0045] AAM Organic (100×): Dissolve 50 mg of vitamin B1, 50 mg of vitamin B5 and 10 mg of vitamin B6 in an appropriate amount of distilled water, and then bring the volume to 1 L with distilled water.

[0046] Iron salt (100×): Dissolve 3.73 g Na2EDTA.2H2O and 2.78 g FeSO4.7H2O in an appropriate amount of distilled water, and then make up to 1 L with distilled water.

[0047] NB solid culture medium: Dissolve 20 mL of 50×N6 large volume stock solution, 10 mL of 100×B5 micro volume stock solution, 1 mL of 1000×B5 organic stock solution, 150 mg of inositol, 300 mg of hydrolyzed casein, 500 mg of glutamine, 600 mg of proline, 30.0 g of sucrose, and 3 g of plant gel in 1 L of distilled water, then adjust the pH to 5.8 and autoclave for 20 min.

[0048] Co-culture medium: Dissolve 20 mL of 50×N6 large volume stock solution, 10 mL of 100×B5 micro volume stock solution, 1 mL of 1000×B5 organic stock solution, 150 mg of inositol, 300 mg of hydrolyzed casein, 500 mg of glutamine, 600 mg of proline, 30.0 g of sucrose, 3 g of plant gel, and 2 mg of 2,4-D in 1 L of distilled water, adjust the pH to 5.4, sterilize at 121℃ for 15 min, and when cooled to 50~60℃, add 10 g of glucose and 20 mg of acetylsuccinone that have been filtered and sterilized with 0.22 μM.

[0049] Delayed screening medium: Dissolve 20 mL of 50×N6 large volume stock solution, 10 mL of 100×B5 micro volume stock solution, 1 mL of 1000×B5 organic stock solution, 150 mg of inositol, 300 mg of hydrolyzed casein, 500 mg of glutamine, 600 mg of proline, 30.0 g of sucrose, 3 g of plant gel, and 2 mg of 2,4-D in 1 L of distilled water, adjust the pH to 5.8, sterilize at 121℃ for 15 min, and add 500 mg of cephalosporin that has been filtered and sterilized with 0.22 μM when the temperature has cooled to 50~60℃.

[0050] First round of screening culture medium: Dissolve 20 mL of 50×N6 large volume stock solution, 10 mL of 100×B5 micro volume stock solution, 1 mL of 1000×B5 organic stock solution, 150 mg of inositol, 300 mg of hydrolyzed casein, 500 mg of glutamine, 600 mg of proline, 30.0 g of sucrose, 3 g of plant gel and 2,4-D 2 mg in 1 L of distilled water, adjust the pH to 5.8, sterilize at 121℃ for 15 min, and add 500 mg of cephalosporin and 50 mg of hygromycin after filtration sterilization at 0.22 μM when the temperature has cooled to 50~60℃.

[0051] Second round of screening culture medium: Dissolve 20 mL of 50×N6 large volume stock solution, 10 mL of 100×B5 micro volume stock solution, 1 mL of 1000×B5 organic stock solution, 150 mg of inositol, 300 mg of hydrolyzed casein, 500 mg of glutamine, 600 mg of proline, 30.0 g of sucrose, 3 g of plant gel and 2,4-D 2 mg in 1 L of distilled water, adjust the pH to 5.8, sterilize at 121℃ for 15 min, and add 50 mg of hygromycin that has been filtered and sterilized with 0.22 μM when the temperature has cooled to 50~60℃.

[0052] Predifferentiation medium: Dissolve 20 mL of 50×N6 large volume stock solution, 10 mL of 100×B5 micro volume stock solution, 1 mL of 1000×B5 organic stock solution, 150 mg of inositol, 300 mg of hydrolyzed casein, 500 mg of glutamine, 600 mg of proline, 30.0 g of sucrose, 3 g of plant gel, 1 mg of 6-BA, 2 mg of NAA and 5 mg of ABA in 1 L of distilled water, adjust the pH to 5.8, sterilize at 121℃ for 15 min, and add 50 mg of hygromycin that has been filtered and sterilized with 0.22 μM solution when cooled to 50~60℃.

[0053] Differentiation medium: Dissolve 20 mL of 50×N6 large volume stock solution, 10 mL of 100×B5 micro volume stock solution, 1 mL of 1000×B5 organic stock solution, 150 mg of inositol, 300 mg of hydrolyzed casein, 500 mg of glutamine, 600 mg of proline, 30.0 g of sucrose, 3 g of plant gel, 2 mg of 6-BA, 1 mg of NAA, and 1 mg of KT in 1 L of distilled water, adjust the pH to 5.8, sterilize at 121℃ for 15 min, and add 50 mg of hygromycin that has been filtered and sterilized with 0.22 μM solution when cooled to 50~60℃.

[0054] Seedling growth medium: Dissolve 25 mL of 20×MS large volume stock solution, 2.5 mL of 200×MS micro volume stock solution, 2.5 mL of 200×MS iron salt stock solution, 2.5 mL of 200×MS organic stock solution, 3 g of plant gel, 0.5 mg of NAA and 0.25 mg of paclobutrazol in 1 L of distilled water, adjust the pH to 5.8, and sterilize at 121℃ for 15 min.

[0055] The solutes and their concentrations in the 20×MS mother liquor were 38.00 g / L KNO3, 8.80 g / L CaCl2·2H2O, 7.40 g / L MgSO4·7H2O, 3.40 g / L KH2PO4, and 33.00 g / L NH4NO3. The solvent was water, and the pH was natural.

[0056] The solutes and their concentrations in the 200×MS trace mother liquor were 4.46 g / L MnSO4·4H2O, 0.166 g / L KI, 1.24 g / L H3BO3, 1.72 g / L ZnSO4·7H2O, 0.050 g / L Na2MoO4·2H2O, 0.005 g / L CuSO4·5H2O, and 0.005 g / L CoCl2·6H2O. The solvent was water, and the pH was natural.

[0057] The solutes and their concentrations in the 200×MS iron salt mother liquor were 5.56 g / L FeSO4·7H2O and 7.46 g / L Na2·EDTA·2H2O, with water as the solvent and natural pH.

[0058] The solutes and their concentrations in the 200×MS organic mother liquor were 20 g / L inositol, 100 mg / L nicotinic acid, 100 mg / L pyridoxine hydrochloride, 100 mg / L thiamine hydrochloride and 400 mg / L glycine, with water as the solvent and natural pH.

[0059] The solutes and their concentrations in the large volume of 50×N6 mother liquor were 141.50 g / L KNO3, 20 g / L KH2PO4, 23.15 g / L (NH4)2SO4, 9.25 g / L MgSO4·7H2O and 8.30 g / L CaCl2·2H2O, with water as the solvent and natural pH.

[0060] The solutes and their concentrations in the 100×B5 trace mother liquor were 0.3 g / L H3BO3, 1 g / L MnSO4·4H2O, 0.0025 g / L CoCl2·6H2O, 0.0025 g / L CuSO4·5H2O, 0.2 g / L ZnSO4·7H2O, 0.025 g / L Na2MoO4·2H2O, and 0.075 g / L KI. The solvent was water, and the pH was natural.

[0061] The solutes and their concentrations in the 1000×B5 organic mother liquor were 2 g / L glycine, 100 g / L inositol, 1 g / L nicotinic acid, 1 g / L pyridoxine hydrochloride, and 10 g / L thiamine hydrochloride. The solvent was water, and the pH was natural.

[0062] In this invention, all raw materials and reagents used can be purchased from the market.

[0063] The present invention will be further illustrated below with reference to the embodiments:

[0064] Example 1: Obtaining the OsCIP1 gene

[0065] 1. Obtaining the OsCIP1 gene

[0066] This invention screened interacting proteins of CTB4a, a protein that has been reported to regulate cold tolerance in rice, using two methods, and identified a gene encoding the CIP1 protein.

[0067] First, the coding sequence of CTB4a was amplified from the pMDC32-CTB4a vector. The CTB4a sequence was then ligated into the pBT3-SUC vector using the Sfi site to construct the Bait strain. Subsequently, this vector and the empty Prey vector were simultaneously introduced into the yeast strain NMY51 using chemical transformation. Positive clones were screened using SD-Leu-Trp-deficient medium. Self-activation verification showed that the Bait vector did not self-activate, allowing for further experiments.

[0068] Kunming white millet was sown in the field at the Shangzhuang Experimental Station in Beijing. After growing under normal conditions for one month, the seedlings were transplanted into paddy fields for normal water and fertilizer management. When the plants reached the heading stage, they were subjected to cold treatment in an artificial climate chamber under alternating light and dark conditions at 16℃; 14 hours of light culture followed by 10 hours of darkness; and a light intensity of 90 μE / m² during the light culture period. 2 / s. Treatment for 7 days. Then, total RNA was extracted from the treated young spikelets, and the RNA was reverse transcribed into cDNA using reverse transcriptase. The cDNA library was cloned into the Prey vector using chemical transformation. The recombinant Prey vector library was amplified and extracted using *E. coli* to ensure its diversity and capacity. The Prey vector containing the cDNA library was then transformed on a large scale into yeast strains containing the Bait vector.

[0069] Positive clones were screened using SD-Leu-Trp-His-Ade medium containing 50 mM 3-AT, plasmids of the positive clones were extracted and verified one-to-one, and finally 38 proteins interacting with CTB4a were obtained.

[0070] This invention uses a second method, namely pull-down technique combined with mass spectrometry, to screen interacting proteins of CTB4a. First, pGEX-4T-1-CTB4a... KD The vector was introduced into E. coli for expression using chemical transformation, and GST-CTB4a was purified. KD Protein was extracted, and empty GST protein was purified as a control. Subsequently, young panicles of Kunming Xiaobaigu rice, after 3 days of cold treatment, were used to extract total protein using protein extraction buffer, under the same plant culture conditions. The extracted total rice protein was then compared with GST-CTB4a. KD The protein and GST protein were incubated for 2 hours, then GST magnetic beads were added and incubated for another 2 hours. The magnetic beads were then removed, and the protein was eluted with elution buffer for mass spectrometry analysis. The mass spectrometry results were compared with the UniProt database (http: / / www.uniprot.org / ), yielding 17 proteins with scores higher than 100.

[0071] By comparing the candidate proteins screened by the two methods, it was found that only one gene, Os08g0126300, encoded a protein that appeared in both screening results simultaneously. Figure 1 As shown, we named this protein CIP1 and used it as the most likely interacting protein of CTB4a for further analysis.

[0072] in, OsCIP1

[0073] The protein encoded by the OsCIP1 gene is OsCIP1, and its amino acid sequence is shown in SEQ ID NO:2: Met Gly Lys Ile Lys Ile Gly Ile Asn Gly Phe Gly Arg Ile Gly Arg Leu Val AlaArg Val Ala Leu Gln Ser Glu Asp Val Glu Leu Val Ala Val Asn Asp Pro Phe IleThr Thr Asp Tyr Met Thr Tyr Met Phe Lys Tyr Asp Thr Val His Gly Gln Trp LysHis Ser Asp Ile Lys Ile Lys Asp Ser Lys Thr Leu Leu Leu Gly Glu Lys Pro ValThr Val Phe Gly Ile Arg Asn Pro Asp Glu Ile Pro Trp Ala Glu Ala Gly Ala GluTyr Val Val Glu Ser Thr Gly Val Phe Thr Asp Lys Glu Lys Ala Ala Ala His LeuLys Gly Gly Ala Lys Lys Val Val Ile Ser Ala Pro Ser Lys Asp Ala Pro Met PheVal Cys Gly Val Asn Glu Asp Lys Tyr Thr Ser Asp Ile Asp Ile Val Ser Asn AlaSer Cys Thr Thr Asn Cys Leu Ala Pro Leu Ala Lys Val Ile His Asp Asn Phe GlyIle Ile Glu Gly Leu Met Thr Thr Val His Ala Ile Thr Ala Thr Gln Lys Thr ValAsp Gly Pro Ser Ser Lys Asp Trp Arg Gly Gly Arg Ala Ala Ser Phe Asn Ile IlePro Ser Ser Thr Gly Ala Ala Lys Ala Val Gly Lys Val Leu Pro Asp Leu Asn GlyLy s Leu Thr Gly Met Ser Phe Arg Val Pro Thr Val AspVal Ser Val Val Asp LeuThr Val Arg Ile Glu Lys Ala Ala Ser Tyr Asp Ala Ile Lys Ser Ala Ile Lys SerAla Ser Glu Gly Lys Leu Lys Gly Ile Ile Gly Tyr Val Glu Glu Asp Leu Val SerThr Asp Phe Val Gly Asp Ser Arg Ser Ser Ile Phe Asp Ala Lys Ala Gly Ile AlaLeu Asn Asp Asn Phe Val Lys Leu Val Ala Trp Tyr Asp Asn Glu Trp Gly Tyr SerAsn Arg Val Ile Asp Leu Ile Arg His Met Ala Lys Thr Gln.

[0074] 2. OsCIP1 Gene expression patterns

[0075] Kunming white millet was sown in the field at the Shangzhuang Experimental Station in Beijing. After growing for one month under normal conditions, the seedlings were transplanted into paddy fields for normal water and fertilizer management.

[0076] Normal processing: Samples of different tissues of Kunming white millet material under normal planting conditions were taken, including roots, stems, mature leaves, leaf sheaths, and ears of grain at 1cm, 3cm, 5cm, 7cm, 10cm, and 15cm. After sampling, the samples were flash-frozen in liquid nitrogen and stored at -80℃ for later use.

[0077] Low temperature stress treatment: Kunming Xiaobaigu materials grown to the booting stage under normal conditions were transplanted to an artificial climate chamber at 18 degrees Celsius. Leaf and ear samples were taken at 1h, 2h, 4h, 8h, 12h, 16h, 24h, 1d, 2d, 3d, 4d, 5d, 6d, and 7d after treatment. The samples were then flash-frozen in liquid nitrogen and stored at -80℃ for later use.

[0078] Total RNA was extracted from all tissue samples using a total RNA extraction kit (Adelaide, Beijing) following the prescribed procedure. First-strand cDNA was synthesized via reverse transcription using M-MLV reverse transcriptase. This first-strand cDNA was used as a template for real-time quantitative analysis using primers. OsActin1 was used as an internal reference gene. ΔΔc(t)The relative expression levels of the genes were calculated. The OsCIP1 amplification primer set consisted of qRT-OsCIP1-F and qRT-OsCIP1-R, and the OsActin1 amplification primer set consisted of Actin1-F and Actin1-R. The sequences are as follows:

[0079] OsCIP1 amplification primers:

[0080] The sequence of qRT-OsCIP1-F is shown in SEQ ID NO:3: 5'-TGACAAGGAGAAGGCTGCTG-3';

[0081] The sequence of qRT-OsCIP1-R is shown in SEQ ID NO:4: 5'-GAGCAAGGCAGTTTGTGGTG-3'.

[0082] OsActin1 amplification primers:

[0083] The sequence of Actin1-F is shown in SEQ ID NO:5: 5'-CACAGGTATTGTGTTGGACTCTG-3';

[0084] The sequence of Actin1-R is shown in SEQ ID NO:6: 5'-AGTAACCACGCTCCGTCAGG-3'.

[0085] The amplification system consisted of 20 μL of 10 μL 2×SYBR Premix ExTaq Buffer; 0.4 μL F and R primers; 2 μL cDNA; 0.4 μL DyeII; and 6.8 μL ultrapure water.

[0086] The amplification conditions were: (1) 95℃, 30s; (2) 95℃, 5s; (3) 95℃, 34s; (4) Steps (2) to (3) were repeated for 40 cycles.

[0087] The experiment was repeated three times.

[0088] OsCIP1 gene tissue expression pattern as follows Figure 2 As shown, the OsCIP1 gene is expressed in different tissues, with a higher expression abundance in leaf sheaths.

[0089] OsCIP1 cold-induced expression analysis, such as Figure 3 As shown, it can be seen that the OsCIP1 gene is induced by low temperature in Xiaobaigu Valley in Kunming, indicating that the OsCIP1 gene is a gene related to low temperature stress.

[0090] Example 2 Functional verification of the OsCIP1 gene

[0091] I. Obtaining and Identifying OsCIP1 Gene-Transgenic Rice

[0092] 1. Construction of recombinant vector ubi::OsCIP1 and obtaining recombinant Agrobacterium EHA105 / ubi::OsCIP1

[0093] (1) Using the cDNA of rice variety IRAT109 as a template, the following formula was used: F-HA-F: 5'-ATCGATACCGTCGACGAGCTCTCTAGA ACTAG TATGGGCAAGATTAAGATCGGAATC-3' (as shown in SEQ ID NO:7, the underlined part is the recognition site of the restriction endonuclease SpeI) and F-HA-R: 5'-AGACTGGTGATTTTTGCGGAGTACCCGG GTACC The primer pair consisting of CTACTGGGTCTTGGCCATGTGG-3' (as shown in SEQ ID NO:8, the underlined part is the recognition site of the restriction endonuclease KpnI) was used for PCR amplification, resulting in a PCR amplification product of approximately 1014 bp.

[0094] (2) The PCR amplification product obtained in step (1) was sequenced. The results showed that the PCR amplification product contained the DNA fragment shown in SEQ ID NO:1.

[0095] (3) The PCR amplification product obtained in step (1) and the vector pCM1307-3FLAG were homologously recombinated to obtain the recombinant vector ubi::OsCIP1.

[0096] The recombinant vector ubi::OsCIP1 was sequenced. Sequencing results showed that the recombinant vector ubi::OsCIP1 was obtained by replacing a small DNA fragment between the restriction endonucleases SpeI and KpnI in the vector pCM1307-3FLAG with the DNA molecule shown in SEQ ID NO:1.

[0097] In the recombinant vector ubi::OsCIP1, the OsCIP1 gene is inserted downstream of the maize ubiquitin promoter. The recombinant vector ubi::OsCIP1 expresses the OsCIP1 protein shown in SEQ ID NO:2.

[0098] (4) The recombinant vector ubi::OsCIP1 was introduced into Agrobacterium tumefaciens EHA105 to obtain recombinant Agrobacterium, which was named EHA105 / ubi::OsCIP1.

[0099] 2. Obtaining T3 generation homozygous transgenic OsCIP1 rice

[0100] The method described in the reference (MR Xu, ZH Xia, WX Zhai, JL Xu, YL Zhou, ZKLi. (2008) Construction of double right-border binary vector carrying non-host gene Rxo1 resistant to rice bacterial leaf streak, Rice Science, 15 (3), 243-246.) was used to transform EHA105 / ubi::OsCIP1 into Towada rice, and then T3 generation homozygous transgenic OsCIP1 rice was obtained through self-pollination. The specific steps are as follows:

[0101] (1) Acquisition of callus

[0102] Mature Towada seeds were shelled, sterilized with alcohol, then with sodium hypochlorite, rinsed once with sterile water, and air-dried for 3 hours to obtain sterile Towada seeds. These sterile seeds were inoculated onto NB solid medium and cultured in the dark at 28°C for 2 weeks to obtain embryogenic callus. The embryogenic callus was subcultured twice to obtain callus tissue. Each culture method involved inoculating the embryogenic callus onto fresh NB solid medium and incubating in the dark at 28°C for 2 weeks.

[0103] (2) Preparation of the infiltration solution

[0104] EHA105 / ubi::OsCIP1 was spread on YEP solid medium containing kanamycin and rifampin and incubated in the dark at 28°C for 2 days; then Agrobacterium was scraped onto AAM liquid medium to obtain OD. 600nm The infection solution is approximately 0.3.

[0105] (3) Co-cultivation

[0106] Select naturally dispersed, bright yellow granular callus tissue with a diameter of about 3-5 mm from the callus tissue obtained in step (1) and place it in an Erlenmeyer flask. Then add the infection solution prepared in step (2) and let it stand for 10 min for infection. After that, use sterile filter paper to absorb the excess infection solution and place the callus tissue on a co-culture medium covered with a layer of filter paper. Co-culture at 20℃ for 2-3 days.

[0107] (4) Screening of resistant callus

[0108] ① Take the callus tissue after completing step (3), first wash it with sterile water by shaking 5-6 times, then wash it with sterile water containing cephalosporin and carbenicillin for 20 minutes, then place it on sterile filter paper to drain for 3 hours, and finally transfer it to delayed screening medium and incubate it in the dark at 28°C for 1 week.

[0109] ②After completing step ①, transfer the callus tissue to the first round of screening medium and incubate in the dark at 28°C for 2 weeks.

[0110] ③ After completing step ②, the callus tissue was transferred to the second round of screening medium and cultured in the dark at 28°C for 2 weeks to obtain resistant callus tissue.

[0111] (5) Differentiation

[0112] ① Transfer the resistant callus obtained from step (4) to a predifferentiation medium and incubate in the dark at 28°C for 2 weeks.

[0113] ②After completing step ①, the resistant callus tissue is transferred to the differentiation medium and cultured under alternating light for 2-3 weeks to obtain transgenic seedlings.

[0114] (6) Strong seedlings

[0115] The transgenic seedlings obtained in step (5) were transferred to a seedling culture medium. After the seedlings took root, they were removed from the culture bottle, the culture medium on the roots was washed off, and the seedlings were hardened off for 1-2 weeks before being transplanted to the field for planting until maturity, thus obtaining T0 generation rice transgenic with the OsCIP1 gene.

[0116] (7) Select positive seedlings of the T0 generation of rice seedlings to be transgenic into the OsCIP1 gene obtained in step (6), and then perform self-pollination to obtain the T1 generation of rice seedlings transgenic into the OsCIP1 gene.

[0117] (8) Select positive seedlings of the T1 generation transgenic OsCIP1 rice obtained in step (7), and then perform self-pollination to obtain the T2 generation transgenic OsCIP1 rice.

[0118] (9) Select positive seedlings of the T2 generation transgenic OsCIP1 rice obtained in step (8), and then perform self-pollination to obtain T3 generation homozygous transgenic OsCIP1 rice.

[0119] The above method for screening positive seedlings is as follows: Genomic DNA of rice seedlings is extracted and used as a template. PCR amplification is performed using primer pairs consisting of hyg(280)-F: 5'-ACGGTGTCGTCCATCACAGTTTGCC-3' (as shown in SEQ ID NO:9) and hyg(280)-R: 5'-TTCCGGAAGTGCTTGACATTGGGGA-3' (as shown in SEQ ID NO:10) to obtain PCR amplification products. Then, the following judgment is made: If a PCR amplification product contains a DNA fragment of approximately 280 bp, the rice seedling corresponding to that PCR amplification product is a positive seedling.

[0120] 3. Real-time quantitative PCR detection of the relative expression level of the OsCIP1 gene in T3 generation homozygous transgenic OsCIP1 rice.

[0121] (1) The seedlings of each T3 generation homozygous transgenic OsCIP1 gene rice that have grown to 10 days were placed in liquid nitrogen for preservation to obtain the corresponding test samples. Take Towada seeds and culture them at 28℃ with alternating light and dark for 10 days to obtain the test rice seedlings; place the test rice seedlings in liquid nitrogen for preservation to obtain the corresponding test samples.

[0122] (2) Total RNA was extracted from the sample to be tested using the Trizo1 method, and then the first-strand cDNA was reverse transcribed using a reverse transcription kit (Aidelai, Beijing). The cDNA was diluted 50 times with sterile water as a template, and the relative expression level of the OsCIP1 gene was detected by real-time quantitative PCR (with OsActin1 gene Os03g0718100 as an internal reference gene).

[0123] The primer set for detecting the OsCIP1 gene is shown in Part 2 of Example 1.

[0124] The primer set for detecting the Actin gene is shown in Part 2 of Example 1.

[0125] Some test results are as follows Figure 4 As shown in the figure. The results showed that, compared with Towada, the relative expression level of the OsCIP1 gene was significantly increased in each T3 generation homozygous transgenic OsCIP1 gene rice. Three T3 generation homozygous transgenic OsCIP1 gene rice lines were named OsCIP1-OE1 (abbreviated as OE-1), OsCIP1-OE2 (abbreviated as OE-2), and OsCIP1-OE3 (abbreviated as OE-3), respectively.

[0126] 4. Identification of cold tolerance in OsCIP1-transformed rice

[0127] Three methods were established to assess the cold resistance of OsCIP1 transgenic materials, such as... Figure 5 The figures shown represent the following conditions: 7 days in a 50cm deep cold water tank (16~17℃) during the booting stage in the field (CS-DW); 7 days in an artificial climate chamber (CS-PT) during the booting stage at 16~17℃; and 7 days under natural low-temperature conditions at high altitude in Yunnan (CS-HAA).

[0128] Wild-type rice (cold-sensitive variety Towada), T3 generation OsCIP1 transgenic rice lines OE-1, OE-2, and OE-3, all in the booting stage, were selected and tagged. After the stress was relieved, the tested materials were replanted in the field to allow rice growth to resume. Upon maturity, the seed setting rate of the tagged panicles was assessed, with the seed setting rate of normally growing plants serving as a control. The relative seed setting rate was calculated (relative seed setting rate = seed setting rate after treatment / seed setting rate under normal conditions). 100% was used as an indicator of cold tolerance. Natural identification in Yunnan was conducted in the Heilongtan area of ​​Yunnan, at an altitude of 1916 meters, with an annual temperature of 19-22℃. The plants grew under naturally low-temperature conditions from sowing to the heading stage, and the seed setting rate of the main ear was examined after maturity. The phenotypic characteristics of the ears after treatment in high-altitude areas of Yunnan are as follows: Figure 6 As shown, the agronomic traits of the tested materials treated during the booting stage changed significantly. Each material was tested in triplicate, with 10 plants per replicate.

[0129] After 7 days of exposure to a 50cm deep cold water pool (16℃) in the field during the booting stage, the growth of T3 generation OsCIP1 transgenic rice lines OE-1, OE-2, and OE-3 was significantly better than that of wild-type rice; the seed setting rate was statistically analyzed as follows: Figure 7 As shown, the relative fruit set rate of Towada was 26.31%, while the relative fruit set rates of OE1, OE2 and OE3 were 60.13%, 72.11% and 47.07%, respectively, all significantly higher than that of Towada.

[0130] Results of 7-day treatment at 16℃ in an artificial climate chamber during the booting stage: Figure 8 As shown, the relative fruit set rate of Towada was 17.49%, while the relative fruit set rates of OE1, OE2 and OE3 were 39.77%, 45.39% and 28.34%, respectively, which were also significantly higher than those of Towada.

[0131] Results of natural low-temperature treatment throughout the entire growth period in Yunnan are as follows: Figure 9 As shown, the relative fruit set rate of Towada was 10.99%, while the relative fruit set rates of OE1, OE2 and OE3 were 69.76%, 57.72% and 70.96%, respectively, which were also significantly higher than those of Towada.

[0132] The above results indicate that low temperature during the booting stage mainly affects the seed setting rate. Under different locations and treatment methods, the seed setting rate of the main panicle of the OsCIP1 overexpressing lines was significantly higher than that of the cold-sensitive parent, Towada. This suggests that the OsCIP1 gene has the function of tolerating low temperature during the booting stage and is a gene with a positive and long-lasting effect, which can reduce the impact of low temperature on the seed setting rate under low temperature stress.

[0133] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A method for improving the cold tolerance of rice during the booting stage, characterized in that, This includes introducing a nucleic acid molecule encoding the OsCIP1 protein shown in SEQ ID NO:2 into a target rice variety to obtain transgenic rice, wherein the transgenic rice exhibits higher cold tolerance during the booting stage than the target rice.

2. The method according to claim 1, characterized in that, The genetically modified rice exhibits higher cold tolerance during the booting stage than the target rice, as evidenced by its higher seed setting rate under low-temperature stress during the booting stage.

3. The method according to claim 1 or 2, characterized in that, The low temperature is 16-17 ℃.

4. The application of the OsCIP1 protein shown in SEQ ID NO:2, the nucleic acid molecule encoding the OsCIP1 protein shown in SEQ ID NO:2, or the recombinant vector, expression cassette, or recombinant bacteria containing the nucleic acid molecule in improving the cold tolerance of rice during the booting stage.

5. The application according to claim 4, characterized in that, The improvement in cold tolerance of rice during the booting stage is reflected in the increased grain filling rate of rice under low temperature stress during the booting stage.

6. The application according to claim 4 or 5, characterized in that, The low temperature is 16-17 ℃.

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