Application of MsMCU1.2 protein and related biological materials thereof in cultivation of anti-freezing plant varieties

Abstract

The invention discloses an application of an MsMCU1.2 protein and a related biological material thereof in cultivation of an anti-freezing plant variety. The MsMCU1.2 protein is any one of the following B1)-B4): B1) a protein with an amino acid sequence as shown in a sequence 2; b2) a fusion protein which is obtained by connecting a label to the N end and / or the C end of the amino acid sequence as shown in the sequence 2 and has the same function; b3) a protein which is obtained by substitution and / or deletion and / or addition of one or more amino acid residues on the amino acid sequence as shown in the sequence 2 and has the same function; and B4) a protein which has 80% or more than 80% of identity with the amino acid sequence shown in the sequence 2 and has the same function with the amino acid sequence shown in the sequence 2. It is found for the first time that the MsMCU1.2 protein and related biological materials thereof can regulate and control the cold resistance of plants and will play an important role in cultivation of anti-freezing plant varieties.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology, and specifically relates to the application of MsMCU1.2 protein and related biological materials in cultivating frost-resistant plant varieties. Background Art

[0002] Alfalfa is an important perennial legume forage grass, widely planted around the world, mainly used as forage and feed. It is widely planted in different temperature zones due to its high yield, rich nutritional value, good palatability, strong nitrogen fixation ability, and strong ecological adaptability. However, the growth and production of alfalfa are affected by a variety of environmental stresses, among which low temperature stress is one of the important factors limiting the growth and production of alfalfa. Under extreme environmental conditions, freezing stress is the main factor that weakens the growth, development, productivity and distribution of alfalfa, especially in the cold northern regions. In addition, abnormal temperature mutations can cause serious economic losses in winter and subsequent spring frost events. The cold environment not only affects the growth rate of alfalfa, but also causes severe physiological damage and even plant death, which seriously affects its yield and quality.

[0003] Traditionally, the main method to improve the cold tolerance of alfalfa is to breed varieties with strong cold tolerance. However, this method is time-consuming and limited by genetic background and environmental factors. With the rapid development of biotechnology, transgenic technology has provided a new way to improve the cold tolerance of alfalfa. Summary of the invention

[0004] The technical problem to be solved by the present invention is how to regulate the cold resistance of plants and cultivate frost-resistant plant varieties.

[0005] In order to solve the above technical problems, the present invention first provides a new use of MsMCU1.2 protein.

[0006] The present invention provides the use of MsMCU1.2 protein in any of the following A1)-A3):

[0007] A1) Regulate plant stress tolerance;

[0008] A2) cultivating transgenic plants with improved stress tolerance;

[0009] A3) Plant breeding;

[0010] The MsMCU1.2 protein is any one of the following B1)-B4):

[0011] B1) The amino acid sequence is the protein shown in SEQ ID NO: 2;

[0012] B2) A fusion protein having the same function obtained by connecting a tag to the N-terminus and / or C-terminus of the amino acid sequence shown in SEQ ID NO: 2;

[0013] B3) a protein having the same function obtained by replacing and / or deleting and / or adding one or more amino acid residues of the amino acid sequence shown in SEQ ID NO: 2;

[0014] B4) A protein that has 80% or more identity with the amino acid sequence shown in SEQ ID NO: 2 and has the same function.

[0015] In the protein described in B2) above, the tag refers to a polypeptide or protein that is fused and expressed with the target protein using DNA in vitro recombination technology to facilitate the expression, detection, tracing and / or purification of the target protein. The tag includes but is not limited to: GST (glutathione sulfhydryl transferase) tag protein, His6 tag protein (His-tag), MBP (maltose binding protein) tag protein, Flag tag protein, SUMO tag protein, HA tag protein, Myc tag protein, GFP (green fluorescent protein), CFP (cyan fluorescent protein), YFP (yellow-green fluorescent protein), mCherry (monomeric red fluorescent protein) or AviTag tag protein.

[0016] In a specific embodiment of the present invention, the tag is a 3×FLAG tag protein. The amino acid sequence of the fusion protein is shown in Sequence 3.

[0017] In the protein described in B3) above, the substitution and / or deletion and / or addition of one or several amino acid residues is a substitution and / or deletion and / or addition of no more than 10 amino acid residues.

[0018] In the protein described in B4) above, the identity refers to the identity of the amino acid sequence. The identity of the amino acid sequence can be determined using a homology search site on the Internet, such as the BLAST page on the NCBI homepage website. For example, in Advanced BLAST2.1, by using blastp as a program, setting the Expect value to 10, setting all filters to OFF, using BLOSUM62 as a matrix, setting the Gap existence cost, Per residue gap cost and Lambda ratio to 11, 1 and 0.85 (default value) respectively, and searching for the identity of a pair of amino acid sequences for calculation, the value of the identity (%) can be obtained. The identity includes amino acid sequences having 80% or more, or 85% or more, or 90% or more, or 91% or more, or 92% or more, or 93% or more, or 94% or more, or 95% or more, or 96% or more, or 97% or more, or 98% or more, or 99% or more homology with the amino acid sequence shown in Sequence 2 of the present invention.

[0019] The proteins described in B1)-B4) above can be synthesized artificially, or their encoding genes can be synthesized first and then expressed biologically.

[0020] In order to solve the above technical problems, the present invention also provides new uses of biomaterials related to the above MsMCU1.2 protein.

[0021] The present invention provides the use of the biomaterial related to the above-mentioned MsMCU1.2 protein in any of the following A1)-A3):

[0022] A1) Regulate plant stress tolerance;

[0023] A2) cultivating transgenic plants with improved stress tolerance;

[0024] A3) Plant breeding;

[0025] The biological material is a nucleic acid molecule encoding the MsMCU1.2 protein or an expression cassette, a recombinant vector or a recombinant microorganism containing the nucleic acid molecule;

[0026] In the above application, the nucleic acid molecule is any one of the following:

[0027] F1) DNA molecule shown in sequence 1;

[0028] F2) A DNA molecule that has 75% or more identity with the nucleotide sequence defined in F1) and encodes the above-mentioned MsMCU1.2 protein.

[0029] The nucleic acid molecule may be DNA, such as cDNA, genomic DNA or recombinant DNA; the nucleic acid molecule may also be RNA, such as mRNA or hnRNA.

[0030] A person skilled in the art can easily mutate the nucleotide sequence encoding the MsMCU1.2 protein of the present invention by using known methods, such as directed evolution and point mutation. Those artificially modified nucleotides having 75% or higher identity with the nucleotide sequence encoding the MsMCU1.2 protein are derived from the nucleotide sequence of the present invention and are equivalent to the sequence of the present invention as long as they encode the MsMCU1.2 protein and have the same function.

[0031] The term "identity" as used herein refers to sequence similarity to a natural nucleic acid sequence. "Identity" includes nucleotide sequences that have 75% or more, or 85% or more, or 90% or more, or 95% or more identity to the nucleotide sequence of the protein composed of the amino acid sequence shown in the coding sequence 2 of the present invention. Identity can be evaluated by the naked eye or by computer software. Using computer software, the identity between two or more sequences can be expressed as a percentage (%), which can be used to evaluate the identity between related sequences.

[0032] The above 75% or more identity may be 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% or more identity.

[0033] In the above application, the expression cassette refers to a DNA capable of expressing the MsMCU1.2 protein in a host cell, and the DNA may include not only a promoter for initiating transcription of MsMCU1.2, but also a terminator for terminating transcription of MsMCU1.2. Further, the expression cassette may also include an enhancer sequence. Promoters that can be used in the present invention include, but are not limited to, constitutive promoters; tissue, organ and development-specific promoters and inducible promoters. Suitable transcription terminators include, but are not limited to, Agrobacterium nopaline synthase terminator (NOS terminator), cauliflower mosaic virus CaMV 35S terminator, tml terminator, pea rbcSE9 terminator, and nopaline and octopine synthase terminators.

[0034] In the above application, the vector refers to a vector that can carry the above nucleic acid molecule into the host cell for amplification and expression. The vector can be a cloning vector or an expression vector, including but not limited to: plasmid, phage (such as lambda phage or M13 filamentous phage, etc.), cosmid (i.e., cosmid), Ti plasmid, viral vector (such as retrovirus (including lentivirus), adenovirus, adeno-associated virus, etc.).

[0035] The recombinant vector refers to a recombinant DNA molecule formed by connecting the above nucleic acid molecule and the vector in vitro. The recombinant vector containing the MsMCU1.2 gene expression cassette can be constructed using an existing plant expression vector. The plant expression vector includes a binary Agrobacterium vector and a vector that can be used for plant microprojectile bombardment, etc. Such as pAHC25, pBin438, pCAMBIA1302, pCAMBIA2301, pCAMBIA1301, pCAMBIA1300, pBI121, pCAMBIA1391-Xa or pCAMBIA1391-Xb, etc. The plant expression vector may also contain the 3′ non-translated region of the foreign gene, i.e., a polyadenylic acid signal and any other DNA fragments involved in mRNA processing or gene expression. The polyadenylic acid signal can guide the addition of polyadenylic acid to the 3' end of the mRNA precursor, such as the non-translated region transcribed at the 3' end of the Agrobacterium crown gall induction (Ti) plasmid gene (such as the nopaline synthase gene Nos) and the plant gene (such as the soybean storage protein gene) all have similar functions. When using the gene of the present invention to construct a plant expression vector, enhancers can also be used, including translation enhancers or transcription enhancers. These enhancer regions can be ATG start codons or adjacent region start codons, etc., but must be the same as the reading frame of the coding sequence to ensure the correct translation of the entire sequence. The sources of the translation control signal and the start codon are extensive and can be natural or synthetic. The translation start region can come from the transcription start region or the structural gene. In order to facilitate the identification and screening of transgenic plant cells or plants, the plant expression vector used can be processed, such as adding genes that can be expressed in plants and encode enzymes or luminescent compounds that can produce color changes (GUS gene, luciferase gene, etc.), antibiotic marker genes (such as nptII gene that confers resistance to kanamycin and related antibiotics, bar gene that confers resistance to the herbicide phosphinothricin, hph gene that confers resistance to the antibiotic hygromycin, dhfr gene that confers resistance to methotrexate, EPSPS gene that confers resistance to glyphosate) or chemical resistance marker genes (such as herbicide resistance genes), mannose-6-phosphate isomerase gene that provides the ability to metabolize mannose. Considering the safety of transgenic plants, no selective marker gene can be added, and transformed plants can be directly screened by adversity.

[0036] In the above application, the microorganism can be yeast, bacteria, algae or fungi. The bacteria can be Agrobacterium, such as Agrobacterium tumefaciens LBA4404.

[0037] The recombinant microorganism refers to a recombinant microorganism with changed functions obtained by operating and modifying the genes of the target microorganism. For example, the recombinant microorganism obtained by introducing the above-mentioned recombinant vector into the target microorganism. The recombinant microorganism can be understood to refer not only to a specific recombinant microorganism, but also to the offspring of such a cell, and due to natural, accidental or intentional mutations and / or changes, the offspring may not be completely identical to the original parent cell, but is still included in the scope of the recombinant microorganism.

[0038] In the above application, the regulation of plant stress tolerance is to improve plant stress tolerance. The improvement of plant stress tolerance is reflected in that when the content and / or activity of MsMCU1.2 protein in the plant is increased, the stress tolerance of the plant is improved.

[0039] Furthermore, the improving of plant stress tolerance is improving plant cold tolerance.

[0040] Furthermore, the improvement of plant cold tolerance is specifically manifested in that when the expression level of the MsMCU1.2 gene in the plant is increased, the survival rate of the plant after freezing stress is increased.

[0041] In the above application, the purpose of plant breeding is to cultivate stress-resistant plant varieties (such as frost-resistant plant varieties).

[0042] In order to solve the above technical problems, the present invention finally provides a method for cultivating transgenic plants with improved stress tolerance.

[0043] The method for cultivating transgenic plants with improved stress tolerance provided by the present invention comprises the following steps: increasing the content and / or activity of the MsMCU1.2 protein in the target plant to obtain a transgenic plant; the stress tolerance of the transgenic plant is higher than that of the target plant.

[0044] In the above method, the stress resistance is cold resistance.

[0045] Furthermore, the stress tolerance of the transgenic plant is higher than that of the target plant, which is reflected in that the survival rate of the transgenic plant after freezing stress is higher than that of the target plant.

[0046] Furthermore, the freezing stress condition may be -10°C treatment for 1.5h.

[0047] In the above method, the method for increasing the content and / or activity of the MsMCU1.2 protein in the target plant is to overexpress the MsMCU1.2 protein in the target plant.

[0048] Furthermore, the overexpression method is to introduce the coding gene of the MsMCU1.2 protein into the target plant.

[0049] Furthermore, the nucleotide sequence of the gene encoding the MsMCU1.2 protein is shown in Sequence 1.

[0050] In any of the above applications or methods, the transgenic plants are understood to include not only the first generation transgenic plants obtained by transforming the MsMCU1.2 gene into a recipient plant, but also its progeny. For transgenic plants, the gene can be propagated in the species, or the gene can be transferred into other varieties of the same species, especially commercial varieties, using conventional breeding techniques. The transgenic plants include seeds, callus, complete plants and cells.

[0051] In any of the above applications or methods, the plant is a monocotyledon or a dicotyledon; further, the dicotyledon may be a legume and / or a crucifer; further, the legume may be alfalfa (such as alfalfa); the crucifer may be Arabidopsis thaliana (such as Columbia ecotype col-0).

[0052] The present invention overexpresses MsMCU1.2 in wild-type Arabidopsis to obtain MsMCU1.2 overexpressing Arabidopsis, and after cold tolerance analysis of the MsMCU1.2 overexpressing Arabidopsis, it is found that overexpression of MsMCU1.2 can improve plant cold tolerance. The present invention is the first to discover that MsMCU1.2 protein and related biomaterials can regulate plant cold tolerance, which will play an important role in breeding frost-resistant plant varieties. BRIEF DESCRIPTION OF THE DRAWINGS

[0053] Figure 1 Analysis of the freezing stress expression pattern of MsMCU1.2.

[0054] Figure 2 Schematic diagram and identification of the structure of MsMCU1.2 overexpression vector.

[0055] Figure 3 The identification of MsMCU1.2 overexpressing Arabidopsis thaliana, wherein WT represents wild-type Arabidopsis thaliana (Columbia ecotype), and 35S:MsMCU1.2 represents homozygous strains of MsMCU1.2 overexpressing Arabidopsis thaliana.

[0056] Figure 4 This is an analysis of the cold tolerance of Arabidopsis thaliana overexpressing MsMCU1.2. WT represents wild-type Arabidopsis thaliana (Columbia ecotype), and 35S:MsMCU1.2 represents homozygous strains of Arabidopsis thaliana overexpressing MsMCU1.2. DETAILED DESCRIPTION

[0057] The present invention is further described in detail below in conjunction with specific embodiments, and the examples provided are only for illustrating the present invention, rather than for limiting the scope of the present invention. The examples provided below can be used as a guide for further improvements by those of ordinary skill in the art, and do not constitute a limitation of the present invention in any way.

[0058] The experimental methods in the following examples, unless otherwise specified, are all conventional methods, and are performed according to the techniques or conditions described in the literature in the field or according to the product instructions. The materials, reagents, etc. used in the following examples, unless otherwise specified, can all be obtained from commercial channels.

[0059] The alfalfa variety Dongnong No. 1 in the following embodiment is recorded in the document "Yin Xiujie, Cui Guowen, Hu Guofu, et al. Breeding report of a new alfalfa variety Dongnong No. 1 [J]. Chinese Journal of Grassland, 2018, 40(01): 29-34."

[0060] The pCAMBIA1300-221-3×FLAG vector in the following examples is described in the document “Xiang Y, Li G, LiQ, Niu Y, Pan Y, Cheng Y, Bian X, Zhao C, Wang Y, Zhang A. Autophagy receptor ZmNBR1 promotes the autophagic degradation of ZmBRI1a and enhances drought tolerance in maize. J Integr Plant Biol. 2024Jun;66(6):1068-1086.”

[0061] The Agrobacterium tumefaciens LBA4404 in the following examples is recorded in the document “C. Chen, X. Sun, H. Duanmu, D. Zhu, Y. Yu, L. Cao, A. Liu, B. Jia, J. Xiao, Y. Zhu. GsCML27, a gene encoding a calcium-binding ef-hand protein from Glycine soja, plays differential roles in plant responses to bicarbonate, salt and osmotic stresses. PLoS One, 2015, 10(11): e0141888.”

[0062] Example 1. Obtaining the MsMCU1.2 gene in alfalfa

[0063] 1. Primer design

[0064] The basic information of alfalfa MsMCU1.2 gene was downloaded from the MODMS online database, and gene-specific primers were designed using PrimerPremier5.0. The primer sequences are as follows:

[0065] MsMCU1.2-FP: 5'-ATGTGGGGTAGGTGGTGTT-3';

[0066] MsMCU1.2-RP: 5'-TTAATCTCTATGTAAAGCATCTTCAAGATC-3'.

[0067] 2. Obtaining cDNA

[0068] Select full Dongnong No. 1 alfalfa seeds, soak them in 50°C hot water for 2 hours to break dormancy, place the soaked alfalfa seeds in a culture dish with filter paper for 2-4 days, transfer them to soil culture when the root length grows to 1-2 cm, place them in an artificial culture room and culture them to the three-leaf stage under the conditions of 28°C light 14h / 24°C dark 10h photoperiod. Take the leaves of the seedlings at the three-leaf stage, extract total RNA using Trizol (Invitrogen), and obtain cDNA by reverse transcription.

[0069] 3. PCR amplification

[0070] Using the cDNA obtained in step 2 as a template, the MsMCU1.2-FP and MsMCU1.2-RP primers designed in step 1 were used to polymerize the DNA using the high-fidelity DNA polymerase KOD One TM PCR Master Mix-Blue (TOYOBO) was used for PCR amplification to obtain a PCR product, which is the full-length CDS region of the MsMCU1.2 gene. Its nucleotide sequence is shown in Sequence 1 in the sequence listing, and the amino acid sequence of the MsMCU1.2 protein encoded by the MsMCU1.2 gene is shown in Sequence 2 in the sequence listing.

[0071] The PCR product was connected to the pEASY-T vector, and the positive clones were sequenced after identification. The vector with the correct sequencing was named pEASY-MsMCU1.2 for subsequent research.

[0072] Example 2: Analysis of the expression pattern of MsMCU1.2 under freezing stress

[0073] According to the method in step 2 of Example 1, 30-day-old alfalfa was obtained. The 30-day-old alfalfa was first acclimated at 4°C for 3 days, and then frozen at -5°C. Leaf samples were taken after 0h, 2h, 4h, and 6h of freezing. The total RNA of the leaves was extracted by Trizol method, and cDNA was obtained by reverse transcription using the reverse transcription kit 5×HiScript III RT SuperMix (Vazyme). The diluted cDNA was used as a template, alfalfa MsActin was used as an internal reference gene, and MsMCU1.2-qRT-F and MsMCU1.2-qRT-R primers were used for qRT-PCR. The primer sequences are as follows:

[0074] MsMCU1.2-qRT-FP:5'-GGTTCTTGATGATGCTGGGGT-3';

[0075] MsMCU1.2-qRT-RP:5'-TGTCAGAAGTCAGTGCCAGT-3';

[0076] MsActin-qRT-FP: 5'-CCCACTGGATGTCTGTAGGTT-3';

[0077] MsActin-qRT-RP: 5'-AGAATTAAGTAGCAGCGCAAA-3'.

[0078] The results are as follows Figure 1 As shown in Figure 2, qRT-PCR results showed that MsMCU1.2 was upregulated by freezing stress, and the expression change was the largest at 4 h of freezing stress ( Figure 1 ). The above results indicate that the expression of MsMCU1.2 is induced by freezing stress.

[0079] Example 3: Acquisition of MsMCU1.2-overexpressing Arabidopsis and analysis of its cold tolerance

[0080] 1. Construction of MsMCU1.2 plant overexpression vector

[0081] 1. Using the pEASY-MsMCU1.2 plasmid in Example 1 as a template, using the primers MsMCU1.2-F and MsMCU1.2-R, and using the high-fidelity DNA polymerase KOD One TM PCR Master Mix-Blue (TOYOBO) was used for PCR amplification to obtain the PCR product and to recover it by gel extraction. The primer sequences are as follows (the lower case is the homology arm sequence):

[0082] MsMCU1.2-F: 5'-acgatgataagggcggtaccATGTGGGGTAGGTGGTGTT-3';

[0083] MsMCU1.2-R: 5'-aggctacgtaggatccTTAATCTCTATGTAAAGCATCTTCAAGATC-3'.

[0084] 2. The pCAMBIA1300-221-3×FLAG vector was double-digested with restriction endonucleases Kpn I and BamH I to obtain the vector digestion product and recover it by gel recovery.

[0085] 3. The vector digestion recovery product obtained in step 2 and the MsMCU1.2 gene recovery product obtained in step 1 were subjected to homologous recombination reaction, incubated at 37°C for 30 minutes and transformed into Escherichia coli (DH5α). After successful transformation, single colonies were identified ( Figure 2 B) The positive colonies were then activated and the plasmids were extracted and sent for sequencing. The recombinant plasmid with correct sequencing was named pCAMBIA1300-221-3×FLAG-MsMCU1.2.

[0086] The recombinant plasmid pCAMBIA1300-221-3×FLAG-MsMCU1.2 is a plasmid obtained by replacing the DNA fragment between the Kpn I and BamHI restriction sites of the pCAMBIA1300-221-3×FLAG vector with the MsMCU1.2 gene shown in sequence 1, while keeping the other sequences of the pCAMBIA1300-221-3×FLAG vector unchanged. The schematic diagram of the structure of the recombinant plasmid pCAMBIA1300-221-3×FLAG-MsMCU1.2 is shown in Figure 2 As shown in A. The recombinant plasmid pCAMBIA1300-221-3×FLAG-MsMCU1.2 expresses MsMCU1.2 protein with a 3×FLAG tag.

[0087] II. Acquisition and identification of MsMCU1.2 overexpressing Arabidopsis

[0088] 1. The recombinant plasmid pCAMBIA1300-221-3×FLAG-MsMCU1.2 was introduced into wild-type Arabidopsis thaliana (Columbia ecotype) by Agrobacterium-mediated inflorescence infection, so that the MsMCU1.2 gene was overexpressed in Arabidopsis thaliana to obtain T0 transgenic Arabidopsis thaliana. The specific steps are as follows: the pCAMBIA1300-221-3×FLAG-MsMCU1.2 recombinant plasmid obtained in step 1 was transformed into Agrobacterium tumefaciens LBA4404 by freeze-thaw method, and the single colony identified correctly by PCR was reactivated, centrifuged at 4000rpm for 10min, the supernatant was discarded, and the bacterial block was resuspended in infection solution (5% sucrose + 0.05% Silwet77) to OD 600nm The inflorescence of Arabidopsis thaliana was removed from the developed siliques and immersed in the infective solution for 1 min. After infection, it was kept in the dark overnight and then resumed normal culture.

[0089] 2. After sterilizing the T0 transgenic Arabidopsis seeds with NaClO, screen them on a 1 / 2MS solid medium containing 25 mg / L hygromycin. Arabidopsis that can grow normally on the resistant medium indicates that it is a resistant seedling that has been successfully genetically transformed. Move it into nutrient soil for cultivation. When the resistant seedlings grow up, take samples and extract DNA for testing. Using genomic DNA as a template, use MsMCU1.2-FP and MsMCU1.2-RP primers for PCR identification. The PCR identification results are as follows: Figure 3 As shown in A. As can be seen from the figure: no band was amplified when wild-type Arabidopsis WT and H2O were used as templates, plasmid pCAMBIA1300-221-3×FLAG-MsMCU1.2 was used as a positive control, and a band was cloned at a maker size of about 1000bp. Four strains (#1, #2, #3, and #4) amplified bands, indicating that the Arabidopsis genome already contains the MsMCU1.2 gene.

[0090] 3. Arabidopsis seedlings that were correctly identified by PCR were harvested individually, and the positive T1 plants were propagated to harvest T2 seeds. The resistant seedlings of T2 plants were screened until the homozygous T3 generation was obtained. The homozygous T3 generation seeds were screened together with WT on 1 / 2MS solid culture medium containing 25 mg / L hygromycin. As shown in 3B, WT did not grow on the resistance plate, while the pCAMBIA1300-221-3×FLAG-MsMCU1.2 transgenic strain was able to grow normally. After screening, 4 T3 generation transgenic Arabidopsis homozygous strains with a hygromycin positivity rate of 100% were successfully obtained.

[0091] 4. Samples were taken from the four T3 transgenic Arabidopsis homozygous lines, and proteins were extracted for Western blot analysis. The results showed that protein bands were detected around 40 kDa in the four T3 transgenic Arabidopsis homozygous lines, which was consistent with the expected size of 41.16 kDa for 3×FLAG-MsMCU1.2, indicating that the exogenous gene MsMCU1.2 can be expressed in transgenic Arabidopsis ( Figure 3 C).

[0092] In summary, 3×FLAG-MsMCU1.2 has been successfully expressed in Arabidopsis, and T3 generation MsMCU1.2 overexpressing Arabidopsis homozygous lines #2 and #3 were selected for subsequent cold tolerance analysis experiments.

[0093] III. Analysis of cold tolerance in MsMCU1.2-overexpressing Arabidopsis

[0094] Test materials: wild-type Arabidopsis thaliana (Columbia ecotype), T3 generation MsMCU1.2 overexpressing Arabidopsis thaliana homozygous lines #2 and #3.

[0095] Experimental method: After the seeds of the test materials were disinfected with NaClO, they were vernalized at 4℃ for 2 days, then sown in 1 / 2MS culture medium and cultured normally for 5 days. After the cotyledons unfolded, they were placed in a 4℃ incubator for cold acclimation for 3 days. After the acclimation was completed, the temperature of the incubator was adjusted to -10℃, and the lid was opened for 1.5h. After the treatment, the temperature of the incubator was adjusted to 4℃, and the culture was continued for 12h. Finally, they were transferred to 22℃ and long-day conditions for recovery culture. After 3 days of recovery culture, the phenotype was observed and the survival rate was calculated.

[0096] Phenotypic observations Figure 4 As shown in A, the results show that compared with the homozygous strain of MsMCU1.2 overexpressing Arabidopsis, the wild-type Arabidopsis plants were more severely damaged after freezing at -10℃. Figure 4 As shown in B, the results showed that the survival rate of the homozygous strains #2 and #3 of Arabidopsis thaliana overexpressing MsMCU1.2 was about 75%, while the survival rate of the WT strain was about 30%, and the survival rate of the homozygous strains #2 and #3 of Arabidopsis thaliana overexpressing MsMCU1.2 was significantly higher than that of the WT strain. These results indicate that the homozygous strains of Arabidopsis thaliana overexpressing MsMCU1.2 are more cold-resistant.

[0097] In summary, MsMCU1.2 overexpression can enhance the tolerance of Arabidopsis to freezing stress.

[0098] The present invention has been described in detail above. It will be apparent to those skilled in the art that the present invention may be implemented in a wide range under equivalent parameters, concentrations and conditions without departing from the spirit and scope of the present invention and without the need for unnecessary experimentation. Although the present invention provides specific embodiments, it should be understood that further improvements may be made to the present invention. In short, according to the principles of the present invention, this application intends to include any changes, uses or improvements to the present invention, including changes made by conventional techniques known in the art that depart from the scope disclosed in this application. Applications of some of the basic features may be made within the scope of the following appended claims.

Claims

1. Application of MsMCU1.2 protein in any of the following A1)-A3): A1) Regulate plant stress tolerance; A2) cultivating transgenic plants with improved stress tolerance; A3) Plant breeding; The MsMCU1.2 protein is any one of the following B1)-B4): B1) The amino acid sequence is the protein shown in SEQ ID NO: 2; B2) A fusion protein having the same function obtained by connecting a tag to the N-terminus and / or C-terminus of the amino acid sequence shown in SEQ ID NO: 2; B3) a protein having the same function obtained by replacing and / or deleting and / or adding one or more amino acid residues of the amino acid sequence shown in SEQ ID NO: 2; B4) A protein that has 80% or more identity with the amino acid sequence shown in SEQ ID NO: 2 and has the same function.

2. Application of biological materials related to MsMCU1.2 protein in any of the following A1)-A3): A1) Regulate plant stress tolerance; A2) cultivating transgenic plants with improved stress tolerance; A3) Plant breeding; The biological material is a nucleic acid molecule encoding the MsMCU1.2 protein or an expression cassette, a recombinant vector or a recombinant microorganism containing the nucleic acid molecule; The MsMCU1.2 protein is any one of the following B1)-B4): B1) The amino acid sequence is the protein shown in SEQ ID NO: 2; B2) A fusion protein having the same function obtained by connecting a tag to the N-terminus and / or C-terminus of the amino acid sequence shown in SEQ ID NO: 2; B3) a protein having the same function obtained by replacing and / or deleting and / or adding one or more amino acid residues of the amino acid sequence shown in SEQ ID NO: 2; B4) A protein that has 80% or more identity with the amino acid sequence shown in SEQ ID NO: 2 and has the same function.

3. The use according to claim 2, characterized in that: The nucleic acid molecule is any of the following: F1) The DNA molecule shown in Sequence 1; F2) A DNA molecule that has 75% or more identity with the nucleotide sequence defined in F1) and encodes the MsMCU1.2 protein.

4. The use according to any one of claims 1 to 3, characterized in that: The regulating plant stress tolerance is to improve plant stress tolerance.

5. The use according to any one of claims 1 to 4, characterized in that: The stress resistance is cold resistance.

6. A method for cultivating transgenic plants with improved stress tolerance, comprising the following steps: increasing the content and / or activity of MsMCU1.2 protein in a target plant to obtain a transgenic plant; the transgenic plant has higher stress tolerance than the target plant.

7. The method according to claim 6, characterized in that: The stress resistance is cold resistance.

8. The method according to claim 6 or 7, characterized in that: The method for increasing the content and / or activity of MsMCU1.2 protein in the target plant is to overexpress the MsMCU1.2 protein in the target plant.

9. The method according to claims 6-8, characterized in that: The overexpression method is to introduce the gene encoding the MsMCU1.2 protein into the target plant; Alternatively, the nucleotide sequence of the gene encoding the MsMCU1.2 protein is as shown in Sequence 1.

10. The use according to any one of claims 1 to 5 or the method according to any one of claims 6 to 9, characterized in that: The plant is a monocotyledon or a dicotyledon.

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