New application of millet SiMPK6 protein or coding gene thereof

Abstract

The invention belongs to the technical field of gene engineering, and particularly discloses application of millet SiMPK6 protein or a coding gene thereof in regulation and control of high light stress resistance of plants. Experiments prove that the SiMPK6 gene positively regulates the stress of plants on highlight, can maintain a relatively high photosynthetic rate under adversity, and can be used for modifying C3 plants and improving the highlight resistance of the C3 plants. The specific operation comprises the following steps: performing codon optimization on a coding gene of the millet SiMPK6 protein aiming at C3 plant preference, and constructing a recombinant expression vector containing a target gene; introducing the recombinant expression vector into host bacteria to obtain recombinant bacteria; transforming the recombinant bacteria into a receptor plant, screening out a positive plant, and culturing to obtain a transgenic plant with enhanced high light stress resistance; the C3 plants include but are not limited to rice, wheat, soybeans, potatoes, cotton and the like.

Description

Technical Field

[0001] The present invention belongs to the technical field of genetic engineering, and particularly relates to the application of millet SiMPK6 protein or its encoding gene in regulating plant high light stress resistance. Background Art

[0002] Light is a crucial energy source for photosynthesis in green plants. Plants rely on light for photosynthesis, but due to their immobility, when ambient light intensity exceeds the range they can utilize, it harms their growth, causing photoinhibition, resulting in reduced ΦPSII and decreased photosynthetic rate. To survive in nature, plants must adapt to rapid changes in light intensity and quality, maintaining an autotrophic lifestyle by increasing their photosynthetic activity to adapt to varying light intensities (Dietzel et al., 2008; Pesaresi et al., 2010). Rapid adaptation to light intensity is particularly crucial for young plants in their early stages of growth, as they require a rapidly developing photosynthetic system (Anderson and Osmond, 1987; Anderson et al., 1995).

[0003] To cope with photoinhibition, plants have evolved a series of adaptive mechanisms for high light levels. These mechanisms allow them to adapt to high light levels on various timescales by altering and optimizing various physiological and biochemical processes (Park et al. 1996; Savitch et al. 2000). Plants have evolved a variety of photoreceptors to sense changes in light conditions, thereby helping them to respond and adjust. For example, cryptochrome 1 (CRY1) is implicated in the formation of reactive oxygen species (ROS) under high light stress (Consentino et al. 2015; Jourdan et al. 2015). Furthermore, CRYs can promote the accumulation of soluble phenolics in leaves, improving their response to high light stress and thereby increasing their photosynthetic efficiency (Brelsford et al. 2019). Furthermore, CRY1 can also induce the accumulation of anthocyanins through the COP1 / SPA1 / HY5 pathway to combat high light stress (Kleine et al. 2007). Under high light stress, phototropin B (PHOT B) is also involved in plant stomatal movement and ROS signaling (Amith et al., 2020). Despite extensive research, the mechanisms by which plants sense excessive irradiance and how this information is transmitted to the nucleus to initiate genetic responses remain elusive.

[0004] Over the course of long-term evolution, plants have developed complex adaptive systems, among which the serine / threonine (Ser / Thr) protein kinase network plays a key role in sensing environmental stimuli and translating them into appropriate physiological responses. Mitogen-activated protein kinases (MAPKs), members of the Ser / Thr protein kinase family, regulate diverse cellular processes through phosphorylation cascades. Activated MAPKs can target a variety of downstream substrates, such as kinases, enzymes, and transcription factors, thereby affecting gene expression, metabolism, cell growth, and division. Protein phosphorylation is a crucial mechanism for regulating fundamental cellular processes in organisms, often through a cascade network. The mitogen-activated protein kinase (MAPK) cascade signaling pathway is a ubiquitous and highly conserved class of serine / threonine (Ser / Thr) protein kinase systems in eukaryotes. It plays a crucial role in plant growth and development, as well as in responses to biotic and abiotic stresses. Typically, a complete MAPK cascade signaling pathway is composed of three key kinase components: MAPKKK, MAPKK, and MAPK. During cellular signal transduction, the MAPK cascade precisely regulates a series of complex physiological processes, including cell differentiation, proliferation, growth, and development, as well as responses to various stress signals, through sequential phosphorylation modifications between kinase components. MAPK, also known as MPK, lies at the very bottom of the MAPK cascade. It is a key protein kinase in the conversion and amplification of cellular signals and serves as the direct link between the cascade signal and downstream proteins. Studies have shown that MAPK family members all possess 11 conserved protein kinase domains, including the "A-Loop" region between subdomains VII and VIII, which contains the TXY (Thr-X-Tyr) activation domain. Plant MAPKs are divided into two subtypes, TEY and TDY, based on the amino acid sequence "X" in the activation domain. The TEY subtype is further subdivided into subfamilies A, B, and C, while the TDY subtype constitutes the evolutionarily more distant D subfamily. Two key members of the A subfamily, AtMPK3 and AtMPK6, are involved in plant responses to various biotic and abiotic stresses and in regulating plant growth and development. AtMPK4 of the B subfamily plays a role in salicylic acid and jasmonic acid signaling pathways. AtMPK1, AtMPK2, AtMPK7, and AtMPK14 of the C subfamily are primarily involved in ABA signal transduction, dehydration stress response, and plant immunity. AtMPK9 and AtMPK12 of the D subfamily participate in the ABA signaling pathway involved in plant growth and development, as well as in the regulation of reactive oxygen species. However, the involvement of MAPKs in high-light regulation mechanisms has not been investigated to date.

[0005] Millet (Setaria italica L.), also known as foxtail millet or foxtail millet, is a diploid annual crop native to my country, a traditional grain and feed crop of the genus Setaria in the Poaceae family. It is known in Chinese as the "first of the five grains." With the vigorous development of my country's economy and society, the agricultural sector has undergone profound changes, with the industrial structure continuously optimized. Millet, with its strong adaptability and stress resistance, can achieve relatively good yields on arid and barren lands, fully demonstrating its unique advantages in utilizing my country's scarce land resources and unique geographical and ecological conditions, and opening up an effective path to national food security. As a C4 plant, millet has high photosynthetic efficiency, nitrogen and water use efficiencies, and strong environmental adaptability, making it an ideal model for studying C4 photosynthesis and crop stress tolerance. Summary of the Invention

[0006] The main technical problem solved by the present invention is to provide a new use of foxtail millet SiMPK6 protein or its encoding gene.

[0007] At the same time, the present invention provides a method for improving the high light stress resistance of plants.

[0008] In order to solve the above technical problems, the present invention provides the following technical solutions:

[0009] A new use of a millet SiMPK6 protein or a gene encoding it, wherein the new use is: use in regulating plant high light stress resistance; the amino acid sequence of the millet SiMPK6 protein is shown in SEQ ID NO: 1.

[0010] As a preferred embodiment of the present invention, the regulation is to overexpress SiMPK6 to improve the plant's resistance to high light stress.

[0011] As a preferred embodiment of the present invention, the plant is a C3 plant, including but not limited to rice, wheat, soybean, potato, cotton, etc.

[0012] More preferably, the gene encoding the foxtail millet SiMPK6 protein needs to be codon-optimized for C3 plant preference.

[0013] A method for improving plant resistance to high light stress comprises the following steps:

[0014] The coding gene of millet SiMPK6 protein is introduced into a recipient plant for overexpression to obtain a transgenic plant with enhanced resistance to high light stress; the amino acid sequence of the millet SiMPK6 protein is shown in SEQ ID NO: 1.

[0015] As a preferred embodiment of the present invention, the introduction into the recipient plant includes: constructing a recombinant expression vector containing the coding gene of the foxtail millet SiMPK6 protein; introducing the recombinant expression vector into the host bacteria to obtain recombinant bacteria; transforming the recombinant bacteria into the recipient plant, screening the positive plants and culturing them to obtain transgenic plants with enhanced resistance to high light stress.

[0016] As a preferred embodiment of the present invention, the recipient plant is a C3 plant, including but not limited to rice, wheat, soybean, potato, cotton, etc.

[0017] More preferably, the gene encoding the foxtail millet SiMPK6 protein is codon-optimized for C3 plant preference.

[0018] Beneficial effects of the present invention:

[0019] The present invention provides the use of the foxtail millet SiMPK6 protein or its encoding gene in regulating plant resistance to high light stress. Experiments have confirmed that the SiMPK6 gene positively regulates plant resistance to high light stress, maintaining a high photosynthetic rate under adverse conditions. It can be used to modify C3 plants and improve their tolerance to high light stress. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 This is a diagram of tissue-specific expression analysis of the SiMPK6 gene.

[0021] Figure 2 This is an analysis diagram of the expression pattern of the SiMPK6 gene under high light stress.

[0022] Figure 3 This is the identification result of positive transgenic plants.

[0023] Figure 4 High light stress treatment of SiMPK6 transgenic Arabidopsis thaliana.

[0024] Figure 5 These are the fluorescence parameter detection results of SiMPK6 transgenic Arabidopsis thaliana after high light stress treatment.

[0025] Figure 6 These are the results of H2O2 content and transcription detection of protective enzyme genes in SiMPK6 transgenic Arabidopsis.

[0026] To more clearly illustrate the technical solutions of the embodiments of the present invention, the above briefly introduces the drawings obtained in the experimental examples. It should be understood that the above drawings only illustrate certain experimental examples of the present invention and should not be construed as limiting the scope of protection of the claims. Those skilled in the art can also derive other relevant drawings based on these drawings without inventive effort. DETAILED DESCRIPTION

[0027] The technical solution of the present invention will be clearly and completely described below in conjunction with specific embodiments and experimental examples. However, it should be understood by those skilled in the art that the embodiments are only used to illustrate the technical solution of the present invention and should not be regarded as limiting the scope of protection of the present invention. Based on the following embodiments, all other embodiments obtained by those of ordinary skill in the art without making creative work, such as modifications, deformations, or simple replacements, should fall within the scope of protection of the present invention.

[0028] Unless otherwise specified, the experimental methods used in the following examples and experimental examples are all conventional methods; the raw materials (including biological materials), reagents, culture media, instruments, etc. used are all commonly used in the art and commercially available to the public unless otherwise specified; the terms and abbreviations involved have the conventional meanings in the art, such as PBS buffer is phosphate buffered saline.

[0029] Example 1

[0030] This embodiment provides a new use of a foxtail millet SiMPK6 protein or a gene encoding it, specifically, its use in regulating high light stress resistance in C3 plants; the amino acid sequence of the foxtail millet SiMPK6 protein is shown in SEQ ID NO: 1.

[0031] Foxtail millet SimPK6 protein:

[0032] MDGGAQPPDTEMTDAGAGAGGGGGHPPQQPAGGGGGMMDNIQATLSHGGRFIQYNIFGNVFEVTAKYKPPILPIGKGAYGIVWTGEQVAIKKIANAFDNKIDAKRTLREIKLLRHMDHENYFLYQILRGLKYIHSANVLHRDLKPSNLLLNANCDLKICDFGLARITSETDFMTE YVVTRWYRAPELLLNSSEYTAAIDVWSVGCIFMELMDRKPLFPGRDHVHQLRLLMELIGTPNEADLDFVNENARRYIRQLPRHARQSFPEKFPHVQPLAIDLVEKMLTFDPRQRITVEGALAHPYLASLHDISDEPVCSMPFSFDFEQHALSEEQMKDLIYQEALAFNPDYQ(SEQ ID NO: 1).

[0033] Example 2

[0034] This embodiment provides a method for improving plant resistance to high light stress, comprising the following steps:

[0035] The coding gene of the millet SiMPK6 protein was codon-optimized according to the preference of the C3 plant rice to construct a recombinant expression vector containing the target gene; the recombinant expression vector was introduced into a host bacterium to obtain a recombinant bacterium; the recombinant bacterium was transformed into rice, and positive plants were screened and cultured to obtain transgenic rice with enhanced resistance to high light stress; the amino acid sequence of the millet SiMPK6 protein is shown in SEQ ID NO: 1.

[0036] In other embodiments of the present invention, the coding gene of millet SiMPK6 protein is codon-optimized and introduced into other C3 plants such as wheat, soybean, potato, cotton, etc. for overexpression to improve the high light tolerance of other C3 plants.

[0037] Experimental example

[0038] 1. Bioinformatics Analysis of the Foxtail Millet SiMPK6 Gene

[0039] The sequence information for the foxtail millet SiMPK6 gene, Setaria italicav2.2 (Phytozome genome ID: 312, NCBI taxonomy ID: 4555), was obtained from the foxtail millet genome database of the U.S. Department of Energy Joint Genome Institute. The coding region of this gene is 1044 base pairs long and encodes a protein consisting of 347 amino acids. Using the Compute pI / MW tool from the Swiss Bioinformatics Institute ExPASy (http: / / web.expasy.org), the protein's molecular weight was predicted to be 39131.67 Da, with an isoelectric point of 5.71. Analysis using Serverl ProtScale software revealed that the foxtail millet SiMPK6 protein is hydrophilic and lacks a signal peptide. This suggests that the protein is unlikely to be localized in chloroplasts, mitochondria, or secreted extracellularly. This localization suggests that the protein may function in specific intracellular compartments and participate in cellular physiological processes. Analysis of the secondary structure of SiMPK6 using Sopma software revealed that it is composed of 44.09% α-helices, 5.76% β-sheets, 14.13% extended strands, and 36.02% random coils. Prediction of conserved domains in foxtail millet SiMPK6 using Pfam software revealed a Pkinase (Clan: CL0016) conserved domain between amino acids 120-301. The presence of this conserved domain suggests that SiMPK6 belongs to the MAPK protein kinase family and may possess typical functions within this family, participating in intracellular signal transduction. Furthermore, the tertiary structure of SiMPK6 shows a high degree of similarity (Seq Identity: 87.70%) to that of Arabidopsis thaliana AtMPK6. Similar tertiary structures often indicate similar functions, providing important insights into the function of SiMPK6. It is speculated that its function in foxtail millet may be similar to that of AtMPK6 in Arabidopsis thaliana. NetPhos 3.1 software analysis revealed the presence of 10 serine, 7 threonine, 8 tyrosine, and numerous potential phosphorylation sites in SiMPK6. Phosphorylation is an important post-translational modification of proteins, and the presence of these phosphorylation sites suggests that SiMPK6 may participate in intracellular signaling regulation through phosphorylation and dephosphorylation, thereby affecting plant growth, development, and stress responses.

[0040] TargetP-2.0 predicted the presence of a signal peptide (SP), mitochondrial transit peptide (mTP), chloroplast transit peptide (cTP), or thylakoid lumen transit peptide (lTP) in the N-terminal presequence of foxtail millet SiMPK6, but the specific type of transit peptide was not clearly identified. Analysis using the glycosylation analysis software NetNGlyc (v1.0) revealed that SiMPK6 contains only one glycosylation site (NSSE) at position 190 (N190). Glycosylation modifications may affect protein folding, sorting, localization, and degradation. The presence of this glycosylation site provides new insights into the function of SiMPK6.

[0041] 2. Cloning of the Foxtail Millet SiMPK6 Gene

[0042] 1. Material preparation and processing

[0043] The millet (Setaria italica) variety Yugu No. 1 was obtained from the Beijing Crop Germplasm Resource Bank of the Beijing Academy of Agricultural and Forestry Sciences. Millet seedlings grown in a greenhouse for 35 days were subjected to high light stress and then individual samples were collected. The high light stress treatment method was as follows: the millet seedlings were placed in a 1200 μM·m -2 ·s -1 Under the conditions of 0h, 1h, 3h, 6h, 12h, and 24h, respectively. -2 ·s -1 The cells were cultured under stress conditions for 24 hours and the phenotypes were observed.

[0044] 2. RNA Extraction and cDNA Synthesis

[0045] Appropriate amounts of millet leaves after the various treatments described above, as well as roots, stems, and leaves of millet cultured under normal conditions, were placed in 1.5 mL centrifuge tubes and quickly frozen in liquid nitrogen. The leaves were removed from liquid nitrogen and quickly ground into powder, which was then transferred to a centrifuge tube containing 1 mL of TRIzol reagent. The mixture was vortexed and allowed to stand at room temperature for 5 min. 200 μL of chloroform was added, the mixture was vigorously shaken and allowed to stand at room temperature for 5 min, centrifuged at 12,000 × g and 4°C for 15 min, and the supernatant was aspirated and transferred to a new centrifuge tube. An equal volume of isopropanol was added, the mixture was inverted and allowed to stand at room temperature for 10 min, centrifuged at 12,000 × g and 4°C for 10 min, and the supernatant was discarded. The precipitate was washed with 70% ethanol, centrifuged at 7,500 × g and 4°C for 5 min, and the supernatant was discarded. The precipitate was air-dried and dissolved in 100 μL of sterile water for determination of concentration and purity. Add 3 volumes of anhydrous ethanol and 1 / 10 volume of 3 M potassium acetate (pH 5.8), mix well, and place at -80°C for at least 30 min. Centrifuge at 7500×g and 4°C for 5 min, discard the supernatant, and dissolve the RNA in sterile water to the desired concentration.

[0046] After removing DNA contamination from the obtained RNA, cDNA was synthesized using Takara's M-MLV reverse transcription kit. 2.5 μg of RNA was contained in a 15 μL reaction system, and cDNA was obtained after 90 minutes of reverse transcription at 42°C. The cDNA was placed on ice for 5 minutes and then stored in a -20°C freezer for later use.

[0047] 3. Gene cloning

[0048] Using the above cDNA as a template, PCR amplification was performed using the following primers (SEQ ID NO: 3-4):

[0049] Upstream primer: 5′-CATATGGACGGCGGGGCGCA-3′;

[0050] Downstream primer: 5′-CTACTGGTAATCAGGGTTGAATGCAA-3′.

[0051] The components of the 30 μL PCR reaction system are as follows: 10× Buffer 3 μL, 10 mmol·L -1 dNTP 3μL, upstream and downstream primers (10μmol·L -1 ), 1 μL each of foxtail millet leaf cDNA, 1 μL, TAKARA polymerase 0.3 μL, and 21.7 μL of ddH2O.

[0052] PCR program: pre-denaturation at 98°C for 3 min; denaturation at 98°C for 30 s, annealing at 59°C for 30 s, extension at 72°C for 80 s, 35 cycles; extension at 72°C for 10 min, and storage at 4°C.

[0053] After the PCR reaction, the PCR product was analyzed by 1.0% agarose gel electrophoresis, yielding a 1044 bp PCR product. This product was purified using a DNA gel recovery kit and ligated into the pEASY-Blunt Zero vector to construct the pT-SiMPK6 cloning vector. Sequencing confirmed that the target gene sequence was correct.

[0054] Foxtail millet SiMPK6 gene (SiMAPK6-Seita.4G069900-1044bp):

[0055]

[0056] 3. Analysis of specific expression of SiMPK6 gene in foxtail millet

[0057] To gain a deeper understanding of the expression pattern of the SiMPK6 gene in foxtail millet, we selected roots, stems, and leaves of foxtail millet as research objects and used qRT-PCR technology to accurately analyze its expression level. The specific procedures are as follows:

[0058] The cDNA obtained by reverse transcription was used as a template and the real-time PCR reaction was performed using the SYBR Premix ExTaq kit. The PCR reaction program was as follows: 95℃ for 30s; 95℃ for 5s, 60℃ for 35s, 40 cycles. After the reaction was completed, the expression of ... -ΔΔCt The data were processed and SiACT was used as a reference gene to be amplified simultaneously with the target gene.

[0059] The primer sequences used for fluorescence quantitative verification are (SEQ ID NO: 5-6):

[0060] SiMPK6-qRT-F: 5'-CCTCTGAGTATACTGCGGCA-3';

[0061] SiMPK6-qRT-R: 5'-CGAAGTCCAGATCAGCCTCA-3'.

[0062] The primer sequences used for the internal reference gene SiACT are (SEQ ID NO: 7-8):

[0063] SiACT-F: 5'-TATCGTTCAAACAGATTTACGGCCT-3';

[0064] SiACT-B: 5'-TAGAGAAGAAGTGACGAAGCCTTG-3'.

[0065] Real-time PCR reaction was carried out using cDNA of millet without any adverse stress as template. The results are as follows Figure 1 As shown in Figure 2, the SiMPK6 gene is expressed in the roots, stems, and leaves of millet seedlings, with the highest expression level in leaves, followed by roots, and the lowest in stems. Real-time PCR reaction was performed using the cDNA of millet under high light stress as a template, and the results are shown in Figure 2. Figure 2 As shown, the SiMPK6 gene responded to high light stress, and its expression level was significantly increased in leaves treated with high light.

[0066] In summary, the SiMPK6 gene is expressed in roots, stems, and leaves during the early jointing period, but its expression levels vary significantly among different tissues. The expression level of SiMPK6 in stems is the lowest, only about half of that in roots; whereas the expression level in leaves is significantly higher than in other tissues, approximately 2.5 times that in roots ( Figure 1 ). This indicates that the SiMPK6 gene has a high transcriptional activity in the leaves of millet in the early stage of jointing, and may play an important role in the growth and development or physiological function of the leaves. At the same time, the millet was treated with high light stress and the changes in the transcriptional level of SiMPK6 were detected by qRT-PCR technology. The results showed that SiMPK6 had a significant response to high light stress. SiMPK6 also showed significant induced expression under high light stress. The expression level of SiMPK6 began to increase after 1 hour of treatment, reached a peak at 6 hours, and then began to decrease, but the expression level at 12 hours and 24 hours was still significantly higher than that of the untreated group ( Figure 2 ).

[0067] 4. Millet SiMPK6 gene improves plant resistance to high light

[0068] To investigate the role of SiMPK6 in regulating stress responses, we constructed SiMPK6-overexpressing Arabidopsis plants for subsequent experiments. The specific procedures were as follows:

[0069] 1. Obtaining recombinant vector

[0070] The SiMPK6 gene was inserted between the KpnⅠ and BamHI sites of the pCAMBIA1300 vector to obtain a recombinant vector.

[0071] 2. Preparation of recombinant bacteria

[0072] 2 μL of the above recombinant vector was added to 100 μL of GV3101 competent cells and mixed. After ice bathing for 30 minutes, the cells were quickly frozen with liquid nitrogen for 1 minute, immediately placed in a 37°C water bath for 5 minutes, and then placed on ice for 5 minutes. 450 μL of LB liquid medium was added and incubated at 28°C and 150 rpm. -1 Pre-culture for 5 hours; centrifuge at 6000 rpm for 1 minute, retain 100 μL of supernatant, resuspend the precipitate and evenly apply it on a plate containing 100 μg mL - 1 Kan and 50 μg mL -1 Rifampicin was cultured on LB solid plates at 28°C for 2 days in the dark. Single colonies grown on the plates were picked and inoculated into a solution containing 100 μg mL -1 Kan and 50 μg mL -1 Rif in LB liquid medium, 220r·min -1 Culture for 2 days; extract the plasmid, and screen the positive clones by enzyme digestion verification.

[0073] 3. Obtaining and verifying SiMPK6-transgenic Arabidopsis plants

[0074] The positive clones were transformed into Arabidopsis thaliana (Col-0) using Agrobacterium-mediated method to obtain T0 generation SiMPK6 Arabidopsis. T0 generation SiMPK6 Arabidopsis were plated on hygromycin medium and positive plants OE-1 and OE-2 (such as Figure 3 Then, positive homozygotes are screened out according to Mendel's law for subsequent processing.

[0075] A. Adversity and coercion

[0076] The wild-type and transgenic plants that had grown uniformly for three weeks were treated as follows:

[0077] The first group was left untreated, watered normally, and cultured under 14 h light / 10 h dark (22°C / 28°C) conditions for 35 days;

[0078] The second group was treated with high light. The seedlings were kept at 42°C for 35 days and the light intensity was 1200 μM·m -2 ·s -1 The cells were cultured for 24 h under the conditions of .

[0079] B. Phenotypic testing

[0080] Observe the phenotypic results as Figure 4 As shown in the figure, it can be seen that under high light stress treatment, SiMPK6 transgenic plants are more tolerant to high light than the wild type WT.

[0081] C. Determination of chlorophyll fluorescence parameters in Arabidopsis

[0082] Chlorophyll fluorescence parameters, including initial fluorescence yield (Fo), maximum fluorescence yield (Fm), steady-state fluorescence yield (Fs), maximum fluorescence yield under light adaptation (Fm'), and minimum fluorescence yield under light adaptation (Fo'), were measured using an FMS-1 fluorometer (Hansatech, UK) after 24 h of high light treatment. The maximum photochemical efficiency of photosystem II (PSII), Fv / Fm = (Fm-Fo) / Fm, the actual photochemical efficiency, ΦPSII = (Fm'-Fs) / Fm', and the non-photochemical quenching coefficient, NPQ, = (Fm-Fm') / Fm' were calculated.

[0083] The results are as follows Figure 5 As shown, the Fv / Fm, ΦPSⅡ, and NPQ of SiMPK6 transgenic plants were higher than those of wild type WT.

[0084] The above experimental results show that the SiMPK6 gene can improve the plant's resistance to high light and maintain high photosynthetic efficiency of the plant.

[0085] D. Detection of superoxide content and transcription of protective enzyme genes in SiMPK6 transgenic plants

[0086] H2O2 was histochemically stained with 3,3′-diaminobenzidine (DAB). The uniformly grown plants were treated with high light stress and then treated with 1 mg ml -1 DAB was vacuum infiltrated in 50 mM Tris-acetate buffer and incubated in the dark at 25°C for 6 h. The cells were then immersed in 75% ethanol at 42°C for 30 min, and H2O2 accumulation was detected using an Olympus motorized microscope.

[0087] The results are as follows Figure 6 As shown, the H2O2 content in SiMPK6 transgenic plants was lower than that in wild-type WT; after 6 h of stress treatment, the expression levels of CAT1, POD, cAPX and SOD in OE-1 and OE-2 were significantly higher than those in WT.

[0088] In summary, in the experiment, WT and overexpressing plants with the same growth status for three weeks were subjected to high light stress treatment for 24 hours. The results showed that the transgenic plants had obvious high light stress tolerance characteristics ( Figure 4 ), compared with WT, the PSII photochemical maximum quantum yield (Fv / Fm), non-photochemical quenching coefficient (NPQ), and actual photochemical efficiency (ΦPSⅡ) values ​​of transgenic plants were higher ( Figure 5 The above results indicate that SiMPK6 may play an important role in the adaptation to adverse stress, that is, the SiMPK6 gene positively regulates the plant's response to high light stress and maintains a higher photosynthetic rate under adverse conditions.

[0089] In addition, under biotic and abiotic stresses, plants rapidly accumulate reactive oxygen species, a process that produces various ROS molecules. When ROS levels exceed cellular homeostasis, oxidative stress occurs, causing lipid peroxidation, protein carbonylation, and DNA chain breaks, which may lead to PCD. To combat this situation, plants have developed an antioxidant defense system to remove excess ROS and maintain intracellular ROS homeostasis. Enzymatic systems include SOD, CAT, POD, and APX, and non-enzymatic systems include small molecule antioxidants such as ascorbic acid (AsA) and glutathione (GSH). To elucidate whether ROS is involved in the high-light-induced photoinhibition of SiMPK6, 3,3′-diaminobenzidine (DAB) staining was used to detect H2O2 accumulation in SiMPK6-transgenic Arabidopsis plants. Compared with WT, weaker DAB staining was observed in SiMPK6 transgenic plants under different stress conditions ( Figure 6 A). The expression of genes related to ROS scavenging enzymes was then evaluated. After 6 h of stress treatment, the expression levels of CAT1, POD, cAPX, and SOD in OE-1 and OE-2 were significantly higher than those in WT ( Figure 6B) These results indicate that the enhanced stress resistance of transgenic Arabidopsis plants expressing SiMPK6 may be related to the upregulation of ROS scavenging enzyme genes induced by SiMPK6 overexpression. The present invention provides potential candidate genes for millet stress resistance gene breeding.

[0090] Although the technical solution of the present invention has been described in detail above using general descriptions, specific embodiments, and experimental examples, it should be noted that the embodiments and experimental examples are only intended to illustrate the technical solution and technical effects of the present invention and should not be construed as limiting the scope of protection of the present invention. Simple variations, modifications, or improvements based on the technical concept of the present invention are all within the scope of protection claimed by the present invention.

Claims

1. A novel use of foxtail millet SimPK6 protein or its encoding gene, characterized in that: The new use is: application in regulating plant high light stress resistance; the amino acid sequence of the millet SiMPK6 protein is shown in SEQ ID NO:

1.

2. The novel use according to claim 1, characterized in that: The regulation is to overexpress SiMPK6 to improve the plant's resistance to high light stress.

3. The novel use according to claim 1, characterized in that: The plant is a C3 plant.

4. The novel use according to claim 3, characterized in that: The C3 plants include but are not limited to rice, wheat, soybean, potato, and cotton.

5. The novel use according to claim 3 or 4, characterized in that: The coding gene of the foxtail millet SiMPK6 protein is codon-optimized for C3 plant preference.

6. A method for improving plant resistance to high light stress, characterized in that: The following steps are involved: The coding gene of millet SiMPK6 protein is introduced into a recipient plant for overexpression to obtain a transgenic plant with enhanced resistance to high light stress; the amino acid sequence of the millet SiMPK6 protein is shown in SEQ ID NO:

1.

7. The method according to claim 6, characterized in that: The introduction into the recipient plant comprises: constructing a recombinant expression vector containing a gene encoding the millet SiMPK6 protein; introducing the recombinant expression vector into a host bacterium to obtain a recombinant bacterium; transforming the recombinant bacterium into a recipient plant, screening out positive plants and then culturing them to obtain transgenic plants with enhanced resistance to high light stress.

8. The method according to claim 6, wherein: The recipient plant is a C3 plant.

9. The method according to claim 8, characterized in that: The C3 plants include but are not limited to rice, wheat, soybean, potato, and cotton.

10. The method according to claim 8 or 9, characterized in that: The coding gene of the foxtail millet SiMPK6 protein is codon-optimized for C3 plant preference.

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