Use of millet siMPK6 protein or its encoding gene

Abstract

The application belongs to the technical field of genetic engineering, and specifically discloses application of millet SiMPK6 protein or a coding gene thereof in regulation of high light stress resistance of plants. It is proved through experiments that the SiMPK6 gene positively regulates the high light stress of plants and can maintain a higher photosynthetic rate under adversity, and can be used for modification of C3 plants and improvement of high light tolerance of C3 plants. The specific operation is as follows: the coding gene of the millet SiMPK6 protein is subjected to codon optimization for C3 plant preference, a recombinant expression vector containing the target gene is constructed, the recombinant expression vector is introduced into a host bacterium to obtain a recombinant bacterium, the recombinant bacterium is transformed into a receptor plant, and after positive plants are screened out, the positive plants are cultured to obtain transgenic plants with enhanced high light stress resistance; the C3 plants include but are not limited to rice, wheat, soybean, potato, cotton and the like.

Description

Technical Field

[0001] This invention belongs to the field of genetic engineering technology, specifically relating to the application of millet SiMPK6 protein or its encoding gene in regulating plant resistance to high light stress. Background Technology

[0002] Light is a crucial energy source for photosynthesis in green plants. Plants rely on light for photosynthesis, but due to their immobility, when the light intensity exceeds their capacity, it can harm their growth, causing photoinhibition, reduced ΦPSII, and decreased photosynthetic rate. To survive in nature, plants must adapt to drastic changes in light intensity and quality, increasing their photosynthetic activity to achieve autotrophic survival (Dietzel et al., 2008; Pesaresi et al., 2010). This rapid adaptation is especially critical for young plants in their early growth stages, as they need to quickly synthesize their photosynthetic systems (Anderson and Osmond, 1987; Anderson et al., 1995).

[0003] To cope with photoinhibition, plants have evolved a series of mechanisms to adapt to high light, enabling them to adapt to external high light conditions by altering and optimizing various physiological and biochemical processes across different time scales (Park et al. 1996; Savitch et al. 2000). Plants have evolved multiple photoreceptors to sense changes in light conditions in the external environment, thus helping them to respond and regulate. For example, under high light stress, cryptochrome 1 (CRY1) is involved in the formation of ROS (Consentino et al., 2015; Jourdan et al., 2015), and CRYs can promote the accumulation of soluble phenolic substances in leaves, improving the leaf response to strong light stress and thus increasing photosynthetic efficiency (Brelsford et al., 2019). Furthermore, CRY1 can also induce anthocyanin accumulation through the COP1 / SPA1 / HY5 pathway to resist high light stress (Kleine et al., 2007). Under high light stress, phototropin B (PHOT B) also participates in stomatal movement and ROS signal transduction in plants (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 long periods of evolution, plants have developed complex adaptive systems, in which the serine / threonine (Ser / Thr) protein kinase network plays a crucial 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 a variety of cellular processes through phosphorylation cascades. Activated MAPKs can target various downstream substrates, such as kinases, enzymes, and transcription factors, thereby influencing gene expression, metabolism, cell growth, and division. Protein phosphorylation is an important mechanism regulating fundamental cellular processes in organisms, typically achieved through cascade networks. The mitogen-activated protein kinase (MAPK) cascade signaling pathway is a ubiquitous and highly conserved serine / threonine (Ser / Thr) protein kinase system in eukaryotes, playing a vital role in plant growth and development and responses to biotic and abiotic stresses. Typically, a complete MAPK cascade signaling pathway consists of three key kinase components: MAPKKK, MAPKK, and MAPK. In cell signal transduction, the MAPK cascade signaling system precisely regulates a series of complex physiological processes, including cell differentiation, proliferation, growth and development, and responses to various stress signals, through the stepwise phosphorylation modification of kinase components. MAPK, also known as MPK, is located at the downstream end of the MAPK cascade system and is a key protein kinase in cell signal transduction and amplification, acting as a direct link between the cascade signal and downstream proteins. Studies have shown that members of the MAPK family possess 11 conserved protein kinase domains, among which the "A-Loop" region between subdomains VII and VIII contains the activation domain TXY (Thr-X-Tyr). Based on the different "X" amino acids in the activation domain, plant MAPKs are divided into two subtypes: TEY and TDY. The TEY subtype is further subdivided into three subfamilies: A, B, and C, while the TDY subtype constitutes the evolutionarily distant D subfamily. Two key members of the A subfamily, AtMPK3 and AtMPK6, not only participate in plant responses to various biotic and abiotic stresses but also regulate plant growth and development. AtMPK4 of the B subfamily plays a role in the salicylic acid and jasmonic acid signaling pathways. AtMPK1, AtMPK2, AtMPK7, and AtMPK14 of the C subfamily are mainly involved in ABA signal transduction, dehydration stress response, and plant immunity. AtMPK9 and AtMPK12 of the D subfamily are involved in the ABA signaling pathway for plant growth and development and the regulation of reactive oxygen species. However, whether MAPK is involved in high-light regulation mechanisms has not yet been reported.

[0005] Millet (Setaria italica L.), also known as foxtail millet, is a diploid annual crop native to my country, belonging to the genus Setaria of the Poaceae family. It is traditionally used for both food and forage, and is considered the "king of grains" in Chinese folk tradition. With the vigorous development of my country's economy and society, the agricultural sector has undergone profound changes, and the industrial structure has been continuously optimized. Millet, with its strong adaptability and resilience, can achieve relatively good harvests even in arid and infertile land, fully demonstrating its unique advantages in utilizing my country's scarce land resources and unique geographical and ecological environment, thus opening up an effective path for my country's food security. As a C4 plant, millet has high photosynthetic efficiency, nitrogen and water use efficiency, and strong environmental adaptability, making it an ideal model for studying C4 photosynthesis and crop stress tolerance traits. Summary of the Invention

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

[0007] Meanwhile, this invention provides a method for improving the resistance of plants to high light stress.

[0008] To solve the above-mentioned technical problems, the present invention provides the following technical solutions: The use of the millet SiMPK6 protein or its encoding gene, wherein the use is: in regulating plant resistance to high light stress; the amino acid sequence of the millet SiMPK6 protein is shown in SEQ ID NO: 1.

[0009] In a preferred embodiment of the present invention, the regulation involves overexpressing SiMPK6 to enhance the plant's resistance to high light stress.

[0010] In a preferred embodiment of the present invention, the plant is a C3 plant, including but not limited to rice, wheat, soybean, potato, cotton, etc.

[0011] More preferably, the gene encoding the millet SiMPK6 protein needs to be codon-optimized for C3 plant preference.

[0012] A method for improving plant resistance to high light stress includes the following steps: The gene encoding the millet SiMPK6 protein was introduced into recipient plants for overexpression, resulting in transgenic plants with enhanced resistance to high light stress; the amino acid sequence of the millet SiMPK6 protein is shown in SEQ ID NO: 1.

[0013] In a preferred embodiment of the present invention, the recipient plant is introduced by: 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 culturing them to obtain a transgenic plant with enhanced resistance to high light stress.

[0014] In 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.

[0015] More preferably, the gene encoding the millet SiMPK6 protein is codon-optimized for C3 plant preference.

[0016] This invention provides the application of the 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 higher photosynthetic rate under adverse conditions. It can be used to modify C3 plants and improve their tolerance to high light stress. Attached Figure Description

[0017] Figure 1 This is a diagram showing tissue-specific expression analysis of the SiMPK6 gene.

[0018] Figure 2 This is a diagram showing the expression pattern of the SiMPK6 gene under high light stress.

[0019] Figure 3 The results indicate that the transgenic plant is positive.

[0020] Figure 4 High light stress treatment for SiMPK6 transgenic Arabidopsis thaliana.

[0021] Figure 5 The results show the fluorescence parameters of SiMPK6 transgenic Arabidopsis thaliana after high light stress treatment.

[0022] Figure 6 The results show the H2O2 content and transcriptional results of the protective enzyme gene in SiMPK6 transgenic Arabidopsis thaliana.

[0023] To more clearly illustrate the technical solutions of the embodiments of the present invention, the accompanying drawings obtained in the experimental examples have been briefly described above. It should be understood that the above drawings only show some experimental examples of the present invention and should not be considered as any limitation on the scope of protection of the claims. For those skilled in the art, other related drawings can be obtained based on these drawings without any creative effort. Detailed Implementation

[0024] The technical solution of the present invention will be clearly and completely described below with reference to specific embodiments and experimental examples. However, those skilled in the art should understand 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 implementation schemes obtained by those skilled in the art without creative effort, such as implementation schemes obtained by modification, variation or simple substitution, should fall within the scope of protection of the present invention.

[0025] Unless otherwise specified, the experimental methods used in the following examples and experimental cases are conventional methods; the raw materials (including biological materials), reagents, culture media, instruments, etc. used are all commonly used in the field and commercially available to the public unless otherwise specified; the terms and abbreviations used have their conventional meanings in the field, such as PBS buffer being phosphate buffer.

[0026] Example 1

[0027] This embodiment provides an application of the millet SiMPK6 protein or its encoding gene, specifically in regulating the resistance of C3 plants to high light stress; the amino acid sequence of the millet SiMPK6 protein is shown in SEQ ID NO: 1.

[0028] Millet SiMPK6 protein: MDGGAQPPDTEMTDAGAGAGGGGGHPPQQPAGGGGGMMDNIQATLSHGGRFIQYNIFGNVFEVTAKYKPPILPIGKGAYGIVWTGEQVAIKKIANAFDNKIDAKRTLREIKLLRHMDHENYFLYQILRGLKYIHSANVLHRDLKPSNLLLNANCDLKICDFGLARITSETDFMTE YVVTRWYRAPELLLNSSEYTAAIDVWSVGCIFMELMDRKPLFPGRDHVHQLRLLMELIGTPNEADLDFVNENARRYIRQLPRHARQSFPEKFPHVQPLAIDLVEKMLTFDPRQRITVEGALAHPYLASLHDISDEPVCSMPFSFDFEQHALSEEQMKDLIYQEALAFNPDYQ (SEQ ID NO: 1).

[0029] Example 2

[0030] This embodiment provides a method for improving the resistance of plants to high light stress, including the following steps: The coding gene for the millet SiMPK6 protein was codon-optimized to target the preference of C3 plant rice, and a recombinant expression vector containing the target gene was constructed. 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.

[0031] In other embodiments of the present invention, the gene encoding the millet SiMPK6 protein is codon-optimized and then introduced into other C3 plants such as wheat, soybean, potato, and cotton for overexpression to improve the high light tolerance of other C3 plants.

[0032] Experimental Example

[0033] I. Bioinformatics Analysis of the SiMPK6 Gene in Millet

[0034] The SiMPK6 gene sequence information of *Setaria italicav2.2* (Phytozome genome ID: 312, NCBI taxonomy ID: 4555) from the *Setaria italicav2.2* genome database of the Joint Genome Institute of the U.S. Department of Energy was obtained. The coding region of this gene is 1044 bp long, encoding a protein composed of 347 amino acids. The molecular weight of the protein was predicted to be 39131.67 Da and the isoelectric point to be 5.71 using the Compute pI / MW method from the Swiss Institute of Bioinformatics ExPASy (http: / / web.expasy.org). Analysis using Serverl ProtScale software showed that the *Setaria italicav2.2* protein is a hydrophilic protein and does not contain a signal peptide. This indicates that the protein is unlikely to be located in chloroplasts, mitochondria, or secreted extracellularly. This localization characteristic suggests that the protein may function in a specific intracellular region, participating in intracellular physiological processes. Analysis using Sopma software revealed that the secondary structure of SiMPK6 consists of 44.09% α-helices, 5.76% β-sheets, 14.13% extended strands, and 36.02% random coils. Pfam software was used to predict conserved domains in the millet SiMPK6 protein, revealing a conserved Pkinase (Clan: CL0016) domain between amino acids 120-301. The presence of this conserved domain indicates that SiMPK6 belongs to the MAPK protein kinase family and may possess the typical functions of this family of proteins, participating in intracellular signal transduction. Furthermore, the tertiary structure of SiMPK6 shows a high degree of similarity to Arabidopsis thaliana AtMPK6 (Seq Identity: 87.70%). Similar tertiary structures often imply similar functions, providing an important reference for studying the function of SiMPK6. Based on this, it is speculated that its function in millet may be similar to that of AtMPK6 in Arabidopsis thaliana. NetPhos 3.1 software analysis revealed that SiMPK6 contains 10 serine, 7 threonine, 8 tyrosine residues, and numerous potential phosphorylation sites. Phosphorylation is an important post-translational modification of proteins, and the presence of these phosphorylation sites suggests that SiMPK6 may participate in intracellular signal regulation through phosphorylation and dephosphorylation processes, thereby influencing plant growth, development, and stress responses.

[0035] TargetP-2.0 predicted the presence of a signal peptide (SP), mitochondrial transport peptide (mTP), chloroplast transport peptide (cTP), or thylakoid coelom transport peptide (lTP) in the N-terminal pro-sequence of the millet SiMPK6 protein, but did not specify the exact type of transport peptide. Glycosylation analysis using NetNGlyc (v1.0) software revealed that SiMPK6 contains only one glycosylation site (NSSE) at position 190 (N190). Glycosylation modification can affect protein folding, sorting, localization, and degradation processes; the presence of this glycosylation site provides a new direction for studying the function of SiMPK6.

[0036] II. Cloning of the Millet SiMPK6 Gene

[0037] 1. Preparation and processing of materials The tested millet variety (Setaria italica) Yugu 1 was obtained from the Millet Resource Bank of the Beijing Crop Germplasm Resource Bank, Beijing Academy of Agricultural and Forestry Sciences. Millet seedlings grown in a greenhouse for 35 days were subjected to high light stress treatment, and individual plants were sampled. The high light stress treatment method was as follows: the millet seedlings were placed in an environment with 1200 μM·m⁻² light. -2 ·s -1 Under the given conditions, treatment times were recorded for 0h, 1h, 3h, 6h, 12h, and 24h. Phenotypic observation of the high-light treatment involved placing Arabidopsis seedlings that had grown to 3 weeks under a light source at 1200 μM·m⁻². -2 ·s -1 Cultured under stress for 24 hours and observed for phenotype.

[0038] 2. RNA extraction and cDNA synthesis Take appropriate amounts of millet leaves after each of the above treatments, as well as roots, stems, and leaves of millet under normal culture conditions, and place them in 1.5 mL centrifuge tubes and quickly freeze them in liquid nitrogen. Remove them from the liquid nitrogen and grind them into powder. Transfer the powder to a centrifuge tube containing 1 mL of TRIzol reagent, vortex to mix, and let stand at room temperature for 5 min. Add 200 μL of chloroform, shake vigorously to mix, and let stand at room temperature for 5 min. Centrifuge at 12000 × g and 4 °C for 15 min, and transfer the supernatant to a new centrifuge tube. Add an equal volume of isopropanol, invert to mix, and let stand at room temperature for 10 min. Centrifuge at 12000 × g and 4 °C for 10 min, and discard the supernatant. Wash the precipitate with 70% ethanol, centrifuge at 7500 × g and 4 °C for 5 min, discard the supernatant, dry the precipitate, dissolve it in 100 μL of sterile water, and determine its concentration and purity. Add 3 volumes of anhydrous ethanol and 1 / 10 volume of 3M potassium acetate (pH 5.8), mix well, and incubate at -80°C for at least 30 minutes; centrifuge at 7500×g and 4°C for 5 minutes, discard the supernatant, and dissolve the RNA in sterile water to the desired concentration.

[0039] After removing DNA contamination from the obtained RNA, cDNA was synthesized using Takara's M-MLV reverse transcription kit. The 15 μL reaction system contained 2.5 μg RNA. After reverse transcription at 42 °C for 90 min, cDNA was obtained. The cDNA was placed on ice for 5 min and then stored at -20 °C for later use.

[0040] 3. Gene cloning Using the above cDNA as a template, PCR amplification was performed using the following primers (SEQ ID NO: 3-4): Upstream primer: 5'-CATATGGACGGCGGGGCGCA-3'; Downstream primer: 5'-CTACTGGTAATCAGGGTTGAATGCAA-3'.

[0041] The components of the 30 μL PCR reaction system are as follows: 3 μL of 10×Buffer, 10 mmol·L⁻¹ -1 dNTP 3 μL, upstream and downstream primers (10 μmol·L⁻¹) -1 1 μL each of millet leaf cDNA, 0.3 μL of TAKARA polymerase, and 21.7 μL of ddH2O.

[0042] PCR program: 98℃ pre-denaturation for 3 min; 98℃ denaturation for 30 s, 59℃ annealing for 30 s, 72℃ extension for 80 s, repeat this process for 35 cycles; 72℃ extension for 10 min, store at 4℃.

[0043] After the PCR reaction, the PCR product was detected by 1.0% agarose gel electrophoresis, yielding a 1044bp PCR product. The PCR product was purified and recovered using a DNA gel extraction kit. The PCR product was ligated into the pEASY-Blunt Zero cloning vector to construct the pT-SiMPK6 cloning vector, and then sequenced. The results showed that the target gene sequence was correct.

[0044] Millet SiMPK6 gene (SiMAPK6-Seita.4G069900-1044bp):

[0045] III. Analysis of SiMPK6 gene-specific expression in millet

[0046] To gain a deeper understanding of the expression pattern of the SiMPK6 gene in millet, roots, stems, and leaves of millet were selected as research subjects. qRT-PCR technology was used to accurately analyze its expression level. The specific procedures are as follows: Using cDNA obtained from reverse transcription as a template, real-time PCR was performed using the SYBR Premix ExTaq kit. The PCR reaction program was as follows: 95℃ for 30 s; 95℃ for 5 s, 60℃ for 35 s, for 40 cycles. After the reaction, the cDNA was analyzed using Formula 2. -ΔΔCt Data processing was performed, with SiACT used as the reference gene and the target gene amplified simultaneously.

[0047] The primer sequences used for quantitative fluorescence verification are (SEQ ID NO: 5-6): SiMPK6-qRT-F: 5'-CCTCTGAGTATACTGCGGCA-3'; SiMPK6-qRT-R: 5'-CGAAGTCCAGATCAGCCTCA-3'.

[0048] The primer sequences used for the internal reference gene SiACT are (SEQ ID NO: 7-8): SiACT-F: 5'-TATCGTTCAAACAGATTTACGGCCT-3'; SiACT-B: 5'-TAGAGAAGAAGTGACGAAGCCTTG-3'.

[0049] Real-time PCR was performed using cDNA from millet seeds that had not undergone any adverse environmental stress as a template. The results are as follows: Figure 1 As shown, 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 was performed using cDNA from millet seedlings under high light stress as a template, and the results are as follows. Figure 2 As shown, the expression level of the SiMPK6 gene is significantly increased in leaves treated with high light stress in response to high light stress.

[0050] In summary, the SiMPK6 gene is expressed in roots, stems, and leaves during the pre-jointing stage, but its expression level varies significantly among different tissues. The expression level of SiMPK6 is lowest in the stem, only about half that in the root; while the expression level in the leaf is significantly higher than in other tissues, approximately 2.5 times that in the root. Figure 1This indicates that the SiMPK6 gene has high transcriptional activity in millet leaves during the early jointing stage, and may play an important role in leaf growth, development, or physiological function. Simultaneously, high light stress treatment of millet and the changes in SiMPK6 transcriptional levels were detected using qRT-PCR. The results showed that SiMPK6 has a significant response to high light stress. SiMPK6 also showed significant induction of expression under high light stress; the expression level of SiMPK6 began to upregulate after 1 h of treatment, reached a peak at 6 h, and then began to decline, but the expression levels at 12 h and 24 h were still significantly higher than those in the untreated group. Figure 2 ).

[0051] IV. The SiMPK6 gene in millet enhances plant resistance to high light intensity.

[0052] To investigate the role of SiMPK6 in regulating stress response, SiMPK6-overexpressing Arabidopsis thaliana plants were constructed for subsequent experiments. The specific procedures are as follows: 1. Obtaining the recombinant vector The SiMPK6 gene was inserted between the KpnⅠ and BamHI sites of the pCAMBIA1300 vector to obtain a recombinant vector.

[0053] 2. Preparation of recombinant bacteria Add 2 μL of the recombinant vector to 100 μL of GV3101 competent cells and mix well. Incubate on ice for 30 min, then flash freeze in liquid nitrogen for 1 min, immediately place in a 37°C water bath for 5 min, and then on ice for 5 min. Add 450 μL of LB liquid medium and incubate at 28°C and 150 rpm. -1 Pre-culture for 5 h; centrifuge at 6000 rpm for 1 min, retain 100 μL of supernatant, resuspend the precipitate and spread it evenly on a medium containing 100 μg / mL. -1 Kan and 50 μg·mL -1 Incubate rifampicin on LB agar plates at 28°C in the dark for 2 days; pick single colonies that grow on the plates and inoculate them onto plates containing 100 μg / mL rifampicin. -1 Kan and 50 μg·mL -1 In Rif's LB liquid medium, 220 r·min -1 Cultured for 2 days; plasmid extracted, enzyme digestion verified and screened positive clones.

[0054] 3. Obtaining and validating SiMPK6 Arabidopsis thaliana plants Positive clones were transformed into Arabidopsis thaliana (Col-0) using Agrobacterium-mediated transformation to obtain T0 generation SiMPK6 Arabidopsis thaliana. The T0 generation SiMPK6 Arabidopsis thaliana were plated on hygromycin medium, and positive plants OE-1 and OE-2 (e.g., ...) were initially screened out. Figure 3(as shown in the figure), and then positive homozygotes are screened out according to Mendel's laws for further processing.

[0055] A. Adversity and stress Wild-type and transgenic plants that grew uniformly for three weeks were treated as follows: The first group was left untreated and watered normally, then placed under 14 hours of light / 10 hours of darkness (22℃ / 28℃) conditions for 35 days. The second group of high-light treatment involved 35-day-old seedlings at 42℃ and 1200 μM·m light. -2 ·s -1 Under these conditions, culture for 24 hours.

[0056] B. Phenotypic detection Observation of phenotypic results as follows Figure 4 As shown in the figure, it can be seen that under high light stress treatment, SiMPK6 transgenic plants are more tolerant of high light than wild-type WT.

[0057] C. Measurement of chlorophyll fluorescence parameters in Arabidopsis thaliana Chlorophyll fluorescence parameters of leaves treated with high light for 24 hours were detected using an FMS-1 fluorometer (Hansatech, UK), 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'). The maximum photochemical efficiency of photosystem II (PSII) was calculated as Fv / Fm = (Fm – Fo) / Fm, the actual photochemical efficiency ΦPSII = (Fm' – Fs) / Fm', and the non-photochemical quenching coefficient NPQ = (Fm – Fm') / Fm'.

[0058] The results are as follows Figure 5 As shown, SiMPK6 transgenic plants had higher Fv / Fm, ΦPSⅡ, and NPQ than wild-type WT.

[0059] The above experimental results indicate that the SiMPK6 gene can enhance the plant's resistance to high light and maintain high photosynthetic efficiency.

[0060] D. Detection of superoxide content and transcription of protective enzyme genes in SiMPK6 transgenic plants. Histochemical staining of H2O2 was performed using 3,3′-diaminobenzidine (DAB). Uniformly growing plants were subjected to high light stress treatment, followed by treatment with 1 mg / ml... -1 DAB was vacuum-wetted in 50 mM Tris-acetate buffer and incubated in the dark at 25°C for 6 h; then immersed in 75% ethanol at 42°C for 30 min, and H2O2 accumulation was detected by an Olympus motorized microscope.

[0061] The results are as follows Figure 6As shown, the H2O2 content of SiMPK6 transgenic plants was lower than that of 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.

[0062] In summary, the experiment showed that transgenic plants with uniform growth status at three weeks WT and overexpression underwent high light stress treatment for 24 hours. The results indicated that the transgenic plants exhibited significant tolerance to high light stress. Figure 4 Compared with WT, transgenic plants exhibit higher values ​​for PSII photochemical maximum quantum yield (Fv / Fm), non-photochemical quenching coefficient (NPQ), and actual photochemical efficiency (ΦPSⅡ). Figure 5 These results indicate that SiMPK6 may play an important role in the adaptation to abiotic stress, namely, the SiMPK6 gene positively regulates the plant's response to high light stress and maintains a higher photosynthetic rate under stress.

[0063] In addition to biotic and abiotic stress, plants rapidly accumulate reactive oxygen species (ROS), a process that produces various ROS molecules. When ROS levels exceed cellular homeostasis, oxidative stress occurs, leading to lipid peroxidation, protein carbonylation, and DNA strand breaks, potentially causing cellular dysplasia (PCD). To combat this, plants have developed an antioxidant defense system to scavenge excess ROS and maintain intracellular ROS homeostasis. Enzymatic systems include SOD, CAT, POD, and APX, while non-enzymatic systems include small molecule antioxidants such as ascorbic acid (AsA) and glutathione (GSH). To clarify whether ROS participates in light-induced photoinhibition in SiMPK6 transgenic Arabidopsis plants, H2O2 accumulation was detected using 3,3′-diaminobenzidine (DAB) staining. Compared to WT, SiMPK6 transgenic plants showed weaker DAB staining under various stress conditions. Figure 6 A). The expression of genes associated with ROS scavenging enzymes was then assessed. After 6 hours of stress treatment, the expression levels of CAT1, POD, cAPX, and SOD in OE-1 and OE-2 significantly exceeded those in WT (Wt). Figure 6 (B) These results suggest that the enhanced stress resistance of SiMPK6-expressing transgenic Arabidopsis plants may be related to the upregulation of ROS scavenging enzyme genes induced by SiMPK6 overexpression. This invention provides potential candidate genes for millet stress-resistance gene breeding.

[0064] Although the technical solution of the present invention has been described in detail above with general descriptions, specific embodiments, and experimental examples, it should be noted that the embodiments and experimental examples are only used to illustrate the technical solution and technical effects of the present invention, and should not be regarded as any limitation on the scope of protection of the present invention. Simple modifications, alterations, or improvements made based on the technical concept of the present invention are all within the scope of protection claimed by the present invention.

Claims

1. The use of the millet SiMPK6 protein-coding gene, characterized in that... : In the process of overexpressing SiMPK6 to improve the resistance of Arabidopsis thaliana to high light stress Applications; the amino acid sequence of the millet SiMPK6 protein is shown in SEQ ID NO:

1.

2. The use according to claim 1, characterized in that... The gene encoding the millet SiMPK6 protein is specifically targeted at Arabidopsis thaliana. Codon optimization.

Images