Application of MYC gene in regulation and control of chloroplast development and composite stress resistance

Abstract

The invention belongs to the technical field of biology, particularly relates to the technical field of plant genetic engineering, and more particularly relates to application of an MYC gene in regulation and control of chloroplast development and composite stress resistance. Specifically, the invention innovatively discovers that the combined action of warm and JA can significantly induce seedling cotyledon chloroplast dysplasia, and deletion / reduction of MYC gene expression can significantly inhibit chloroplast dysplasia of plants under the combined action of warm and JA. The discovery provides guidance for remarkably improving plant seedling chloroplast development in a complex dynamic environment and improving the seedling survival rate and photosynthetic productivity, can promote optimization of a chloroplast artificial regulation and control technology, and realizes directional design of efficient photosynthetic organs in facility agriculture and an artificial photosynthetic system.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of biotechnology, particularly relates to the technical field of plant genetic engineering, and more particularly relates to the application of MYC gene in preventing chloroplast development abnormalities under plant complex stress. BACKGROUND

[0002] Chloroplast is a key organelle responsible for photosynthesis in plant cells, and its development and function directly affect the plant's light energy utilization, carbon assimilation and stress resistance. Chloroplast development is a complex process regulated by multiple genes, involving protoplast differentiation, inner membrane system establishment, photosynthetic protein complex assembly and environmental signal integration. Chloroplast development abnormalities can lead to decreased photosynthetic efficiency, energy metabolism disorder, and thus affect plant growth, yield and environmental adaptability. At present, the molecular regulation network of chloroplast development, especially the interaction mechanism between key transcription factors and signal pathways, is not clear. Analyzing the core regulatory mechanism of chloroplast development not only has important significance for revealing the biological basis of plant organelle differentiation, but also provides potential targets for improving photosynthetic efficiency and stress resistance breeding of crops, which meets the strategic needs of national agricultural sustainable development and food security.

[0003] Jasmonates (JAs) as a class of lipid-derived plant hormones, its signaling pathway shows complex and precise regulation in plant temperature response. The core of JA signaling pathway is COI1-JAZ-MYC2 module. Under normal conditions, JAZ protein inhibits the activity of transcription factors such as MYC2; when JA accumulates, SCF COI1 ubiquitin ligase mediates JAZ degradation, releasing MYC2 to regulate downstream target genes [1] .

[0004] Under the background of global warming, high temperature stress has become a key factor limiting crop productivity. In recent years, research has found that JA is no longer a "special signal molecule" for mechanical damage and pathogen defense, but through cross-talk with abscisic acid (ABA), salicylic acid (SA), auxin (IAA) and other hormones, it becomes a core coordinator for plant response to environmental temperature.

[0005] Therefore, the co-occurrence of high temperature stress and the activation of JA signaling pathway and the JA accumulation mediated by it is a common complex stress scenario faced by plants in modern agriculture. For example, in natural environment, summer drought and heat waves (activate JA pathway), or high temperature / warm temperature accompanied by disease and pest outbreaks (activate JA pathway); in facility agriculture (greenhouse or shed), high temperature and humidity and disease (activate JA pathway) environment caused by improper environmental management; and specific agricultural activities (spraying, pruning, etc.) under high temperature weather.

[0006] However, the influence of the common synergistic scenario of high temperature / warm temperature conditions accompanied by JA accumulation on the growth and development of plants, especially chloroplast development and functional implementation, has not been reported. SUMMARY

[0007] On the basis of the prior art, the present application discloses the molecular mechanism of warm high temperature (28°C or 30°C) and jasmonic acid (JA) signal synergistically regulating chloroplast development of Arabidopsis seedlings, and innovatively finds that the combined action of warm temperature and JA can significantly induce cotyledon yellowing of seedlings, which is manifested as significant reduction of total chlorophyll content and photosystem II (PSII) photosynthetic efficiency; impaired chloroplast occurrence and development; impaired chloroplast ultrastructure, including disordered thylakoid membrane system, increased starch grains and disintegrated grana lamella structure. In addition, the present application also first clarifies the dual regulation function of JA core transcription factor MYC (especially MYC2) in this process - under high temperature and JA combined stress, MYC2 negatively regulates chloroplast development by activating the senescence pathway and inhibiting the expression of chloroplast development genes, while its mutants (myc2-2 and myc2 / 3 / 4 triple mutants) exhibit significant stress resistance advantages under the same stress, including green cotyledon, stable chloroplast structure and maintained photosynthetic efficiency.

[0008] In this regard, the present application includes but is not limited to the following:

[0009] In one aspect, the present application provides the use of MYC genes in preventing abnormal chloroplast development of plant seedlings, characterized in that the expression of MYC genes in plants is reduced / inactivated, the MYC genes including MYC2 genes.

[0010] In another aspect, the present application provides the use of reduced / inactivated MYC gene expression in preventing abnormal chloroplast development of plant seedlings, the MYC genes including MYC2 genes.

[0011] In another aspect, the present application provides the use of a recombinant vector for reducing / inactivating MYC gene expression in plants in preventing abnormal chloroplast development of plant seedlings, the MYC genes including MYC2 genes.

[0012] In yet another aspect, the present application provides the use of a recombinant engineering bacterium in preventing abnormal chloroplast development of plant seedlings, the recombinant engineering bacterium comprising a recombinant vector for reducing / inactivating MYC gene expression in plants, the MYC genes including MYC2 genes, preferably the engineering bacterium is Agrobacterium.

[0013] In yet another aspect, the present application provides a method for preventing abnormal chloroplast development of plant seedlings, the method comprising: introducing into the plant a recombinant vector for reducing / inactivating MYC gene expression in plants or a recombinant engineering bacterium comprising the recombinant vector, the MYC genes including MYC2 genes.

[0014] In yet another aspect, the present application provides a method for obtaining a plant with improved traits, comprising: introducing into the plant a recombinant vector or a recombinant engineering bacterium comprising the recombinant vector which reduces / inactivates the expression of MYC genes in plants, wherein the MYC genes comprise a MYC2 gene.

[0015] In one aspect, the improved traits according to the present application refer to reduced abnormal chloroplast development in plant seedlings.

[0016] In one aspect, the abnormal chloroplast development according to the present application is caused by high temperature or warm temperature conditions accompanied by JA accumulation.

[0017] In one aspect, the high temperature or warm temperature according to the present application is 25-35℃, preferably 28-30℃, more preferably 28℃ or 30℃, and most preferably 28℃.

[0018] In one aspect, the plant according to the present application is Arabidopsis thaliana.

[0019] In one aspect, the engineering bacterium according to the present application is Agrobacterium. Preferably, the Agrobacterium is Agrobacterium GV3101.

[0020] In one aspect, the present application reduces / inactivates the expression of a MYC2 gene in plants or reduces / inactivates the expression of a MYC2 gene, a MYC3 gene and a MYC4 gene in plants; preferably, the amino acid sequence encoded by the MYC2 gene is as shown in SEQ ID NO: 2, the amino acid sequence encoded by the MYC3 gene is as shown in SEQ ID NO: 4, and / or the amino acid sequence encoded by the MYC4 gene is as shown in SEQ ID NO: 6.

[0021] In one aspect, the nucleotide sequence of the coding region of the MYC2 gene according to the present application is as shown in SEQ ID NO: 1, the nucleotide sequence of the coding region of the MYC3 gene is as shown in SEQ ID NO: 3, and / or the nucleotide sequence of the coding region of the MYC4 gene is as shown in SEQ ID NO: 5.

[0022] In one aspect, the amino acid sequence encoded by the MYC2 gene according to the present application is as shown in SEQ ID NO: 2.

[0023] In one aspect, the nucleotide sequence of the coding region of the MYC2 gene according to the present application is as shown in SEQ ID NO: 1.

[0024] In one aspect, the present application prevents abnormal chloroplast development in plant seedlings by simultaneously reducing / inactivating the expression of a MYC2 gene, a MYC3 gene and a MYC4 gene.

[0025] In one aspect, the amino acid sequence encoded by the MYC3 gene of the present application is shown as SEQ ID NO: 4.

[0026] In one aspect, the nucleotide sequence of the coding region of the MYC3 gene of the present application is shown as SEQ ID NO: 3.

[0027] In one aspect, the amino acid sequence encoded by the MYC3 gene of the present application is shown as SEQ ID NO: 6.

[0028] In one aspect, the nucleotide sequence of the coding region of the MYC3 gene of the present application is shown as SEQ ID NO: 5.

[0029] The beneficial technical effects of the present application include but are not limited to the following:

[0030] (1) The present application first discovers a new pathway of "temperature-JA signal" coupling regulation of chloroplast development, breaks through the limitations of single environmental factor or hormone research, and helps to understand the productivity and adaptability of plants under complex and variable climate and farming conditions;

[0031] (2) Further reveals the environmental-dependent functional conversion mechanism of MYC transcription factors, i.e., from promoting development to inhibiting development under the combined action of high temperature / warm temperature-JA, which provides a new target for resistance breeding of crops under stress caused by high temperature / warm temperature-JA combined action.

[0032] (3) Finally, it is confirmed that MYC mutant plants can be used as resistance materials for stress caused by high temperature / warm temperature-JA combined action, which can significantly improve chloroplast development of seedling, increase seedling survival rate and photosynthetic capacity in complex dynamic environment, promote the optimization of chloroplast artificial regulation technology, and realize the directional design of high-efficiency photosynthetic organs in facility agriculture and artificial photosynthesis system. BRIEF DESCRIPTION OF DRAWINGS

[0033] Figure 1 Showing high temperature strengthening JA-induced cotyledon yellowing.

[0034] Figure 2 Showing high temperature and JA synergistically regulate seedling cotyledon development.

[0035] Figure 3 Showing differential gene expression analysis of high temperature and JA co-regulation in Col-0.

[0036] Figure 4 Showing MYC specifically regulates high temperature and JA-mediated seedling cotyledon development.

[0037] Figure 5 Showing MYC negatively regulates high temperature and JA-mediated seedling chloroplast development.

[0038] Figure 6 MYC was shown to regulate chloroplast development genes expression in a dual mode under the synergistic effect of high temperature and JA. DETAILED DESCRIPTION

[0039] The following examples facilitate a better understanding of the present application, but do not limit the present application. The experimental methods in the following examples are all conventional methods unless otherwise specified. The test materials used in the following examples are all commercially available from conventional biochemical reagent manufacturers unless otherwise specified.

[0040] Example 1 Warm temperature and JA regulate cotyledon development of seedlings

[0041] To further analyze the molecular mechanism of warm temperature and JA interaction regulating cotyledon development of seedlings, this study used a controllable culture system. Wild type seeds were sown in MS medium with or without the addition of Methyl Jasmonate (MeJA, a derivative of JA-ILE, Catalog No. 392707, manufacturer Sigma-Aldrich) (0 μM / 25 μM / 50 μM). After 4°C vernalization treatment, temperature gradients (18°C / 22°C / 25°C / 28°C / 30°C) were set for comparison culture. The specific experimental scheme is as follows:

[0042] Wild type Arabidopsis Col-0 seeds were sown in MS medium with or without the addition of MeJA (0 μM / 25 μM / 50 μM). After 3 days of 4°C vernalization, they were transferred to different temperatures (18°C / 22°C / 25°C / 28°C / 30°C) in a full light incubator (light intensity 40 μmol m -2 s -1 ) for 6 days. A digital camera (Olympus M.zuiko) was used to take clear photos of the whole dish to show the overall growth trend of the seedlings; a body microscope (E-M5 II) was used to take clear photos of the cotyledons. Chlorophyll content determination: After taking photos, 20 seedlings with consistent growth state were selected and their fresh weight (FW) was accurately weighed using an analytical balance. The samples were placed in 1 mL of pre-cooled 80% (v / v) acetone extraction solution and extracted under 4°C in the dark for 16-18 h. After extraction, the supernatant was obtained by centrifugation at 12,000 rpm for 5 min at 4°C. The optical density (OD) of chlorophyll a and chlorophyll b was measured at characteristic wavelengths of 663 nm and 647 nm, respectively, using a UV-visible spectrophotometer. The formula for calculating the content of chlorophyll is as follows:

[0043] Chlorophyll (a+b) (μg / g FW) = (7.15 × A 663 + 18.71 × A647 )÷FW

[0044] The experimental results show that in the control group without MeJA application, the cotyledon of seedlings shows slight chlorosis with the increase of temperature, but the phenotypic difference does not reach a statistically significant level Figure 1 A-C). In the 25 μM MeJA treatment group, the degree of cotyledon yellowing increases with the increase of temperature, and the chlorophyll content decreases significantly Figure 1 A-C). When the MeJA concentration is increased to 50 μM, the temperature-dependent chlorophyll degradation effect is further intensified, especially in the temperature range of 28-30℃, the regulatory effect of JA on seedling cotyledon yellowing is significantly enhanced.

[0045] Through systematic experimental screening of temperature gradient and JA concentration gradient, this study determines the warm temperature (28℃) and 25 μM MeJA as the experimental treatment conditions, and carefully characterizes and quantitatively analyzes the phenotypic characteristics. The specific experimental methods are as follows:

[0046] Maximum photochemical efficiency Fv / Fm determination: Arabidopsis seedlings grown at 22℃, 22℃+JA, 28℃ and 28℃+JA for 6-7 days, after 15 min dark treatment, the chlorophyll fluorescence kinetics curve determination and chloroplast fluorescence imaging analysis were performed using the modulated chlorophyll fluorescence imaging system (Imaging-PAM). The fluorescence parameters were collected and processed by ImagingWin software, and the corresponding fluorescence image data were exported for subsequent analysis.

[0047] Chlorophyll autofluorescence detection: Arabidopsis seedling leaves were placed on glass slides. Olympus FV1000MPE two-photon fluorescence lifetime system was used for sample observation and microscopic imaging with a 63x water immersion objective. The fluorescence detection parameters were set as follows: GFP excitation wavelength 488 nm, emission spectrum collection range 490-585 nm. Image acquisition was performed using Z-axis layer scanning mode (interlayer distance 0.5 μm), after multi-focus plane image fusion, FV10-ASW software (v4.2) was used for image analysis and data export. The specific experimental methods refer to the description in the article published in Cell in 2024 (Frangedakis et al., 2024, Cell 187, 1-18). [2]

[0048] Chloroplast ultrastructure observation: refer to the description in the article published in Nature Communications in 2024 by Tachibana R et al. [3]

[0049] ​​The results showed that: compared with the control condition (22℃), 22℃+JA treatment significantly induced the etiolation phenotype of cotyledon, which showed that the cotyledon area was significantly reduced, the chlorophyll content and the maximum photochemical efficiency of PSII (Fv / Fm) were significantly reduced( Figure 2 A-C). At 28℃, the seedlings showed a slight etiolation of cotyledon, accompanied by a significant increase in cotyledon area and a significant decrease in chlorophyll content( Figure 2 A-C). Notably, the synergistic treatment of 28℃ and JA (28℃+JA) led to more significant phenotypic changes: the seedling cotyledon color became significantly lighter, showing a light green or even light yellow phenotype, the leaf area was significantly reduced, the chlorophyll content and Fv / Fm were extremely significantly reduced( Figure 2 A-C). At the same time, we observed that compared with 22℃+JA, the chloroplast development of seedlings under 28℃+JA treatment was severely damaged, the chloroplast arrangement was loose, the chloroplast number and area were significantly reduced, and the chlorophyll fluorescence was significantly weakened( Figure 2 D). The results of transmission electron microscopy (TEM) showed that compared with 22℃+JA, under 28℃+JA treatment, the chloroplast volume became smaller and further swelled and deformed, the thylakoid membrane ruptured, the grana thylakoid and stroma thylakoid arrangement was scattered, the stroma lamella was broken, and the grana stacking was significantly reduced( Figure 2 E). The above data all showed that warm temperature and JA treatment could independently induce the yellowing process of seedling cotyledon, and there was a significant additive effect between the two, suggesting that there might be a synergistic mechanism between temperature signal and JA signal pathway in regulating the yellowing process of seedling cotyledon.

[0050] Example 2 Synergistic regulation of stress response by warm temperature and JA

[0051] To further analyze the molecular mechanism of warm temperature and JA-induced leaf yellowing, we selected Col seedlings cultured at 22℃ or 28℃ under full light for 6 days (MS medium with or without 25μM MeJA), isolated the cotyledon part, extracted RNA, and performed RNA-seq sequencing. The screening criteria of expression fold (Fold Change) ≥2 and FDR <0.05 were used to analyze the differentially expressed genes (DEGs). The specific experimental scheme is as follows:

[0052] Cotyledons of Arabidopsis thaliana seedlings grown at 22℃, 22℃+JA, 28℃, and 28℃+JA for 6-7 days were harvested and frozen in liquid nitrogen. Total RNA was extracted using the Arabidopsis thaliana total RNA extraction kit (Aikerui Biotechnology Co., Ltd.), digestion, and reverse transcription kit (Thermo Scientific reverse transcription kit), and RNA digestion was performed to remove gDNA. The prepared RNA was sent to Biomarker Biotechnology Corporation for subsequent library construction, sequencing, and quality control. Data analysis and graphing: Biomarker Cloud Platform (…) was used. https: / / plotted on the international.biocloud.net / zh / mysample / list Figure 3 Venn diagrams and volcano diagrams in the image. Using the David online database (…). https: / / david.ncifcrf.gov / conversion.jsp GeneOntology (GO) cluster analysis was performed. GO enrichment analysis was then conducted on specific biological processes using... ChiPlot ( https: / / www.chiplot.online / #BioPlot Online plotting software is used for data visualization to form... Figure 3 Bubble chart in the image.

[0053] The results showed that compared with the normal growth temperature (22℃), there were 1958 DEGs under the warm temperature of 28℃, 1446 DEGs after applying JA at 22℃ (22℃+JA), and 2576 DEGs under the same conditions at 28℃ (28℃+JA). Furthermore, Venn diagram cross-pollination revealed that 365 genes were co-regulated under the three conditions of 22℃+JA, 28℃, and 28℃+JA. Figure 3 A).

[0054] In addition, to elucidate the biological processes of the specific effects of combined warm temperature and JA treatment, this study conducted GO enrichment analysis on the specific regulatory genes of Col-0 under three treatment conditions: 22℃+JA, 28℃, and 28℃+JA.

[0055] The results showed that among the 520 DEGs specifically regulated by 22℃+JA treatment, significant enrichment was observed in biological processes such as hypoxia response, osmotic stress response, JA response, and photosignal response; among the 580 DEGs specifically regulated by 28℃ treatment, the main pathways involved were hypoxia response, oxidative stress response, and ABA signaling response; while among the 811 DEGs specifically regulated by 28℃+JA treatment, significant enrichment was observed in processes such as defense, ion homeostasis regulation, and reactive oxygen species metabolism. Figure 3B-D). Further GO enrichment analysis of the up-regulated and down-regulated genes specifically regulated by 28°C+JA showed that the up-regulated genes were mainly clustered in the biological processes of "response to wounding", "JA response", "ABA response", "salt stress", "dehydration", etc., while the down-regulated genes were mainly clustered in the biological processes of "response to light stimulus", "growth and development", "tissue development", etc. Figure 3 E and F).

[0056] Notably, compared with single-factor (JA or 28°C) treatment, the synergistic effect of double factors (28°C+JA) more significantly activated the defense-related pathways, indicating that the high-temperature environment might enhance the sensitivity of plants to JA signals through epigenetic modification. In addition, by comparison, 22°C+JA treatment specifically induced differential expression of genes related to secondary metabolite synthesis, including "thioglucoside biosynthesis" and "indole compound metabolism" pathways, which is highly consistent with the biological function of JA in defense response. Notably, the 28°C+JA treatment group was specifically enriched in processes such as "hypoxia response", "heat shock protein complex assembly", "ABA signaling pathway", and "dehydration response" ( Figure 3 G-I). These data suggest that high temperature might enhance the sensitivity of the JA signaling pathway by regulating JA synthesis and metabolism-related genes, thereby triggering excessive defense responses, leading to cotyledon yellowing and impaired development, and other phenotypic characteristics.

[0057] Example 3 MYC negatively regulates warm temperature and JA synergistically mediated chloroplast development

[0058] JAZ proteins, as the core regulatory factors of jasmonic acid (JA) signaling pathway, due to the functional redundancy among JAZ family members, a single jaz mutant usually only shows slight or negligible phenotype, while five mutants (jazQ) or eleven mutants (jazU) exhibit more significant JA-related phenotypes, including over-activation of JA-dependent defense genes, enhanced resistance to herbivorous insects and pathogens, and growth inhibition— all of which are consistent with the characteristics of constitutive JA signaling. Given its significant phenotypic effects, we selected the jaz multiple mutants for further verification.

[0059] MYC transcription factors (especially MYC2) are the core regulatory factors of JA-dependent seedling morphogenesis and leaf development. To clarify their role in high-temperature-JA interaction, we analyzed the phenotypes of myc2-2 loss-of-function mutant, myc2 / 3 / 4 triple mutant (myc2 myc3 myc4), and 35S:MYC2-Myc overexpression lines under combined treatment. Under 22°C, 22°C+JA or 28°C conditions, no morphological differences were observed among the lines Figure 4A-C). However, under 28°C+JA co-treatment, cotyledons of myc2-2 and myc2 / 3 / 4 mutants remained green and had significantly higher chlorophyll content than wild-type Col-0, while 35S:MYC2-Myc seedlings showed accelerated chlorophyll degradation Figure 4 A-C). Although jazQ and jazU were highly sensitive to high temperature and JA, their cotyledons only showed slight yellowing Figure 4 A-C). These results suggest that MYC plays a dominant role in high temperature and JA-mediated cotyledon development in seedlings. The specific acquisition methods of MYC loss-of-function or overexpression materials are as follows:

[0060] myc2-2 mutant, seed stock number: SALK_083483, mutant gene: At1g32640 (MYC2 / JASMONATE-INSENSITIVE 1, JIN1), background strain: Arabidopsis thaliana Columbia (Col-0). Mutation type: T-DNA insertion mutation, which is generated by inserting a foreign T-DNA sequence into the Arabidopsis genome through Agrobacterium-mediated transformation. Insertion site: The T-DNA is inserted into the 3rd exon (Exon 3) of the MYC2 gene. The insertion mutant can be referred to Clough, S. J. and Bent, A. F. (1998), The Plant Journal, 16, 735-743 [4] constructed according to the method disclosed in Clough, S. J. and Bent, A. F. (1998), The Plant Journal, 16, 735-743.

[0061] According to the SALK sequence tag information, the insertion site is located at +1289 bp of the coding region (CDS) (A is +1 relative to the transcription start site ATG). Effect of mutation on gene function: The T-DNA is inserted into a key exon that encodes a protein domain, completely destroying the coding sequence of the exon. This usually results in the production of a truncated and non-functional protein, or triggers mRNA nonsense-mediated degradation (NMD), resulting in a significant reduction or even complete absence of mRNA levels. Therefore, myc2-2 is considered a loss-of-function mutant.

[0062] myc2 / 3 / 4 mutant, common name: myc2 myc3 myc4, myc2 / 3 / 4, JIN1-9JAZ2D myc3 myc4, or myc234 triple mutant. Background strain: Arabidopsis thaliana Columbia (Col-0). The triple mutant was obtained by crossing three independent T-DNA insertion mutants (as shown in Table 1 below) and screened for genotype identification. The triple mutant was obtained by Zhu et al., 2015 in Plant physiology [5]The research content published above has been constructed and described in detail, aiming to reveal the redundant function of MYC protein in JA signal.

[0063] Table 1 Information of three independent T-DNA insertion mutants

[0064]

[0065]

[0066] 35S:MYC2-Myc overexpression line: the coding sequence (CDS) of MYC2 gene is driven by CaMV 35S strong promoter, and Myc expression tag is fused at the C terminal. Arabidopsis is transformed by Agrobacterium-mediated floral dip method, transgenic positive plants are screened, and homozygous lines are obtained. The specific method steps are as follows:

[0067] Expression vector construction: ① Cloning CDS sequence of MYC2 gene: specific primers are designed, necessary enzyme digestion sites are introduced, cDNA of Arabidopsis (Col-0) seedlings is used as template, high-fidelity DNA polymerase KOD FX (TOYOBO) is used, and the target fragment is amplified by PCR. The PCR reaction system is: 25 μL 2×KOD mix, 1.5 μL 10 μM forward primer F, 1.5 μL 10 μM reverse primer R, 2 μL template, 20 μL ddH2O. The PCR reaction program is: pre-denaturation (95℃, 3 min), denaturation (95℃, 10 s), annealing (58℃, 30 s), extension (68℃, 30 s-60 s), 30-35 cycles. After amplification, the final product is analyzed by 1% agarose gel electrophoresis to verify the specificity of the amplified fragment. The target fragment is cut by ultraviolet imaging instrument, and the nucleic acid fragment is purified according to the instructions of Zhongke Ruite DNA recovery kit. ② Select intermediate vector (pEASY-Blunt Simple (TransGen Biotech)) and expression vector (pCAMBIA1300-Myc). The correctly sequenced MYC2 CDS is cloned into pEASY vector by TA cloning to construct pEASY-MYC2. Select a target vector pCAMBIA1300-Myc with 35S promoter, C terminal Myc tag and plant selection marker (hygromycin resistance). The pEASY-MYC2 and pCAMBIA1300-Myc are digested with the corresponding endonuclease (EcoR1 / SalI) to obtain linearized vectors, and then in vitro ligation is performed using T4 ligase (T4-DNA ligase) to finally obtain the expression vector 35S:MYC2-Myc. The constructed plasmid is transformed into Agrobacterium competent cells (GV3101 strain) by Agrobacterium transformation method.

[0068] Arabidopsis thaliana genetic transformation: ① Agrobacterium culture and infection solution preparation: pick Agrobacterium single clone containing the correct recombinant plasmid, and expand culture in LB liquid medium containing the corresponding antibiotic. Centrifugal collection of bacterial cells, resuspended to OD 600 ≈0.8 with transformation infiltration medium (1 / 2MS salt + 5% sucrose + 0.02% Silwet L-77). ② Floral Dip method transformation: dip the flowering wild type inflorescence of Arabidopsis thaliana into the prepared Agrobacterium infection solution for about 30 seconds. After 24 hours of light-protected and moisture-protected culture, return to normal light and temperature conditions for culture, repeat infection 3-4 times, until the seeds (T0 generation seeds) are harvested.

[0069] Screening and identification of transgenic positive plants: ① Resistance screening: sow the harvested T0 generation seeds on 1 / 2MS medium containing hygromycin. After about 7-10 days of culture, the surviving green seedlings are potential transgenic positive seedlings. ② Progeny segregation analysis: individually harvest the preliminarily identified positive T1 generation plants (transgenic positive plants are usually heterozygous) to obtain T2 generation seeds. Sow the T2 generation seeds on the screening plate again, and observe the resistance and non-resistance segregation ratio. The single plant that meets the 3:1 segregation ratio is considered to be a single insertion site.

[0070] ③ Selection of homozygous lines: in the T2 generation, select the T2 generation family in which all the resistant seedlings survive, and the corresponding T3 generation seeds should be 100% resistant, i.e. homozygous transgenic lines.

[0071] jaz mutant material preparation information is as follows:

[0072] 1. jazQ five mutant. Full name: jazQ or jaz1 / 3 / 4 / 9 / 10. Component genes: JAZ1 (At1g19180), JAZ3 (At3g17860), JAZ4 (At1g48500), JAZ9 (At1g70700) and JAZ10 (At5g13220). Background: Arabidopsis thaliana Columbia (Col-0). First characterization: constructed and reported by Campos et al. in Nature Communications [6] 2016. Mutant alleles: jaz1-2 (JIC-SM.22668), jaz3-4 (GK-097F09), jaz4-1 (SALK_141628), jaz9-4 (GK-265H05) and jaz10-1 (SAIL_92_D08).

[0073] 2. jazU decuple mutant. Full name: jazU or jaz decuple. Component genes: contains 5 genes with jazQ knocked out, and additionally 5 genes knocked out: JAZ2, JAZ5, JAZ6, JAZ7, and JAZ8. Total of 10 JAZ genes (JAZ1-7 and JAZ9-11) knocked out. JAZ12 and JAZ13 not knocked out. Background: Arabidopsis thaliana Col-0. First characterization: by Guo et al. 2018 in PNAS [7] The above report is one of the ultimate genetic materials for studying the redundant functions of JAZ. Mutant alleles: constructed by multiple years of crossing, backcrossing, and genotype identification from multiple SALK or SAIL series T-DNA insertion mutants.

[0074] To further explore the biological function of MYC in the process of cotyledon development of seedlings under high temperature and JA mediation, the research team conducted photosynthetic function analysis and chloroplast ultrastructure observation using the myc triple mutant (myc2 / 3 / 4) as experimental material.

[0075] Chlorophyll fluorescence curve and photosynthetic parameter determination: (for specific experimental methods, refer to Liu et al. 2024 in Plant Journal [8] ): Arabidopsis seedlings grown at 22℃ / 28℃ for 6-7 days, dark treatment for 15 min, then determination. Modulated chlorophyll fluorescence imaging system (Imaging-PAM) was used for chlorophyll fluorescence kinetic curve determination and chloroplast fluorescence imaging analysis. During the experiment, 80 μmol m -2 s -1 of actinic light was continuously applied as background light. To obtain the maximum fluorescence yield (Fm') under light adaptation, the system applied a 2800 μmol m -2 s -1 of saturated pulse light with 20 s intervals. All fluorescence parameters were collected and processed by ImagingWin software, and the corresponding fluorescence image data were exported for subsequent analysis.

[0076] Under 22℃, 22℃+JA, and 28℃, myc2 / 3 / 4 and wild type Col-0 showed no significant difference, so the phenotype under 28℃+JA was analyzed. Under double treatment, myc2 / 3 / 4 showed higher photosynthetic capacity than wild type Col-0, specifically in terms of maximum photochemical efficiency (Fv / Fm), actual photosynthetic efficiency (ΦPSII), and electron transport efficiency (ETR), myc2 / 3 / 4 mutant plants were significantly higher than wild type Col-0 plants, while the redox state of QA electron acceptor of PSII (1-qP) of myc2 / 3 / 4 mutant plants was significantly lower than Col-0 Figure 5A-E).

[0077] Cell microscopic observation found that, compared with 22℃+JA, there were significant differences between myc2 / 3 / 4 mutant plants and wild type Col-0 plants under 28℃+JA, wherein the chloroplasts in mesophyll cells of myc2 / 3 / 4 mutant plants were still distributed densely, the number, area and chlorophyll autofluorescence intensity of chloroplasts were significantly higher than those of wild type Col-0 plants, and the myc2 / 3 / 4 mutant plants could maintain better chloroplast development state under complex stress conditions Figure 5 F-H). Chloroplast ultrastructure observation showed that under complex stress, although the chloroplasts of myc2 / 3 / 4 mutant plants were swollen and deformed to a certain extent, the chloroplast volume became smaller, but the thylakoid membrane remained basically intact, the grana thylakoids and matrix thylakoids were still arranged in order, and there were relatively more grana stacking numbers Figure 5 I), which indicated that under complex stress conditions, myc mutation could greatly alleviate the abnormal development of chloroplasts caused by complex stress and basically ensure the normal function of chloroplasts.

[0078] Example 4 MYC regulates the expression of genes related to leaf development

[0079] To clarify the molecular mechanism of MYC transcription factors in the high temperature-JA synergistically regulated development of cotyledons of seedlings, the specific regulated genes of myc2 / 3 / 4 triple mutant under 28℃+JA treatment were systematically analyzed in this study.

[0080] The results showed that the DEGs regulated by MYC reached 1840 (398 up-regulated and 1442 down-regulated; Figure 6 A), and the volcano plot showed that about one-third of the genes in the mutant were significantly up-regulated, and two-thirds of the genes were significantly down-regulated Figure 6 B). Based on the analysis of the cotyledon development phenotype of the mutant, the genes co-regulated by MYC and 28℃&JA were analyzed, and it was found that the enriched signal pathways were “mechanical damage response”, “JA-mediated signal pathway” and “dehydration response” etc. Figure 6

[0081] ​In addition, GO-BP enrichment analysis of up-regulated and down-regulated genes specific to MYC regulation was performed, and it was found that MYC positively regulated "mechanical damage response", "JA-mediated signal pathway", "pathogen defense", "ABA signal transduction" and other pathways. Further GO function annotation of the core regulatory pathway found that MYC-activated target genes not only included JA signal classic factors such as LOX2 (Lipoxygenase Gene 2), AOS (Allene Oxide Synthase), JAZs, etc., but also regulated senescence-related transcription factors NAP (NAC-Like, Activated by AP3 / PI), WRKY53 / 6, leaf senescence genes such as SAG21, SAG20, SOC1 (SUPPRESSOR OF OVEREXPRESSION OF CONSTANS1), SRG1 (Senescence-Related Gene 1), and chlorophyll degradation genes NYC1 (Non-Yellow Coloring 1), MES16, CYP89A9, etc. Figure 6 D) On the contrary, GATA9 / 12 (GATA transcription factor 9 / 12), CRF1 and GRF6, etc. were identified as positive regulators of chloroplast development in MYC2 negatively regulated target genes Figure 6 E) In summary, these experimental results show that MYC plays a pivotal role in integrating environmental high temperature and JA signal regulation of cotyledon development by activating negative regulators of leaf development on the one hand and inhibiting positive regulators of leaf development on the other hand.

[0082] The technical features of the above-described embodiments can be combined in any manner. To make the description concise, not all possible combinations of the technical features in the above-described embodiments are described, but as long as the combinations of the technical features do not contradict, they should be considered within the scope of the present disclosure.

[0083] The above-described embodiments only express several embodiments of the present application, which are described in detail and specifically, but should not be construed as limiting the scope of the patent. It should be noted that for ordinary skilled persons in the art, without departing from the concept of the present application, several modifications and improvements can be made, which are within the scope of the present application. Therefore, the scope of protection of the present patent should be subject to the appended claims.

[0084] The above-described embodiments only express several embodiments of the present application, which are described in detail and specifically, but should not be construed as limiting the scope of the patent. It should be noted that for ordinary skilled persons in the art, without departing from the concept of the present application, several modifications and improvements can be made, which are within the scope of the present application. Therefore, the scope of protection of the present patent should be subject to the appended claims.

[0085] References

[0086] [1] ALI M S, BAEK K-H. Jasmonic acid signaling pathway in response to abiotic stresses in plants [J]. International Journal of Molecular Sciences, 2020, 21(2): 621.

[0087] [2] Frangedakis E, Yelina NE, Billakurthi K, et al. MYB-related transcription factors control chloroplast biogenesis. Cell. 2024; 187(18): 4859-4876.e22. doi: 10.1016 / j.cell.2024.06.039.

[0088] [3] Tachibana R, Abe S, Marugami M et al. BPG4 regulates chloroplast development and homeostasis by suppressing GLK transcription factors and involving light and brassinosteroid signaling [J]. Nature Communications, 2024, 15: 370.

[0089] [4] Clough SJ, Bent AF. Floral dip: a simplified method for Agrobacterium-mediated transformation of Arabidopsis thaliana. Plant J. 1998 Dec; 16(6): 735-43.

[0090] [5] Zhu X, Chen J, Xie Z, et al. Jasmonic acid promotes degreening via MYC2 / 3 / 4- and ANAC019 / 055 / 072-mediated regulation of major chlorophyll catabolic genes. Plant J. 2015; 84(3):597-610.

[0091] [6] Campos ML, Yoshida Y, Major IT et al. Rewiring of jasmonate and phytochrome B signalling uncouples plant growth-defense tradeoffs [J]. Nature Communications, 2016, 7: 12570.

[0092] [7] Guo Q, Yoshida Y, Major IT et al. JAZ repressors of metabolic defense promote growth and reproductive fitness in Arabidopsis [J]. Proceedings of the National Academy of Sciences of the United States of America, 2018, 115(45): E10768-E10777.

[0093] [8] Liu L, Si L, Zhang L, et al. Metabolomics and transcriptomics analysis revealed the response mechanism of alfalfa to combined cold and saline-alkali stress. Plant J. 2024; 119(4): 1900-1919. doi: 10.1111 / tpj.16896.

[0094] SEQUENCE LISTING

[0095] SEQ ID NO: 1 Nucleotide sequence of the coding region of the MYC2 gene

[0096]

[0097] SEQ ID NO: 2 Amino acid sequence of the protein encoded by the MYC2 gene

[0098] MTDYRLQPTMNLWTTDDNASMMEAFMSSSDISTLWPPASTTTTTATTETTPTPAMEIPAQAGFNQETLQQRLQALIEGTHEGWTYAIFWQPSYDFSGASVLGWGDGYYKGEEDKANPRRRSSSPPFSTPADQEYRKKVLRELNSLISGGVAPSDDAVDEEVTDTEWFFLVSMTQSFACGAGLAGKAFATGNAVWVSGSDQLSGSGCERAKQGGVFGMHTIACIPSANGVVEVGSTEPIRQSSDLINKVRILFNFDGGAGDLSGLNWNLDPDQGENDPSMWINDPIGTPGSNEPGNGAPSSSSQLFSKSIQFENGSSSTITENPNLDPTPSPVHSQTQNPKFNNTFSRELNFSTSSSTLVKPRSGEILNFGDEGKRSSGNPDPSSYSGQTQFENKRKRSMVLNEDKVLSFGDKTAGESDHSDLEASVVKEVAVEKRPKKRGRKPANGREEPLNHVEAERQRREKLNQRFYALRAVVPNVSKMDKASLLGDAIAYINELKSKVVKTESEKLQIKNQLEEVKLELAGRKASASGGDMSSSCSSIKPVGMEIEVKIIGWDAMIRVESSKRNHPAARLMSALMDLELEVNHASMSVVNDLMIQQATVKMGFRIYTQEQLRASLISKIG

[0099] SEQ ID NO: 3 Nucleotide sequence of the coding region of the MYC3 gene:

[0100]

[0101] SEQ ID NO: 4 Amino acid sequence of the protein encoded by the MYC3 gene:

[0102] MNGTTSSINFLTSDDDASAAAMEAFIGTNHHSSLFPPPPQQPPQPQFNEDTLQQRLQALIESAGENWTYAIFWQISHDFDSSTGDNTVILGWGDGYYKGEEDKEKKKNNTNTAEQEHRKRVIRELNSLISGGIGVSDESNDEEVTDTEWFFLVSMTQSFVNGVGLPGESFLNSRVIWLSGSGALTGSGCERAGQGQIYGLKTMVCIATQNGVVELGSSEVISQSSDLMHKVNNLFNFNNGGGNNGVEASSWGFNLNPDQGENDPALWISEPTNTGIESPARVNNGNNSNSNSKSDSHQISKLEKNDISSVENQNRQSSCLVEKDLTFQGGLLKSNETLSFCGNESSKKRTSVSKGSNNDEGMLSFSTVVRSAANDSDHSDLEASVVKEAIVVEPPEKKPRKRGRKPANGREEPLNHVEAERQRREKLNQRFYSLRAVVPNVSKMDKASLLGDAISYINELKSKLQQAESDKEEIQKKLDGMSKEGNNGKGCGSRAKERKSSNQDSTASSIEMEIDVKIIGWDVMIRVQCGKKDHPGARFMEALKELDLEVNHASLSVVNDLMIQQATVKMGSQFFNHDQLKVALMTKVGENY

[0103] SEQ ID NO: 5 Nucleotide sequence of the coding region of the MYC4 gene:

[0104]

[0105] SEQ ID NO: 6 Amino acid sequence of the protein encoded by the MYC4 gene:

[0106] MSPTNVQVTDYHLNQSKTDTTNLWSTDDDASVMEAFIGGGSDHSSLFPPLPPPPLPQ VNEDNLQQRLQALIEGANENWTYAVFWQSSHGFAGEDNNNNNTVLLGWGDGYYK GEEEKSRKKKSNPASAAEQEHRKRVIRELNSLISGGVGGGDEAGDEEVTDTEWFFL VSMTQSFVKGTGLPGQAFSNSDTIWLSGSNALAGSSCERARQGQIYGLQTMVCVAT ENGVVELGSSEIIHQSSDLVDKVDTFFNFNNGGGEFGSWAFNLNPDQGENDPGLWI SEPNGVDSGLVAAPVMNNGGNDSTSNSDSQPISKLCNGSSVENPNPKVLKSCEMVN FKNGIENGQEEDSSNKKRSPVSNNEEGMLSFTSVLPCDSNHSDLEASVAKEAESNR VVVEPEKKPRKRGRKPANGREEPLNHVEAERQRREKLNQRFYSLRAVVPNVSKMDK ASLLGDAISYISELKSKLQKAESDKEELQKQIDVMNKEAGNAKSSVKDRKCLNQESS VLIEMEVDVKIIGWDAMIRIQCSKRNHPGAKFMEALKELDLEVNHASLSVVNDLM IQQATVKMGNQFFTQDQLKVALTEKVGECP.

Claims

1. Use of MYC genes to prevent abnormal chloroplast development in plant seedling leaves, characterized in that, Reducing / inactivating MYC gene expression in plants, the MYC gene including MYC2 gene.

2. Use of reducing / inactivating MYC gene expression in preventing abnormal chloroplast development in seedling leaves of plants, the MYC gene including MYC2 gene.

3. Use of recombinant vector for reducing / inactivating MYC gene expression in plants in preventing abnormal chloroplast development in seedling leaves of plants, the MYC gene including MYC2 gene.

4. Use of a recombinant engineered bacterium in preventing abnormal chloroplast development in leaves of plant seedlings, characterized in that, The recombinant engineering bacteria comprise a recombinant vector for reducing / inactivating MYC gene expression in plants, the MYC gene including MYC2 gene, preferably the engineering bacteria is Agrobacterium.

5. A method for preventing abnormal chloroplast development in leaves of plant seedlings, characterized by, The method comprises: introducing into the plant a recombinant vector for reducing / inactivating MYC gene expression in plants or a recombinant engineering bacteria comprising the recombinant vector, the MYC gene including MYC2 gene.

6. A method for obtaining a plant with improved traits, comprising, The method comprises: introducing into the plant a recombinant vector for reducing / inactivating MYC gene expression in plants or a recombinant engineering bacteria comprising the recombinant vector, the MYC gene including MYC2 gene.

7. Use according to any one of claims 1 to 4 and method according to claim 5 or 6, characterized in that, The chloroplast development is damaged by high temperature or warm condition accompanied by JA accumulation.

8. Use according to any one of claims 1 to 4 and method according to claim 5 or 6, characterized in that, The plant is Arabidopsis thaliana.

9. Use or method according to claim 8, characterized in that, Reducing / inactivating MYC2 gene expression in plants or reducing / inactivating MYC2 gene, MYC3 gene and MYC4 gene expression in plants; preferably the amino acid sequence encoded by the MYC2 gene is as shown in SEQ ID NO: 2, the amino acid sequence encoded by the MYC3 gene is as shown in SEQ ID NO: 4, and / or the amino acid sequence encoded by the MYC4 gene is as shown in SEQ ID NO:

6.

10. Use or method according to claim 9, characterized in that, The nucleotide sequence of the coding region of the MYC2 gene is as shown in SEQ ID NO: 1, the nucleotide sequence of the coding region of the MYC3 gene is as shown in SEQ ID NO: 3, and / or the nucleotide sequence of the coding region of the MYC4 gene is as shown in SEQ ID NO: 5.

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