Plant mutant for maintaining photosynthetic efficiency under high temperature stress and application thereof

Abstract

The application discloses a plant mutant for maintaining photosynthetic efficiency under high-temperature stress and application thereof, and belongs to the technical field of plant genetic engineering. WTG1 The mutant is a mutant in which a single base mutation occurs in a splicing site of a gene, which leads to abnormal translation of the gene and protein function defects, WTG1 The gene encodes a protein having an amino acid sequence as shown in SEQ ID NO. 1, the mutant shows dark green leaf color under normal growth conditions, under high-temperature stress, the survival rate and chlorophyll content of the mutant are significantly higher than those of a wild type, and the mutant can maintain a relatively high photosynthetic efficiency, which reveals that the mutant can stabilize plant photosynthetic efficiency and high-temperature adaptation capability; the application provides a key gene target and a new technical path for crop high-temperature resistance breeding, provides a new perspective for a chloroplast response process to environmental stress, provides a new dimension for a chloroplast protein sorting mechanism and functional regulation of photosynthesis, and has important application value.

Description

Technical Field

[0001] This invention relates to the field of plant genetic engineering technology, specifically to a plant mutant that maintains photosynthetic efficiency under high temperature stress and its application. Background Technology

[0002] The global climate system is undergoing a long-term trend characterized primarily by warming, leading to frequent heat stress events. Extreme high temperatures are a significant limiting factor affecting crop yield and plant growth and development. Current research has identified various plant adaptation mechanisms in response to high-temperature stress, such as the synthesis of heat shock proteins (HSPs) under heat stress, which assist in protein folding and prevent irreversible protein aggregation, as well as state transitions (Wang, Chen et al. 2018, Wu, Chen et al. 2023).

[0003] Chloroplasts are crucial sites for photosynthesis in plants and also serve as sensors for thermal changes, coordinating complex signal networks to respond to plant stress (Danilova, Kudryakova et al. 2018). The light response in chloroplasts is primarily catalyzed by two reaction centers: photosystem II and photosystem I. The highly abundant light-harvesting antenna protein LHCI attaches to the PSI core, while LHCII attaches to the PSII core (Wu, Chen et al. 2023). Studies have shown that antenna proteins not only capture light energy but are also important targets for heat-induced damage (Pospíšil and Yamamoto 2017). During heat stress, plants induce ROS production, and LHCII and LHCI dissociate from the reaction centers to form aggregates, restricting photosynthetic electron transport and reducing light energy conversion efficiency (Tang, Wen et al. 2007). If the binding stability between LHCI and the photosystem reaction centers is improved under heat stress, efficient energy transfer between the photosynthetic antenna and the reaction centers can be effectively maintained, thereby enhancing the plant's heat tolerance.

[0004] In summary, the regulation of protein levels in the photosystem reaction center is a crucial regulatory target for plant heat stress. Regulating antenna proteins through genetic engineering is an important strategy for modifying plant light adaptability. Summary of the Invention

[0005] Technical problem to be solved: In view of the above-mentioned technical problems, the present invention provides a plant mutant that maintains the photosynthetic efficiency of plants under high temperature stress and its application, which can effectively solve the technical problems existing in the prior art, such as damage to the photosynthetic system of plants caused by high temperature stress, resulting in abnormal growth and development, and thus causing a decline in yield and quality.

[0006] Purpose of the invention: The purpose of this invention is to provide a method for targeted modification.WTG1 The technical methods for using genes to maintain photosynthetic efficiency in plants under high temperature stress include providing an improved plant mutant and its derived biological materials, revealing new applications in improving plant high temperature tolerance, and providing new genetic resources and breeding pathways for the selection of crop stress-resistant varieties.

[0007] To achieve the above objectives, the present invention adopts the following technical solution: a plant mutant that maintains photosynthetic efficiency under high-temperature stress, wherein the plant mutant contains... WTG1 That is, the AT5G53080 gene has undergone a mutation that causes a defect in the function of its encoded protein, and the amino acid sequence of the WTG1 protein is shown in SEQ ID NO.1.

[0008] As a preferred technical solution of this application, the... WTG1 The nucleotide sequence of the AT5G53080 gene is shown in SEQ ID NO.2.

[0009] As a preferred technical solution of this application, the mutant is WTG1 That is, a mutant with a single base mutation of AT5G53080, the mutation being... WTG1 Single-base mutations that occur in the nucleotide sequence of a gene.

[0010] As a preferred technical solution of this application, the single-base mutation occurs WTG1 The splicing donor site of the fourth intron of the gene is mutated from G to A, resulting in the intron being retained in the mature mRNA and translated.

[0011] As a preferred technical solution of this application, the plant is Arabidopsis thaliana.

[0012] Biological materials containing any of the above-mentioned plant mutants that maintain photosynthetic efficiency under high temperature stress, wherein the biological material is an expression vector, a bacterial strain, a transgenic cell line, or a plant seed.

[0013] The application of any of the above-described plant mutants that maintain photosynthetic efficiency under high-temperature stress in the breeding of high-temperature tolerant plant varieties.

[0014] As a preferred technical solution of this application, the high temperature tolerance is manifested as an increase in the survival rate of plants and / or an increase in chlorophyll content under high temperature stress conditions.

[0015] A method for cultivating heat-tolerant plants includes inducing heat into the target plant. WTG1 Mutations in homologous genes lead to functional defects in their encoded proteins, thereby resulting in plants with increased heat tolerance; as shown. WTG1The protein encoded by the homologous gene has at least 80% homology with the amino acid sequence shown in SEQ ID NO.1.

[0016] As a preferred technical solution of this application, the mutation includes making the WTG1 The splicing site of homologous genes may change, or premature stop codons or frameshift mutations may be introduced through gene editing technology.

[0017] Explanation of the principle: This invention uses forward genetics to screen for a mutant with dark green leaves and high temperature tolerance. ， Map-based cloning and sequencing confirmed that the mutated gene encodes a novel TPR protein. WTG1 Mutations at the splice site of this gene lead to protein functional defects. The WTG1 protein is located in the chloroplast stroma and interacts physically with cpFtsY, a key receptor protein in the chloroplast signal recognition particle pathway. The WTG1 protein can significantly enhance the GTP hydrolase activity of cpFtsY. wtg1 In the mutant, due to the functional defect of WTG1, the GTPase activity of cpFtsY decreased, which led to an adaptive adjustment in the transport and integration efficiency of LHCPs. Specifically, the contents of LHCI and LHCII were reduced, thus maintaining a high photochemical efficiency and ultimately exhibiting a phenotype with significantly increased heat tolerance, survival rate and chlorophyll content.

[0018] The plant mutant for maintaining photosynthetic efficiency under high-temperature stress described in this application and its application have the following technical advantages compared with the prior art: 1. This invention obtains a model with severely yellowed leaves through forward genetics. Fv / Fm Normal mutants are named srps17-41-7 After determining that the mutation was a recessive mutation controlled by a single gene, map-based cloning and high-throughput sequencing were performed on the gene to identify... srps17-41-7 The mutated gene in the double mutation is AT5G53080. ， Right now WTG1 This application discloses for the first time a WTG1 Gene function defect mutants wtg1 The mutant grows normally at room temperature, but under high temperature stress, its survival rate is significantly higher than that of the wild type, and its chlorophyll content is also significantly higher than that of the wild type, showing excellent high temperature tolerance. 2. wtg1In the mutant, a single-base mutation occurred at the end of the fourth intron of the gene, changing from G to A. The splicing conserved sequence GT-AG was disrupted, and the fourth intron was translated completely, resulting in protein functional defects. WTG1 protein is a TPR protein located in the chloroplast stroma. It interacts with cpFtsY, enhancing the GTP hydrolase activity of cpFtsY and participating in the cpSRP protein sorting pathway. LHCP is a specific substrate for the cpSRP pathway. wtg1 The content decreased significantly under high temperature stress. wtg1 The mutant exhibits stronger tolerance, with significantly higher plant survival rate and chlorophyll content than WT. This application reveals for the first time that WTG1 protein acts as a regulator of the cpSRP pathway, a chloroplast protein sorting pathway, and regulates the transport of light-harvesting antenna proteins by enhancing the GTP hydrolase activity of cpFtsY. This discovery provides a new perspective for understanding the mechanism of plant high-temperature adaptation and points to a new target for improving crop stress resistance through genetic engineering. 3. This application not only protects the mutant itself, but also covers multi-level application schemes such as using the mutant gene information for the construction of biological materials, molecular marker-assisted breeding, and the creation of new germplasm directly through gene editing. This invention provides a new dimension for elucidating the molecular interaction mechanism and regulatory pathway of chloroplast response to abiotic stress, and provides innovative ideas for agricultural research fields such as crop variety improvement and grain yield increase, and has extremely high industrial transformation value. Attached Figure Description

[0019] Figure 1 This is a phenotypic diagram of the wild type and mutant of the present invention and wtg1 Schematic diagram of mutation sites, where A shows the phenotypic and fluorescence pseudocolor images of wild-type and mutant; B shows... wtg1 Phenotypic diagram, mutation site diagram and corresponding sequencing peak diagram (G mutation to A); Figure 2 This invention relates to stable Arabidopsis thaliana transformation plants ( 35S:WTG1-GFP Fluorescence localization map of WTG1-GFP in leaf cells; Figure 3 This invention describes a three-step method for separating chloroplast stroma and thylakoid membrane components, and uses specific antibodies to detect the results. Coomassie brilliant blue staining (CBB) is used as a loading control. Stroma represents chloroplast stroma, Thylakoid represents thylakoid membrane, RbcL is a chloroplast stroma control protein, and LHCII is a thylakoid membrane control protein. The left image is a protein immunoblotting analysis image, and the right image is a Coomassie brilliant blue staining image. Figure 4 This is a chloroplast protein content analysis diagram of the present invention, and an immunoblotting analysis of wild-type cells grown for 7 days. wtg1mutants rps17 mutants and srps17-41-7 Determination of chloroplast protein content in double mutants; Figure 5 This is a diagram showing the yeast two-hybrid experiment analysis of WTG1 and cpFtsY according to the present invention; Figure 6 This is a BIFC experimental analysis diagram of WTG1 and cpFtsY proteins of this invention; Figure 7 This is a pull-down experimental analysis diagram of WTG1 and cpFtsY proteins of this invention; Figure 8 This is a diagram showing the interaction results between WTG1 protein and cpFtsY in this invention. A represents the domain division diagram of cpFtsY. According to domain function, cpFtsY can be divided into a leader peptide (1-40 aa), domain A (41-149 aa), and domain B (150-366 aa). Domain A contains a membrane-targeting sequence (MTS), and domain B is related to GTP binding and hydrolysis. B represents the yeast two-hybrid analysis between the domains of WTG1 and cpFtsY. Figure 9 This is a concentration gradient diagram of the GTPase activity of WTG1 against cpFtsY according to the present invention; Figure 10 This is a time gradient diagram of the GTPase activity of WTG1 against cpFtsY according to the present invention; Figure 11 This is the present invention. wtg1 The results of high-temperature stress treatment on mutants are shown in the figure, where A represents WT and wtg1 Phenotypic diagram of mutants treated at 44℃ for 3 hours and recovered for 2 days; B represents WT and wtg1 Statistical chart of lethality after high-temperature treatment, where C represents WT and wtg1 A graph showing the chlorophyll content measured after high-temperature treatment. Detailed Implementation

[0020] The technical solution of the present invention will be clearly and completely described below with reference to the accompanying drawings and specific embodiments. The described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0021] A plant mutant that maintains photosynthetic efficiency under high-temperature stress, wherein the plant mutant contains WTG1 Specifically, the AT5G53080 gene has undergone a mutation that results in a functional defect in the protein it encodes, and the amino acid sequence of the WTG1 protein is shown in SEQ ID NO.1: MVFSNSLLSSTTSLTTWANQSSSQSSLSPRYSTWQCVCLRNQKRKPKLYLIPARHFLSTPIDSVSSSSITASRYATSGVSEVQRSTSSNNVTEMEEFEMELQELFNEVKSMVKIGKESDAMDLLRANYVAVKEELDSGLKGIEQAAVLDIIALGYMAVGDLKPVPALLDMINKIVDNLKDSEPLLDSVLMHVGSMYSVIGKFENAILVHQRAIRILENRYGKCNTLLVTPLLGMAKSFASDGKATKAIGVYERTLTILERNRGSESEDLVVPLFSLGKLLLKEGKAAEAEIPFTSIVNIYKKIYGERDGRVGMAMCSLANAKCSKGDANEAVDIYRNALRIIKDSNYMTIDNSILENMRIDLAELLHFVGRGDEGRELLEECLLINERFKGKNHPSMATHLINLAASYSRSKNYVEAERLLRTCLNIMEVSVGSEGQSITFPMLNLAVTLSQLNRDEEAEQIALKVLRIREKAFGEDSLPVGEALDCLVSIQARLGRDDGEILGLLKRVMMIQEKEFGPSAQELIVTLQKIIHFLEKLEMKDDKFKFRRRLALLRERYKQSLSY.

[0022] WTG1 That is, the nucleotide sequence of the AT5G53080 gene is shown in SEQ ID NO. 2:

[0023] Example 1, wtg1 Obtaining mutants and identifying their phenotypic characteristics: First of all rps17 Based on the mutant, the recovered strain was obtained through EMS mutagenesis screening. rps17 Phenotype srps17- 41-7 After determining that the mutation was a recessive mutation controlled by a single gene, map-based cloning and high-throughput sequencing were performed on the gene to identify... srps17-41-7 The mutated gene in the double mutation is AT5G53080. ， Right now WTG1 Then will srps17-41-7 The mutant was crossed with wild-type WT and removed rps17 Mutant background plants were used, and whole-genome sequencing analysis of the remaining WT background plants revealed that the mutation site was located in... WTG1 A point mutant is obtained by mutating G to A at the conserved splice site at the end of the fourth intron of a gene. wtg1 Subsequently, specific primers were designed based on the sequence near the point mutation (forward primer as shown in SEQ ID NO.3: 5'-ATGGTGTTCTCTAACTCTCTCC-3', reverse primer as shown in SEQ ID NO.4: 5'-CTCTTAGCCATACCAAGT-3'), and the mutation was confirmed by RT-PCR and sequencing of the PCR products. wtg1 In mutants WTG1 The conserved GT-AG sequence of the gene was disrupted, confirming that this mutation caused abnormal intron splicing, affecting gene splicing. The fourth intron was translated completely, resulting in WTG1 functional deficiency. Sequencing results are as follows: Figure 1 As shown in B.

[0024] WTG1, a novel TPR protein, exists as a tandem array of 3-16 motifs. Generally, proteins with multiple copies of TPR motifs typically act as scaffold proteins, coordinating protein assembly into multi-subunit complexes. TPR proteins in plastids consistently play crucial roles in chloroplast gene expression, protein turnover, photosystem assembly and repair, chlorophyll biosynthesis, and thylakoid membrane biogenesis. Phenotypic observations show that… wtg1 The mutant has dark green leaves, plant size is not significantly different from the wild type, Fv / Fm is slightly higher than the wild type, and it is heat-tolerant. Its phenotype is as follows. Figure 1 As shown in B.

[0025] Example 2, Subcellular localization of WTG1 protein: The CDS sequence of the WTG1 protein was cloned into the stable transformation expression vector PRI101-GFP, and the fluorescence distribution of WTG1-GFP was observed using laser confocal microscopy. The results are as follows: Figure 2 As shown, the green fluorescence of the WTG1-GFP fusion protein completely overlaps with the auto-red fluorescence of the chloroplast, indicating that WTG1 is located in the chloroplast.

[0026] Subsequently, a three-step method was used to separate the chloroplast stroma and thylakoid membrane, and Western blot analysis of proteins was performed using specific antibodies. The results are as follows: Figure 3 As shown, the WTG1-GFP fusion protein is mostly enriched in chloroplast stroma components, with a small amount detected on the thylakoid membrane of chloroplasts. RBCL and LHCII serve as marker proteins for stroma and thylakoid proteins, respectively. In summary, WTG1 is located in the chloroplast stroma.

[0027] Example 3, wtg1 The expression level of LHCP in the middle limbic system differed from that in the wt limbic system. To explore wtg1 Changes in the protein content of each subunit of the thylakoid membrane complex in mutants were analyzed using specific antibodies against wild-type and wild-type mutants grown for 7 days. wtg1 mutants rps17 mutants and srps17-41-7 Western blot analysis was performed on the double mutants, including D1, D2, Ctyb6, PsaN, and LHCII. The results are as follows: Figure 4 As shown, compared to WT, wtg1 The expression levels of marker proteins for PSI, Ctb6f complex, and ATP synthase remained unchanged, but the levels of LHC1 and LHCII were significantly reduced. LHCP is a specific substrate protein in the chloroplast signal recognition particle (cpSRP) pathway, indicating that WTG1 may be involved in LHCP transport. To further investigate whether WTG1 affects other transport pathways, this application performed Western blot analysis on PsbO (a substrate protein in the chloroplast secretion (cpSec) pathway) and OE23 and PsaN (substrate proteins in the chloroplast diarginine (cpTat) pathway). The results showed... wtg The expression levels of cpSec and cpTat substrate proteins were no different from those in the wild type. In summary, WTG1 protein may be related to the cpSRP protein sorting pathway.

[0028] Example 4: WTG1 participates in chloroplast protein transport: The cpSRP pathway is mainly composed of four recognition particles: cpSRP54 / 43, cpFtsY, and Alb3. cpSRP54 / 43 binds to the substrate protein LHCP to form a soluble transient complex, preventing lipid protein aggregation. cpFtsY fully exerts its protein targeting function, while the membrane protein Alb3 promotes the integration of the substrate protein into the thylakoid membrane. This application utilizes various interaction experiments to verify the protein-protein interactions. First, yeast two-hybrid technology is used to... CDSCloned into the pGBK-AD-bHLH104 vector, WTG1 CDS The expression vector was cloned into the pGBKT7-BD vector. Yeast transformation experiments were then performed using the fusion expression vector, and yeast growth and plaque bluening were observed. Results are as follows: Figure 5 As shown, WTG1 interacts with cpFtsY, but not with cpSRP54 / 43 or Alb3. Further analysis via BIFC (…) Figure 6 Pull-down Figure 7 The experiment further validated this finding. cpFtsY contains a leader peptide and an N-domain containing a membrane-targeting sequence (MTS). The tethering of cpFtsY to the membrane is mediated by the amphiphilic helix at the N-terminus, GFP binding, and the required NG domain for hydrolysis. This application divides it into domain A (41-149 aa) and domain B (150-366 aa) according to function, and a yeast two-hybrid experiment was performed again. The results are as follows... Figure 8 As shown in Figure B, the interaction strength between cpFtsY domain A and WTG1 is greater than that between domain B. This result provides insights for subsequent experiments. In summary, WTG1 participates in chloroplast protein transport through its interaction with cpFtsY.

[0029] Example 5, WTG1 enhances the GTP hydrolase activity of cpFtsY: First, fusion expression vectors WTG1-pGEX-4T-2-GST and cpFtsY-pETMALc-H-MBP were constructed. These vectors were then transformed into the prokaryotic expression systems Transetta and BL21, respectively. After small-scale induction, high-efficiency transformants were selected for large-scale purification, yielding the fusion proteins WTG1-GST and cpFtsY-MBP. This invention utilizes the principle of GTPase hydrolysis of GTP to form GDP and release a dihydrogen phosphate (Pi). Changes in enzyme activity are reflected by detecting the absorbance at A630 nm of the green complex formed by the reaction of Pi with a specific yellow dye.

[0030] This invention establishes a concentration gradient, selecting 0, 2.5, 5, 7.5, and 10 μg of the fusion protein for co-incubation with GTP. The results are as follows... Figure 9 As shown, there is a linear relationship between protein concentration and phosphate release in the GTP hydrolysis reaction. The amount of Pi released by co-incubation of WTG1 and cpFtsY is higher than that released by cpFtsY alone. This indicates that WTG1 enhances the GTP hydrolase activity of cpFtsY. To avoid interference from the enzymatic activity of WTG1 and the tag protein itself, different concentrations of samples were co-incubated with GTP. The results are as follows. Figure 9 and Figure 10As shown, the tag protein and WTG1 do not cause Pi release, indicating that they do not have enzymatic activity and the product does not change drastically with concentration and time gradient.

[0031] To ensure that the measured rate represents the initial rate of true enzyme activity and to avoid errors caused by substrate consumption, product inhibition, or enzyme inactivation, this invention measures enzyme activity at a fixed protein concentration (5 μg) with time gradients of 0, 1, 2, and 3 hours. The results are as follows: Figure 10 As shown, WTG1 significantly enhanced the GTPase activity of cpFtsY in a time-dependent manner, as evidenced by the higher accumulation rate of Pi in the co-incubation system compared to the cpFtsY-only reaction system. In conclusion, WTG1 enhances the GTPase activity of cpFtsY.

[0032] Example 6, wtg1 Mutant response to high temperature stress: Wild-type and wild-type plants grown on petri dishes for 7 days were selected. wtg1 The mutant seedlings were subjected to high-temperature stress treatment, placed in a 45℃ dark incubator for 3 hours, and then transferred to 22℃ normal light conditions for 3 days to recover. Phenotypic characteristics were observed, survival rate was recorded, and chlorophyll content was measured. Results are as follows: Figure 11 As shown, after being subjected to high-temperature stress at 44℃, wtg1 The survival rate, chlorophyll a, and chlorophyll b content of the mutant plants were significantly higher than those of the wild-type WT, indicating that... wtg1 It has high temperature resistance.

[0033] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

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