Autophagy-related protein ZmATG8c and its coding gene in regulating heat tolerance of plants

Abstract

The application belongs to the field of plant genetic engineering breeding, and provides application of autophagy-related protein ZmATG8c and a coding gene thereof in regulating heat tolerance of plants. The application first finds that corn ZmATG8c protein and the coding gene thereof can significantly affect the heat tolerance of plants (preferably corn), overexpression of the gene in wild-type plants significantly increases the survival rate of the obtained transgenic plants after heat treatment compared with the wild type; gene editing of the gene editing plants destroying the coding sequence of the gene and the encoded protein significantly reduces the survival rate after heat treatment compared with the wild type. The application has important significance for improving crop yield under heat stress.

Description

Technical Field

[0001] This invention belongs to the field of plant genetic engineering breeding, specifically, it relates to the application of autophagy-related protein ZmATG8c and its encoding gene in regulating plant heat tolerance. Background Technology

[0002] Global warming is leading to increasingly frequent extreme heat events. It is predicted that by the end of the 21st century, the global average surface temperature will rise by approximately 3.35 ± 2.35 °C (IPCC, 2021). As sessile organisms, plants are susceptible to heat stress, which alters their physiological properties and inhibits growth and yield. Unfortunately, crops such as rice and maize frequently encounter high-temperature stress throughout their life cycle, potentially leading to catastrophic yield losses (Kan et al. 2023; Liu et al. 2023). Therefore, it is urgent to discover new heat-resistant genes in crops and create new heat-resistant germplasm materials.

[0003] Autophagy is one of the mechanisms that can alleviate heat stress; autophagosomes accumulate in Arabidopsis and tomato plants under heat stress (Zhou et al. 2013; Zhou et al. 2014; Yang et al. 2015). Furthermore, mutations in the ATG5 or ATG7 genes in Arabidopsis and tomato suppress autophagy and reduce heat tolerance, while overexpression of MdATG18a enhances the basal heat tolerance of apples by promoting autophagy (Zhou et al. 2013; Zhou et al. 2014; Huo et al. 2020). A recent study showed that after heat stress, damaged Golgi apparatus is transported to vacuoles and degraded, thus aiding in its recovery and remodeling; this process is accomplished by ATG8-mediated atypical autophagy (Zhou et al. 2023). ATG8 is a highly conserved ubiquitin-like protein that regulates the autophagy pathway by binding to the autophagy membrane and multiple proteins, including cargo receptors and core autophagy components (Zess et al. 2019). ATG8 is also involved in selective autophagy, in which it interacts with autophagy receptors to facilitate the recognition of cargo that needs to be degraded. The interaction between the receptor protein NBR1 and ATG8 mediates selective autophagy in plants under heat stress in order to maintain protein homeostasis under heat stress conditions (Zhou et al. 2013; Jung et al. 2020). Stress induced by heat shock or proteasome inhibitors can induce the accumulation of cytoplasmic aggregates associated with NBR1 and ATG8. In the atg7 mutant, the accumulation levels of these aggregates in the cytoplasm are higher than in the wild type, suggesting that the aggregates are degraded via autophagy (Jung et al. 2020). The NBR1-mediated selective autophagy pathway degrades heat shock protein 90 (HSP90) and peptidylproline cis-trans isomerase FKBP1 (ROF1) during the heat stress recovery phase, thereby inhibiting the transcriptional activity of HSFA2 (heat shock transcription factor A2), which in turn suppresses the persistence of HSP synthesis and enhances protection against potential impending heat stress (Thirumalaikumar et al. 2020). Despite recent significant advances in Arabidopsis, functional redundancy among ATG8 isoforms is generally considered to exist due to the increased copy number and high sequence conservation of ATG8 in higher plants (especially crops) (Kellner et al. 2017; Zess et al. 2019). Therefore, clarifying the contribution of specific ATG8 isoforms to heat tolerance in plants, particularly crops, is considered extremely challenging. Summary of the Invention

[0004] The purpose of this invention is to provide the application of autophagy-related protein ZmATG8c and its encoding gene in regulating plant heat tolerance.

[0005] To achieve the objectives of this invention, in a first aspect, this invention provides any of the following applications of the ZmATG8c gene:

[0006] (1) Used to regulate plant heat resistance;

[0007] (2) Used to prepare transgenic plants;

[0008] (3) Used for plant genetic breeding.

[0009] Furthermore, the regulation described in (1) is positive regulation, that is, improving the heat resistance of plants.

[0010] In this invention, the ZmATG8c gene is a gene encoding either (a) or (b) the following protein:

[0011] (a) A protein consisting of the amino acid sequence shown in SEQ ID NO:1;

[0012] (b) A protein derived from (a) with the sequence shown in SEQ ID NO:1 substituted, deleted or added with one or more amino acids and having the same function.

[0013] Furthermore, the plant (such as a crop) is a monocotyledonous or dicotyledonous plant, preferably a grass, and more preferably corn, sorghum, rice and wheat.

[0014] When the plant in question is corn, improving plant heat resistance refers to improving the heat resistance of corn seedlings and during the reproductive growth stage.

[0015] In a second aspect, the present invention provides a method for improving the heat resistance of plants, the method comprising: increasing the activity and / or content of autophagy-related protein ZmATG8c in a recipient plant, or enhancing the expression of the gene encoding autophagy-related protein ZmATG8c, to obtain a target plant with improved heat resistance compared to the recipient plant.

[0016] In this invention, the autophagy-related protein ZmATG8c is:

[0017] (a) A protein consisting of the amino acid sequence shown in SEQ ID NO:1;

[0018] (b) A protein derived from (a) with the sequence shown in SEQ ID NO:1 substituted, deleted or added with one or more amino acids and having the same function.

[0019] Furthermore, the methods for enhancing gene expression can be selected from the following 1) to 5), or any combination thereof:

[0020] 1) By importing a plasmid containing the gene;

[0021] 2) By increasing the copy number of the aforementioned genes on plant chromosomes;

[0022] 3) By altering the promoter sequence of the aforementioned genes on plant chromosomes;

[0023] 4) By operatively linking a strong promoter to the gene;

[0024] 5) By introducing enhancers.

[0025] Thirdly, the present invention provides a method for reducing the heat resistance of maize, the method comprising: modifying the maize ZmATG8c gene to cause the gene to lose or weaken its function.

[0026] Furthermore, the method for modifying the ZmATG8c gene can be at least one of self-mutation, site-directed mutagenesis, or homologous recombination.

[0027] In one specific embodiment of the present invention, the method includes: using a gene editing method to knock out the DNA fragment containing the DNA fragment shown in SEQ ID NO:3 from 1629-2361 bp and its reverse fragment from the maize genome.

[0028] Fourthly, the present invention provides the application of transgenic plants obtained according to the method in plant breeding.

[0029] Furthermore, breeding methods include, but are not limited to, transgenic, hybridization, backcrossing, self-pollination, or asexual reproduction.

[0030] By employing the above technical solution, the present invention has at least the following advantages and beneficial effects:

[0031] This invention is the first to discover that the maize ZmATG8c protein and its encoding gene can significantly affect the heat tolerance of plants (preferably maize). Overexpression of this gene in wild-type plants resulted in transgenic plants with a significantly increased survival rate after heat treatment compared to wild-type plants. Gene-edited plants with disrupted coding sequences and encoded proteins showed a significantly reduced survival rate after heat treatment compared to wild-type plants. The application of this invention is of great significance for improving crop yields under heat stress. Attached Figure Description

[0032] Figure 1 This is the result of routine PCR identification of ZmATG8c overexpressing transgenic plants in a preferred embodiment of the present invention.

[0033] Figure 2 This is the result of routine PCR identification of ZmATG8c gene-edited plants in a preferred embodiment of the present invention.

[0034] Figure 3The results of real-time PCR identification of ZmATG8c overexpressing transgenic plants in a preferred embodiment of the present invention are shown. *** indicates P < 0.001.

[0035] Figure 4 The results of quantitative real-time PCR identification of ZmATG8c gene-edited plants in a preferred embodiment of the present invention are shown. Here, ns indicates no significant difference.

[0036] Figure 5 The phenotypes of ZmATG8-OE and KO under thermal stress are shown in a preferred embodiment of the present invention.

[0037] Figure 6 The figures show the statistical results of plant survival rate in a preferred embodiment of the present invention. Different lowercase letters indicate significant effects.

[0038] Figure 7 The results of electrolyte leakage rate measurement are shown in a preferred embodiment of the present invention. Different lowercase letters indicate significant effects.

[0039] Figure 8 This is a schematic diagram of the process for introducing molecular marker-assisted traits, i.e., backcross traits, in a preferred embodiment of the present invention. Detailed Implementation

[0040] This invention aims to provide the application of the maize autophagy-associated protein ZmATG8c in improving plant heat tolerance. The CDS sequence of the maize autophagy-associated protein gene ZmATG8c is shown in SEQ ID NO:2; its encoded amino acid sequence is shown in SEQ ID NO:1; the heat tolerance refers to heat tolerance during the seedling stage and the reproductive growth stage.

[0041] In the above applications, the plant refers to an agricultural crop, preferably corn or sorghum.

[0042] Furthermore, improving the heat resistance of maize refers to its heat resistance during the seedling and reproductive growth stages.

[0043] The present invention also includes nucleic acid molecular expression cassettes containing the autophagy-associated protein ZmATG8c sequence, recombinant vectors, microbial strains or transgenic plants and their application in improving plant heat resistance.

[0044] Those skilled in the art can readily mutate the nucleotide sequence encoding the ZmATG8c protein of this invention using known methods, such as directed evolution and point mutation. Artificially modified nucleotides that possess 75% or higher identity to the nucleotide sequence of the ZmATG8c protein isolated in this invention, provided they encode a protein similar to ZmATG8c and have the function of the ZmATG8c protein, are all derived from and equivalent to the nucleotide sequence of this invention.

[0045] The term "identity" as used herein refers to sequence similarity to a natural nucleic acid sequence. "Identity" includes nucleotide sequences that have 75% or higher, 85% or higher, 90% or higher, or 95% or higher identity with the nucleotide sequence of a protein constituting the amino acid sequence shown in SEQ ID NO:1, the coding sequence of this invention. Identity can be evaluated visually or using computer software (sequence alignment software). Using sequence alignment software, the identity between two or more sequences can be expressed as a percentage (%), which can be used to evaluate the similarity between related sequences.

[0046] The aforementioned 75% or higher degree of identity can be 80%, 85%, 90%, or 95% or higher degree of identity.

[0047] The expression cassette (ZmATG8c gene expression recombinant vector) containing a nucleic acid molecule encoding the ZmATG8c protein described in the above applications refers to plasmid DNA capable of expressing the ZmATG8c protein in host cells. This DNA may include not only a promoter to initiate ZmATG8c gene transcription but also a terminator to terminate ZmATG8c gene transcription. Furthermore, the expression cassette may also include an enhancer sequence.

[0048] Promoters that can be used in this invention include, but are not limited to, constitutive promoters, tissue-, organ-, and development-specific promoters, and inducible promoters. Examples of promoters include, but are not limited to, the constitutive promoter 35S of cauliflower mosaic virus; the constitutive promoter Ubquitin from maize; the chemically inducible promoter Pathogenesis Related 1 (PR1) from tobacco (induced by salicylic acid and BTH (benzothiazole-7-thiohydroxy acid S-methyl ester); the tomato protease inhibitor II promoter (PIN2) or the LAP promoter (both induced by methyl jasmonic acid); the heat shock promoter (US Patent 5,187,267); and seed-specific promoters, such as the millet seed-specific promoter pF128 (CN101063139B (Chinese Patent 200710099169.7)). They can be used alone or in combination with other plant promoters. All references cited herein are incorporated herein by reference in their entirety. Suitable transcription terminators include, but are not limited to: Agrobacterium carmine synthase terminator (NOS terminator), cauliflower mosaic virus CaMV 35S terminator, tml terminator, pea rbcS E9 terminator, and carmine and octopine synthase terminator.

[0049] Recombinant vectors containing the ZmATG8c gene expression cassette can be constructed using existing expression vectors. These plant expression vectors include binary Agrobacterium vectors and vectors suitable for plant gene gun bombardment transformation, such as pAHC25, pBin438, pCAMBIA1302, pCAMBIA2301, pCAMBIA1301, pCAMBIA1300, pBI121, pCAMBIA1391-Xa, PSN1301, or pCAMBIA1391-Xb (CAMBIA). The plant expression vectors may also contain the 3' untranslated region of the exogenous gene, i.e., the polyadenylated nucleotide signal and any other DNA fragments involved in mRNA processing or gene expression. The polyadenylation signal can guide polyadenylation to be added to the 3' end of the mRNA precursor. Similar functions are found in the untranslated regions transcribed at the 3' end of Agrobacterium crown gall tumor-inducing (Ti) plasmid genes (such as the carmine synthase gene Nos) and plant genes (such as the soybean storage protein gene).

[0050] When constructing plant expression vectors using the genes of this invention, enhancers, including translational enhancers or transcriptional enhancers, can also be used. These enhancer regions can be upstream or adjacent regions of the ATG start codon, but must be identical to the reading frame of the coding sequence to ensure correct translation of the entire sequence. The sources of the translation control signals and start codons are wide-ranging, and can be natural or synthetic. The translation initiation region can originate from the transcription initiation region or structural genes. To facilitate the identification and screening of transgenic plant cells or plants, the plant expression vectors used can be processed, such as by adding genes that can be expressed in plants encoding enzymes or luminescent compounds that produce color changes (GUS genes, green fluorescent protein genes, etc.), antibiotic marker genes (such as the nptII gene conferring resistance to kanamycin and related antibiotics, the bar gene conferring resistance to the herbicide phosphinic acid, the hph gene conferring resistance to the antibiotic hygromycin, and the EPSPS gene conferring resistance to glyphosate), or chemical reagent resistance marker genes (such as herbicide resistance genes), and mannose-6-phosphate isomerase genes that provide the ability to metabolize mannose. From the perspective of the safety of transgenic plants, it is possible to select transformed plants directly by stress without adding any selective marker genes.

[0051] In the above applications, the vector can be a plasmid, a granule, a bacteriophage, or a viral vector. Specifically, the plasmid can be a NEWMOL vector or a PTF101.1 vector.

[0052] The recombinant vector may specifically be NEWMOL-ZmATG8c. NEWMOL-ZmATG8c is a recombinant vector obtained by replacing the DNA fragment between the BamHI and SacI recognition sequences of the NEWMOL vector with the ZmATG8c coding sequence shown in SEQ ID NO:2. NEWMOL-ZmATG8c is driven by the maize Ubquitin promoter and can express the ZmATG8c protein shown in SEQ ID NO:1.

[0053] The recombinant vector can be a recombinant vector that targets the DNA fragment and its reverse fragment shown at positions 1629-2361 bp in SEQ ID NO:3, and can inhibit ZmATG8c gene expression or disrupt the translation of ZmATG8c into the complete protein. Specifically, the recombinant vector can be pCXB053-ZmATG8c, which consists of two sgRNAs targeting the ZmATG8c genomic DNA inserted into the vector pCXB053. The targeted ZmATG8c genomic DNA sequence is located at positions 1629-2361 bp in SEQ ID NO:3, where the first 23 bp of this 733 bp sequence is target site 1, and the last 23 bp is target site 2 (targeting the antisense strand). pCXB053-ZmATG8c can be used to inhibit ZmATG8c gene expression or disrupt the translation of ZmATG8c into the complete protein.

[0054] In the above applications, the microorganisms can be bacteria or fungi. Among them, bacteria can be Escherichia coli, such as Rosetta(DE3)pLysS; fungi can be Agrobacterium or yeast, such as Agrobacterium EHA105, or yeast strain Pichia pastoris.

[0055] The present invention also provides a product for regulating plant heat tolerance, the product containing ZmATG8c or the biomaterial.

[0056] The product may use ZmATG8c or the biomaterial as its active ingredient, or it may combine ZmATG8c or the biomaterial with a substance having the same function as its active ingredient.

[0057] In this invention, the plant can be a monocotyledonous plant or a dicotyledonous plant, specifically a grass family plant, and more specifically corn, sorghum, rice and wheat.

[0058] The present invention also provides a method for improving the heat resistance of plants, the method comprising: increasing the activity and / or content of ZmATG8c in a recipient plant, or promoting the expression of the gene encoding ZmATG8c, to obtain a target plant with improved heat resistance compared to the recipient plant.

[0059] The present invention also provides a method for cultivating plants with improved heat resistance, the method comprising: increasing the activity and / or content of ZmATG8c in a recipient plant, or promoting the expression of the gene encoding ZmATG8c, to obtain a target plant with improved heat resistance compared to the recipient plant.

[0060] In this invention, the target plant can be a transgenic plant with increased expression of autophagy-related proteins compared to the recipient plant, obtained by introducing an overexpression T-DNA fragment of ZmATG8c into the recipient plant through backcross trait introduction.

[0061] The gene encoding ZmATG8c may be the nucleic acid molecule shown in SEQ ID NO:2.

[0062] Furthermore, the target plant is a transgenic plant with increased expression of the autophagy-related protein ZmATG8c compared to the recipient plant, obtained by introducing a gene overexpressing the autophagy-related protein ZmATG8c into the recipient plant.

[0063] In this invention, the recipient plant can be a monocotyledonous plant or a dicotyledonous plant, specifically a grass family plant.

[0064] In this invention, the gene encoding ZmATG8c can be modified as follows before being introduced into the recipient plant to achieve better expression:

[0065] 1) Modify and optimize according to actual needs to enable efficient gene expression; for example, the codons can be changed to conform to the plant preference while maintaining the amino acid sequence encoded by ZmATG8c described in this invention; during the optimization process, it is best to maintain a certain GC content in the optimized coding sequence to best achieve high-level expression of the introduced gene in the plant, wherein the GC content can be 35%, more than 45%, more than 50%, or more than about 60%;

[0066] 2) Modify the gene sequence adjacent to the initiation methionine to enable efficient translation initiation; for example, by using a known effective sequence in plants for modification;

[0067] 3) Linked to promoters of various plant expression to facilitate their expression in plants; the promoters may include constitutive, inducible, temporally regulated, developmentally regulated, chemically regulated, tissue-selective, and tissue-specific promoters; the selection of promoters will vary with the time and space requirements of expression, and also depends on the target species; for example, tissue or organ-specific expression promoters, depending on the stage of development of the target receptor; although it has been shown that many promoters derived from dicotyledons are functional in monocotyledons and vice versa, ideally, dicotyledonous promoters are selected for expression in dicotyledons, and monocotyledonous promoters are selected for expression in monocotyledons;

[0068] 4) Linking with a suitable transcription terminator can also improve the expression efficiency of the gene of the present invention; for example, tml from CaMV, E9 from rbcS; any known and available terminator that functions in plants can be linked with the gene of the present invention.

[0069] 5) Introduce enhancer sequences, such as intron sequences (e.g., derived from Adh1 and Bronze1) and viral leader sequences (e.g., derived from TMV, MCMV, and AMV).

[0070] The encoding gene of ZmATG8c can be introduced into recipient plants using a recombinant expression vector containing the encoding gene of ZmATG8c. Specifically, the recombinant expression vector can be NEWMOL-ZmATG8c.

[0071] The recombinant expression vector can be introduced into plant cells using conventional biotechnological methods such as Ti plasmid (T-DNA), plant virus vector, direct DNA transformation, microinjection, and electroporation.

[0072] The target plant is understood to include not only first-generation plants containing the ZmATG8c protein or its encoding gene that have been altered, but also their progeny. For the target plant, the gene can be inherited within the species, or it can be transferred into other varieties of the same species using conventional breeding techniques, particularly commercial varieties. The target plant includes seeds, callus tissue, intact plants, and cell lines.

[0073] In this invention, the heat resistance specifically refers to the plant's resistance to temperatures higher than those required for its normal growth. In one specific embodiment of this invention, the heat resistance is resistance at 42°C.

[0074] In one specific embodiment of the present invention, the plant is a seedling. Specifically, the seedling is a plant at the three-leaf-one-heart stage.

[0075] This invention also provides the application of using maize autophagy-related protein ZmATG8c via transgenic overexpression technology to cultivate a new heat-resistant line with a significantly higher survival rate than wild-type plants after heat stress.

[0076] This invention also provides the application of using maize autophagy-related protein ZmATG8c via transgenic overexpression technology to cultivate a new heat-resistant line with significantly higher plant yield than wild-type plants after heat stress.

[0077] The present invention also provides an application of introducing transgenic materials containing ZmATG8c overexpression into conventional (non-transgenic) commercial varieties or breeding materials using conventional breeding (backcross trait introduction) methods to improve their heat resistance.

[0078] Experiments have shown that the ZmATG8c gene and its encoding gene of this invention can affect the heat tolerance of plants: the survival rate of transgenic plants after heat treatment was significantly increased compared with wild-type plants after introducing this gene into wild-type plants; the survival rate of transgenic plants after heat treatment was significantly decreased compared with wild-type plants after disrupting the coding sequence of this gene. This indicates that the ZmATG8c gene and its encoded autophagy-related protein of this invention can affect the heat tolerance of plants.

[0079] The following examples are used to illustrate the present invention, but are not intended to limit the scope of the invention. Unless otherwise specified, the examples are conducted under conventional experimental conditions, such as those described in Sambrook et al., Molecular Cloning: a Laboratory Manual (Sambrook J & Russell DW, 2001), or as recommended by the manufacturer's instructions.

[0080] Example 1: The effect of the ZmATG8c gene and its encoded protein on maize heat tolerance.

[0081] This embodiment provides a gene encoding an autophagy-related protein, named ZmATG8c, derived from the maize (Zea mays) inbred lines KN5585 or B73. The genomic sequence of this gene in the maize KN5585 or B73 inbred lines is shown in SEQ ID NO:3, its CDS sequence is shown in SEQ ID NO:2, and its protein sequence is shown in SEQ ID NO:1. The ZmATG8c gene and its encoded ZmATG8c protein can affect the heat tolerance of maize. The specific detection method is as follows:

[0082] 1. Construction of the recombinant carrier:

[0083] Using cDNA from the wild-type KN5585 inbred line as a template, PCR was performed using primers C-BamHI-F: 5'-tctgcaggtcgactctagaggatccATGGCCAAGACGAGCTCGTTC-3' and C-SacI-R: 5'-gatcggggaaattcgagctcCTAGAGGAGCCCGAAGGTGTTC-3'. The PCR product with the correct sequence was ligated into the intermediate vector pCE2 TA (Nanjing Novizan, catalog number: C601-01). After obtaining the recombinant plasmid, the DNA fragment with sticky ends was obtained by restriction endonuclease digestion with BamHI (NEB, #R0136V) and SacI-HF (NEB, #R3156V). This fragment was then ligated into the vector NEWMOL, which had been treated with the same endonuclease, using T4 DNA ligase (Promega, M180B). The recombinant vector with the correct sequence obtained by ligation was named NEWMOL-ZmATG8c. NEWMOL-ZmATG8c is a recombinant vector obtained by replacing the DNA fragment between the BamHI and SacI recognition sequences of the vector NEWMOL with the ZmATG8c coding sequence shown in SEQ ID NO:2. NEWMOL-ZmATG8c can express the ZmATG8c protein shown in SEQ ID NO:1.

[0084] To generate CRISPR / Cas9-mediated ZmATG8c maize mutants, dual gRNA targets (target 1 and target 2) were selected to target specific sequences in exons 4 and 6 of ZmATG8c, respectively. The CRISPR / Cas9 vector used was pCXB053. pCXB053-ZmATG8c disrupts the coding sequence of this gene, resulting in complete or incomplete expression of the ZmATG8c protein, thereby impairing its function.

[0085] 2. Obtaining transgenic plants:

[0086] The NEWMOL-ZmATG8c and pCXB053-ZmATG8c obtained in step 1 were introduced into Agrobacterium EHA105 to obtain recombinant Agrobacterium-infected immature embryos of the maize inbred line KN5585. Specifically, approximately 1 mm immature maize embryos were peeled and placed into 2 ml centrifuge tubes containing 1.8 mL of Agrobacterium suspension. Approximately 150 immature embryos were processed within 30 minutes. The suspension was then removed, and the maize embryos settled in the tubes. 1.0 ml of Agrobacterium suspension was then added, and the tubes were allowed to stand for 5 minutes. The immature embryos in the centrifuge tubes were then resuspended and poured onto co-culture medium. Excess Agrobacterium suspension on the surface was removed with a pipette, and the tubes were co-cultured in the dark at 23°C for 3 days. After co-culture, the immature embryos were transferred to resting medium and cultured in the dark at 28°C for 6 days. Then, they were placed on selection medium containing diammonium phosphate for selection culture. Two generations of selection were performed, with each generation lasting 15 days. The resistant callus tissue was then transferred to a medium without selection agents and cultured for one generation under 16 hours of light per day. After that, it was transferred to a differentiation medium and cultured at 25°C, 5000 lx, and light for 3 weeks. The differentiated seedlings were then transferred to a rooting medium and cultured at 25°C, 5000 lx, and light until rooting. The ZmATG8c overexpression and gene-edited plants obtained from NEWMOL-ZmATG8c and pCXB053-ZmATG8c were named ZmATG8c-OE and ZmATG8c-KO, respectively. These seedlings were transferred to plug trays for growth and transplanted to the field or greenhouse when they reached about 10 cm in height for self-pollination and fruit setting.

[0087] 3. Identification of transgenic plants:

[0088] Genomic DNA from the ZmATG8c-OE plants obtained in step 2 was amplified by PCR using Bar-F and Bar-R. The PCR products were then subjected to electrophoresis. The size and presence of the resulting fragments determined whether the transgenic plants were positive. Wild-type KN5585 and an empty vector were used as controls. Primer sequences are as follows:

[0089] Bar-F: 5'-TGCACCATCGTCAACCACTACAT-3'

[0090] Bar-R: 5'-AGAAACCCACGTCATGCCAGT-3'

[0091] The results showed that the bands of the wild-type KN5585 (WT) product differed from those of the empty vector and ZmATG8c-OE plant; WT showed no band, while the bands of the empty vector and ZmATG8c-OE plant were of uniform size. The obtained ZmATG8c-OE plant was subjected to PCR amplification of its genomic DNA using primers composed of C-BamHI-F and C-SacI-R. The results showed that the sequence of the obtained PCR product contained the correct coding sequence of the ZmATG8c gene shown in SEQ ID NO:2. Figure 1 ).

[0092] Genomic DNA from the ZmATG8c-KO plants obtained in step 2 was amplified by PCR using T1-F and T2-R primers. The PCR products were then subjected to electrophoresis, and the size of the resulting fragments was used to determine whether the gene-edited plants were positive. Wild-type KN5585 was used as a control. The primer sequences are as follows:

[0093] T1-F: 5'-TGTGCAGCCTACTTGACCTG-3'

[0094] T2-R: 5'-GCACAGGTTCAGGGAAGCAA-3'

[0095] The results showed that the band sizes of the products from wild-type KN5585 (WT) and ZmATG8c-KO plants differed. The WT band was approximately 1242 bp in size, while the ZmATG8c-KO band was approximately 548 bp or 543 bp (the size of the knocked-out sequence varied among different lineages). Figure 2 The PCR amplification products of the obtained ZmATG8c-KO plants were sequenced, and the results showed that the sequence of the obtained PCR product contained the correct genomic sequence of the ZmATG8c gene shown in SEQ ID NO:3.

[0096] To understand the relationship between transgenic plants and heat tolerance, genetically stable T3 generation overexpression plants (OE) and gene-edited plants (KO) were selected, with wild-type (KN5585, WT) as a control. RNA was extracted from leaves, and the expression level of the target gene was detected by quantitative real-time PCR (qRT-PCR). In the ZmATG8c-OE material analysis, the maize ZmEF1α gene was used as an internal reference gene. In the maize ZmATG8c-OE lines (OE 1 and OE 2), the mRNA abundance of ZmATG8c was 7.78-11.54 times that of the control plants. Figure 3 ).

[0097] The expression level of ZmATG8c in ZmATG8c-KO plants was not significantly different from that in wild-type plants. Figure 4However, sequencing results showed 105bp deletion and 6bp insertion (KO 1) and 104bp deletion (KO 2) at the cDNA level, consistent with the expected genomic DNA editing results based on conventional PCR identification. Figure 2 The amino acid sequences deduced from the sequencing results indicate that both strains exhibit premature termination mutations at the N-terminus of their proteins. These results demonstrate the effectiveness of the ZmATG8c-OE and ZmATG8c-KO materials created in this study.

[0098] 4. Phenotypic identification of transgenic plants

[0099] Homozygous maize plants of different genotypes (WT, OE1, OE2, KO1, and KO2) grown for two weeks at 28℃ (light, 16h / d) / 22℃ (dark, 8h / d) were transplanted into a 4-day continuous heat treatment at 42℃ (light 16h / d, dark 8h / d) for photographic recording. They were then allowed to recover at 28℃ for 4 days to determine survival rates. Almost all KO line plants died, approximately 40% of wild-type plants survived, while the transgenic overexpression lines showed almost no mortality and maintained upright and relatively good growth. Figure 5 and Figure 6 Furthermore, after 24 hours of heat stress, KO plants exhibited a relatively higher electrolyte leakage (REL) rate compared to WT and OE plants. Figure 7 The above results indicate that ZmATG8c positively regulates the heat tolerance of maize seedlings.

[0100] Example 2: Creation of heat-resistant maize inbred lines

[0101] In this embodiment, ZmATG8-OE was introduced into a commercial maize inbred line to create a heat-resistant maize inbred line using conventional breeding methods (backcross trait introduction).

[0102] Backbone inbred lines, such as Zheng 58, Chang 7-2, PH4CV, and PH6WC, were used as recurrent parents. Using a backcross trait introduction method, the ubiquitin promoter-driven ZmATG8c overexpression lines (OE 1 and OE 2), obtained through the Agrobacterium-mediated transgenic method provided in this invention, were introduced as donor parents into the aforementioned backbone inbred lines. Backcrossing removed the genetic background influence of the donor parents. Simultaneously, the use of the molecular marker ZmATG8c-S developed in this invention for screening accelerated the backcross trait introduction process, and accurate identification reduced unnecessary workload and costs. The specific backcross trait introduction process is as follows: Figure 8 As shown.

[0103] Although the present invention has been described in detail above with general descriptions and specific embodiments, modifications or improvements can be made to it, which will be obvious to those skilled in the art. Therefore, all such modifications or improvements made without departing from the spirit of the present invention fall within the scope of protection claimed by the present invention.

Claims

1. Overexpression ZmATG8c Any of the following applications of genes: (1) Used to improve the heat resistance of plants; (2) Used to prepare transgenic plants with improved heat resistance; (3) Used for plant genetic breeding, wherein the breeding is for improving the heat resistance of plants; in, ZmATG8c The gene is a gene that encodes a protein with an amino acid sequence as shown in SEQ ID NO:1; The plant in question is corn; The improvement of plant heat resistance refers to improving the heat resistance of corn seedlings and reproductive growth stages.

2. A method for improving the heat resistance of plants, characterized in that, The method includes: increasing the activity and / or content of autophagy-related protein ZmATG8c in the recipient plant, or enhancing the expression of the gene encoding autophagy-related protein ZmATG8c, to obtain a target plant with improved heat resistance compared to the recipient plant; The amino acid sequence of the autophagy-related protein ZmATG8c is shown in SEQ ID NO:1; The plant in question is corn.

3. The method according to claim 2, characterized in that, The methods for enhancing gene expression are selected from the following 1) to 5), or any combination thereof: 1) By importing a plasmid containing the gene; 2) By increasing the copy number of the aforementioned genes on plant chromosomes; 3) By altering the promoter sequences of the aforementioned genes on plant chromosomes; 4) By operatively linking a strong promoter to the gene; 5) By introducing enhancers.

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