Use of ltg1 and its encoding gene in regulating cold resistance of plant seeds

Abstract

The present application relates to the technical fields, especially to LTG1 and the application of the coding gene thereof in regulating the cold resistance of plant seeds. The present application finds that the LTG1 protein and the coding gene thereof are related to the cold resistance of plant seeds, and the protein and the coding gene thereof can be used to cultivate low-temperature germination-resistant plants and can be used as a marker for screening low-temperature germination-resistant plants, thereby laying a foundation for the cultivation of transgenic plants.

Description

Technical Field

[0001] This invention relates to the field of technology, and more particularly to the application of LTG1 and its encoding gene in regulating the cold resistance of plant seeds. Background Technology

[0002] Seeds are the foundation of grain production. Good seed emergence (full, uniform, and robust seedlings) is a prerequisite for high and stable crop yields. However, seeds often face stress during the germination and emergence stage, resulting in problems such as slow emergence, weak seedlings, and missing seedlings. The main environmental factors affecting seed emergence are temperature, water, and oxygen. Water can be regulated through irrigation, and oxygen content can be increased by loosening the soil. However, temperature is the most difficult to control under field conditions. Improving the temperature tolerance of seeds for emergence has become the main measure to overcome the effects of temperature. Low temperatures slow down seed metabolism and make imbibition difficult, leading to slow emergence, uneven emergence, weak seedlings, or even no emergence in the field, which can cause serious yield losses. Therefore, elucidating the molecular mechanism of seed germination at low temperatures and genetically improving seedling low-temperature tolerance is of great significance for improving the field emergence rate of seeds and ensuring stable agricultural yields.

[0003] Seed vigor refers to the overall potential of seeds to germinate rapidly and uniformly and develop into normal seedlings under a wide range of field conditions. Common methods for determining seed vigor include the Hiltner test, low-temperature germination test, artificial accelerated aging test, freezing test, cold soaking test, and combined stress test. Studies have shown that the low-temperature germination test has the strongest correlation with the field emergence rate and is the most reliable method for evaluating seed vigor. Therefore, researching and improving the low-temperature emergence rate of seeds is a key aspect of improving seed quality.

[0004] Corn is an important global food, economic, feed, and renewable energy crop, and it is also my country's largest food crop. Originating in the tropics, corn is a warm-season crop, and its seed germination and emergence are particularly sensitive to low temperatures. Generally, the minimum temperature required for corn seed germination is 5-15℃. If the average daily temperature remains below 8℃ for 3-4 days after sowing, it can cause seed powdering or rotting, resulting in incomplete emergence and severe yield reduction. In recent years, the phenomenon of low corn emergence rates and reduced grain yields due to early spring low-temperature stress has shown an increasing trend. Statistics show that Northeast China experiences an average of two low-temperature years every seven years, leading to an average corn yield reduction of 15%. As the dividing line between different maturity types of corn shifts northward, the risk of low-temperature damage to corn in the Hetao region of my country is also increasing. Therefore, improving the low-temperature emergence capacity of corn seeds is of great significance for achieving high and stable grain yields in my country. Furthermore, the acquisition and application of low-temperature-tolerant corn varieties can enable earlier sowing, extend the growing season, and increase yield; as well as expand the planting range of corn (extending to higher latitude regions) and the sown area, thus providing a guarantee for national food security. In-depth analysis of the genetic mechanisms of maize seedling emergence at low temperatures and the discovery of key genes and QTLs controlling this trait are prerequisites for improving the low-temperature germination rate of maize seeds through genetic means. Summary of the Invention

[0005] In view of this, the technical problem to be solved by the present invention is to provide the application of LTG1 and its encoding gene in regulating the cold resistance of plant seeds.

[0006] This invention provides the application of at least one of the following (I) to (V) in regulating the cold resistance of plant seeds:

[0007] I) LTG1 protein;

[0008] II) Proteins that have one or more amino acids substituted, deleted, or added to the amino acid sequence of LTG1 protein and have the same or similar functions as LTG1.

[0009] The nucleic acids encoding the proteins described in III), I), or II);

[0010] IV) A nucleic acid molecule in which one or more nucleotides are substituted, deleted or added in the nucleotide sequence encoding the nucleic acid described in III) and that can encode the same or similar functional protein;

[0011] V) substances that can regulate the level or activity of at least one of I) to V).

[0012] In this invention, the plant is a dicotyledonous plant or a monocotyledonous plant. In some embodiments, the monocotyledonous plant is a grass (Poaceae). The grass includes, but is not limited to, corn, wheat, barley, oats, rye, rice, sorghum, millet, foxtail millet, or barnyard grass. In some specific embodiments, the grass is a species of the genus *Zea*. More specifically, the plant is corn.

[0013] In this invention, the LTG1 and its encoding gene are derived from dicotyledonous or monocotyledonous plants. In some embodiments, the LTG1 and its encoding gene are derived from grasses (Poaceae). In some specific embodiments, the LTG1 and its encoding gene are derived from the genus *Zea*. More specifically, the LTG1 and its encoding gene are derived from maize (*Zea mays*). In this invention, the LTG1 protein from maize is denoted as ZmLTG1, and the nucleic acid encoding ZmLTG1 is denoted as ZmLTG1.

[0014] In some embodiments, the LTG1 protein is ZmLTG1, whose amino acid sequence is shown in SEQ ID NO:1;

[0015] Alternatively, a fusion protein can be obtained by attaching a tag to the N-terminus and / or C-terminus of the protein shown in SEQ ID NO:1;

[0016] Or it may be a protein whose amino acid sequence shown in SEQ ID NO.1 has been modified by substitution and / or deletion and / or addition of one or more amino acid residues and is related to the cold resistance of plant seeds;

[0017] It may be a protein derived from corn that has more than 90% identity with SEQ ID NO.1 and is associated with cold resistance in plant seeds.

[0018] In some embodiments, the gene encoding LTG1 is ZmLTG1, which encodes ZmLTG1. The present invention does not limit the nucleic acid sequence of ZmLTG1.

[0019] In some specific embodiments, the nucleic acid sequence of ZmLTG1 is shown in SEQ ID NO:2;

[0020] Alternatively, a nucleic acid obtained by attaching a tag to the 3' end and / or 5' end of the nucleic acid shown in SEQ ID NO:2;

[0021] Or it may be a nucleic acid obtained by substituting and / or deleting and / or adding one or more nucleotides to the nucleic acid sequence shown in SEQ ID NO.2;

[0022] It may be a nucleic acid that has more than 90% identity with SEQ ID NO.1 and encodes the same functional protein;

[0023] Or it could be a nucleic acid that can hybridize with the nucleic acid sequence described above.

[0024] This invention demonstrates that LTG1 inactivation leads to a decrease in the cold resistance of plant seeds, while overexpression of LTG1 can improve the cold resistance of plant seeds. Therefore, the cold resistance of plants can be regulated by controlling LTG1. In this invention, regulating the cold resistance of plant seeds includes regulating the low-temperature germination ability of plant seeds. In this invention, the low-temperature germination ability includes: increasing the seed germination rate, increasing the dry weight of the seed radicle, increasing the germination index, and / or shortening the average germination time under low-temperature conditions. In this invention, the low-temperature condition is ≥0℃, preferably ≥5℃. In some embodiments, the low-temperature condition is 10℃~18℃, more specifically, it is 10℃ or 18℃.

[0025] Furthermore, the present invention provides an agent for improving the cold resistance of plant seeds, comprising at least one of the following a) to f):

[0026] a) The LTG1 protein or a nucleic acid molecule encoding the LTG1 protein;

[0027] b) Expression vectors containing nucleic acids encoding the LTG1 protein;

[0028] c) Hosts containing b) or hosts whose genomes integrate the coding nucleic acid of the LTG1 protein;

[0029] d) The promoter or enhancer of the LTG1 gene;

[0030] e) Inducers that promote LTG1 gene expression;

[0031] f) Substances that enhance the activity of LTG1 protein.

[0032] The formulation provided by this invention includes substances that overexpress LTG1 protein or its encoded nucleic acid, as well as substances that enhance the activity of LTG1 protein. This invention does not limit the source of the LTG1 protein or the nucleic acid encoding LTG1 protein. For example, it can be derived from any species of grass. In this invention, LTG1 protein derived from maize is used as an example to verify the regulation of cold resistance.

[0033] In this invention, the host is preferably Agrobacterium. An expression vector containing nucleic acid encoding the LTG1 protein is transformed into a plant explant using an Agrobacterium-mediated transgenic method. The plant explant includes an immature embryo.

[0034] In this invention, the promoter or enhancer is derived from maize or other plants. This invention does not limit the source of the promoter or enhancer. Any substance that can increase the expression level of the LTG1 gene or enhance its activity is within the scope of protection of this invention.

[0035] The inducer or substance that enhances the activity of LTG1 protein described in this invention can be derived from the plant itself or given exogenously; this invention does not limit the source of the inducer.

[0036] Furthermore, the present invention also provides a method for improving the cold resistance of plant seeds, which includes increasing the level and / or activity of endogenous LTG1 protein in plants, or causing plants that do not contain LTG1 or have LTG1 inactivated to express LTG1 protein.

[0037] The method described in this invention can be implemented through genetic engineering, or through hybridization breeding, mutation breeding, polyploid breeding, haploid breeding, cell engineering breeding, etc., and this invention does not limit the specific methods used. In this embodiment of the invention, the level of endogenous LTG1 protein in plants is increased through genetic engineering to enhance the cold resistance of plant seeds.

[0038] Furthermore, the present invention also provides an SNP molecular marker for identifying the cold resistance of plant seeds, which is a single base variation at position 2493 in the LTG1 encoding gene.

[0039] Experiments showed that the LTG1 protein coding region of maize 220 has a C base at position 2493, and its LTG1 gene promoter region has a 1839 bp insertion sequence, indicating high cold resistance and the ability of seeds to germinate at low temperatures. The LTG1 protein coding region of maize PH4CV has a T base at position 2493, and its LTG1 gene promoter region has a 2107 bp insertion sequence, indicating low temperature sensitivity and low germination rate of seeds under low temperature conditions.

[0040] Furthermore, the present invention also provides an InDel molecular marker for identifying cold resistance in plant seeds, used to identify sequence variations involving transposon insertions in the LTG1 gene promoter.

[0041] The experiments of this invention show that by identifying the haplotype of the promoter of the ZmLTG1 gene in different maize inbred lines, it is possible to determine whether the germplasm has cold resistance.

[0042] Furthermore, the present invention also provides preparations for identifying the cold resistance of plant seeds, comprising: preparations for detecting the transcriptional level of the LTG1 gene; and / or preparations for detecting the expression level or activity of the LTG1 protein.

[0043] In some embodiments, the agent for identifying cold resistance includes a primer-probe combination for detecting the aforementioned SNP molecular markers. This invention does not limit the primers or probes used for detection; any primer or probe capable of targeting the aforementioned SNP molecular markers is acceptable. As an example, in an embodiment of this invention, the nucleic acid sequence of the upstream primer is shown in SEQ ID NO:3; the nucleic acid sequence of the downstream primer is shown in SEQ ID NO:4; and the nucleic acid sequence of the probe is shown in SEQ ID NO:5.

[0044] In other embodiments, the agent for identifying cold resistance includes a primer set for detecting the InDel molecular markers as described above, comprising two upstream primers and two downstream primers. This invention does not limit the primers or probes used for detection; any primer capable of targeting the aforementioned InDel molecular markers is acceptable. As an example, in embodiments of this invention, the nucleic acid sequences of the two upstream primers are shown in SEQ ID NO:6 or 8, respectively; and the nucleic acid sequences of the two downstream primers are shown in SEQ ID NO:7 or 9, respectively.

[0045] Furthermore, the present invention also provides a method for identifying the cold resistance of plant seeds, which uses the preparations described above to identify the germplasm.

[0046] The germplasm includes the seeds, seed coats, plumules, hypocotyls, radicles, cotyledons, leaves, roots, or stems of plants.

[0047] In some embodiments, the identification includes detection of SNP sites as described above. This invention does not limit the interpretation of the results in this scheme; PCR bands can be observed, with the presence or absence of bands as the criterion, or the amplification products can be sequenced, with the genotype of the SNP site as the criterion. In some specific embodiments, the identification includes detection of the sample using the primers shown in SEQ ID NO:3-4 and the probe shown in SEQ ID NO:5. The detection includes amplification of the sample DNA. In this embodiment, the criteria for identification include:

[0048] If the SNP site is C, the seed has strong cold resistance, and haplotype 220 is preferred.

[0049] In other embodiments, the identification includes detection of the InDel molecular marker as described above. In some specific embodiments, primers as described above (SEQ ID NO: 6-9) are used to detect the sample. The detection includes amplification of the sample DNA. In this embodiment, the criteria for identification include:

[0050] SEQ ID NO:7 and SEQ ID NO:8 are used as primers for amplification. If an amplification product is present, the sample is from germplasm that can germinate at low temperatures, preferably haplotype 220.

[0051] Using SEQ ID NO:6 and SEQ ID NO:9 as primers for amplification, the presence of amplification products indicates that the sample is derived from low-temperature sensitive germplasm, preferably the PH4CV haplotype. This invention has discovered that the LTG1 protein and its encoding gene are related to the cold resistance of plant seeds. This protein and its encoding gene can be used to cultivate plants that germinate under low temperatures, and can also be used as markers for screening plants that germinate under low temperatures, laying the foundation for the cultivation of transgenic plants. Attached Figure Description

[0052] Figure 1 Germination phenology of parents 220 and PH4CV used to construct the genetic population under different temperature conditions;

[0053] Figure 2 To utilize genetic populations for fine mapping and cloning of the ZmLTG1 gene;

[0054] Figure 3 Genotyping of Mu mutants and overexpressing transgenic maize;

[0055] Figure 4 To verify the function of the ZmLTG1 gene using Mu mutant maize and wild-type maize;

[0056] Figure 5 To verify the function of the ZmLTG1 gene using transgenic and non-transgenic maize overexpression;

[0057] Figure 6 To investigate the causes of functional variations in the ZmLTG1 gene;

[0058] Figure 7 This is an example of using InDel molecular markers to detect the promoter haplotype of the maize inbred line ZmLTG1 gene. Detailed Implementation

[0059] This invention provides the application of LTG1 and its encoding gene in regulating the cold resistance of plant seeds. Those skilled in the art can refer to this document and appropriately modify the process parameters to achieve the desired results. It is particularly important to note that all similar substitutions and modifications are obvious to those skilled in the art and are considered to be included in this invention. The methods and applications of this invention have been described through preferred embodiments. Those skilled in the art can clearly modify or appropriately change and combine the methods and applications described herein without departing from the content, spirit, and scope of this invention to realize and apply the technology of this invention.

[0060] Unless otherwise defined in this invention, the scientific and technical terms associated with this invention shall have the meanings understood by one of ordinary skill in the art.

[0061] In this invention, the terms "comprising," "including," and "having" are used interchangeably to indicate the inclusiveness of a solution, meaning that the solution may contain elements other than those listed. It should also be understood that the use of "comprising," "including," and "having" herein also provides for solutions "consisting of...".

[0062] In this invention, "and / or" as used herein includes the meaning of "and", "or", and "all or any other combination of elements linked by the term".

[0063] In this invention, "at least one" means one or more, and "more than one" means two or more. "At least one of the following" or similar expressions refer to any combination of these items, including any combination of a single item or a plurality of items.

[0064] It should be understood that in the various embodiments of this application, the order of the above processes does not imply the order of execution. Some or all steps may be executed in parallel or sequentially. The execution order of each process should be determined by its function and internal logic, and should not constitute any limitation on the implementation process of the embodiments of this application.

[0065] In this invention, the “LTG1” protein is derived from maize (Zea mays) and can improve the germination ability of maize seeds at low temperatures. The protein ZmLTG1 that improves the germination ability of maize seeds at low temperatures, as described in this invention, specifically includes (1) or (2) or (3) or (4): a protein consisting of the amino acid sequence shown in SEQ ID NO.1 of the sequence listing; a fusion protein obtained by attaching a tag to the N-terminus and / or C-terminus of the protein described in (1); a protein derived from (1) by substituting and / or deleting and / or adding one or more amino acid residues of the amino acid sequence of SEQ ID NO.1 of the sequence listing, and which is related to plant low-temperature germination. It is a protein derived from maize that has more than 90% similarity to (1) and is related to plant low-temperature germination.

[0066] (SEQ ID NO:1)

[0067] In this invention, "nucleic acid" includes any compound and / or substance comprising a polymer of nucleotides. Each nucleotide consists of a base, particularly a purine or pyrimidine base (i.e., cytosine (C), guanine (G), adenine (A), thymine (T), or uracil (U)), a sugar (i.e., deoxyribose or ribose), and a phosphate group. Typically, nucleic acid molecules are described by the sequence of bases, whereby the bases represent the primary structure (linear structure) of the nucleic acid molecule. The sequence of bases is typically represented as 5′ to 3′. In this document, the nucleic acid can be DNA, such as cDNA, genomic DNA, or recombinant DNA; or it can be RNA, such as mRNA, hnRNA, or tRNA, etc. Nucleic acid molecules can be linear or circular. Furthermore, the term nucleic acid molecule includes both sense and antisense strands, as well as single-stranded and double-stranded forms. Moreover, the nucleic acid molecules described herein may contain naturally occurring or non-naturally occurring nucleotides. Examples of non-naturally occurring nucleotides include modified nucleotide bases having derived sugar or phosphate backbones bonded or chemically modified residues. Nucleic acid molecules also encompass DNA and RNA molecules, which are suitable as vectors for the direct expression of the proteins of the present invention in vitro and / or in vivo, such as in a host. In the present invention, the nucleic acid encoding ZmLTG1 has one of the following nucleotide sequences: (1) the nucleotide sequence described in SEQ ID No. 2 of the sequence listing; (2) a polynucleotide sequence encoding the protein sequence of SEQ ID No. 1 of the sequence listing; (3) a DNA molecule derived from maize and having more than 90% identity with (1) or (2) and encoding the protein; (4) a DNA molecule that hybridizes to the nucleotide sequence defined in (1) or (2) or (3) and encoding the protein.

[0068] The hybridization conditions included hybridization in a solution of 2×SSC and 0.1% SDS at 68°C, followed by washing the membrane twice for 5 min each time, and then hybridization in a solution of 0.5×SSC and 0.1% SDS at 68°C, followed by washing the membrane twice for 15 min each time.

[0069]

[0070] In this invention, "tag" or "expression tag" refers to a specific label used to mark and identify the expression product during gene expression. These tags can be specific amino acid sequences or protein domains, and they can be associated with post-translational modifications, localization, transport, and other processes of the expression product. As an example, the tags and their sequences are shown in Table 1:

[0071] Table 1: Label Sequence

[0072] Poly-Arg 5-6 (usually 5) RRRRR Poly-His 2-10 (usually 6) HHHHHH FLAG 8 DYKDDDDK C-MYC 10 EQKLISEEDI HA 9 YPYDVPDYA GFP 36 MVSKGEELFTGVVPILLEFVTAAGITHGMDELYRS*

[0073] In this invention, "vector" refers to a nucleic acid molecule capable of amplifying another nucleic acid linked to it. This term includes vectors as self-replicating nucleic acid structures and vectors integrated into the genome of a host cell into which the vector has been introduced. Some vectors are capable of directing the expression of nucleic acids operatively linked to them. Such vectors are referred to herein as "expression vectors" or "recombinant vectors," wherein the recombinant vector is a recombinant expression vector or a recombinant cloning vector.

[0074] In this invention, the recombinant expression vector can be constructed using existing expression vectors. The expression vector may also contain the 3' untranslated region of the exogenous gene, i.e., containing a polyadenylated signal and any other DNA fragment involved in mRNA processing or gene expression. The polyadenylated signal can guide the addition of polyadenylated nucleotides to the 3' end of the mRNA precursor. When constructing the recombinant expression vector using the gene, any type of enhancing, constitutive, tissue-specific, or inducible promoter can be added before its transcription initiation nucleotide; these can be used alone or in combination with other promoters. Furthermore, when constructing the recombinant expression vector using the gene of this invention, enhancers, including translational enhancers or transcriptional enhancers, can also be used. These enhancer regions can be ATG start codons or adjacent region start codons, etc., but must be identical to the reading frame of the coding sequence to ensure correct translation of the entire sequence. 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 express enzymes or luminescent compounds that produce color changes (GUS gene, GFP gene, luciferase gene, etc.), antibiotic resistance markers (gentamicin markers, kanamycin markers, etc.), or chemical reagent resistance marker genes (such as herbicide resistance genes). Considering the safety of transgenic plants, no selective marker genes may be added, and transformed plants can be directly selected by stress screening. In a specific embodiment, the recombinant expression vector is a plasmid obtained by inserting a DNA fragment with the nucleic acid sequence shown in SEQ ID No. 2 into the multiple cloning site of the vector pCAMBIA3301.

[0075] In this invention, an "expression cassette" refers to a specific region of the genome that contains a complete gene and its regulatory elements. Expression cassettes can autonomously regulate gene expression, thereby achieving fine-grained control over gene expression. The expression cassette described in this invention includes, as previously mentioned, the nucleic acid encoding ZmLTG1, and at least one of the following elements: promoter, enhancer, transcription factor binding site, etc.

[0076] In this invention, "host cell" refers to a cell in which exogenous nucleic acids have been introduced, including the progeny of such cells. Host cells include "transformers" and "transformed cells," which include primary transformed cells and their progeny, regardless of the number of passages. Progeny cells may not be completely identical to parental cells in their nucleic acid contents and may contain mutations. This includes mutant progeny cells with the same function or biological activity as those screened or selected in the initially transformed cells.

[0077] In this invention, "similarity" can be calculated as follows: To determine the percentage of "similarity" between two amino acid sequences or two nucleic acid sequences, the sequences are aligned for optimal comparison purposes (e.g., vacancies may be introduced in one or both of the first and second amino acid sequences or nucleic acid sequences for optimal alignment, or non-homologous sequences may be discarded for comparison purposes). Then, amino acid residues or nucleotides at corresponding amino acid or nucleotide positions are compared. When a position in the first sequence is occupied by the same amino acid residue or nucleotide at the corresponding position in the second sequence, the molecules are identical at that position. In this invention, the similarity is preferably 95% or more; more specifically, 96% or more; more specifically, 97% or more; more specifically, 98% or more; more specifically, 99% or more.

[0078] This invention provides the application of the protein, the coding gene, or the recombinant vector, expression cassette, transgenic cell line, or host bacterium as described above in enhancing the low-temperature germination ability of plant seeds.

[0079] This invention also provides a method for cultivating transgenic plants, which involves introducing the encoding gene as described above, or the recombinant vector, expression cassette, transgenic cell line, or host bacterium as described above, into a target plant to obtain a transgenic plant; compared with the target plant, the transgenic plant has enhanced seed germination ability at low temperatures.

[0080] In this invention, "SNP" refers to single nucleotide polymorphism, a base change caused by variation at a single nucleotide site, which can serve as a genetic marker. This invention provides a KASP marker for identifying the SNP encoding gene of ZmLTG1, namely, a competitive allele-specific polymerase chain reaction (KASP). KASP is a novel SNP-based genotyping technique that utilizes fluorescence detection after PCR amplification for genotyping. The primers are as follows:

[0081] SNP30-335M-VIC-F GAAGGTCGGAGTCAACGGATTCCCACCATGACTGTGGTGTCT SNP30-R GGGCTGGCCCGCCTCCAT

[0082] In this invention, "InDel molecular markers" refer to insertion / deletion markers, which are genetic variation markers based on the insertion or deletion of nucleotide fragments of different sizes in the genome. This invention provides InDel molecular markers for identifying the promoter of the ZmLTG1 encoding gene, as follows:

[0083] IDP30-R CAACGGAGGATCCCATCTCATAG 220-F GCGATCTAATACAGCAAAACACC PH4CV-R ATGCCGCAAACTTATTTCGCA

[0084] Unless otherwise specified, the experimental methods used in the following examples are conventional methods. Unless otherwise specified, the materials and reagents used in the following examples are commercially available. The low-temperature resistant germination material 220 and the low-temperature sensitive material PH4CV used in the following examples are described in the literature LiX, Wang G, Fu J, Li L, Jia G, Ren L, Lubberstedt T, Wang G, Wang J and GuR (2018) QTL Mapping in Three Connected Populations Reveals a Set of Consensus Genomic Regions for Low Temperature Germination Ability in Zea mays L. Front. Plant Sci. 9:65. These materials are available to the public from China Agricultural University.

[0085] The inbred line CAL used for genetic transformation in the following examples is described in the literature DuX, Fang T, Liu Y, Huang L, Zang M, Wang G, Liu Y and Fu J (2019) Transcriptome Profiling Predicts New Genes to Promote Maize Callus Formation and Transformation. Front. Plant Sci. 10:1633. This material is available to the public from China Agricultural University.

[0086] The pCAMBIA3301 vector (bivalent expression vector pCAMBIA3301) is described in the following literature: Liu, Y., Zhang, Y., Liu, Y. et al. Metabolic effects of glyphosate on transgenic maize expressing aG2-EPSPS gene from Pseudomonas fluorescens. J. Plant Biochem. Biotechnol. 24, 233–241 (2015).

[0087] The test materials used in this invention are all common commercially available products. The invention is further illustrated below with reference to embodiments:

[0088] Example 1

[0089] Germination phenotypic observations of the parents 220 and PH4CV used to construct the genetic population at room temperature and low temperature are shown in the figure. Figure 1

[0090] This invention selects the low-temperature tolerant maize inbred line 220 and the low-temperature sensitive inbred line PH4CV (also known as 335M) for population construction. 220 was collected from Northeast China and has the genetic background of Dan 337. It has a loose plant type, many female ears, flint kernels, small kernels, and a relatively high embryo-to-kernel ratio, resulting in faster germination at low temperatures. Inbred line PH4CV is the male parent of the excellent maize single-cross hybrid Xianyu 335. It has a shorter plant height than 220, a compact plant type, semi-dent kernels, and larger kernels, but a slower germination rate at low temperatures. The experimental method used was sand culture. First, cleaned fine sand was thoroughly dried and sterilized at 130℃. Then, deionized water was added and thoroughly mixed to achieve a sand moisture content of 16%. A 3cm layer of moist sand was then placed at the bottom of the germination box. Forty seeds with good appearance were selected as one replicate, and at least three biological replicates were set up for each inbred line. Disinfect seeds with 1% NaClO for 10 minutes, rinse thoroughly with deionized water, sow seeds sequentially with the embryo side facing upwards, and seal with a resealable bag to prevent rapid moisture loss. Incubate in the dark at 25℃, 18℃, and 10℃. After 32 hours, 36 hours, and 7 days of incubation, count the number of germinating seeds daily until the germination rate stabilizes. Finally, weigh the seedling roots (RFW). The formulas for calculating germination rate (GR), germination index (GI), and mean germination time (MGT) are as follows:

[0091]

[0092]

[0093]

[0094] Where, N i N represents the number of germinating seeds at each time point, N represents the number of seeds sown in each repetition, and T represents the number of seeds sown at each time point. i The time of germination.

[0095] from Figure 1 It can be seen that:

[0096] At 25℃, the final average germination rate of 220 was 100%, the germination index was 1.115, and the fresh weight of seedling roots was 3.729 grams, while the final average germination rate of PH4CV was 96.7%, the germination index was 0.929, and the fresh weight of seedling roots was 3.121 grams. At 18℃, the final average germination rate of 220 was 100%, the germination index was 0.914, and the fresh weight of seedling roots was 3.026 grams, while the final average germination rate of PH4CV was 95.8%, the germination index was 0.621, and the fresh weight of seedling roots was 1.965 grams. At 10℃, the final average germination rate of 220 was 100%, the germination index was 0.289, and the fresh weight of seedling roots was 1.318 grams, while the final average germination rate of PH4CV was 94.2%, the germination index was 0.227, and the fresh weight of seedling roots was 0.967 grams. Figure 1 Regardless of whether the germination rate, germination index, and seedling root fresh weight time of 220 were better than those of PH4CV, it indicates that the germination phenotype of 220 is superior to that of PH4CV.

[0097] Example 2: Discovery of ZmLTG1 protein and its encoding gene

[0098] Genetic populations were constructed using the cold-tolerant maize inbred line 220 and the cold-sensitive inbred line PH4CV (also known as 335M). Initial mapping yielded a QTL named qCTG1, which was mapped to the space between Umc1754 and Indel8 on maize chromosome 1. Then, using the flanking markers Umc1754 and Indel8 of qCTG1, 151 exchange plants were selected from 14,400 BC2F2 plants. These exchange plants were self-pollinated, and their offspring seeds (BC2F2:3) were used for low-temperature germination phenotype identification. Simultaneously, six new DNA markers were identified within the qCTG1 region, which could classify the 151 materials into 12 exchange types. Based on the low-temperature phenotype of the exchange plants, the qCTG1 region was narrowed down to a range of 908 kb, between markers SSR1 and Indel15.

[0099] Next, using approximately 8,000 BC3F2 monoplants, we screened and obtained 18 monoplants that exchanged within the interval Umc1754-Indel15. After self-pollination, we used the seeds of their offspring to identify the low-temperature germination phenotype. Simultaneously, we continued screening for polymorphic markers within the Umc1754-Indel15 interval, obtaining 8 new markers. These markers allowed us to classify the exchanged monoplants into 6 types. Based on genotype and phenotype, we narrowed the interval down to approximately 107 kb, containing 3 genes (results are shown below). Figure 2 (As shown).

[0100] Based on the maize B73 reference genome and functional annotations, ORF1 is an unannotated gene; ZmLTG1 is a gene encoding a plant lectin receptor protein kinase; only a portion of ORF2 is located within its mapping region, and it encodes a serine / threonine protein kinase with unknown function. Literature review indicates that lectin proteins play a crucial role throughout the plant life cycle, acting as vital components in cell-cell communication, plant development, and defense responses. Recent studies have shown that lectin receptor protein kinases have significant functions in responding to stress signals, resisting pests and diseases, and regulating plant development. Therefore, ZmLTG1 has been preliminarily identified as a candidate gene.

[0101] Example 3 verifies the gene function of ZmLTG1

[0102] 1. Verification using Mu transposon insertion mutants

[0103] (1) Mu transposon insertion mutant obtained

[0104] First, Mu insertion mutants of ZmLTG1 were obtained from the US maize UniformMu mutant library and the Chinese maize ChinaMu mutant library, respectively. The insertion sites of the Mu transposons were chr1:182807744 and chr1:182807937, with genetic backgrounds of W22 and B73, respectively. Backcrossing into W22 and B73 cells purified the genetic backgrounds, yielding BC1F1 generation seeds. Then, the BC1F1 seeds were self-pollinated to obtain homozygous mutants and wild-type BC1F2 generation seeds, named zmltg1-1 and zmltg1-2, respectively. Further self-pollination of the BC1F2 generation seeds expanded the population to obtain BC1F3 seeds. Germination experiments were conducted using BC1F3 seeds. The mutants were identified using PCR. The Mu insertion sites and PCR primers are as follows: Figure 3 As shown in Figure A, the identification results are as follows: Figure 3 As shown in C.

[0105] PCR identification method: Take plant leaves and extract genomic DNA. For the zmltg1-1 mutant with W22 background, PCR amplification was performed using three sets of primers: F1+R1, F1+Mu1, and R1+Mu1. If no amplification product was found with F1+R1, but amplification products were found with both F1+Mu1 and R1+Mu1, the plant was a homozygous Mu insertion mutant. If amplification products were found with F1+R1, and amplification products were found with both F1+Mu1 and R1+Mu1, the plant was a heterozygous Mu insertion mutant. If amplification products were found with F1+R1, but no amplification products were found with either F1+Mu1 or R1+Mu1, the plant was a homozygous plant without Mu insertion. For the zmltg1-2 mutant with a B73 background, PCR amplification was performed using three primer sets: F2+R2, F2+Mu2, and R2+Mu2. If no amplification product was found with F2+R2, but amplification products were found with both F2+Mu2 and R2+Mu2, the plant was a homozygous Mu insertion mutant. If amplification products were found with F2+R2, and amplification products were found with both F2+Mu2 and R2+Mu2, the plant was a heterozygous Mu insertion mutant. If amplification products were found with F2+R2, but no amplification products were found with either F2+Mu2 or R2+Mu2, the plant was a homozygous plant without a Mu insertion. The primers are as follows:

[0106] F1:TGTGAGGACAAGGCGGTAAG

[0107] R1:GAACCCGTCGTAGCGCTG

[0108] Mu1:AGAGAAGCCAACGCCAWCGCCTCYATTTCGTC

[0109] F2:GAGGACCAGAGCGTGGTGTTTC

[0110] R2:AGTACCTCTACCCGACCCTGAATC

[0111] Mu2:GAAGCCAACGCCAWCGCCTCYATTTCGTCGAAT

[0112] (2) Identification of the low-temperature germination ability of Mu transposon inserted mutant seeds

[0113] Germination experiments were conducted using homozygous Mu insertion mutants and wild-type seeds under different temperature treatments, following the same sand culture method as described in Example 1. The results are as follows: Figure 4 As shown, the details are as follows:

[0114] At 25℃, the final average germination rate of WT (W22 background) was 95.7%, the fresh weight of seedling roots was 0.894g, and the germination index was 0.675. The final average germination rate of zmltg1-1 was 90.9%, the fresh weight of seedling roots was 0.592g, and the germination index was 0.595. There were significant differences in seedling root fresh weight and germination index between WT and zmltg1-1, but no significant difference in final germination rate. The final average germination rate of WT (B73 background) was 97.1%, the fresh weight of seedling roots was 0.86g, and the germination index was 0.721. The final average germination rate of zmltg1-2 was 80.8%, the fresh weight of seedling roots was 0.588g, and the germination index was 0.564. There were significant differences in final germination rate, seedling root fresh weight, and germination index between WT and zmltg1-2. Figure 4 ).

[0115] At 18℃, the final average germination rate of WT (W22 background) was 89.8%, the fresh weight of seedling roots was 0.558g, and the germination index was 0.385. The final average germination rate of zmltg1-1 was 79.8%, the fresh weight of seedling roots was 0.448g, and the germination index was 0.314. The germination index of WT was significantly different from that of zmltg1-1, but the final germination rate and fresh weight of seedling roots were not significantly different. The final average germination rate of WT (B73 background) was 82.1%, the fresh weight of seedling roots was 0.493g, and the germination index was 0.35. The final average germination rate of zmltg1-2 was 57.9%, the fresh weight of seedling roots was 0.314g, and the germination index was 0.242. The final germination rate, fresh weight of seedling roots, and germination index of WT were significantly different from those of zmltg1-2. Figure 4 ).

[0116] At 10℃, the final average germination rate of WT (W22 background) was 88.9%, the fresh weight of seedling roots was 0.378g, and the germination index was 0.134. The final average germination rate of zmltg1-1 was 67%, the fresh weight of seedling roots was 0.223g, and the germination index was 0.094. The final germination rate, fresh weight of seedling roots, and germination index of WT were significantly different from those of zmltg1-1. The final average germination rate of WT (B73 background) was 86.2%, the fresh weight of seedling roots was 0.521g, and the germination index was 0.143. The final average germination rate of zmltg1-2 was 53.1%, the fresh weight of seedling roots was 0.292g, and the germination index was 0.081. The final germination rate, fresh weight of seedling roots, and germination index of WT were significantly different from those of zmltg1-2. Figure 4 ).

[0117] Overall, regardless of whether the temperature was normal (25℃), 18℃, or low (10℃), the final germination rate, seedling root fresh weight, and germination index of WT were superior to those of zmltg1-1 and zmltg1-2. The germination phenotype of WT was also superior to those of zmltg1-1 and zmltg1-2. These results indicate that the candidate gene ZmLTG1 can influence seed germination.

[0118] 2. Verify the function of ZmLTG1 using overexpressed transgenic materials.

[0119] (1) Cloning of the ZmLTG1 gene

[0120] First, total RNA was extracted. Seeds germinated at room temperature for 3 days were used, endosperm and seed coat removed, and then ground in liquid nitrogen. Total RNA was extracted using the RNAprep Pure Polysaccharide-Polyphenol Plant Total RNA Extraction Kit (DP441) from Tiangen Biotech. Then, whole-genome cDNA was obtained by reverse mixing using the Genestar StarScript II RT Mix with gDNA Remover StarScript II kit. Next, the ZmLTG1 gene was amplified from the whole-genome cDNA using LTG1-F and LTG1-R high-fidelity enzymes. Sequencing showed that the nucleic acid fragment amplified by the primer pairs had the nucleic acid sequence shown in SEQ ID NO.2 of the sequence listing, which is the coding region nucleic acid sequence of the ZmLTG1 gene. The primer sequences are as follows:

[0121] LTG1-F:GGACTCTTGACCATG ATGTCCATGGCGTCGCCTGTCCTC

[0122] LTG1-R:ATTCGAGCTGGTCAC TCATCTGGGTCCCGAGAGCTGCTG

[0123] (2) Construction of expression carrier

[0124] First, the pCAMBIA3301 vector was double-digested with NcoI and BstPI restriction endonucleases, and the digested products were purified using the Tiangen Common DNA Product Purification Kit (DP204); at the same time, the ZmLTG1 gene amplified above (1) was purified; then, the 2X MultiF Seamless Assembly from ABclonal was used. The Mix recombinase was used to recombine the vector and the ZmLTG1 gene, and the reaction was carried out at 50°C for 30 minutes. The recombinant product was then added to DH5α competent cells and incubated on ice for 30 minutes. After heat shock at 42°C for 45 seconds, the cells were immediately cooled on ice for 2-3 minutes. 900 μL of antibiotic-free LB medium was added, and the cells were incubated at 37°C for 1 hour (200-250 rpm). Kanamycin-resistant LB plates were preheated at 37°C. After centrifugation at 5,000 rpm for 5 minutes, 900 μL of supernatant was discarded, and the cells were resuspended. The resuspended cells were then lightly spread evenly on plates containing the corresponding antibiotic using a sterile spreader. The cells were incubated upside down at 37°C for 12-16 hours. Single clones were picked and colony PCR was performed using primers 35S-F and LTG1-R2 to identify positive clones. Sequencing of positive clones showed that the inserted vector sequence matched the nucleic acid sequence shown in SEQ ID No. 2 of the sequence listing. The recombinant plasmid was named pCAMBIA3301-35S-ZmLTG1. Figure 3 (As shown in Figure B). The 35S-F and LTG1-R2 primers are as follows:

[0125] 35S-F:CTGCCGACAGTGGTCCCAAA;

[0126] LTG1-R2:GACGATGGAGTTGAACATGGTTGAG

[0127] (3) Obtaining transgenic plants

[0128] The pCAMBIA3301-35S-ZmLTG1 vector was transformed into Agrobacterium strain EHA105. Immature embryos from the maize inbred line CAL were transformed using the Agrobacterium-mediated transformation method (Chenet et al., 2018). After the callus tissue grew into seedlings, DNA was extracted from samples and identified by PCR using primers 35S-F and LTG1-R2. Using CAL as a negative control, the amplified products were detected by electrophoresis on an agarose gel. The results showed that no corresponding DNA fragment was detected in the untransfected maize material CAL negative control group. Three of the ten selected experimental group plants showed a band of approximately 540 bp; these plants with amplified products were transgenic positive plants, named OE1, OE2, and OE3, respectively. The remaining plants without detected amplified products were transgenic negative plants (NT). This indicates that the ZmLTG1 gene has been integrated into the genome of the maize material CAL in the above experimental group. Figure 3 (D).

[0129] The T0 generation transgenic plants that tested positive were self-pollinated to obtain T1 generation seeds (T1 generation seeds were not obtained from OE2). After sowing, the T1 generation seeds were further genotyped using primers 35S-F and LTG1-R2. The results showed that the T1 generation plants of both OE1 and OE3 exhibited genotype segregation, being OE1 and NT1, and OE3 and NT3, respectively. The T1 generation plants were then self-pollinated to obtain T2 generation seeds.

[0130] Then, the expression levels of OE1 and NT1, and OE3 and NT3 were analyzed using RT-qPCR technology. First, total RNA was extracted. Seeds germinated at low temperature for 10 days were used, endosperm and seed coat removed, and then ground in liquid nitrogen. Total RNA was extracted using the RNAprep Pure Polysaccharide and Polyphenol Plant Total RNA Extraction Kit (DP441) from Tiangen Biotech. Then, whole-genome cDNA was obtained by reverse PCR using the Genestar StarScript II RT Mix with gDNA Remover StarScript II kit. Finally, expression levels were detected using the 2×HQ SYBR qPCR Mix (Low ROX) from Zhuangmeng International. The instrument used was ABI QuantStudio. TM 6. Real-time PCR instrument, primers used were ZmLTG1-qPCR-F and ZmLTG1-qPCR-R, internal reference gene was GAPDH, and 2^(- ΔΔ Expression levels were calculated using the CT method. Results showed that the expression level of ZmLTG1 in OE1 was significantly higher than that in NT1, while the expression level of ZmLTG1 in OE3 was not significantly different from that in NT3. Figure 3 The mean ± SEM of three biological replicates was calculated.

[0131] ZmLTG1-qPCR-F:TGCCTTCCTCACCTACTGGT

[0132] ZmLTG1-qPCR-R:GAGGAGCCTGTAAACGGTGT

[0133] GAPDH-F:CCATCACTGCCACACAGAAAAC

[0134] GAPDH-R:AGGAACACGGAAGGACATACCAG

[0135] (4) Identification of the low-temperature germination ability of transgenic plant seeds

[0136] Germination experiments were conducted using the obtained T2 generation transgenic seeds under different temperature treatments, following the same sand culture method as shown in Example 1. The results are as follows: Figure 4 As shown, the details are as follows:

[0137] At 25℃, the final average germination rate of OE1 was 99.3%, the fresh weight of seedling roots was 0.806g, and the germination index was 0.85. The final average germination rate of NT1 was 90.7%, the fresh weight of seedling roots was 0.551g, and the germination index was 0.685. Significant differences were found between OE1 and NT1 in terms of final germination rate, seedling root fresh weight, and germination index. The final average germination rate of OE3 was 94.2%, the fresh weight of seedling roots was 0.507g, and the germination index was 0.599. The final average germination rate of NT3 was 98.5%, the fresh weight of seedling roots was 0.485g, and the germination index was 0.604. No significant differences were found between OE3 and NT3 in terms of final germination rate, seedling root fresh weight, germination index, and germination index. Figure 5 ).

[0138] At 18℃, the final average germination rate of OE1 was 97.1%, the fresh weight of seedling roots was 0.933g, and the germination index was 0.477. The final average germination rate of NT1 was 83.9%, the fresh weight of seedling roots was 0.686g, and the germination index was 0.368. Significant differences were found between OE1 and NT1 in terms of final germination rate, fresh weight of seedling roots, and germination index. The final average germination rate of OE3 was 89.7%, the fresh weight of seedling roots was 0.523g, and the germination index was 0.396. The final average germination rate of NT3 was 96%, the fresh weight of seedling roots was 0.458g, and the germination index was 0.392. No significant differences were found between OE3 and NT3 in terms of final germination rate, fresh weight of seedling roots, and germination index. Figure 5 (A)

[0139] At 10℃, the final average germination rate of OE1 was 95%, the fresh weight of seedling roots was 0.613g, and the germination index was 0.178. The final average germination rate of NT1 was 67.5%, the fresh weight of seedling roots was 0.334g, and the germination index was 0.094. Significant differences were found between OE1 and NT1 in terms of final germination rate, fresh weight of seedling roots, and germination index. The final average germination rate of OE3 was 90.2%, the fresh weight of seedling roots was 0.596g, and the germination index was 0.154. The final average germination rate of NT3 was 85.5%, the fresh weight of seedling roots was 0.436g, and the germination index was 0.134. No significant differences were found between OE3 and NT3 in terms of final germination rate, fresh weight of seedling roots, and germination index. Figure 5 (A)

[0140] Regardless of whether the temperature was normal (25℃), 18℃, or low (10℃), the final germination rate, seedling root fresh weight, and germination index of OE1 seedlings overexpressing the ZmLTG1 gene were superior to those of NT1 seedlings. However, OE3 seedlings without ZmLTG1 gene overexpression showed no difference in final germination rate, seedling root fresh weight, and germination index compared to NT3 at 25℃. These results further demonstrate that increasing the expression level of candidate genes can improve seed germination.

[0141] In summary, the germination results of the Mu mutant and the overexpressing transgenic lines under different temperature conditions demonstrate that the ZmLTG1 gene controls maize seed germination.

[0142] Example 4: Investigation into the causes of functional variations in the ZmLTG1 gene

[0143] (1) ZmLTG1 gene expression analysis

[0144] RNA was extracted from 220 and PH4CV dry seeds during the imbibition saturation stage (14h), radicle emergence stage (48h), and plumule emergence stage (72h) at 25℃, and during the imbibition saturation stage (2 days), radicle emergence stage (9d), and plumule emergence stage (15d) at 10℃, respectively. The RNA was reverse-engineered into cDNA and then detected by qPCR. The ZmGAPDH gene was used as an internal control. The specific experimental steps were the same as those for the ZmLTG1 gene cloning in Example 3.

[0145] Experimental results showed that the expression level of the ZmLTG1 gene differed significantly during the low-temperature swelling saturation stage, and the expression level of the ZmLTG1 gene in the 220 material was significantly higher than that in the PH4CV material. Figure 6 (A). Based on this, it is speculated that the reason why the 220 germinated plants have a better phenotype than PH4CV at low temperature may be due to the difference in the expression level of the ZmLTG1 gene.

[0146] (2) ZmLTG1 gene promoter sequence and activity analysis

[0147] To verify the above hypothesis, we analyzed the promoter region and gene region of this gene. The results showed that compared with the reference genome B73, there was a 1839 bp sequence insertion 400 bp upstream of the ATG in the ZmLTG1 gene in gene 220, and a 2107 bp sequence insertion 600 bp upstream of the ATG in the ZmLTG1 gene in gene PH4CV. Furthermore, compared with PH4CV, gene region 220 had 4 SNP variations. Figure 6 Significant sequence differences in the promoter region may lead to alterations in gene expression; therefore, promoter activity analysis was performed. The 2000 bp promoter of the ZmLTG1 gene in B73, the 3832 bp promoter of the ZmLTG1 gene in 220, the 4112 bp promoter of the ZmLTG1 gene in PH4CV, and the mutant B73 promoter (with 24 bp deleted at the insertion site of the 220 type transposon) were amplified, constructed into the pGreenⅡ800 vector, and transformed into protoplasts of etiolated maize seedlings. After 18 hours of dark incubation, LUC and REN values ​​were measured. The results showed that the activity of the 220 promoter was higher than that of the PH4CV, B73, and mutant B73 promoters. Figure 6 (C). This indicates that type 220 insertion sequences affect gene expression.

[0148] (3) Transgenic ZmLTG1 gene promoter and gene sequence

[0149] Build Pro 220 ::ZmLTG PH4CV and Pro PH4CV ::ZmLTG 220 The vector was inserted into pCAMBIA3301 and transformed into CAL material, following the same procedure as in Example 3 where the function of ZmLTG1 was verified using overexpressed transgenic material. After obtaining T2 generation transgenic positive seeds, germination experiments were conducted at 25℃, 18℃, and 10℃, with results as follows... Figure 6 As shown in DG, Pro at room temperature 220 ::ZmLTG PH4CV and Pro PH4CV ::ZmLTG 220 No significant difference; as temperature decreases, Pro... 220 ::ZmLTG PH4CV The final germination rate, germination index, and average germination time were all superior to Pro. PH4CV ::ZmLTG 220 This result indicates that the ZmLTG1 gene, in the presence of a 220-type promoter, can enhance the germination ability of maize at low temperatures.

[0150] Example 5: Detection of promoter haplotype of maize inbred line ZmLTG1 gene using InDel molecular markers

[0151] (1) Design and synthesize the following primers

[0152] Sequencing revealed different types of transposon insertions in the ZmLTG1 gene promoter of different maize inbred lines, classifying them into three haplotypes: Hap(B73) (no transposon insertion), Hap(220) (1839 bp transposon insertion), and Hap(PH4CV) (2107 bp transposon insertion). To address this, a universal primer pair IDP30-F+IDP30-R was designed for the promoter, a specific primer 220-F for the Hap(220) transposon, and a specific primer PH4CV-R for the Hap(PH4CV) transposon. These three primer sets were used to identify different transposon types: IDP30-F+IDP30-R, 220-F+IDP30-R, and IDP30-F+PH4CV-R (e.g., ...). Figure 7 (As shown in A).

[0153]

[0154]

[0155] (2) PCR preparation system and procedure

[0156] ①IDP30-F+IDP30-R

[0157]

[0158] ②220-F+IDP30-R

[0159]

[0160] ③IDP30-F+PH4CV-R

[0161]

[0162] (3) Electrophoretic identification

[0163] Identification was performed using 1.5% agarose gel electrophoresis at 130V for 25 minutes. The results are as follows: Figure 7As shown in Figure B, PCR and electrophoresis using IDP30-F+IDP30-R both yielded bright bands. The Hap(B73) band was 464 bp, the Hap(220) band was 2301 bp, and the Hap(PH4CV) band was 2574 bp. PCR and electrophoresis using 220-F+IDP30-R also showed a bright band of 548 bp for the Hap(220) type. PCR and electrophoresis using IDP30-F+PH4CV-R also showed a bright band of 1602 bp for the Hap(PH4CV) type. This demonstrates that the three primer sets IDP30-F+IDP30-R, 220-F+IDP30-R, and IDP30-F+PH4CV-R can successfully identify the haplotypes of the promoter of the ZmLTG1 gene in different maize inbred lines.

[0164] The above are merely preferred embodiments of the present invention. It should be noted that those skilled in the art can make various improvements and modifications 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.

Claims

1. Application of overexpression of ZmLTG1 protein with amino acid sequence as shown in SEQ ID NO:1 or its encoded nucleic acid in improving cold resistance of maize seeds.

2. The application according to claim 1, characterized in that, The improvement of corn seed cold resistance includes: improving the ability to germinate at low temperatures.

3. A preparation for improving the cold resistance of corn seeds, characterized in that, Includes at least one of the following: a) to c) a) The ZmLTG1 protein or its encoded nucleic acid, with an amino acid sequence as shown in SEQ ID NO:1; b) An expression vector containing nucleic acid encoding the ZmLTG1 protein; c) Hosts containing b) or hosts whose genomes integrate the coding nucleic acid of the ZmLTG1 protein.

4. A breeding method for improving the cold resistance of maize seeds, characterized in that, To increase the level and / or activity of endogenous ZmLTG1 protein in maize, or to express ZmLTG1 protein in maize that does not contain ZmLTG1 or has ZmLTG1 inactivation; the amino acid sequence of said ZmLTG1 is shown in SEQ ID NO:

1.

5. A preparation for identifying the cold resistance of maize seeds, characterized in that, include: A primer set for detecting the InDel molecular marker; the primer set for detecting the InDel molecular marker includes two upstream primers and two downstream primers: The nucleic acid sequences of the two upstream primers are shown in SEQ ID NO:6 and 8, respectively; The nucleic acid sequences of the two downstream primers are shown in SEQ ID NO:7 and 9, respectively.

6. A method for identifying the cold resistance of maize seeds, characterized in that, The sample was identified using the formulation described in claim 5.

7. The method according to claim 6, characterized in that, The criteria for identification include: SEQ ID NO:7 and SEQ ID NO:8 were used as primers for amplification. If an amplification product was present, the sample was derived from germplasm that can germinate at low temperatures. If SEQ ID NO:6 and SEQ ID NO:9 are used as primers for amplification, and the presence of amplification products indicates that the sample is derived from low-temperature sensitive germplasm.

Images