Application of transcription factor bZIP29 in regulation and control of plant seed germination

Abstract

The invention relates to the technical field of biology, in particular to application of a transcription factor bZIP29 in regulation and control of plant seed germination. Through research and analysis of the action mechanism of bZIP29, it is found that ZmbZIP29 can positively regulate and control rapid germination of corn seeds, rapid germination of the corn seeds can be promoted by overexpressing the ZmbZIP29 gene or improving the ZmbZIP29 gene or protein level, and seed germination can be delayed by knocking out and knocking down the ZmbZIP29 gene or inhibiting the ZmbZIP29 gene or protein level. It is shown that the transcription factor ZmbZIP29 can promote rapid germination or delay germination of plant seeds, and therefore the transcription factor ZmbZIP29 can be used for guiding breeding work of high-activity plant varieties and has very important scientific research value and application prospects.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and in particular to application of transcription factor bZIP29 in regulating plant seed germination. Background Art

[0002] Corn is currently the world's most produced crop and is crucial to ensuring global food security. As my country's largest grain crop, corn occupies a strategic position in the agricultural economy. Used primarily as feed, industrial processed products, and a staple food, corn has become a key pillar for maintaining the development of animal husbandry, the manufacture of bio-based materials, and the security of food reserves. In recent years, the average annual growth rate of corn consumption in my country has exceeded the growth rate of corn production over the same period. The gap between supply and demand has forced an urgent need to improve yields per unit area. However, the quality of arable land in my country is deteriorating and its area is decreasing year by year, while the area cultivated with corn has not increased significantly. Therefore, increasing corn yields per unit area is a key measure for ensuring food security in my country.

[0003] Cultivating high-quality, high-vibrant corn seeds facilitates mechanized operations and increases yields in modern agricultural production. Highly vigorous seeds germinate quickly and emerge uniformly in the field, adapting to diverse environmental conditions. Seed germination is the beginning of a plant's life cycle and the starting point for optimal plant growth. Therefore, identifying the key genes and regulatory mechanisms that regulate corn seed germination can effectively improve seed vigor and increase corn yield.

[0004] Seed germination begins with rapid water absorption, the seed coat and endosperm soften at a suitable temperature, and ends with the plumule and radicle protruding from the seed coat. The ability of seeds to use reserve materials during germination, such as the mobilization of seed reserves, is an important parameter for assessing germination vigor (Soltani, A., A. Gholipoor and E. Zeinali, Seed reserve utilization and seedling growth of wheat as affected by drought and salinity. ENVIRONMENTAL AND EXPERIMENTAL BOTANY, 2006. 55(1-2): p. 195-200.). Dry matter mobilization during the imbibition period regulates seed germination, for example, storing starch to provide energy for early seed germination. Transcription factors, as core regulatory factors, play an important role in regulating seed germination. In maize, the AP2 / ERF transcription factor ZmEREB92 is inhibited during germination, releasing the expression of ZmAMYa2 and ZmEIL7, thereby increasing starch degradation and ethylene signaling in seeds (Fu, J., et al., ZmEREB92 plays an anegative role in seed germination by regulating ethylene signaling and starch mobilization in maize. PLOS GENETICS, 2023. 19(11): p. 28.). In rice, the R2R3 MYB transcription factor CSA optimizes seed germination by balancing glucose and ABA metabolism during early seed germination by inhibiting Amylase 3A (Sun, L., et al., Carbon Starved Anther modulates sugar and ABA metabolism to protect rice seed germination and seedling fitness. PLANTPHYSIOLOGY, 2021. 187(4): p. 2405-2418.). However, there is a lack of systematic research on the mechanism of how to efficiently utilize storage materials to promote germination in corn.

[0005] The transcription factor ZmbZIP29, a member of the bZIP family, is a key transcription factor in maize. Studies have shown that this transcription factor plays a crucial role in the initial filling of the maize endosperm. It effectively promotes the formation and accumulation of storage materials, profoundly impacting maize seed development and quality. Maize seed germination, the initial stage of its life cycle, not only directly influences emergence rate, seedling uniformity, and field vigor, but also significantly impacts subsequent growth and development, ultimately yield. Currently, ZmbZIP29 has not been shown to play a role in maize seed germination. Summary of the Invention

[0006] In view of this, the present invention provides the use of transcription factor bZIP29 in regulating plant seed germination.

[0007] The present invention provides any one of the following uses in regulating plant seed germination:

[0008] I), transcription factor bZIP29;

[0009] II), a substance that regulates the level or activity of the transcription factor bZIP29 described in I);

[0010] III), a nucleic acid encoding the transcription factor bZIP29 described in I);

[0011] IV), a substance that regulates the expression and / or transcription of the nucleic acid fragment described in III).

[0012] In the present invention, the regulation includes positive regulation and negative regulation. The positive regulation is: upregulating the expression of the nucleic acid fragment, increasing the protein level of the transcription factor bZIP29 and / or enhancing the protein activity of the transcription factor bZIP29, which can promote plant seed germination. The negative regulation is: downregulating and / or knocking down the expression of the transcription factor bZIP29 gene, reducing the protein level of the transcription factor bZIP29, and / or inhibiting the protein activity of the transcription factor bZIP29, which can delay plant seed germination.

[0013] Furthermore, bZIP29 regulates plant seed germination by regulating the starch degradation pathway. The regulation of the starch degradation pathway includes regulating the starch consumption rate. The seed germination is early seed germination.

[0014] In the present invention, the plant includes a grass plant. Specifically, the grass plant includes corn, rice, wheat or sorghum. In a specific embodiment of the present invention, the plant is specifically corn.

[0015] In a specific embodiment of the present invention, the bZIP29 is derived from Zea mays and is referred to as ZmbZIP29. ZmbZIP29 is used as an example to verify the application of ZmbZIP29 in regulating plant seed germination.

[0016] The amino acid sequence of the transcription factor ZmbZIP29 is selected from any one of the following:

[0017] (1) the amino acid sequence shown in SEQ ID NO: 1;

[0018] (2) an amino acid sequence in which one or more amino acids are replaced, deleted, added or modified in the amino acid sequence shown in (1) and has the same or similar function as (1);

[0019] (3) An amino acid sequence having at least 90% identity with the amino acid sequence shown in (1) or (2).

[0020] In the present invention, the sequence of the nucleic acid encoding the transcription factor ZmbZIP29 is selected from any one of the following:

[0021] I. the nucleotide sequence shown in SEQ ID NO: 2;

[0022] II. A nucleotide sequence in which one or more bases are replaced, deleted, added or modified in the amino acid sequence shown in I and which has the same or similar function as I;

[0023] III. A nucleotide sequence that is at least 90% identical to the nucleotide sequence shown in I or II.

[0024] In the amino acid sequences and nucleotide sequences of the present invention, the at least 90% identity refers to sequences with at least 90% identity, at least 91% identity, at least 92% identity, at least 93% identity, at least 94% identity, at least 95% identity, at least 96% identity, at least 97% identity, at least 98% identity, at least 99% identity, at least 99.5% identity, at least 99.6% identity, at least 99.7% identity, at least 99.8% identity, and at least 99.9% identity.

[0025] Experiments have shown that ZmbZIP29 can positively regulate plant seed germination, that is, promote rapid seed germination. In a specific embodiment of the present invention, ZmbZIP29 takes corn ZmbZIP29 (i.e., ZmbZIP29) as an example, and utilizes the zmbzip29 KO mutant of ZmbZIP29 knockout constructed in the early stage and the OE1 and / or OE2 overexpression lines overexpressing ZmbZIP29 (Yang et al., ABA-induced phosphorylation of basic leucine zipper 29, ABSCISIC ACID INSENSITIVE19, and Opaque2 by SnRK2.2 enhances gene transactivation for endosperm filling in maize. THE PLANT CELL, 2022.34: p.1933-1956.). Corn seeds of wild-type WT, zmbzip29 KO mutant and OE1 and OE2 overexpression lines were subjected to germination experiments. The results showed that the germination rate of seeds in the OE1 and OE2 overexpression lines was significantly higher than that of the WT seeds at 30 to 72 hours, but there was no significant difference in the final germination rate. The OE1 and OE2 overexpression lines had a higher germination index (GI) and a lower mean germination time (MGT) than the WT seeds. This suggests that ZmbZIP29 can positively regulate the rapid germination of maize seeds. Overexpression of ZmbZIP29, agents that promote the expression of the transcription factor ZmbZIP29 gene, and agents that increase the protein level and / or enhance the protein activity of the transcription factor ZmbZIP29 can promote rapid germination of maize seeds, which is of great significance for the breeding of high-vigor maize varieties.

[0026] The present invention also provides a product for promoting plant seed germination, comprising at least one of the following:

[0027] A, transcription factor ZmbZIP29 or a nucleic acid fragment encoding transcription factor ZmbZIP29;

[0028] B, an expression cassette containing the nucleic acid fragment described in A;

[0029] C. A recombinant vector containing the expression cassette described in B;

[0030] D. transforming or transfecting the host cell with the recombinant vector described in C;

[0031] E. Reagents that promote the expression of the transcription factor ZmbZIP29 gene;

[0032] F. A preparation for increasing the protein level of transcription factor ZmbZIP29 and / or enhancing the protein activity of transcription factor ZmbZIP29.

[0033] The present invention also provides a method for promoting seed germination, comprising: using the product of the present invention to promote and / or enhance the expression and / or transcription of the transcription factor ZmbZIP29 gene, increase the level of the plant endogenous transcription factor ZmbZIP29, and / or enhance the activity of the transcription factor ZmbZIP29.

[0034] The present invention also provides a product for delaying plant seed germination, comprising at least one of the following:

[0035] a. An expression vector or lentiviral vector for knocking down or knocking out the transcription factor ZmbZIP29 gene;

[0036] b. transforming or transfecting host cells with the expression vector or lentiviral vector described in a;

[0037] c. Substances that interfere with and / or inhibit the expression and / or transcription of the transcription factor ZmbZIP29 gene.

[0038] In the product for delaying seed germination of the present invention, the substance that interferes with and / or inhibits the expression and / or transcription of the transcription factor ZmbZIP29 gene includes any one of the following: RNAi, siRNA, miRNA, antisense oligonucleotides, gRNA, inhibitors and / or antagonists of the transcription factor ZmbZIP29 targeting the transcription factor ZmbZIP29.

[0039] In a specific embodiment of the present invention, the gRNA for knocking out the ZmbZIP29 gene has a nucleic acid sequence of GTCGCCGCCCGACGTCGTGG (SEQ ID NO. 3): (Yang et al., ABA-induced phosphorylation of basic leucine zipper 29, ABSCISIC ACID INSENSITIVE 19, and Opaque2 by SnRK2.2 enhances gene transactivation for endosperm filling in maize. THE PLANT CELL, 2022. 34: p. 1933-1956.).

[0040] The present invention also provides a method for promoting or delaying seed germination, using the product of the present invention to knock out or knock down the expression of the transcription factor ZmbZIP29 gene, inhibit or interfere with the transcription of the transcription factor ZmbZIP29 gene, and reduce the level of the plant endogenous transcription factor ZmbZIP29 and / or the activity of the transcription factor ZmbZIP29.

[0041] Based on the above research results, ZmbZIP29 can be used as a marker for breeding high-vigor plant varieties. Therefore, the present invention also provides preparations for breeding high-vigor plant varieties, including preparations for detecting the level or activity of the transcription factor ZmbZIP29 protein, or preparations for detecting the expression of the gene encoding ZmbZIP29.

[0042] The present invention also provides the use of the product for promoting plant seed germination or the preparation for breeding high-vigor plant varieties in breeding high-vigor plant varieties.

[0043] A method for breeding high-vigor plant varieties, comprising: detecting the protein level or activity of the transcription factor ZmbZIP29, or detecting the expression level of the gene encoding ZmbZIP29, using the preparation for breeding high-vigor plant varieties of the present invention;

[0044] Alternatively, the product for promoting plant seed germination of the present invention is used to promote and / or increase the expression and / or transcription of the transcription factor ZmbZIP29 gene, increase the level of the plant endogenous transcription factor ZmbZIP29, and / or enhance the activity of the transcription factor ZmbZIP29.

[0045] The present invention provides the use of the transcription factor ZmbZIP29 in regulating plant seed germination. The present invention has found that ZmbZIP29 can positively regulate the rapid germination of corn seeds. Overexpressing the ZmbZIP29 gene or increasing the level of the ZmbZIP29 gene or protein can promote rapid germination of corn seeds, while knocking out or knocking down the ZmbZIP29 gene or inhibiting the level of the ZmbZIP29 gene or protein can delay seed germination. This indicates that the transcription factor ZmbZIP29 is of great significance for breeding high-vigor corn varieties. BRIEF DESCRIPTION OF THE DRAWINGS

[0046] Figure 1 The figure shows the germination phenotype of transgenic seeds of the maize transcription factor ZmbZIP29 in Example 1. WT, zmbzip29-1, and zmbzip29-2 are the wild type and zmbzip29 KO mutant materials of the gene mediated by CRISPR / Cas9; A represents the seed phenotype of the mutant and the wild type 78 hours after germination; B represents the germination rate statistics of the mutant and the wild type 30 to 78 hours after germination; C represents the final germination rate of the mutant and the wild type 78 hours after germination; D and E represent the germination index GI and the average germination time MGT of the mutant and the wild type, respectively.

[0047] Figure 2This is the seedling phenotype of the maize transcription factor ZmbZIP29 transgenic material 7 days after germination in Example 1. In the figure, WT, zmbzip29-1, and zmbzip29-2 are the wild-type and zmbzip29 KO mutant materials mediated by CRISPR / Cas9 of this gene; A represents the phenotype of the wild-type and mutant seedlings on the seventh day after germination; B represents the dry weight of the aboveground stems of the wild-type and mutant seedlings; C represents the dry weight of the underground roots of the wild-type and mutant seedlings; C represents the length of the aboveground stems of the wild-type and mutant seedlings; and D represents the length of the underground roots of the wild-type and mutant seedlings.

[0048] Figure 3 Figure 2 shows the germination phenotype of transgenic seeds of the maize transcription factor ZmbZIP29 in Example 2. WT, OE1, and OE2 are the wild-type and overexpression line materials of this gene; A represents the seed phenotype of the overexpression line and the wild type 72 hours after germination; B represents the germination rate statistics of the overexpression line and the wild type 30 to 72 hours after germination; C represents the final germination rate of the overexpression line and the wild type 72 hours after germination; D and E represent the average germination time MGT and germination index GI of the overexpression line and the wild type.

[0049] Figure 4 This is a schematic diagram of the maize transcription factor ZmbZIP29 regulating seed germination through the starch decomposition pathway in Example 3. In the figure, WT, zmbzip29-1, and zmbzip29-2 are the wild-type and zmbzip29 KO mutant materials of this gene mediated by CRISPR / Cas9; wherein A represents the change in total starch content in the embryo of the mutant and the wild-type at the germination stage; B represents the change in total starch content in the endosperm of the mutant and the wild-type at the germination stage; C represents the starch consumption level in the embryo of the mutant and the wild-type at the germination stage; D represents the starch consumption level in the endosperm of the mutant and the wild-type at the germination stage; E represents the α-amylase level in the embryo of the mutant and the wild-type at the germination stage; and F represents the α-amylase level in the endosperm of the mutant and the wild-type at the germination stage. DETAILED DESCRIPTION

[0050] The present invention provides the use of the transcription factor bZIP29 in regulating plant seed germination. Those skilled in the art can refer to the contents of this article and appropriately improve the process parameters to achieve the desired effect. It should be noted that all similar substitutions and modifications are obvious to those skilled in the art and are considered to be included in the present invention. The methods and applications of the present invention have been described through preferred embodiments, and relevant personnel can obviously modify or appropriately change and combine the methods and applications herein without departing from the content, spirit and scope of the present invention to implement and apply the technology of the present invention.

[0051] The present invention provides the use of ZmbZIP29 protein or nucleic acid encoding ZmbZIP29 protein in promoting rapid seed germination.

[0052] The ZmbZIP29 protein is specifically as shown in (1) or (2) or (3) or (4):

[0053] (1) The amino acid sequence of the protein shown in SEQ ID NO. 1;

[0054] (2) a fusion protein obtained by connecting a tag to the N-terminus and / or C-terminus of the protein described in (1);

[0055] (3) A protein whose amino acid residue sequence shown in SEQ ID NO. 1 has been substituted, deleted and / or added with one or more amino acid residues and has the same or similar function as the protein shown in (1);

[0056] (4) A protein derived from corn that is more than 98% identical to (1) and is associated with plant germination.

[0057] The present invention has no special restrictions on the source of the ZmbZIP29 protein described in (1), (2), (3), or (4) above. It can be artificially synthesized, or its encoding gene can be synthesized first and then expressed biologically.

[0058] The nucleic acid encoding the ZmbZIP29 protein can be DNA, such as cDNA, genomic DNA or recombinant DNA; or RNA, such as mRNA, hnRNA or tRNA. The nucleic acid has one of the following nucleotide sequences:

[0059] (1) the nucleotide sequence shown in SEQ ID NO. 2;

[0060] (2) a nucleotide sequence encoding the protein shown in SEQ ID NO. 1;

[0061] (3) a nucleotide sequence that can hybridize with the DNA sequence defined in (1) or (2) under stringent conditions;

[0062] (4) A DNA sequence that has more than 90% homology with the DNA sequence defined in (1) or (2) or (3) and encodes a protein with the same function; specifically, the homology is more than 95%; more specifically, more than 96%; more specifically, more than 97%; more specifically, more than 98%; and more specifically, more than 99%.

[0063] In the present invention, stringent conditions refer to: hybridization and membrane washing twice in a 2×SSC, 0.1% SDS solution at 68°C for 5 minutes each time, and hybridization and membrane washing twice in a 0.5×SSC, 0.1% SDS solution at 68°C for 15 minutes each time.

[0064] In the present invention, there is no particular limitation on the method for overexpressing the ZmbZIP29 gene; any commonly used method in the art may be used, such as plasmid transfection, chemical methods, gene gun techniques, microinjection, viral vector-mediated gene transfer, homologous recombination, zinc finger nucleases, TALENs, CRISPR, and the like. In the present invention, methods for knocking out ZmbZIP29 include, but are not limited to, homologous recombination, zinc finger nucleases, TALENs, CRISPR, and the like. In a specific embodiment of the present invention, ZmbZIP29 is knocked out using CRISPR, and overexpression is achieved using the pTF102 overexpression vector driven by a 27-kD zein promoter.

[0065] In the present invention, "including", "comprising", and "having" are used interchangeably to indicate the inclusiveness of the solution, meaning that the solution may contain other elements in addition to the listed elements. It should also be understood that the use of "including", "comprising", and "having" in this document also provides "consisting of" solutions.

[0066] In the present invention, when used herein, "and / or" includes the meanings of "and", "or" and "all or any other combinations of elements linked by the corresponding terms".

[0067] In the present invention, "at least one" means one or more, and "plurality" means two or more. "At least one," "at least one item," or similar expressions refer to any combination of these items, including any combination of single or plural items.

[0068] The sequence involved in the present invention is as follows:

[0069] Amino acid sequence of transcription factor:

[0070] MASSASTTSGGDERPRAPHADACAGTAPPQAHVEWAASMQAYYAA GGQPYAWHAAQQECEELAQKVTDLTVVNGTLRSELDELKKACEDMEAE NSQLIGELEQSEAPSVVTTLSIQIDTTKAHHRSSDQHGNKNNTGSNG*(SEQ ID NO: 1)

[0071] Nucleic acid sequence of transcription factor:

[0072] ATGGCGTCGTCCGCCTCCACGACGTCGGGCGGCGACGAGCGCCCGCGCGCGCCTCACGCCGACGCGTGCGCTGGGACGGCGCCGCCGCAGGCGCACGTGGAGTGGGCCGCCTCGATGCAGGCCTACTACGCCGCCGGTGGGCAGCCTTACGCCTGGCACGCCGCGCAGCAAGAATGTGAGGAACTAGCGCAGAAGGTAACTGACCTGACCGTC GTCAACGGCACGCTCAGATCAGAACTCGACGAGCTTAAGAAGGCCTGTGAAGACATGGAAGCAGAGAATTCACAGCTAATTGGTGAACTGGAGCAGTCCGAGGCGCCTAGCGTTGTGACGACTCTGAGCATCCAGATTGACACAACGAAGGCGCATCATAGAAGCAGTGACCAGCATGGTAATAAAAACAACACTGGTAGCAATGGGTAG(SEQ ID NO:2)

[0073] In the present invention, the ZmbZIP29 coding gene is first synthesized and then biologically expressed to obtain the ZmbZIP29 protein.

[0074] In a specific embodiment of the present invention, the N-terminus and / or C-terminus of the ZmbZIP29 protein further comprises a tag.

[0075] The tag includes at least one of polyArg, poly-His, FLAG, C-MYC, HA, and GFP. The specific tag sequences of polyA and poly-His are shown in Table 1.

[0076] Table 1 Tag sequences

[0077]

[0078] Unless otherwise specified, the experimental methods used in the following examples are conventional methods.

[0079] Unless otherwise specified, the materials and reagents used in the following examples can be obtained from commercial sources.

[0080] The zmbzip29 transgenic materials used in the following examples include zmbzip29 knockout mutants zmbzip29-1 and zmbzip29-2, and overexpression lines OE1 and OE2 (overexpressing zmbzip29), see Yang, T., et al., ABA-induced phosphorylation of basic leucine zipper29, ABSCISIC ACIDINSENSITIVE 19, and Opaque2 by SnRK2.2 enhances gene transactivation for endosperm filling in maize. PLANT CELL, 2022. 34(5): p. 1933-1956. zmbzip29 can also be introduced into corn according to methods commonly used in the art to obtain transgenic materials overexpressing zmbzip29.

[0081] The present invention will be further described below in conjunction with the embodiments:

[0082] Example 1 Germination test of zmbzip29 KO mutant material

[0083] The test method is as follows:

[0084] (1) Thirty plump, neat seeds were selected from the zmbzip29 KO mutants zmbzip29-1 and zmbzip29-2 and the corresponding wild-type WT materials harvested in the current season, and three biological replicates were set up. It was observed that the dry seeds of the zmbzip29 KO mutant were smaller than those of the WT. First, the seeds were completely immersed in NaClO (sodium hypochlorite) solution for 10 minutes and then rinsed with deionized water three times to ensure that the NaClO on the seed surface was basically removed.

[0085] (2) The experimental method adopted was the sand culture method. To prepare the experiment, fine sand was required to filter out impurities and sterilize at high temperature at 130°C. The cooled sand was evenly mixed with deionized water to keep the water content of the sand at about 16%. The seeds were kept with the embryo side facing up, and each repetition was evenly sown in a 3 cm thick sand bed in a 5×6 manner. Finally, the germination box was sealed with a self-sealing bag to prevent water evaporation and placed in a constant temperature incubator at 25°C.

[0086] (3) The number of radicles protruding from the seed coat was observed at 6-hour intervals. The radicles appeared after 30 hours, and all the seeds germinated after 78 hours. The germination phenotype at 78 hours was recorded by taking photos. The germination rate, final germination rate, germination index GI and average germination time MGT of the germination stage were calculated. The results showed that Figure 1As shown in the results, the germination rate of zmbzip29KO mutant seeds was significantly lower than that of WT at 30h to 78h, and there was no significant difference in the final germination rate between zmbzip29KO mutant and WT. The zmbzip29KO mutant had lower GI and higher MGT than WT, proving that ZmbZIP29 positively regulates seed germination.

[0087] (4) Observe the morphology of seedlings 7 days after germination and take photos to record them. Ten seedlings were used as one replicate, and there were 3 replicates in total. The stem and root lengths of the seedlings were measured, and then they were dehydrated at high temperature to constant weight. The dry weights of the stems and roots were recorded separately. The results showed that Figure 2 As shown, the volume of zmbzip29 KO mutant seedlings was significantly smaller than that of WT, the stem length and root length of zmbzip29 KO mutant seedlings were shorter than those of WT, and the dry weight of the stem and root was lighter than that of WT, proving that ZmbZIP29 regulates seed germination and affects seedling growth.

[0088] Example 2 Germination test of ZmbZIP29 overexpression line material

[0089] The test method is as follows:

[0090] (1) Thirty plump, neat seeds were selected from the OE1 and OE2 overexpression lines and the corresponding wild-type WT materials harvested in the current season, each serving as a replicate, with three biological replicates. It was observed that the dry seeds of the OE1 and OE2 overexpression lines were larger than those of the WT. First, the seeds were completely immersed in a NaClO (sodium hypochlorite) solution for 10 minutes, followed by three rinses with deionized water to ensure that the NaClO on the seed surface was substantially removed.

[0091] (2) Preparation of the experiment requires filtering impurities from fine sand and sterilizing it at 130°C. After cooling, the sand and deionized water are evenly stirred to keep the water content of the sand at about 16%. The seeds are kept with the embryo side facing up and sown evenly in a 3 cm thick sand bed in a 5×6 pattern for each replicate. Finally, the germination box is sealed with a ziplock bag to prevent water evaporation and placed in a constant temperature incubator at 25°C.

[0092] (3) The number of radicles protruding from the seed coat was observed at 6-hour intervals. The radicles appeared after 30 hours, and all the seeds germinated after 72 hours. The germination phenotype of the seeds was recorded by taking photos. The germination rate, final germination rate, germination index GI and average germination time MGT of the germination stage were calculated. The results showed that Figure 3 As shown in the results, the germination rate of seeds in the OE1 and OE2 overexpression lines was significantly higher than that in the WT at 30h to 72h, and there was no significant difference in the final germination rate between the OE1 and OE2 overexpression lines and the WT. The OE1 and OE2 overexpression lines had higher GI and lower MGT than the WT, proving that ZmbZIP29 positively regulates seed germination.

[0093] Example 3 Starch Level and α-amylase Activity Detection Test of Early Germination Seeds of zmbzip29 KO Mutant

[0094] The test method is as follows:

[0095] (1) 180 plump, neat seeds of the zmbzip29KO mutant and the corresponding wild-type WT material harvested that season were selected as one replicate, with three biological replicates. First, the seeds were completely immersed in a NaClO (sodium hypochlorite) solution for 10 minutes and then rinsed three times with deionized water to ensure that the NaClO on the seed surface was substantially removed. The germination test method was the same as the sand culture method described in Example 1.

[0096] (2) Samples were collected at 0 h, 6 h, 12 h, 24 h, and 36 h after germination. Thirty seeds were randomly selected from a replicate at each time point. The seeds were divided into embryo and endosperm parts and tested using a starch detection kit and an α-amylase activity detection kit.

[0097] (3) The results show that if Figure 4 As shown, the starch content in the dry seed embryo of the zmbzip29 KO line was significantly lower than that of the WT in the embryo, and the content was the same at 6h, but increased significantly thereafter; the starch degradation rate of the zmbzip29 KO line was significantly lower during the period from 0h to 6h, but was the same during the period from 6h to 24h, and was higher at 36h; the amylase activity of the zmbzip29 KO line was significantly lower than that of the WT during the period from 0h to 6h, but reached a similar level from 12h to 24h, and decreased at 36h.

[0098] In the endosperm, total starch content and amylase activity were reduced in the zmbzip29 KO line compared to the WT line at all time points. The starch consumption rate in the zmbzip29 KO line was significantly reduced from 0 to 24 hours, but remained similar from 24 to 36 hours. These results suggest that the slower starch consumption rate caused by the ZmbZIP29 mutation, rather than low starch content, is the primary cause of the slower germination in ZmbZIP29 seeds.

[0099] In summary, the present invention provides the function of ZmbZIP29 gene in positively regulating maize seed germination. The CRISPR / Cas9-mediated zmbzip29 KO mutant material is used to explore the mechanism of regulating early maize seed germination, which is of great significance for breeding high-vigor maize varieties.

[0100] The above are only preferred embodiments of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principles of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. Use of any of the following in regulating plant seed germination: I), transcription factor bZIP29; II), a substance that regulates the level or activity of the transcription factor bZIP29 described in I); III), a nucleic acid encoding the transcription factor bZIP29 described in I); IV), a substance that regulates the expression and / or transcription of the nucleic acid described in III).

2. The use according to claim 1, characterized in that The regulation includes positive regulation and negative regulation; the positive regulation is: upregulating the expression of the nucleic acid, increasing the protein level of the transcription factor bZIP29 and / or enhancing the protein activity of the transcription factor bZIP29, which can promote plant seed germination; The negative regulation is: down-regulating and / or knocking down the expression of the transcription factor bZIP29 gene, reducing the level of the transcription factor bZIP29 protein, and / or inhibiting the activity of the transcription factor bZIP29 protein, which can delay plant seed germination.

3. The use according to claim 1, characterized in that The regulating plant seed germination includes: regulating starch degradation pathway and / or regulating starch consumption rate.

4. The use according to any one of claims 1 to 3, characterized in that The bZIP29 is ZmbZIP29, and the amino acid sequence of the transcription factor ZmbZIP29 is selected from any one of the following: (1) the amino acid sequence shown in SEQ ID NO: 1; (2) an amino acid sequence in which one or more amino acids are replaced, deleted, added or modified in the amino acid sequence shown in (1) and has the same or similar function as (1); (3) an amino acid sequence having at least 90% identity with the amino acid sequence shown in (1) or (2); The sequence of the nucleic acid encoding the transcription factor bZIP29 is selected from any one of the following: I. the nucleotide sequence shown in SEQ ID NO: 2; II. A nucleotide sequence in which one or more bases are replaced, deleted, added or modified in the amino acid sequence shown in I and which has the same or similar function as I; III. A nucleotide sequence that is at least 90% identical to the nucleotide sequence shown in I or II.

5. The use according to claim 1, characterized in that The plants include plants of the grass family.

6. The use according to claim 5, characterized in that The grass plant includes corn, rice, wheat or sorghum.

7. A product for promoting plant seed germination, characterized in that: Include at least one of the following: A, transcription factor ZmbZIP29 or a nucleic acid fragment encoding transcription factor ZmbZIP29; B, an expression cassette containing the nucleic acid fragment described in A; C. A recombinant vector containing the expression cassette described in B; D. transforming or transfecting the host cell with the recombinant vector described in C; E. Reagents that promote the expression of the transcription factor ZmbZIP29 gene; F. A preparation for increasing the protein level of transcription factor ZmbZIP29 and / or enhancing the protein activity of transcription factor ZmbZIP29.

8. A method for promoting seed germination, characterized in that: include: The product according to claim 6 promotes and / or increases the expression and / or transcription of the transcription factor ZmbZIP29 gene, increases the level of the plant endogenous transcription factor ZmbZIP29, and / or enhances the activity of the transcription factor ZmbZIP29.

9. A product for delaying the germination of plant seeds, characterized in that Include at least one of the following: a. An expression vector or lentiviral vector for knocking down or knocking out the transcription factor ZmbZIP29 gene; b. transforming or transfecting host cells with the expression vector or lentiviral vector described in a; c. Substances that interfere with and / or inhibit the expression and / or transcription of the transcription factor ZmbZIP29 gene.

10. The product according to claim 8, characterized in that The substance that interferes with and / or inhibits the expression and / or transcription of the transcription factor ZmbZIP29 gene includes any one of the following: RNAi, siRNA, miRNA, antisense oligonucleotide, gRNA, inhibitors and / or antagonists of the transcription factor ZmbZIP29.

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