Application of maize ZmTAR4 gene in regulating callus related traits and / or IAA content

Abstract

The present application relates to the field of plant genetic engineering and molecular breeding technology, in particular to the application of corn ZmTAR4 gene in regulating callus related traits. The present application identifies a key gene ZmTAR4 through whole genome association analysis and weighted gene co-expression network analysis, which encodes a protein with tryptophan amino transferase activity. CRISPR / Cas9 knockout experiment proves that the gene is a gene necessary for positively regulating corn young embryo endogenous auxin (IAA) synthesis and embryogenic callus (EC) induction. Further, through overexpression experiment, it is proved that in corn materials with low induction rate, improving the expression level of ZmTAR4 can significantly improve the induction rate, growth amount and embryonic structure number of embryogenic callus, which fully verifies the positive regulation effect of the gene from the functional gain perspective.

Description

Technical Field

[0001] This invention relates to the fields of plant genetic engineering and molecular breeding technology, and in particular to the application of the maize ZmTAR4 gene in regulating callus-related traits and / or IAA content. Background Technology

[0002] Maize is one of the world's most important food and feed crops. With the development of biotechnology, transgenics and gene editing have become core methods for improving maize traits. However, the successful application of these technologies is highly dependent on the recipient material's ability to efficiently generate embryogenic callus (EC). Currently, maize genetic transformation generally suffers from severe genotype dependence, with the EC induction rate of most superior backbone inbred lines being extremely low. This has become a key bottleneck restricting the industrialization of maize biotechnology breeding.

[0003] Plant hormones, especially auxins (IAAs), play a central regulatory role in cell dedifferentiation and callus formation. Although previous studies have identified some relevant genetic loci, progress in cloning and functional verification of key genes has been slow, making it difficult to directly apply them to genetic engineering improvement. Therefore, identifying and verifying the major genes regulating maize EC induction ability and elucidating their molecular mechanisms is of great significance for creating widely adaptable and efficient genetic transformation recipients and promoting the development of maize biotechnology breeding. Summary of the Invention

[0004] The purpose of this invention is to provide the application of the maize ZmTAR4 gene in regulating callus-related traits and / or IAA content, so as to solve the problems existing in the prior art.

[0005] To achieve the above objectives, the present invention provides the following solution: This invention provides the application of ZmTAR4 protein, its encoding gene, or biological materials containing the encoding gene in regulating callus-related traits and / or IAA content in plants. The amino acid sequence of the ZmTAR4 protein is shown in SEQ ID NO.2; the nucleotide sequence of the encoding gene is shown in SEQ ID NO.1.

[0006] Optionally, by increasing the activity of the ZmTAR4 protein or the expression level of the encoding gene in the plant, the effect of increasing plant callus-related traits and / or IAA content can be achieved.

[0007] Optionally, the callus-related traits include one or more of embryogenic callus induction rate, callus diameter, and callus fresh weight, and the plant includes maize; The biomaterials include recombinant vectors and recombinant bacteria.

[0008] This invention provides a method for improving callus-related traits and / or IAA content in plants, comprising the step of increasing the activity of ZmTAR4 protein or the expression level of the encoding gene in the plant; the amino acid sequence of the ZmTAR4 protein is shown in SEQ ID NO.2; the nucleotide sequence of the encoding gene is shown in SEQ ID NO.1.

[0009] Optionally, the callus-related traits include one or more of the following: embryogenic callus induction rate, callus diameter, and callus fresh weight.

[0010] This invention provides the application of ZmTAR4 protein, its encoding gene, or biological materials containing the encoding gene in cultivating plants with increased callus-related traits and / or IAA content. The amino acid sequence of the ZmTAR4 protein is shown in SEQ ID NO.2; the nucleotide sequence of the encoding gene is shown in SEQ ID NO.1.

[0011] Optionally, by increasing the activity of the ZmTAR4 protein or the expression level of the encoding gene in the plant, the effect of improving plant callus-related traits can be achieved.

[0012] Optionally, the callus-related traits include one or more of embryogenic callus induction rate, callus diameter, and callus fresh weight, and the plant includes maize; The biomaterials include recombinant vectors and recombinant bacteria.

[0013] This invention provides a method for cultivating plants with increased callus-related traits and / or IAA content, comprising the step of increasing the activity of ZmTAR4 protein or the expression level of the encoding gene in the plant; the amino acid sequence of the ZmTAR4 protein is shown in SEQ ID NO.2; the nucleotide sequence of the encoding gene is shown in SEQ ID NO.1.

[0014] Optionally, the callus-related traits include one or more of the following: embryogenic callus induction rate, callus diameter, and callus fresh weight. The present invention discloses the following technical effects: 1. This invention is the first to clone and identify the key gene ZmTAR4 that regulates EC induction rate from a natural maize population through forward genetics (GWAS and WGCNA combined analysis), and its necessity was verified by CRISPR / Cas9 knockout experiments.

[0015] 2. This invention confirmed the tryptophan aminotransferase activity of ZmTAR4 protein through in vitro enzyme activity experiments, and elucidated its molecular mechanism of regulating callus formation by catalyzing key steps in auxin synthesis.

[0016] 3. This invention demonstrates through overexpression experiments that increasing the expression of ZmTAR4 in materials with low callus induction rate can significantly improve the induction rate, growth, embryonic state, and IAA content of embryogenic callus. This fully verifies the positive regulatory role of the gene from the perspective of functional gain, providing a direct and effective strategy for the targeted improvement of maize genetic transformation efficiency through genetic engineering.

[0017] 4. This invention forms a complete technical system, providing a clear and effective molecular target for the targeted improvement of maize genetic transformation efficiency through genetic engineering methods (such as overexpression and gene editing to enhance expression). Attached Figure Description

[0018] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0019] Figure 1 The results of the identification and association analysis of the ZmTAR4 gene are shown below. A shows the correlation analysis between IAA content and REC in the embryos and callus of 50 inbred lines; BC shows the significant SNPs identified in the association analysis of IAA content in the embryos (B) and callus cultured for 10 days (C); D shows the hierarchical clustering tree of the weighted co-expression network analysis; E shows the correlation analysis between the blue module and IAA content. The numbers above the colored blocks represent the correlation coefficients, and the numbers in parentheses below represent the significance. p Values: F represents the gene expression pattern in the sample from the blue module; G represents the 20 biological process classifications of gene enrichment in the blue module; H represents the co-expression network of the core gene ZmTAR4 in the blue module; I represents the candidate gene association analysis of the ZmTAR4 gene, with the significance threshold represented by the red dashed line, the gene structure in the middle, the exons represented by blue bars, and the linkage disequilibrium blocks between gene markers at the bottom; J represents the comparison of IAA content between the two haplotypes. P<0.01; Figure 2 This study analyzed the activities of two haplotype promoters of the ZmTAR4 gene; in A, the fluorescence signal of the HapG promoter in tobacco leaves was stronger than that of the HapA promoter; in B, the relative LUC activities of different haplotype promoters in tobacco leaves were analyzed. P<0.01; Figure 3This study aims to determine the spatiotemporal expression pattern of the ZmTAR4 gene and the activity of the ZmTAR4 enzyme. Specifically, A shows the expression pattern analysis of the ZmTAR4 gene in callus tissues from different tissue sites and at different stages of two haplotype materials; B shows the presence of a transmembrane domain at the 5' end of the ZmTAR4 protein; C shows Coomassie brilliant blue staining of the His-ZmTAR4 recombinant protein (M, Marker, lanes 1-2, His-ZmTAR4); and D shows the kinetic determination of the His-ZmTAR4 recombinant protease activity. P<0.01; Figure 4 shows the reduced hormone content and weakened callus induction phenotype of ZmTAR4 knockout materials; where A represents the ZmTAR4 gene structure and CRISPR / Cas9 editing of the three transformation events; B represents the IAA content in callus tissue of ZmTAR4 knockout lines and their wild-type at different time points; C represents the pyruvate (IPA) content in callus tissue induced by wild-type B104 and ZmTAR4 knockout materials for 25 days; D is the peak diagram of IPA in genetic materials detected by LC-MS, with Std representing IPA standards; E represents wild-type. The phenotypes of shoot length in B104 and the three knockout lines on day 5 of callus induction; F and G show the statistical analysis of budding rate and shoot length in wild-type B104 and the three knockout lines on day 5 of callus induction; H shows the differences in callus appearance in wild-type B104 and the three knockout lines at different induction stages, with a scale bar of 10 mm; I shows the statistical analysis of callus induction rate at 25 days of induction; J shows the comparison of callus appearance at 25 days of induction; K shows the callus diameter at 25 days of induction; L shows the fresh weight of callus at 25 days of induction. Data in the figures are expressed as mean ± standard deviation. P<0.05, P<0.01, P<0.001; Figure 5 The ZmTAR4 overexpression lines exhibited enhanced callus induction phenotypes. A shows the shoot length (LS) phenotype of the wild-type (YH7) and three ZmTAR4 overexpression (OE) lines on day 5 of callus induction; B shows the His-tagged ZmTAR4 protein detected by Western blot, confirming overexpression of this protein in the transgenic line (YH7 background); C shows the differences in callus morphology between YH7 and OE lines at different induction stages (scale bar: 10 mm); D shows the quantitative analysis of multiple callus-related traits 25 days after induction: shoot length (LS), budding rate (RSF), embryogenic callus induction rate (REC), IAA content (IAA), callus diameter (CD), and callus fresh weight (FW); E shows representative images of callus from the YH7 and OE lines 25 days after induction. P<0.05, P<0.01, P<0.001. Detailed Implementation

[0020] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0021] It should be understood that the terminology used in this invention is merely for describing particular embodiments and is not intended to limit the invention. Furthermore, with respect to numerical ranges in this invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Any stated value or intermediate value within a stated range, as well as each smaller range between any other stated value or intermediate value within said range, is also included in this invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0022] Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. While only preferred methods and materials have been described herein, any methods and materials similar or equivalent to those described herein may be used in the implementation or testing of this invention. All references to this specification are incorporated by way of citation to disclose and describe methods and / or materials associated with those references. In the event of any conflict with any incorporated reference, the content of this specification shall prevail.

[0023] Various modifications and variations can be made to the specific embodiments described in this specification without departing from the scope or spirit of the invention, as will be apparent to those skilled in the art. Other embodiments derived from this specification will also be apparent to those skilled in the art. This specification and embodiments are merely exemplary.

[0024] The terms “include,” “including,” “have,” “contain,” etc., used in this article are all open-ended terms, meaning that they include but are not limited to.

[0025] Unless otherwise specified, all techniques or conditions described in the examples were performed in accordance with the techniques or conditions described in the literature in this field or according to the product instructions. Reagents or instruments whose manufacturers are not specified are all commercially available conventional products.

[0026] The CDS nucleotide sequence of the ZmTAR4 gene involved in this invention is shown in SEQ ID NO.1, as follows: The amino acid sequence of the ZmTAR4 protein is shown in SEQ ID NO.2, as follows: MEAAEERPRRRPAGLGVLALLCSSLLLNVLFLAYYYFLSPPSQLADGGSCGLSWALRAARDAEALAATDSCSGHGQVFLDGVVGEDGRPGCECNRCFDGPHCSIRTPNCTADATSGDPLFL EPYWKRHAAASAVLVPGWHRLSYATTDGLFQSVELENHIRRLHRAVGNAVVDGKRLVFGAGSTQLINALVHALSPDANAAAASPPARVVATAPYYPPYRTQTAMFDGREYRWEGTTAAAWAN ASRNSSSFIEFVTSPNNPDALLRAPVLRGSAVIADHAYYWPHFTHIAAPADEDVMLFTMSKPSGHAGSRLGWALIRDEKVAKRAYEYVQSSIMGASRDTQLRMLEIVKVMLANLHGEEDIFA FGHDVMRTRWRRLSAVVSRSRRISLQRINPQYCTYFNRVREPSPAYAWVKCEREEDDDCYEALLKARIITRSGAGYDASSRYTRVSLLKSDDDFEVLVERVTDLVNAENYDDDVPRGSSSM.

[0027] The promoter region sequence of the ZmTAR4 gene is shown in SEQ ID NO.3 (HapG) or SEQ ID NO.4 (HapA), as follows.

[0028] g AAAAGAAACATCAACAGACCAGCTTACGTTCTTTTAATTTGCGTGCTGGATACATGTAACCGGAAATACGTGCACATACAAGTCGCTGTAAACCAATCATCGGCGTTCCTTGCAGGGCTGGATCAATTAGCGCCGGTCTCTAACCTTGCGGGTCGGATTTGTTAGTTCATGACGAGAAACAAAGTTATACCACGTTGTTGAATATCGAACGGTGATGGAGTATTAGAGTTGCTCGCAGGAAAAACTATATATTACGGCTTTTGAAAGCACGCCGAGACGTACGTCCAACAATAATCCAACA (SEQ ID NO.3), wherein the lowercase part is the position of significant variation; a AAAAGAAACATCAACAGACCAGCTTACGTTCCTTTAATTTGCGTGCTGGATACATGTAACCGGAAATACGTGGCAGATACAAGTCGCTGTAAAACCAATCATCGGCGTTCCTTGCAGGGCTGGATCAATTAGCACCGGTCTCTAACCTTGCGGGTCGG ATTTGTTAGTTCATGACGAGAAACGAAGCTACACCACGTTGTTGAACATCGAACGGTGATGGAGTGTTAGAGTTGCTCGCAGGAAAAACAATATATTACGGCTTTTGAAGGCACGCCGAGACGTATACGTCCAAGTCCAACAAATAATCCAACA (SEQ ID NO.4), where the lowercase part is the significant mutation position.

[0029] Example 1: Identification of ZmTAR4, a key gene regulating IAA content and callus induction rate in maize immature embryos. This embodiment details the methods and results for identifying ZmTAR4, a key gene regulating maize embryonic auxin (IAA) content and embryogenic callus (EC) induction rate, and its major genetic variations.

[0030] 1. Phenotypic association analysis and genome-wide association analysis (GWAS) First, 50 inbred lines were randomly selected from 330 maize natural variant populations, and the IAA content in their immature embryos and callus induced for 10 days was measured. Correlation analysis was performed between IAA content and embryogenic callus induction rate (REC). The results showed that IAA content was significantly positively correlated with REC (IAA_0d in immature embryos: r=0.681; IAA_10d in callus: r=0.742). p <0.001)( Figure 1 The A in the figure confirms that the IAA content can be used as a reliable indicator for screening materials with high induction rate.

[0031] Based on this, IAA content was determined in the entire population (n=330) of immature embryos, and GWAS analysis was performed using a mixed linear model (MLM) based on 1,004,950 SNP markers covering the entire genome. In the immature embryo stage (IAA_0d), 83 significantly associated SNP loci were identified (p<2.17E-05), which could explain 3.31%–13.42% of the phenotypic variation. The loci with the strongest association signals were concentrated on chromosome 3. Figure 1 (BC in the middle).

[0032] 2. Weighted Gene Co-expression Network Analysis (WGCNA) to Identify Candidate Gene Modules Transcriptome data from the integrated population during callus induction were analyzed. Weighted co-expression network analysis (WGCNA) was performed on 837 genes located within all significantly associated SNP linkage disequilibrium (LD) regions and differentially expressed during induction. Five characteristic modules were obtained, among which the "blue" module (containing 154 genes) showed a highly significant negative correlation with IAA_0d content (module-trait correlation coefficient r = -0.69). p =5e-08)( Figure 1 DF in the module). GO enrichment analysis showed that the genes in this module were significantly enriched in auxin-related biological processes such as "auxin-activated signaling pathways" and "cellular responses to auxin stimulation". Figure 1 Network analysis revealed that the gene Zm00001d043651, encoding tryptophan aminotransferase (homologically related to Arabidopsis thaliana AtTAA1), is located at the core node of this module, thus identifying it as a key candidate gene and naming it ZmTAR4 (G). Figure 1 (H in the text).

[0033] 3. Candidate gene association analysis identifies major-effect SNPs in promoter regions. To further refine the localization, 75 inbred lines were randomly selected from the population, and the coding region and upstream 2000 bp promoter region of the ZmTAR4 gene were resequencing. Association analysis between intragene variation and phenotype (GLM+PCA model) was performed using TASSEL 5.0 software. p ≤0.01). The results showed that a SNP (S3_206034247, G / A) located 301 bp upstream of the start codon ATG was significantly correlated with the content of IAA_0d in the immature embryo. Figure 1 The difference between the sequence information shown in SEQ ID NO.3 and the sequence information shown in SEQ ID NO.4 is that the bases at position 29 are G and A, respectively. Figure 2 Based on this locus, the materials were divided into two haplotypes: HapG (43 copies) carrying the G allele and HapA (32 copies) carrying the A allele. Phenotypic comparison confirmed that the IAA content in the immature embryos of HapG type materials was significantly higher than that of HapA type materials. p <0.01)( Figure 1 (J), which directly reveals that the SNP site in this promoter region is a key natural variation that regulates ZmTAR4 expression and IAA synthesis.

[0034] Example 2: Verification of ZmTAR4 promoter function and enzymatic activity This embodiment aims to verify the functional differences of the key SNPs (G / A) in the ZmTAR4 promoter region identified in Example 1, and to confirm the biochemical activity of the ZmTAR4 protein, thereby elucidating its regulatory mechanism at the molecular and biochemical levels.

[0035] 1. Analysis of ZmTAR4 promoter activity and spatiotemporal expression patterns To elucidate the regulatory function of the promoter region SNP (S3_206034247, G / A), promoter sequences (SEQ ID NO.3 and SEQ ID NO.4) of two haplotypes (HapG and HapA) of ZmTAR4 were cloned into the dual-luciferase reporter vector pGreenII-0800-LUC. Promoter activity was detected in tobacco leaves using an Agrobacterium-mediated transient transformation system. The results showed that the luciferase activity driven by the HapG promoter carrying the G allele was significantly higher than that driven by the HapA promoter carrying the A allele (p<0.01). Figure 2 The AB in the figure directly proves that this SNP is a functional variant that leads to differences in promoter transcriptional activity.

[0036] Furthermore, the expression pattern of ZmTAR4 in materials carrying different haplotypes (50 inbred lines randomly selected from 330 maize natural variant populations in Example 1) was analyzed using qRT-PCR. At the silking stage, ZmTAR4 was highly expressed in the tassel, root, and callus tissues, and the expression level in the roots of the HapG type material was significantly higher than that of the HapA type. Figure 3 A). During callus induction (0-15 days), the expression level of ZmTAR4 was significantly upregulated over time, and throughout the induction period, the expression level of HapG-type material was consistently significantly higher than that of HapA-type material. Figure 3 (A in the original text). This result is consistent with the promoter activity data above, indicating that the superior allele (G) in the promoter region can respond to developmental signals by enhancing the transcriptional level of ZmTAR4.

[0037] 2. Heterologous expression and enzyme activity assay of ZmTAR4 protein To verify the biochemical function of ZmTAR4, its coding sequence was analyzed. Protein structure prediction showed that amino acids 13-35 at the N-terminus constitute a transmembrane domain. Figure 3(B in the original text). To this end, a prokaryotic expression vector pCold-TF-ZmTAR4-His was constructed encoding its cytoplasmic region (amino acids 36-395). This vector (based on pCold-TF, with the coding sequence of amino acids 36-395 of the ZmTAR4 protein (SEQ ID NO.2) inserted at the BamHI restriction site) was used. After induction of expression in *E. coli* BL21(DE3), soluble recombinant ZmTAR4 protein was purified by nickel column affinity chromatography. SDS-PAGE analysis showed a single enriched band at approximately 90 kDa, consistent with the expected molecular weight. Figure 3 (C in the middle).

[0038] The catalytic function was verified using an in vitro enzyme activity assay. First, a 500 mmol / L borate buffer (pH 8.5) was prepared as the reaction buffer. Then, L-tryptophan (final concentration 300 μmol / L) as the substrate, pyridoxal phosphate (10 μmol / L) as the cofactor, and 1 μg of purified soluble recombinant ZmTAR4 protein were added sequentially. After incubation at 37 °C for 3 min, 1 mmol / L sodium pyruvate was added to initiate the reaction. The reaction mixture without sodium pyruvate served as a control. The characteristic absorbance change of the reaction product indole-3-pyruvate (IPA) at 330 nm was dynamically monitored using a spectrophotometer. The results showed that the OD of the experimental group... 330 The absorbance value continued to rise within 1 hour, while the absorbance value of the control group remained stable (0.164±0.012). Figure 3 (D in the original text). This result confirms that the recombinant ZmTAR4 protein possesses tryptophan aminotransferase activity, which can catalyze a key step in the auxin biosynthesis pathway.

[0039] Therefore, this embodiment demonstrates, through a promoter activity reporter system, the regulatory role of the key SNP (G / A) on the transcriptional level of the ZmTAR4 gene, with the G allele (HapG) exhibiting a significantly stronger driving force. Simultaneously, in vitro enzymatic experiments confirmed for the first time that the maize ZmTAR4 protein possesses tryptophan aminotransferase activity. These two results collectively validate the function of the ZmTAR4 gene at the molecular and biochemical levels, providing a direct mechanistic explanation for its influence on callus-induced phenotypes through regulation of auxin synthesis.

[0040] Example 3: Genetic verification (knockout) of ZmTAR4 gene function This embodiment uses CRISPR / Cas9 gene editing technology to create ZmTAR4 knockout mutants, and reversely verifies the core function of the ZmTAR4 gene in regulating auxin synthesis and inducing embryogenic callus (EC) from the genetic and phenotypic levels.

[0041] 1. Creation and identification of ZmTAR4 knockout mutants Using maize inbred line B104 as the genetic background, specific sgRNAs (Target 1: AGAGGAAGTAGTAGTACGCG (SEQ ID NO.5); Target 2: GAGTGCAATCGCTGCTTCGA (SEQ ID NO.6)) were designed targeting the first exon of the ZmTAR4 gene and constructed into the vector pYLCRISPR / Cas9Pubi-B. T0 generation transformed plants were obtained through Agrobacterium-mediated transformation. PCR amplification of the target sequence and sequencing analysis identified three independent homozygous knockout lines, named KO#1, KO#2, and KO#3. Sequencing results showed that all three lines produced effective frameshift mutations at the target site (A in Figure 4), confirming them as ZmTAR4 loss-of-function mutants.

[0042] 2. Analysis of endogenous hormone levels in knockout mutants Wild-type B104 and three knockout lines (KO#1-3) embryos were inoculated into modified N6 induction medium for callus culture. Endogenous hormones in wild-type B104 and the three knockout lines (KO#1-3) were quantitatively analyzed at different time points during callus induction. Enzyme-linked immunosorbent assay (ELISA) results showed that during induction, the auxin (IAA) content of the wild-type material steadily increased from 0.386 μg / g at the embryonic stage (day 0) to 2.847 μg / g at day 35. In contrast, the IAA content of all knockout lines was significantly reduced at all time points (p<0.05). Specifically, at day 25 of induction, the IAA content of the knockout line KO#2 (0.817 μg / g) was only 38.4% of that of the wild type (Figure 4, B). Meanwhile, liquid chromatography-mass spectrometry (LC-MS) analysis showed that the content of indole-3-pyruvate (IPA), the direct precursor of IAA, in the knockout callus was reduced by an average of 19.78% compared with the wild type (CD in Figure 4). These results directly confirm that knocking out ZmTAR4 severely hinders the biosynthetic pathway from tryptophan to IPA and then to IAA.

[0043] 3. Knockout mutant embryogenic callus-induced phenotypic defects Systematic tissue culture phenotypic identification of wild-type and knockout lines: Tissue Culture: Wild-type B104 and three knockout lines (KO#1-3) were self-pollinated, and three immature ears were collected from the middle row of each material. A total of 108 immature embryos, 1.2-1.5 mm in length, were selected from the middle of each ear. The specific method is as follows: About 10-12 days after pollination, ears with suitable embryo size were cleaned by removing the husks in the tissue culture room, disinfected by spraying with 75% alcohol, and then placed in a clean bench. A scalpel was heated to red-hot over an alcohol lamp. After cooling, excess endosperm was scraped off with a blade, and then the immature embryos were carefully picked out with the tip of the scalpel. These immature embryos were placed in three separate petri dishes containing modified N6 induction medium (with 2 mg / L 2,4-D added), with the scutellum facing upwards. They were aseptically cultured in the dark at 28°C. The modified N6 induction medium formula is as follows: 4 g / L N6 basal medium (+vitamin), 2 mg / L 2,4-D, 100 mg / L inositol, 2.76 mg / L proline, 30 g / L sucrose, 100 mg / L acid-hydrolyzed casein, 8 g / L agar powder, pH 5.8, and 25 μM silver nitrate added after sterilization.

[0044] Identification: (1) In the early stage of induction (5 days), the germination rate of the knockout line (27.0-31.9%) was significantly lower than that of the wild type (60.3%), and the average bud length (1.67-1.93 mm) was reduced by 32.0-41.2% compared with the wild type (2.84 mm) (EG in Figure 4).

[0045] (2) During the mid-to-late stages of induction (10-25 days), the wild type can form typical, firm granular embryogenic callus, while the knockout line only produces soft, non-embryogenic callus (H in Figure 4).

[0046] (3) Quantitative analysis showed that at 25 days of induction, the callus induction rate of the wild type reached 83.7%, significantly higher than that of the knockout line (24.3-35.7%). Meanwhile, the average diameter of the callus in the knockout line (2.75-3.37 mm) was 76.7-116.7% smaller than that of the wild type (5.96 mm), and the fresh weight decreased by 54.0-68.2% (IL in Figure 4). These data collectively indicate that the loss of ZmTAR4 function leads to the maize embryo losing its ability to efficiently form embryogenic callus.

[0047] In summary, ZmTAR4 is a key gene essential for maize embryos to initiate dedifferentiation and form high-quality embryogenic callus by catalyzing a crucial step in the auxin synthesis pathway, thereby regulating the level of endogenous IAA in cells. This result provides the most direct functional validation for its use as a molecular target for improving genetic transformation efficiency.

[0048] Example 4: Genetic verification (overexpression) of ZmTAR4 gene function This embodiment constructs a ZmTAR4 overexpression vector and transforms it into maize material with low induction rate, thus positively verifying the core role of this gene in improving the induction rate of embryogenic callus (EC) from the perspective of functional gain.

[0049] 1. Creation and identification of ZmTAR4 overexpression lines Using the inbred line YH7 (a modified B73 line provided by Beijing Bomeixingao Biotechnology Co., Ltd., with a 20-year commitment from the applicant to release the gene for 20 years from the application date), which has a low embryogenic callus induction rate, as the recipient, the full-length coding sequence of the ZmTAR4 gene (LOC100383587) cloned from the inbred line B73 was constructed into the plant expression vector pCAMBIA3301 driven by the maize embryo-specific globulin-1 promoter (pGlb1). Immature embryos of YH7 were transformed using Agrobacterium-mediated transformation. After resistance selection and molecular identification, three independent transgenic lines were obtained, named OE1, OE2, and OE3. Western blot analysis confirmed that the expression level of ZmTAR4 protein in these three lines was significantly higher than that in wild-type YH7 (…). Figure 5 (B in the text) was confirmed as a stable ZmTAR4 overexpression line.

[0050] 2. Overexpression lines induce enhanced phenotype in embryogenic callus. Systematic tissue culture phenotypic identification of wild-type YH7 and three overexpression lines (same as in Example 3): (1) In the early induction period (5 days), the average bud length of the immature embryos of the overexpression lines (OE1: 4.75 mm; OE2: 3.67 mm; OE3: 2.87 mm) was significantly shorter than that of the wild-type YH7 (7.58 mm), showing a trend of earlier dedifferentiation initiation. Figure 5 (A and D in the text).

[0051] (2) In the mid-to-late stages of induction (15-25 days), wild-type YH7 mainly forms soft, non-embryonic callus, while overexpression lines can form a large amount of tightly structured, granular embryogenic callus. Figure 5 (C in the middle).

[0052] (3) Quantitative analysis showed that, after 25 days of induction, the embryogenic callus induction rates of the overexpression lines OE1-OE3 were 41.71%, 36.88%, and 39.24%, respectively, all significantly higher than the 14.88% of the wild-type YH7. Simultaneously, the average callus diameter (6.54-7.88 mm) and fresh weight (increased by 17.81%-46.37% compared to YH7) of the overexpression lines were also significantly better than those of the wild-type ( Figure 5(DE in the text). These data collectively indicate that increasing the expression level of ZmTAR4 can significantly enhance the ability of maize immature embryos to form embryogenic callus.

[0053] (4) At 25 days after induction, the IAA content in the callus of the overexpression lines OE1-OE3 was 0.296, 0.223 and 0.225 ug / g, respectively, which was significantly increased by 37.3%-82.5% compared with 0.162 ug / g of wild type YH7.

[0054] In summary, overexpression of the ZmTAR4 gene in materials with low induction rates can significantly improve the induction efficiency and growth status of embryogenic callus. Combining the results of Example 2 (enzyme activity verification) and Example 3 (knockout verification), which demonstrate that this gene encodes a key enzyme for auxin synthesis and that its loss of function leads to phenotypic defects, this example fully confirms from a gain-of-function perspective that the ZmTAR4 gene is a key gene positively regulating maize embryogenic callus formation, providing a direct strategic basis for improving maize genetic transformation efficiency through genetic engineering.

[0055] The embodiments described above are merely preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Various modifications and improvements made by those skilled in the art to the technical solutions of the present invention without departing from the spirit of the present invention should fall within the protection scope defined by the claims of the present invention.

Claims

1. The application of ZmTAR4 protein, its encoding gene, or biological material containing said encoding gene in regulating callus-related traits and / or IAA content in plants, characterized in that, The amino acid sequence of the ZmTAR4 protein is shown in SEQ ID NO.2; the nucleotide sequence of the encoding gene is shown in SEQ ID NO.

1.

2. The application according to claim 1, characterized in that, By increasing the activity of the ZmTAR4 protein or the expression level of the encoding gene in the plant, the effect of increasing the plant callus-related traits and / or IAA content can be achieved.

3. The application according to claim 1 or 2, characterized in that, The callus-related traits include one or more of embryogenic callus induction rate, callus diameter, and callus fresh weight, and the plant includes maize; The biomaterials include recombinant vectors and recombinant bacteria.

4. A method for increasing the content of related traits and / or IAA in plant callus, characterized in that, The method includes steps to increase the activity of the ZmTAR4 protein in the plant or the expression level of the encoding gene in the plant; the amino acid sequence of the ZmTAR4 protein is shown in SEQ ID NO.2; and the nucleotide sequence of the encoding gene is shown in SEQ ID NO.

1.

5. The method according to claim 4, characterized in that, The callus-related traits include one or more of the following: embryogenic callus induction rate, callus diameter, and callus fresh weight.

6. The application of the ZmTAR4 protein, its encoding gene, or biological material containing the encoding gene in cultivating plants with increased callus-related traits and / or IAA content, characterized in that, The amino acid sequence of the ZmTAR4 protein is shown in SEQ ID NO.2; the nucleotide sequence of the encoding gene is shown in SEQ ID NO.

1.

7. The application according to claim 6, characterized in that, By increasing the activity of the ZmTAR4 protein or the expression level of the encoding gene in the plant, the effect of improving plant callus-related traits can be achieved.

8. The application according to claim 6, characterized in that, The callus-related traits include one or more of embryogenic callus induction rate, callus diameter, and callus fresh weight, and the plant includes maize; The biomaterials include recombinant vectors and recombinant bacteria.

9. A method for cultivating plants with enhanced callus-related traits and / or improved IAA, characterized in that, The method includes steps to increase the activity of the ZmTAR4 protein in the plant or the expression level of the encoding gene in the plant; the amino acid sequence of the ZmTAR4 protein is shown in SEQ ID NO.2; and the nucleotide sequence of the encoding gene is shown in SEQ ID NO.

1.

10. The method according to claim 9, characterized in that, The callus-related traits include one or more of the following: embryogenic callus induction rate, callus diameter, and callus fresh weight.

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