Application of ZmFBP6 gene in regulating maize tassel branch development

Abstract

The application belongs to the technical field of corn breeding, and particularly relates to application of a ZmFBP6 gene in regulating corn tassel branch development. The sequence of the ZmFBP6 gene is shown as SEQ ID NO. 1, and the amino acid sequence of the ZmFBP6 gene coding protein is shown as SEQ ID NO. 2. By inhibiting the expression of the ZmFBP6, specifically by introducing sgDNA shown as any one of SEQ ID NO. 3-4 into corn, the number of corn tassel branches can be reduced; the application has guiding significance for genetic improvement of corn tassels, modification of corn plant type or cultivation of new high-yield varieties resistant to dense planting.

Description

Technical Field

[0001] This invention belongs to the field of maize breeding technology, specifically involving the application of the ZmFBP6 gene in regulating the branching development of maize tassels. Background Technology

[0002] Maize plant architecture determines the spatial distribution of the population and affects planting density, ultimately impacting maize yield. Therefore, coordinating maize plant architecture and increasing planting density is a crucial way to increase maize yield. The selection of plant architecture has evolved from geometric to physiological morphology, and finally to the ideal plant architecture. In 1968, Donald first proposed the concept of the ideal crop plant architecture, defining it as the plant architecture that minimizes the resource and environmental requirements for dry matter production per unit of crop, while still coordinating individual and population yields with increasing planting density, thereby improving biological yield and economic coefficient. The ideal maize plant architecture, achieved through various breeding methods, optimizes the size and spatial distribution of various organs within the maize plant, thereby increasing planting density and ultimately improving maize yield. To meet the ever-increasing demand for maize, the maize plant architecture continues to evolve, with the ideal maize plant architecture gradually evolving into a biological morphology characterized by upward-pointing upper leaves, reduced leaf angles, lower ear position, smaller tassels, and fewer tassel branches.

[0003] The maize tassel is an important agronomic trait derived from the wild ancestor of maize, teosinte, and participates in the composition of the maize plant architecture, influencing the photosynthesis of the upper leaves. The size and number of branches of the tassel directly affect the light interception of the maize population, potentially blocking 6.5%–28.8% of the light from the upper leaves, thus affecting the yield of individual plants and the entire population. Studies have shown that, with the overall nutrient absorption of maize remaining constant, the female and male ears develop simultaneously, thus competing for nutrients. Appropriately reducing the size and number of branches of the tassel can increase yield. Compared to undried maize, artificially detasseled maize can increase yield by 5%–19%. From 1967 to 1991, in hybrids bred by Pioneer, the tassel size was reduced by 36%, but the yield increased threefold. Therefore, breeders tend to select smaller tassels with fewer branches to increase yield.

[0004] However, there are currently few known functional genes that regulate the number of branches on maize tassels. Summary of the Invention

[0005] To address the aforementioned technical problems, this invention provides the application of the ZmFBP6 gene in regulating the branching development of maize tassels.

[0006] The present invention adopts the following technical solution:

[0007] One of the objectives of this invention is to provide the application of the ZmFBP6 gene in regulating the branching development of maize tassels, the sequence of which is shown in SEQ ID NO.1.

[0008] Furthermore, the regulation of maize tassel branching development is to reduce the number of maize tassel branches.

[0009] Furthermore, by inhibiting the expression of the ZmFBP6 gene, the number of male branches in maize ears is reduced.

[0010] In one specific embodiment of the present invention, inhibiting the expression of the ZmFBP6 gene can reduce the number of male branches in maize. However, there are no significant differences between the mutant and the wild type in traits such as ear length, ear diameter, 100-grain weight, plant height, ear height, and number of internodes, proving that ZmFBP6 is the gene that controls the number of male branches and has no effect on other agronomic traits.

[0011] A second objective of this invention is to provide an sgRNA for targeted knockout of the ZmFBP6 gene.

[0012] Furthermore, the sequence of the DNA molecule is shown in any one of SEQ ID NO. 3 to 4.

[0013] A third objective of this invention is to provide an expression cassette containing the DNA molecule.

[0014] A fourth objective of this invention is to provide a recombinant vector comprising the expression cassette.

[0015] In a specific embodiment of the present invention, the sgRNA and pBUE411 vector are ligated using 10×Bsa I restriction enzyme and high concentration of T4 ligase, E. coli are transformed and single clones are selected for identification. Correctly ligated single clones are selected, cultured, and plasmids are extracted to obtain the recombinant vector.

[0016] A fifth objective of the present invention is to provide recombinant bacteria comprising the recombinant vector.

[0017] In a specific embodiment of the present invention, the recombinant bacteria is Agrobacterium competent cells GV3101.

[0018] The sixth objective of this invention is to provide the application of the sgRNA, the DNA molecule, the expression cassette, the recombinant vector, or the recombinant bacteria in reducing the number of branches on maize tassels.

[0019] The seventh objective of this invention is to provide a method for reducing the number of male tassel branches in maize, comprising: knocking out or mutating the ZmFBP6 gene in maize, and screening for new maize varieties with reduced male tassel branches.

[0020] The present invention has the following beneficial effects:

[0021] This invention identifies a key gene, ZmFBP6, in maize that regulates the number of tassel branches. By inhibiting the expression of ZmFBP6, the number of tassel branches in maize can be reduced. This invention has guiding significance for the genetic improvement of maize tassels, the modification of maize plant architecture, or the breeding of high-yielding new varieties tolerant to dense planting. Attached Figure Description

[0022] Figure 1 Phenotypic identification and comparison of Mu transposon insertion mutants and wild type in terms of tassel branch number and other agronomic traits; A: Identification of Mu rotor mutants; B: Phenotypic identification of tassel branch number in mutants and wild type; C: Statistical analysis of tassel branch number in mutants; D: Comparison of spikelet length, spikelet diameter, 100-grain weight, plant height, spike position height, and internode number between mutants and wild type. Student's t-test, ** indicates significant difference at P≤0.01.

[0023] Figure 2 Phenotypic identification and comparison of ZmFBP6 knockout mutant and wild type in terms of tassel branch number and agronomic traits.

[0024] Figure 3 The expression levels of ZmFBP6 in different tissues were analyzed. V4 to V18 refer to leaf age, that is, from the 4-leaf stage to the 18-leaf stage.

[0025] Figure 4 Subcellular localization of ZmFBP6; A: Subcellular localization of empty GFP vector; B and C: Subcellular localization of ZmFBP6 in tobacco. GFP: Green fluorescent protein; Chloroplast: Chloroplast autofluorescence; Nucleus: Nuclear marker; Bright field; Merged: Fitted; Scale bar = 20 μm. Detailed Implementation

[0026] The present invention will now be described in detail with reference to the accompanying drawings and specific embodiments, but this should not be construed as limiting the invention. Unless otherwise specified, the technical means used in the following embodiments are conventional means well known to those skilled in the art, and the materials, reagents, etc. used in the following embodiments are commercially available unless otherwise specified.

[0027] This invention provides evidence of a new use for the ZmFBP6 gene, the nucleotide sequence of which is shown in SEQ ID NO.1, the amino acid sequence of the protein it encodes is shown in SEQ ID NO.2, and the nucleotide sequences of sgRNA-1 and sgRNA-2 for targeted knockout of the ZmFBP6 gene are shown in SEQ ID NO.3 to 4, respectively.

[0028] SEQ ID NO.1:

[0029] ATGCATCCCAAGGCACGAATCCACGCCGACCCGGTGCGCGAGGTCGACCACTTTGACTGCCTGCCGGATTCGCTCGTCCTGCTGATCCTGAACAAGGTCGAGGACGTGCGATCGCTCGGCCGGTGCTACGCCGTGTCCAAGCGGCTTTGTGGGCTTGTCCCCCTTGTCCATGATGTGTATGTCAAGATTGACCGCGTCGTGGCGGTCGACGGCGACGCTGAGGACGCGCTCAACCTGTCATCGCCGAAGCCTCGGAACATCTTCTCGCACTTCCTGAAGCTGATGCTCTTCACCATCATCAAGCCTTTCCACAACATGCGCAACCCTAATGGTACTGGGAGGTCATTGTTCCCGCAGCTCTCGCACCACTCGCCAGCGCAGGTGCTCAGGAACTTCACGCACATCAGGAATCTTCGGATCGAGCTCCCTTCTGGGGATGTGGGTACTGAGGAGGGAGTTCTCCTGAAATGGCGAGCAGAGTATGGGAGCACACTTCAGAGTTGTGTGATCCTGGGGGGTACCCGGGTTGACCGCAAGCCTGTTGGTGGAGAGCATGAGCAGTCTTTGGAGGATAATGGAAGCATGCCAGAGTCTTTCTATACCAATGGTGGGCTGAAACTTCGTGTCGTCTGGACAATCAGCTCTCTAATTGCGGCATCAACGAGGCACTATCTTCTCCGCTCAATCATCAATGACCATTCCACACTTCTGAGCTTGGTACTGACAGATGCCGATGGCCAAGGCACATTGTGCATGGGAGCAGAGCAGCTGAAGGAGTTCAGAGAGAACCAGTTGTCGGCCTCAGCATGTTCCAACAGGACTCAGGTCCCAGCCTGCAACATGAAGCTCAAGTATGCTCCATATCTTGAACTGCCTGGTGGTATGGCGTTGCAAGGCGCCACTTTGGTGGCTATCAAACCCTCACCTGACGGAAGCAATGGAAGCCACCCTAACCGTAAAGAAGCCGACGCATTTGTCTTTGGAGCATTTGATGGGCCATTTAAGGCTGCTGTGAAGGCTTTGATGAAGAGGCGCACTTACCTTCTGGAGATGAATGGCTTCTAG。

[0030] SEQ ID NO.2:

[0031] MHPKARIHADPVREVDHFDCLPDSLVLLILNKVEDVRSLGRCYAVSKRLCGLVPLVHDVYVKIDRVVAVDGDAEDALNLSSPKPRNIFSHFLKLMLFTIIKPFHNMRNPNGTGRSLFPQLSHHSPAQVLRNFTHIRNLRIELPSGDVGTEEGVLLKWRAEYGSTLQSCVILGGTRVDRKPVGGEHEQSLEDNGSMPESFYTNGGLKLRVVWTISSLIAASTRHYLLRSIINDHSTLLSLVLTDADGQGTLCMGAEQLKEFRENQLSASACSNRTQVPACNMKLKYAPYLELPGGMALQGATLVAIKPSPDGSNGSHPNRKEADAFVFGAFDGPFKAAVKALMKRRTYLLEMNGF。

[0032] SEQ ID NO.3:

[0033] 5'-GACAAGCCCACAAAGCCGCTTGG-3'.

[0034] SEQ ID NO.4:

[0035] 5'-AGAGCATGAGCAGTCTTTGGTGG-3'.

[0036] Example 1: Functional verification and molecular mechanism study of ZmFBP6 gene

[0037] 1. Materials and Methods

[0038] 1.1 Experimental Materials and Cultivation

[0039] The Mu transposon mutant was provided by Maize Genetics Cooperation Stock Center (http: / / maizecoop.cropsci.uiuc.edu / ) and planted at the North Experimental Field of Shenyang Agricultural University (Liaoning, Shenyang, 41.48°N, 123.25°E) and the Hainan Southern Breeding Base of Shenyang Agricultural University (Hainan, Sanya, 18.15°N, 109.30°E). The transgenic knockout material was genetically transformed by Boyuan Biotechnology. The T1 and T2 generation transgenic materials were planted at the North Experimental Field of Shenyang Agricultural University (Liaoning, Shenyang, 41.48°N, 123.25°E), with rows 4m long and 0.6m apart, 16 plants per row, and field management was the same as in the main field.

[0040] 1.2 Phenotypic Investigation and Data Analysis of Mu Transposon Mutants and Knockout Materials

[0041] Phenotypic analysis was conducted when maize vegetative growth ended and leaf growth ceased. The phenotypes investigated included: (1) Leaf length (LL): from the junction of the leaf and leaf sheath to the leaf apex, including the leaf length of the ear-mounted leaf (LLA), ear-positioned leaf (LLM), and ear-below leaf (LLB); (2) Leaf width (LW): the widest part of the leaf, including the leaf width of the ear-mounted leaf (LWA), ear-positioned leaf (LWM), and ear-below leaf (LWB); (3) Leaf angle (LA): the acute angle between the main vein and the vertical stem, including the leaf angle of the ear-mounted leaf (LAF), the leaf angle of the ear-mounted leaf (LAS), and the leaf angle of the ear-mounted leaf (LAT); (4) Tassel branch number (TBN): branches with more than one floret were counted as effective branches. The phenotype of each individual plant in the F2:3 ear row was investigated and the mean was calculated as the phenotype of the F2:3 ear row.

[0042] Descriptive statistical analysis and Pearson correlation analysis were performed on leaf length, leaf width, leaf angle, and number of tassel branches of the parents, F2, and F2:3 using SPSS Statistics 24.0 software (SPSS Corporation, Chicago, Illinois, USA). Statistical results were plotted using R language (https: / / www.r-project.org / ).

[0043] 1.3 Identification of Mu transposon mutants

[0044] DNA was extracted from the mutant, and primers were designed based on the insertion site of the Mu transposon. The upstream primer (537MuF) and downstream primer (537MuR) were used for PCR detection with primers specifically targeting the terminal inverted repeat (TIR) ​​sequence of the Mu mutant. Three sets of PCR tests were performed: 537MuF with TIR, 537MuR with TIR, and 537MuF and 537MuR. Water and wild-type were added as negative and positive controls, respectively. Agarose gel electrophoresis was used to screen for mutants, and self-crossing was performed to obtain homozygous mutants for propagation.

[0045] 537MuF(SEQ ID NO.5):GTGGAGAGCATGAGCAGTCT;

[0046] 537MuR (SEQ ID NO. 6): AAGGTAAGTGCGCCTCTTCA;

[0047] TIR (SEQ ID NO. 7): CGCCTCCATTTCGTCGAATCCCCT.

[0048] 1.4 Construction and identification of knockout transgenic lines

[0049] Based on the CDS sequence of the candidate gene ZmFBP6, a 5'-NGG-3' sequence was designed, and sgRNA-1 and sgRNA-2 were designed. Amplification was performed using the intermediate vector pCBC-MT1T2 (Catalog: 50593) as a template. Two PCR products containing the target sequence were purified and recovered. The PCR products and the pBUE411 vector were ligated using 10×BsaI restriction enzyme and high-concentration T4 ligase. The ligation was performed on *E. coli*, and single colonies were selected for identification. Correctly ligated single colonies were selected, cultured, and the plasmid was extracted and stored at -20℃. Using the B104 maize inbred line as material, recombinant *Agrobacterium* was used for infection. Genetic transformation experiments were performed by Boyuan Company. DNA was extracted from the T1 generation knockout material, and specific primers (T1-F / R, T2-F / R) were designed approximately 150 bp before and after the two target sites. After confirming the size of the PCR product using agarose gel electrophoresis, sequencing was performed, and the results were compared with the B104 genome to confirm whether mutations occurred. At the same time, Cas9 protein was amplified in the edited material, and materials without Cas9 protein were screened for self-crossing.

[0050] T1-F (SEQ ID NO.8):GATTTGACACGCGTTTCTTC;

[0051] T1-R (SEQ ID NO.9):GCTTCTTTACGGTTAGGGTG;

[0052] T2-F (SEQ ID NO. 10): GCCATGCAGGTTTGCTTAACC;

[0053] T2-R (SEQ ID NO. 11): AGCTTAGCTAAATATGCCATGC.

[0054] Construction of the 1.5ZmFBP6 gene expression profile

[0055] Using the reference genome sequence of maize inbred line B73 as a template, the expression level of ZmFBP6 gene in different tissues was amplified and analyzed.

[0056] 1.6ZmFBP6 Subcellular Localization

[0057] 1.6.1 Carrier Construction

[0058] First, the signal peptide of ZmFBP6 was predicted using the online tool SignalP5.0 (https: / / services.healthtech.dtu.dk / service.php?SignalP-5.0). After confirming the absence of a signal peptide, the target gene ZmFBP6 (sequence shown in SEQ ID NO.1) was cloned. The target gene was then purified using gel extraction and ligated into the pMD18-T vector. The reaction system is shown in Table 1.

[0059] Table 1. Systems linked to the pMD18-T vector

[0060] Element Volume (μL) pMD18-T Vector 1 Target gene 1 Sterilized water 3 Solution I 5

[0061] The prepared system was placed in a PCR instrument and reacted at 16℃ for 30 min. The vector containing the target gene was added to *E. coli* competent cells (DH5α) that had been thawed on ice, mixed well, and placed on ice for 30 min. Afterward, it was placed in a 42℃ water bath for 90 s, immediately removed, and placed on ice for 2 min. 500 μL of antibiotic-free LB medium was added, and the mixture was activated at 37℃ and 200 rpm for 4 hours. After the tube became turbid, it was evenly spread onto LB agar (containing kanamycin) and incubated upside down overnight. Single clones were picked and added to LB liquid medium. After culturing for 3 hours, PCR amplification was performed. The bacterial cultures with correct band positions were sent to the company for first-generation sequencing. For single clones with correct sequences, the target gene plasmid was extracted. pCAMBIA1300-GFP was linearized using restriction endonucleases KpnⅠ and XbaⅠ. The reaction system is shown in Table 2.

[0062] Table 2 Double enzyme digestion reaction system

[0063] Element Usage pCAMBIA1300-GFP 1000ng Kpn I 1μL Xal I 1μL 10×NEBuffer 10μL ddH2O Fill to 50μL

[0064] Linearized pCAMBIA1300-GFP and the target gene ZmFBP6 after PCR were purified by gel extraction and homologous recombination using a seamless cloning kit. The reaction system is shown in Table 3.

[0065] Table 3 Seamless Cloning Reaction System

[0066] Element Volume (μL) 2×Flex Seamless Clon 5 Linearization of pCAMBIA1300-GFP (10-80 ng) 2 Target gene 1 <![CDATA[ddH2O]]> 1

[0067] 1.6.2 Agrobacterium-mediated transformation

[0068] Thaw Agrobacterium competent cells GV3101 on ice. Add 2 μL of homologous recombinant vector to the thawed Agrobacterium competent cells, incubate on ice for 30 min, flash freeze in liquid nitrogen for 5 min, incubate in a 37°C water bath for 10 min, incubate on ice for 5 min, add antibiotic-free LB medium to centrifuge tubes, and incubate at 28°C and 200 rpm for 3 h. Spread the turbid liquid onto solid LB medium containing kanamycin and rifampin, and incubate upside down at 28°C for 3 days. Pick single clones and amplify them. After PCR detection, perform first-generation sequencing. Select bacterial cultures with correct sequencing results, add 30% glycerol to the total volume, mix well, and store in an ultra-low temperature freezer.

[0069] 1.6.3 Instantaneous transformation of tobacco leaves

[0070] Agrobacterium-mediated propagation was carried out in 25 mL centrifuge tubes. The supernatant was discarded after centrifugation, and the bacterial culture was resuspended using the resuspension buffer. The OD value was adjusted to OD0.05. 600 =1, and the OD value of the pSoup-p19 bacterial culture was adjusted to OD using the same method. 600 =1, the bacterial suspension containing the target plasmid, the bacterial suspension containing the P2300-35S-H2B-mCherry plasmid, and the bacterial suspension containing pSoup-p19 were mixed at a volume ratio of 1:1:1 and incubated in the dark at 28℃ for 3 hours. The resuspension preparation system is shown in Table 4:

[0071] Table 4 Resuspension System

[0072] Element volume 1M MES-KOH (pH 5.6) 1mL 1M MgCl2 1mL 1M Acetyleugenone Stock Solution (AS) 15μL <![CDATA[ddH2O]]> Adjust the volume to 100 mL

[0073] Select healthy leaves, make a hole with a syringe, and inject the bacterial solution into the leaves. After injecting 3-4 leaves, cover the tobacco with a plastic bag to keep it moist, and incubate in the dark for 3 days. Observe the fluorescence signal using a confocal microscope.

[0074] 2 Results and Analysis

[0075] 2.1 Phenotypic identification of Mu transposon mutants

[0076] Based on the insertion location of the Mu transposon provided by MaizeGDB, a pair of specific primers (Mu537F, Mu537R) were designed approximately 150 bp above and below it. Identification was performed using the Mu537F / Mu537R and Mu transposon-specific primers TIR. Figure 1 A). The results showed that the homozygous mutant had an average of 5.6 tassel branches, while the wild type had an average of 8.8 tassel branches. The number of tassel branches in the mutant was significantly less than that in the wild type. Figure 1 B, Figure 1C). Further comparison of other agronomic traits between the wild type and the mutant revealed no significant differences between the mutant and the wild type in traits such as ear length, ear diameter, 100-grain weight, plant height, ear height, and number of internodes. Figure 1 D- Figure 1 I). Therefore, it is preliminarily demonstrated that ZmFBP6 is the gene that controls the number of male spike branches and has no effect on other agronomic traits.

[0077] 2.2 Phenotypic Identification of ZmFBP6 Knockout Line

[0078] To further demonstrate that ZmFBP6 is the gene regulating the number of male spike branches, two sgRNAs were designed in the ZmFBP6 exon, and gene editing was performed on the inbred line B104 using the CRISPR-Cas9 system. Based on PCR results, two homozygous knockout events were obtained: zmfbp6-1 and zmfbp6-2. zmfbp6-1 showed an addition of one base, while zmfbp6-2 showed a deletion of three bases, both resulting in frameshift mutations. Figure 2 A). An investigation of the number of tassel branches in wild-type and knockout lines revealed that the wild-type had 5.8 branches, zmfbp6-1 had 2.7 branches, and zmfbp6-2 had 2.9 branches. The wild-type had significantly more tassel branches than the knockout lines zmfbp6-1 and zmfbp6-2. Therefore, ZmFBP6 is considered to be the gene regulating the number of tassel branches in maize. Figure 2 B,C).

[0079] In addition, to further investigate whether ZmFBP6 regulates other agronomic traits, spike length, spike diameter, 100-grain weight, plant height, spike height, and internode number were investigated. The results showed no significant differences between the wild type and the knockout lines zmfbp6-1 and zmfbp6-2. Figure 2 D- Figure 2 I).

[0080] 2.3 Study on the expression pattern of ZmFBP6 gene

[0081] To further investigate the expression pattern of ZmFBP6, samples were taken from different tissues of the maize inbred line B73 and qRT-PCR experiments were performed. The results showed that ZmFBP6 was expressed in maize roots, stems, leaves, tassels, and silks, with the highest expression level in the tassels at stage V3, followed by the silks. Figure 3 The expression levels of ZmFBP6 in different tissues were analyzed. ZmFBP6 was cloned during the high expression period, and the subcellular localization vector ZmFBP6-GFP was constructed. The green fluorescence signal was then observed. Figure 4Using online methods to predict that ZmFBP6 might be localized in chloroplasts, colocalization was initially performed using chloroplast autofluorescence. However, the green fluorescence signal of ZmFBP6-GFP did not colocalize with chloroplast autofluorescence. Figure 4 B), and it may be located in the cell nucleus. Co-localization of ZmFBP6-GFP with nuclear Maker was again performed, and the results showed that ZmFBP6-GFP and nuclear Maker were located at the same location, indicating that ZmFBP6 is located in the cell nucleus. Figure 4 C).

[0082] Although preferred embodiments of the invention have been described, those skilled in the art, upon learning the basic inventive concept, can make other changes and modifications to these embodiments. Therefore, the appended claims are intended to be interpreted as including both the preferred embodiments and all changes and modifications falling within the scope of the invention.

[0083] Obviously, those skilled in the art can make various modifications and variations to this invention without departing from its spirit and scope. Therefore, if these modifications and variations fall within the scope of the claims of this invention and their equivalents, this invention also intends to include these modifications and variations.

Claims

1. ZmFBP6 The application of genes in regulating the branching development of maize tassels is characterized by, The ZmFBP6 The gene sequence is shown in SEQ ID NO.1, and it is obtained by inhibiting the above. ZmFBP6 Gene expression reduces the number of branches on the maize tassel.

2. A method for reducing the number of branches on a maize tassel, characterized in that, include: The corn as described in claim 1 ZmFBP6 Gene knockout was used to screen for new maize varieties with reduced tassel branch numbers, thus introducing the aforementioned gene knockout gene into the maize cultivar. ZmFBP6 Gene knockout is achieved using a CRISPR-Cas9 system consisting of sgRNA and Cas9, and the sequence of the DNA molecule encoding the sgRNA is shown in any one of SEQ ID NO. 3~4.

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