Maize gene ZmHB14a, cloning method and application

Abstract

The present application relates to a maize ZmHB14a gene, a cloning method and application.The nucleotide sequence of the gene is shown in SEQ ID No.1, the encoded amino acid sequence is shown in SEQ ID No.2, the gene can regulate maize inflorescence meristem development and ear length and ear row, and can be used for maize breeding, thereby providing a new genetic resource for improving crop quality and yield.

Description

Technical Field

[0001] This invention belongs to the field of agricultural biotechnology, and specifically relates to a ZmHB14a gene and its cloning method, and its application in the development of maize inflorescence meristem and the control of ear length and ear row. Background Technology

[0002] As the world's largest food crop, maize (Zea mays) directly impacts food security and energy supply. The FAO predicts that global maize demand will increase by more than 67% by 2050 compared to current levels. However, in recent years, factors such as climate change, frequent pests and diseases, and the incomplete realization of genetic improvement potential have combined to make the current yield growth rate (approximately 1.5% annually) insufficient to meet this demand, leading to a widening yield gap. As the core organ responsible for maize yield, the development of the female ear directly determines the number of kernels per ear and the kernel abortion rate. Its development involves key biological events such as maintaining meristematic tissue activity, floret differentiation efficiency, and sink-source coordination. For gramineous crops, inflorescence length and the number of branches largely determine yield. Currently, considerable research interest is focused on increasing ear length and the number of rows per ear. Studies have shown that an increase of 1 cm in ear length or one additional row per ear can increase yield by 3%-5%. Traditional methods for improving maize ear traits rely on phenotypic selection, which is time-consuming and easily influenced by the environment.

[0003] In recent years, with the rapid development of functional genomics and molecular design, significant progress has been made in the study of spikelet development regulation: transcription factors such as KNOX and YABBY have been shown to influence spikelet formation by regulating the activity of meristematic stem cells; the spatiotemporal distribution gradients of plant hormones (such as auxin and cytokinin) have been shown to dominate the polar differentiation of spikelet primordia. However, existing research has largely focused on model crops such as Arabidopsis thaliana and rice, while the unique inflorescence structure and complex spikelet differentiation process of maize still have many unknown regulatory nodes. Therefore, elucidating the molecular regulatory network of maize spikelet development has become a key scientific problem in overcoming yield bottlenecks. Summary of the Invention

[0004] The purpose of this invention is to provide a gene that regulates the size of maize inflorescence meristem and the length and number of rows of ears. Mutation of this gene leads to an increase in the size of maize inflorescence meristem and an increase in ear length and the number of rows of ears.

[0005] This invention is achieved through the following technical solution:

[0006] The present invention provides the maize ZmHB14a gene, the nucleotide sequence of which is shown in SEQ ID No.1.

[0007] Furthermore, the amino acid sequence encoding the maize ZmHB14a gene is shown in SEQ ID No. 2.

[0008] This invention provides a method for cloning the maize ZmHB14a gene described above, comprising the following steps:

[0009] An F2 population was constructed using the *Tegus davidii* fragment introgression line TM89 and MO17. Based on the remaining heterozygous lines, QTL-qIMS3 was finely mapped to determine the target region. QTL-qIMS3 is located at position 112.3-145.1 Mb on maize chromosome 3.

[0010] Within the target region, candidate genes were identified by combining searches in public RNA-seq databases and cDNA detection results from female ears of TM89 and MO17.

[0011] Plant DNA was extracted and amplified by PCR. The cloned gene ZmHB14a of maize was obtained by sequencing correctly.

[0012] Furthermore, the fine localization involves designing InDel markers within the QTL-qIMS3 region, screening superior exchange individuals through electrophoresis and KASP genotyping, and positioning the QTL-qIMS3 region within the 326kb target segment.

[0013] More preferably, the CTAB method is used to extract DNA from single plants.

[0014] This invention also provides the application of the above-mentioned maize ZmHB14a gene in any of the following:

[0015] (1) Application in regulating maize inflorescence meristems;

[0016] (2) Application in regulating maize ear length;

[0017] (3) Application in regulating the number of rows in maize ears;

[0018] (4) Application in regulating maize yield.

[0019] Furthermore, the application is achieved by inhibiting or knocking out the expression of the ZmHB14a gene.

[0020] This invention also provides the application of the above-mentioned ZmHB14a gene in marker-assisted breeding or trait improvement breeding of maize, wherein the marker-assisted breeding or trait improvement breeding of maize has any of the following objectives:

[0021] (1) Regulating the size of maize inflorescence meristems;

[0022] (2) Regulating corn ear length;

[0023] (3) Adjust the number of rows in the corn ear;

[0024] (4) Regulate corn yield.

[0025] Furthermore, the application involves gene editing to knock out the ZmHB14a gene in the plant.

[0026] Furthermore, the gene editing employed the CRISPR / Cas9 method.

[0027] The present invention has the following advantages and effects:

[0028] This invention provides a gene that can regulate the development of maize inflorescence meristem and ear length and row number, enriching the genetic resources for maize yield formation. Furthermore, the ZmHB14a gene directly affects ear length and grain yield by regulating meristem cell proliferation, and can simultaneously regulate meristem size, ear length, and ear row number, breaking through the limitations of single-trait improvement. This invention elucidates the genetic basis of ZmHB14a's regulation of maize inflorescence meristem size, and is also of significant value for theoretical research on the molecular mechanisms of maize ear development. Attached Figure Description

[0029] Figure 1 Phenotypic diagrams of MO17 and TM89 are shown, where: A: Chromosome diagrams of MO17 and TM89 (white segments represent MO17 chromosome segments, black segments represent teosinte segments), young spikelets (bar=250μm) and mature spikelets (bar=3cm), MO17 (left) and TM89 (right); B: Statistical comparison of spikelet and mature spikelet traits of MO17 and TM89; *** indicates p <0.001;

[0030] Figure 2 This study aims to finely map maize qIMS3 and identify candidate genes. A represents fine mapping of qIMS3; t-tests are used for significance testing; *, **, and *** represent significance at the 0.05, 0.01, and 0.001 levels, respectively; BC represents validation of differentially expressed genes in candidate regions.

[0031] Figure 3 Functional validation of candidate gene ZmHB14a; where A: schematic diagram of ZmHB14a gene editing; B: comparison images of young and mature panicles of hb14a knockout mutant and wild type; CF: statistical comparison of panicle traits of hb14a knockout mutant and wild type; *** indicates p < 0.001. Detailed Implementation

[0032] The present invention will be further illustrated below with reference to specific embodiments, but these embodiments do not limit the present invention in any way. Unless otherwise specified, the reagents, methods, and equipment used in the present invention are conventional reagents, methods, and equipment in this technical field. Unless otherwise specified, the reagents and materials used in the following embodiments are commercially available.

[0033] The following embodiments are used to illustrate the present invention, but are not intended to limit the scope of the invention. Any modifications or substitutions made to the methods, steps, or conditions of the present invention without departing from the spirit and substance of the invention are within the protection scope of the present invention.

[0034] Example 1: Cloning of maize ZmHB14a

[0035] Experimental materials: TM89, a line with a replacement of the teosinte fragment (the genome within the QTL-qIMS3 region is entirely composed of the teosinte fragment, and other regions are basically reverted to the recurrent parent MO17 genotype); MO17.

[0036] 1.1 Fine-grained localization population construction in qIMS3

[0037] The applicant's research group previously detected a QTL-qIMS3 controlling the size of inflorescence meristem at a location of 112.3-145.1 Mb on chromosome 3. This QTL also significantly affects ear length and kernel number per row in maize. Using the teosinte fragment replacement line TM89 as the male parent and MO17 as the female parent, an F2 population was constructed using TM89 and MO17 to perform fine mapping of qIMS3.

[0038] Specifically, in the winter of 2021, an F2 population of 572 individual plants (TM89 × MO17 F1) was constructed through self-pollination at the Huazhong Agricultural University Nanfan Base in Sanya, Hainan Province (18 °N, 109 °E). The plants were 3 m long, with a plant spacing of 25 cm and a row spacing of 60 cm, and were managed under normal field conditions. Ears were harvested at maize maturity, air-dried, and abnormal ears were removed. Ear traits such as ear length, ear diameter, number of rows per ear, and number of kernels per row were assessed for each individual plant. A t-test was used to examine the significance of differences in traits between chromosome segments originating from MO17 and *Tegus spp.* within the specified intervals.

[0039] In the spring and winter of 2022, seven types of exchanged plants screened from the F2 population in 2021 were planted in Gansu (37°N, 100°E) and Sanya, Hainan (18°N, 109°E) for progeny testing. The materials used for progeny testing were planted in 10 rows, 3 m long, with a plant spacing of 25 cm, 12 plants per row, and a row spacing of 60 cm, under normal field management. Ears were harvested at maize maturity, air-dried, and abnormal ears were removed. Ear traits such as ear length, ear diameter, number of rows per ear, and number of kernels per row were examined for each plant. A t-test was used to examine the significance of differences in traits between chromosome segments originating from MO17 and *Tegus spp.* within the specified intervals.

[0040] In the spring of 2023, seven types of exchanged individual plants selected from the progeny test population in 2022 were planted in Xiangyang (32 °N, 112 °E). Progeny testing was conducted, with each material planted in 6 rows, 3 m row length, 25 cm plant spacing, and 60 cm row spacing, under normal field management. Ears were harvested at maize maturity, air-dried, and abnormal ears were removed. Ear traits such as ear length, ear diameter, number of rows per ear, and number of kernels per row were assessed for each individual plant. A t-test was used to examine the significance of differences in traits between chromosome segments originating from MO17 and *Tetrandrus spp.* within the specified intervals.

[0041] like Figure 1 As shown, TM89 and MO17 differ significantly in inflorescence meristem size and spike length.

[0042] 1.2 Fine positioning of qIMS3

[0043] Using the IBM population public database in the MaizeGDB database, we checked for the presence of common InDel markers within this interval. Since insertions and deletions had occurred in the 5'UTR and 3'UTR regions of the gene, primers were designed in the UTR region of the gene, referencing the B73 genome sequence within the QTL confidence interval. Amplification was performed using MO17 and TM89 as templates. Sanger sequencing was used to screen for insertion / deletion sites. InDel markers were designed for large insertions and deletions, and kasp markers were designed for SNP sites. Primers were designed online using Primer3 Plus, and the PCR product size was approximately 150 bp.

[0044] By utilizing the two KASP markers M designed 2 / 3 and four indel markers M 0 / 1 / 4 / N A scan of the F2 population constructed from TM89×MO17 revealed seven types of exchanged individuals. These seven types of exchanged individuals were used for multi-site planting to test offspring, and the results were further analyzed using the encrypted marker M. 5 / 6 / 7 / 8 Seven new recombination types were obtained, and offspring were tested at multiple locations. Ultimately, qIMS3 was located within a 326kb region. Figure 2 As shown in A in the diagram.

[0045] 1.3 Differential Gene Analysis of Candidate Target Segments

[0046] Within the 326Kb target region identified in section 1.2, candidate genes were determined by combining searches in public RNA-seq databases and detection results of female ear cDNA from TM89 and MO17. Then, plant DNA was extracted and subjected to PCR amplification; those sequenced correctly were clones of the maize gene ZmHB14a.

[0047] Genotyping analysis involves DNA extraction and PCR amplification. Single-plant DNA extraction uses the CTAB method, with the following specific steps:

[0048] 1. Add 700 μL of CTAB (83.5 mM Tris-HCl, pH 8.0, 16.7 mM EDTA pH 8.0, 1.17M NaCl, 1.67% CTAB) to a mortar, take an appropriate amount of fresh young leaves and grind them on ice until homogenized, then transfer them to a 2 mL centrifuge tube.

[0049] 2. Incubate in a 65°C water bath for 40 minutes, inverting and mixing several times during the process;

[0050] 3. After cooling to room temperature, add an equal volume of chloroform:isoamyl alcohol (24:1, V / V), gently shake to mix for about 15 min, and then centrifuge at 12000 rpm for 10 min.

[0051] 4. Transfer 200 μL of supernatant to another new 1.5 mL centrifuge tube, add twice the volume of ice-cold ethanol, gently shake to mix, and let stand for 30 min.

[0052] 5. Centrifuge at 12000 rpm for 10 min, discard the supernatant. Wash the precipitate twice with 75% ethanol;

[0053] 6. Centrifuge to remove supernatant, let dry at room temperature, add 100 μL ddH2O to dissolve, and store at -20℃ for later use.

[0054] The PCR amplification reaction system is shown in Table 1, and the PCR reaction procedure is shown in Table 2.

[0055] Table 1 PCR reaction system

[0056]

[0057] Table 2 PCR reaction procedure

[0058] The genotyping of individual plants was performed using a combination of 4% agarose gel electrophoresis and KASP typing at Wuhan Jingtai Biotechnology Co., Ltd.

[0059] Specifically, the process for identifying the candidate gene ZmHB14a is as follows: Within the 326 kb target region, the maize reference genome (B73 RefGen_V4.0) showed three candidate genes: Zm00001d041488, Zm00001d041489, and Zm00001d041491. The expression levels of these three genes in different tissues were analyzed using a public RNA-seq database. Only Zm00001d041489 was found to be specifically highly expressed in the inflorescence meristem and young spikelets, while the other two genes were not expressed at all. Furthermore, qPCR was performed using cDNA from 3-5 mm female spikes of TM89 and MO17, revealing that only Zm00001d041489 was significantly expressed in TM89 / MO17 young spikelets, while the other two genes were not significantly expressed (e.g., ...). Figure 2 (BC in the text). Based on this, it is inferred that Zm00001d041489 is a candidate gene for qIMS3. Since Zm00001d041489 is homologous to AtHB14 in Arabidopsis thaliana, it is named ZmHB14a.

[0060] Example 2: Functional verification of maize ZmHB14a

[0061] To determine the gene function of ZmHB14a, the gene was edited using CRIPSR / Cs9 to obtain a ZmHB14a knockout mutant, which was then used in field trials and phenotypic identification. Genotyping analysis involved DNA extraction and PCR amplification. DNA was extracted from individual plants using the CTAB method, as detailed in Example 1.

[0062] In the spring and winter of 2023, homozygous T2 generation mutants and wild-type mutants with different knockout types were planted in Wuhan (30 °N, 114 °E) and Sanya, Hainan Province (18 °N, 109 °E), respectively. Each knockout type was planted in 12 rows, including 6 rows of wild-type and 6 rows of homozygous mutants. Rows were 3 m long, with a plant spacing of 25 cm and a row spacing of 60 cm, and 12 plants per row. Normal field management was implemented. All materials were open-pollinated. After harvest, uniform, plump, mature ears were selected from each row for phenotypic measurements, including ear length, ear diameter, number of rows per ear, and number of kernels per row. A t-test was used to examine the significance of differences in each trait between the two materials.

[0063] refer to Figure 3 Phenotypic analysis of the ZmHB14a knockout mutant and its corresponding wild type at two time points in one year showed that the ZmHB14a knockout mutant had significantly larger inflorescence meristems in the early stage than the wild type, and exhibited longer ear length and more ear rows at maturity. Based on these results, the application of this gene in breeding processes can be guided to effectively improve maize yield.

[0064] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the technical principles of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

[0065] The technical features of the above embodiments can be combined in any way. For the sake of brevity, not all possible combinations of the technical features in the above embodiments are described. However, as long as there is no contradiction in the combination of these technical features, they should be considered to be within the scope of this specification.

[0066] The embodiments described above are merely illustrative of several implementations of the present invention, and while the descriptions are relatively specific and detailed, they should not be construed as limiting the scope of the invention patent. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of the present invention, and these all fall within the protection scope of the present invention. Therefore, the protection scope of this invention patent should be determined by the appended claims.

Claims

1. The maize ZmHB14a gene, characterized by, The nucleotide sequence of the ZmHB14a gene is shown in SEQ ID No.

1.

2. The maize ZmHB14a gene according to claim 1, characterized in that, The encoded amino acid sequence is shown in SEQ ID No.

2.

3. The cloning method for the maize ZmHB14a gene as described in claim 1 or 2, characterized in that: Includes the following steps: An F2 population was constructed using the introgression lines TM89 and MO17 of the teosinte fragment. The QTL-qIMS3 was finely mapped using the remaining heterozygous lines to determine the target segment. The QTL-qIMS3 is located at position 112.3-145.1 Mb on chromosome 3 of maize. Within the target region, candidate genes were identified by combining searches in public RNA-seq databases and cDNA detection results from female ears of TM89 and MO17. Plant DNA was extracted and amplified by PCR. The cloned gene ZmHB14a of maize was obtained by sequencing correctly.

4. The cloning method according to claim 3, characterized in that, The fine localization involves designing InDel markers within the QTL-qIMS3 region, and then using electrophoresis and KASP typing to select superior exchange individuals to locate the QTL-qIMS3 region within the 326kb target segment.

5. The cloning method according to claim 3, characterized in that, DNA was extracted from individual plants using the CTAB method.

6. The use of the maize ZmHB14a gene as described in claim 1 or 2 in any of the following: (1) Application in regulating maize inflorescence meristems; (2) Application in regulating maize ear length; (3) Application in regulating the number of rows in maize ears; (4) Application in regulating maize yield.

7. The application according to claim 6, characterized in that, The application is achieved by inhibiting or knocking out the expression of the ZmHB14a gene.

8. The application of the ZmHB14a gene as described in claim 1 or 2 in marker-assisted breeding or trait improvement breeding of maize, wherein the marker-assisted breeding or trait improvement breeding of maize has any of the following objectives: (1) Regulating the size of maize inflorescence meristem; (2) Regulating corn ear length; (3) Adjust the number of rows in the corn ear; (4) Regulate corn yield.

9. The application according to claim 8, characterized in that, The application involves gene editing to knock out the ZmHB14a gene in the plant.

10. The application according to claim 9, characterized in that, The gene editing described uses the CRISPR / Cas9 method.

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