Application of SPL23 gene in regulating the number of branches on maize tassels

Abstract

This invention discloses the application of the SPL23 gene in regulating the number of tassel branches in maize, including: applying a miR529-insensitive form of the SPL23 gene to regulate the number of tassel branches in maize. The method for increasing the number of tassel branches in maize is characterized by: constructing a vector containing a miR529-insensitive form of the SPL23 gene; and transforming the constructed miR529-insensitive form of the SPL23 gene into maize to obtain maize plants with an increased number of tassel branches. This invention, by constructing miR529-insensitive SPL23 plants, enables the SPL23 gene to resist miR529 degradation and thus undergo overexpression, significantly promoting tassel development and increasing the number of tassel branches, providing an effective approach for the genetic improvement of maize varieties with fewer male tassels.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology, and in particular relates to the application of the SPL23 gene in regulating the number of branches on maize tassels. Background Technology

[0002] Corn is one of the world's most widely cultivated major food and feed crops, and also an important raw material for industrial processing. Corn starch and straw can be used to process ethanol, which is one of the effective ways to solve the energy crisis. With the gradual increase in global demand for food, the importance of corn in future food production is receiving increasing attention.

[0003] The tassel trait in maize is one of the important yield traits. A suitable tassel structure is key to developing high-yielding and high-quality maize varieties and is also a key research direction in modern molecular breeding. Inbred lines of maize varieties with few tassels show a gradual decrease in the number of tassel branches in their offspring. Varieties with too few tassel branches typically have less pollen, a shorter pollen shedding period, and weaker resistance to abiotic stresses such as high temperatures, easily leading to grain filling problems. Furthermore, currently discovered genes regulating tassel branching development are mostly quantitative trait loci (QTLs), with a limited number of individual genes. Moreover, many of the discovered individual genes affect other agronomic traits, such as plant height and ear development, thus hindering their direct application to genetic improvement. In the North China Plain and the Yellow River Basin of my country, summers are easily affected by continental warm high-pressure ridges and solar radiation, resulting in prolonged periods of high temperatures. Maize varieties with few tassels have poor resistance to heat damage, leading to reduced summer maize harvests. Therefore, identifying key genes regulating tassel branching development and maintaining a reasonable tassel structure is an important breeding goal for ensuring maize yield. Summary of the Invention

[0004] This invention provides a method for increasing the number of male tassel branches in maize using the SPL23 gene.

[0005] An SPL23 gene, characterized in that the miR529-insensitive form of the SPL23 gene is applied to regulate the number of male tassel branches in maize.

[0006] A method for increasing the number of branches on a maize tassel includes:

[0007] Construct a vector for the miR529-insensitive form of the SPL23 gene;

[0008] Transforming the miR529-insensitive form of the SPL23 gene into maize resulted in maize plants with an increased number of tassel branches.

[0009] Furthermore, it also includes:

[0010] By taking advantage of codon degeneracy, the miR529 target site of the SPL23 gene was edited, resulting in a base mutation in the specific sequence of the SPL23 gene mRNA without changing the types of amino acids, thus obtaining the miR529-insensitive form of the SPL23 gene rSPL23.

[0011] Furthermore, it also includes:

[0012] rSPL23 transgenic maize plants were obtained by transforming maize inbred line C01 using Agrobacterium-mediated transformation.

[0013] Furthermore, it also includes:

[0014] Base mutations in the SPL23 gene were designed using SnapGene Viewer software, homology sequence alignment, and codon degeneracy.

[0015] The mutated rSPL23 gene sequence was used to construct a miR529-insensitive SPL23 gene vector in the cloning island plant vector using the commercial In-Fusion PCR Cloning Kit.

[0016] Furthermore, the base mutation sequence of the SPL23 gene was designed to be the 5'-TCTCTCTCTTCT-3' sequence at 3163-3174 bp of the SPL23 gene, with the mutation point being 5'-CTTGAGCCTCCT-3'.

[0017] The target plant includes, but is not limited to, monocotyledonous or dicotyledonous plants. More preferably, the target plant is corn.

[0018] This invention discloses the application of the SQUAMOSA promoter-binding protein-like 23 (SPL23) gene in regulating maize tassel development, specifically the application of the miR529 insensitive form of the SPL23 gene in regulating the number of branches on maize tassels. This invention constructs a miR529-insensitive form of the SPL23 gene (i.e., the miR529-resistant SPL23 gene, abbreviated as rSPL23). Utilizing codon degeneracy, a base alteration is made in the SPL23 gene, preventing the SPL23 transcript from being recognized and cleaved by miR529 without affecting its protein composition, thus achieving overexpression of the SPL23 gene. Results show that the number of tassel branches in the miR529-insensitive rSPL23 transgenic plants is significantly increased. This indicates that the miR529-insensitive form of the SPL23 gene can induce an increased tassel branching phenotype in transgenic maize plants. This invention provides a theoretical basis for increasing the number of tassel branches in maize and for breeding and improving maize varieties with fewer male tassels, and has significant application prospects. Attached Figure Description

[0019] To more clearly illustrate the technical solutions in the embodiments of the present invention, the accompanying drawings used in the description of the embodiments will be briefly introduced below. Obviously, the accompanying 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.

[0020] Figure 1 A schematic diagram illustrating the target site of miR529 recognizing the SPL23 gene transcript, provided for embodiments of the present invention;

[0021] Figure 2 A schematic diagram of the rSPL23 carrier spectrum provided for an embodiment of the present invention;

[0022] Figure 3 A schematic diagram showing the comparison of the SPL23 gene sequence between wild-type (WT) and rSPL23 transgenic plants provided for embodiments of the present invention;

[0023] Figure 4 A schematic diagram of the phenotype of the transgenic plant provided for an embodiment of the present invention. Detailed Implementation

[0024] To enable those skilled in the art to better understand the present invention, the technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings.

[0025] An SPL23 gene, using a miR529-insensitive form of the SPL23 gene to regulate the number of male tassel branches in maize.

[0026] A method for increasing the number of branches on a maize tassel includes:

[0027] Step 1: Construct a vector insensitive to miR529 on the SPL23 gene;

[0028] Specifically, by taking advantage of the degeneracy of codons, the miR529 target site of the SPL23 gene is edited, causing a base mutation in the specific sequence of the SPL23 gene mRNA without changing the types of amino acids, thus obtaining a miR529-insensitive form of the SPL23 gene (rSPL23).

[0029] Specifically, base mutations in the SPL23 gene were designed using SnapGene Viewer software, homologous sequence alignment, and codon degeneracy.

[0030] The mutated rSPL23 gene sequence was used to construct a miR529-insensitive SPL23 gene vector in the cloning island plant vector using the commercial In-Fusion PCR Cloning Kit.

[0031] SQUAMOSA promoter-binding protein-like (SPL) proteins are a class of transcription factors unique to plants and widely distributed in green plants. SPLs play important regulatory roles in multiple physiological and biochemical processes, including plant growth regulation, developmental stage transitions, morphogenesis, fruit development, and responses to abiotic stress. MicroRNAs (miRNAs) are a class of small, non-coding RNAs approximately 21 nucleotides in length, ubiquitously present in organisms. They play a crucial role in regulating plant organ development, signal transduction, and responses to abiotic stress. miRNAs primarily participate in post-transcriptional gene regulation, and in plants, they can induce mRNA degradation or translational repression through specific base pairing with target mRNAs.

[0032] The degeneracy of the genetic code refers to the fact that in several triplet genetic codes encoding the same amino acid, the first and second bases are mostly the same, only the third base is different. For example, ACU, ACC, ACA, and ACG are all codons for threonine, while UGU, UGC, UGA, and UGG are all codons for valine. Thus, if a point mutation occurs at the third base of a codon, it does not affect the type of amino acid translated, nor does it affect the final protein product.

[0033] Step 2: Transform the constructed miR529-insensitive form of the SPL23 gene into maize to obtain maize plants with increased tassel branching.

[0034] Specifically, rSPL23 transgenic maize plants were obtained by transforming maize inbred line C01 using Agrobacterium-mediated transformation.

[0035] One embodiment of the present invention is as follows:

[0036] Step 1: Construction of the miR529-insensitive vector for the maize SPL23 gene;

[0037] First, using SnapGene Viewer software and homologous sequence alignment and codon degeneracy, base mutations were designed in the SPL23 gene. The 5'-TCTCTCTCTTCT-3' sequence at 3163–3174 bp of the SPL23 gene was mutated to 5'-CTTGAGCCTCCT-3'. miR529 recognizes target sites of the SPL23 gene transcript, such as... Figure 1 As shown, Figure 1miR529 identifies target sites and encoded amino acids of SPL23 and rSPL23 transcripts. Red text indicates rSPL23 mutant bases. This represents a codon reading frame. A, adenine; G, guanine; C, cytosine; U, uracil. Ser, serine; Cys, cysteine; Ala, alanine; Leu, leucine.

[0038] Step 2: The mutated rSPL23 sequence was cloned into a plant vector using a commercial In-Fusion PCR Cloning Kit. The resulting miR529-insensitive SPL23 gene vector, after being sequenced correctly, was used for subsequent genetic transformation.

[0039] The constructed vector has the sequence shown in SEQ ID NO:(3), and its map is shown in... Figure 2 As shown, rSPL23 transgenic maize plants were obtained by transforming maize inbred line C01 using Agrobacterium-mediated transformation.

[0040] Results: The SPL23 gene in rSPL23 transgenic plants was amplified by PCR, and the PCR product was sequenced and compared with the wild-type sequence. Results are as follows. Figure 3 As shown, Figure 3 Sequence alignment of the SPL23 gene between wild-type (WT) and rSPL23 transgenic plants. MT represents rSPL23 base mutant plants. Red text indicates the mutated bases in rSPL23. Figure 4 This represents the phenotype of the transgenic plant.

[0041] This invention overcomes the deficiencies and shortcomings of existing technologies in the fertility of maize varieties with few male reproductive organs under adverse stresses such as high temperature and drought, and provides an application of the SPL23 gene in increasing the number of male reproductive branches in maize ears. In this invention, utilizing the characteristic that miR529 can target and specifically degrade SPL23 mRNA, by mutating the site of SPL23 gene recognized by miR529 without altering the amino acid sequence, transgenic plants overexpressing SPL23 in a miR529-insensitive form were constructed. These plants exhibited a phenotype with a significantly increased number of male reproductive branches, while other agronomic traits remained largely unchanged. This invention, by constructing miR529-insensitive SPL23 plants, enables the SPL23 gene to be overexpressed in resistance to miR529 degradation, thereby significantly promoting male reproductive development and increasing the number of male reproductive branches, providing an effective approach for the genetic improvement of maize varieties with few male reproductive organs.

[0042] This invention utilizes the degeneracy of codons to edit the SPL23 gene, with the sequence shown in SEQ ID NO:(1) at the miR529 target site, causing a base mutation in the specific sequence of the SPL23 gene mRNA, but without changing the types of amino acids, resulting in the sequence shown in SEQ ID NO:(2). This sequence was then transformed into maize to obtain rSPL23 transgenic plants. The results showed that the number of tassel branches in the rSPL23 mutant plants increased, demonstrating the application value of SPL23 overexpression after escaping the inhibition of miR529 in increasing the number of tassel branches.

[0043] The following gene sequences are shown below:

[0044] (1)SEQ ID NO:(1)SPL23 gene sequence:

[0045] (2)SEQ ID NO:(2)rSPL23 gene sequence:

[0046] (3)SEQ ID NO:(3)rSPL23 vector sequence

[0047] Obviously, the above embodiments are merely illustrative examples for clear explanation and are not intended to limit the implementation. Those skilled in the art will recognize that other variations or modifications can be made based on the above description. It is neither necessary nor possible to exhaustively list all possible implementations here. However, obvious variations or modifications derived therefrom are still within the scope of protection of this invention.

[0048] The above description is only a partial 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 principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. An SPL23 gene, characterized in that, The miR529-insensitive form of the SPL23 gene was applied to regulate the number of male tassel branches in maize.

2. A method for increasing the number of branches on a maize tassel, characterized in that, include: Construct a vector for the miR529-insensitive form of the SPL23 gene; Transforming the miR529-insensitive form of the SPL23 gene into maize resulted in maize plants with an increased number of tassel branches.

3. The method according to claim 2, characterized in that, Also includes: By taking advantage of codon degeneracy, the miR529 target site of the SPL23 gene was edited, resulting in a base mutation in the mRNA sequence of the SPL23 gene without changing the types of amino acids, thus obtaining the miR529-insensitive form of the SPL23 gene, rSPL23.

4. The method according to claim 2, characterized in that, Also includes: rSPL23 transgenic maize plants were obtained by transforming maize inbred line C01 using Agrobacterium-mediated transformation.

5. The method according to claim 2, characterized in that, Also includes: Base mutations in the SPL23 gene were designed using SnapGene Viewer software, homology sequence alignment, and codon degeneracy. The mutated rSPL23 gene sequence was used to construct a miR529-insensitive SPL23 gene vector in the cloning island plant vector using the commercial In-Fusion PCR Cloning Kit.

6. The method according to claim 2, characterized in that, The designed base mutation sequence for the SPL23 gene is the -TCTCTCTCTTCT-3' sequence at 3163-3174 bp of the SPL23 gene, with the mutation point being 5'-CTTGAGCCTCCT-3'.

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