Application of GhPPR3391 gene in regulating plant anther development

Abstract

The present invention discloses the application of the GhPPR3391 gene in regulating plant anther development, belonging to the field of cotton biotechnology breeding. The nucleotide sequence of the GhPPR3391 gene is shown in SEQ ID NO.1. The GhPPR3391 gene fragment disclosed in the present invention plays a key role in regulating cotton male sterility. GhPPR3391 gene knockout leads to cotton male sterility, which is manifested as shortened filaments, complete indehiscence of anthers, abnormal pollen development, and inactive pollen stained with benzidine. The present invention knocks out the GhPPR3391 gene to produce a completely sterile mutant, thereby creating a new cotton male sterile line for producing hybrid seeds, providing important technical support for cotton hybrid breeding, and can be used as a favorable gene resource for cultivating cotton germplasm, with very important applications in agricultural production.

Description

Technical Field

[0001] The present invention relates to the field of cotton biotechnology breeding, and in particular to the application of the GhPPR3391 gene in regulating plant anther development. Background Art

[0002] Cotton, a key cash crop and a vital raw material for the textile industry, exhibits significant hybrid vigor. Hybrid vigor is a ubiquitous genetic phenomenon in the biological world and is widely used in agricultural breeding, particularly in crop improvement, such as hybrid rice and hybrid cotton. Male sterile lines provide an effective tool for hybrid breeding, preventing selfing, thereby ensuring smooth outcrossing and enhancing the expression of hybrid vigor.

[0003] Male sterility refers to the failure of pollen to develop normally, resulting in the inability to produce male gametes (pollen) for sexual reproduction. Anther development is associated with multiple protein families. Down-regulation of the Arabidopsis AGP-encoding gene FLA3 causes abnormal development of the pollen inner wall and sterility. In Arabidopsis, TPD1 and EMS1 jointly regulate the development of tapetum cells. Overexpression of TPD1 leads to delayed tapetum degradation, hindering anther development and ultimately causing pollen abortion. Proteins such as MYB26 and CBSX2 can affect anther dehiscence by controlling the thickness of the inner wall of the anther chamber. Down-regulation of the cotton PME21 gene leads to thickening and delayed degradation of the early anther tapetum, abnormal pollen grain cell wall structure, and difficulty in pollen germination. During anther development, PPR proteins mainly influence pollen maturation and germ cell development by regulating mitochondrial and chloroplast gene expression. The cotton gene GhPPR3391 has a gene ID of Gh_D05G3391. The CDS of this gene is 756 bp long and encodes PPR protein. There is no report on the relationship between GhPPR3391 gene and male sterile lines. Summary of the Invention

[0004] The present invention aims to provide a method for regulating plant anther development using the GhPPR3391 gene to address the aforementioned problems in the prior art. Biotechnology is used to deactivate the GhPPR3391 gene, thereby rendering the plant male sterile. The invention also creates a nuclear male sterile cotton line for producing hybrid seeds, providing important technical support for cotton hybrid breeding.

[0005] To achieve the above object, the present invention provides the following solutions:

[0006] One of the technical solutions of the present invention is the use of the GhPPR3391 gene in regulating plant anther development. The nucleotide sequence of the GhPPR3391 gene is shown in SEQ ID NO.1.

[0007] The second technical solution of the present invention is sgRNA, whose nucleotide sequence is shown in SEQ ID NO.2.

[0008] The third technical solution of the present invention is the use of the sgRNA in preparing male sterile plants.

[0009] The fourth technical solution of the present invention is a method for preparing a plant male sterile line, which uses the sgRNA to silence or knock out the GhPPR3391 gene of the plant to be treated, inhibit the anther development of the plant, and prepare a male sterile plant.

[0010] The fifth technical solution of the present invention is the application of the GhPPR3391 gene in cotton hybrid breeding.

[0011] Based on the above technical solution, the present invention has the following technical effects:

[0012] The GhPPR3391 gene fragment disclosed in the present invention plays a key role in regulating male sterility in cotton. Knockout of the GhPPR3391 gene leads to male sterility in cotton, manifested by shortened filaments, complete indehiscence of anthers, abnormal pollen development, and lack of pollen viability when stained with benzidine. By knocking out the GhPPR3391 gene, the present invention produces a completely sterile mutant, enabling the creation of a new cotton male sterile line for producing hybrid seeds. This provides important technical support for cotton hybrid breeding and can serve as a beneficial genetic resource for cultivating cotton germplasm, with significant applications in agricultural production. BRIEF DESCRIPTION OF THE DRAWINGS

[0013] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for use in the embodiments. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative work.

[0014] Figure 1 Phenotypic analysis of T2 plants generated by gene editing with GhPPR3391. Figure A shows an overall image of an anther from the recipient material Jin668 (hereafter referred to as CK:Jin668), while Figures B, C, and D show overall images of anthers from a PPR3391 mutant plant. Figure E shows an anther from a CK:Jin668 plant, and Figures F, G, and H show anthers from a PPR3391 mutant plant.

[0015] Figure 2 The benzidine staining images of pollen viability of individual plants of CK:Jin668 and PPR3391 mutants; the pollen of CK:Jin668 was evenly stained dark red, while the staining of the PPR3391 mutant was abnormal, with no pollen coloration.

[0016] Figure 3 This is the sequence editing diagram of the PPR3391 mutant single strain in Example 3, where the sgRNA target site and PAM region are highlighted in green and red backgrounds, respectively, and the number of base deletions is shown on the right.

[0017] Figure 4 This is the amino acid editing diagram of the PPR3391 mutant in Example 3. The underlined amino acids are the normally encoded amino acids, and the amino acids in the edited pairs undergo frameshift mutations.

[0018] Figure 5 Scanning electron microscopic observations of anthers and pollen grains of the CK:Jin668 and PPR3391 mutants. The anthers of CK:Jin668 dehisced normally, and the pollen grains were plump and nearly spherical. In contrast, the anthers of the edited mutant did not dehisce, and the pollen grains were irregularly round, with shrunken and concave grains. DETAILED DESCRIPTION

[0019] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as limiting the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0020] It should be understood that the terms described herein are intended only to describe particular embodiments and are not intended to limit the present invention. In addition, for numerical ranges herein, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Each smaller range between any intermediate value within a stated value or stated range and any other stated value or intermediate value within the stated range is also encompassed by the present invention. The upper and lower limits of these smaller ranges may be independently included or excluded within the scope.

[0021] Unless otherwise indicated, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art. Although only preferred methods and materials are described herein, any methods and materials similar or equivalent to those described herein may also be used in the practice or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials associated with the documents. In the event of any conflict with any incorporated document, the contents of this specification shall prevail.

[0022] It will be apparent to those skilled in the art that various modifications and variations may be made to the specific embodiments of the present invention without departing from the scope or spirit of the invention. Other embodiments will be apparent to those skilled in the art from the present invention. The present description and examples are intended to be illustrative only.

[0023] The words “include,” “including,” “have,” “contain,” etc. used in this document are open-ended terms, meaning including but not limited to.

[0024] Unless otherwise specified, the technical solutions described in the present invention are all conventional solutions in the field, and the reagents or raw materials used, unless otherwise specified, are purchased from commercial channels or have been disclosed.

[0025] The embodiment of the present invention provides the use of the GhPPR3391 gene in regulating plant anther development. The nucleotide sequence of the GhPPR3391 gene is shown in SEQ ID NO.1.

[0026] In some specific embodiments, silencing or knocking out the GhPPR3391 gene inhibits anther development in plants and produces male sterile plants.

[0027] In some specific embodiments, the plant comprises cotton.

[0028] The present invention also provides an sgRNA, the nucleotide sequence of which is shown in SEQ ID NO. 2. The target sequence is specifically 5'-CTAAGGTTGATGGAAGGCAG-3'.

[0029] The embodiments of the present invention also provide the use of the sgRNA in preparing male sterile plants.

[0030] The embodiment of the present invention also provides a method for preparing a plant male sterile line, using the sgRNA to silence or knock out the GhPPR3391 gene of the plant to be treated, inhibiting the anther development of the plant, and preparing a male sterile plant.

[0031] The embodiments of the present invention also provide application of the GhPPR3391 gene in cotton hybrid breeding.

[0032] In some specific embodiments, the sgRNA is connected to a CRISPR / Cas9 vector and transformed into a target cotton plant through Agrobacterium-mediated method to obtain a cotton male sterile line.

[0033] The method of the present invention can produce a cotton male sterile mutant with shortened filaments, indehiscent anthers, and inactive pollen grains. Furthermore, Sanger sequencing of the transgenic T2 plants revealed that the GhPPR3391 gene was edited, manifested by base deletions that altered the amino acid sequence.

[0034] Example 1

[0035] The 1GhPPR3391 gene is located on chromosome 5 of the TM-1D genome of upland cotton. The gene is 1400 bp long and its nucleotide sequence is shown in SEQ ID NO. 1. Specifically,

[0036]

[0037] Design of 2sgRNA

[0038] The conserved region of upland cotton GhPPR3391 was selected to design the target site sgRNA (shown in SEQ ID NO.2).

[0039] Table 1 sgRNA sequences of genes

[0040]

[0041] Note: The underlined bases in Table 1 are PAM (protospacer adjacent motif).

[0042] The construction of CRISPR / Cas9 vectors and Agrobacterium-mediated transformation of cotton refer to the specific steps of Jin Shuangxia's doctoral dissertation "Optimization of cotton genetic transformation system and creation of mutants".

[0043] Example 2

[0044] Phenotypic analysis of edited plants

[0045] The edited plants showed male sterility compared to the CK:Jin668 plants. The anthers of the CK:Jin668 plants had abundant pollen grains attached to their surfaces, while the anther filaments of the gene-edited plants were significantly shorter, the anthers did not crack, and there were no pollen grains on the anther surface. Figure 1 ). Benzidine staining was used to stain the pollen grains. The pollen grains of CK:Jin668 plants were round, large, and plump, and were evenly stained red. After knocking out the anthers of sterile plants, a few pollen grains were found, which showed no color after benzidine staining ( Figure 2 ).

[0046] The specific steps of benzidine staining observation are as follows:

[0047] Viable pollen contains active peroxidase, which can use hydrogen peroxide to oxidize various polyphenols and aromatic amines to produce red substances, dyeing the pollen red.

[0048] Preparation of 0.5% benzidine solution: Dissolve 0.2g of benzidine in 100mL of 50% ethanol and place in a brown bottle to prepare 0.5% benzidine solution;

[0049] Preparation of 0.5% α-naphthol solution: Dissolve 0.15g of α-naphthol in 100mL of 50% ethanol (first dissolve it in a small amount of 95% ethanol to form a paste and then add 50% ethanol). Place in a brown bottle to prepare 0.5% α-naphthol solution.

[0050] Preparation of 0.25% Na2CO3 solution: Dissolve 0.25g Na2CO3 in 100mL distilled water to prepare 0.25% Na2CO3 solution;

[0051] Mix equal volumes of the above three solutions to prepare reagent 1, and store it in the dark at room temperature for later use.

[0052] Hydrogen peroxide with a volume fraction of 0.3% is reagent 2.

[0053] During the flowering period of cotton, after the anthers shed pollen, representative flowers were photographed. After photographing, pollen was collected in a 2 mL centrifuge tube. 100 μL of reagent 1 and 100 μL of reagent 2 were added to the tube and mixed. The tube was stained at room temperature for 10 min. During this period, the mixture was pipetted 2 to 3 times to fully release the bubbles generated by the reaction.

[0054] After staining, place 2-3 drops onto a clean glass slide and observe and photograph using a fully motorized research-grade fluorescence microscope (Olympus SZX16). Vigorous pollen appears dark red, weak pollen appears light red, and dead pollen remains unstained.

[0055] Example 3

[0056] Target site sequencing and mutation type analysis of edited plants

[0057] Primers were designed 100-200 bp upstream and downstream of the target site, and Sanger sequencing was used to detect the editing type of the mutant.

[0058] The relevant primer sequences used for amplification are as follows:

[0059] Forward primer F1: 5′-AACTTTTTCAACTTTGATAAATGGGC-3′ (SEQ ID NO. 3);

[0060] Reverse primer R1: 5'-CAACCATTTCATTCAAAAGCCTT-3' (SEQ ID NO. 4).

[0061] The sequencing results showed that there were four types of editing in the edited plants, namely, deletion of 1 base, deletion of 2 bases and deletion of 5 bases.

[0062] The deletion of one base is the deletion of position 17 of the sequence shown in SEQ ID NO.1;

[0063] The two bases deleted are the 16th and 17th bases of the sequence shown in SEQ ID NO.1;

[0064] The deletion of 5 bases is the deletion of positions 13-17 of the sequence shown in SEQ ID NO.1;

[0065] Example 4

[0066] Ultrastructural analysis of anthers and pollen grains of edited plants

[0067] The ultrastructure of anthers and pollen grains of the edited mutant plants was observed using a scanning electron microscope (hitachi SU8100). It was found that compared with CK:Jin668, the mutant plants all showed anthers that did not dehisce, and the pollen grains were shriveled and concave.

[0068] In summary, the present invention uses biotechnology to knock out the GhPPR3391 gene, resulting in loss of gene function and male sterility. Specifically, a specific sgRNA and CRISPR / Cas9 system are used to knock out the GhPPR3391 gene in cotton, creating a new sterile line material of upland cotton, which is of great significance for the creation of cotton male sterile germplasm, the utilization of hybrid vigor and population improvement.

[0069] Obviously, the above embodiments of the present invention are merely examples for the purpose of clearly illustrating the present invention and are not intended to limit the implementation methods of the present invention. A person skilled in the art will be able to make other variations or modifications based on the above description. It is not necessary and impossible to enumerate all implementation methods here. Any modifications, equivalent substitutions, and improvements made within the spirit and principles of the present invention shall be included within the scope of protection of the claims of the present invention.

Claims

1. Application of the GhPPR3391 gene in regulating plant anther development, characterized in that: The nucleotide sequence of the GhPPR3391 gene is shown in SEQ ID NO. 1; silencing or knocking out the GhPPR3391 gene inhibits plant anther development and prepares male sterile plants; the plant is cotton.

2. sgRNA, characterized in that Its nucleotide sequence is shown in SEQ ID NO.

2.

3. Use of the sgRNA according to claim 2 in preparing a male sterile plant, wherein the sgRNA targets and silences or knocks out the plant GhPPR3391 gene, the plant is cotton, and the nucleotide sequence of the GhPPR3391 gene is shown in SEQ ID NO.

1.

4. A method for preparing a plant male sterile line, characterized in that: The sgRNA according to claim 2 is used to silence or knock out the GhPPR3391 gene of the treated plant, thereby inhibiting the anther development of the plant and preparing a male sterile plant; the nucleotide sequence of the GhPPR3391 gene is shown in SEQ ID NO.1, and the plant is cotton.

5. Application of the GhPPR3391 gene in cotton hybrid breeding. The nucleotide sequence of the GhPPR3391 gene is shown in SEQ ID NO.

1. Silencing or knocking out the GhPPR3391 gene inhibits cotton anther development and produces male-sterile cotton.

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