Method for obtaining haploid pennisetum

Abstract

This invention belongs to the field of plant genetic engineering and provides a method for obtaining haploid *Pennisetum purpureus*. The method involves selecting at least one of three target sites on the PaDMP8 gene of *Pennisetum purpureus* 'Liqiu', constructing a gene-editing vector based on the target site, and transforming the gene-editing vector into *Pennisetum purpureus* 'Liqiu' to obtain haploid plants. This invention, through gene-editing technology, knocks out the PaDMP8 gene in *Pennisetum purpureus* 'Liqiu', constructing a haploid induction line for the first time, and successfully obtaining haploid plants through this induction line. This demonstrates the feasibility of haploid induction using the PaDMP8 gene in *Pennisetum purpureus*, providing a solid foundation for double haploid breeding technology of *Pennisetum purpureus*.

Description

Technical Field

[0001] This invention belongs to the field of plant genetic engineering, and specifically relates to a method for obtaining haploid Napier grass. Background Technology

[0002] Napier grass, as a high-quality forage, ornamental grass, energy grass, and ecological grass, is widely used globally due to its strong resistance to adverse conditions and high yield. Currently, Napier grass breeding mainly relies on conventional methods such as introduction and domestication, hybridization, and mutagenesis. However, these techniques have limitations such as long cycles and poor selectivity. Therefore, exploring new Napier grass breeding technologies is particularly important.

[0003] Double haploid breeding technology involves obtaining plant haploids through various methods and then doubling their chromosomes to create 100% pure lines (DH lines) for application in genetics and breeding research. Double haploid breeding technology can rapidly obtain homozygous individuals, significantly shortening the breeding cycle and improving genetic stability. Traditional methods for obtaining haploids include anther culture, unpollinated ovary or ovule culture, and distant hybridization. These methods are highly genotype-dependent, have low regeneration rates, and are prone to producing chimeras. New technologies such as gene editing are gradually improving the efficiency and applicability of haploid induction.

[0004] Constructing haploid induction lines using gene editing technology can improve the efficiency of haploid induction and provide strong support for double haploid breeding in plants. Currently identified genes related to haploid induction include CENH3 (centromere histone H3), MTL / PLA1 (MATRILINEAL / PLASTID TARGRTING LIPASE1), DMP (DOMAIN OF UNKNOWNFUNCTION 679MEMBRANE PROTEIN), and SGP (SINGLE GRAIN PROTEIN). Among these, DMP8 encodes a cell-specific membrane protein with the DUF679 domain. This protein plays a crucial role in sperm-egg fusion; its loss of function during double fertilization leads to defects in sperm-egg fusion. Therefore, loss-of-function mutations in DMP8 can induce haploid production, providing an important theoretical basis for haploid breeding. The function of DMP8 is widely conserved in seed plants, and this characteristic has been verified in a variety of plants. However, the function of the DMP8 gene in Napier grass has not been fully studied and verified, and the DMP8 gene has never been used in the breeding of Napier grass. Summary of the Invention

[0005] To address the aforementioned problems in the existing technology, this invention provides a method for obtaining haploid *Pennisetum alopecuroides*. The method includes obtaining haploid *Pennisetum alopecuroides* plants by knocking out the PaDMP8 gene.

[0006] In a preferred embodiment, the amino acid sequence of the protein edited by the PaDMP8 gene is as shown in Sequence 1; or: based on Sequence 1, an amino acid sequence having the same function as the protein encoded by the PaDMP8 gene is obtained by substitution, deletion, and / or addition of one or more amino acid residues.

[0007] In a preferred embodiment, the nucleotide sequence of the PaDMP8 gene is as shown in Sequence 2; or: based on Sequence 2, a nucleotide sequence encoding a protein having the same function as the protein encoded by the PaDMP8 gene is obtained by substitution, deletion, and / or addition of one or more nucleotides.

[0008] In a preferred embodiment, the gene knockout includes: selecting at least one target site on the PaDMP8 gene of Napier grass, constructing a gene editing vector based on the target site, and transforming the gene editing vector into Napier grass to obtain haploid plants.

[0009] In a preferred embodiment, the gene editing vector includes one or two sgRNAs targeting the target site; or: includes three sgRNAs targeting the target site.

[0010] In a preferred embodiment, the framework vector of the gene editing vector is selected from: pYLCRISPR / Cas9Pubi-H, pYL-CRISPR / Cas9 Pubi-N, pYL-CRISPR / Cas9Pubi-B, pYLgRNA-OsU3, pYLgRNA-OsU3 / LacZ*, pYLgRNA-OsU6a, pYLgRNA-OsU6a / LacZ*, pYLgRNA-OsU6b, and pYLgRNA-OsU6c.

[0011] In a preferred embodiment, the expression cassette of the gene editing vector includes: a rice U3 promoter, tRNA, sgRNA for target 1, sgRNA scaffold, tRNA, sgRNA for target 2, sgRNA scaffold, tRNA, sgRNA for target 3, sgRNA scaffold, and a terminator.

[0012] In a preferred embodiment, the gene editing vector uses the pYL-CRISPR / Cas9 Pubi-N vector as a framework vector, and contains, from upstream to downstream, the following components in sequence: rice U3 promoter, tRNA, sgRNA for target 1, sgRNA scaffold, tRNA, sgRNA for target 2, sgRNA scaffold, tRNA, sgRNA for target 3, sgRNA scaffold, and terminator.

[0013] In a preferred embodiment, the variety of Napier grass is "Liqiu".

[0014] Compared with the prior art, the present invention has the following beneficial effects:

[0015] This invention knocks out the DMP8 gene in "Liqiu" Napier grass using gene editing technology, and for the first time constructs a haploid induction line of "Liqiu" Napier grass. Haploid plants of Napier grass were successfully obtained through the induction line, proving the feasibility of haploid induction of PaDMP8 gene in Napier grass, and providing a solid foundation for double haploid breeding technology of Napier grass. Attached Figure Description

[0016] Figure 1 The bar chart shows the expression levels of the PaDMP8 gene in different tissues of *Phragmites australis* in Example 1.

[0017] Figure 2 This is an electrophoresis diagram of the PaDMP8 gene PCR amplification reaction in Example 2.

[0018] Figure 3 This is a photomicrograph of the YFP fluorescence signal of the PaDMP8 gene in Example 4.

[0019] Figure 4 Phenotypic photographs and bar charts of the PaDMP8 transgenic Arabidopsis thaliana dmp8 / 9 double mutant from Example 5 are shown. Specifically: A. A comparison photograph of plant morphology between the dmp8dmp9 mutant and the reintroduced line; B. A photograph of pod development in the dmp8dmp9 mutant; C. A photograph of pod development in the reintroduced line; D. Images of pods from the dmp8dmp9 mutant and the reintroduced line after clearing treatment; E. A bar chart showing the number of seeds in the pods of the dmp8dmp9 mutant and the reintroduced line.

[0020] Figure 5 Photographs (A) of the genetic transformation and regeneration of *Pennisetum affine* at different times in Example 7, and electrophoresis diagram of the PCR amplification reaction of transgenic positive *Pennisetum affine* plants (B).

[0021] Figure 6 This is a sequencing result of the transgenic positive plant of *Pennisetum affine* from Example 7.

[0022] Figure 7 This is a flow cytometry result of haploid plants of *Pennisetum affine* from Example 8. Detailed Implementation

[0023] To make the technical solution, objectives, and advantages of the present invention clearer, the present invention will be further described in detail below through specific embodiments. It should be understood that the specific embodiments described herein are for illustration and explanation only and are not intended to limit the present invention.

[0024] This invention provides a method for obtaining haploid Napier grass, the method comprising: obtaining haploid Napier grass plants by gene knockout of the PaDMP8 gene (i.e., Napier grass DMP8 gene).

[0025] The amino acid sequence of the protein encoded by the PaDMP8 gene is shown in Sequence 1; or:

[0026] Based on sequence 1, an amino acid sequence with the same function as the protein encoded by the PaDMP8 gene is obtained by substitution, deletion, and / or addition of one or more amino acid residues.

[0027] Sequence 1:

[0028] MAGIRTPNHPQPRATACPHLYIAQQGSSKQTTSSPLLGNVARTVARSPIRPRTRPCIPPTPTPTPTPAPCPSTSAAAAPPLARRPPCRRRHLRRARGVQSTLARTSMLANFLPTGTLLAFEVALPAASGSGRDGVGGSCSAAGA ATLRALLALCAAACFLLHFTDSFRAPDGEVYYGVVTPRGLSLLRTGLGVEVPRDDRYRLAFIDVVHAAMSVLVFAAVALADDRVSGCLLPGHREEMGKVMESFPLVVGAVCSGLFLVFPNTRYGIGCLAVWRQQRPQSTDQTMAV.

[0029] The PaDMP8 gene described above is numbered Pal04G026190 in the database, and its nucleotide sequence is shown in Sequence 2; or:

[0030] Based on sequence 2, a nucleotide sequence encoding a protein with the same function as the protein encoded by the PaDMP8 gene is obtained by substitution, deletion, and / or addition of one or more nucleotides.

[0031] Sequence 2:

[0032] .

[0033] In a preferred embodiment, the above-mentioned gene knockout includes: selecting at least one of three target sites on the PaDMP8 gene of "Liqiu" Napier grass, constructing a gene editing vector based on the target site, and transforming the gene editing vector into "Liqiu" Napier grass to obtain haploid plants.

[0034] In a preferred embodiment, the target point includes at least one of the following three:

[0035] Target 1: 5'-TTGGCGTTGGCGGTATGCAT-3';

[0036] Target point 2: 5'-CTCGCGACGTTCCCGAGGAG-3';

[0037] Target 3: 5'-TGCCTTGCTGGGCTATGTAG-3'.

[0038] In a preferred embodiment, one or two of the above three target sites' sgRNAs can be constructed into the same gene editing vector, or the sgRNAs of the above three target sites can be constructed into the same gene editing vector simultaneously.

[0039] In a preferred embodiment, the expression cassette of the gene editing vector includes: a rice U3 promoter, tRNA, sgRNA for target 1, sgRNA scaffold, tRNA, sgRNA for target 2, sgRNA scaffold, tRNA, sgRNA for target 3, sgRNA scaffold, and terminator.

[0040] The nucleotide sequence composition of the tRNA is shown in Sequence 3, and the nucleotide sequence composition of the sgRNA scaffold is shown in Sequence 4.

[0041] Sequence 3:

[0042] TGCACCAGCCGGGAATCGAACCCGGGTCTGTACCGTGGCAGGTA CTATTCTACCACTAGACCACTGGTGCTTTGTT;

[0043] Sequence 4:

[0044] GCACCGACTCGGTGCCACTTTTTCAAGTTGATAACGGACTAGCCTTATTTTAACTTGCTATTTCTAGCTCTAAAAC. In a preferred embodiment, the gene editing vector described above uses the CRISPR / Cas9 system, preferably a CRISPR / Cas9 multi-gene vector as the framework, and can be selected from any one or more of the following:

[0045] pYLCRISPR / Cas9Pubi-H (carrying the hygromycin resistance gene (HPT)), pYL-CRISPR / Cas9 Pubi-N (carrying the kanamycin resistance gene (NPTII)), pYL-CRISPR / Cas9 Pubi-B (carrying the herbicide resistance gene (Bar)), pYLgRNA-OsU3, pYLgRNA-OsU3 / LacZ*, pYLgRNA-OsU6a, pYLgRNA-OsU6a / LacZ*, pYLgRNA-OsU6b, pYLgRNA-OsU6c.

[0046] The above-mentioned CRISPR / Cas9 multi-gene vector is described in the "Methods for Construction and Mutation Analysis of Plant CRISPR / Cas9 Multi-Gene Editing Vectors" [J], Zeng Dongchang, Ma Xingliang, Xie Xianrong, et al., Science in China: Life Sciences, 2018, 48(07): 783-794.

[0047] In a more preferred embodiment, the gene editing vector is based on the pYL-CRISPR / Cas9 Pubi-N vector and contains, from upstream to downstream, the following components in sequence: rice U3 promoter, tRNA, sgRNA of target site 1, sgRNA scaffold, tRNA, sgRNA of target site 2, sgRNA scaffold, tRNA, sgRNA of target site 3, sgRNA scaffold, and terminator.

[0048] In a more preferred embodiment, the method for constructing the gene-editing vector includes:

[0049] S1: Conventional PCR amplification reaction:

[0050] Using the pYLsgRNA-OsU3-tRNA (POT) vector as a template, PCR amplification was performed using primers F1 and R1 to obtain fragment 1 (512 bp), and PCR amplification was performed using primers F4 and R2 to obtain fragment 4 (131 bp). Using the pYLsgRNA-tRNA (PT) vector as a template, PCR amplification was performed using primers F2 and R2 to obtain fragment 2 (205 bp), and PCR amplification was performed using primers F3 and R3 to obtain fragment 3 (239 bp).

[0051] Fragment 1 includes: rice U3 promoter, tRNA, and a portion of target site 1; Fragment 2 includes: another portion of target site 1's sgRNA, sgRNA scaffold, and tRNA; Fragment 3 includes: sgRNA scaffold, tRNA, and a portion of target site 3's sgRNA; Fragment 4 includes: another portion of target site 3's sgRNA, sgRNA scaffold, and terminator.

[0052] The pYLsgRNA-OsU3-tRNA (POT) and pYLsgRNA-tRNA (PT) vectors mentioned above are described in "tRNA-sgRNA / Cas9 system-mediated gene editing of perennial ryegrass grassland mytoplasm" published by Yao Jiaming in the Journal of Grassland Science in 2023.

[0053] S2: Overlap PCR ligation method:

[0054] Using fragments 1 and 2 as templates, primers F5 and R5 were added to perform a PCR amplification reaction to ligate fragments 1 and 2 together to form fragment 5; using fragments 3 and 4 as templates, primers F6 and R6 were added to perform a PCR amplification reaction to ligate fragments 3 and 4 together to form fragment 6.

[0055] Fragment 5 includes: rice U3 promoter, tRNA, sgRNA of target 1, sgRNA scaffold, tRNA, and a portion of sgRNA of target 2; Fragment 6 includes: another portion of sgRNA of target 2, sgRNA scaffold, tRNA, sgRNA of target 3, sgRNA scaffold, and terminator.

[0056] S3: Kinmen Assembly Method: The CRISPR expression vector pYL-CRISPR / Cas9 Pubi-N, fragment 5, and fragment 6 are subjected to a cleavage-ligation reaction to obtain the assembled product, which is the gene editing vector.

[0057] The above-mentioned "Liqiu" Napier grass variety has the superior variety number: S-BV-PA-007-2021, provided by the Institute of Grassland and Flower Industry, Beijing Academy of Agricultural and Forestry Sciences, and can also be purchased from Beijing Academy of Agricultural Sciences Seed Technology Co., Ltd. Anyone may freely obtain the "Liqiu" Napier grass variety from the above sources for the purpose of this invention.

[0058] 'Liqiu', a type of Pennisetum alopecuroides, is an excellent ornamental grass with a beautiful plant shape, excellent resistance, and a long green period, making it widely used in landscape construction, gardening, and ecological restoration. This invention aims to verify the function of the DMP8 gene in 'Liqiu' and explore its application potential in double haploid breeding technology. By verifying the function of the PaDMP8 gene in the Arabidopsis dmp8 / 9 mutant, the mechanism of action of this gene in plant reproductive development was further understood. Furthermore, the DMP8 gene was knocked out in 'Liqiu' Pennisetum alopecuroides using gene editing technology, and a haploid induction line for 'Liqiu' Pennisetum alopecuroides was constructed for the first time. Haploid plants of Pennisetum alopecuroides were successfully obtained through this induction line, demonstrating the feasibility of haploid induction using the PaDMP8 gene in Pennisetum alopecuroides, and providing a solid foundation for double haploid breeding technology of Pennisetum alopecuroides.

[0059] Unless otherwise specified, all reagents and materials used in the following examples are products that can be obtained from commercial channels; unless otherwise specified, all testing and detection methods used in the following examples are conventional testing and detection methods in the field and can be obtained from textbooks, reference books or academic journals.

[0060] Examples 1-5 below are used to describe the study on the function of the DMP8 gene in *Pennisetum purpureus*.

[0061] Example 1

[0062] This embodiment is used to illustrate the expression pattern analysis of DMP8 in "Liqiu" foxtail grass.

[0063] 1. RNA extraction and reverse transcription from different tissues

[0064] (1) Select different tissues of “Liqiu” foxtail grass, including stems, leaves and anthers, and quickly freeze them in liquid nitrogen after sampling.

[0065] (2) RNA was extracted from different tissues of *Pennisetum affine* using the FastPure Universal Plant Total RNA Isolation Kit (Vazyme, China). The specific steps are as follows:

[0066] Take an appropriate amount of plant tissue that has been ground in liquid nitrogen and immediately add 600 μL of Buffer EL. Vortex vigorously for 30 seconds to ensure thorough mixing of the sample and lysis buffer. Centrifuge at 12,000 rpm for 5 minutes. Transfer the supernatant to FastPure gDNA-Filter Columns III (FastPure gDNA-Filter Columns III is already in the collection tube), centrifuge at 12,000 rpm for 30 seconds, discard FastPure gDNA-Filter Columns III, and collect the filtrate. Add 0.5 times the volume of the filtrate to the collection tube and vortex to mix for 15 seconds. Transfer the mixture to FastPure RNA Columns V (FastPure RNA Columns V is already in the collection tube), centrifuge at 12,000 rpm for 30 seconds, and discard the filtrate. Add 700 μL of Buffer RWA to FastPure RNA Columns V, centrifuge at 12,000 rpm for 30 seconds, and discard the filtrate. Add FastPure... Add 500 μL of Buffer RWB to RNAColumns V, centrifuge at 12000 rpm for 30 seconds, discard the filtrate, and repeat this step twice. Place FastPure RNAColumns V back into the collection tube and centrifuge at 12000 rpm for 2 minutes. Place FastPure RNAColumns V into a new centrifuge tube, add 30-100 μL of N-Nase-free ddH2O to the center of the adsorption membrane, incubate for 1 minute, and centrifuge at 12000 rpm for 1 minute to elute total RNA. Repeat this step twice to obtain RNA from different parts of "Liqiu" foxtail grass.

[0067] (3) Using the extracted RNA as a template, it was reverse transcribed into cDNA using the PrimeScript RT Master Mix (Takara, Japan) kit.

[0068] The reverse transcription system consisted of 2 μL 5×PrimeScript RT Master Mix, 2 μL RNA, and 6 μL LNase FreedH2O.

[0069] The reverse transcription procedure was as follows: reverse transcription reaction at 37℃ for 15 min, followed by reverse transcriptase inactivation reaction at 85℃ for 5 sec.

[0070] 2. Quantitative Real-Time PCR

[0071] (1) Based on the PaDMP8 sequence (database number Pal04G026190), gene-specific primers qPaDMP8-F / R for real-time quantitative PCR were designed using Primer Premier 5.0 software, with PaActin gene as the internal reference gene. The primer sequences are as follows:

[0072] qPaDMP-F:5'-GGTACCGGCTCGCCTTCATC-3';

[0073] qPaDMP-R:5'-TCACCTTCCCCATCTCCTCG-3';

[0074] qPaActin-F:5'-CTGAGCGGGAAATTGTGAGG-3';

[0075] qPaActin-R:5'-CATGGATGGCTGGAAGAGGA-3';

[0076] The cDNA extracted in the previous step was diluted 10 times and used as a template for quantitative real-time PCR.

[0077] Prepare a 25 μL real-time PCR reaction system on an ice bath, including 12.5 μL 2×TB Green Premix ExTaq (Takara, Japan), 1 μL each of forward and reverse primers, 1 μL cDNA template, and 9.5 μL ddH2O.

[0078] (2) The procedure for quantitative real-time PCR reaction is as follows: pre-denaturation at 95℃ for 30 seconds, PCR amplification for 40 cycles, each cycle including 95℃ for 5 seconds and 60℃ for 30 seconds.

[0079] 3. Data Analysis

[0080] Use 2 -ΔΔCt Relative quantitative analysis was performed using the method, and the mean and standard deviation of the obtained relative expression data were calculated. SPSS software was used for significance analysis of differences, and Origin was used for plotting.

[0081] 4. Expression analysis of PaDMP8 gene in different tissues of 'Liqiu' Napier grass

[0082] The expression pattern of the PaDMP8 gene in the stems, leaves, and anthers of *Pennisetum 'Liqiu'* was analyzed. The results showed (e.g.) Figure 1 PaDMP expression was highest in pollen (9.90), followed by leaves (2.35) and lowest in stems (0.72). This indicates that PaDMP8 expression in pollen was significantly higher than in leaves and stems.

[0083] The PaDMP8 gene showed the highest expression level (9.90) in pollen, indicating that its core function is closely related to male reproductive processes and may regulate double fertilization through its transmembrane domain. Mutations in the maize ZmDMP gene lead to haploid embryo formation (HIR 0.1–0.3%), and the high expression level of the PaDMP8 gene in pollen suggests that its functional loss may also disrupt the double fertilization balance, preferentially triggering fertilization in the central cell, thus providing a target for haploid induction technology in *Pennisetum purpureus*.

[0084] Example 2

[0085] This embodiment describes a method for obtaining the "Liqiu" Napier grass DMP8 gene through PCR amplification to obtain the pMD19-T-PaDMP8 plasmid vector.

[0086] 1. Total RNA extraction and reverse transcription

[0087] Total RNA was extracted from *Phragmites australis* 'Liqiu' using the FastPure Universal Plant Total RNA Isolation Kit (Vazyme, China), with the specific steps described in Example 1.

[0088] 2. Gene cloning and purification

[0089] (1) Based on the genome data of “Liqiu” Napier grass (PRJNA952524), this invention uses TBtools to use the DMP8 identified in maize as a reference to perform Blast screening of homologous sequences and selects the gene sequence (Pal04G026190) with the smallest E-value (3e-83) as a candidate gene.

[0090] (2) Using Primer Premier 5.0, PaDMP8 gene cloning-specific primers were designed based on the gene sequence. Using the obtained "Liqiu" Napier cDNA as a template, the ORF region of PaDMP8 was amplified according to the following reaction. The specific primer sequences are as follows:

[0091] PaDMP-F:5'-TTGTTATGCGTAGAAGCAGCCG-3'

[0092] PaDMP-R:5'-GCAATTTGGAACGGATGGAGTA-3'

[0093] The target gene fragment was amplified by PCR using 2×Taq Plus PCR Mix (TIANGEN, Beijing). The reaction system was as follows: 25 μL:

[0094] 12.5 μL 2×Taq Plus PCR Mix, 1 μL template cDNA, 1 μL each of forward and reverse primers, and 9.5 μL ddH2O.

[0095] The amplification reaction procedure is as follows:

[0096] Pre-denaturation at 94℃ for 3 min, denaturation at 94℃ for 30 s, annealing at 55℃ for 30 sec, extension at 72℃ for 1 min, cycled 30 times, and finally extended at 72℃ for 5 min.

[0097] The amplification products were obtained by PCR reaction and detected by 1% agarose gel electrophoresis (180V, 8min). The bands were observed in a gel imaging system (e.g., Figure 2 The target band is clearly visible.

[0098] (3) The PCR reaction solution with the correct band size was purified using the FastPure Gel DNA Extraction Mini Kit (Vazyme, China). The steps are as follows:

[0099] Transfer the PCR product to a 1.5 mL centrifuge tube and bring the sample volume to 100 μL with sterile water. Add 500 μL of Buffer GDP and vortex to mix. Place the adsorption column in the collection tube and transfer the sol solution from the previous step into the adsorption column. Centrifuge at 12000 rpm for 30 seconds. Discard the filtrate. Place the adsorption column in the collection tube and add 700 μL of Buffer GW to the adsorption column. Centrifuge at 12000 rpm for 30 seconds. Repeat this step twice. Discard the filtrate and place the adsorption column in the collection tube. Centrifuge at 12000 rpm for 2 minutes. Place the adsorption column in a new centrifuge tube and add 20-30 μL of Elution Buffer to the center of the adsorption column. Let it stand for 2 minutes and centrifuge at 12000 rpm for 2 minutes. Repeat this step twice. Discard the adsorption column to obtain the purified PaDMP8 target fragment product.

[0100] 3. Ligation of T-vectors, E. coli transformation, and plasmid extraction

[0101] (1) The PaDMP8 cloning vector was constructed using the pMD19-T Vector Cloning Kit (Takara, Japan). The specific steps are as follows:

[0102] The reaction system consisted of 1 μL pMD19-T Vector, 5 μL SolutionI, and 4 μL PaDMP8 target fragment purified product; the reaction conditions were 16℃ for 30 min.

[0103] (2) The above ligation product was transformed into Escherichia coli DH5α competent cells (TIANGEN, Beijing). The specific operation steps are as follows:

[0104] Thaw competent E. coli cells in an ice bath (conversion efficiency is highest when in an ice-water mixture); add 10 μL of ligation product to 50 μL of thawed competent cells, gently shake the centrifuge tube to mix, and incubate on ice for 30 min; incubate in a 42°C metal bath for 1 min, then immediately place in an ice bath for 2 min; add 700 μL of LB medium, place in a constant temperature shaker, and incubate at 37°C and 180 rpm for 1 h.

[0105] (3) Centrifuge the bacterial culture at 5000 rpm for 1 min, retain about 100 μL of supernatant, and mix the bacterial cells with the supernatant using a pipette tip. Spread the mixture onto LB agar plates containing 100 mg / L Amp and incubate at 37°C upside down for 12-14 h. After the culture medium has grown, select positive single clones from the LB solid plate and place them into centrifuge tubes containing 1 mL of LB liquid medium containing Amp antibiotic. Incubate at 37°C, 200 rpm, and shake for 4-5 h.

[0106] (4) Then, using the shaken bacterial culture as a template, and the universal primers M13F and M13R as detection primers, prepare the PCR reaction solution according to the following reaction system, and verify the size of the inserted target gene by colony PCR.

[0107] 10μL reaction system: 5μL 2×Taq Plus PCR Mix, 1μL bacterial template, 1μL each of upstream and downstream M13 primers, and 2μL ddH2O.

[0108] The reaction conditions were: 94℃ pre-denaturation for 3 min, 94℃ denaturation for 30 s, 55℃ annealing for 30 sec, 72℃ extension for 1 min, 30 cycles, and a final extension at 72℃ for 5 min.

[0109] (5) After PCR, the bacterial solution with the same length as the target band was detected by 1% agarose gel electrophoresis. 200 μL of the bacterial solution was sent for sequencing (Ruibo Xingke, China). After obtaining the sequencing results, the sequencing results were compared with the original sequence using DNAMAN software.

[0110] After confirming the correctness of the target gene sequence, the remaining bacterial culture with the correct sequence (800 μL) was mixed with an equal amount of 50% glycerol and stored at -20°C for later use.

[0111] (6) Take about 20 μL of bacterial culture and add it to 20 ml of liquid LB medium containing 100 mg / L Amp antibiotic. Incubate overnight at 37°C with shaking at 200 rpm to increase the number of Escherichia coli per unit volume for subsequent plasmid extraction.

[0112] (7) Plasmids were extracted using the FastPure Plasmid Mini Kit (Vazyme, China). The steps are as follows:

[0113] Take 1-5 mL of overnight cultured bacterial suspension and centrifuge at 10000 rpm for 1 min. Discard the culture medium and invert the tube onto absorbent paper to remove any remaining liquid. Add 250 μL of Buffer P1 to the centrifuge tube containing the bacterial precipitate and vortex to mix. Add 250 μL of Buffer P2 and gently invert to mix 8-10 times to ensure complete lysis of the bacteria. Add 350 μL of Buffer P3 and immediately gently invert to mix 8-10 times. Centrifuge at 12000 rpm for 10 min. Place the adsorption column in a collection tube and transfer the supernatant to the adsorption column. Centrifuge at 12000 rpm for 30 sec and discard the waste liquid. Add 600 μL of Buffer PW2 to the adsorption column and centrifuge at 12000 rpm for 30 sec. Discard the waste liquid. Repeat this step twice. Then, return the adsorption column to the collection tube and centrifuge at 12000 rpm for 1 min. Place the adsorption column in a new centrifuge tube and add 30-100 μL of Elution. Add buffer to the center of the adsorption column membrane, let stand for 2 min, centrifuge at 12000 rpm for 1 min to elute the plasmid. Repeat this step twice to obtain a large number of pMD19-T vectors (pMD19-T-PaDMP8 plasmid vectors) containing the PaDMP8 fragment. That is, the PaDMP8 fragment of "Liqiu" Napier grass has been cloned. The above method of cloning the target gene in cDNA by PCR and constructing the cloning vector, and confirming its correctness by sequencing, can be considered as successful cloning of the target gene.

[0114] Example 3

[0115] This embodiment describes a method for constructing an overexpression vector of "Liqiu" pampas grass DMP8, including the following steps.

[0116] 1. Amplification of the target fragment

[0117] (1) Add a BglⅡ restriction endonuclease site to the 5' end and design specific primers with vector homologous arm sequences as follows:

[0118] PaDMP8-Bgl-F:

[0119] 5'-cacgggggactcttgaccatggtaATGGCGGGCATTCGAACTC-3';

[0120] PaDMP8-Bgl-R:

[0121] 5'-ggtacacgcgtactagtcagatcTTAAACTGCCATAGTCTGG-3'.

[0122] (2) 25 μL reaction system:

[0123] 12.5 μL 2×Taq Plus PCR Mix, 1 μL pMD19-T-PaDMP8 plasmid (obtained in Example 2), 1 μL each of upstream and downstream specific primers, and 9.5 μL ddH2O.

[0124] The reaction conditions were: 94℃ pre-denaturation for 3 min; 94℃ denaturation for 30 s, 55℃ annealing for 30 sec, 72℃ extension for 1 min, 30 cycles; 72℃ final extension for 5 min.

[0125] (3) After PCR, the bands were detected by 1% agarose gel electrophoresis and observed in the gel imaging system. The PCR reaction solution with the correct band size was purified by FastPure Gel DNA Extraction Mini Kit (Vazyme, China) (see Example 2 for the method) to obtain the target fragment, namely the PaDMP8 fragment with added restriction sites.

[0126] 2. Preparation of linearized carriers

[0127] (1) The plant expression vector pCAMBIA3302 was selected, and the vector was digested with restriction enzyme BglⅡ (Takara, Japan).

[0128] The enzyme digestion reaction system consisted of 1 μL BglⅡ, 2 μL 10×Buffer, 1 μL carrier, and 16 μL ddH2O; the reaction conditions were 37℃ for 1 h.

[0129] The pCAMBIA3302 vector was obtained from the China Plasmid Vector Culture Collection Center. Anyone may freely obtain the vector from the inventor to achieve the purpose of this invention.

[0130] (2) The digested vectors were separated by 1% agarose gel electrophoresis. The undigested vectors were used as controls. The bands were observed in the gel imaging system. The digestion was successful if the distance between the electrophoretic band and the well was greater than that of the undigested vector.

[0131] (3) The enzyme-digested reaction solution was purified using the FastPure Gel DNA Extraction Mini Kit (Vazyme, China) (see Example 2 for the method) to obtain the linearized vector.

[0132] 3. Ligation of the target gene and the vector

[0133] The purified linearized vector and the target gene fragment were seamlessly ligated using the ClonExpress II One Step Cloning Kit (Vazyme, China). The resulting recombinant plant expression vector, 3302Y3-PaDMP8, was obtained after the reaction.

[0134] The reaction system consisted of: 2 μL linearized vector, 8 μL insert fragment (i.e., PaDMP8 fragment with added restriction sites), 4 μL 5×CEⅡBuffer, 2 μL ExnaseⅡ, and 2 μL ddH2O.

[0135] The reaction conditions were: 37℃ for 30 min.

[0136] 4. Escherichia coli transformation and screening

[0137] (1) The recombinant plant expression vector 3302Y3-PaDMP8 was transformed into competent Escherichia coli cells (the transformation method is described in Example 2).

[0138] (2) The transformed bacteria were spread on LB solid medium containing Kana and incubated overnight upside down at 37°C. Positive single colonies were picked into centrifuge tubes containing 1 mL LB liquid medium containing Kana antibiotic and cultured at 37°C and 200 rpm for 4-5 hours with shaking.

[0139] (3) Colony PCR was used to verify the size of the inserted target gene. The bacterial culture with shaking was used as a template and the universal primer 3302Y3-F / R was used as the detection primer. (See Example 2 for the method).

[0140] (4) After PCR, the bacterial solution with the same length as the target band was detected by 1% agarose gel electrophoresis. 200 μL of the bacterial solution was sent for sequencing (Ruibo Xingke, China). After obtaining the sequencing results, the sequencing results were compared with the original sequence using DNAMAN software.

[0141] (5) Take about 20 μL of bacterial culture and add it to 20 ml of liquid LB medium containing 100 mg / L Kana antibiotic. Incubate overnight at 37°C with shaking at 200 rpm to increase the number of Escherichia coli per unit volume. Use a kit to extract plasmid 3302Y3-PaDMP8 (see Example 1 for the method).

[0142] Example 4

[0143] This embodiment describes the procedure for obtaining the subcellular localization of the DMP8 gene of "Liqiu" foxtail grass.

[0144] 1. Transformation of Agrobacterium competent cells

[0145] (1) Take 5 μL of the constructed 3302Y3-PaDMP8 plasmid and add it to 100 mL of EHA105 Agrobacterium competent cells. Incubate on ice for 30 min. Quick freeze in liquid nitrogen for 5 min and immediately transfer to a 37℃ water bath for 5 min. Add 1 mL of LB liquid medium and culture at 28℃ in the dark with shaking for 2-3 h. Centrifuge at 5000 rpm for 1 min and discard the supernatant.

[0146] (2) Resuspend the remaining 100 μL of liquid with a pipette tip, spread it on LB solid medium containing 50 mg / L Rif and 100 mg / L Kana antibiotics, and incubate upside down at 28°C for 48–72 h; pick positive colonies into centrifuge tubes containing LB liquid medium containing 50 mg / L Rif and 100 mg / L Kana antibiotics, and incubate at 28°C, 200 rpm, shaking for 24 h.

[0147] (3) Using colony PCR verification, the PCR reaction solution with the correct band size was sent to the company for sequencing. The Agrobacterium tumefaciens solution with the correct sequencing result was added to an equal volume of 50% glycerol and then frozen for storage.

[0148] 2. Injection of Bunsen Tobacco

[0149] (1) Use young, healthy tobacco plants that have grown for 21 days as plant material.

[0150] (2) Take 100 μL of Agrobacterium tumefaciens bacterial solution and add it to 30 mL of liquid LB medium. Shake the medium at 28°C in the dark until the OD600 is about 0.2-0.4.

[0151] (3) Prepare the permeation solution and resuspend the Agrobacterium tumefaciens culture.

[0152] The preparation method of the above permeate is as follows (250 mL): Weigh 2.44 g MES (final concentration 50 mM) and dissolve it in 250 mL of distilled water, adjust the pH to 5.6; then add 1.25 g glucose and 0.06 g NaH2PO4, autoclave, and cool to room temperature for later use; after cooling, discard 12.5 mL of the solution and add 12.5 mL of 20×AB salt solution.

[0153] The above 20×AB salt solution formula (100mL) is: 2g NH4Cl, 0.6g MgSO4·7H2O, 0.3g KCl, 0.005g FeSO4·7H2O and 0.02g CaCl2. After high temperature and high pressure sterilization, it is stored at room temperature.

[0154] (4) Select tobacco plants with good growth, draw up the resuspended Agrobacterium tumefaciens solution with a 1mL syringe without needle, inject the solution into the lower epidermis of the tobacco, and make a label.

[0155] 3. YFP signal observation

[0156] (1) 48 hours after injection, the leaves of Tobacco Bunseni that were injected with Agrobacterium were torn off, pressed into glass slides, and the distribution of YFP signals in the leaves was observed using a laser confocal microscope and photographed.

[0157] (2) Subcellular localization results of the PaDMP8 gene showed that the fluorescent signal was distributed in the cell membrane (e.g., Figure 3 The subcellular localization of the PaDMP8 gene was consistent with that of the ZmDMP8 gene, indicating its consistent intracellular expression location. In previous studies, haploid inducible lines were successfully obtained after knocking out the maize DMP8 gene. The subcellular localization of the PaDMP8 gene was consistent with that of the maize DMP8 gene, suggesting that the Napier grass DMP8 gene and the maize DMP8 gene are homologous genes with similar functions, providing a basis for the application of this gene in Napier grass—specifically, for haploid induction. Furthermore, the fluorescence signal of PaDMP8 is located in the cell plasma membrane, suggesting that this gene is involved in membrane fusion and double fertilization, and has the potential for application in haploid breeding.

[0158] Example 5

[0159] This embodiment describes a method for verifying the function of the DMP8 gene in *Pennisetum affine* by transferring the DMP8 gene into the Arabidopsis thaliana dmp8 / 9 double mutant using the plant expression vector 3302Y3-PaDMP8 constructed in Example 3.

[0160] 1. Transformation of Agrobacterium competent cells

[0161] (1) Take 5 μL of the constructed 3302Y3-PaDMP8 plasmid and add it to 100 mL of GV3101 Agrobacterium competent cells. Incubate on ice for 30 min; freeze in liquid nitrogen for 5 min; immediately transfer to a 37℃ water bath for 5 min; add 1 mL of LB liquid medium and incubate at 28℃ in the dark with shaking for 2-3 h.

[0162] (2) Centrifuge at 5000 rpm for 1 min, discard the supernatant, resuspend the remaining 100 μL of liquid with a pipette tip, spread it on LB solid medium containing 50 mg / L Rif and 100 mg / L Kana antibiotics, and incubate upside down at 28 ℃ for 48-72 h; pick positive colonies into centrifuge tubes containing LB liquid medium containing 50 mg / L Rif and 100 mg / L Kana antibiotics, and incubate at 28 ℃, 200 rpm, shaking for 24 h.

[0163] (3) Using colony PCR verification, the PCR reaction solution with the correct band size was sent to the company for sequencing. The Agrobacterium tumefaciens solution with the correct sequencing result was added to an equal volume of 50% glycerol and then frozen for storage.

[0164] 2. Arabidopsis thaliana infection

[0165] (1) Take 100 μL of Agrobacterium cryopreservation solution containing the target vector (3302Y3-PaDMP8 plasmid) and add it to 100 mL of liquid LB medium. At the same time, add 100 μL of 50 mg / L Kana and 100 μL of 50 mg / L Rif. Incubate at 28 °C in the dark until the OD600 reaches about 0.8. Centrifuge the shaken bacterial solution at 5000 rpm for 10 min at 4 °C, discard the supernatant and keep the bacterial cells. Add an appropriate amount of the prepared 1 / 2 MS medium to suspend the bacterial cells so that the OD600 of the final working solution is about 0.8. Add 0.5 μL of Silwet L-77 and 1 μL of 100 mg / L acetylsuccinone to each 1 mL of suspension.

[0166] (2) Immerse the Arabidopsis thaliana flower buds completely in the prepared inoculum for about 30 seconds.

[0167] (3) Place the infected Arabidopsis thaliana in an artificial climate chamber and keep it in darkness for 12 hours. Then restore normal light and water it in time. Infect it again after one week. The whole experiment requires 2-3 infections. Harvest the seeds after they mature.

[0168] 3. Resistance screening of transgenic Arabidopsis thaliana

[0169] The seeds harvested in the previous step are sieved through a 40-mesh sieve, vernalized at 4℃ for 2 days, and then evenly sown on the nutrient substrate. They are then placed in an artificial climate chamber for cultivation. One week after germination, glufosinate is sprayed for screening. Resistant plants can be isolated after about 3 weeks. The resistant plants are then transferred to a new culture medium for cultivation. After obtaining positive plants, they are cultivated into stable T3 generation seeds.

[0170] 4. Phenotypic observation and index determination

[0171] T3 generation seeds were planted, and the phenotype of the transgenic plants was observed, such as... Figure 4 As shown: Figure 4 A compares the plant type of the dmp8dmp9 mutant and the reintroduced line. By comparing the two plants in the picture, it can be seen that the height of the reintroduced plant is greater than that of the mutant plant, and the number of branches of the reintroduced plant is greater than that of the mutant. Figure 4 B represents the pod development of the dmp8dmp9 mutant, whose pods are thin and short. Figure 4 C represents the pod development of the reintroduced plant; compared to the mutant, the pods of the reintroduced plant are thicker and longer. Figure 4 D shows the transparent images of the pods of the dmp8dmp9 mutant (Figure 1) and the reintroduced lines (Figures 2-4). The mutant pods are short and thin with fewer and sparser seeds. The reintroduced plants show an increase in pod length, width, and seed setting rate. Figure 4E represents the seed count analysis in the pods of the dmp8dmp9 mutant (column 1) and the reintroduced lines (columns 2-4). The number of seeds in the reintroduced lines PaDMP8-74 and PaDMP8-80 was significantly higher than that in the mutant.

[0172] like Figure 4 As shown, in the Arabidopsis dmp8 / 9 double mutant, the seed number was significantly reduced due to defects in the fertilization process, exhibiting a clear abortion phenotype. Through the remediation experiment, it was observed that PaDMP8 could restore the seed number of the Arabidopsis dmp8 / 9 double mutant, indicating that PaDMP8 and AtDMP8 / 9 have a certain degree of functional conservation. PaDMP8 participates in maintaining the normal function of the fertilization process during plant reproduction, thereby affecting seed formation.

[0173] Experimental results indicate that the PaDMP8 gene may also play a role in haploid induction, providing a possibility for haploid induction using the PaDMP8 gene and laying the foundation for further research on double haploid breeding technology of Pennisetum acutum.

[0174] Examples 6-8 below describe the application of the DMP8 gene in the cultivation of haploid Napier grass, that is, the method of cultivating haploid Napier grass by knocking out the DMP8 gene of Napier grass.

[0175] Example 6

[0176] This embodiment describes the construction of the DMP8 gene editing vector for "Liqiu" foxtail grass.

[0177] Based on the 5'-N(20)-NGG-PAM-3' structure, three target sites were designed on the PaDMP8 sequence; the tRNA-sgRNA / Cas9 (PTG / Cas9) system provided by Professor Xu Bin's research group at Nanjing Agricultural University was used to perform gene editing on multiple targets.

[0178] The meaning of the above 5'-N(20)-NGG-PAM-3' (5'-20nt-NGG) structure is as follows: the 5'-3' direction represents the reading direction of the DNA double strand; N represents the four bases AGCT; N(20) refers to the 20nt sequence, which is the target sequence (the complementary region of sgRNA, corresponding to the guiding region of crRNA); NGG is an AGG, GGG, CGG, or TGG sequence; NGG is the PAM (Protospacer Adjacent Motif) sequence. The recognition mechanism of PAM is that Cas9 will unwind the DNA and initiate cleavage upstream of PAM only when PAM is detected. Therefore, this structure represents a typical target structure, with PAM (NGG) located at the 3' end of the non-complementary strand, and the target sequence selected is the 20nt sequence upstream of PAM.

[0179] The amino acid sequence of the protein edited by the PaDMP8 gene is shown below (Sequence 1):

[0180] MAGIRTPNHPQPRATACPHLYIAQQGSSKQTTSSPLLGNVARTVARSPIRPRTRPCIPPTPTPTPTPAPCPSTSAAAAPPLARRPPCRRRHLRRARGVQSTLARTSMLANFLPTGTLLAFEVALPAASGSGRDGVGGSCSAAGA ATLRALLALCAAACFLLHFTDSFRAPDGEVYYGVVTPRGLSLLRTGLGVEVPRDDRYRLAFIDVVHAAMSVLVFAAVALADDRVSGCLLPGHREEMGKVMESFPLVVGAVCSGLFLVFPNTRYGIGCLAVWRQQRPQSTDQTMAV.

[0181] The specific sequences of the three target sites on the PaDMP8 sequence are as follows:

[0182] Target 1: 5'-TTGGCGTTGGCGGTATGCAT-3';

[0183] Target point 2: 5'-CTCGCGACGTTCCCGAGGAG-3';

[0184] Target 3: 5'-TGCCTTGCTGGGCTATGTAG-3'.

[0185] Since the aforementioned target points were designed based on the inverse complementary sequence of Sequence 2, they are shown in reverse order and are also inversely complementary in Sequence 2 below. The inverse complementary sequences of the above three target points are marked with boxes in the following Pal04G026190 sequence (Sequence 2) (target point 3, target point 2, and target point 1, respectively), and the inverse complementary sequences of the PAM sequences of each target point are underlined:

[0186] >Pal04G026190

[0187] ATGGCGGGCATTCGAACTCCGAACCACCCCCAACCGCGCGCAACCGCA

[0188] TGCCCGCA CCTCTACATAGCCCAGCAAGGCA GCAGCAAGCAAACCACT

[0189] TCCTCT CCACTCCTCGGGAACGTCGCGAG AA C AG TT GC A C GCTCTCCG

[0190] ATCCGGCCAAGAACGCG CCCATGCATACCGCCAACGCCAA CGCCAACG

[0191] CCAACGCCAACGCCGGCGCCGTGTCCGTCGACATCCGCCGCGGCCGCT

[0192] CCGCCCCTCGCGCGCCGACCTCCTTGCCGCCGCCGCCACCTCCGCCG

[0193] GCGCGGGGCGTCCAGAGCACGCTGGCTAGGACGTCGATGCTGGCCAAC

[0194] TTCCTCCCCACGGGCACGCTCCTGGCCTTCGAAGTGGCGCTCCCGGCG

[0195] GCGTCCGGCTCCGGCCGGGACGGCGTCGGCGGCTCCTGCTCCGCCGCC

[0196] GGCGCCCGCGACGCTCCGAGCCCTCCTCGCGCTCTGCGCCGCCGCCTGC

[0197] TTCCTCCTCCACTTCACCGACAGCTTCCGCGCGCCAGACGGGGAGGTG

[0198] TACTACGGCGTGGTCACGCCGCGGGGCCTCTCGCTGCTCAGGACCGGG

[0199] CTCGGCGTCGAGGTGCCCCGGGACGACAGGTACCGGCTCGCCTTCATC

[0200] GACGTCGTGCACGCGGCCATGTCGGTGCTCGTCTTCGCGGCCGTCGCG

[0201] CTCGCCGACGACAGGGTCTCCGGCTGCCTCCTCCCCGGACACCGCGAG

[0202] GAGATGGGGAAGGTGATGGAGAGCTTCCCGCTCGTTGTGGGGCCGTG

[0203] TGCAGCGGCCTCTTCCTCGTGTTCCCCAACACCCGCTACGGCATCGGTT

[0204] GCTTGGCTGTTTGGAGACAGCAGAGACCACAGAGCACAGACCAGACTATGGCAGTTTAA.

[0205] Of the three target sites mentioned above, knocking out just one would prevent the expression of the PaDMP8 gene. However, due to the uncertainty of gene editing efficiency, all three target sites were selected on this gene to improve gene editing efficiency. PaDMP8 non-expression yields a haploid inducible line. Haploids can be obtained in the offspring through self-pollination of the inducible line or by hybridization using it as the male parent.

[0206] The DMP8 gene editing vector constructed in this embodiment uses the pYL-CRISPR / Cas9 Pubi-N vector as a framework and contains, from upstream to downstream: rice U3 promoter, tRNA, sgRNA for target 1, sgRNA scaffold, tRNA, sgRNA for target 2, sgRNA scaffold, tRNA, sgRNA for target 3, sgRNA scaffold, and terminator.

[0207] The nucleotide sequence of the above tRNA is as follows (Sequence 3):

[0208] TGCACCAGCCGGGAATCGAACCCGGGTCTGTACCGTGGCAGGTA CTATTCTACCACTAGACCACTGGTGCTTTGTT;

[0209] The nucleotide sequence of the above sgRNA scaffold is as follows (Sequence 4):

[0210] GCACCGACTCGGTGCCACTTTTTCAAGTTGATAACGGACTAGCCTTATTTTAACTTGCTATTTCTAGCTCTAAAAC.

[0211] 1. The specific steps for constructing gene editing vectors include:

[0212] (1) Conventional PCR amplification: Using pYLsgRNA-OsU3-tRNA (POT) vector as template, primers F1 and R1 were used for PCR amplification to obtain fragment 1 (512bp), primers F4 and R2 were used for PCR amplification to obtain fragment 4 (131bp); using pYLsgRNA-tRNA (PT) vector as template, primers F2 and R2 were used for PCR amplification to obtain fragment 2 (205bp), primers F3 and R3 were used for PCR amplification to obtain fragment 3 (239bp).

[0213] The aforementioned pYLsgRNA-OsU3-tRNA (POT) and pYLsgRNA-tRNA (PT) vectors are both described in Yao Jiaming's 2023 publication in the *Acta Grasslandica Sinica*, "TRNA-sgRNA / Cas9 System-Mediated Gene Editing of Perennial Rye Grassland Mycoplasma". Anyone may freely obtain these two vectors from the inventors for the purposes of this invention. The pYLsgRNA-OsU3-tRNA (POT) vector (3301 bp) uses the pYLsgRNA-OsU3 vector (i.e., pYLsgRNA-OsU3m vector) as its framework, and from upstream to downstream, it contains: a rice U3 promoter, tRNA, a aminocapenicillin resistance gene, an sgRNA scaffold, and a terminator. The above-mentioned pYLsgRNA-tRNA(PT) vector (3294bp) uses the pYLsgRNA-OsU3 vector as a framework and contains, from upstream to downstream, the following components: rice U3 promoter, ampicillin resistance gene, sgRNA scaffold, tRNA, and terminator.

[0214] Fragments 1-4 mentioned above are not in the CDS sequence, but all contain the gene editing target sequence mentioned above. Their function is to assemble the target sequence into the gene editing vector through a 3-step PCR amplification reaction.

[0215] PCR amplification reaction 30μL reaction system: 15μL 2×Phanta Max buffer, 0.6μL Phanta Max Super-Fidelity DNA polymerase, 1μL POT plasmid template, 1.2μL each of upstream and downstream specific primers, and 10.4μL ddH2O.

[0216] The reaction conditions were: 95℃ pre-denaturation for 3 min; 95℃ denaturation for 15 s, 60℃ annealing for 15 sec, 72℃ extension for 30 sec, for 35 cycles; and 72℃ final extension for 5 min.

[0217] After the above PCR amplification reaction is completed, product 1 contains fragment 1, product 2 contains fragment 2, product 4 contains fragment 3, and product 4 contains fragment 4.

[0218] Fragment 1 includes: rice U3 promoter, tRNA, and part of the sgRNA of target site 1; Fragment 2 includes: another part of the sgRNA of target site 1, sgRNA scaffold, and tRNA; Fragment 3 includes: sgRNA scaffold, tRNA, and part of the sgRNA of target site 3; Fragment 4 includes: another part of the sgRNA of target site 3, sgRNA scaffold, and terminator.

[0219] (2) Overlap PCR ligation method:

[0220] Using products 1 and 2 as templates, primers F5 and R5 were added to perform a PCR amplification reaction to ligate products 1 and 2 together, forming product 5. Product 5 contains fragment 5, which includes: rice U3 promoter, tRNA, sgRNA of target site 1, sgRNA scaffold, tRNA, and a portion of sgRNA of target site 2.

[0221] Using products 3 and 4 as templates, primers F6 and R6 were added to perform a PCR amplification reaction to ligate products 3 and 4 together, forming product 6. Product 6 contains fragment 6, which includes: another part of the sgRNA of target site 2, sgRNA scaffold, tRNA, sgRNA of target site 3, sgRNA scaffold, and terminator.

[0222] 30 μL reaction system: 15 μL 2×Phanta Max buffer, 0.6 μL dNTP Mix, 0.6 μL PhantaMax Super-Fidelity DNA polymerase, 1 μL product 1 and 1 μL product 2 (or 1 μL product 3 and 1 μL product 4), 1.2 μL each of forward and reverse specific primers, and 9.4 μL ddH2O.

[0223] The reaction conditions were: 95℃ pre-denaturation for 3 min; 95℃ denaturation for 15 s, 60℃ annealing for 15 sec, 72℃ extension for 30 sec, for 30 cycles; and 72℃ final extension for 5 min.

[0224] (3) Kinmen assembly method: The CRISPR expression vector pYL-CRISPR / Cas9 Pubi-N, product 5 and product 6 were subjected to a cleavage-ligation reaction to obtain the assembled product.

[0225] 10 μL reaction system: 1 μL 10×cutsmar, 1 μL 10×T4 DNA ligase Buffer, 2.5 μL product 5, 2.5 μL product 6, 2 μL pYL-CRISPR / Cas9 Pubi-N, 0.5 μL BsaI, 0.5 μL T4 ligase, reaction conditions: 37℃ for 5 min.

[0226] The pYL-CRISPR / Cas9 Pubi-N vector (Genbank accession number MG719602) is described in "Methods for Construction and Mutation Analysis of Plant CRISPR / Cas9 Multigene Editing Vectors" [J], Zeng Dongchang, Ma Xingliang, Xie Xianrong, et al., Science in China: Life Sciences, 2018, 48(07): 783-794. Anyone may freely obtain this vector from the inventors for the purposes of this invention.

[0227] The primer sequences used are as follows:

[0228] F1:5'-CTCCGTTTTACCTGTGGAATC-3';

[0229]

[0230] (The box contains the inverse complementary sequence of target point 1)

[0231]

[0232] (Target point 1 is shown in the box)

[0233] R2:5'-CGGAGGAAAATTCCATCCAC-3'.

[0234] F3:5'-GGCTCGTATGTTGTGTGG-3';

[0235]

[0236] (The box shows the inverse complementary sequence of target 3)

[0237]

[0238] (Target point 3 is shown in the box)

[0239] F5:5'-TT.CAGAGGTCTCTACCGTGGAATCGGCAGCAAA-3';

[0240]

[0241] (The box contains the inverse complementary sequence of a portion of the target 2 sequence).

[0242]

[0243] (The part of the sequence shown in the box represents target point 2)

[0244] R6:5'-AGCGTGGGTCTCGCTCGTCCATCCACTCCAAGC-3'.

[0245] The inverse complementary sequence of the partial sequence of target 2 in R5 and the partial sequence of target 2 in F6 can be integrated into the sequence of target 2, and the nucleotide bases of the repeating part of the two are GTTC.

[0246] 2. E. coli transformation and plasmid extraction

[0247] The assembled product was directly transformed into competent E. coli cells, and single clones were selected for sequencing verification. Specific operational steps are detailed in Example 3. The verified vector was the DMP8 gene editing vector.

[0248] 3. Agrobacterium-mediated transformation

[0249] The constructed DMP8 gene editing vector plasmid was transformed into GV3101 Agrobacterium, and the specific steps are described in Example 5. Single clones were picked and sequenced, and colony PCR was used for verification. PCR reaction solutions with correct band sizes were sent to the company for sequencing. Agrobacterium culture with correct sequencing results was added to an equal volume of 50% glycerol and then frozen for storage.

[0250] Example 7

[0251] This embodiment describes the genetic transformation and detection of "Liqiu" foxtail grass by Agrobacterium containing the DMP8 gene editing vector obtained in Example 6.

[0252] 1. Agrobacterium-mediated genetic transformation and plant regeneration of Napier grass

[0253] The method for genetic transformation and plant regeneration of *Pennisetum alopecuroides* used in this patent is based on the description by Mu Tong in "Optimization of Agrobacterium-mediated Transformation System for *Pennisetum alopecuroides* and Obtaining Transgenic Plants". Specifically, the steps include:

[0254] (1) Preparation of recipient material: Callus tissue was induced by mature and plump “Liqiu” wolfberry seeds. After the callus tissue was subcultured for 2 generations, it could be used for infection once the callus tissue growth was stable.

[0255] (2) Agrobacterium tumefaciens suspension with an OD600 of 0.4–0.6 was used to infect callus tissue of *Phragmites australis* cultured for 2 days. After infection, the callus tissue was inoculated onto a selection medium containing a bactericide. After a period of culture, the differentiated rootless seedlings were divided into individual plants and inoculated onto a rooting medium to root. Figure 5 A). The four pictures in the figure show plants at 0 days, 14 days, 28 days, and 48 days after the end of infection.

[0256] 2. Identification of transgenic positive plants

[0257] (1) After the rooted seedlings were hardened off for 10 days, they were transplanted into nutrient soil, leaf tissues were cut, and DNA was extracted by CTAB method.

[0258] (2) Using the DNA from the leaves of the resistant plants to be tested as a template and the DNA from the leaves of the untransformed Napier grass as a negative control, the resistant plants obtained after infection were detected by using the vector-specific primers Cas9-F / R.

[0259] The primer sequences are as follows:

[0260] Cas9-F: 5'-GCTACGCCGGTTACATTGAC-3';

[0261] Cas9-R: 5'-GCTCGTTGTAGACCGTGAAGTA-3'.

[0262] 10μL reaction system: 5μL 2×Taq Plus PCR Mix, 1μL DNA template, 1μL each of forward and reverse primers, 2μL ddH2O.

[0263] The reaction conditions were: 94℃ pre-denaturation for 3 min; 94℃ denaturation for 30 s, 55℃ annealing for 30 sec, 72℃ extension for 1 min, 30 cycles; 72℃ final extension for 5 min.

[0264] The DNA of the transgenic plant amplified to the target band, while the negative control showed no amplification band. Figure 5 B) Preliminary evidence suggests that these strains are positive plants.

[0265] 3. Screening of gene-edited plants

[0266] (1) PCR amplification was performed on the three target sites and adjacent sequences. The primer sequences are as follows:

[0267] seqDMP-F:

[0268] 5'-TCGTCGGCAGCGTCAGATGTGTATAAGAGACAGCATTCGAACTC CGAACCACC-3';

[0269] seqDMP-R:

[0270] 5'-GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAGTGCCCGTGG GGAGGAAGTTG-3'.

[0271] 25μL reaction system: 12.5μL 2×Taq Plus PCR Mix, 1μL DNA template, 1μL each of forward and reverse primers, 9.5μL ddH2O.

[0272] The reaction conditions were: 94℃ pre-denaturation for 3 min; 94℃ denaturation for 30 s, 55℃ annealing for 30 sec, 72℃ extension for 1 min, 30 cycles; 72℃ final extension for 5 min.

[0273] (2) Sequence alignment analysis revealed that gene editing occurred at the third target site in strains 20, 21, and 23. Figure 6 Among them, lines 20, 21, and 23 all underwent base substitutions and fragment deletions, proving that three gene-edited lines were obtained.

[0274] Example 8

[0275] This embodiment describes the method and detection results of haploid induction of "Liqiu" foxtail grass.

[0276] 1. Self-pollinate the gene-edited T0 generation plants, and harvest T1 generation seeds after self-pollination.

[0277] Plants No. 20, 21, and 23 were designated as T0 generation plants, totaling three plants. The harvested seeds were designated as T1 generation seeds. Plant No. 20 yielded 300 seeds, plant No. 21 yielded 120 seeds, and plant No. 23 yielded 300 seeds.

[0278] 2. The harvested T1 generation seeds were sown, and ploidy was determined by flow cytometry after seedling emergence. Two haploid plants were detected in the T1 generation seeds of line 21, with a haploid induction rate of 1.67%.

[0279] The flow cytometry method for identification was referenced from Jia Ming's "Induction and Identification of Polyploids in 'Four Seasons' Green Sedge":

[0280] (1) Take 1cm 2 For the young leaves of the plant to be tested, place them in a petri dish, add 1 mL of cell extraction solution, chop the leaves with a blade, add another 1 mL of cell extraction solution, and let stand for 5 minutes.

[0281] (2) Filter the extract into a 1.5 mL centrifuge tube using a 400 mesh filter membrane, centrifuge at 4℃ and 1100 r / min for 6 min, discard the supernatant, add 200 μL of staining solution, and stain at 4℃ in the dark for 20 min.

[0282] (3) Two haploid plants were screened using flow cytometry (BD AccuriC6). Figure 7).

[0283] By knocking out the DMP8 gene in the 'Liqiu' Napier grass using gene editing technology, a haploid induction line for the first time was constructed. Haploid lines were then obtained by hybridizing the haploid induction line with the maternal parent plant. This result demonstrates the feasibility of haploid induction using the PaDMP8 gene in Napier grass. This lays a solid foundation for the further development of DH technology in Napier grass breeding programs.

[0284] The above description is merely a preferred embodiment of the present invention and is not intended to limit the invention. Various modifications and variations can be made to the present invention by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. A method for obtaining haploid *Phragmites australis*, comprising: Through gene knockout PaDMP8 Genetic modification yielded haploid plants of *Pennisetum affine*; The PaDMP8 The amino acid sequence of the gene-edited protein is shown in Sequence 1.

2. The method for obtaining haploid *Phragmites australis* according to claim 1, characterized in that: The PaDMP8 The nucleotide sequence of the gene is shown in Sequence 2.

3. The method for obtaining haploid *Phragmites australis* according to claim 1 or 2, characterized in that: The gene knockout includes: through the following methods in *Phragmites australis*. PaDMP8 At least one of the target sites is selected in the gene, and a gene editing vector is constructed based on the target site. The gene editing vector is then transformed into Napier grass to obtain haploid plants.

4. The method for obtaining haploid *Phragmites australis* according to claim 3, characterized in that: The target point includes at least one of the following three: Target point 1: 5'-TTGGCGTTGGCGGTATGCAT- 3'; Target point 2: 5'-CTCGCGACGTTCCCGAGGAG- 3'; Target point 3: 5'-TGCCTTGCTGGGCTATGTAG- 3'.

5. The method for obtaining haploid *Phragmites australis* according to claim 4, characterized in that: The gene editing vector includes one or two sgRNAs targeting the target site; or: includes three sgRNAs targeting the target site.

6. The method for obtaining haploid *Phragmites australis* according to claim 5, characterized in that: The framework vector for the gene editing vector is selected from: pYLCRISPR / Cas9Pubi-H, pYL-CRISPR / Cas9 Pubi-N, pYL-CRISPR / Cas9 Pubi-B, pYLgRNA-OsU3, pYLgRNA-OsU3 / LacZ*, pYLgRNA-OsU6a, pYLgRNA-OsU6a / LacZ*, pYLgRNA-OsU6b, and pYLgRNA-OsU6c.

7. The method for obtaining haploid *Pennisetum affine* according to claim 5, characterized in that: The expression cassette of the gene editing vector comprises, in sequence: rice U3 promoter, tRNA, sgRNA for target 1, sgRNA scaffold, tRNA, sgRNA for target 2, sgRNA scaffold, tRNA, sgRNA for target 3, sgRNA scaffold, and terminator.

8. The method for obtaining haploid *Phragmites australis* according to claim 6, characterized in that: The gene editing vector uses the pYL-CRISPR / Cas9 Pubi-N vector as a framework vector, and contains, from upstream to downstream, the following components: rice U3 promoter, tRNA, sgRNA for target 1, sgRNA scaffold, tRNA, sgRNA for target 2, sgRNA scaffold, tRNA, sgRNA for target 3, sgRNA scaffold, and terminator.

9. The method for obtaining haploid *Phragmites australis* according to claim 1, characterized in that: The variety of the grass mentioned is "Liqiu".

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