A method for preparing humidity-sensitive male sterile maize and related genes

Abstract

The present invention relates to the field of biotechnology, and particularly to a method for preparing humidity-sensitive male-sterile maize and related genes. The method includes: reducing the activity of a protein in maize, reducing the content of the protein in maize, inhibiting the expression of the coding gene of the protein in maize, or knocking out the coding gene of the protein in maize, to obtain humidity-sensitive male-sterile maize; the protein is as follows (A1) or (A2): (A1) a protein with an amino acid sequence shown in SEQ ID NO: 2 or 4; (A2) a protein with the same function obtained by substitution and / or deletion and / or addition of one or several amino acids to the amino acid sequence shown in SEQ ID NO: 2 or 4. The humidity-sensitive male-sterile maize material prepared by the method of the present invention shows sterility or extremely low seed-setting rate under natural environment, while its fertility can be restored under high humidity environment, and has good application prospects in the two-line hybrid breeding of maize.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and particularly relates to a method for preparing humidity-sensitive male sterile maize and related genes thereof. Background Art

[0002] Maize is the world's largest food crop and occupies a very important position in the feed industry and the energy field. In recent years, with the development of the economy, the sown area and yield of maize have increased year by year, and its planting area and yield rank first among all crops in China. Maize is the first crop to use male sterile lines for seed production. However, due to the narrow germplasm resources of cytoplasmic male sterile lines, the widespread application of this technology led to the large-scale epidemic of southern leaf blight in the United States in the 1970s, causing serious economic losses. Therefore, this technology had to withdraw from commercial applications, and still needs to produce hybrid seeds by manual emasculation. In recent years, with the development of biotechnology, people hope to change the existing mode of maize commercial seed production by developing nuclear male sterile lines based on transgenic technology, reduce labor demand, and thus save costs. Currently, dozens of maize male sterile mutants have been discovered, but due to the backward basic research, only a few genes have been cloned. This situation has brought great obstacles to the development of new technologies. Therefore, it is necessary to quickly discover genes related to stable male nuclear sterility to meet the needs of commercial seed production.

[0003] The two-line method of maize is the earliest method applied to the production of maize hybrids with nuclear male sterility. In the process of using the two-line method to produce hybrids, homozygous sterile plants need to be crossed with heterozygous fertile plants. Among the offspring obtained in the seed production field, 50% of the sterile plants are used for producing hybrid seeds, and the remaining 50% of the plants need to be removed; while among the offspring obtained in the field for cultivating dual-purpose plants, 50% of the heterozygous plants are retained, and the remaining 50% of the plants need to be removed. The biggest drawback of this method is that half of the plants need to be removed, with low resource utilization rate, wasting land and labor, and it is not easy to distinguish sterile plants from fertile plants, bringing many difficulties to production. Therefore, developing maize environment-sensitive male sterile mutants is the key to promoting the two-line method of maize hybrid breeding. Although temperature-sensitive male sterile materials and light-sensitive male sterile materials have also been reported in maize, they are all limited by strict temperature conditions and light durations, and the fertility in production is greatly affected by environmental fluctuations, resulting in unsafe seed production. Summary of the Invention

[0004] The technical problem to be solved by the present invention is how to prepare humidity-sensitive male sterile maize. To solve the above technical problem, the present invention provides a method for preparing humidity-sensitive male sterile maize.

[0005] The method for preparing humidity-sensitive male sterile maize provided by the present invention includes: reducing the activity of a protein in maize, reducing the content of the protein in maize, inhibiting the expression of the coding gene of the protein in maize, or knocking out the coding gene of the protein in maize, to obtain humidity-sensitive male sterile maize;

[0006] The name of the protein is UGT702A4, and it is as follows (A1) or (A2):

[0007] (A1) A protein with an amino acid sequence shown in SEQ ID NO: 2 or 4;

[0008] (A2) A protein with the same function obtained by substitution and / or deletion and / or addition of one or several amino acids in the amino acid sequence shown in SEQ ID NO: 2 or 4.

[0009] In the above method, the coding gene of the protein includes any one of the following nucleotide sequences (b1)-(b3):

[0010] (b1) The nucleotide sequence shown in SEQ ID NO: 1 or 3;

[0011] (b2) A nucleotide sequence having more than 75% identity with the nucleotide sequence defined in (b1) and encoding the protein;

[0012] (b3) The nucleotide sequence encoding the protein.

[0013] As used herein, the term "identity" refers to the sequence similarity with the natural nucleic acid sequence. The above-mentioned more than 75% identity can be 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more than 99% identity.

[0014] The above method can be achieved by site-directed editing of the coding gene of the protein through a CRISPR gene editing system.

[0015] The CRISPR gene editing system can be a CRISPR-Cas9 system; the target sequence of the coding gene of the protein used in the CRISPR-Cas9 system can be target sequence 1 or target sequence 2;

[0016] The target sequence 1 is as follows (c1), (c2) or (c3):

[0017] (c1) The nucleotide sequence shown in SEQ ID NO: 5;

[0018] (c2) A nucleotide sequence derived from (c1) that has more than 75% identity with the nucleotide sequence defined by (c1);

[0019] (c3) A nucleotide sequence derived from (c1) that hybridizes with the nucleotide sequence defined by (c1) under stringent conditions;

[0020] The target sequence 2 is one of the following (d1), (d2), or (d3):

[0021] (d1) The nucleotide sequence shown in SEQ ID NO:6;

[0022] (d2) A nucleotide sequence derived from (d1) that has more than 75% identity with the nucleotide sequence defined by (d1);

[0023] (d3) A nucleotide sequence derived from (d1) that hybridizes with the nucleotide sequence defined by (d1) under stringent conditions.

[0024] The stringent conditions can be hybridization in a solution of 2×SSC, 0.1% SDS at 68°C and washing the membrane twice, 5 minutes each time, and then hybridization in a solution of 0.5×SSC, 0.1% SDS at 68°C and washing the membrane twice, 15 minutes each time.

[0025] The method can obtain humidity-sensitive male sterile maize by introducing the coding gene of sgRNA targeting the target sequence 1 or the target sequence 2 and the coding gene of Cas9 into maize.

[0026] In the above method, the coding gene of the sgRNA can be achieved by introducing a recombinant vector containing the coding gene of the sgRNA into maize.

[0027] The recombinant vector can be pCXB053-UGT702A4-spacer1-spacer2, which is a recombinant vector obtained by inserting the target sequence 1 and the target sequence 2 between the BsaI restriction sites of the pCXB053 plasmid. pCXB053-UGT702A4-spacer1-spacer2 expresses Cas9, sgRNA1, and sgRNA2, where sgRNA1 contains the RNA sequence encoded by the target sequence 1 and sgRNA2 contains the RNA sequence encoded by the target sequence 2.

[0028] In the above method, the water loss rate of the pollen of the humidity-sensitive male sterile maize is higher than that of the pollen of the wild-type maize; the fertility of the humidity-sensitive male sterile maize can be restored by the method of artificial humidity-assisted pollination.

[0029] The water loss rate can be the water loss rate under the humidity condition of 60% ≥ RH ≥ 30%.

[0030] The present invention also provides a product for regulating maize fertility, which is used to reduce the activity of UGT702A4 protein in maize, reduce the content of UGT702A4 protein in maize, inhibit the expression of the coding gene of UGT702A4 protein in maize, or knockout the coding gene of UGT702A4 protein in maize.

[0031] The product can be reagents and / or instruments required for site-directed editing of the coding gene of UGT702A4 protein using the CRISPR / Cas9 system.

[0032] The reagent can be Reagent 1, Reagent 2, Reagent 3, Reagent 4, or Reagent 5;

[0033] The Reagent 1 is R1) or R2):

[0034] R1) sgRNA targeting the target sequence 1;

[0035] R2) sgRNA targeting the target sequence 2;

[0036] The Reagent 2 is a composition composed of the Reagent 1 and Cas9;

[0037] The Reagent 3 is a recombinant vector containing the coding gene of R1) or R2);

[0038] The Reagent 4 is a composition composed of the Reagent 3 and a recombinant vector containing the coding gene of Cas9;

[0039] The Reagent 5 is a recombinant vector containing the coding gene of R1) and / or R2) and the coding gene of Cas9.

[0040] The application of UGT702A4 protein or the coding gene of UGT702A4 protein in regulating maize fertility also belongs to the protection scope of the present invention.

[0041] The present invention has obtained maize humidity-sensitive male sterile materials by controlling the expression of maize UGT702A4 gene and its encoded protein. When the environmental relative humidity value is 40-60%, the pollen grains of CRISPR homozygous mutants ugt-3, ugt-4, and ugt-5 of maize UGT702A4 gene rapidly lose water in the air after being isolated, and all show complete shriveling 3 minutes after isolation, while the pollen grains of wild-type maize KN5585 have no morphological changes 3 minutes after isolation and require more than 60 minutes to completely shrivel. When the ear matures, the wild-type maize KN5585 shows normal seed setting, while the mutants ugt-3, ugt-4, and ugt-5 show sterility or the number of seeds set on a single ear does not exceed 10 grains ( Figure 2) Artificial humidity-assisted pollination was carried out on maize. Under high humidity (RH > 90%) conditions, the seed setting rate of the wild type KN5585 was 89.6%, while the seed setting rates of the mutants ugt-3, ugt-4, and ugt-5 were 81.6%, 80.3%, and 82% respectively. Figure 3 ) Therefore, the maize humidity-sensitive male sterile material prepared by the method of the present invention shows sterility or extremely low seed setting in the natural environment, but can restore fertility in a high humidity environment, and has good application prospects in the two-line hybrid breeding of maize. Description of the Drawings

[0042] Figure 1 is the mutation site of the UGT702A4 gene in the CRISPR / Cas9-edited mutant of maize. Among them, ugt-1, ugt-2, ugt-3, ugt-4, and ugt-5 are different types of UGT702A4 gene knockout allelic mutants, and WT is the wild type of maize KN5585.

[0043] Figure 2 is the phenotypic observation of the CRISPR / Cas9-edited mutant of maize UGT702A4 gene. Among them, (A) shows the morphological characteristics of the pollen grains of the wild type of maize KN5585 and the CRISPR / Cas9-edited homozygous mutants ugt-3, ugt-4, and ugt-5 just after isolation (0 m) and 3 minutes after isolation (3 m) under natural conditions, where m represents minutes; (B) shows the pollen water loss rate curves of the wild type of maize KN5585 and the CRISPR / Cas9-edited homozygous mutants ugt-3, ugt-4, and ugt-5, where m represents minutes; (C) shows the seed-bearing ear types of the wild type of maize KN5585 and the CRISPR / Cas9-edited homozygous mutants ugt-3, ugt-4, and ugt-5, and the scale bars are all 2 cm.

[0044] Figure 3 is the seed setting situation of the CRISPR / Cas9-edited mutant of maize UGT702A4 gene after artificial humidity-assisted pollination. Among them, (A) shows the seed-bearing ear types of the wild type of maize KN5585 and the CRISPR / Cas9-edited homozygous mutants ugt-3, ugt-4, and ugt-5 after artificial humidity-assisted pollination, where the scale bars are all 2 cm and RH represents humidity; (B) shows the seed setting rates (%) of the wild type of maize KN5585 and the CRISPR / Cas9-edited homozygous mutants ugt-3, ugt-4, and ugt-5 after artificial humidity-assisted pollination.

[0045] Figure 4 is the plasmid map of pCXB053.

[0046] Figure 5 This is the plasmid map of pSG-ZmU6. Detailed implementation mode

[0047] The present invention will be further described below in conjunction with embodiments. It should be understood that the following embodiments are only for the explanation and illustration of the present invention and do not limit the scope of the present invention in any way.

[0048] The pCXB053 plasmid is a gene editing backbone vector provided by Weimi Biotechnology (Jiangsu) Co., Ltd.

[0049] The pSG-ZmU6 plasmid is a common vector containing elements such as gRNA and ZmU6, and is provided by Weimi Biotechnology (Jiangsu) Co., Ltd.

[0050] The KOD FX high-fidelity enzyme was purchased from TOYOBO.

[0051] The FastPure Gel DNA Extraction Mini Kit was purchased from Nanjing Novoprotein Science and Technology Co., Ltd., product number: DC301-01.

[0052] The BsaI enzyme was purchased from New England Biolabs, product number: R0535.

[0053] The T4 DNA ligase was purchased from New England Biolabs, product number: M0202.

[0054] The 2×Taq Master Mix Dye was purchased from CWBIO Co., Ltd., product number: CW0682L.

[0055] The 2×GoldStar MasterMix (Dye) was purchased from CWBIO Co., Ltd., product number: CW0960M.

[0056] Unless otherwise specified, the reagents used in the following examples are all conventional reagents in the art, which can be obtained commercially or prepared according to the conventional methods in the art; the experimental methods and conditions used are all conventional experimental methods and conditions in the art, and relevant experimental manuals, well-known literatures or manufacturer's instructions can be referred to. Unless otherwise specified, the quantitative tests in the following examples are all set with three repeated experiments, and the results are averaged. Unless otherwise defined, the meanings of all technical and scientific terms used herein are the same as those commonly understood by those of ordinary skill in the art to which the present invention belongs.

[0057] Example 1. Obtaining of humidity-sensitive male sterile materials in maize

[0058] The inventors of the present application discovered a gene related to humidity-sensitive male sterility in maize - the UGT702A4 gene. The gene number of the UGT702A4 gene in maize B73 v4 is Zm00001d048106. In maize inbred line B73, the coding sequence (CDS) of the UGT702A4 gene is shown in SEQ ID NO:1, and the amino acid sequence of the UGT702A4 protein encoded by it is shown in SEQ ID NO:2. In maize inbred line KN5585, the coding sequence (CDS) of the UGT702A4 gene is shown in SEQ ID NO:3, and the amino acid sequence of the UGT702A4 protein encoded by it is shown in SEQ ID NO:4.

[0059] Use the CRISPR / Cas9 gene editing technology to create maize UGT702A4 gene mutants.

[0060] 1. Target sequence

[0061] The target sequences used are target sequence 1 and target sequence 2.

[0062] Target sequence 1: 5’-CGCAGCAGATCAGCAAAGAG CGG -3’ (SEQ ID NO:5),

[0063] Target sequence 2: 5’-GGGCGGGGTGACCGAAGCGG CGG -3’ (SEQ ID NO:6),

[0064] Wherein the PAM motif is underlined.

[0065] 2. Construction of the double-target knockout vector

[0066] Use the pSG-ZmU6 plasmid (provided by Weimi Biotechnology Co., Ltd.) as a template, and perform polymerase chain reaction (PCR) with target sequence primers to amplify the gRNA fragment. The target sequence primers used are as follows:

[0067] gRNA-F (5’-3’):

[0068] CAATGGTCTCAATTG GCAGCAGATCAGCAAAGAG GTTTTAGAGCTAGAAATAG (SEQ ID NO:7);

[0069] gRNA-R (5’-3’):

[0070] TTGGGGTCTCTAAAC CCGCTTCGGTCACCCCGCC CAATTCGGTGCTTGCGGCTC (SEQ ID NO:8).

[0071] Among them, the forward primer gRNA-F contains the 2nd to 20th nucleotides of target sequence 1 (underlined), and the reverse primer gRNA-R contains the reverse complementary sequence of the 2nd to 20th nucleotides of target sequence 2 (underlined). The maize U6 promoter (ZmU6 promoter) (SEQ ID NO: 9) is introduced in front of target sequence 2 by PCR, so that the amplified gRNA fragment (SEQ ID NO: 10) contains "sgRNA1-ZmU6 promoter-target sequence 2".

[0072] The amplification system is as follows:

[0073] pSG-ZmU6 plasmid 1 μl gRNA-F (10 μM) 1.5 μl gRNA-R (10 μM) 1.5 μl Buffer 20 μl KODFX high-fidelity enzyme 1 μl dNTPs (2 mM) 10 μl <![CDATA[ddH2O]]> 15 μl Total volume 50 μl

[0074] The amplification program is: pre-denaturation at 94°C for 3 min; denaturation at 98°C for 30 s, annealing at 58°C for 30 s, extension at 72°C for 1 min, for 30 cycles; final extension at 72°C for 5 min. After the PCR is completed, 1 μl of the product is taken for agarose gel electrophoresis detection, and the size of the target band (gRNA fragment) is about 700 bp. The gRNA fragment in the gel is recovered using the FastPure Gel DNA Extraction Mini Kit (Vazyme Biotech Co., Ltd., Nanjing, DC301-01) according to the kit operation method.

[0075] The pCXB053 plasmid (from Weimi Biotechnology Co., Ltd.) and the recovered gRNA fragment are digested with BsaI enzyme (New England Biolabs, R0535) respectively. The digestion system is as follows:

[0076] pCXB053 plasmid / gRNA fragment 1 μg BsaI enzyme 1 μl 10×CutSmartBuffer 5 μl <![CDATA[Add ddH2O]]> Up to 50 μl

[0077] Digestion condition: digest at 37°C for 2 hours.

[0078] The linearized pCXB053 plasmid and gRNA fragment obtained by digestion are recovered using the FastPure Gel DNA Extraction Mini Kit (Vazyme Biotech Co., Ltd., Nanjing, DC301-01) according to the kit operation method respectively.

[0079] The pCXB053 plasmid itself contains the ZmU6 promoter. The gRNA fragment containing "sgRNA1-ZmU6 promoter-target sequence 2" is ligated to the pCXB053 plasmid using T4 DNA ligase (New England Biolabs, M0202) to obtain a dual-target knockout vector containing "ZmU6 promoter-sgRNA1-ZmU6 promoter-sgRNA2". The dual-target knockout vector can drive the expression of target sequence 1 and target sequence 2 respectively.

[0080] The ligation system is as follows:

[0081] gRNA fragment digestion product 6 μl Linearized pCXB053 plasmid 1 μl T4 DNA ligase 1 μl Buffer 2 μl <![CDATA[ddH2O]]> 10 μl Total volume 20 μl

[0082] After mixing the ligation system, place it in a 4°C refrigerator for overnight ligation. Transfer the ligation product into Escherichia coli DH5α by heat shock method. The method is as follows: Take out 60 μl of Escherichia coli DH5α competent cells from -80°C, place them on ice to thaw. After the competent cells are completely thawed, quickly add 5 μl of the ligation product, gently flick to mix, then let it stand on ice for 30 min, perform heat shock at 42°C for 90 s, and gently place (do not shake) on ice for 2 - 3 min. Add 500 μl of LB liquid medium without antibiotics, shake culture at 30°C with a shaker at 200 rpm for 1 h to recover the bacteria. Take 200 μl of the bacterial solution and spread it on an LB solid medium containing 100 μg / ml kanamycin. After culturing at 37°C for 24 h, pick single colonies and shake culture for 4 - 6 hours, and take the bacterial solution for PCR identification. The primers used are QC2: 5’-TCCCAGTCACGACGTTGTAA-3’ (SEQ ID NO:11) and QC4: 5’-TCAAACAAGTGTGACAAAAA-3’ (SEQ ID NO:12). The size of the amplification product is about 1350 bp or 750 bp. Among them, the 750 bp is the amplification product of the empty vector, and the 1350 bp is the amplification product of the double-target knockout vector. Use 2×Taq Master Mix Dye (ComWin Biotech, CW0682L) for amplification. The system is as follows:

[0083] 2×TaqMix 5 μl QC2 (10 μM) 0.4 μl QC4 (10 μM) 0.4 μl Bacterial solution 1 μl <![CDATA[ddH2O]]> 3.2 μl Total volume 10 μl

[0084] The amplification program is: pre-denaturation at 94°C for 2 min; denaturation at 94°C for 30 s, annealing at 50°C for 30 s, extension at 72°C for 45 s, 35 cycles; final extension at 72°C for 5 min. Select the band with a size of 1350 bp by agarose gel electrophoresis for Sanger sequencing, and the sequencing primer is QC4. After sequencing identification, extract the plasmid of the positive clone with the correct sequence to obtain the recombinant plasmid pCXB053-UGT702A4-spacer1-spacer2. This recombinant plasmid contains target sequence 1 and target sequence 2, and expresses Cas9, sgRNA1 and sgRNA2. Among them, sgRNA1 contains the RNA sequence encoded by target sequence 1, and sgRNA2 contains the RNA sequence encoded by target sequence 2.

[0085] 3. Agrobacterium transformation

[0086] The above recombinant plasmid pCXB053-UGT702A4-spacer1-spacer2 was transformed into Agrobacterium tumefaciens EHA105 by electroporation (voltage 2000V). Single colonies were picked for culture and directly subjected to colony PCR (the sequencing primer was the above QC4) to identify positive clones, and the recombinant strain EHA105-pCXB053-UGT702A4-spacer1-spacer2 was obtained.

[0087] 4. Genetic transformation of maize immature embryos

[0088] Using the maize inbred line KN5585 as the transformation receptor material, male sterile maize materials were obtained by infecting maize immature embryos with Agrobacterium tumefaciens. The strain used was the above recombinant strain EHA105-pCXB053-UGT702A4-spacer1-spacer2. Genetic transformation was entrusted to Weimi Biotechnology Co., Ltd. The method was as follows: Using freshly dissected KN5585 maize immature embryos about 1 mm in size (provided by Weimi Biotechnology Co., Ltd.) as materials, the dissected maize immature embryos were placed in a 2 ml plastic centrifuge tube containing 1.8 ml of suspension, and about 150 immature embryos were treated within 30 min; the suspension was aspirated, and the remaining maize immature embryos were in the tube, then 1.0 ml of Agrobacterium suspension was added and left for 5 min. The immature embryos in the centrifuge tube were suspended and then poured onto the co-culture medium, and the excess Agrobacterium suspension on the surface was aspirated with a pipette, and co-cultured in the dark at 23 °C for 3 days. After co-culture, the immature embryos were transferred to the resting medium and cultured in the dark at 28 °C for 6 days, then placed on the selection medium containing bialaphos and screened for two weeks, and then screened on a new selection medium for 2 weeks. The resistant calli were transferred to the differentiation medium and cultured at 25 °C, 5000 lx, under light for 3 weeks. The seedlings differentiated were transferred to the rooting medium and cultured at 25 °C, 5000 lx, under light until rooting. The seedlings were transferred to a plug tray for growth, and the DNA of T0 generation plants was extracted and positive detection of the Bar gene was performed by PCR. The primers used were Bar-F: 5’-TGCACCATCGTCAACCACTACAT-3’ (SEQ ID NO:13) and Bar-R: 5’-AGAAACCCACGTCATGCCAGT-3’ (SEQ ID NO:14), and the amplified fragment size was 269 bp. Amplification was carried out using 2×Taq Master Mix Dye (ComWin Biotech, CW0682L), and the reaction system was as follows:

[0089] 2×TaqMix 5 μl Bar-F (10 μM) 0.4 μl Bar-R (10 μM) 0.4 μl DNA of T0 plants 1 μl <![CDATA[ddH2O]]> 3.2 μl Total volume 10 μl

[0090] The amplification program was: pre-denaturation at 94 °C for 2 min; denaturation at 94 °C for 30 s, annealing at 52 °C for 30 s, extension at 72 °C for 9 s, for 35 cycles; final extension at 72 °C for 5 min.

[0091] The formulation of the above suspension: Prepared with ddH2O, containing 4 g of N6 (including N6 vitamin) per liter, 2 mg of 2,4-D, 100 mg of inositol, 0.7 g of L-proline, 68.4 g of sucrose, 36 g of glucose, 1 mL of AgNO3 (10 g / L), and 1 mL of As (100 M), with pH = 5.2.

[0092] The formulation of the above co-culture medium: Prepared with ddH2O, containing 4 g of N6 (including N6 vitamin) per liter, 2 mg of 2,4-D, 30 g of sucrose, 8 g of agar, 1 mL of AgNO3 (10 g / L), 1 mL of As (100 M), and 3 mL of L-cysteine (100 g / L), with pH = 5.8.

[0093] The formulation of the above resting medium: Prepared with ddH2O, containing 4 g of N6 (including N6 vitamin) per liter, 1 mL of N6 vitamin (1000×), 1.5 mg of 2,4-D, 0.7 g of L-proline, 30 g of sucrose, 5 μM of AgNO3, 0.5 g of MES, 100 mg of cefotaxime, 100 mg of vancomycin, and 8 g of agar, with pH = 5.8.

[0094] The formulation of the above screening medium: The resting medium containing 1.5 mg / L of bialaphos.

[0095] The formulation of the above differentiation medium: Prepared with ddH2O, containing 4.43 g of MS salts containing MS vitamin (including inositol) per liter, 30 g of sucrose, 4 g of phytagel, and 1 mL of Cefo (250 g / L), with pH = 5.8.

[0096] The formulation of the above rooting medium: Prepared with ddH2O, containing 2.22 g of MS per liter, 30 g of sucrose, 51.55 mg of MS vitamin, and 4 g of phytagel, with pH = 5.8.

[0097] 5. Screening of mutant lines without Cas9

[0098] Sequencing verification was performed on the Bar gene of the T0 generation plants with positive PCR detection results above. Positive plants with correct sequences were selected for self-crossing or test-crossing to obtain T1 generation seeds. By growing T1 generation seeds in the greenhouse with additional generations, samples were taken respectively during the seedling stage for identification. Lines containing target sequence 1 or target sequence 2 and without Cas9 protein were screened out and self-crossed for propagation. Self-crossing propagation of homozygous mutants requires artificial pollination with humidity preservation, and the specific method is referred to below. At the same time, lines containing Cas9 protein or with no editing at both target sites were removed. Greenhouse culture conditions: 16 hours of light, 8 hours of darkness, 25 °C during the day, and 18 °C at night.

[0099] The target sequence 1 and the target sequence 2 are approximately 800 bp apart on the genome. Therefore, primers were designed to cover this interval for a single PCR amplification. The primers used were Check-F: 5’-GCGCATGTACTTCATCCCGT-3’ (SEQ ID NO:15) and Check-R: 5’-AAAAATCTCAGCATCCCGCC-3’ (SEQ ID NO:16), and the length of the amplified fragment was 1459 bp. Amplification was carried out using the 2×GoldStar MasterMix (Dye) kit (ComWin Biotech, CW0960M). The reaction system was as follows:

[0100] 2×Goldmix 15 μl Check-F (10 μM) 1.2 μl Check-R (10 μM) 1.2 μl DNA of T1 generation plants 1 μl <![CDATA[ddH2O]]> 11.6 μl Total volume 30 μl

[0101] The amplification program was: pre-denaturation at 95°C for 10 min; denaturation at 95°C for 30 s, annealing at 52°C for 30 s, extension at 72°C for 1 min, for 35 cycles; final extension at 72°C for 5 min. The PCR products were subjected to first-generation sequencing, using Check-F and Check-R as the forward and reverse sequencing primers respectively, to analyze sequence insertions or deletions.

[0102] The method for detecting the Cas9 protein mainly involves PCR amplification of a partial coding sequence of the protein and agarose gel electrophoresis of the products. If the T1 generation plants do not contain the Cas9 protein (or vector backbone), the corresponding bands cannot be detected, indicating that the offspring of this line are stable and will not segregate. Conversely, if the T1 generation plants contain the Cas9 protein (or vector backbone), the corresponding bands can be detected, indicating that the vector still has gene knockout function, such as off-target effects. The detection primers were Cas9-F: 5’-CACCATCTACCACCTGAGAA-3’ (SEQ ID NO:17) and Cas9-R: 5’-CGAAGTTGCTCTTGAAGTTG-3’ (SEQ ID NO:18), and the length of the amplified fragment was 371 bp. Amplification was carried out using 2×Taq Master Mix Dye (ComWin Biotech, CW0682L). The reaction system was as follows:

[0103] 2×Taqmix 5 μl Cas9-F (10 μM) 0.4 μl Cas9-R (10 μM) 0.4 μl DNA of T1 generation plants 0.5 μl <![CDATA[ddH2O]]> 3.7 μl Total volume 10 μl

[0104] The amplification program was: pre-denaturation at 94°C for 2 min; denaturation at 94°C for 30 s, annealing at 52°C for 30 s, extension at 72°C for 18 s, for 35 cycles; final extension at 72°C for 2 min.

[0105] By detecting the gene editing sites of the T1 generation plants, different types of UGT702A4 gene knockout allelic mutants were obtained, named ugt-1, ugt-2, ugt-3, ugt-4, and ugt-5 respectively. AsFigure 1 As shown, compared with the UGT702A4 coding sequence of maize KN5585 wild type, ugt-1 lacks 792 bp between the 349th base and the 1142nd base; ugt-2 lacks 4 bp between the 347th base and the 352nd base, and inserts 1 bp between the 1141st base and the 1142nd base; ugt-3 inserts 1 bp between the 351st base and the 352nd base, and inserts 1 bp between the 1141st base and the 1142nd base; ugt-4 lacks 33 bp between the 317th base and the 351st base, and inserts 1 bp between the 1141st base and the 1142nd base; ugt-5 inserts 1 bp between the 351st base and the 352nd base, and lacks 32 bp between the 1122nd base and the 1155th base. All the above mutation types result in premature termination of UGT702A4 protein translation or truncated UGT702A4 protein. Further, Cas9 sequence detection was performed on these mutants, and finally homozygous mutant lines without Cas9 protein were obtained, namely ugt-3, ugt-4, and ugt-5.

[0106] 6. Pollen phenotype observation

[0107] Pollen grains of wild-type maize KN5585 and the above homozygous mutants ugt-3, ugt-4, and ugt-5 were observed. An optical microscope Olympus CX21 (Olympus, Japan) equipped with an image sensor CCD (coupled device) was used to take real-time photos of pollen grains. The method is as follows: Fresh pollen grains during the maize flowering period were shaken onto a glass slide and directly observed under a 10× microscope. Photos of pollen grains just after detachment (i.e., 0 m, m represents minutes) and pollen grains at different times after detachment were taken and recorded through the OPTPro software. The relative humidity value of the environment during pollen grain observation was controlled at 40 - 60% (adjusted by an air conditioner, and the real-time humidity was observed using an Aiwosi W8 high-precision digital hygrometer). The shriveling rate (pollen water loss rate) of pollen grains at different times after detachment was statistically analyzed, that is, the percentage of shriveled pollen grains in the field of view at different times in all pollen grains in the field of view was statistically analyzed. More than 10 plants of each material were observed, and all the observed materials grew in a culture room.

[0108] Observation of pollen grains under a microscope revealed that when pollen grains were just released from anthers (just detached from the body, denoted as 0m), there were no significant differences between mutants ugt-3, ugt-4, and ugt-5 and the wild type. However, 3 minutes after pollen grain release (3 minutes after detachment from the body, denoted as 3m), the pollen grains of mutants ugt-3, ugt-4, and ugt-5 all showed complete shriveling, while the pollen grains of the wild type KN5585 showed almost no morphological changes ( Figure 2 -A). Continued observation showed that it took more than 60 minutes for the pollen grains of the wild type KN5585 to completely shrivel ( Figure 2 -B).

[0109] 7. Analysis of natural seed setting rate in the field

[0110] In 2022 in Xiangshan, Haidian, Beijing, plots were designed to test the seed setting rates of different types of mutants under natural conditions, and the wild type maize KN5585 was designed as a control. The row spacing of each plot was 60 cm, the plant spacing was 15 cm, there were 5 plants in each row, and each plot was replicated three times. Barriers were set between plots of different materials to avoid cross-pollination among materials during the flowering period. The water and fertilizer management of each plot was the same. After entering the flowering period, the plants in each plot completed pollination in the natural environment. At the same time, a hygrometer (Aivs W8 high-precision digital hygrometer and thermometer) was used to record the humidity value in the field. Missing data that were not recorded were represented by NA. When the seeds matured, 5-6 plants in the middle of each plot were selected for seed setting rate statistics. The seed setting rate was the percentage of the number of seeds set on each ear to the total number of all ovules. To ensure the authenticity of the seed setting rate of each mutant and prevent the seed setting rate of each mutant plot from being too large due to cross-pollination by foreign pollen, homozygosity detection was carried out on the seeds set on all mutant ears. If the seeds were of heterozygous genotype, they were removed when calculating the seed setting rate. The detection primers were the above-mentioned Check-F and Check-R, and the PCR system and amplification program were the same as described above.

[0111] After the plants entered the flowering period, the humidity changes in the field were shown in Table 1. Through field observation, it was found that usually during high humidity or rainy days, the anthers of both mutants ugt-3, ugt-4, and ugt-5 and the wild type KN5585 could not dehisce. When the ears matured, the wild type KN5585 showed relatively normal seed setting, while mutants ugt-3, ugt-4, and ugt-5 showed sterility or the number of seeds set on a single ear did not exceed 10 ( Figure 2 -C). The reason was that during the pollen dispersal period of the mutants, the pollen grains lost water and became inactivated rapidly after being detached from the body, resulting in their inability to complete normal fertilization.

[0112] Table 1 Field humidity in the plot during the flowering period

[0113]

[0114]

[0115] 8. Artificial Humidification Pollination of Mutants

[0116] To improve the seed setting rate of ugt-3, ugt-4, and ugt-5 mutants, observations were started after the mutants entered the flowering stage. When all the filaments had grown out, a spray bottle was held in hand and small water droplets were sprayed onto the filaments to evenly cover them. Immediately after that, the fresh pollen grains when the anthers of the mutants just cracked were shaken off and collected, and the filaments that had been sprayed with water were immediately artificially pollinated. Then, the pollinated filaments together with the female ears were quickly covered with transparent plastic self-sealing bags (size: 14 cm × 17 cm) for humidity preservation (humidity > 90%) to prevent the pollen grains of the mutants from losing water before hydrating with the filaments and enabling them to have normal fertilization ability. The pollination time point was generally selected at 8 - 10 am (peak pollen shedding period), and each female ear was continuously pollinated for 2 - 3 days. At the same time, the wild-type maize KN5585 was used as a control, and three different individual plants of each material were selected for the experiment. When the seeds matured, the corresponding seed setting rate analysis was carried out.

[0117] The results are as Figure 3 shown. In an environment with high humidity (humidity > 90%), the seed setting rate of the wild-type KN5585 was 89.6%, and the seed setting rates of the mutants ugt-3, ugt-4, and ugt-5 were 81.6%, 80.3%, and 82% respectively. It is worth noting that the mutants ugt-3, ugt-4, and ugt-5 showed extremely low seed setting in the natural environment, but were able to recover fertility under high humidity, indicating that the UGT702A4 gene mediates the regulation of maize fertility by environmental humidity. The mutants with loss-of-function of the UGT702A4 gene are excellent maize humidity-sensitive male sterile materials and have good application prospects in the two-line hybrid breeding of maize. SEQ ID NO:1 (1521bp)

[0118] Coding sequence (5’-3’) of the UGT702A4 gene in maize B73 line

[0119] ATGCGACCGTCGCCATCTTCCGTAGCGTCGTCGGGGGACACGGCGGCGCCGCGCATGTA

[0120] CTTCATCCCGTTCCCGACGCCAGGGCACGCGCTGCCGATGTCGGACCTCGCCCGCCTTT

[0121] TCGCGTCCCGCGGCGCCGACACCACGCTTGTCCTCACGCGCGGCAACGCCGCCAGGCT

[0122] CGGTGGCCCCGTCGCCCGCGCGGCCGCCACTGGCCTCCGCATCCGTATCGTCGCGCTCA

[0123] CTCTGCCCGCAGAGGCCGCCGGGCTCGCGGGTGGCCACGAGAGCGCCGACGACCTTCC

[0124] CAACCGCGAGCTCGCTGGGCCTTTCGCGGTCGCCGTTGACCTTCTCGCG CCGCTCTTTG

[0125] CTGATCTGCTGCG CCGCCAGCCTGCCGACGCCATCGTGTTCGACGGTGTCCTCCCGTGG

[0126] GCTGCCACTGCGGCCCCGGAGCTCGGCATCCCGCGGTACGCGTTCACCGGCACGGGGT

[0127] GCTTCGCGCTCTCGGTGCAGCGTGCTCTGCTGCTCCACAGCCCGCAAAATGGCGTGGCG

[0128] TCGGACACCGAGCCGTTCCTCGTGCCGGGCCTCCCCGACGCGGTGCGGCTCACCAGGT

[0129] CGAGGCTCGCCGAGGCGACGCTCCCGGGCGCGCACTCGCGCGAGTTCTTGAGCCGCAT

[0130] GTTCGACGCCGAGCGCGTCACGGCCGGGTGGGTCGTCAACTCGTTCGCGGACCTCGAG

[0131] CAGAGGTACATCGAACACTACGAGAAGGACACGGGGAAGCCGGTGTTCGCCGTCGGGC

[0132] CGGTCTGCCTTGTCAACGGCGACGGCGACGACGCCCTGGAGCGCGGCCGCGGCGGGGA

[0133] CTCCAGTACGGCCGCCGAGGCCGCGCGCGTGCTGAGGTGGCTGAACACGAAACCCGCA

[0134] CGGTCGGTGGTCTACGTCTGCTTCGGCAGCCTCACCAGGTTCCCGCGTGAGCAGGTGGC

[0135] GGAGCTCGGCATGGGCCTCGCCGACTCCGGCGCGAACTTCGTGTGGGTCGTCGGGGAC

[0136] AAGGACGCGCCGCAGCTCCCGGACATCGACGGCGCGGCGCCCGGCCGCGGGCTGGTGG

[0137] TCAGGGGGTGGGCCCCGCAGGTGGCGGTGCTGCGGCACGCGGCGGTGGGCGCGTTCGT

[0138] GACGCACTGCGGGTG GGGCGGGGTGACCGAAGCGGCGG CGGCGGGCGTCCCGGTGCT

[0139] GGCGTGGCCGGTGTTCGCAGAGCAGTTCTACAACGAGGCGCTGGTGGTGGGGCTCGCG

[0140] GGCACGGGCGTCTCCATGGGCGCGGAGAGGGGGTACGTGTGGGGAGGCGAGGCGCTG

[0141] GGCGGGGTGGTGGTGGGCAGGGCGGCAGTGGCGGAGCGGGTGCGCAGCGCCATGGCG

[0142] GACGAGGAGCTGCGGGGGAGGGCTGGACGGGTTGGCGAGCGCGCGCGGCGCGCGGTG

[0143] GAGGCGGGAGGGTCATCGTACGAGGCCGTGGGCGCGCTGCTGGAGGATGTGCTGCGGC

[0144] CTCAACGCCAGGTCCAGGATCTGGATGCCGTCCGGGAGACGAGGCGGGATGCTGAGAT

[0145] TTTTAATTAA

[0146] SEQ ID NO:2(506aa)

[0147] Amino acid sequence (N-C) of UGT702A4 protein in maize B73 line

[0148] MRPSPSSVASSGDTAAPRMYFIPFPTPGHALPMSDLARLFASRGADTTLVLTRGNAARLGGP

[0149] VARAAATGLRIRIVALTLPAEAAGLAGGHESADDLPNRELAGPFAVAVDLLAPLFADLLRRQP

[0150] ADAIVFDGVLPWAATAAPELGIPRYAFTGTGCFALSVQRALLLHSPQNGVASDTEPFLVPGLP

[0151] DAVRLTRSRLAEATLPGAHSREFLSRMFDAERVTAGWVVNSFADLEQRYIEHYEKDTGKPV

[0152] FAVGPVCLVNGDGDDALERGRGGDSSTAAEAARVLRWLNTKPARSVVYVCFGSLTRFPRE

[0153] QVAELGMGLADSGANFVWVVGDKDAPQLPDIDGAAPGRGLVVRGWAPQVAVLRHAAVG

[0154] AFVTHCGWGGVTEAAAAGVPVLAWPVFAEQFYNEALVVGLAGTGVSMGAERGYVWGGE

[0155] ALGGVVVGRAAVAERVRSAMADEELRGRAGRVGERARRAVEAGGSSYEAVGALLEDVLR

[0156] PQRQVQDLDAVRETRRDAEIFN

[0157] SEQ ID NO:3(1515bp)

[0158] Coding sequence (5'-3') of UGT702A4 gene in maize KN5585 line

[0159] ATGCGACCGTCGCCATCTTCCGTAGCGTCGTCGGGGGACACGGCGGCGCCGCGCATGTA

[0160] CTTCATCCCGTTCCCGACGCCAGGGCACGCGCTGCCGATGTCGGACCTCGCCCGCCTTT

[0161] TCGCGTCCCGCGGCGCAGACACCACGCTTGTCCTCACGCGCGGCAACGCCGCCAGGCT

[0162] CGGTGGCCCCGTCGCCCGCGCGGCCGCCACTGGCCTCCGCATCCGTATCGTCGCGCTCA

[0163] CTCTGCCCGCAGAGGCCGCCGGGCTCACGGGTGGCCACGAGAGCGCCGACGACCTTCC

[0164] CAACCGCGAGCTCGCCGGGCCTTTCGCGATCGCCGTTGACCTTCTCGCG CCGCTCTTTG

[0165] CTGATCTGCTGCG CCGCCAGCCTGCCGACGCCATCGTGTTCGACGGTGTCCTCCCGTGG

[0166] GCTGCCACTGCGGCCCCGGAGCTCGGCATCCCGCGGTACGCGTTCACCGGCACGGGGT

[0167] GCTTCGCGCTCTCGGTGCAGCGTGCTCTGCTGCTCCACAGCCCGCAAAATGGCGTGGCG

[0168] TCGGACACCGAGCCGTTCCTCGTGCCGGGCCTCCCCGACGCGGTGCGGCTCACCAGGT

[0169] CGAGGCTCGCCGAGGCGACGCTCCCGGGCGCGCACTCGCGCGAGTTCTTGAGCCGCAT

[0170] GTTCGACGCCGAGCGCGTCACGGCCGGGTGGGTCGTCAACTCGTTCGCGGACCTCGAG

[0171] CAGAGGTACATCGAACACTACGAGAAGGACACGGGCAAGCCGGTGTTCGCCGTCGGGC

[0172] CGGTCTGCCTTGTCAACGGCGACGGCGACGACGCCCTGGAGCGCGGCCGCGGCGGGGA

[0173] CTCCAGTACGGCCGCCGAGGCCGCGCGCGTGCTGAGGTGGCTGAACACGAAACCCGCC

[0174] CGGTCGGTGGTCTACGTATGCTTCGGCAGCCTCACCAGGTTCCCGCGTGAGCAGGTGGC

[0175] GGAGCTCGGCATGGGCCTCGCCGACTCCGGCGCGAACTTCGTGTGGGTCGTCGGGGAC

[0176] AAGGACGCGCCGCAGCTCCCGGACATCGACGGCGCGGCGCCGGGCCGCGGGCTGGTG

[0177] GTCAGGGGGTGGGCCCCGCAGGTGGCGGTGCTGCGGCACGCGGCGGTGGGCGCGTTCG

[0178] TGACGCACTGCGGGTG GGGCGGGGTGACCGAAGCGGCGG CGGCGGGCGTCCCAGTGC

[0179] TGGCGTGGCCGGTATTCGCAGAGCAGTTCTACAACGAGGCGCTGGTGGTGGGGCTCGC

[0180] GGGCACGGGCGTCTCCATGGGCGCGGAGAGGGGGTACGTGTGGGGAGGCGAGGCGCT

[0181] GGGCGGGGTGGTGGTGGACAGGGCGGAAGTGGCGGAGCGGGTGCGCAGCGCCATGGC

[0182] GAACGAGGCGCTGCGGGGGAGGGCGGGGCAGGTTGGCGAGCGCGCGCGGCGCGCGGT

[0183] GGAGGCGGGAGGGTCGTCGTACGAGGCCGTGGGCGCGCTGCTGGAGGATGTGCTGCGG

[0184] CCTCAACGCCAGGTCCAGGATGCCGTCCGGGAGACGAGGCGGGATGCTGAGATTTTTAA

[0185] TTAA

[0186] SEQ ID NO:4(504aa)

[0187] Amino acid sequence (N-C) of UGT702A4 protein from maize KN5585 line

[0188] MRPSPSSVASSGDTAAPRMYFIPFPTPGHALPMSDLARLFASRGADTTLVLTRGNAARLGGP

[0189] VARAAATGLRIRIVALTLPAEAAGLTGGHESADDLPNRELAGPFAIAVDLLAPLFADLLRRQP

[0190] ADAIVFDGVLPWAATAAPELGIPRYAFTGTGCFALSVQRALLLHSPQNGVASDTEPFLVPGLP

[0191] DAVRLTRSRLAEATLPGAHSREFLSRMFDAERVTAGWVVNSFADLEQRYIEHYEKDTGKPV

[0192] FAVGPVCLVNGDGDDALERGRGGDSSTAAEAARVLRWLNTKPARSVVYVCFGSLTRFPRE

[0193] QVAELGMGLADSGANFVWVVGDKDAPQLPDIDGAAPGRGLVVRGWAPQVAVLRHAAVG

[0194] AFVTHCGWGGVTEAAAAGVPVLAWPVFAEQFYNEALVVGLAGTGVSMGAERGYVWGGE

[0195] ALGGVVVDRAEVAERVRSAMANEALRGRAGQVGERARRAVEAGGSSYEAVGALLEDVLR

[0196] PQRQVQDAVRETRRDAEIFN

[0197] SEQ ID NO:5(23bp)

[0198] Target sequence 1 (5’-3’)

[0199] CGCAGCAGATCAGCAAAGAG CGG

[0200] SEQ ID NO:6 (23bp)

[0201] Target sequence 2 (5’-3’)

[0202] GGGCGGGGTGACCGAAGCGG CGG

[0203] SEQ ID NO:7 (53bp)

[0204] gRNA-F (5’-3’)

[0205] CAATGGTCTCAATTG GCAGCAGATCAGCAAAGAG GTTTTAGAGCTAGAAATAG

[0206] SEQ ID NO:8 (54bp)

[0207] gRNA-R (5’-3’)

[0208] TTGGGGTCTCTAAAC CCGCTTCGGTCACCCCGCC CAATTCGGTGCTTGCGGCTC

[0209] SEQ ID NO:9 (500bp)

[0210] Maize U6 promoter (5’-3’)

[0211] GCTGTTTTTGTTAGCCCCATCGAATCCTTGACATAATGATCCCGCTTAAATAAGCAACCTC

[0212] GCTTGTATAGTTCCTTGTGCTCTAACACACGATGATGATAAGTCGTAAAATAGTGGTGTC

[0213] CAAAGAATTTCCAGGCCCAGTTGTAAAAGCTAAAATGCTATTCGAATTTCTACTAGCAGT

[0214] AAGTCGTGTTTAGAAATTATTTTTTTATATACCTTTTTTCCTTCTATGTACAGTAGGACACA

[0215] GTGTCAGCGCCGCGTTGACGGAGAATATTTGCAAAAAAGTAAAAGAGAAAGTCATAGC

[0216] GGCGTATGTGCCAAAAACTTCGTCACAGAGAGGGCCATAAGAAACATGGCCCACGGCC

[0217] CAATACGAAGCACCGCGACGAAGCCCAAACAGCAGTCCGTAGGTGGAGCAAAGCGCTG

[0218] GGTAATACGCAAACGTTTTGTCCCACCTTGACTAATCACAAGAGTGGAGCGTACCTTATA

[0219] AACCGAGCCGCAAGCACCGAATT

[0220] SEQ ID NO:10(702bp)

[0221] gRNA fragment (5’-3’)

[0222] CAATGGTCTCAATTG GCAGCAGATCAGCAAAGAG GTTTTAGAGCTAGAAATAGGTTTT

[0223] AGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGCA

[0224] CCGAGTCGGTGCTTTTTTGTTTTCCTGCAGGGCTGTTTTTGTTAGCCCCATCGAATCCTTG

[0225] ACATAATGATCCCGCTTAAATAAGCAACCTCGCTTGTATAGTTCCTTGTGCTCTAACACAC

[0226] GATGATGATAAGTCGTAAAATAGTGGTGTCCAAAGAATTTCCAGGCCCAGTTGTAAAAG

[0227] CTAAAATGCTATTCGAATTTCTACTAGCAGTAAGTCGTGTTTAGAAATTATTTTTTTATATA

[0228] CCTTTTTTCCTTCTATGTACAGTAGGACACAGTGTCAGCGCCGCGTTGACGGAGAATATT

[0229] TGCAAAAAAGTAAAAGAGAAAGTCATAGCGGCGTATGTGCCAAAAACTTCGTCACAGA

[0230] GAGGGCCATAAGAAACATGGCCCACGGCCCAATACGAAGCACCGCGACGAAGCCCAAA

[0231] CAGCAGTCCGTAGGTGGAGCAAAGCGCTGGGTAATACGCAAACGTTTTGTCCCACCTTG

[0232] ACTAATCACAAGAGTGGAGCGTACCTTATAAACCGAGCCGCAAGCACCGAATTGAGCCG

[0233] CAAGCACCGAATTG GGCGGGGTGACCGAAGCGG GTTTAGAGACCCCAA

[0234] SEQ ID NO:11(20bp)

[0235] QC2:5’-TCCCAGTCACGACGTTGTAA-3’

[0236] SEQ ID NO:12(20bp)

[0237] QC4:5’-TCAAACAAGTGTGACAAAAA-3’

[0238] SEQ ID NO:13(23bp)

[0239] Bar-F:5’-TGCACCATCGTCAACCACTACAT-3’

[0240] SEQ ID NO:14(21bp)

[0241] Bar-R:5’-AGAAACCCACGTCATGCCAGT-3’

[0242] SEQ ID NO:15(20bp)

[0243] Check-F:5’-GCGCATGTACTTCATCCCGT-3’

[0244] SEQ ID NO:16 (20bp)

[0245] Check-R: 5’-AAAAATCTCAGCATCCCGCC-3’

[0246] SEQ ID NO:17 (20bp)

[0247] Cas9-F: 5’-CACCATCTACCACCTGAGAA-3’

[0248] SEQ ID NO:18 (20bp)

[0249] Cas9-R: 5’-CGAAGTTGCTCTTGAAGTTG-3’

Claims

1. A method for preparing humidity-sensitive male sterile maize, comprising: Reducing the activity of the protein in maize, reducing the content of the protein in maize, inhibiting the expression of the coding gene of the protein in maize, or knocking out the coding gene of the protein in maize to obtain humidity-sensitive male-sterile maize; the amino acid sequence of the protein is as shown in SEQ ID NO: 2 or 4.

2. The method according to claim 1, characterized in that: The nucleotide sequence of the coding gene of the protein is as shown in SEQ ID NO: 1 or 3.

3. The method according to claim 2, characterized in that: The method is achieved by site-directed editing of the coding gene of the protein through the CRISPR gene editing system.

4. The method according to claim 3, characterized in that: The CRISPR gene editing system is the CRISPR-Cas9 system; the target sequence of the coding gene of the protein used in the CRISPR-Cas9 system is target sequence 1 or target sequence 2; the nucleotide sequence of target sequence 1 is as shown in SEQ ID NO: 5; the nucleotide sequence of target sequence 2 is as shown in SEQ ID NO:

6.

5. The method according to claim 4, characterized in that: The method obtains humidity-sensitive male-sterile maize by introducing the coding gene of sgRNA targeting target sequence 1 or target sequence 2 and the coding gene of Cas9 into maize.

6. The method according to any one of claims 1-5, characterized in that: The water loss rate of the pollen of the humidity-sensitive male-sterile maize is higher than that of the pollen of the wild-type maize; the fertility of the humidity-sensitive male-sterile maize can be restored by the method of artificial humidity-assisted pollination.

7. A product for preparing humidity-sensitive male sterile maize, the product being used to reduce the activity of the protein described in claim 1 in maize, reduce the content of the protein in maize, inhibit the expression of the coding gene of the protein in maize or knockout the coding gene of the protein in maize.

8. The product according to claim 7, characterized in that: The product is the reagent and / or instrument required for site-directed editing of the coding gene of the protein using the CRISPR / Cas9 system.

9. The product according to claim 8, characterized in that: The reagent is reagent 1, reagent 2, reagent 3, reagent 4, or reagent 5; The reagent 1 is R1) or R2): R1) sgRNA targeting target sequence 1 described in claim 4; R2) sgRNA targeting target sequence 2 described in claim 4; The reagent 2 is a composition composed of the reagent 1 and Cas9; The reagent 3 is a recombinant vector containing the coding gene of R1) or R2); The reagent 4 is a composition composed of the reagent 3 and a recombinant vector containing the coding gene of Cas9; The reagent 5 is a recombinant vector containing the coding gene of R1) and / or R2) and the coding gene of Cas9.

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