The male sterility gene ZmC3H41 and its application in creating male-sterile maize lines

Abstract

This invention discloses a male infertility gene. ZmC3H41 This invention, and its application in creating male-sterile maize lines, belongs to the field of plant biotechnology breeding. This invention utilizes CRISPR / Cas9 gene editing technology to perform site-directed mutations in this gene in wild-type maize, causing abnormal pollen development and male abortion, demonstrating… ZmC3H41 Genes play a regulatory role in male reproductive development in maize. Through offspring screening, stable male-sterile maize lines without transgenic components can be obtained, which is of great significance for controlling male fertility and producing hybrid seeds in maize. This invention also addresses the obtained... c3h41 Functional molecular markers were designed for the male sterility mutant gene, which have important application value in the breeding of male sterile maize lines, sterile hybridization seed production, and molecular marker-assisted selection.

Description

Technical Field

[0001] This invention belongs to the field of plant biotechnology breeding, specifically involving male sterility genes. ZmC3H41 And its application in the creation of male-sterile maize lines. Background Technology

[0002] Corn is my country's largest grain crop in terms of both planting area and total yield, with an annual planting area exceeding 600 million mu (approximately 40 million hectares). Among the three major grain crops, corn is the one whose heterosis has been utilized earliest and most thoroughly. However, the corn seed production industry remains in a labor-intensive stage, primarily relying on manual detasseling, resulting in high costs, huge resource consumption, and difficulty in guaranteeing seed quality. Using male-sterile materials for hybrid seed production can save the cost of manual or mechanical detasseling, improve seed purity and yield, and has significant industrial application value.

[0003] Plant male sterility can be divided into nuclear male sterility (GMS), controlled by nuclear genes, and cytoplasmic male sterility (CMS), which is synergistically regulated by nuclear and cytoplasmic genes. CMS lines suffer from poor genetic diversity, homogeneous cytoplasm, and susceptibility to disease. GMS, controlled solely by nuclear genes, can overcome these drawbacks. Therefore, in production applications, creating more GMS lines and cloning the corresponding GMS genes is particularly important. Compared with model plants Arabidopsis thaliana and rice, the number of cloned and identified GMS genes and created male sterile materials in maize is relatively small. CRISPR / Cas9 (Clustered, Regularly Interspaced, Short Palindromic Repeats-associated Endonuclease 9) gene editing technology, due to its low cost, ease of operation, and high mutation induction rate, is increasingly widely used in plant gene function research, crop genetic improvement, and breeding, showing great promise. Using CRISPR / Cas9 technology to discover and identify candidate genes for male sterility in maize and to create male sterile materials can rapidly enrich maize GMS gene and sterile material resources, and promote the popularization and application of maize sterilization breeding and seed production. Summary of the Invention

[0004] To address the shortcomings of existing technologies, the purpose of this invention is to provide a male sterility gene. ZmC3H41 Its application in creating male-sterile maize lines can be used to create male-sterile maize lines, which can then be applied to maize hybridization breeding and seed production.

[0005] To achieve the above objectives, the present invention provides ZmC3H41The application of a gene in controlling male reproductive development in maize is characterized in that the nucleotide sequence of the gene is shown in SEQ ID NO.1 and the amino acid sequence of the gene is shown in SEQ ID NO.2.

[0006] On the other hand, the present invention also provides a method for creating male-sterile maize lines, characterized by inhibiting the growth of male-sterile maize... ZmC3H41 Gene expression and / or activity were selected from male-sterile maize plants.

[0007] In some implementations, the methods for inhibiting gene expression and / or activity include any one of gene editing, RNA interference, and T-DNA insertion.

[0008] In some implementations, the gene editing described above uses the CRISPR / Cas9 method.

[0009] In some implementations, the CRISPR / Cas9 method includes: in ZmC3H41 A CRISPR / Cas9 vector target (MT) was designed at exon 1 of the gene, and the DNA sequence of the target is shown in SEQ ID NO.3.

[0010] On the other hand, the present invention also provides a way to obtain c3h41 The method for obtaining male-sterile lines will be achieved through the methods described above. c3h41 Male-sterile lines are hybridized and backcrossed with target materials to obtain the desired results. c3h41 Male infertility traits and gene mutations.

[0011] The present invention also includes those obtained by any of the above methods. c3h41 The application of sterile lines in hybridization breeding and seed production. The application in hybridization breeding and seed production refers to... c3h41 The sterile line is used as the maternal parent in crosses with other paternal parents, or the resulting... c3h41 Male-sterile lines are hybridized and backcrossed with other target materials to obtain the desired results. c3h41 Male infertility traits and gene mutations.

[0012] Furthermore, this invention also provides two male-sterile maize lines. c3h41 The molecular marker primers, the sequences of primers ZmC3H41-F1 and ZmC3H41-R1 are shown in SEQ ID NO.4 and SEQ ID NO.5, respectively; the sequences of primers ZmC3H41-F2 and ZmC3H41-R2 are shown in SEQ ID NO.6 and SEQ ID NO.7, respectively.

[0013] The advantages and beneficial effects of this invention are as follows: ZmC3H41 ( Zm00001eb014140 The regulation of male reproductive development in maize by the gene and its encoded protein is previously unreported. This invention utilizes the CRISPR / Cas9 method to mutate the maize gene. ZmC3H41 ( Zm00001eb014140 ), discovered ZmC3H41 ( Zm00001eb014140 The gene's regulatory function on maize tassel development. Using CRISPR / Cas9 gene editing and the resulting male-sterile mutants, male-sterile lines in maize can be created, which can then be applied to maize hybrid breeding and seed production. (Targeting...) c3h41 Cosegregating molecular markers developed for male-sterile lines can be used for identifying fertility alleles in plants, screening target plants in marker-assisted breeding, and identifying seed purity. Attached Figure Description

[0014] Figure 1 for ZmC3H41 Analysis of gene expression patterns in anthers at different developmental stages of maize S5, sporogenous cell stage; S6, microsporocyte stage; S7, meiosis initiation stage; S8a, meiosis I, didic stage; S8b, meiosis II, tetrad stage; S8b-9, tetrad-monuclear microsporus stage; S9, mononuclear microsporus stage; S9-10, mononuclear microsporus-microsporus vacuolization stage; S10, microsporus vacuolization stage; S11, first unequal mitosis of microsporus, binuclear microsporus stage; S12, second mitosis of microsporus, trinuclear microsporus stage.

[0015] Figure 2 for pCas9-ZmC3H41 Physical map of site-directed mutagenesis expression vector pCas9-ZmC3H41 From the left to the right boundary of the T-DNA are herbicide resistance genes. Bar Expression cassette; nuclease-encoding gene Cas9 The expression box; ZmC3H41 Expression cassettes of gene targets (MT).

[0016] Figure 3 Wild type ZmC3H41 Gene structure and DNA sequence analysis of its sterile mutant wild type ZmC3H41 (WT) - ZmC3H41 The gene is 1840 bp in length and includes 2 exons and 1 intron. c3h41 mutant ZmC3H41-Cas9-1One base (C) is deleted at 135 bp in the first exon, and one base (T) and 34 bases (CGCTGGAGACACTGCTCAACTCGACCTCATCGTG) occur between 284 bp and 317 bp. ZmC3H41-Cas9-2 One base (A) is inserted at position 135 bp in the first exon, and one base (C) is deleted at position 314 bp.

[0017] Figure 4 Wild type and c3h41 Phenotypic analysis of tassels, anthers, and pollen grains of homozygous mutants The top row shows wild-type corn (WT) and ZmC3H41-Cas9-1 and ZmC3H41-Cas9-2 Phenotypic comparison of mutant male ears; second row shows WT and... ZmC3H41-Cas9-1 and ZmC3H41-Cas9-2 Phenotypic comparison of mutant anthers; bottom row: WT and... ZmC3H41-Cas9-1 and ZmC3H41-Cas9-2 Comparison of I2-KI staining of mutant pollen grains.

[0018] Figure 5 Wild type and c3h41 Scanning electron microscopy (SEM) analysis of anthers of homozygous mutants From left to right: Wild-type (WT) anthers as a whole; c3h41 The anthers as a whole; the WT (top) after peeling and c3h41 (Bottom) Anthers; mature pollen grains of WT (top) and c3h41 Shriveled pollen grains (bottom); WT (top) and c3h41 (Bottom) Cuticle of the anther's outer epidermis; WT (top) and c3h41 (Below) Ustite of the inner epidermis of the anther.

[0019] Figure 6 To utilize co-separation markers ZmC3H41-Cas9-1 Genotyping of F2 generation plants from sterile lines Six plants were co-isolated using the marker ZmC3H41-F1 / R1. ZmC3H41-Cas9-1 PCR and agarose gel electrophoresis results of F2 generation plants from the sterile line: a 190 bp band was amplified in homozygous wild-type (AA) plants; C3H41 / c3h41 Two bands, 190 bp and 157 bp, were amplified in heterozygous (Aa) plants; c3h41 / c3h41 A 157 bp band was amplified in the homozygous mutant (aa) plant.

[0020] Figure 7 To utilize co-separation markers ZmC3H41-Cas9-2 Genotyping of F2 generation plants from sterile lines The co-segregating marker ZmC3H41-F2 / R2 was used on 11 plants. ZmC3H41-Cas9-2 PCR and polyacrylamide gel electrophoresis (PAGE) identification results of F2 generation plants of the sterile line: a 55 bp band was amplified in homozygous wild-type (AA) plants; C3H41 / c3h41 Two bands, 55 bp and 54 bp, were amplified in heterozygous (Aa) plants; c3h41 / c3h41 A 54 bp band was amplified in the homozygous mutant (aa) plant. Detailed Implementation

[0021] The following embodiments are used to illustrate the present invention, but do not limit the scope of the invention. Any modifications or substitutions made to the methods, steps, or conditions of the present invention without departing from the spirit and essence of the invention are within the scope of the invention. Unless otherwise specified, the synthesis and sequencing of primers and genes used in the embodiments were performed by Beijing Ruiboxingke Biotechnology Co., Ltd. Other biochemical reagents, unless otherwise specified, are conventional commercially available reagents, and the technical means used in the embodiments are conventional means well known to those skilled in the art.

[0022] Example 1: Corn ZmC3H41 ( Zm00001eb014140 Gene sequence and expression pattern analysis The query for corn in the maizeGDB database (https: / / www.maizegdb.org / ) yielded results. ZmC3H41 ( Zm00001eb014140, Zm00001d028828 The gene, in B73, has the nucleic acid sequence shown in SEQ ID NO.1, and its function is annotated as C3H transcription factor 341. C3H41 Its encoded protein contains 397 amino acids, and its sequence is shown in SEQ ID NO.2.

[0023] C3H transcription factors are involved in the regulation of numerous physiological processes in plants, because ZmC3H41 There is no publicly available research data on the actual function of this gene in maize. To investigate the relationship between this gene and male reproductive development in maize, this invention first used qRT-PCR to analyze the expression pattern of this gene at different stages of maize anther development. The specific steps are as follows:

[0024] 1. Sampling and developmental stage identification of maize anthers Anther samples of different lengths were collected from tassels of maize inbred line B73 at different developmental stages, according to the length of the anthers. Twenty fresh anthers of similar length were collected from each sample. Three of them were fixed in FAA solution (Coolaber, China) and their specific developmental stage was determined by resin semi-thin sectioning experiment. The remaining 17 anthers were immediately frozen in liquid nitrogen for RNA extraction.

[0025] The anthers fixed for resin sectioning were dehydrated using a gradient of ethanol (50%, 70%, 90%, 100%) for 15-30 minutes per step. During dehydration, the anthers could be preserved long-term in 70% ethanol. To facilitate later embedding, 0.1% eosin could be added to 90% ethanol for staining. To ensure thorough dehydration, the material needed to be dehydrated 2-3 times in anhydrous ethanol. Resin replacement was then performed, with the anthers placed sequentially in ethanol-to-Spurr resin solutions at volume ratios of 3:1, 1:1, and 1:3 for 2-4 hours, and finally in pure resin overnight. After resin replacement, the anthers were placed in a mold, 200 µL of Spurr resin was added, and the mold was placed in an oven at 70°C for overnight polymerization. The mold was then trimmed, and sections were prepared using a Leica microtome to a thickness of 2 µm. The sections were then picked up with forceps, placed in sterile water in the center of a glass slide, and incubated overnight at 42°C. Immerse the glass slide containing the fixed sample in 0.1% toluidine blue staining solution for 1 minute, then rinse with deionized water, place on a slide stage, and dry before use for microscopic observation; alternatively, it can be mounted for long-term preservation. Analyze the resin sections and determine the specific developmental stage of each sample based on the cytological characteristics of 14 different developmental stages of maize (Stage 1-Stage 14: S1-S14).

[0026] 2. qRT-PCR analysis Total RNA was extracted from maize anthers at different developmental stages (S5-S12) using Trizol reagent (Invitrogen, USA); cDNA was then synthesized using 5X All-in-One RT Master Mix (ABM, Canada); quantitative reverse transcription polymerase chain reaction (RT-PCR) was performed using TBGreen™ PreMix Ex Taq™ (TaKaRa, Japan) on a QuantStudio 5 Real-Time PCR System (ABI, USA). The amplification primers were: ZmC3H41-qF (SEQ ID NO.3): 5'-ATTCTCAGCCACGCCCACAT-3'; ZmC3H41-qR (SEQ ID NO.4): 5'-ACCGAACACGGAGCCACT-3'; ZmUBI2 As a reference gene, its amplification primers are: UBI2-F (SEQ ID NO.5): 5'- CGACAACGTGAAGGCGAAGA -3'; UBI2-R (SEQ ID NO.6): 5'-ACGCAGATACCCAGGTACAGC-3'; Each developmental stage included three biological replicates, and each sample included three technical replicates; data were collected using 2... -ΔΔCt The methods were analyzed, and the quantitative results are presented in the form of mean ± standard deviation (Means ± SD).

[0027] ZmC3H41 The gene exhibits a pattern of anther development-specific expression: higher expression in the early stage of maize anther development (S6), and lower expression levels maintained in the middle and late stages of anther development. Figure 1 ).

[0028] Example 2: Corn ZmC3H41 ( Zm00001eb014140 The function of genes and the creation of male-sterile maize lines using CRISPR / Cas9. To clarify corn ZmC3H41 ( Zm00001eb014140 Regarding the function of maize, this invention selects the maize inbred line Xiang 249 as the recipient material for gene editing and uses the CRISPR / Cas9 gene editing method to mutate it. Zm00001eb014140 Gene sequence, knocking out the function of this gene in maize.

[0029] 1. ZmC3H41 Construction of CRISPR / Cas9 gene editing vector The gene editing vector of the present invention is pBUE411-MT1T2-Cas9 The basic carrier of this carrier is pBUE411- Cas9 The intermediate carrier is pCBCmT1T2 This invention provides gRNA. The process involves designing target sites on primers, obtaining MT-sgRNA via PCR, and then ligating it into a basic vector via enzyme digestion. The specific construction procedure is as follows: (1) Design of target gRNA. ZmC3H41 ( Zm00001eb014140 The gene sequence of the sgRNA was input into http: / / crispr.hzau.edu.cn / cgi-bin / CRISPR2 / CRISPR for target design. The DNA sequences of the target regions selected in this invention are shown in SEQ ID NO.7 and SEQ ID NO.8. The sgRNA backbone sequence of this invention is derived from the intermediate vector. pCBCmT1T2Obtained by direct amplification.

[0030] MT1 (SEQ ID NO.7):CCTCATTGTGCGTCTCCGA; MT2 (SEQ ID NO.8):CATCGTGCGTCTCCGACAG; (2) MT-sgRNA was obtained by designing target sites on primers and then amplifying by PCR. Primers ZmC3H41-MT-F and ZmC3H41-MT-R were used to amplify the intermediate vector. pCBCmT1T2 This was used to obtain a fragment of sgRNA containing the target, with a product length of 891 bp. The PCR system and conditions are as follows: template DNA (intermediate vector) pCBCmT1T2 ≥30 ng / μL) 1.2 μL; Primer F / R: 1.2 μL each; Sterile ddH2O: 11.4 μL; 2X MCLAB enzyme (product number: I5HM-200): 15 μL. The PCR temperature program is as follows: ① 98 ℃ for 2 minutes; ② 98 ℃ for 10 seconds; ③ 58 ℃ for 30 seconds; ④ 72 ℃ for 30 seconds; ⑤ Cycle 34 times from ② to ④; ⑥ 72 ℃ for 5 minutes; ⑦ 25 ℃ for 10 minutes. Finally, recover the PCR product. The primer sequences required for vector construction are as follows: ZmC3H41-MT-F (SEQ ID NO.9): 5'-ATATATGGTCTCTGGCGACCTCATTGTGCGTCTCCGAGTTTTAGAGCTAGAAATAGCAA -3' ZmC3H41-MT-R (SEQ ID NO.10): 5'-ATTATTGGTCTCTAAACCATCGTGCGTCTCCGACAGTGCTTCTTGGTGCCGC-3' (3) Construct the backbone vector by enzyme digestion and ligation. pBUE411-Cas9 Vectors and recovered target-carrying sgRNA fragments are used BsaI Digestion was performed, and T4 ligase was added to ligate the vector and sgRNA fragment. The 15 μL restriction enzyme ligation system was as follows: sgRNA fragment: 2 μL, pBUE411-Cas9 vector (≥60 ng / μL): 2 μL, 10 x NEB Buffer: 1.5 μL. BsaI Endonuclease (product number: #R3733S): 1 μL, T4 ligase (product number: #M0202M): 1 μL, sterile ddH2O: 6 μL.

[0031] Figure 2 The target gene is shown. ZmC3H41( Zm00001eb014140 ) targets, marker genes Cas9 and bar With skeleton carrier pBUE411-Cas9 Constructed expression carrier pCas9-ZmC3H41 .

[0032] 2. Agrobacterium-mediated genetic transformation of maize The above-constructed pCas9-ZmC3H41 The vector was transferred into Agrobacterium EHA105 via heat shock and identified by PCR. The Agrobacterium containing the knockout vector was then stored in glycerol at -80 °C. Freshly peeled immature embryos (approximately 1.5 mm in diameter) of the maize inbred line Xiang 249 were used as recipient material. The peeled maize embryos were placed in 2 mL plastic centrifuge tubes containing 1.8 mL of suspension for no more than 1 hour, with approximately 100 embryos per tube. The suspension was removed, and the embryos were washed twice with fresh suspension, leaving a small amount at the bottom of the tube to submerge them. The tubes were then heat-shocked at 43 °C for 2 minutes, followed by an ice bath for 1 minute. The remaining wash solution was aspirated with a pipette, and 1.0 mL of Agrobacterium infection solution was added. The tubes were gently shaken for 30 seconds and then incubated in the dark for 8 minutes. Next, the embryos and infection solution were poured onto a co-culture medium, mixed well, and excess infection solution was aspirated with a pipette. All embryos were cultured with their scutes facing upwards at 23 °C in the dark for 3 days. After co-culture, the immature embryos were transferred to recovery medium using sterile forceps and cultured at 28 ℃ for 7-14 days, during which time any emerging shoots should be removed promptly. After recovery culture, the immature embryos were placed on a selection medium containing 1.5 mg / L Bialaphos for three rounds of selection, each round lasting two weeks, and then transferred to a selection medium containing 2 mg / L Bialaphos for two more rounds of selection, each round lasting two weeks. The resistant callus was transferred to propagation medium and cultured in the dark at 28 ℃ for two weeks. Subsequently, the propagated resistant callus was transferred to induction medium and cultured in the dark at 28 ℃ for two weeks. Then, it was transferred to differentiation medium and cultured under light at 25 ℃ and 5000 lx for two weeks. After culture, the differentiated seedlings were separated into individual seedlings and placed in rooting medium at 25 ℃ and 5000 lx under light until rooting. The seedlings were then transferred to small nutrient pots for further growth. Once established, they were transplanted into a greenhouse, and the offspring seeds were harvested after 3-4 months.

[0033] 3. Detection of CRISPR / Cas9 mutation results in T0 generation plants To determine the CRISPR / Cas9 mutation results in T0 generation plants, the following steps were taken: This invention first uses the CTAB method to extract DNA from maize leaves. The specific method is as follows: Cut seedling leaves approximately 2 cm in length and place them in a 2 mL centrifuge tube containing steel balls; immerse the centrifuge tube containing the leaves in liquid nitrogen for 5 minutes, then crush the leaf sample using a grinder; add 700 μL of CTAB extraction buffer (containing 1% β-mercaptoethanol) to the centrifuge tube and mix vigorously; preheat in a 65 ℃ constant temperature water bath for 20-30 min (during which time remove and invert 1-2 times, paying attention to the correspondence of the experimental sample numbers); after the centrifuge tube cools to room temperature, add 700 μL of chloroform:isoamyl alcohol (24:1) extraction solution, shake vigorously for 30 seconds, and then let stand at room temperature for a short time; centrifuge at 12000 rpm for 5 min at 4 ℃, and take 500 μL of the supernatant after centrifugation into a new 1.5 mL container. In a 1 mL centrifuge tube, add an equal volume of isopropanol to the tube containing the supernatant, gently vortex to mix, and let stand at room temperature for about 10 min. Then, place the centrifuge tube containing the sample in a 4 ℃ centrifuge at 12000 rpm for 10 min. Gently aspirate the supernatant, discard it, and retain the precipitate. Add 800 μL of 75% ethanol, wash the precipitate twice, centrifuge at 10000 rpm for 5 min, and discard the supernatant. Allow the sample to air dry at room temperature for 2-4 hours to obtain DNA precipitate. Add an appropriate amount of sterile water to dissolve the DNA, gently vortex to fully dissolve the DNA. Store the DNA sample at -20 ℃. Detect the DNA concentration using Nanodrop and dilute to 10 ng / L for use as a PCR template.

[0034] Then according to ZmC3H41 ( Zm00001eb014140 ) Design PCR primers based on gene sequences.

[0035] Target: MT; Product size: 486 bp; Primer sequences are as follows: ZmC3H41-TF (SEQ ID NO.11): 5'-TTCGATGGTCGCTACGGC-3'; ZmC3H41-TR (SEQ ID NO. 12): 5'-GGTCCTGGGTGAAGAGTTGTTT-3'.

[0036] Genomic DNA was extracted and amplified using the following PCR parameters: Reaction system: 15 μL MIX conventional PCR system, 0.5 μL forward primer, 0.5 μL reverse primer, 1 μL DNA, 5.5 μL sterile ddH2O, 7.5 μL 2x Taq mix (product number: 10103ES).

[0037] Reaction procedure: Conventional PCR: annealing at 58 ℃, extension for 1 minute, 32 cycles.

[0038] Next, the PCR products were recovered and ligated into a T vector for sequencing. By sequencing the DNA sequences of the target regions of multiple T0 generation independent positive transformation events, it was determined whether gene editing had occurred in the target regions. Ultimately, it was found that the target region sequences of two T0 transformation events had changed, and both were homozygous mutations. The sequences before and after editing are shown below. Figure 3 As shown, there are 2 corresponding ones. c3h41 homozygous mutant: ZmC3H41-Cas9-1 and ZmC3H41-Cas9-2 Sequence alignment with wild type showed... ZmC3H41-Cas9-1 and ZmC3H41- Cas9-2 A deletion mutation occurred at the target site, and... ZmC3H41 Compared to the nucleotide sequence of the gene in SEQ ID NO.1, ZmC3H41-Cas9-1 exist ZmC3H41 The first exon has a 135 bp site where one C base is deleted, and a 1 T base is inserted between 284 bp and 317 bp, resulting in a 34-base deletion: CGCTGGAGACACTGCTCAACTCGACCTCATCGTG. ZmC3H41- Cas9-2 One A base is inserted at the 135 bp site of the first exon, and one C base is deleted at the 314 bp site.

[0039] For 2 c3h41 Comparative analysis of amino acid sequences in homologous mutants revealed that, compared with the unedited WT, the mutant strains... ZmC3H41-Cas9-1 and ZmC3H41-Cas9-2 The deletion of nucleotides at the target site caused frameshift mutations in all amino acids, and subsequent amino acids all underwent premature termination. Therefore, the ZmC3H41 protein in these transformants exhibited a loss of function.

[0040] 4. Genotyping of F1 generation plants Because greenhouse-grown T0 generation maize plants often exhibit uncoordinated development of female and male ears, and because fertility is also affected when the edited gene is associated with male development, this invention uses wild-type pollen from the maize inbred line Xiang 249 as the genetic material for propagating T0 generation plants and ensuring the inheritance of the obtained gene-edited type. ZmC3H41-Cas9-1 and ZmC3H41-Cas9-2 The T0 generation plants are pollinated to obtain F1 generation seeds, and the resulting plants are F1 generation plants.

[0041] The F1 generation plants included two segregation types, one of which was... Cas9 - Positive plants (transgenic plants), another type is Cas9- To prevent the continuous editing of the wild-type alleles of Xiang 249 introduced through hybridization by sgRNA and Cas9, thus causing complexity in mutation types, we need to select plants from the F1 generation that do not contain sgRNA or Cas9 through genotyping. Cas9 The genes, but containing the T0 generation mutation type, can produce non-transgenic F2 generation plants after self-pollination. The genotyping steps for F1 generation plants are as follows: After extracting leaf DNA using the CTAB method described above, the first step is to utilize... Cas9 Gene-specific primers Cas9-F and Cas9-R were used for PCR amplification.

[0042] Cas9-F (SEQ ID NO.13): 5'-CCCGGACAATAGCGATGT-3; Cas9-R (SEQ ID NO. 14): 5'-GAGTGGGCCGACGTAGTA-3'.

[0043] The PCR reaction system was the same as above; the reaction procedure was as follows: standard PCR: annealing at 58℃, extension for 1 minute, 32 cycles. After agarose gel electrophoresis, the PCR products were distinguished based on the results. Cas9 -positive plants and Cas9 -Negative plants.

[0044] Further targeting Cas9 - For negative plants, PCR amplification was performed using the primers ZmC3H41-TF and ZmC3H41-TR for detecting the MT target. After purification of the PCR product, it was ligated into the T vector and sequenced. The genetic information of the T0 generation mutation type was determined based on the sequencing results.

[0045] Example 3 c3h41 Phenotypic analysis of sterile lines The above-described Example 2 identified that it does not contain Cas9 The F1 generation plants of the gene were self-pollinated to produce F2 generation seeds, two types. c3h41 Mutation type ( ZmC3H41-Cas9-1 and ZmC3H41-Cas9-2 One self-pollinated single ear from each F2 line was sown in panicle rows, and phenotypic analysis was conducted at maturity. In both F2 lines, the ratio of fertile to sterile plants conformed to a 3:1 segregation, further indicating... c3h41 The sterility trait in the sterile line is controlled by a single recessive gene, and then stable non-transgenic lines are obtained by targeting the F2 generation. c3h41 A detailed phenotypic comparison was conducted between the sterile line and the wild type.

[0046] 1. Observation of the viability of male spikes, anthers, and pollen. In terms of vegetative growth and female ear development, c3h41Sterile line ( ZmC3H41-Cas9-1 and ZmC3H41-Cas9- 2 The plants of the wild type are basically no different from those of the wild type; in terms of male inflorescence development, the wild type can produce males normally, the anthers can dehisce and release pollen normally, and it can produce seeds normally after self-pollination, while... c3h41 Although the sterile line can produce males normally, it cannot flower normally. The anthers and glumes are almost unable to dehisce, and the anthers are smaller and almost not exposed compared to the wild type. Figure 4 Further I2-KI staining of wild-type and mutant pollen revealed that wild-type pollen developed normally and turned black after staining, but mutant pollen could not fill with starch. Figure 4 This indicates that... ZmC3H41 ( Zm00001eb014140 Genes control male development in maize, and males created through gene editing methods... c3h41 The sterile line is a pollen grain abortion type and exhibits sterility characteristics.

[0047] 2. Scanning electron microscopy (SEM) observation of anthers To analyze in depth c3h41 The cytological characteristics of wild-type and mutant anthers were analyzed by scanning electron microscopy (SEM). Mature (S13) wild-type and mutant anthers were harvested and immediately fixed in FAA (Coolaber, China) solution, with the volume of the fixative not less than 20 times the volume of the studied material. For mutant anthers, perforations in the anther wall were made using a dissecting needle to improve fixative penetration, or repeated vacuuming was performed until the anthers sank to the bottom of the fixative. After fixation at room temperature for 2 hours, the material was stored at 4°C, or sequentially dehydrated in 50%, 60%, 70%, 80%, 90%, and 100% ethanol, maintaining each gradient for 15 minutes. The material could also be stored overnight in 70% ethanol. After dehydration, the samples were subjected to critical point carbon dioxide drying and then gold plating for observation. Findings... c3h41 The cuticle of the anther epidermis and the Ubstein bodies of the inner epidermis of the mutant were not significantly different from those of the wild type. However, c3h41 The pollen grains of the mutant were mostly shriveled, and the starch filling was abnormal. Figure 5 The above results indicate that... ZmC3H41 ( Zm00001eb014140 Gene mutations can affect pollen starch filling, ultimately leading to pollen abortion.

[0048] Example 4 c3h41 Development and application of cosegregating molecular markers for the identification of sterile lines

[0049] 1. Development of co-separated molecular markers In this invention, for the two obtained c3h41The mutation sites of the sterile lines were identified by primer design using Primer 5.0 software, and two pairs of co-segregating molecular markers, ZmC3H41-F1 / R1 and ZmC3H41-F2 / R2, were developed. Combined with PCR, agarose gel electrophoresis, and polyacrylamide gel electrophoresis (PAGE) detection methods, the genotype of the mutants could be separated based on the obtained bands and their size.

[0050] The co-separating molecular marker ZmC3H41-F1 / R1 comprises a first primer ZmC3H41-F1 and a second primer ZmC3H41-R1; this marker can specifically detect maize. ZmC3H41-Cas9-1 Mutants and mutant genes in maize male-sterile materials derived from them c3h41 And can distinguish between wild type at the same time. C3H41 Genes and mutants c3h41 Genes; targeting mutated genes c3h41 A 157 bp band was amplified in the middle, while in the wild type... C3H41 The gene amplified into a 190 bp band. The primer sequences are as follows: ZmC3H41-F1 (SEQ ID NO.15): 5'-GACATGGGGATCGCCGC-3'; ZmC3H41-R1 (SEQ ID NO. 16): 5'-CGCCATCTTCGGGCTACC-3'.

[0051] The co-separating molecular marker ZmC3H41-F2 / R2 comprises a first primer ZmC3H41-F2 and a second primer ZmC3H41-R2; this marker can specifically detect maize. ZmC3H41-Cas9-2 Mutants and mutant genes in maize male-sterile materials derived from them c3h41 And can distinguish between wild type at the same time. C3H41 Genes and mutants c3h41 Genes; targeting mutated genes c3h41 A 54 bp band was amplified in the middle, while in the wild type... C3H41 The gene was amplified into a 55 bp band. The primer sequences are as follows: ZmC3H41-F2 (SEQ ID NO. 17): 5'-CGCCGCTGGAGACACTGC-3'; ZmC3H41-R2 (SEQ ID NO. 18): 5'-CCACGGCTGTCGGAGACG-3'.

[0052] 2. Application of co-separated molecular markers To verify the effectiveness of the above-mentioned markers, the F2 strain obtained in Example 3 was used as material for testing. c3h41Allele detection. The DNA extraction method, PCR amplification system and conditions were the same as in Example 2. The PCR products were separated by agarose gel electrophoresis.

[0053] Theoretically, ZmC3H41-F1 / R1 and ZmC3H41-F2 / R2 in C3H41 / C3H41 In homozygous wild-type (AA) DNA, bands of 190 bp and 55 bp can be amplified. c3h41 / c3h41 In the homozygous mutant material (aa), bands of 157 bp and 54 bp were amplified, respectively, while... C3H41 / c3h41 In hybrid (Aa) materials, the corresponding two bands can be amplified simultaneously. The verification results of the ZmC3H41-F1 / R1 and ZmC3H41-F2 / R2 molecular markers are as follows: Figure 6 , Figure 7 As shown, the results indicate that the designed functional molecular markers completely met expectations in detecting F2 plants. C3H41 / C3H41 Homozygous wild type (AA) C3H41 / c3h41 Heterozygous (Aa) and c3h41 / c3h41 The homozygous mutant material (aa) amplified bands of corresponding sizes, which can be used as... C3H41 , c3h41 An ideal marker for allele detection.

[0054] These molecular markers help identify mutant genotypes before flowering and pollination, enabling hybridization and backcrossing to breed male-sterile lines under different genetic backgrounds, and have important application value.

Claims

1. A method for creating male-sterile maize lines, characterized in that, Knockout of maize using gene editing technology ZmC3H41 Genes were used to obtain male-sterile maize plants; the aforementioned ZmC3H41 The nucleotide sequence of the gene is SEQ ID NO.1, and its encoded amino acid sequence is SEQ ID NO.

2.

2. The method for creating a male-sterile maize line according to claim 1, characterized in that, The gene editing was performed using the CRISPR / Cas9 method.

3. The method for creating a male-sterile maize line according to claim 2, characterized in that, The CRISPR / Cas9 method includes designing a CRISPR / Cas9 vector target at the first exon of the gene, the DNA sequence of which is shown in SEQ ID NO.7 and SEQ ID NO.

8.

4. The method for creating male-sterile maize lines as described in claim 3. c3h41 Mutant genes ZmC3H41- Cas9-1 and ZmC3H41-Cas9-2, Its characteristic is that, as described in claim 1 ZmC3H41 Compared to the nucleotide sequence of the gene SEQ ID NO.1, ZmC3H41-Cas9-1 exist ZmC3H41 One C base is deleted at position 135 bp, and one T base is inserted between 284 bp and 317 bp, while 34 bases are deleted: CGCTGGAGACACTGCTCAACTCGACCTCATCGTG. ZmC3H41- Cas9-2 One base A is inserted at the 135 bp site, and one base C is deleted at the 314 bp site.

5. Primers ZmC3H41- F1 and primers ZmC3H41- R1 in identifying the claim 1 ZmC3H41 Gene and the mutant gene of claim 4 ZmC3H41-Cas9-1 The application of this is characterized by, The primers ZmC3H41- The F1 sequence is SEQ ID NO.15, and the primers... ZmC3H41- The R1 sequence is SEQ ID NO.16, using ZmC3H41- F1 and ZmC3H41- R1 primer pair for genotype: C3H41 / C3H41 A 190 bp band can be amplified in homozygous wild-type DNA, and in genotypes of c3h41- Cas9-1 / c3h41-Cas9-1 A 157 bp band can be amplified in homozygous mutant DNA, while in genotypes... C3H41 / c3h41-Cas9-1 The heterozygous DNA can simultaneously amplify the two bands mentioned above, 190 bp and 157 bp.

6. Primers ZmC3H41- F2 and ZmC3H41- R1 in identifying the claim 1 ZmC3H41 Gene and the mutant gene of claim 4 ZmC3H41-Cas9-2 The application of this is characterized by, The primers ZmC3H41- The F2 sequence is SEQ ID NO. 17, and the primers... ZmC3H41- The R1 sequence is SEQ ID NO.16, using ZmC3H41- F2 and ZmC3H41- R1 primer pair for genotype: C3H41 / C3H41 A 55bp band can be amplified in homozygous wild-type DNA, and in genotypes of c3h41-Cas9- 2 / c3h41-Cas9-2 A 54 bp band can be amplified in homozygous mutant DNA, while in genotypes of... C3H41 / c3h41- Cas9-2 In the heterozygous DNA, two bands of 55 bp and 54 bp can be amplified simultaneously.

7. A method for obtaining different genetic backgrounds c3h41 The method for male-sterile lines is characterized by, Obtained by any one of the methods described in claims 1, 2, and 3 c3h41 Male-sterile lines are hybridized and backcrossed with target materials to obtain the desired results. c3h41 Male infertility genes and male infertility traits.

8. Obtained by the method according to any one of claims 1, 2, 3 and 7 c3h41 Application of male-sterile lines in maize hybrid breeding and seed production.

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