Application of ZmGalAT1 and its encoding protein in maize fertility control

Abstract

The application belongs to the field of plant biotechnology breeding, and relates to application of ZmGalAT1 gene and coded protein thereof in maize fertility control. galat1 A maize male sterility mutant is obtained by EMS mutagenesis ZmGalAT1 , and a gene for controlling maize male flower development is identified by map-based cloning galat1 . The gene is site-mutated in wild-type maize by CRISPR / Cas9 gene editing technology, and three maize male sterility mutants are created GalAT1 . The mutant maize male flowers are completely aborted, and the sterile mutants without transgenic components can be obtained through offspring screening, and are further applied to maize sterilization breeding and seed production. Functional molecular markers are designed for the three male sterility mutants, and the markers have important application value in maize male sterile line breeding, sterile hybrid seed production and molecular marker assisted selection.

Description

Technical Field

[0001] This invention belongs to the field of plant biotechnology breeding and relates to controlled fertility. ZmGalAT1 Genes and their infertility mutation genes galat1 ,as well as ZmGalAT1 Application of encoded proteins in maize fertility control. Background Technology

[0002] corn( Zea mays Corn (L.) is an important food crop of the Poaceae family and is widely cultivated globally. It serves not only as a staple food for humans, but its byproducts are also an important source of animal feed. Furthermore, corn kernels have significant value in the industrial sector. Therefore, increasing corn yield plays a crucial role in promoting the development of modern agricultural economy. [1] In crop breeding, heterosis is a common genetic phenomenon, referring to the superior growth vigor, yield, and stress resistance of offspring from crosses between different lines compared to their homozygous parents. Hybrid maize often exhibits higher yields, stronger reproductive capacity, and better resistance to diseases, pests, and stresses. Due to these advantages, heterosis has been widely applied in plant breeding and has become an important means of improving crop productivity. [2] .

[0003] As a monoecious crop with separate male and female flowers, maize requires emasculation of the female parent during hybridization breeding and seed production. Commercial maize hybrids were first introduced in 1924, and the emasculation technique for the female parent has undergone numerous optimizations. Currently, the main emasculation methods include manual emasculation, mechanical emasculation, chemical emasculation, and male sterility (MS). However, traditional methods all have significant drawbacks: manual emasculation is costly and inefficient, and delays can affect seed purity; mechanical emasculation causes physical damage to the plant, hindering normal growth and development; chemical emasculation is not only toxic to the plant but may also lead to incomplete emasculation. In contrast, male sterility offers significant advantages. This technique utilizes naturally or artificially induced male-sterile plants (i.e., plants with abnormal pollen development and inability to pollinate normally) as the female parent, ensuring the purity of hybrid seeds without the need for manual emasculation. Because this technique has no side effects on the environment or plants and can significantly reduce seed production costs, it has become an important way to improve the utilization rate of heterosis and yield in maize. [3] [4] .

[0004] By integrating map-based cloning technology with CRISPR / Cas9 gene editing technology, new maize male sterility genes can be rapidly identified. [5]By combining systematic cytological observations, we can gain a deeper understanding of the morphological changes in male flower development. This will not only help elucidate the molecular mechanisms of male flower development in maize but also rapidly expand the maize nucleus-male sterility gene pool and related germplasm resources. This research strategy will strongly promote the application and dissemination of maize sterility breeding and seed production technologies, ultimately helping to overcome the two major bottlenecks that have long plagued my country's maize seed industry: the lack of stable sterile lines and the difficulty in breeding groundbreaking varieties.

[0005] This invention relates to a male-sterile mutant of maize obtained through EMS mutagenesis. galat1 Using map-based cloning as a material, a gene controlling male flower development in maize was identified. ZmGalAT1 Genes were obtained, and three types were obtained through CRISPR / Cas9 technology. galat1 Allelic male sterility mutants provide valuable genetic material for elucidating the molecular mechanisms of male flower development in maize, and offer excellent genes and key germplasm resources for improving the utilization of heterosis in maize. Summary of the Invention

[0006] To address the shortcomings of existing technologies, the purpose of this invention is to provide... ZmGalAT1 The application of its encoded proteins in maize fertility control can be used to create male-sterile lines, which can be applied to maize hybrid breeding and seed production, improve the utilization efficiency of maize heterosis, and ultimately increase maize yield per unit area.

[0007] To achieve the above objectives, this invention provides a male fertility gene for maize. ZmGalAT1 and its infertility mutation gene galat1 The characteristic is that the maize male fertility gene ZmGalAT1 The nucleotide sequence is SEQ ID NO.1, and the sequence of the protein ZmGalAT1 encoded by it is SEQ ID NO.2.

[0008] This invention obtains a male-sterile mutant gene in maize through EMS mutagenesis. galat1 Its characteristic is that the mutation is caused by the wild type ZmGalAT1 The mutation, caused by the deletion of a single base (C) at position +3039 of exon 7, results in premature termination of translation of the ZmGalAT1 protein, which is only 365 amino acids long. This leads to the inclusion of mutated nucleotide sequences. galat1 Although the mutant can produce males normally, its anthers are shriveled and no pollen is produced, ultimately exhibiting a complete male sterility phenotype; the mutant gene... galat1 The full-length DNA and amino acid sequences are shown in SEQ ID NO.3 and SEQ ID NO.4.

[0009] On the other hand, the present invention also provides ZmGalAT1Application of the gene or ZmGalAT1 protein in the cultivation of male-sterile plants; This invention uses gene editing to inhibit ZmGalAT1 Gene expression and / or activity were used to obtain male-sterile maize lines.

[0010] On the other hand, the present invention also provides a method for creating corn galat1 A method for producing allelic male-sterile mutants, characterized in that the method is a CRISPR / Cas9-based gene editing method, targeting the maize genome... ZmGalAT1 By performing site-directed mutations in genes, their fertility function is lost, resulting in different types of male-sterile maize lines with different mutation types.

[0011] The CRISPR-Cas9 system described above expresses the gene for the Cas9 protein and the gene for sgRNA. The target sequence of the sgRNA is shown in SEQ ID NO.5 of the sequence listing, located at... ZmGalAT1 The 7th exon region of the gene.

[0012] Furthermore, the present invention provides corn galat1 Three allelic male-sterile mutants, characterized in that, galat1 Allelic mutants include ZmGalAT1-Cas9-1, ZmGalAT1-Cas9-2 and ZmGalAT1-Cas9-3 ;in ZmGalAT1-Cas9-1 A deletion of 17 bases occurs at position 3029 bp-3045 bp in the 7th exon; ZmGalAT1-Cas9-2 Nineteen bases are deleted at position 3024 bp-3042 bp in the 7th exon; ZmGalAT1-Cas9-3 Thirteen bases are deleted at position 3027 bp-3039 bp in the 7th exon.

[0013] This invention also provides three maize male sterility mutant genes. ZmGalAT1-Cas9-1, ZmGalAT1-Cas9-2 and ZmGalAT1-Cas9-3 Functional markers;

[0014] The sequences of the mutant functional molecular marker primers ZmGalAT1-F and ZmGalAT1-R are shown in SEQ ID NO. 6 and SEQ ID NO. 7, respectively. This functional marker can simultaneously distinguish between wild-type and wild-type mutants. ZmGalAT1 Genes and galat1-Cas9-1 Mutant genes.

[0015] The advantages and beneficial effects of this invention are as follows: ZmGalAT1 The application of its encoded protein in maize fertility control is previously unreported. This invention employs a map-based cloning method, first using the obtained maize male-sterile mutant material… galat1 In this study, a new gene regulating male flower development in maize was isolated. ZmGalAT1 ,Discover ZmGalAT1The mutation results in abnormal flowering, shriveled anthers, and no pollen production, ultimately manifesting as complete male sterility without pollen. Cloning this gene provides resources and pathways for the artificial creation of male-sterile lines. This invention utilizes CRISPR / Cas9 gene editing to site-directedly mutate the ZmGalAT1 protein-encoding gene, which can be used for maize fertility control and hybrid seed production. This invention targets three genes obtained after CRISPR / Cas9 editing. galat1 Functional molecular markers developed from allelic male sterility mutant genes can be applied to allele identification, target plant screening, and seed purity assessment in maize male-sterile line breeding and seed production. Therefore, this invention is of great significance in maize male sterility breeding and seed production. Attached Figure Description

[0016] Figure 1 wild-type (WT) and mutant maize galat1 phenotype

[0017] A and B, WT and mutants galat1 The tassel phenotype; C and D, WT and mutants galat1 Anther phenotypes; E and F, WT and mutants galat1 I2-KI staining phenotype of anthers. Scale bar = 5cm (AB), 2mm (CD), 100μm (EF).

[0018] Figure 2 For corn ZmGalAT1 Fine mapping and map-based cloning of genes

[0019] A, Infertility galat1 The segregation ratio of polymorphic molecular markers between population DNA and fertile population DNA; 52 male-sterile plants and 52 male-fertile plants in the B, F2 population were used for maize. ZmGalAT1 The initial location of the gene, initially... ZmGalAT1 The gene is located on chromosome 7 between SSR markers umc1450 and umc1324; C, ZmGalAT1 Gene fine mapping was performed within a 146.3 kb interval between SSR markers P2-2 and P2-3; D, four gene models predicted within the fine mapping interval; E, wild-type and... galat1 In mutants ZmGalAT1 Gene structure and sequencing analysis.

[0020] Figure 3 Wild type (WT) and galat1 In mutants Zm00001eb319550 Sequence alignment of gene mutation sites ±100 bp

[0021] Figure 4 for ZmGalAT1Analysis of gene expression patterns at different developmental stages of maize anthers

[0022] S5, sporogenous cell stage; S6, microspore mother cell stage; S7, meiosis initiation stage; S8a, meiosis I, didigrin stage; S8b, meiosis II, tetrad stage; S9, uninucleate microspore stage; S10, microspore vacuolization stage; S11, first unequal mitosis of microspores, binucleate microspore stage; S12, second mitosis of microspores, trinucleate microspore stage; S13, complete starch filling.

[0023] Figure 5 for pCas9-ZmGalAT1 Physical map of site-directed mutagenesis expression vector

[0024] pCas9-ZmGalAT1 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; ZmGalAT1 Expression cassettes for gene target 2 (MT2) and target 1 (MT1). The target sequences of the two expression cassettes are identical.

[0025] Figure 6 Wild type ZmGalAT1 and ZmGalAT1-Cas9 Gene structure and DNA sequence analysis of sterile mutants

[0026] wild type ZmGalAT1 (WT) -ZmGalAT1 The length from the start codon (ATG) to the stop codon (TAG) is 4216 bp, consisting of 9 exons and 8 introns; galat1 Allelic mutants ZmGalAT1-Cas9-1 The mutant has 17 bases deleted at position 3029bp-3045bp in the 7th exon. ZmGalAT1-Cas9-2 The mutant has a 19-base deletion at position 3024 bp-3042 bp in the 7th exon. ZmGalAT1-Cas9-3 : ZmGalAT1-Cas9-3 Thirteen bases are deleted at position 3027 bp-3039 bp in the 7th exon.

[0027] Figure 7 Wild type (WT) and galat1 Phenotypic analysis of tassels, anthers, and pollen grains from three homozygous allelic mutants

[0028] The top row shows corn Wt and ZmGalAT1-Cas9-1 , ZmGalAT1-Cas9-2 and ZmGalAT1-Cas9-3 Phenotypic comparison of homozygous mutant tassels; second row shows WT and... ZmGalAT1-Cas9-1 , ZmGalAT1-Cas9-2 andZmGalAT1- Cas9-3 Phenotypic comparison of anthers from homozygous mutants; bottom row shows WT and... ZmGalAT1-Cas9-1 , ZmGalAT1-Cas9-2 and ZmGalAT1-Cas9-3 Comparison of I2-KI staining of pollen grains from homozygous mutants.

[0029] Figure 8 To utilize molecular markers ZmGalAT1-Cas9-1 Genotyping of F2 generation plants of sterile mutant

[0030] Molecular marker ZmGalAT1-F / R on 12 strains ZmGalAT1-Cas9-1 PCR and agarose gel electrophoresis results of the F2 generation of sterile mutant plants: a 120 bp band was amplified in homozygous wild-type (AA) plants; GALAT1 / galat1 Two bands, 120 bp and 103 bp, were amplified in heterozygous (Aa) plants; galat1 / galat1 A 103 bp band was amplified in the homozygous mutant (aa) plant.

[0031] Figure 9 To utilize molecular markers ZmGalAT1-Cas9-2 Genotyping of F2 generation plants of sterile mutant

[0032] Molecular marker ZmGalAT1-F / R on 12 strains ZmGalAT1-Cas9-2 PCR and agarose gel electrophoresis results of the F2 generation of sterile mutant plants: a 120 bp band was amplified in homozygous wild-type (AA) plants; GALAT1 / galat1 Two bands, 120 bp and 101 bp, were amplified in heterozygous (Aa) plants; galat1 / galat1 A 101 bp band was amplified in the homozygous mutant (aa) plant.

[0033] Figure 10 To utilize molecular markers ​ Genotyping of F2 generation plants of sterile mutant

[0034] Molecular marker ZmGalAT1-F / R on 12 strains ​ PCR and agarose gel electrophoresis results of the F2 generation of sterile mutant plants: a 120 bp band was amplified in homozygous wild-type (AA) plants; ​ Two bands, 120 bp and 107 bp, were amplified in heterozygous (Aa) plants; ​ / ​ A 107 bp band was amplified in the homozygous mutant (aa) plant. Detailed Implementation

[0035] 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 Sangon Biotech (Shanghai) 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.

[0036] Example 1: Male-sterile mutant of maize ​ The acquisition

[0037] By screening the Ethyl Methane Sulfonate (EMS)-induced mutant library of maize inbred line Zheng 58 in our laboratory, a completely male-sterile mutant was obtained and named [name missing]. ​ The male sterility trait of this mutant was stably inherited after multiple generations of hybridization with the wild type in Beijing and Sanya, Hainan, and was unaffected by the environment. Observations and comparisons of plant morphology throughout the entire growth cycle between the mutant and the wild type under various conditions, including the Pinggu Experimental Base of Beijing University of Science and Technology and the Sanya Experimental Base in Hainan, revealed no significant differences, with the mutant exhibiting only the male sterility trait.

[0038] Example 2: Plant phenotypic identification and pollen fertility observation

[0039] ​ The mutant showed virtually no difference from the wild type in vegetative growth and female ear development; however, in male ear development, the wild type could tassel normally, with anthers dehiscing and pollen shedding normally, and could produce seeds normally after self-pollination, while... ​ Although the mutant can produce males normally, it cannot flower normally; the anthers are shriveled and not exposed. ​ A- ​ D); Further I2-KI staining of pollen from wild-type and mutant plants revealed that wild-type pollen developed normally and the pollen grains turned black after staining, but the mutant plants produced no pollen. ​ E, 1F).

[0040] Example 3: ​ Analysis of gene location, cloning, and mutation sites

[0041] Using inbred line B73 as the male parent and mutant ​ In the cross, the F1 generation was fertile, while the F2 generation showed fertility segregation, as shown in Table 1. The segregation of normal fertile plants (F) and sterile plants (S) in the F2 population conformed to the segregation ratio of 3:1 for a single gene, that is, the male sterility phenotype of the mutant is a typical single-gene recessive inheritance.

[0042] Table 1 Corn ​Genetic analysis of mutant segregation

[0043]

[0044] Select ​ The ×B73 F2 population contained 52 male-sterile plants and 52 male-fertile plants, used for maize. ​ Preliminary gene localization study. The SSR markers used are shown in Table 2.

[0045] Table 2 is used for ZmGalAT1 SSR markers for gene localization

[0046]

[0047] Sequencing analysis based on BSA (Bulked Segregant Analysis) revealed that the associated regions of chromosome 7 exhibited the highest polymorphic marker ratio. Figure 2 A). ZmGalAT1 The gene was initially located between the SSR markers umc1450 and umc1324 on chromosome 7. Figure 2 B), further refined to locate the interval of approximately 146.3 kb between markers P2-2 and P2-3 ( Figure 2 C), within this interval, 4 candidate genes are predicted ( Figure 2 D); Cloning and sequencing analysis were performed on these four candidate genes, compared with the wild type, galat1 mutants in Zm00001eb319550 The deletion of one base (C) at position +3039 bp in the gene results in a frameshift mutation, causing premature termination of protein translation. Figure 2 E and Figure 3 Excluding the untranslated regions at both ends, ZmGalAT1 The gene contains 4216 nucleotides, including 9 exons, and encodes a galacturonyltransferase protein. This invention names this gene... ZmGalAT1 .

[0048] Example 4: ZmGalAT1 spatiotemporal expression analysis

[0049] To investigate the relationship between this gene and male reproductive development in maize, this invention first used qPCR to analyze the expression pattern of this gene at different stages of maize anther development. The specific steps are as follows:

[0050] 1. Sampling and developmental stage identification of maize anthers

[0051] 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.

[0052] 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).

[0053] 2. qPCR analysis

[0054] Total RNA was extracted from maize anthers at different developmental stages (S5-S13) using Trizol reagent (Invitrogen, USA); cDNA was then synthesized using 5X All-in-One RT Master Mix (ABM, Canada); and ZmGalAT1 quantitative reverse transcription polymerase chain reaction was detected using TBGreen™ PreMix Ex Taq™ (TaKaRa, Japan) on a QuantStudio5 Real-Time PCR System (ABI, USA).

[0055] The ZmGalAT1 amplification primers are: qGalAT1-F (SEQ ID NO. 28): 5'-TCTTCTTCGTCGTGCGCAAT-3'; qGalAT1-R (SEQ ID NO. 29): 5'-CTGCTGCTTCCTGATTGCCA-3';

[0056] ZmUbiqutin As a reference gene, its amplification primers are:

[0057] Ubiqutin-F (SEQ ID NO. 30): 5'-CGACAACGTGAAGGCGAAGA-3';

[0058] Ubiqutin-R (SEQ ID NO. 31): 5'-ACGCAGATACCCAGGTACAGC-3'.

[0059] 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).

[0060] ZmGalAT1 The gene exhibits a pattern of anther development-specific expression: it has the highest expression level during the S6 stage of maize anther development, then decreases, gradually increases from the S10-S13 stages, and reaches a second expression peak at the S13 stage. Figure 4 ).

[0061] Example 5: ZmGalAT1 Functional verification of genes and creation of maize male sterility mutants using CRISPR / Cas9 method

[0062] To clarify corn ZmGalAT1 Regarding its function in maize, this invention employs CRISPR / Cas9 gene editing to mutate [the maize sample]. Zm00001eb319550 Gene sequence was determined, and the function of the gene in maize was knocked out. This invention utilizes the maize inbred line Xiang 249 as the recipient material for gene editing, selecting... ZmGalAT1 Design of target regions for CRISPR / Cas9 gene editing in conserved gene regions.

[0063] 1. ZmGalAT1 Construction of CRISPR / Cas9 gene editing vector

[0064] 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 pCBCmT1T2This 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:

[0065] (1) Design of target gRNA. ZmGalAT1 ( Zm00001eb319550 The gene sequence of CRISPR2 is input into http: / / crispr.hzau.edu.cn / cgi-bin / CRISPR2 / CRISPR for target design. This invention selects... ZmGalAT1 The target site MT was designed at exon 7 of the gene, and the DNA sequence of the target region is shown in SEQ ID NO. 9. The sgRNA backbone sequence of this invention was derived from the intermediate vector. pCBCmT1T2 Obtained by direct amplification.

[0066] MT (SEQ ID NO. 5): tgaggaacggaaattccca.

[0067] (2) MT-sgRNA was obtained by designing target sites on primers and then amplifying by PCR. Primers ZmGalAT1-MT1-F and ZmGalAT1-MT2-R were used to amplify the intermediate vector. pCBCmT1T2 This method is used to obtain sgRNA fragments containing two identical targets, with a product length of 965 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 was 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, the PCR products were recovered.

[0068] The primer sequences required for vector construction are as follows:

[0069] ZmGalAT1-MT1-F (SEQ ID NO.32): 5'-ATATATGGTCTCTGGCGATGAGGAACGGAAATTCCCAGTTTTAGAGCTAGAAATAGCAA-3';

[0070] ZmGalAT1-MT2-R (SEQ ID NO. 33): 5'-ATTATTGGTCTCTAAACTGGGAATTTCCGTTCCTCATGCTTCTTGGTGCCGC-3'.

[0071] (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 10 μL restriction enzyme ligation system was as follows: sgRNA fragment: 1 μL, pBUE411-Cas9 vector (≥60 ng / μL): 1 μL, 10 x NEB Buffer: 1 μL. BsaI Endonuclease (product number: #R3733S): 0.5 μL, T4 ligase (product number: #M0202M): 0.25 μL, sterile ddH2O: 6.25 μL.

[0072] Figure 5 The target gene is shown. ZmGalAT1 ( Zm00001eb319550 The target, marker genes Cas9 and bar, and the backbone vector pBUE411-Cas9 Constructed expression carrier pCas9-ZmGalAT1 .

[0073] 2. Agrobacterium-mediated genetic transformation of maize

[0074] The above-constructed pCas9-ZmGalAT1The vectors were transferred into Agrobacterium EHA105 via heat shock and identified by PCR. The bacterial culture was then stored at -80 °C in a 1:1 volume mixture of Agrobacterium and glycerol. Freshly peeled immature embryos of the maize inbred line Xiang 249 (approximately 1.2-1.5 mm in diameter) were used as recipient materials. The peeled maize embryos were placed in 2 mL plastic centrifuge tubes containing 1.8 mL of suspension for no more than one hour, with approximately 100 embryos per tube. The suspension was removed, and the embryos were washed twice with fresh suspension, leaving a small amount of suspension at the bottom of the tube to submerge the embryos. The tubes were then heat-shocked at 43 °C for 2 minutes, followed by an ice bath for 1 minute. The remaining wash solution at the bottom of the tube 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, pour the embryos and infection solution from the centrifuge tubes onto the co-culture medium, shake well, and then use a pipette to remove excess infection solution. Ensure all embryos have their scutellaria facing upwards and co-culture at 23 °C in the dark for 3 days. After co-culture, use sterile forceps to transfer the embryos to recovery medium and culture at 28 °C for 7-14 days. During this process, carefully remove any sprouts that appear on the embryos. After recovery culture, place the embryos on 1.5 mg / L Bialaphos selection medium for 3 rounds of selection, each round lasting 2 weeks. Then transfer them to 2 mg / L Bialaphos selection medium for 2 rounds of selection, each round lasting 2 weeks. Transfer the resistant callus to propagation medium and culture at 28 °C in the dark for 2 weeks. Then transfer the propagated resistant callus to induction medium and culture at 28 °C in the dark for 2 weeks. Finally, transfer it to differentiation medium and culture at 25 °C, 5000 lx under light for 2 weeks. After the culture is completed, the differentiated seedlings are separated into individual seedlings and placed in a rooting medium. They are cultured at 25 ℃, 5000 lx, and under light until they root. The seedlings are then transferred to small nutrient pots for growth. After they have survived, they are transplanted into a greenhouse. The offspring seeds are harvested after 3-4 months.

[0075] 3. Detection of CRISPR / Cas9 mutation results in T0 generation plants

[0076] To determine the CRISPR / Cas9 mutation results in T0 generation plants, the following steps were taken:

[0077] 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 500 μL of CTAB extraction buffer (containing 1% β-mercaptoethanol) to the centrifuge tube and vigorously vortex to mix; 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 500 μL of chloroform:isoamyl alcohol (24:1) extraction solution, shake vigorously for 30 seconds, and then let stand at room temperature for a while; centrifuge at 12000 rpm for 5 min at 4 ℃, and take 500 μL of the supernatant after centrifugation into a new 1.5 mL centrifuge tube; add an equal volume of isopropanol to the centrifuge tube containing the supernatant and gently vortex to mix; let stand at room temperature for 10 minutes. After approximately 10 minutes, place the centrifuge tube containing the sample in a 4 °C centrifuge at 12,000 rpm for 10 minutes. Gently aspirate the supernatant, discard the supernatant, and retain the precipitate. Add 800 μL of 75% ethanol, wash the precipitate twice, centrifuge at 10,000 rpm for 5 minutes, and discard the supernatant. Allow the sample to air dry at room temperature for 2-4 hours to obtain DNA precipitate. Dissolve the precipitate in an appropriate amount of sterile water, gently vortex, and ensure complete DNA dissolution. Store the DNA sample at -20 °C. Detect the DNA concentration using Nanodrop and dilute to 10 ng / L for use as a PCR template.

[0078] Then according to ZmGalAT1 ( Zm00001eb319550 ) Design PCR primers based on gene sequence.

[0079] The primer sequences are as follows:

[0080] ZmGalAT1-T-F1 (SEQ ID NO.34): 5'-ATCACAGGTATGCGTCTGCATC-3';

[0081] ZmGalAT1-T-R1 (SEQ ID NO. 35): 5'-AGACATGCCAAACATCACCATTAGA-3'.

[0082] Genomic DNA was extracted and amplified using the following PCR parameters:

[0083] 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).

[0084] Reaction procedure: Conventional PCR: annealing at 58 ℃, extension for 30 s, 35 cycles.

[0085] PCR product size: 524 bp.

[0086] 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 regions of three T0 transformation events had changed sequences, all of which were homozygous mutations. The sequences before and after editing are shown below. Figure 6 As shown, there are 3 corresponding ones. galat1 Allelic homozygous mutants: ZmGalAT1-Cas9-1 , ZmGalAT1-Cas9-2 and ZmGalAT1-Cas9-3 Sequence alignment with the wild type showed that... ZmGalAT1-Cas9-1 , ZmGalAT1-Cas9-2 and ZmGalAT1-Cas9-3 All of them had deletion mutations at the target site. ZmGalAT1-Cas9-1 A 17-base deletion occurs between 3029 bp and 3045 bp in exon 7; mutant. ZmGalAT1- Cas9-2 A 19-base deletion occurs at position 3024 bp-3042 bp in exon 7; mutant. ZmGalAT1-Cas9-3 Thirteen bases are deleted at position 3027 bp-3039 bp in the 7th exon.

[0087] For 3 galat1 Comparative analysis of the amino acid sequences in the allelic mutants revealed that, compared with the unedited WT, the mutant strains... ZmGalAT1-Cas9-1 , ZmGalAT1-Cas9-2 and ZmGalAT1-Cas9-3 The deletion of the encoded nucleotide at the target site caused amino acid deletions and frameshift mutations. Therefore, in these transformants... Zm00001eb319550 The functions of all proteins were lost.

[0088] 4. Genotyping of F1 generation plants

[0089] 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. ZmGalAT1-Cas9-1 , ZmGalAT1-Cas9-2 and ZmGalAT1-Cas9-3 The T0 generation plants are pollinated to obtain F1 generation seeds, and the resulting plants are F1 generation plants.

[0090] The F1 generation plants included two segregation types, one of which was... Cas9 - Positive plants (transgenic plants), another type is Cas9 - To prevent sgRNA and Cas9 from continuously editing wild-type alleles introduced through pollination, thus creating a complexity of 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:

[0091] After extracting leaf DNA using the CTAB method described above, the first step is to utilize... Cas9 Gene-specific primers were used for PCR amplification. The primer sequences are as follows:

[0092] Cas9-F (SEQ ID NO.36): 5'-CCCGGACAATAGCGATGT-3;

[0093] Cas9-R (SEQ ID NO. 37): 5'-GAGTGGGCCGACGTAGTA-3'.

[0094] 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.

[0095] Further targeting Cas9 - For negative plants, PCR amplification was performed using the primers ZmGalAT1-T-F1 and ZmGalAT1-T-R1, which are used to detect the above-mentioned 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.

[0096] Example 6: Corn ZmGalAT1-Cas9 Phenotypic analysis of male sterility mutants

[0097] The above-described Example 5 identified that it does not contain Cas9 F1 generation plants of the gene were self-crossed to obtain F2 generation seeds, with three mutation types ( ZmGalAT1-Cas9-1 , ZmGalAT1-Cas9-2 and ZmGalAT1-Cas9-3One self-pollinated single ear from each F2 line was sown in panicle rows, and phenotypic analysis was conducted at maturity. In all three F2 lines, the ratio of fertile to sterile plants conformed to a 3:1 segregation, further indicating... ZmGalAT1-Cas9 The sterility trait in the sterile mutant is controlled by a single recessive gene, and then stable non-transgenic strains are obtained by targeting the F2 generation. ZmGalAT1-Cas9 The sterile mutant and the wild type were subjected to detailed observation of the tassel, anther, and pollen viability.

[0098] In terms of vegetative growth and female ear development, ZmGalAT1-Cas9-1 , ZmGalAT1-Cas9-2 and ZmGalAT1- Cas9-3 The sterile mutant plants were essentially no different from the wild type; however, in terms of tassel development, the wild type could tassel normally, with anthers dehiscing and pollen shedding normally, and could produce seeds normally after self-pollination, while the three... ZmGalAT1-Cas9 Although the sterile mutant can produce males normally, it cannot flower normally. The anthers and glumes do not split open, the anthers are significantly smaller, and they are browned, shriveled, and not exposed. Figure 7 Further I2-KI staining of pollen from wild-type and mutant plants revealed that wild-type pollen developed normally and turned black after staining, while the mutant plants did not form pollen grains. Figure 7 This indicates that... ZmGalAT1 ( Zm00001eb319550 Genes control male development in maize, and males created through gene editing methods... ZmGalAT1-Cas9 The sterile mutant is a pollen-free sterile line, exhibiting complete sterility.

[0099] Example 7: ZmGalAT1-Cas9 Development and application of co-segregating functional molecular markers for the identification of sterility mutants

[0100] 1. Development of co-separated molecular markers

[0101] In this invention, for the three obtained ZmGalAT1-Cas9 The mutation sites of the sterile mutants were identified by primer design using Primer 5.0 software, and a pair of co-segregating functional molecular markers, ZmGalAT1-F / R, were developed. Combined with PCR and agarose gel electrophoresis detection methods, the genotype of the mutants could be separated based on the obtained bands and their size.

[0102] The co-separating molecular marker ZmGalAT1-F / R comprises a first primer ZmGalAT1-F and a second primer ZmGalAT1-R; this marker can specifically detect maize. ZmGalAT1-Cas9-1 , ZmGalAT1-Cas9-2 and ZmGalAT1-Cas9-3 Mutants and mutant genes in maize male-sterile materials derived from them galat1-Cas9-1 , galat1-Cas9-2 and galat1- Cas9-3 And can distinguish between wild type at the same time. ZmGalAT1 Genes and mutants galat1-Cas9-1 , galat1-Cas9-2 and galat1-Cas9-3 Genes; targeting mutated genes galat1-Cas9-1 A 103 bp band was amplified, targeting the mutated gene. galat1-Cas9-2 A 101 bp band was amplified, targeting the mutated gene. galat1-Cas9-3 A 107 bp band was amplified in the middle, while in the wild type... ZmGalAT1 The gene was amplified into a 120 bp band. The primer sequences are as follows:

[0103] ZmGalAT1-F (SEQ ID NO.6): 5'-AACAATTGATTACTATATCCTGCCCCT -3';

[0104] ZmGalAT1-R (SEQ ID NO. 7): 5'-ACAGAGGCTGCCAAAACATTATCA-3'.

[0105] 2. Application of co-separated molecular markers

[0106] To verify the effectiveness of the above-mentioned markers, the F2 strain obtained in Example 6 was used as material for testing. GalAT1 Allele 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.

[0107] Theoretically, ZmGalAT1-F / R in GALAT1 / GALAT1 A 120bp band can be amplified in homozygous wild-type (AA) DNA. galat1-Cas9 / galat1-Cas9 In the homozygous mutant material (aa), bands of 103 bp, 101 bp, and 107 bp were amplified, respectively, while... GALAT1 / galat1-Cas9 In hybrid (Aa) materials, the corresponding two bands can be amplified simultaneously. The validation results of the ZmGalAT1-F / R molecular marker are as follows: Figure 8 , Figure 9 and Figure 10 As shown, the results indicate that the detection results of this functional molecular marker in F2 plants are entirely in line with expectations. GALAT1 / GALAT1 Homozygous wild type (AA) GALAT1 / galat1-Cas9 Heterozygous (Aa) and galat1-Cas9 / galat1-Cas9 The homozygous mutant material (aa) amplified bands of corresponding sizes, which can be used as... GalAT1 , galat1-Cas9 An ideal marker for allele detection.

[0108] 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.

[0109] References:

[0110] [1] Liu HJ, Liu J, Zhai Z, Dai M, Tian F, Wu Y, Tang J, Lu Y, Wang H, Jackson D, Yang X, Qin F, Xu M, Fernie AR, Zhang Z, Yan J. Maize2035: A decadal vision for intelligent maize breeding Mol Plant. 2025 Feb 3;18 (2):313-332. doi:10.1016 / j.molp.2025.01.012. PMID:39827366.

[0111] [2] Wan X, Wu S, Li

[0112] [3] An X,Dong Z,Tian Y,Xie K,Wu S,Zhu T,Zhang D,Zhou Y,Niu C,Ma B,HouQ,Bao J,Zhang S,Li Z,Wang Y,Yan T,Sun Plant. 2019 Mar 4;12 (3):343-359. doi:10.1016 / j.molp.2019.01.011. PMID:30684599.

[0113] [4] Wu Y,Fox TW,Trimnell MR,Wang L,Xu RJ,Cigan AM,Huffman GA,GarnaatCW,Hershey H,Albertsen MC. Development of a novel recessive genetic malesterility system for hybrid seed production in maize and other cross-pollinating crops Plant Biotechnol J. 2016;14 (3):1046-54. doi:10.1111 / pbi.12477. PMID:26442654.

[0114] [5] Qi X,Zhang C,Zhu J,Liu C,Huang C,Li X,Xie C. Genome EditingEnables Next-Generation Hybrid Seed Production Technology Mol Plant. 2020 Spt7;13 (9):1262-1269. doi:10.1016 / j.molp.2020.06.003. PMID:32645290。

Claims

1. Inhibit corn ZmGalAT1 The application of genes in controlling male reproductive development in maize; characterized by, Inhibit corn ZmGalAT1 Gene expression and / or activity, selecting male-sterile maize plants; The corn ZmGalAT1 The gene is located on chromosome 7 of maize, and its nucleotide sequence is shown in SEQ ID NO.

1. Its encoded protein sequence is shown in SEQ ID NO.

2.

2. A mutant gene for male sterility in maize galat1 Its characteristics are, The infertility mutant gene galat1 The corn described in claim 1 ZmGalAT1 The male sterility mutation in maize is caused by the deletion of one C base at position +3039 of exon 7; galat1 The full-length DNA sequence is SEQ ID NO.3, and its encoded amino acid sequence is SEQ ID NO.

4.

3. The inhibition according to claim 1 ZmGalAT1 The application of genes in controlling male reproductive development in maize is characterized by, Methods for inhibiting gene expression and / or activity include gene editing and RNA interference.

4. The inhibition according to claim 3 ZmGalAT1 The application of genes in controlling male reproductive development in maize is characterized by, The gene editing was performed using the CRISPR / Cas9 gene editing method.

5. The inhibition according to claim 4 ZmGalAT1 The application of genes in controlling male reproductive development in maize is characterized by, The CRISPR / Cas9 gene editing method includes: designing a CRISPR / Cas9 vector target at the 7th exon of the gene, wherein the DNA sequence of the target is shown in SEQ ID NO.

5.

6. A creative corn galat1 The method for male-sterile lines is characterized by, The method described is the CRISPR / Cas9 gene editing method as described in claim 5, that is, by editing the maize genome... ZmGalAT1 Genes were subjected to site-directed mutagenesis to obtain maize allelic male-sterile mutants with different mutation types, wherein the male-sterile mutants include mutants. ZmGalAT1- Cas9-1, ZmGalAT1-Cas9-2 and ZmGalAT1-Cas9-3 Compared with the maize ZmGalAT1 gene described in claim 1, the mutant... ZmGalAT1-Cas9-1 In ZmGalAT1 The gene has a 17-base deletion at position 3029bp-3045bp in the 7th exon; mutant ZmGalAT1-Cas9-2 In ZmGalAT1 The gene has a 19-base deletion at position 3024bp-3042bp in the 7th exon; mutant ZmGalAT1-Cas9-3 In ZmGalAT1 The gene has a 13-base deletion at position 3027bp-3039bp in the 7th exon.

7. The male sterility gene obtained by the method for creating male-sterile maize galat1 lines according to claim 6. ZmGalAT1-Cas9-1 , ZmGalAT1-Cas9-2 and ZmGalAT1-Cas9-3 Its characteristics are, Compared to the maize ZmGalAT1 gene described in claim 1, ZmGalAT1-Cas9-1 A deletion of 17 bases occurs at position 3029bp-3045bp in the 7th exon; ZmGalAT1-Cas9-2 Nineteen bases are deleted from the 3024bp-3042bp position in the 7th exon; ZmGalAT1-Cas9-3 Thirteen bases are deleted at position 3027bp-3039bp in the 7th exon.

8. A type of maize with different genetic backgrounds galat1 The method for male-sterile lines is characterized by, Using inclusion galat1 Male-sterile lines containing male-sterile genes are hybridized and backcrossed with target materials to obtain male-sterile genes. galat1 Male infertility genes and galat1 The trait of male infertility; galat1 Male sterility genes include the maize male sterility mutant gene as described in claim 2. galat1 And / or the male sterility genes ZmGalAT1-Cas9-1, ZmGalAT1-Cas9-2 and ZmGalAT1-Cas9-3 as described in claim 7.

9. The method obtained by claim 8 galat1 Application of male-sterile lines in hybridization breeding and seed production.

10. The corn according to claim 9 galat1 The application of male-sterile lines in hybridization breeding and seed production is characterized by, Targeting the mutant alleles of maize galat1 Design functional tags, applied to galat1 Marker-assisted selection in hybridization breeding and seed production of male-sterile lines; the sequences of the functional molecular marker primers ZmGalAT1-F and ZmGalAT1-R are shown in SEQ ID NO.6 and SEQ ID NO.7, respectively.

Images