WO2023169490A1 - Gène clé pour commander la transformation du maïs denté en maïs corné - Google Patents

Gène clé pour commander la transformation du maïs denté en maïs corné Download PDF

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WO2023169490A1
WO2023169490A1 PCT/CN2023/080384 CN2023080384W WO2023169490A1 WO 2023169490 A1 WO2023169490 A1 WO 2023169490A1 CN 2023080384 W CN2023080384 W CN 2023080384W WO 2023169490 A1 WO2023169490 A1 WO 2023169490A1
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seq
fln1
corn
transcription factor
gene
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巫永睿
王海海
黄永财
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中国科学院分子植物科学卓越创新中心
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    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01H—NEW PLANTS OR NON-TRANSGENIC PROCESSES FOR OBTAINING THEM; PLANT REPRODUCTION BY TISSUE CULTURE TECHNIQUES
    • A01H5/00—Angiosperms, i.e. flowering plants, characterised by their plant parts; Angiosperms characterised otherwise than by their botanic taxonomy
    • A01H5/10—Seeds
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01H—NEW PLANTS OR NON-TRANSGENIC PROCESSES FOR OBTAINING THEM; PLANT REPRODUCTION BY TISSUE CULTURE TECHNIQUES
    • A01H6/00—Angiosperms, i.e. flowering plants, characterised by their botanic taxonomy
    • A01H6/46—Gramineae or Poaceae, e.g. ryegrass, rice, wheat or maize
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/415—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from plants
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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
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    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6888—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms
    • C12Q1/6895—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms for plants, fungi or algae

Definitions

  • the invention belongs to the technical field of corn breeding, and specifically relates to a key gene that controls the transformation of dent corn into hard corn and its application in improving and creating hard corn germplasm resources.
  • the first purpose of the present invention is to provide a method for improving and creating durum corn germplasm resources, including the following steps: making ARF (auxin response factor, auxin response factor) in the corn genome
  • ARF auxin response factor, auxin response factor
  • the function of the gene encoding the transcription factor ZmARF17 is lost or down-regulated, that is, the expression of the ARF transcription factor is inactivated or down-regulated), leading to the transformation of dent corn into hard-kernel corn, or the enhancement of the hard-kernel phenotype.
  • amino acid sequence of the above-mentioned ARF transcription factor ZmARF17 (FLN1 for short) can be SEQ ID NO:1, and the NCBI accession number is Zm00001d014013; the nucleotide sequence of the encoding gene of the ARF transcription factor ZmARF17 is SEQ ID NO:2, which is named in this article Fln1.
  • SEQ ID NO:1 and SEQ ID NO:2 are respectively the amino acid sequence and coding gene sequence of ZmArf17 protein in dent corn B73.
  • the gene encoding the ARF transcription factor and its three homologous genes are mutated at the same time, and their mutants cause the original function to be inactivated or down-regulated.
  • the three homologous genes are SEQ ID NO: 7.
  • ZmArf2 NCBI accession number is Zm00001d032683, ZmArf2 gene CDS region
  • ZmArf19 with nucleotide SEQ ID NO:9 NCBI accession number is Zm00001d014507, ZmArf19 gene CDS region
  • ZmArf21 with nucleotide SEQ ID NO:9 NCBI accession number is Zm00001d000358, ZmArf21 gene CDS region.
  • the knockout of the ARF transcription factor encoding gene in method (1), the substitution of gene mutants in method (2), and the mutation of genes in method (3) can all be implemented through gene editing technology.
  • the gene editing uses CRISPR-Cas9 system, CRISPR-Cas12 system, CRISPR-Cpf1 system, CRISPR-Cas related transposition system INTEGRATE system or CAST system.
  • the ARF transcription factor gene mutant is represented as fln1-m; the ZmArf2 gene is represented as arf2 WT , and the ZmArf2 gene mutant is represented as arf2m or arf2 cr ; the ZmArf19 gene is represented as arf19 WT , and the ZmArf19 gene mutant is represented as arf19m or arf19 cr ; ZmArf21 gene is represented as arf21 WT , and ZmArf21 gene mutant is represented as arf21m or arf21 cr , where "cr" is the abbreviation of the gene editing system CRISPR-Cas, which means gene mutation is achieved through gene editing technology.
  • method (2) can be selected from the following group:
  • the interacting protein of the ARF transcription factor described in (2-3) is Pericarp Color1, MYB40, the homologous gene of the transcription factor P1 that regulates zeaxanthin metabolism.
  • the mutant of the ARF transcription factor gene in the above mode (3) and (4) is selected from the following group:
  • Nucleotide SEQ ID NO:3, represented as fln1-m1 the mutation method is that the mutation of G to A at position 995 in SEQ ID NO:2 causes Trp 332 to become a stop codon;
  • Nucleotide SEQ ID NO:4 represented as fln1-m2
  • the mutation method is that the mutation of C to T at position 466 in SEQ ID NO:2 causes Gln 156 to become a stop codon;
  • Nucleotide SEQ ID NO:5 expressed as fln1-m3, its mutation method is the deletion of base G at position 487 in SEQ ID NO:2, resulting in early termination;
  • Nucleotide SEQ ID NO:6 expressed as fln1-m4, its mutation method is the deletion of two bases CG at positions 486 and 487 in SEQ ID NO:2, resulting in early termination.
  • mutants of the three homologous genes in the above method (4) are selected from the following group:
  • the mutation mode is that the deletion of base G at position 478 in SEQ ID NO:7 (NCBI accession number Zm00001d032683, ZmArf2 gene CDS region) leads to premature termination of translation;
  • the mutation mode is that the deletion of base G at position 475 in SEQ ID NO:8 (NCBI accession number Zm00001d014507, ZmArf19 gene CDS region) leads to premature termination of translation;
  • the mutation mode is the deletion of base G at position 496 in SEQ ID NO:9 (NCBI accession number Zm00001d000358, CDS region of ZmArf21 gene), which leads to premature termination of translation.
  • the above-mentioned method of improving and creating durum corn germplasm resources can be implemented on the parents of hybrid corn, Zheng 58 (female parent) and Chang 7-2 (male parent).
  • mutant gene detection methods For the detection of the mutants SEQ ID NOs:3-6 of the above-mentioned ARF transcription factor genes in the corn genome, and the mutants arf2 cr , arf19 cr and arf21 cr of the three homologous genes, the following mutant gene detection methods can be used:
  • forward primer fln1-F AAGGGTCCTGGTGGGTACAT; reverse primer fln1-R: TGCTTAGCGTGGGACTGAC.
  • the PCR product is 638bp; the sequencing primer is fln1-F.
  • PCR testing can be performed using ordinary PCR MIX and programs.
  • the amplified sequence is SEQ ID NO:10, which is used as a fln1-m1 molecular marker.
  • the forward primer arf17 cr -F TACATTCCTACTCCGCTTTG
  • the reverse primer arf17 cr -R CCTGCCCTACCCTCATAC.
  • the PCR product is 1798bp
  • the sequencing primer is arf17 cr -sequencing: CGCTCCCGTGTCTACTA
  • the target sequence is GTGCTCGCCAAGGACGTGCA.
  • PCR detection can use KOD FX Neo high-fidelity enzyme (KFX-201T, TOYOBO) and its PCR program.
  • sequence analysis website is: http://skl.scau.edu.cn/dsdecode/ ; the template sequence is SEQ ID NO:12, which is used as a fln1-m3 or fln1-m4 molecular marker.
  • the above-mentioned mutation gene detection method can use a kit to perform DNA sequencing, thereby determining that the corn genome contains the above-mentioned gene mutant (mutation site). Therefore, another aspect of the present invention also provides a kit for implementing the above-mentioned mutation gene detection method, which includes the above-mentioned primers for detecting SEQ ID NOs: 3-6, arf2 cr , arf19 cr and arf21 cr , Or DNA/RNA probes, or DNA/RNA probe microarray chips.
  • the second object of the present invention is to provide a polynucleotide selected from:
  • (C) A nucleotide sequence complementary to the nucleotide sequence described in any one of (A) or (B).
  • the third aspect of the present invention is to provide the use of the above-mentioned polynucleotide, which is used to introduce into corn to replace the ARF transcription factor coding gene in the corn genome, causing the transformation of dent corn to hard kernel corn, or enhancing the hard kernel surface. type.
  • the present invention first discovered that the ARF transcription factor ZmARF17 (NCBI accession number Zm00001d014013) has the function of controlling the formation of hard kernel/dent corn, and the functional loss of its encoding gene Fln1, or the allelic variation fln1-m (selected from SEQ ID NOs: 3- 6)
  • the corn kernels can be changed into hard-kernel corn or the hard-kernel phenotype can be enhanced. Therefore, the regulation of gene Fln1 can be used to improve and create durum corn germplasm resources, greatly speeding up the breeding speed of durum corn genetic improvement and germplasm resource innovation, and avoiding the long-term, large-scale field exploration process with unclear prospects. Wide application prospects and huge economic value.
  • Figure 1 shows the phenotypic photos of the reciprocal cross of the hard-grained dent inbred line.
  • Figure 2 shows the mature grain phenotype and variation statistical results of dent corn B73 and EMS mutant (fln1-m1).
  • A E: B73; B, F: fln1-m1; C, G: B73x fln1-m1; D, H: fln1-m1x B73;
  • I-K The top angle I of the mature grains planted in Shanghai, the reciprocal mature grains
  • the seed length is J
  • the seed width of the mature seeds of the reciprocal cross is K
  • the length-to-width ratio of the mature seeds of the reciprocal cross is L
  • M-P the top angle of the mature seeds planted in Sanya is M
  • the length of the mature seeds of the reciprocal cross is N
  • the mature seeds of the reciprocal cross are length N.
  • the seed width is O; the length-to-width ratio of the mature seeds of the reciprocal cross is P.
  • Figure 3 shows the grain phenotype and statistical results of the fln1-m1 mutant at different development stages.
  • A-D B73 seeds A, fln1-m1 seeds B, longitudinal section C of B73 seeds, longitudinal section D of fln1-m1 seeds planted at 12, 15, 20, 25, 30 days after pollination (DAP) at Songjiang Farm in Shanghai.
  • E-G Seed length E, seed width F, and seed coat length G at different development stages planted in Shanghai
  • H-J Seed length H, seed width I, and seed coat length J at different development stages planted in Sanya. Take 6 ears and no less than 50 seeds from each ear for seed testing. The length of the seed coat is measured on 6 ears, and no less than 6 kernels are measured on each ear. **,P ⁇ 0.01as determined by Student’s t test.
  • Figure 4 shows the cytological changes in the testa and endosperm of mutant fln1-m1.
  • Picture A Transmission electron microscope section of the top testa of B73 seeds; B: Transmission electron microscope section of the top testa of fln1-m1 seeds; C: Semi-thin section of the distal embryo side of B73 seeds; D: Distal embryo of fln1-m1 seeds Semi-thin section from the side; E: Semi-thin section from the embryonic side of B73 seeds Section; F: semi-thin section of the near-embryo side of fln1-m1 seeds; G: length of top seed coat cells; H: length of seed coat cells on the far-embryo side; I: length of seed coat cells on the near-embryo side; J: whole Number of seed coat cells; K: Semi-thin and transmission electron microscope observations of the top endosperm of seeds at different development stages, showing different filling abilities.
  • Figure 5 shows the BSA localization detection of gene Fln1.
  • A F 2:3 localization map of B73 and fln1-m1; B, ED6 step-by-step on 10 chromosomes; C, ED6 fitting results.
  • Figure 6 shows the expression pattern analysis of the candidate gene ZmArf17.
  • A the variation of ZmArf17 within the BSA localization interval
  • B the expression level of ZmArf17 in the seed coat and endosperm at different development stages, three biological replicates
  • C Western Blot analysis of ZmARF17 expression in the endosperm and seed coat at different development stages.
  • Expression level D, in situ hybridization analysis of ZmArf17 in 12DAP seed species
  • E negative control of in situ hybridization
  • F analysis of GFP transgenic maize driven by ZmArf17 promoter.
  • Figure 7 shows tissue expression pattern analysis of ZmArf17.
  • A the expression level of ZmArf17 in different tissues of maize (relative expression level);
  • B the expression level of ZmArf17 in B73 and fln1-m1 seed coats at different development stages (relative expression level).
  • Figure 8 shows a combination of CRISPR knockout mutants of ZmArf17 and its three homologous genes.
  • the figure shows the phenotypes of single gene mutants of arf2, arf17, arf19, and arf21, the phenotypes of different combinations of double mutations, triple mutations, and the phenotypes of quadruple mutations.
  • the red line indicates the type of edit that deleted the base.
  • Figure 9 shows the detection results of mutant fln1-m1, arf17-2 and arf17 cr alleles.
  • the bold part is the sequence of the mutation site in the wild type, and the dotted line shows the gene editing site.
  • Figure 10 shows the RNA-seq analysis profiles of the testa of dent corn B73 and EMS mutant fln1-m1.
  • A the intersection of B73 and fln1-m1 at 12, 20, and 30DPA seed coat RNA-seq
  • B KEGG analysis of the intersection of B73 and fln1-m1 at 12, 20, and 30DPA seed coat RNA-seq
  • C B73 and Expression of phenylpropanoid metabolism pathway genes in seed coat RNA-seq of fln1-m1 at 12, 20, and 30 DPA
  • D quantitative PCR verification of phenylpropanoid metabolism pathway genes under different seasonal planting conditions. Data are from three biological replicates.
  • Figure 11 shows the metabolome analysis of the testa of dent corn B73 and EMS mutant fln1-m1.
  • the testa of 15 and 25 DAP was used for metabolome analysis, and the intersection analysis of differential metabolites in the two periods showed that the differential metabolites were specifically enriched in the flavonoid metabolic pathway. Data are from three biological replicates.
  • Figure 12 shows the changes in auxin content in the seed coat of mutant fln1-m1 seeds.
  • PC placental chalaza area;
  • Ped pedicel tissue;
  • En endosperm;
  • Upper seed coat tissue excluding basal tissue;
  • Basal seed basal tissue including PC and Ped. **,P ⁇ 0.01 as determined by Student's t test.
  • Figure 13 shows the analysis spectrum of direct binding of FLN1 and P1 to inhibit its transcriptional activation activity.
  • A BiFC analysis
  • B Luciferase complementation experiment
  • C Co-IP experiment
  • D Luciferase transcription activation experiment of CHS promoter
  • E Luciferase transcription activation experiment of DFR promoter
  • F LUC transcription activation experiment in tobacco system
  • G EMSA experiment proves that MYB40 can directly bind to the CHS promoter sequence, and FLN1 can enhance the binding function of MYB40
  • H EMSA experiment proves that MYB40 can directly bind to the CHS promoter sequence, and FLN1 can Enhance the binding function of MYB40.
  • Figure 14 shows the phenotypes of wild-type dent corn B73 and its mutant fln1-m1 grown at the Experimental Base of Northeast Agricultural University in Harbin.
  • Figure 15 shows the creation of durum hybrids by introducing the gene fln1-m1 into Zheng 58 and Chang 7-2, the parents of the hybrid Zhengdan 958.
  • the phenotype of the plants after introducing the gene fln1-m1 into the parents of the main cultivar Zhengdan 958 has no obvious changes compared to before improvement;
  • B the hybrid created after using fln1-m1 to improve the parents of Zhengdan 958.
  • the hard-grain phenotype was significantly enhanced;
  • C 100-grain weight; D, moisture content; E, cob length; F, number of panicle rows, G, number of grains in a row.
  • Hard-kernel and dent-shaped corns are important germplasm resources for modern hybrid corn breeding and are also important agronomic traits. Since hard-kernel dent corn is controlled by micro-effect polygenes and the genetic mechanism is complex, the QTL that controls its formation is difficult to clone and has not yet been reported; at the same time, the formation mechanism of hard-kernel corn is also unclear. Therefore, the genetic improvement of durum corn and the expansion of genetic germplasm through conventional breeding methods are not only time-consuming and labor-intensive, but also inefficient, and the progress is very slow and the direction is unclear.
  • the basis for this invention includes the following processes and analysis.
  • durum mutants After crossing pollen from A619, the grains in the entire ear were durum type; conversely, A619 was used as the female parent. , the pollen of the hybrid hard-grain parent, the grains of the entire ear are dent-shaped; but the ears harvested from F1 are all dent-shaped. These genetic phenomena show that the durum type and dent type are determined by the maternal genotype, and the durum type is recessive relative to the dent type. We then sorted out and analyzed the EMS mutants accumulated over the years and obtained a series of stable genetic durum mutants. Among them, a durum mutant on the B73 background had the most obvious phenotype and the best genetic stability, and was named fln1-m1.
  • auxin response factor (ARF) transcription factor ZmARF17 gene in the localization interval: NCBI accession number Zm00001d014013.
  • a G to A base mutation occurred in the coding region.
  • Trp 332 becomes a stop gain codon, causing protein translation to terminate prematurely.
  • the gene expression pattern shows that compared to roots, stems, leaves, endosperm and embryos, ZmArf17 is predominantly highly expressed in seed coat tissues at different developmental stages; and the expression level in the seed coat of the fln1-m1 mutant is significantly reduced; Western blot experiments also It shows that ZmARF17 protein is enriched in the testa and also in fln1-m1 Barely detectable in the seed coat.
  • ZmArf17 has three other homologous genes: ZmArf2, ZmArf19, and ZmArf21.
  • CRISPR CRISPR to knock out these four genes simultaneously, and then isolated the single, double, triple and quadruple mutations of these four genes. Accurately identify the genotypes of different combinations of materials, and then strictly self-cross them to examine the phenotypes. It was found that in all combinations, as long as it contains a ZmArf17 loss-of-function mutation, it can lead to a durum phenotype; however, loss-of-function mutations in the other three genes, regardless of single, double or triple mutations, cannot lead to the formation of durum corn.
  • ZmArf17 is the key gene Fln1 that regulates hard corn formation. It was also found that when these four homologous genes ZmArf2, ZmArf17, ZmArf19, and ZmArf21 were mutated simultaneously, the hard-core phenotype was more obvious.
  • FLN1 affects auxin content and seed coat development by regulating the synthesis of flavonoids
  • the key enzymes of the flavonoid synthesis pathway are CHS (chalcone synthase gene), DFR (dihydro flavonol reductase gene) and two UGTs (UDP-glycosyltransferase gene, UDP).
  • CHS chalcone synthase gene
  • DFR dihydro flavonol reductase gene
  • UGTs UDP-glycosyltransferase gene
  • FLN1 and MYB40 combine to inhibit its transcriptional activation activity and regulate the flavonoid metabolism pathway.
  • MYB40 can activate the CHS and DFR promoters, but FLN1 cannot activate the expression of these two genes; when FLN1 and MYB40 are co-transferred, P1 will be inhibited. Activating activity on CHS and DFR promoters. Further EMSA experiments were performed to prove that MYB40 can bind to CHS and DFR promoters in vitro, and adding FLN1 can enhance the DNA binding ability of MYB40. These results indicate that FLN1 negatively regulates the flavonoid metabolism pathway in corn seed coat by directly binding to MYB40 and inhibiting its transcriptional activation activity on flavonoid synthesis genes, thereby affecting the development of the seed coat.
  • fln1-m has very strong function and good genetic stability. Introducing fln1-m hybridization into different corn inbred lines can turn their kernels into hard kernels. The theory is valid for most inbred lines, which will greatly expand the germplasm resources of hard kernels and dents. Moreover, the genetic stability of fln1-m is very good, and the phenotype is very stable in Northeast China, Huanghuaihai and Sanya.
  • durum corn using fln1-m The time required to improve durum corn using fln1-m is short. Since the genes and mechanisms that control the formation of durum corn are unclear, conventional genetic improvement of durum corn requires large-scale field investigations at multiple sites over many years to obtain stable materials.
  • fln1-m When using fln1-m to improve hard corn, you only need to cross it with other inbred lines, then self-cross for one generation, and obtain fln1-m homozygous plants through the molecular marker identification we developed; Cross or cross other corn pollen, the seeds produced are all hard-grained. Moreover, during the import process, at least one ear only needs to be retained for hybridization and selfing to obtain stable hard grain material, which can greatly save workload.
  • fln1-m to improve durum corn is simple and suitable for large-scale operations.
  • the technology of corn hybridization and selfing is relatively simple and can be mastered by ordinary workers.
  • the corn leaf DNA extraction, PCR reaction and sequencing involved are all routine molecular experiments and can be completed in ordinary laboratories and sequencing companies.
  • fln1-m can be used to create durum hybrid corn.
  • the temperature generally refers to room temperature (15-30°C).
  • Molecular biology experiments in the examples include plasmid construction, enzyme digestion, ligation, competent cell preparation, transformation, culture medium preparation, etc., mainly refer to "Molecular Cloning Experiment Guide” (Third Edition), J. Sambrook, Edited by D.W. Russell (USA), translated by Huang Peitang et al., Science Press, Beijing, 2002). If necessary, specific experimental conditions can be determined through simple experiments.
  • PCR amplification experiments were performed according to the reaction conditions provided by the reagent supplier or the kit instructions. If necessary, it can be adjusted through simple experiments.
  • Ear preparation Usually 50-100 corn plants are mutated each time, and the ears are trimmed one afternoon in advance to make the silk spinning neat the next day. When EMS is applied, the silk spinning is about 1-2cm.
  • EMS/pollen solution In the morning, first collect pollen in a large bag from male flowers (loose powder for more than half an hour). During this period, prepare EMS chemical mutagen (prepared in step 2), and then filter and collect pollen with a fine mesh bag. To 40mL of EMS mutagen solution, add 5-8mL of pollen to obtain an EMS/pollen solution. Mutagenize for 45 minutes. During this period, shake continuously or moderately to disperse the pollen evenly into the EMS mutagen.
  • Pollen the ears of corn after EMS treatment: Use a brush dipped in the EMS/pollen solution and apply it to the ears of corn that spin well. After the ears are coated, put the bag in place immediately to avoid other pollen contamination. After completing the EMS mutagenesis, spray stopping agent on the body.
  • the stopping agent is 10% sodium thiosulfate (containing 1% Tween20), which can quickly deactivate EMS into low/harmless substances.
  • B73 is a typical dent-shaped corn, with a concave top and a small concave angle (see A, E, and I in Figure 2);
  • fln1-m1 is a hard-kernel type, with a convex top and a larger angle (B and F in Figure 2).
  • B73 is used as the female parent to cross fln1-m1, and the grain phenotype is similar to B73, which is dent (C, G, I in Figure 2);
  • fln1-m1 is used as the female parent to cross B73, and the grain phenotype is Similar to fln1-m1, it is a duplex type (D, H, and I in Figure 2).
  • D, H, and I in Figure 2
  • the hard kernel phenotype is consistent with the genetic rules controlled by maternal genotype.
  • the seed length of the reciprocal hard grain is significantly shorter than that of the dent, the seed width does not change much, and the aspect ratio decreases (G-L in Figure 2).
  • the phenotypes and test data of the reciprocal crosses grown in Sanya were consistent with those grown in Shanghai (I-L in Figure 2).
  • Example 3 Study on seed coat development and cell biology phenotype of mutant fln1-m1
  • Example 4 BSA sequencing and cloning of the key gene Fln1 that controls durum corn formation
  • the ears with the extreme dent phenotype and the ears with the extreme hard kernel phenotype were selected, and DNA was extracted from the leaves corresponding to the numbers for BSA mixed pool sequencing analysis. DNA extraction, library construction, sequencing and bioinformatics analysis were entrusted to Shanghai Ouyi Biomedical Technology Co., Ltd. The BSA sequencing results showed that the only peak appeared on chromosome 5 (BD in Figure 5). Further analysis found that there was an ARF transcription factor ZmARF17 gene Zm00001d014013 in the localization interval, and a G to A (C to T) mutation in the coding region (fln1-m1 ), causing Trp 332 to become a stop codon Stop gain, leading to premature termination of protein translation (A in Figure 6).
  • the gene expression pattern shows that ZmArf17 is predominantly highly expressed in seed coat tissues at different developmental stages compared to roots, stems, leaves, endosperm, embryos and seed coats (A in Figure 7); and in the seed coat of the fln1-m1 mutant The expression level decreased significantly (B in Figure 6; B in Figure 7); Western blot experiments also showed that ZmARF17 protein was enriched in the testa and was almost undetectable in the fln1-m1 testa (C in Figure 6). At the same time, in situ hybridization experiments and fluorescence experiments of transgenic corn with ZmArf17 promoter driving GFP also showed that ZmArf17 is mainly highly expressed in the seed coat (D-F in Figure 6). Therefore, we used ZmArf17 as a candidate gene for subsequent research.
  • ZmArf17 has three other homologous genes: ZmArf2, ZmArf19, and ZmArf21.
  • CRISPR CRISPR to knock out these four genes simultaneously, and then isolated the single, double, triple and quadruple mutations of these four genes.
  • the CRISPR knockout caused the deletion of base G at position 478 in Zm00001d032683, ZmArf2 CDS region (i.e., SEQ ID NO: 7), resulting in early termination of translation.
  • ZmArf2 CDS region i.e., SEQ ID NO: 7
  • the vector is transformed into Agrobacterium tumefaciens EHA105 strain after sequencing verification;
  • the vector is used to transform corn high II B x A lines immature embryos using Agrobacterium-mediated method to obtain CRISPR transgenic corn.
  • the forward primer arf2 cr -F ACGAAGAGGAGGAGGAGT
  • the reverse primer arf2 cr -R TCAGGTGGGCTACAAACA.
  • the PCR product is 1349bp
  • the sequencing primer is arf2 cr -sequencing: CGCTCCCGTGTCTACTA
  • the target sequence is GTGCTTGCCAAGGACGTGCA.
  • KOD FX Neo high-fidelity enzyme KFX-201T, TOYOBO
  • the sequence analysis website is: http://skl.scau.edu.cn/dsdecode/ ; the template sequence is SEQ ID NO:13.
  • ZmArf17, ZmArf19 and ZmArf21 mutants were constructed through CRISPR gene editing technology, and the maize mutant genes were identified.
  • the ZmArf17 cr method includes CRISPR knockout, which causes the deletion of base G at position 487 of the B73 reference genome ZmArf17 (CDS region of Zm00001d014013, SEQ ID NO: 2), resulting in premature termination (fln1-m3); causing both positions 486 and 487 of the CDS region Deletion of CG bases leads to premature termination (fln1-m4).
  • the ZmArf19 cr method is CRISPR knockout to make Zm00001d014507, and the deletion of base G at position 475 of the ZmArf19 CDS region (ie SEQ ID NO: 8) leads to premature termination of translation.
  • the ZmArf21 cr method uses CRISPR to knock out Zm00001d000358, and the deletion of base G at position 496 in the ZmArf21 CDS region (ie SEQ ID NO: 9) leads to premature termination of translation.
  • ZmArf17 is the key gene Fln1 that regulates hard corn formation.
  • Reverse transcription of RNA Use Pomega's reverse transcription kit (ImProm-II TM Reverse Transcription System) to perform reverse transcription of RNA according to the standard method in the kit.
  • BIO-RAD fluorescence quantitative analyzer CFX Use the BIO-RAD fluorescence quantitative analyzer CFX to detect gene expression using a two-step PCR amplification method. Reaction conditions: pre-denaturation 95°C, 30s; amplification: 95°C, 5s, 60°C, 35s, 40 Cycle; termination: 95°C, 15s; 60°C, 60s; 95°C, 15s.
  • RT-qPCR results showed that the expression of these genes changed in different stages.
  • CHS chalcone synthase gene
  • DFR dihydro flavonol reductase gene
  • UGTs UDP-glycosyltransferase genes
  • the protoplasts were terminated with W5 buffer (154mM NaCl, 125mM CaCl 2 , 5mM KCl, 5mM Glucose, 0.03% MES, pH 5.7), and the protoplasts were washed and collected. Then MMG (0.4M Mannitol, 15mM MgCl2, 0.1% MES, pH 5.7) was added to adjust the protoplast concentration. Add 10 ⁇ g plasmid and 110 ⁇ L PEG (45% PEG4000, 0.2M Mannitol, 100mM CaCl 2 ) to each 100 ⁇ L protoplast buffer for transformation. After reacting at room temperature for 15 minutes, add 440 ⁇ L W5 buffer to terminate. After removing the supernatant, add 1ml WI (20mM KCl, 0.6M Mannitol, 4mM MES pH5.7) and incubate the protoplasts for 16h.
  • W5 buffer 154mM NaCl, 125mM CaCl 2 , 5mM
  • Protoplasts were captured on a laser confocal scanning microscope LSM880 to capture the YFP signal.
  • the Reporter Assay System extracts the total protein and performs the reaction, and then analyzes the ratio of LUC and REN on a photometer (Promega 20/20).
  • Promoter probe preparation Label the 100 bp region of the target promoter with the CAAC element with the 5' end of fluorescein FAM (primers synthesized by Shanghai Boshang Biotechnology Co., Ltd.).
  • Electrophoresis In a 4% polyacrylamide gel, use 0.5M Tris-borate-EDTA for electrophoresis at 4°C and 110V constant voltage for about 80 minutes.
  • Example 10 Examining the performance of fln1m durum corn under different ecological conditions

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Abstract

L'invention concerne un procédé d'amélioration et de création de ressources de germoplasme de maïs corné, comprenant les étapes suivantes consistant à : permettre à une fonction de gène de codage d'un facteur de transcription ARF, ZmARF17, dans un génome de maïs d'être perdue ou à son expression d'être régulée à la baisse, de telle sorte que le maïs denté est transformé en maïs corné ou le phénotype corné est amélioré.
PCT/CN2023/080384 2022-03-11 2023-03-09 Gène clé pour commander la transformation du maïs denté en maïs corné WO2023169490A1 (fr)

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