Gene PIN1 for converting purslane seeds into hard seeds

Abstract

The invention discloses a method for converting portulaca oleracea into hard-grain corn, which comprises the following steps of: A, down-regulating expression of auxin transporter gene PIN1 in portulaca oleracea genome, inactivating, weakening function or knocking out, so that the portulaca oleracea is converted into the hard-grain corn; or B. enabling the 467th tryptophan codon TGG of the overexpressed auxin transporter gene PIN1 of the portulaca mays to be mutated into a PIN1W467X mutant of a termination codon, the mutant does not express the complete auxin transporter gene PIN1 any more, and the expressed protein loses the original PIN1 function or changes the original PIN1 function, so that the portulaca mays is converted into the dura grain phenotype.

Description

Technical Field

[0001] The present invention belongs to the technical field of corn breeding, and in particular relates to a gene PIN1 mutant for converting dent corn into flint corn and its application in creating flint corn germplasm resources. Background Art

[0002] Maize (Zea mays L.) is my country's top crop. Flint and dent maize are two important maize germplasm resources and are also important agronomic traits. The most obvious difference is kernel shape (Unterseer, S. et al. A comprehensive study of the genomic differentiation between temperate dent and flint maize. Genome Biol 17, doi: ARTN 137 10.1186 / s13059-016-1009-x, 2016.). Both types have unique geographical adaptability and are widely used in maize breeding.

[0003] Dent kernels have severely underfilled endosperm at the top, causing them to collapse inward during dehydration, resulting in a dent shape. Flint kernels, on the other hand, have a well-filled endosperm at the top, providing sufficient support during dehydration to prevent them from collapsing and instead remain convex, forming a flint kernel. Dent and flint kernels have different characteristics. Dent kernels are generally longer, which promotes yield, but they contain more floury endosperm, making them susceptible to breakage. Flint kernels, on the other hand, are shorter but contain more hard endosperm, making them less susceptible to breakage and facilitating mechanical harvesting, transportation, and storage (Zhang, H. & Xu, G. Physicochemical properties of vitreous and floury endosperm flours in maize. Food Sci Nutr 7, 2605-2612, doi:10.1002 / fsn3.1114, 2019). Therefore, breeding efforts aim to combine the advantages of dent and flint kernels to cultivate ideal flint-shaped corn.

[0004] However, dent and flint corn are controlled by micro-effect polygenes, and the molecular genetic mechanism is complex. Currently, there are not many genes reported to control these two grain types. In breeding, selection can only be made based on experience, which is very blind and time-consuming and labor-intensive, and has low efficiency. We have cloned a gene Fln1 (ZmARF17, NCBI No. Zm00001d014013) that can determine the dent of corn flint kernels for the first time, referring to patent document CN2022102361297. Later, through Fln1, we cloned another key transcription factor MYB40 (NCBI No. Zm00001d040621) that controls flint kernel dent corn, referring to patent document CN2023100025953. Summary of the Invention

[0005] Further research on Fln1 and MYB40 has recently uncovered another key gene, PIN1, that regulates the flint / dent phenotype in maize. Mutating PIN1 in dent corn B73 can convert dent kernels to flint. Crossing the PIN1 mutant with dent corn kernels reveals the phenotype in the F2 generation, facilitating subsequent genetic improvement. Furthermore, identification of the material is straightforward, requiring only conventional PCR screening. Therefore, the discovery of the new function of PIN1 makes genetic improvement and germplasm resource innovation in flint dent corn more efficient and convenient, and has extremely important application and economic value. Based on this important discovery, the present invention includes the following technical solutions.

[0006] A method for converting dent corn into flint corn comprises the following steps:

[0007] A. downregulating, inactivating, weakening or knocking out the auxin transporter gene PIN1 in the dent corn genome, thereby converting dent corn to flint corn; or

[0008] B. Overexpression of the auxin transporter gene PIN1 in dent corn by mutating the 467th tryptophan (Trp or W) codon TGG to a stop codon (X) W467X A mutant that no longer expresses the complete auxin transporter gene PIN1, and the expressed protein loses the original PIN1 function or changes the original PIN1 function; or

[0009] C. Replace the PIN1 gene in the dent corn genome with the PIN1 gene described in step B. W467X Mutant replacement.

[0010] In one embodiment, the stop codon in step B or step C is TGA, that is, the 467th tryptophan (Trp or W) codon TGG is mutated to TGA, and the corresponding gene PIN1 mutant is PIN1 W467X The nucleotide sequence of mutant 1 is SEQ ID NO: 1; or

[0011] The stop codon is TAG, that is, the 467th tryptophan (Trp or W) codon TGG is mutated to TAG, and the corresponding gene PIN1 mutant is PIN1 W467X Mutant 2; or

[0012] The stop codon is TAA, that is, the 467th tryptophan (Trp or W) codon TGG is mutated to TAA, and the corresponding gene PIN1 mutant is PIN1 W467X Mutant Three.

[0013] For example, step A can be implemented as follows:

[0014] (1) Knockout of the PIN1 gene in the dent maize genome;

[0015] (2) downregulating the expression level of the gene PIN1 in the dent corn genome; or

[0016] (3) Replace the PIN1 gene in the dent corn genome with a PIN1 mutant whose coding function is lost or downregulated.

[0017] Wherein, the above method (2) can be selected from the following group:

[0018] (2-1) Mutation of the PIN1 promoter region results in downregulation of PIN1 expression;

[0019] (2-2) mutation of the upstream regulatory factor of PIN1 gene leads to downregulation of the expression level of PIN1 gene; or

[0020] (2-3) The function of the PIN1 gene was altered by introducing an interacting protein of the auxin transporter PIN1 into dent corn.

[0021] Preferably, the interacting protein of the ARF transcription factor in the above method (2-3) is the transcription factor MYB40 (NCBI accession number: Zm00001d040621) or the transcription factor ARF (NCBI accession number: Zm00001d014013) that regulates zeaflavone metabolism.

[0022] In one embodiment, the above step B or step C is achieved by gene editing technology on the dent corn genome; or by plasmid transformation method, i.e., comprising PIN1 W467XThe mutant plasmid is transformed into dent corn via Agrobacterium-mediated transformation; or gene editing is combined with plasmid transformation, and the gene editing technology can be selected from the following groups: homologous double crossover, TALEN system, CRISPR-Cas9 system, CRISPR-Cpf1 system, CRISPR-Cas12 system, CRISPR-BEST system, MUCICAT, MuGENT (multiplex genome editing by natural transformation), etc.

[0023] The second aspect of the present invention provides a polynucleotide selected from the group consisting of:

[0024] PIN1 mentioned above W467X Mutant 1, i.e., a PIN1 mutant in which the tryptophan codon TGG at position 467 is mutated to the stop codon TGA, with the nucleotide sequence being SEQ ID NO: 1;

[0025] PIN1 mentioned above W467X Mutant 2, i.e., a PIN1 mutant in which the tryptophan codon TGG at position 467 is mutated to the stop codon TAG, and the nucleotide sequence is that G at position 1400 in SEQ ID NO: 1 is replaced by A; or

[0026] PIN1 mentioned above W467X Mutant three is a PIN1 gene mutant in which the tryptophan codon TGG at position 467 is mutated to the stop codon TAA, and the nucleotide sequence is that GG at positions 1400-1401 in SEQ ID NO: 1 is replaced by AA.

[0027] Accordingly, another aspect of the present invention provides a plasmid comprising the above-mentioned polynucleotide.

[0028] Preferably, the plasmid is a gene expression plasmid, which is used to be transformed into dent corn through Agrobacterium-mediated method to express the polynucleotide.

[0029] Furthermore, the above-mentioned plasmid can be a gene editing system plasmid, which is used to integrate the above-mentioned polynucleotide into the corn genome through the gene editing system.

[0030] Those skilled in the art can use the above polynucleotides to be introduced into dent corn to create hard-kernel germplasm.

[0031] Another aspect of the present invention provides a method for detecting the gene PIN1 in the corn genome. W467X The method for preparing a mutant, namely the nucleotide sequence SEQ ID NO: 1, comprises the following steps:

[0032] The maize genome was PCR amplified and sequenced using the forward primer pin1-F and the reverse primer pin1-R using conventional PCR MIX and procedures, wherein,

[0033] Forward primer pin1-F: GCGATGCGACTCCAGACAGA (SEQ ID NO: 2),

[0034] Reverse primer pin1-R: ACACCTGTTCTGTTCTTGTTGGAC (SEQ ID NO: 3).

[0035] The PCR amplification product is 556 bp, and the nucleotide sequence is SEQ ID NO: 4:

[0036] GCGATGCGACTCCAGACAGAGGAGGCAGCAGAGAGGGTTTTGAGATCCACGGACCGAGGCTGAACATGGCCATGCCGAGCCCGGCGTCGGAGAGGATCGAGATGGACTTGAGGATGATCGCCGGCA TCTCGAAGTTCCACCTGCGTATGCGTTCGTGTCCAGAAACGAAACAGGTTAGCGAAAGATGCAACGCCCACTACTAATTTCAAGCACGCCGCGCCGGTGACGCTGGTTACCTGAAGCAGACGAGCGA T CAGATGAGGCCGATGAGGCTGGAGTAGGTGTTGGGGTTACGGATGAGTTTGCGCCACACCATGATGAGGATCAGCCGCGTCATCACGCTCGTCGGTGGCATCGCCGCGGGCGCCGCCACGGCGCCTGCATTCCCCTGCGCAGCCGCAGCCGCC GCCGCCGCCTTCTCGTCGCCGGCTTCCGCGTCCCTCTCCGCCGCGCCCCTGTTCCCGAAGCTGAAGTCGTCCCGCTCCGCGAAGTCCTCGCCCCGCTCCTTCCTCCCGTCCGCCGCAACTAAAACGTCCAACAAGAACAGAACAGGTGT(SEQ ID NO:4).

[0037] That is, the 254th position of the mutant amplified sequence is T, while the 254th position of the original gene PIN1 amplified sequence is C.

[0038] Another aspect of the present invention provides a kit for implementing the above-mentioned detection of the gene PIN1 in the corn genome.W467X The method for mutant 1 comprises primers corresponding to SEQ ID NO: 1, or DNA / RNA probes, or a microarray chip of DNA / RNA probes.

[0039] The above kit is mainly used to detect the gene PIN1 in corn W467X To express the mutant 1, corn genomic DNA was extracted as a template, and PCR was performed on pin1-F and pin1-R using the above primers. If an amplified band as shown in nucleotide sequence SEQ ID NO: 4 was amplified, it indicated that the gene PIN1 W467X Mutant 1 was expressed in maize.

[0040] This study, published in Nature Communications, discovered for the first time that the auxin transporter gene PIN1 has a novel function in regulating the hard / dent phenotype. Mutating the tryptophan codon TGG at position 467 of this gene to the stop codon TGA can convert dent corn kernels into hard kernels. Therefore, regulating PIN1 can be used to improve and create hard-kernel corn germplasm resources, accelerating breeding for genetic improvement and germplasm innovation in hard-kernel corn, and avoiding the lengthy, large-scale field trial process. This approach holds great promise for future applications. BRIEF DESCRIPTION OF THE DRAWINGS

[0041] Figure 1 Shown are RNA-seq and RT-qPCR data of seed coats of wild-type B73 maize and fln1 mutants at different developmental stages.

[0042] Figure 2 Figure 2 shows the co-regulation of PIN1C expression by the transcription factors ARFTF17 and MYB40. (a) EMSA analysis of ARFTF17 and MYB40 binding to the PIN1-pro promoter; (b) Luciferase complementation assay of ARFTF17, MYB40, and the PIN1-pro promoter. ARFTF17, MYB40, and PIN1-pro were co-transfected into maize protoplasts, and P values ​​were determined using a two-tailed Student's t-test.

[0043] Figure 3 Figure 1 shows PIN1 gene expression analysis and IAA content determination in the seed coats of maize B73 and pin1 mutants. (a) Identification of the pin1 mutant; (b) Statistical analysis of PIN1 gene expression in the seed coats of B73 and pin1 mutant kernels 14 days after pollination; (c) Statistical analysis of IAA content in the seed coats of B73 and pin1 mutant kernels 14 days after pollination. P values ​​were determined using a two-tailed Student's t-test.)

[0044] Figure 4The following table shows the phenotypes of mature kernels of maize B73 and pin1 mutants. (a) Homozygous B73 and pin1 ears, scale bar 1 cm; (b) Longitudinal section of mature kernels from homozygous B73 and pin1 ears, scale bar 2 mm; (c) Statistical graph of the degree of concavity at the top of kernels from homozygous B73 and pin1 ears, with downward concavity defined as positive and upward convexity defined as negative. P values ​​were determined using a two-tailed Student's t-test; (d) Statistical graph of seed coat length of kernels from homozygous B73 and pin1 ears, with P values ​​determined using a two-tailed Student's t-test.) DETAILED DESCRIPTION

[0045] The gene PIN1 encodes an auxin transporter protein, its gene number is Zm00001d044812; NCBI GeneID: 103637824; Gene symbol is LOC103637824.

[0046] The protein sequence of PIN1 in the wild-type B73 maize genome is:

[0047] MITGTDFYHVMTAMVPLYVAMILAYGSVRWWRIFTPDQCSGINRFVALFAFVPLLSFHFISTNNPYTMNIRFIAADTLQKLIVLALLTAWSYLSRRGCLEWTITLFSLSTLPNTLVMGIPLLKGMYGDFSGSLMVQIVVLQCIIWYTLMLF MFEYRGARILITEQFPDTAGAIASIVVDPDVVSLDGRNDAIETEAEVKEDGKIHVTVRRSNASRSDIYSRRSMGFSSTTPRPSNLTNAEIYSLQSSRNPTPRGSSFNHTDFYSMVGRSSNFAAGDAFGLRTGATPRPSNYEEDPQGKANK YGQYPAPNPAMAAQPAKGLKKAANGQAKGEDGKDLHMFVWSSSASPVSSDVFGNGAAEYNDAAAVKEVRMAVASPRKVAADGRKERGEDFAERDDFSFGNRGAAERDAEAGDEKAAAAAAAAQGNAGAVAAPAAMPPTSVMTRLILIMVWR KLIRNPNTYSSLIGLIWSLVCFRWNFEMPAIILKSISILSDAGLGMAMFSLGLFMALQPRIIACGNKVATFAMAVRFLTGPAVMAAASFAVGLRGTLLHVAIVQAALPQGIVPFVFAKEYGVHPDILSTAVIFGMLIALPITLVYYILMGL

[0048] For simplicity of description, the names of proteins, such as proteins, and the names of their encoding genes (DNA) are sometimes used interchangeably herein. Those skilled in the art will understand that they represent different substances in different contexts. For example, when describing the function or class of a transcription factor, the term "PIN1" refers to the protein; when describing it as a gene, it refers to the gene encoding the auxin transporter, and so on. This is well known to those skilled in the art.

[0049] In addition, for the sake of convenience, the uppercase and lowercase abbreviations of genes are sometimes mixed and used. They represent the same meaning. For example, pin1 and PIN1 represent the same gene. Alternatively, the lowercase abbreviations are used to represent mutants, which is easy for technical personnel in this field to understand.

[0050] In our research on Fln1 and MYB40, we discovered a new function of the gene PIN1, including the following process and analysis.

[0051] (1) Discovery of the key gene PIN1 controlling hard grain dent corn

[0052] Previously, we cloned two key genes controlling maize hard kernel production, Fln1 (ARFTF17) and MYB40, and found that the FLN1 and MYB40 transcription factors interact to regulate flavonoid synthesis, auxin content, and seed coat development. In further studies, we analyzed RNA-seq data from the seed coats of wild-type B73 and fln1 mutants at different developmental stages and found that not only was the expression of the flavonoid synthesis pathway gene Fln1 upregulated in the seed coat, but the expression of the auxin transporter gene PIN1 was significantly downregulated at different developmental stages. These results were also replicated in quantitative RT-PCR experiments under different planting environments ( Figure 1 The above results indicate that PIN1 may be a downstream regulatory gene of FLN1.

[0053] (2) ARFTF17 and MYB40 jointly regulate the expression of PIN1

[0054] To verify the above inference, EMSA experiments were first used to prove that MYB40 can bind to the PIN1 promoter in vitro, and the addition of ARFTF17 can enhance the DNA binding ability of MYB40 ( Figure 2 To further verify whether the protein interaction between MYB40 and ARFTF17 can regulate the expression of the PIN1 gene, the two transcription factors were co-expressed with the PIN1 promoter in maize leaf protoplasts. It was found that ARFTF17 could not directly regulate the PIN1 promoter, while MYB40 inhibited the expression of the PIN1 promoter. When ARFTF17 and MYB40 were co-transfected with the PIN1 promoter, it was found that the inhibition of MYB40 on the PIN1 promoter was significantly suppressed by the addition of ARFTF17 ( Figure 2 (middle b) These results suggest that ARFTF17 can directly bind to MYB40, thereby inhibiting MYB40's negative regulation of the PIN1 promoter and positively regulating PIN1 expression. Consequently, PIN1 expression is significantly downregulated in the seed coat of ARFTF17 mutants.

[0055] (3) PIN1 mutation can reduce auxin content

[0056] The above studies indicate that PIN1 is a downstream gene in the ARFTF17-MYB40 regulatory module and may be involved in the formation of hard-grain dent grains. Therefore, a mutant pin1 (EMS4-0b3dd4) with premature termination mutation was obtained from the B73 mutant library MEMD (http: / / elabcaas.cn / memd / public / index.html# / ). Three homozygous mutants were identified from the F2 line ( Figure 3 (a) After planting the homozygous mutant, the seed coat of the grain was taken 14 days after pollination to measure the gene expression and auxin content. The results showed that the expression level of PIN1 in the seed coat was significantly reduced ( Figure 3 b), and the auxin content also decreased significantly (Figure 3c).

[0057] (4) PIN1 mutation can transform the grains of dentata into hard grains

[0058] At the same time, we conducted a systematic study on the grain phenotype of pin1 and found that the grain shape of pin1 changed significantly from dent type to hard type ( Figure 4 Middle a), the endosperm at the top of the grain is full and not concave but remains convex, and the seed coat length is shortened ( Figure 4 These results indicate that mutations in the auxin transporter gene PIN1 reduce auxin content in the maize seed coat, inhibiting seed coat development and causing dent kernels to transform into hard kernels. This is the third key gene we have discovered controlling hard dent kernel formation, following Fln1 (ARFTF17) and MYB40. This not only clarifies the molecular regulatory module for hard dent kernel formation in maize but also provides new genetic resources for genetic improvement of hard dent kernels, with significant application value.

[0059] Gene sequencing revealed that the mutation of the PIN1 gene was that the codon TGG for the 467th amino acid tryptophan (Trp or W) was mutated to the termination codon TGA (i.e., the 1401st base G in the CDS sequence of the PIN1 gene in B73 was mutated to A), resulting in premature termination. The mutant pin1 is PIN1 W467X Exhibits a hard-grained phenotype.

[0060] Based on this discovery, a method for identifying mutant PIN1 in the maize genome is provided. W467X The method may include the following steps: extracting genomic DNA from biological materials, performing PCR amplification using specific primers pin1-F and pin1-R, wherein:

[0061] pin1-F: 5'-GCGATGCGACTCCAGACAGA-3' (SEQ ID NO: 2),

[0062] pin1-R: 5'-ACACCTGTTCTGTTCTTGTTGGAC-3' (SEQ ID NO: 3).

[0063] PCR amplified a 556 bp band with the following nucleotide sequence:

[0064] GCGATGCGACTCCAGACAGAGGAGGCAGCAGAGAGGGTTTTGAGATCCACGGACCGAGGCTGAACATGGCCATGCCGAGCCCGGCGTCGGAGAGGATCGAGATGGACTTGAGGATGATCGCCGGCA TCTCGAAGTTCCACCTGCGTATGCGTTCGTGTCCAGAAACGAAACAGGTTAGCGAAAGATGCAACGCCCACTACTAATTTCAAGCACGCCGCGCCGGTGACGCTGGTTACCTGAAGCAGACGAGCGA C / T CAGATGAGGCCGATGAGGCTGGAGTAGGTGTTGGGGTTACGGATGAGTTTGCGCCACACCATGATGAGGATCAGCCGCGTCATCACGCTCGTCGGTGGCATCGCCGCGGGCGCCGCCACGGCGCCTGCATTCCCCTGCGCAGCCGCAGCCG CCGCCGCCGCCTTCCTCGTCGCCGGCTTCCGCGTCCCTCTCCGCCGCGCCCCTGTTCCCGAAGCTGAAGTCGTCCCGCTCCGCGAAGTCCTCGCCCCGCTCCTTCCTCCCGTCCGCCGCAACTAAAACGTCCAACAAGAACAGAACAGGTGT. That is, the C at position 254 in the PCR product of the original gene PIN1 was mutated to T.

[0065] The above mutant PIN1 W467X The discovery provides effective genetic resources and materials for improving dent corn into flint corn. When this mutant is introduced into a conventional corn inbred line, the offspring, regardless of whether they are in direct or reciprocal crosses, will produce flint-type ears as long as they carry the homozygous PIN1 mutation or reduced expression.

[0066] The technical solution of the present invention has the following technical effects when applied to corn breeding.

[0067] 1. The PIN1 gene is highly potent and genetically stable. Crossing PIN1 homozygous mutant corn with dent corn can convert dent kernels into hard kernels. This is theoretically effective for most inbred lines, significantly expanding the germplasm resources for hard and dent varieties. Furthermore, genetic stability is exceptional, with phenotypes remaining highly stable at the Shanghai Songjiang, Heilongjiang, and Sanya breeding bases at the Center for Excellence in Molecular Plant Sciences, Chinese Academy of Sciences.

[0068] 2. Improving flint corn using pin1 mutants is simple and suitable for large-scale operations. Hybridization and selfing techniques for corn are relatively simple and can be mastered by ordinary workers. Using the primers we developed to identify pin1 mutants, pin1 genotypes can be easily identified. The DNA extraction and PCR reactions involved are routine biochemical experiments that can be performed in an ordinary laboratory.

[0069] 3. Using the pin1 mutant for flint corn improvement can significantly reduce costs. Compared to traditional corn genetic improvement, using the pin1 mutant for flint corn improvement offers stronger results, better stability, shorter production times, and reduced workload, leading to significant cost savings, similar to the fln1 mutant.

[0070] The technical solutions of the present invention are described and verified by the following examples. It should be understood that the following examples are only used to illustrate the present invention and are not used to limit the scope of the present invention.

[0071] Example

[0072] The examples herein involve the addition amounts, contents and concentrations of various substances, wherein the percentages mentioned, unless otherwise specified, are by mass percentages.

[0073] In the examples herein, if no specific description is given for the reaction temperature or the operating temperature, the temperature generally refers to room temperature (15-30° C.).

[0074] Materials and methods

[0075] Corn self-pollination, hybridization, genetic modification, and field breeding are carried out according to conventional breeding methods.

[0076] The primer synthesis and gene sequencing in the examples were all completed by Shanghai Boshang Biotechnology Co., Ltd.

[0077] The molecular biology experiments in the examples, including plasmid construction, enzyme digestion, ligation, competent cell preparation, transformation, culture medium preparation, etc., were performed primarily with reference to Molecular Cloning: A Laboratory Manual (3rd edition), edited by J. Sambrook and D.W. Russell (USA), translated by Huang Peitang et al., Science Press, Beijing, 2002. Specific experimental conditions can be determined by simple experiments when necessary.

[0078] PCR amplification experiments should be performed according to the reaction conditions provided by the reagent supplier or the kit instructions. Adjustments can be made through simple experiments if necessary.

[0079] The primers used in PCR in the examples are listed in the table below:

[0080] Table 1: Primers for mutant identification:

[0081] pin1-F GCGATGCGACTCCAGACAGA pin1-R ACACCTGTTCTGTTCTTGTTGGAC

[0082] In the table, the suffix "-F" in the name represents forward direction; "-R" represents reverse direction. The same applies to the following.

[0083] Table 2: Vector construction primer list:

[0084]

[0085] Table 3: List of quantitative RT-PCR primers:

[0086]

[0087]

[0088] Example 1: RNA-seq data and RT-qPCR data infer that PIN1 is a downstream regulatory gene of FLN1

[0089] In spring, corn cobs of maize B73 (hereinafter referred to as B73) and fln1 mutant maize (hereinafter referred to as fln1 mutant or fln1) planted in Shanghai Songjiang breeding base were taken at 12 days (12DAP), 20DAP, and 30DAP after self-pollination. The seed coats of the well-grown kernels in the middle part were peeled off and placed in 15mL centrifuge tubes. They were quickly frozen in liquid nitrogen, fully ground with a sample grinder, and quickly transferred to a -80℃ ultra-low temperature freezer for storage. RNA was extracted and RNA-seq analysis was performed at Shanghai Ouyi Company. The seed coat materials of B73 and fln1 mutant planted in Sanya Damao base were taken at 15DAP and 25DAP after self-pollination, and RNA was extracted using the method of Shanghai Yisheng Biological Company. qPCR RT-qPCR analysis was performed using the Green Master Mix (No Rox) kit. RNA-seq data analysis revealed that genes involved in the flavonoid biosynthesis pathway were upregulated in the fln1 seed coat, while the expression of the auxin transporter gene PIN1 was significantly downregulated at different developmental stages; quantitative RT-qPCR experiments under different planting environments reproduced these results (see Figure 1 ), and the quantitative primers were PIN1-QF and PIN1-QR. This suggests that PIN1 may be a downstream regulatory gene of FLN1.

[0090] RNA extraction

[0091] (1) Place approximately 50 mg of sample in a 2 mL Eppendorf tube, add 400 μL of RNA extraction SDS buffer, shake thoroughly, and place on ice. See the table below for SDS buffer:

[0092]

[0093] (2) Add 400 μL of phenol-chloroform (phenol is saturated in NaAc / HAc, mixed in a 1:1 ratio, the pH of phenol is 4.2, placed at 4°C, and the lower layer is used after stratification), shake vigorously for 30 seconds, place on ice for 5 minutes, shake during this period, centrifuge at 12000 rpm at 4°C for 10 minutes, and aspirate 350 μL of supernatant into a new 1.5 mL centrifuge tube.

[0094] (3) Add 1 mL of Trizol extract to the supernatant tube, shake thoroughly, and place on ice for 5 min. Then add 200 μL of chloroform to the mixture, shake thoroughly, place on ice for 5 min, centrifuge at 12,000 rpm and 4°C for 10 min, and aspirate 500 μL of the supernatant (do not aspirate the middle layer and the organic phase below) into a new centrifuge tube.

[0095] (4) Add 500 μL of isopropanol to the supernatant, shake thoroughly, place on ice for 10 min, centrifuge at 12,000 rpm at 4°C for 10 min, and discard the supernatant.

[0096] (5) Add 1 mL of 70% ethanol solution and flick the pellet to make it float. Place the pellet on ice for 1 min, then centrifuge at 12,000 rpm at 4°C for 5 min and discard the supernatant.

[0097] (6) Briefly centrifuge and remove excess liquid with a small pipette tip. Allow the RNA pellet to dry at room temperature for approximately 2 minutes and add 100 μL of ddH2O to dissolve the pellet.

[0098] (7) Using QIAGEN's RNeasy Plus Mini Kit, the RNA was purified by column chromatography according to the standard protocol of the kit, which involved the removal of DNA using DNaseI, and finally dissolved in 30 μL of RNase-free H2O.

[0099] Quantitative RT-PCR

[0100] (1) RNA reverse transcription: Pomega's reverse transcription kit (ImProm-II TM RNA was reverse transcribed using the PCR amplification system (PCR amplification system) according to the standard method in the kit.

[0101] (2) Specific primers were designed by NCBI and specificity analysis was performed, using TUA4 (Zm00001d013367) as the internal reference gene.

[0102] (3) According to Shanghai Yisheng Biotechnology Co., Ltd. qPCR Green Master Mix (No Rox) kit standard method was used for quantitative analysis. Three technical replicates were performed for each sample. The cDNA samples after reverse transcription were uniformly diluted 8 times and quantitative analysis was performed using a 20 μL reaction system. GreenMaster Mix 10 μL, primers 0.5 μL each, diluted cDNA 3 μL, ddH2O 6 μL were used for the reaction.

[0103] (4) Gene expression was detected by two-step PCR amplification using a BIO-RAD CFX fluorescence quantitative analyzer. The reaction conditions were as follows: pre-denaturation at 95°C for 30 s; amplification at 95°C for 5 s, then 60°C for 35 s, for 40 cycles; and termination at 95°C for 15 s, then 60°C for 60 s, and finally 95°C for 15 s.

[0104] (5) The quantitative data were analyzed using EXCELL 2010 and the △△CT method.

[0105] Example 2: EMSA experiments demonstrated that MYB40 can bind to the PIN1 promoter in vitro

[0106] The coding sequence of ZmARF17 was amplified using primers ZmARF17-PET30F and ZmARF17-PET30R and cloned into the pET30a vector to produce a His-tagged fusion protein. The coding sequence of MYB40 was amplified using primers MYB40 pCOLDF and MYB40 pCOLDR and cloned into the pCold vector to produce a GST-tagged fusion protein at the NH terminus. The vectors were transformed into Escherichia coli BL21(DE3) and induced with 0.5 mM isopropyl β-D-thiogalactopyranoside (IPTG) at 20°C for 20 hours to produce recombinant proteins. The recombinant ZmARF17-His and MYB40-GST proteins were purified on Ni-NTA agarose (QIAGEN). Simultaneously, DNA fragments PIN1-P and P3 (2X) of the PIN1 promoter were amplified using primers PIN1-EMSA-F and PIN1-EMSA-R. 5' end labeling was performed with fluorescein amide (FAM). 0.05 pmol of labeled probe was incubated with 100 ng of purified recombinant protein at room temperature for 30 minutes in a 20 μL binding reaction mixture (1 μL 1 mg / ml salmon sperm DNA, 20 mM Tris-HCl, pH 7.9, 5% glycerol, 0.04 mg / ml bovine serum albumin, 2 mM MgCl2, 0.2 mM dithiothreitol, and 40 mM KCl, ultrapure water to a final volume of 20 μL). For competition analysis, 10x, 50x, and 100x unlabeled probe were added to the reaction mixture, respectively. Electrophoresis was performed on a native 4% polyacrylamide gel with 0.5 M tris-borate-EDTA at 4°C and a constant voltage of 110°C for 80 minutes. Fluorescence was detected using a Starion FLA-9000 instrument (FujiFilm, Japan). This indicates that MYB40 can bind to the PIN1 promoter in vitro, and the addition of ARFTF17 can enhance the DNA binding ability of MYB40 ( Figure 2 (a)

[0107] Example 3: Luciferase complementation assay verifies that ARFTF17 can positively regulate PIN1 gene expression

[0108] The CDS of ZmARF17 and ZmMYB40 were amplified using ARFTF17-62SKF and ARFTF17-62SKR, and MYB40-62SKF and MYB40-62SKR, respectively, and then cloned into the 35S-driven effector plasmid 62SK to obtain ZmARF17:62SK and ZmMYB40:62SK. The DNA sequence of PIN1pro was cloned upstream of the reporter vector pGreenII0800LUC using PIN1-LUCF and PIN1-LUCR to obtain PIN1pro:LUC. The plasmids were then extracted using the NucleoBond Xtra Midi (50) plasmid extraction kit, and the plasmid concentration was adjusted to 1 μg / μL. B73 seedlings were grown in the dark at 28°C for 7 days, and then the leaves were cut into 1 mm slices and incubated at 22°C for 3 h in lysis buffer (1.5% Cellulase R10, 0.5% Macerozyme R10, 0.4 M mannitol, 20 mM KCl, 20 mM MES pH 5.7, 10 mM CaCl2, 0.1% BSA, 5 mM β-mercaptoethanol). The reaction was then terminated using W5 buffer (154 mM NaCl, 125 mM CaCl2, 5 mM KCl, 5 mL glucose, 0.03% MES, pH 5.7) and protoplasts were washed and collected. MMG buffer (0.4 M mannitol, 15 mM MgCl2, 0.1% MES, pH 5.7) was added to the protoplasts to adjust the concentration to 1 x 10 6 / mL. 10μg of plasmid and 110μL of PEG (45% PEG4000, 0.2M mannitol, 100mM CaCl2) were added to each 100μL of protoplasts for transformation. After incubation at room temperature for 15 minutes, 440μL of W5 buffer was added to terminate the reaction. After removing the supernatant, 1mL of WI (20mM KCl, 0.6M mannitol, 4mM MES pH5.7) was added and the protoplasts were incubated for 16 hours. After the protoplasts were incubated for 16 hours, Total protein was extracted and reacted using the Reporter Assay System, and the ratio of LUC to REN was analyzed on a Promega 20 / 20 luminometer. Empty 62SK and PIN1pro:LUC were used as negative controls. The experiment showed that ARFTF17 can directly bind to MYB40, thereby inhibiting the negative regulatory function of MYB40 on the PIN1 promoter, thereby positively regulating PIN1 expression ( Figure 2 (b)

[0109] Example 4: Identification of PIN1 mutants in B73

[0110] The pin1 maize strain (EMS4-0b3dd4) with premature termination mutation was planted and self-pollinated to obtain F2. DNA was extracted from the leaves of F2 plants. PCR amplification was performed on the F2 plants using identification primers pin1-F and pin1-R. The PCR products were sequenced and the mutation sites were analyzed using SnapGene software. Single plants homozygous for the three mutation sites were identified ( Figure 3 (a)

[0111] Genomic DNA extraction and PCR identification:

[0112] Extract maize DNA using the CTAB method for subsequent identification. Place maize leaves in a 2 mL tube, add steel balls, treat with liquid nitrogen, and grind (60 Hz, 60 s); after grinding, add 0.6 mL of CTAB extraction buffer and mix well; place in a 65 ° C oven for 60 min, mixing every 10-15 min; take out and place at room temperature for 5-10 min, add an equal volume of chloroform: isoamyl alcohol (24:1) to the centrifuge tube, seal and shake for 5 min; centrifuge at 13000 rpm at room temperature for 15 min, aspirate the supernatant into a new 1.5 mL centrifuge tube; add an equal volume of isopropanol, mix by inversion, and place at -20 ° C for 20 min; centrifuge at 12000 rpm at room temperature for 1 min, and discard the supernatant. Wash the DNA pellet 1 to 2 times with 1 mL of 75% ethanol, centrifuging at 12,000 rpm for 1 min each time. Pour off the ethanol; briefly centrifuge, aspirate excess liquid, and air-dry the DNA pellet at room temperature; add 0.3 mL of ddH2O to dissolve the DNA pellet.

[0113] PCR detection reaction was carried out using the PCR Master Mix (With Dye) kit, prepare 20 μL reaction system: PCR Master Mix 10 μL, primers 1 μL each, DNA 2 μL, ddH2O 6 μL, specific detection primers designed by NCBI, reaction conditions are: pre-denaturation 95 ° C 5 min, amplification 95 ° C 30 s, Tma 30 s, 72 ° C 1 min, 35 cycles, termination 72 ° C 10 min, 16 ° C 1 min.

[0114] Example 5: Analysis of PIN1 expression in mutant seed coats

[0115] After planting the homozygous mutant, the seed coat of the grain was sampled 14 days after pollination and gene expression analysis was performed (refer to Example 1 for the method). The results showed that the expression level of PIN1 in the seed coat of pin1 was significantly decreased ( Figure 3 b), the specific method is carried out with reference to Example 1.

[0116] Example 6: Analysis of IAA content in the seed coat of pin1 mutants

[0117] The IAA content was determined in the seed coats of B73 and pin1 mutant maize 14 days after pollination. 50 mg of the sample was extracted in 1 ml of 80% methanol solution for 4 hours and then centrifuged at 13523 g for 10 minutes at 4°C. The liquid supernatant was placed at -20°C overnight and centrifuged at 13523 g for 10 minutes at 4°C before analysis. The extracts were analyzed on a UPLC instrument equipped with an electrospray ionization (ESI) source (AB SCIEX). The instrument was controlled and data were acquired using an Analyst 1.6.3 software (AB SCIEX), and data were processed using MultiQuant 3.0.2 software (ABSCI EX). Plant hormones were quantified by calculating the area of ​​each peak and comparing it to a standard curve. To quantify IAA content, a standard curve was created using 5, 20, 50, 100, 200, and 500 ng / ml IAA (Agrisera). The results showed that the auxin content in the pin1 seed coat was significantly decreased ( Figure 3 (c)

[0118] Example 7: PIN1 mutation can convert dent grains into hard grains

[0119] The seeds of B73 and pin1 mutants were examined. Mature seeds of B73 and pin1 in good condition were sliced ​​by hand and photographed under a stereo microscope (Leica M165FC). The top depression angle and seed coat length of the slices were measured and statistically analyzed using Imag J software. It was found that the shape of pin1 seeds changed significantly from dent type to hard type ( Figure 4 In middle a), the endosperm at the top of the grain is filling and filling, the hard endosperm content increases, the top of the grain does not sink but remains convex, and the seed coat length becomes shorter ( Figure 4 (middle bd).

[0120] Although the above embodiment only verifies the function of PIN1 using corn as an example, those skilled in the art should understand that without violating the concept of the present invention, those skilled in the art can make various changes or modifications on this basis, such as applying it to other grain improvements or new variety creation, and the equivalent forms of various deformations or modifications made thereby should also fall within the scope of the present invention.

Claims

1. A method for converting dent corn into flint corn, characterized in that: The steps include: A. downregulating, inactivating, weakening or knocking out the auxin transporter gene PIN1 in the dent corn genome; or B. Overexpression of the auxin transporter gene PIN1 in dent corn by mutating the tryptophan codon TGG at position 467 to a stop codon W467X mutant; or C. Replace the PIN1 gene in the dent corn genome with the PIN1 gene described in step B. W467X Mutant replacement.

2. The method according to claim 1, wherein The stop codon in step B or step C is TGA, that is, the 467th tryptophan codon TGG is mutated to TGA, and the corresponding gene PIN1 mutant is PIN1 W467X The nucleotide sequence of mutant 1 is SEQ ID NO: 1; or The stop codon is TAG, that is, the 467th tryptophan codon TGG is mutated to TAG, and the corresponding gene PIN1 mutant is PIN1 W467X Mutant 2; or The stop codon is TAA, that is, the 467th tryptophan codon TGG is mutated to TAA, and the corresponding gene PIN1 mutant is PIN1 W467X Mutant Three.

3. The method according to claim 1, wherein Step A is carried out as follows: (1) Knockout of the PIN1 gene in the dent maize genome; (2) downregulating the expression level of the gene PIN1 in the dent corn genome; or (3) Replace the PIN1 gene in the dent corn genome with a PIN1 mutant whose coding function is lost or downregulated.

4. The method according to claim 3, wherein Method (2) is selected from the following group: (2-1) Mutation of the PIN1 promoter region results in downregulation of PIN1 expression; (2-2) mutation of the upstream regulatory factor of PIN1 gene leads to downregulation of the expression level of PIN1 gene; or (2-3) The function of the PIN1 gene was altered by introducing an interacting protein of the auxin transporter PIN1 into dent corn.

5. The method according to claim 4, wherein The interacting protein of the ARF transcription factor in method (2-3) is the transcription factor MYB40 or the transcription factor ARF that regulates zeaflavone metabolism.

6. A polynucleotide selected from the group consisting of: PIN1 as claimed in claim 2 W467X Mutant 1, i.e., a PIN1 mutant in which the tryptophan codon TGG at position 467 is mutated to the stop codon TGA, with the nucleotide sequence being SEQ ID NO: 1; PIN1 as claimed in claim 2 W467X Mutant 2, i.e., a PIN1 mutant in which the tryptophan codon TGG at position 467 is mutated to the stop codon TAG; or PIN1 as claimed in claim 2 W467X Mutant three is a PIN1 gene mutant in which the tryptophan codon TGG at position 467 is mutated to the stop codon TAA.

7. The use of the polynucleotide according to claim 6, characterized in that Used to create hard-grain germplasm by introducing into dent corn.

8. A method for detecting the gene PIN1 in the maize genome as claimed in claims 2 and 6. W467X The method for preparing a mutant, namely the nucleotide sequence SEQ ID NO: 1, comprises the following steps: The maize genome was PCR amplified and sequenced using a conventional PCR MIX program using forward primer pin1-F and reverse primer pin1-R. The PCR amplification product was 556 bp and the nucleotide sequence was SEQ ID NO: 4, wherein: Forward primer pin1-F: GCGATGCGACTCCAGACAGA (SEQ ID NO: 2), Reverse primer pin1-R: ACACCTGTTCTGTTCTTGTTGGAC (SEQ ID NO: 3).

9. A kit for implementing the method according to claim 8, characterized in that: It includes corresponding primers for detecting SEQ ID NO: 1, or DNA / RNA probes, or a microarray chip of DNA / RNA probes.

10. The kit according to claim 10 is used to detect the gene PIN1 according to claims 2 and 6 in corn. W467X The use of a mutant in overexpression, characterized in that Maize genomic DNA was extracted as a template, and PCR was performed on pin1-F and pin1-R using the primers described in claim 8 to amplify the amplified band shown in nucleotide sequence SEQ ID NO: 4, indicating that the gene PIN1 W467X Overexpression of mutant.

Images