Use of negative regulation of ipcks gene in reducing phytic acid content in plants

Abstract

The application belongs to the technical field of plant genetic engineering, and particularly relates to application of negative regulation IPCKs gene in reducing phytic acid content in plants. The application provides application of IPCKs gene in reducing phytic acid content in plants, and by knocking out IPCKs gene in plants, i.e. knocking out at least four of IPCK1, IPCK2, IPCK3, IPCK4, IPCK5 and IPCK6 genes in target plants, reducing expression amount of IPCKs gene in plants or directly affecting the function of the coded protein, the phytic acid content in plants, especially in plant seeds, is significantly reduced, and it is further determined that the loss of IPCKs function can reduce the phytic acid content in plants, which has important significance for reducing the phytic acid content in plant seeds by biological means, and provides important technical basis and support for low-phytic-acid plant breeding.

Description

Technical Field

[0001] This invention belongs to the field of plant genetic engineering technology, specifically involving negative regulation. IPC classes Application of genes in reducing phytic acid content in plants. Background Technology

[0002] Myo-inositol-1,2,3,4,5,6-hexakisphosphate (InsP6 or IP6), also known as phytic acid, is the main form of phosphorus stored in plants, especially seeds (particularly cereals, legumes, and oilseeds), playing a crucial role in maintaining phosphorus homeostasis within plants. Because IP6 contains six negatively charged phosphate groups, it has a strong chelating ability for minerals (cations such as magnesium, calcium, and iron) and proteins. Monogastric animals lack the corresponding phytase, making it difficult for them to absorb and utilize the stable salts and protein complexes chelated by phytic acid. Ultimately, these are excreted into the environment in feces. Therefore, IP6 is considered an "anti-nutrient," reducing the utilization rate of minerals and other nutrients by animals, and further contributing to eutrophication of water bodies due to undigested phytates in excrement. Thus, IP6 has numerous negative impacts on human and animal nutrition and the ecological environment. Resolving the contradiction between the high presence of IP6 in seeds and environmental pollution, as well as the low utilization rate of human nutrients, has become a major challenge for scientists.

[0003] In recent years, addressing the issue of high IP6 content at the source of crop variety selection has been an important breeding goal for breeders. Germplasm innovation in crops with low phytic acid content, and the study of the genetic characteristics of mutants and the localization and functional analysis of related genes have become new research hotspots and solutions. However, how to obtain plant germplasm with low phytic acid content remains unknown. Summary of the Invention

[0004] The purpose of this invention is to provide negative regulation. IPC classes The application of genes in reducing phytic acid content in plants can reduce the phytic acid content in plants, especially plant seeds, and provide technical support for low-phytic acid breeding.

[0005] This invention provides negative regulation IPC classes The application of genes in reducing phytic acid content in plants, the IPC classes Genes include IPC1, IPC2, IPC3, IPC4, IPC5 and IPC6 At least four of them; the IPC1, IPC2, IPC3, IPC4, IPC5 and IPC6 The nucleotide sequences are shown in SEQ ID NO:1 to SEQ ID NO:6, respectively.

[0006] Preferred, negative regulation IPC classes Genes reduce the IPC classes Gene expression or reduction in plants IPC classes The realization of gene-encoded proteins.

[0007] This invention also provides a way to reduce the concentration of certain substances in plants. IPC classes Gene expression or reduction in plants IPC classes The application of genetically encoded protein-rich biomaterials in reducing phytic acid content in plants; IPC classes Genes include IPC1, IPC2, IPC3, IPC4, IPC5 and IPC6 At least four of them; the IPC1, IPC2, IPC3, IPC4, IPC5 and IPC6 The nucleotide sequences are shown in SEQ ID NO:1 to SEQ ID NO:6, respectively.

[0008] Preferably, the biomaterial is at least one of the following A1 to A10: A1: Used to eliminate the applications described in the above technical solutions. IPC classes Nucleic acid molecules of genes; A2: A recombinant expression vector containing the nucleic acid molecule described in A1; A3: Recombinant microorganisms containing the nucleic acid molecules described in A1; A4: Recombinant microorganisms containing the recombinant expression vector described in A2; A5: Transgenic plant cell lines containing the nucleic acid molecules described in A1; A6: Transgenic plant cell lines containing the recombinant expression vector described in A2; A7: Transgenic plant tissue containing the nucleic acid molecules described in A1; A8: Transgenic plant tissue containing the recombinant expression vector described in A2; A9: Transgenic plant organs containing the nucleic acid molecules described in A1; A10: Transgenic plant organs containing the recombinant expression vector described in A2.

[0009] Preferably, the nucleic acid molecules in A1 include those for knockout. IPC5 Nucleic acid molecules used for knockout IPC6 Multiple nucleic acid molecules and T-DNA sequences, used for knockout IPC5 Nucleic acid molecules and those used for knockout IPC6 The nucleotide sequences of the nucleic acid molecules are shown in SEQ ID NO:11 and SEQ ID NO:12, respectively.

[0010] Preferred, for knockout IPC5The upstream and downstream primers of the primers are shown in SEQ ID NO:9 and SEQ ID NO:10, respectively; Used for knocking IPC6 The upstream and downstream primers of the primers are shown in SEQ ID NO:11 and SEQ ID NO:12, respectively.

[0011] Preferably, the plant includes Arabidopsis thaliana; the plant includes plant seeds.

[0012] The present invention also provides a biomaterial, wherein the biomaterial is at least one of the following A1 to A4: A1: Used to eliminate the applications described in the above technical solutions. IPC classes Nucleic acid molecules of genes; A2: A recombinant expression vector containing the nucleic acid molecule described in A1; A3: Recombinant microorganisms containing the nucleic acid molecules described in A1; A4: Recombinant microorganisms containing the recombinant expression vector described in A2.

[0013] This invention also provides a method for cultivating low-phytate plants, comprising: reducing the phytate content in the target plant. IPC classes Gene expression, or reduction of the expression of the target plant. IPC classes The content of the gene-encoded protein was used to obtain the low-phytate plant.

[0014] Preferred, reducing the content of the target plant IPC classes Gene expression or reduction in the target plant IPC classes The content of the gene-encoded protein is determined by introducing a knockout gene into the target plant. IPC classes Nucleic acid molecules of genes.

[0015] Beneficial effects: This invention provides negative regulation IPC classes The application of genes in reducing phytic acid content in plants involves knocking out phytic acid in plants. IPC classes Genes, that is, genes that are knocked out in the target plant IPC1 , IPC2 , IPC3 , IPC4 , IPC5 and IPC6 At least four of the genes reduce IPC classes Gene expression in plants may directly affect the function of encoded proteins, leading to a significant reduction in phytic acid content, especially in plant seeds, and thus clarifying... IPC classes The loss of function can reduce the phytic acid content in plants, which is of great significance for reducing the phytic acid content in plant seeds through biological means, and provides an important technical foundation and support for breeding low-phytic acid plants. Attached Figure Description

[0016] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the accompanying drawings used in the embodiments will be briefly described below.

[0017] Figure 1 In Example 1 ipc classes Schematic diagram of mutant T-DNA insertion information; Figure 2 For the three-primer method in Example 1 IPC classes The results of homozygosity testing of the mutants; Figure 3 This is a comparison of the target gene expression levels between wild-type and T-DNA insertion mutant lines in Example 1; Figures 4 - 6 The CRISPR vector map in Example 2; Figure 7 In Example 4 IPC5 A schematic diagram showing the location information of CRISPR knockout mutations; Figure 8 This is a comparison chart of phytic acid content in seeds of wild-type and mutant lines in Example 5. Detailed Implementation

[0018] This invention provides negative regulation IPC classes The application of genes in reducing phytic acid content in plants, the IPC classes Genes include IPC1 , IPC2 , IPC3 , IPC4 , IPC5 and IPC At least four of them; the ​ , ​ , ​ , ​ , ​ and ​ The nucleotide sequences are shown in SEQ ID NO:1 to SEQ ID NO:6, respectively. The present invention... ​ Gene selection includes ​ , ​ , ​ and ​ or ​ , ​ , ​ , ​ and ​ or ​ , ​ , ​ , ​ , ​ and ​ More preferably ​ , ​ ,​ , ​ and ​ .

[0019]

[0020] The negative regulation described in this invention ​ Gene selection is achieved by reducing the aforementioned ​ Gene expression or reduction in plants ​ The realization of gene-encoded proteins.

[0021] This invention also provides a way to reduce the concentration of certain substances in plants. ​ Gene expression or reduction in plants ​ The application of genetically encoded protein-rich biomaterials in reducing phytic acid content in plants; ​ Genes include ​ ​ and ​ At least four of them, further preferably including ​ , ​ , ​ and ​ or ​ , ​ , ​ , ​ and ​ or ​ , ​ , ​ , ​ , ​ and ​ More preferably ​ , ​ , ​ , ​ and ​ The ​ and ​ The nucleotide sequences are shown in SEQ ID NO:1 to SEQ ID NO:6, respectively.

[0022] In this invention, the biomaterial is preferably at least one of the following A1 to A10: A1: used for knocking out the application described in the above technical solution. ​ A1: A nucleic acid molecule containing a gene; A2: A recombinant expression vector containing the nucleic acid molecule described in A1; A3: A recombinant microorganism containing the nucleic acid molecule described in A1; A4: A recombinant microorganism containing the recombinant expression vector described in A2; A5: A transgenic plant cell line containing the nucleic acid molecule described in A1; A6: A transgenic plant cell line containing the recombinant expression vector described in A2; A7: A transgenic plant tissue containing the nucleic acid molecule described in A1; A8: A transgenic plant tissue containing the recombinant expression vector described in A2; A9: A transgenic plant organ containing the nucleic acid molecule described in A1; A10: A transgenic plant organ containing the recombinant expression vector described in A2.

[0023] In this invention, the method used to knock out the above-described technical solution ​The nucleic acid molecules of the gene preferably include those used in the CRISPR knockout technique described above. ​ Multiple nucleic acid molecules and T-DNA sequences of the gene, more preferably those used in the CRISPR knockout technique described above. ​ The nucleic acid molecule and T-DNA sequence of the gene. The present invention describes a technique for CRISPR knockout as described above. ​ The preferred nucleic acid molecules for gene knockout include those for use in gene knockout. ​ Nucleic acid molecules and those used for knockout ​ Nucleic acid molecules; the ones used for knockout ​ Nucleic acid molecules and those used for knockout ​ The preferred nucleotide sequences of the nucleic acid molecules are shown in SEQ ID NO:7 and SEQ ID NO:8, specifically 5'-TTGTCAACCTTCATTCATAGAGG-3' and 3'-CAGCACCAAGCAACTTAGCAAGG-5', respectively. This invention does not impose any specific limitations on the T-DNA sequence; any sequence designed using conventional methods in the art that can achieve knockout... ​ The T-DNA sequences of all genes fall within the scope of protection of this invention, such as the four T-DNA insertion mutants purchased in the embodiments of this invention. ​ (SALK_047485C) ​ (SAIL_916_B10). ​ (SALK_026210C) and ​ (SAIL_913_F05) was obtained by inserting a T-DNA sequence into the target plant, Arabidopsis thaliana, at the specific insertion site as shown below. ​ As shown.

[0024] In this invention, it is used for CRISPR knockout. ​ The preferred nucleotide sequences of the upstream and downstream primers are shown in SEQ ID NO:9 and SEQ ID NO:10, respectively, specifically 5'-GATTGTTGTCAACCTTCATTCATAGAGG-3' and 5'-AAACCCTCTATGAATGAAGGTTGACAAC-3'; used for CRISPR knockout. ​ The preferred nucleotide sequences of the upstream and downstream primers are shown in SEQ ID NO:11 and SEQ ID NO:12, respectively, specifically 5'-GGTCACAGCACCAAGCAACTTAGCAAGG-3' and 5'-AAACCCTTGCTAAGTTGCTTGGTGCTGT-3'. This invention relates to the method for CRISPR knockout. ​ and ​The preferred annealing conditions for the primers are: 37℃, 30 min; 95℃, 5 min; 25℃, 6 min, with a descent rate of 0.2℃ / s.

[0025] In this invention, the amount of [something] in plants is reduced. ​ Gene expression or reduction in plants ​ Preferred methods for reducing phytic acid content in plants using genetically encoded protein-rich biological materials include: [the following methods are used to describe methods for reducing phytic acid content in plants by] knocking out [the following proteins]. ​ , ​ , ​ and ​ Tetramutant of a gene ​ Import and remove the above ​ Nucleic acid molecules of genes and knockout ​ Nucleic acid molecules of genes. The tetramutant mutant described in this invention. ​ The preferred method for obtaining it is to use the mutant ​ (SALK_047485C) ​ (SAIL_916_B10). ​ (SALK_026210C) and ​ The mutant described in this invention was obtained by hybridization with (SAIL_913_F05). ​ (SALK_047485C) ​ (SAIL_916_B10). ​ (SALK_026210C) and ​ (SAIL_913_F05) was purchased from the Arabidopsis Seed Resource Center (http: / / abrc.osu.edu). The hybridization method described in this invention is preferably artificial pollination, specifically: Selecting a time when the Arabidopsis plant's pollen is highly viable, carefully remove the sepals and petals from the fully opened male parent flower using tweezers to expose the stamens. Then, select a female parent flower that is showing white petals but not yet fully open, and remove the petals and stamens using tweezers to expose the style. Finally, repeatedly smear the male parent's stamens onto the female parent's style several times to complete the hybridization.

[0026] The recombinant expression vector of the present invention preferably contains the knockout of the [specific ingredient]. ​ Nucleic acid molecules of genes and knockout ​ The nucleic acid molecule of the gene; the initial vector of the recombinant expression vector preferably includes a Cas9 plasmid, more preferably pEx-pAtUBQ-Cas9; the knockout of the gene ​ Nucleic acid molecules of genes and knockout ​ The nucleic acid molecule of the gene is preferably inserted into the pEx-pAtUBQ-Cas9. ​ III and ​ Between the I restriction sites, the knockout of the ​ Nucleic acid molecules of genes and knockout​ Nucleic acid molecules of genes through ​ Ligation is performed at the I restriction site. The present invention does not specifically limit the construction steps of the recombinant expression vector; conventional operating procedures in the art can be followed.

[0027] The initial microorganism in the recombinant microorganisms of the present invention preferably includes Agrobacterium, and more preferably GV3101.

[0028] The transgenic plant cell lines, transgenic plant tissues, and transgenic plant organs described in this invention do not include propagation material. Preferably, the transgenic plant cell lines, transgenic plant tissues, and transgenic plant organs described in this invention are obtained by transferring the recombinant microorganisms into the target plant. This invention does not specifically limit the method for transferring the recombinant microorganisms into the target plant; conventional transgenic methods in the art can be used, such as the vacuum transformation method used in the embodiments of this invention.

[0029] In this invention, the ​ Gene-encoded proteins preferably include ​ - ​ The proteins encoded by the gene are composed of 494, 512, 466, 493, 467, and 484 amino acids, respectively, and are preferably represented by SEQ ID NO: 13~18, as follows: SEQ ID NO:13:MTSQLKRTLTKRYGVLELWEIIVIALFAAFIVILVLSVWLSFRKKSKRSNATTLPVTQSPRFTEEIKEISVDHGSSNNNGTSYQTLDEKFVEDIENGDKFSGSLEKKPLVGS HLPPSTPSTTAPSPLLGLPEVSHIGWGHWFTLRDLQLATNHFSKESIIGDGGYGVVYHGTLTNKTPVAVKKLLNNPGQADKDFRVEVEAIGHVRHKNLVRLLGYCVEGTHRMLVYEYMN NGNLEQWLHGDMIHKGHLTWEARIKVLVGTAKALAYLHEAIEPKVVHRDIKSSNILMDDNFDAKLSDFGLAKLLGADSNYVSTRVMGTFGYVAPEYANSGLLNEKSDVYSYGVVLLEA ITGRYPVDYARPKEEVHMVEWLKLMVQQKQFEEVVDKELEIKPTTSELKRALLTALRCVDPDADKRPKMSQVARMLESDEYPVMPREERRRRRNQNAETHRESTDTNKDNDITTDAKI; SEQ ID NO:14:MGSGLNDTLSRNYNGLELWEIIIVILSAIFVVVLAISLWLTFRRKTSRSSSNLIPVSRQIPPSVPEEIKEIRVDEVSSSSNGGNGYPSISEKFGDKEPEKGIKAESENGDSSRSGSFNHLEKKDGSSVSSANPLTAPSLSPEGLFSHLGWGHWFTLRDLQMATNQFSRDNIIGDGGYGVVYRGNLVNGTPVAVKKLLNNLGQADKDFRVEVEAIGHVRHKNLVRLLGYCMEGTQRMLVYEYVNNGNLEQWLRGDNQNHEYLTWEARVKILIGTAKALAYLHEAIEPKVVHRDIKSSNILIDDKFNSKISDFGLAKLLGADKSFITTRVMGTFGYVAPEYANSGLLNEKSDVYSFGVVLLEAITGRYPVDYARPPEVHLVEWLKMMVQQRRSEEVDPNLETKPSTSALKRTLLTALRCVDPMSEKRPRMSQVARMLESEEYPIAREDRRRRSQNGTTRDSPPRNSTDTDKSEYHDLKPEGG; SEQ ID NO:15:MPPESSLNAEMSKKISFFGLKGLKLWVWVCLVVGVFIVMILCILSLWITFRRKSRRSSSKFPFNQIPHVSKDIRVDRAGFQNPHPESLYIEMNDKSTGKTMMSHLGRTKSSDNDTLSQCSSVNHHERACSSHSGEEGGFGSAGRQYGGGPVTASPLVGLPEISHLGWGHWFTLRDLELATNRFAPVNVLGEGGYGVVYRGKLVNGTEVAVKKLLNNLGQAEKEFRVEVEAIGHVRHKNLVRLLGYCIEGVHRMLVYEYVNSGNLEQWLHGAMRQHGNLTWEARMKIITGTAQALAYLHEAIEPKVVHRDIKASNILIDDEFNAKLSDFGLAKLLDSGESHITTRVMGTFGYVAPEYANTGLLNEKSDIYSFGVLLLEAITGRDPVDYGRPANEVNLVEWLKMMVGTRRAEEVVDPRLEPRPSKSALKRALLVSLRCVDPEAEKRPRMSQVARMLESDEHPFHKERRNKRSKTAGMEIVETKDESLGPSGSETKP; SEQ ID NO:16:MSSESSLSADMSKKVSFLGLKGMKLWVLICLVVGTFVVLVFCILSLWIAFRRKSRRSSHKLLPFSQIPRVAKDIRVDDRVGFQNHNENLSITNADKSSDRNSGKMMSYLGRTKSSDNDSISQCSSVHHHERACSSHSGEDGSFGAAWRQNSLSQGGLVTASPLVGLPEISHLGWGHWFTLRDLQLATNRFAAENVIGEGGYGVVYKGRLINGNDVAVKKLLNNLGQAEKEFRVEVEAIGHVRHKNLVRLLGYCIEGVNRMLVYEYVNSGNLEQWLHGAMGKQSTLTWEARMKILVGTAQALAYLHEAIEPKVVHRDIKASNILIDDDFNAKLSDFGLAKLLDSGESHITTRVMGTFGYVAPEYANTGLLNEKSDIYSFGVLLLETITGRDPVDYERPANEVNLVEWLKMMVGTRRAEEVVDSRIEPPPATRALKRALLVALRCVDPEAQKRPKMSQVVRMLESDEHPFREERRNRKSRTASMEIVETTEESADTSKGPGHSENTTKPEKTHV; SEQ ID NO:17:MAGLKIWQAIFITIALIIIVVLSVLSFCLIWKKKSRRSKTLSLPIIQTPVVSKEIKEVRIEHVVSTSSNFDPQDENNNESDKFLLNLEMEKNRENGLSSSRSGSGKEGYLCVANRSTSSLYEMATPSPSPLSGLPESHLGWGHWFTLRDLEIATNRFSKENVIGEGGYGVVYRGELVNGSLVAVKKILNHLGQAEKEFRVEVDAIGHVRHKNLVRLLGYCIEGTNRILVYEYMNNGNLEEWLHGAMKHHGYLTWEARMKVLTGTSKALAYLHEAIEPKVVHRDIKSSNILIDDRFNAKISDFGLAKLLGDGKSHVTTRVMGTFGYVAPEYANTGLLNEKSDVYSFGVLVLEAITGRDPVDYARPANEVNLVEWLKMMVGSKRLEEVIDPNIAVRPATRALKRVLLTALRCIDPDSEKRPKMSQVVRMLESEEYPVPREERRVRRTQEENSDTDRSRPVSRSQSKRL。

[0030] SEQ ID NO:18:MGGDLKSQLSRESHVFGLKVWEVIGIAVALLIIAILSVLSCCLTSKKKSRRSKTGLPVIQTPPVVSKEIREVRVEHVSASNFAPGEGILLTIQDKNNKDSEKVMVHLDMRKKRSSSGR SGSFHHLEIIDKHSDSAEEVSASSSLYNIATPSPLSGLPESHLGWGHWFTLRDLETATNRFSKENVIGEGGYGVVYRGELMNGTPVAVKKILNQLGQAEKEFRVEVDAIGHVRHKNLVRLLGYCI EGTHRILVYEYVNNGNLEQWLHGAMRQHGYLTWEARMKVLIGTSKALAYLHEAIEPKVVHRDIKSSNILINDEFNAKVSDFGLAKLLGAGKSHVTTRVMGTFGYVAPEYANSGLLNEKSDVYSFG VVLLEAITGRDPVDYGRPAHEVNLVDWLKMMVGTRRSEEVVDPNIEVKPPTRSLKRALLTALRCVDPDSDKRPKMSQVVRMLESEEYPIPREDRRRSRTREGSMEINSDTTDMSTPVSRSQSKRQ.

[0031] In this invention, the plant preferably includes Arabidopsis thaliana, and more preferably includes Arabidopsis thaliana seeds.

[0032] This invention also provides a method for cultivating low-phytate plants, comprising: reducing the phytate content in the target plant. ​ Gene expression, or reduction of the expression of the target plant. ​ The content of the gene-encoded protein is used to obtain the low-phytate plant. The plant described in this invention preferably includes Arabidopsis thaliana. ​ The nucleotide sequence of the gene and the ​ The amino acid sequence of the gene-encoded protein has been described in the above technical solution and will not be repeated here.

[0033] The present invention also provides a biomaterial, wherein the biomaterial is at least one of the following A1 to A4: A1: used to knock out the application described in the above technical solution. ​ A1: A nucleic acid molecule of a gene; A2: A recombinant expression vector containing the nucleic acid molecule described in A1; A3: A recombinant microorganism containing the nucleic acid molecule described in A1; A4: A recombinant microorganism containing the recombinant expression vector described in A2. The biological materials described in this invention have been specifically defined and described in the above technical solutions, and will not be repeated here.

[0034] The low-phytate plant of this invention is produced by introducing a knockout agent into the target plant. ​ Plants obtained from the nucleic acid molecules of genes described in this invention. ​ The preferred nucleic acid molecules for gene knockout include those for use in gene knockout. ​ Nucleic acid molecules used for knockout ​ Multiple nucleic acid molecules and T-DNA sequences, more preferably for knockout ​ Nucleic acid molecules used for knockout ​ The nucleic acid molecules and T-DNA sequences. In this invention, the low-phytate plant is preferably obtained by knocking out... IPCK1 , IPCK2 , IPCK3 and IPCK4 Tetramutant of a gene T-4m Import and remove the above IPCK5 Nucleic acid molecules of genes and knockout IPCK6 Plants obtained from the nucleic acid molecules of genes. The tetramutant mutant described in this invention. T-4m The preferred method for obtaining it is to use the mutant IPCK1 (SALK_047485C) ipck2 (SAIL_916_B10). ipck3 (SALK_026210C) and ipck4 The mutant described in this invention was obtained by hybridization with (SAIL_913_F05). ipck1 (SALK_047485C) ipck2 (SAIL_916_B10). ipck3 (SALK_026210C) and ipck4 (SAIL_913_F05) was purchased from the Arabidopsis Seed Resource Center (http: / / abrc.osu.edu). The hybridization method described in this invention is preferably artificial pollination. This invention does not specifically limit the method of introduction; conventional transgenic procedures in the art can be used.

[0035] This invention knocks out the plant IPCKs Genes, that is, genes that are knocked out in the target plant IPCK1 , IPCK2 , IPCK3 , IPCK4 , IPCK5 and IPCK6 At least four of the genes reduce IPCKs The expression level of genes in plants can directly affect the function of encoded proteins, significantly reducing the phytic acid content in plants, especially in seeds, thus obtaining low-phytic acid plant germplasm and providing important technical support for the breeding of low-phytic acid plants.

[0036] To further illustrate the present invention, the technical solutions provided by the present invention will be described in detail below with reference to the accompanying drawings and embodiments, but these should not be construed as limiting the scope of protection of the present invention.

[0037] Example 1 T-DNA insertion mutant ipcks Homozygosity identification and tetramutants T-4m Construction: Four T-DNA insertion mutants were purchased from the Arabidopsis Seed Resource Center (http: / / abrc.osu.edu). ipck1 (SALK_047485C) ipck2 (SAIL_916_B10). ipck3 (SALK_026210C) and ipck4 (SAIL_913_F05), Insertion position information is as follows Figure 1 As shown, the homozygosity of the T-DNA insertion mutant was identified using the three-primer method. The PCR identification system followed the instructions for the Vazyme high-fidelity enzyme. The identification results showed that all mutants were homozygous. Figure 2 As shown, LB and LP refer to the upstream primers designed for the three-primer identification method, and RP refers to the downstream primer. The band size obtained by LP+RP amplification is about 800-900bp, and the band size obtained by LB+RP amplification is about 500bp.

[0038] The identification primers are: IPCK 1-LP: 5'-AAGACCGAAATCAGATAACTTTGC-3' (SEQ ID NO: 19); IPCK 1-RP: 5'-TCCTCTTTCCACTGATCTTTAGG-3' (SEQ ID NO: 20); IPCK 2-LP: 5'-AACCAAGCCGCCTAACTAGAC-3' (SEQ ID NO: 21); IPCK 2-RP: 5'-TTCAGTTCTCAGACCAAACGC-3' (SEQ ID NO: 22); IPCK 3-LP: 5'-TTTAGGCTACCTCATTAGCTGGAC-3' (SEQ ID NO: 23); IPCK3-RP: 5'-TAGTTCTCACTCTGGTGAAGATGG-3' (SEQ ID NO: 24); IPCK 4-LP: 5'-TTCCCAAGTGAGATTTCCATG-3' (SEQ ID NO: 25); IPCK 4-RP: 5'-CCATTGCTTCTACGCTTTCTG-3' (SEQ ID NO: 26).

[0039] Wild-type Col-0 (Columbia wild-type) and IPCK1 - IPCK4 RNA was extracted from young seedlings of the mutant plant, reverse transcribed, and then detected by real-time quantitative PCR using TOYOBO's SYBR Green Realtime PCR Master Mix. Actin2 Genes were used as internal controls; the detection system and primers used are as follows: The primers used for real-time quantitative PCR were: q IPCK 1-F: 5'-AAAGCCCTCGCTTTACCGAA-3' (SEQ ID NO: 27); q IPCK 1-R: 5'-CGCCATTCTCGATGTCCTCA-3' (SEQ ID NO: 28); q IPCK 2-F: 5'-ATGGCTCCGTGGAGACAATC-3' (SEQ ID NO: 29); q IPCK 2-R: 5'-CTTTGGCTCAATCGCCTCG-3' (SEQ ID NO: 30); q IPCK 3-F: 5'- CGCAGGCGCTTGCTTATTTA -3' (SEQ ID NO: 31); q IPCK 3-R: 5'-AGCATATTCGGGTGCGACAT-3' (SEQ ID NO: 32); q IPCK 4-F: 5'-CTCACTTGGGAAGCACGGAT-3' (SEQ ID NO: 33); q IPCK 4-R: 5'-ACTCTCACCCGAGTCCAAGA-3' (SEQ ID NO: 34); qActin2-F: 5'-GGTAACATTGTGCTCAGTGGTGG-3' (SEQ ID NO:35); qActin2-R: 5'-AACGACCTTAATCTTCATGCTGC-3' (SEQ ID NO: 36).

[0040] The reaction procedure for real-time quantitative PCR is as follows: Pre-denaturation: 95℃, 1 min; PCR cycling: 95℃, 15 sec; 60℃, 15 sec; 72℃, 45 sec (40 cycles).

[0041] The reaction system (10 μL) for real-time quantitative PCR is as follows: SYBR Green Realtime PCR Master Mix 5μL, q IPCK sF (10 μM) 0.1 μL, q IPCK 0.1 μL of sR (10 μM), 4.6 μL of ultrapure water, and 0.2 μL of reverse product (1000 ng).

[0042] After testing, it was found that, compared with the wild type, the expression levels of the corresponding target genes in each T-DNA insertion mutant were significantly suppressed, such as... Figure 3 As shown.

[0043] Further, through artificial pollination and homozygosity testing, a homozygous T-DNA insertion tetramutant mutant was obtained and named... T-4m .

[0044] The specific process of artificial pollination is as follows: Select a time when the pollen of the Arabidopsis thaliana plants is highly vigorous. Carefully remove the sepals and petals from the fully opened male parent flowers using tweezers to expose the stamens. Then, select unopened but budding female parent flowers and remove the petals and stamens using tweezers to expose the style. Finally, repeatedly smear the male parent's stamens onto the female parent's style several times to complete the hybridization.

[0045] By IPCK1 and IPCK2 Single mutant hybridization and IPCK3 and IPCK4 First, construct double mutants by hybridizing single mutants. IPCK1 IPCK2 and IPCK3 IPCK4 Then, by crossing these two homozygous double mutants again, a quadruple mutant is finally obtained. T-4m .

[0046] Example 2 Genes involved in regulating phytic acid synthesis in plant seeds IPCK5 and IPCK6CRISPR vector construction for genes: Target primers were designed online and then annealed under the following conditions: 37℃ for 30 min; 95℃ for 5 min; 25℃ for 6 min, with a descent rate of 0.2℃ / s.

[0047] The upstream and downstream primers for the target are as follows: IPCK 5. Upstream primer: 5'-GATTGTTGTCAACCTTCATTCATAGAGG-3' (SEQ ID No: 9); IPCK 5. Downstream primer: 5'-AAACCCTCTATGAATGAAGGTTGACAAC-3' (SEQ ID No: 10); IPCK 6. Upstream primer: 5'-GGTCACAGCACCAAGCAACTTAGCAAGG-3' (SEQ ID No: 11); IPCK 6. Downstream primer: 5'-AAACCCTTGCTAAGTTGCTTGGTGCTGT-3' (SEQ ID No: 12).

[0048] 18T-pAtU6chim (vector spectrum see...) Figure 4 ), 18T-U3bchim (carrier spectrum see Figure 5 The carriers were respectively passed through Bbs I. Digestion with enzymes: The digestion system followed the Thermo FastDigest instructions. Then, gel extraction was performed, following the MAGEN HiPure Gel Pure DNA Mini Kit instructions, to obtain the digested linear vector. The annealed primers were then ligated to the digested linear vector using T4 ligase (NEB, reaction conditions as per instructions). IPCK 5 connected to 18T-pAtU6chim; IPCKThe ligation product was transferred to *E. coli* DH5α via heat shock and evenly spread onto an antibiotic-resistant plate containing 50 mg / L ampicillin (Amp). The plate was incubated overnight at 37°C, and single clones were picked the following day for sequencing verification. Using recombinant 18T-pAtU6chim and 18T-U3bchim plasmids carrying the target sequence as templates, PCR was performed using the M13F / R primer pair (specifically: M13F: 5'-TGTAAAACGACGGCCAGT-3' (SEQ ID No: 37); M13R: 5'-CAGGAAACAGCTATGACC-3' (SEQ ID No: 38)). The target sequence carrying the target was amplified using the Vazyme high-fidelity enzyme manual (2× Phanta MaxMaster Mix). The gel-recovered product was digested with the appropriate restriction endonuclease to recover the target sequence. Hind III and Xho I enzyme digestion of the recovered product using 18T-pAtU6chim as a template; Xho I and Xba The recovered product was digested with enzyme I using 18T-U3bchim as a template. The pEx-pAtUBQ-Cas9 empty vector plasmid (see diagram) Figure 6 )pass Hind III and Xba The linear vector was obtained by I-recovery and ligated with the two enzyme digestion products obtained in the previous step via T4 ligation. The ligation product was transformed into *E. coli* DH5α via heat shock and evenly spread on an antibiotic-resistant plate containing 50 mg / L kanamycin (Kana). The plate was incubated overnight at 37°C, and single colonies were picked the following day for sequencing verification. The final product contained... IPCK5 and IPCK6 Cas9 knockout plasmids targeting the sequence.

[0049] Example 3 Transformation methods of Arabidopsis thaliana 0.5 μg of the Cas9 knockout plasmid prepared in Example 2 was transformed into Agrobacterium ( Agrobacterium tumefaciens In GV3101 competent cells, after sequentially incubating on ice for 5 min, in liquid nitrogen for 5 min, in a 37°C water bath for 5 min, and on ice for 5 min, antibiotic-free LB liquid culture medium was added and activated in a shaker at 28°C for 1 h to obtain an Agrobacterium strain containing a binary plasmid vector. The prepared GV3101 strain containing the binary plasmid vector was then used to transform Arabidopsis thaliana, with the following specific steps: The GV3101 strain containing the binary plasmid vector was cultured overnight at 28°C with shaking in LB medium containing 50 mg / L kanamycin (Kan) and 50 mg / L rifampin (Rif). When the absorbance reached 1.0 at OD600, the cells were collected by centrifugation at 4000 rpm for 15 min and resuspended in 1 / 2 MS medium containing 50 g / L sucrose. Cells that had bolted and partially completed flowering were selected. T-4m Using mutant Arabidopsis thaliana as transgenic material, mature pods were removed, while flowers and buds were retained. A vacuum transformation method was employed, in which the above-prepared bacterial solution was immersed in the mutant. After vacuum extraction for 5 minutes, the mixture was cultured in the dark at 23°C for 24 hours. Resistant seedlings were obtained after one week of selection on 1 / 2 MS medium containing 50 mg / L hygromycin. These seedlings were then transplanted into soil and cultured before harvesting the first-generation (T1) transgenic seeds. The T1 seeds were further selected on 1 / 2 MS medium containing 50 mg / L hygromycin to obtain homozygous T2 transgenic material.

[0050] Example 4 Methods for detecting target gene knockout DNA was extracted from young seedlings of wild-type and knockout transgenic plants, amplified by PCR, and sequenced to identify the target gene mutation. The PCR system and primers used for sequencing are as follows: The PCR amplification primers used were: IPCK 5-PCR-F: 5'- GTGTAATATAAAAGATCGGACAGT -3' (SEQ ID NO: 39); IPCK 5-PCR-R: 5'-TATCCTGTTTGTGCCTTCAATGCAG-3' (SEQ ID NO: 40); IPCK 6-PCR-F: 5'-TGTTCTGAATAATGTAATTGACTA-3' (SEQ ID NO: 41); IPCK 6-PCR-R: 5'-AGAACAACACCAAAGCTGTAGACA-3' (SEQ ID NO: 42).

[0051] The PCR amplification reaction procedure is as follows: The amplification system was referenced from the Vazyme high-fidelity enzyme instructions (2× Phanta Max Master Mix). The PCR amplification reaction program was as follows: pre-denaturation: 94℃, 2 min; denaturation: 98℃, 10 s, annealing: 58℃, 30 s, extension: 72℃, 1 min (34 cycles); final extension: 72℃, 7 min.

[0052] Sequencing revealed that, compared to wild-type lines, CRISPR knockout transgenic plants showed [significant differences]. IPCK5 The CDS region of the gene inserts a C / G base between 142-143 bp, such as... Figure 7 As shown, but IPCK6 Unable to obtain homozygous mutation sites, further observation revealed that homozygous hexamutase mutants were lethal to embryos; therefore, only pentamutase homozygous mutants were obtained, named [name missing]. C-5m ,illustrate IPCK Factors 1-6 not only affect phytic acid synthesis, but are also very important for the normal development of plants.

[0053] Example 5 IPCKs Genes are indeed involved in the phytate synthesis pathway in plant seeds, as shown in the following comparative experiment: Detection of phytic acid content in seeds: Weigh 0.1g of dried seeds, grind them in a mortar with liquid nitrogen, and then quickly add 5mL of pre-cooled 1M perchloric acid solution. Collect the solution in a 50mL centrifuge tube, place it horizontally in an ice box, and incubate it on a shaker for 15min. Then centrifuge at 12000g and 4℃ for 10min. Transfer the supernatant to a new 50mL centrifuge tube, filter it through a 0.45µm filter into a 10mL centrifuge tube containing 30mg TiO2Beads, mix well, and continue to incubate on an ice shaker for 20-30min. Centrifuge at 5000g and 4℃ for 10min, discard the supernatant, wash the Beads 3-5 times with pre-cooled 1M perchloric acid, and elute the Beads 5 times with 500μL of 10% ammonia solution. Collect the eluent and freeze dry it into powder. Finally, add 50μL of 10% ammonia solution to dissolve the freeze-dried powder to obtain the final enriched IP6 for later use.

[0054] Prepare a 33.3% concentration SDS-polyacrylamide gel (5.285 mL 40% methylhydrazine, 635 μL 10× TBE (108 g Tris, 55 g boric acid, 7.44 g EDTA-Na2, dissolved by sonication), 367 μL ddH2O, 45 μL 10% APS, 5 μL TEMED), add 1× TBE electrophoresis buffer, and bury the electrophoresis tank in ice to maintain a low temperature. Run the gel at 300 V for 20 min. Take an appropriate amount of IP6 collection solution, add 5 μL of 6× Dye solution (10 mM Tris-HCl (pH 7.0); 1 mM EDTA-Na2, 30% glycerol, 0.1% Orange G, diluted to 50 mL by sonication) and 10 μL of 5× Buffer (150 mM HEPES (pH 6.8), 250 mM NaCl, 30 mM MgSO4, 5 mM NaF, 5 mM DTT) and mix well. At the same time, take 10 µM of synthesized IP6 (Sichem) as a reference for the IP6 position. After loading the sample, run at 100 V at low temperature for 5-8 h until the orange indicator band is 2 / 3 of the way from the top. The entire gel was then removed and immersed in Toluidine Blue staining solution (20% methanol, 2% glycerol, 0.05% Toluidine Blue, ddH2O to a final volume of 10 mL) for 5 minutes. The gel was then washed 2-3 times with washing solution (20% methanol, 2% glycerol, ddH2O to a final volume of 10 mL). Finally, the gel was observed and photographed using a white light transmission spectrometer, and the brightness of the bands was analyzed and compared using ImageJ software.

[0055] The results showed that the T-DNA insertion tetramutant mutant... T-4m The phytic acid content in the seeds was significantly lower than that of the wild type, and the five-mutant mutant obtained by CRISPR knockout was... C-5m The phytic acid content in the seeds is relatively high T-4m Further decline. See details. Figure 8 (Where C-5m-1 and C-5m-2 represent two biological repeats), indicating that the gene IPCKs It does indeed participate in the regulation of phytic acid content in plant seeds.

[0056] Although the above embodiments have provided a detailed description of the present invention, they are only some embodiments of the present invention, and not all embodiments. People can obtain other embodiments based on these embodiments without creative effort, and these embodiments all fall within the protection scope of the present invention.

Claims

1. Knockout IPCKs The application of genes in reducing phytic acid content in Arabidopsis seeds, the aforementioned IPCKs Genes include two combinations: 1) and / or 2): 1) IPCK1, IPCK2, IPCK3 and IPCK4 ; 2) IPCK1, IPCK2, IPCK3 and IPCK4 and IPCK5 ; The IPCK1, IPCK2, IPCK3, IPCK4 and IPCK5 The nucleotide sequences are shown in SEQ ID NO:1 to SEQ ID NO:5, respectively.

2. Knockout of Arabidopsis thaliana IPCKs Application of gene-based biomaterials in reducing phytic acid content in Arabidopsis seeds; The IPCKs Genes include two combinations: 1) and / or 2): 1) IPCK1, IPCK2, IPCK3 and IPCK4 ; 2) IPCK1, IPCK2, IPCK3 sum IPCK4 sum IPCK5 ; The IPCK1, IPCK2, IPCK3, IPCK4 and IPCK5 The nucleotide sequences are shown in SEQ ID NO:1 to SEQ ID NO:5, respectively; The biomaterial is at least one of the following A1 to A10: A1: Used to eliminate the application described in claim 1 IPCKs Nucleic acid molecules of genes; A2: A recombinant expression vector containing the nucleic acid molecule described in A1; A3: Recombinant microorganisms containing the nucleic acid molecules described in A1; A4: Recombinant microorganisms containing the recombinant expression vector described in A2; A5: Transgenic plant cell lines containing the nucleic acid molecules described in A1; A6: Transgenic plant cell lines containing the recombinant expression vector described in A2; A7: Transgenic plant tissue containing the nucleic acid molecules described in A1; A8: Transgenic plant tissue containing the recombinant expression vector described in A2; A9: Transgenic plant organs containing the nucleic acid molecules described in A1; A10: Transgenic plant organs containing the recombinant expression vector described in A2.

3. The application according to claim 2, characterized in that, The nucleic acid molecules in A1 include those used for knockout. IPCK5 The nucleic acid molecules and T-DNA sequences, used for knockout IPCK5 The nucleotide sequence of the nucleic acid molecule is shown in SEQ ID NO:

7.

4. The application according to claim 3, characterized in that, Used for knocking IPCK5 The upstream and downstream primers of the primers are shown in SEQ ID NO:9 and SEQ ID NO:10, respectively.

5. A method for cultivating low-phytate Arabidopsis thaliana, characterized in that, include: Knockout target Arabidopsis thaliana IPCKs Genes were used to obtain the low-phytate Arabidopsis thaliana; The IPCKs Genes include two combinations: 1) and / or 2): 1) IPCK1, IPCK2, IPCK3 and IPCK4 ; 2) IPCK1, IPCK2, IPCK3 sum IPCK4 sum IPCK5 ; The IPCK1, IPCK2, IPCK3, IPCK4 and IPCK5 The nucleotide sequences are shown in SEQ ID NO:1 to SEQ ID NO:5, respectively.

6. The method according to claim 5, characterized in that, The target of the knockout was Arabidopsis thaliana. IPCKs The gene-based method involves introducing the gene knockout gene into the target Arabidopsis thaliana. IPCKs Nucleic acid molecules of genes.

7. A low-phytate Arabidopsis thaliana, characterized in that, In the genome IPCKs The gene was knocked out; IPCKs Genes include two combinations: 1) and / or 2): 1) IPCK1, IPCK2, IPCK3 and IPCK4 ; 2) IPCK1, IPCK2, IPCK3 sum IPCK4 sum IPCK5 ; The IPCK1, IPCK2, IPCK3, IPCK4 and IPCK5 The nucleotide sequences are shown in SEQ ID NO:1 to SEQ ID NO:5, respectively.

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