HPPD gene variant and application thereof in enhancing tolerance of plants to HPPD inhibitor herbicides

Abstract

The invention discloses one or more sgRNAs, an expression cassette containing the sgRNAs, a recombinant vector, a recombinant cell or a recombinant strain, an HPPD mutant promoter and an HPPD mutant gene of the HPPD mutant promoter. The invention also discloses an application of the product in improving the tolerance of plants to HPPD inhibitor herbicides. According to the invention, a gene editing technology is utilized for the first time, a plurality of single-chain guide RNAs with gene specificity are designed, a rice HPPD gene expression regulation region is deleted, and the expression level of the HPPD gene in the obtained rice mutant is improved, so that the tolerance of the obtained mutant to HPPD inhibitor herbicides is enhanced. When the constructed gene editing vector containing the four editing sites is used for transforming a plant, the tolerance of the transgenic plant to the HPPD inhibitor herbicide is improved, and the application value of the HPPD gene variant in improving the tolerance of the plant to the HPPD inhibitor herbicide is proved.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology and relates to HPPD gene variants and their application in enhancing plant tolerance to HPPD inhibitor herbicides. Specifically, it relates to the application of enhancing plant tolerance to HPPD inhibitor herbicides by editing the promoter of the HPPD gene in plants. Background Technology

[0002] Rice (Oryza sativa L.) is one of the most important food crops for mankind. About half of the world's population relies on rice as their staple food. In particular, rice occupies an extremely important position in my country's grain production. Improving the yield and quality of rice is an important issue related to the national economy and people's livelihood.

[0003] With the increasing scale and mechanization of agricultural production, weed infestation has become one of the most significant factors contributing to crop yield reduction. Paddy field weed infestation is a major threat to rice production, severely impacting yield reductions by more than 30% and affecting rice uniformity and quality. Reports indicate that annual yield reductions due to paddy field weeds average over 20%.

[0004] Traditional manual weeding and cultivation methods such as tilling are labor-intensive and sometimes ineffective. Applying chemical herbicides offers better results and higher efficiency. Especially with the advent of highly effective, low-toxicity, and broad-spectrum herbicides, herbicide application has become the most effective means of weed control.

[0005] HPPD inhibitor herbicides were initially used as selective herbicides in cornfields to control various broadleaf weeds and some annual grass weeds. HPPD inhibitor herbicides are fast-acting, have a short residual period, and a broad spectrum of weed control, making them a promising candidate for weed control in rice paddies. HPPD inhibitor herbicides act on 4-hydroxyphenylpyruvate dioxygenase (HPPD) in plants, competitively inhibiting its activity. HPPD catalyzes the conversion of 4-hydroxyphenylpyruvate (HPPA) to homogentisate (HGA), a key enzyme in the tyrosine to plastisol and tocopherol synthesis pathway. Plastisol is the electron acceptor in the phytoene desaturase reaction, a key enzyme in the carotenoid synthesis pathway, and also an electron acceptor in photosynthetic system II. Plastidone and tocopherol are both important antioxidants that scavenge reactive oxygen free radicals in plant tissues. Therefore, the application of mesotrione to inhibit HPPD activity leads to a deficiency of plastidone and tocopherol, affecting carotenoid synthesis, impairing plant photosynthesis, and ultimately causing plant bleaching and death.

[0006] However, most wild-type rice varieties are sensitive to HPPD inhibitor herbicides, and their use can easily cause phytotoxicity. Therefore, in order to control weeds in paddy fields using HPPD inhibitor herbicides, it is necessary to create rice germplasm resistant to these herbicides. There are three main strategies for creating herbicide-resistant crops: The first is to overexpress the target gene of the herbicide in the plant, enabling the crop to tolerate a certain level of herbicide. For example, overexpressing the glyphosate target gene EPSPS in the plant allows the crop to tolerate more than twice the amount of glyphosate. The second is to modify one or more amino acids of the herbicide target protein, so that the herbicide cannot bind to the target protein, while the normal physiological function of the target protein is not affected, thus giving the plant resistance to the corresponding herbicide. This resistance is usually called target resistance. For example, multiple point mutations in ALS and ACCase can resist the corresponding herbicides. The third is non-target resistance. Generally, this involves introducing the gene encoding the enzyme that can degrade, modify, or inactivate the herbicide absorbed into the plant into the plant, thus inactivating the herbicide and preventing it from binding to the plant's target gene.

[0007] Regarding HPPD target resistance, some studies have utilized gene editing-mediated inversion to drive the rice HPPD gene with the rice UBQ gene, thereby enhancing the resistance of transgenic rice to HPPD inhibitor herbicides; or by editing the HPPD gene to alter the coding region or 3' untranslated region (3'UTR) of the HPPD gene to confer resistance to HPPD inhibitor herbicides. However, no reports have yet demonstrated that altering the promoter region or 5'UTR of the HPPD gene can increase HPPD gene expression levels and thus enhance rice resistance to HPPD inhibitor herbicides.

[0008] Gene editing technology, which has been developed in recent years, can be used to efficiently perform directed evolution on the coding or transcriptional regulatory regions of target genes, in order to screen for mutants with changes in the expression level or function of target genes. This is a powerful means to accelerate the creation of new plant germplasm. Summary of the Invention

[0009] Objective of the Invention: The technical problem to be solved by the present invention is to conduct directed evolution on the sequence upstream of the translation start site of the rice HPPD gene. It was found that deleting the specific DNA segment upstream of the translation start site of the rice HPPD gene can significantly increase the expression level of the rice HPPD gene and enhance the tolerance of rice to HPPD inhibitor herbicides. This provides technical ideas and gene resources for breeding rice varieties resistant to HPPD inhibitor herbicides and controlling weeds in paddy fields.

[0010] The technical problem that this invention also aims to solve is to provide one or more sgRNAs, expression cassettes, recombinant expression vectors, recombinant cell lines or recombinant bacteria that can perform targeted editing of the HPPD gene to delete the expression regulatory region of the rice HPPD gene, thereby enhancing the herbicide resistance of the plant.

[0011] Another technical problem to be solved by this invention is to provide HPPD mutant promoters and mutant genes obtained through gene editing mediated by Cas9 and sgRNA.

[0012] The technical problem to be solved by this invention is to provide the application of sgRNA, the expression cassette, the recombinant vector, the recombinant cell or recombinant strain, the HPPD mutant promoter, and the HPPD mutant gene in regulating plant tolerance to HPPD inhibitor herbicides.

[0013] The final technical problem to be solved by this invention is to provide a method for obtaining plants with improved tolerance to HPPD inhibitor herbicides.

[0014] Technical solution: In order to solve the above technical problems, the present invention provides one or more sgRNAs, wherein the sgRNA sequence is selected from one or more of the sequences such as SEQ ID NO.1 to 43.

[0015] The present invention also includes expression cassettes, recombinant vectors, recombinant cells or recombinant strains, which contain the sgRNA described above.

[0016] The present invention also includes an HPPD mutant promoter, the sequence of which is shown in SEQ ID NO.45.

[0017] This invention also includes an HPPD mutant gene, which is based on a mutation in the expression regulatory region of the sgRNA in rice. Specifically, it corresponds to a base deletion or mutation at positions 3680143–3680147, and / or positions 3679979–3680080, and / or positions 3679912–3679972 of the rice GenBank accession number AP014958.1 sequence. Preferably, the HPPD mutant gene specifically corresponds to position 36799 of the rice GenBank accession number AP014958.1 sequence. Base deletions or mutations occur at positions 98–3680011, and / or positions 3679999–3680016, and / or positions 3679995–3680013, and / or positions 3680049–3680058, and / or positions 3679911–3679919, and / or positions 3679914–3679922, and / or positions 3679939–3679948, and / or positions 3679945–3679954, and / or positions 3679946–3679955, and / or positions 3679947–3679956.

[0018] Preferably, the missing region is a segment of the GenBank:AP014958.1 sequence, specifically bits 3680143–3680147, 3679979–3680080, and 3679912–3679972, with the missing region sequences shown in TCTAA, SEQ ID NO.46, and SEQ ID NO.47, respectively.

[0019] The present invention also includes the sgRNA, the expression cassette, the recombinant vector, the recombinant cell or recombinant strain, the HPPD mutant promoter, and the application of the HPPD mutant gene in regulating plant tolerance to HPPD inhibitor herbicides.

[0020] The application includes selecting one or more editing target sites upstream of the translation start site ATG of the rice HPPD gene, determining each pair of oligo sequences corresponding to each editing target site, introducing restriction enzyme sites at the 5' end of each pair of oligo sequences, designing primer pairs, and constructing one or more gene editing target sites on the same knockout vector.

[0021] The application involves selecting a first, second, third, and fourth editing target site upstream of the translation initiation site ATG of the rice HPPD gene, determining the first pair of oligo sequences corresponding to the first editing target site, the second pair of oligo sequences corresponding to the second editing target site, the third pair of oligo sequences corresponding to the third editing target site, and the fourth pair of oligo sequences corresponding to the fourth editing target site, respectively, introducing restriction enzyme sites at the 5' end of each pair of oligo sequences, designing four pairs of primer sequences, and constructing the gene editing at the four target sites on the same knockout vector.

[0022] Specifically, the first edit site is positions 3679902 to 3679921 of the rice GenBank:AP014958.1 sequence, the second edit site is positions 3679958 to 3679977 of the rice GenBank:AP014958.1 sequence, the third edit site is positions 3680048 to 3680067 of the rice GenBank:AP014958.1 sequence, and the fourth edit site is positions 3680127 to 3680146 of the rice GenBank:AP014958.1 sequence.

[0023] Wherein, the HPPD gene promoter in rice undergoes a mutation, and the resulting deletion, mutation site, or binding element is partially or entirely located within the sequence segment. Specifically, the HPPD gene promoter corresponds to positions 3680143–3680147, and / or positions 3679979–3680080, and / or positions 3679912–3679972 of the rice GenBank accession number AP014958.1 sequence, where a base deletion or mutation occurs. Preferably, the HPPD mutant gene specifically corresponds to position 3680143–3680147 of the rice GenBank accession number AP014958.1 sequence. Base deletions or mutations occur at positions 79998–3680011, and / or positions 3679999–3680016, and / or positions 3679995–3680013, and / or positions 3680049–3680058, and / or positions 3679911–3679919, and / or positions 3679914–3679922, and / or positions 3679939–3679948, and / or positions 3679945–3679954, and / or positions 3679946–3679955, and / or positions 3679947–3679956;

[0024] Specifically, positions 3679947–3679956 are AP2 transcription factor family binding sites, positions 3679946–3679955 are AP2 transcription factor family binding sites, positions 3679945–3679954 are AP2 transcription factor family binding sites, positions 3679939–3679948 are Homeodomain transcription factor family binding sites, positions 3679914–3679922 are calmodulin transcription factor binding sites, and positions 3679911–3679919 are calmodulin transcription factor binding sites.

[0025] Furthermore, positions 3680049–3680058 are binding sites for the TCP transcription factor family, positions 3679999–3680016 are binding sites for the bHLH transcription factor family, positions 3679995–3680013 are binding sites for the bZIP transcription factor family, and positions 3679998–3680011 are binding sites for the bHLH transcription factor family.

[0026] Preferably, the missing region is a segment of the GenBank:AP014958.1 sequence, specifically bits 3680143–3680147, 3679979–3680080, and 3679912–3679972, with the missing region sequences shown in TCTAA, SEQ ID NO.46, and SEQ ID NO.47, respectively.

[0027] This invention also includes a method for obtaining plants with increased tolerance to HPPD inhibitor herbicides. The method involves altering some or all bases at positions 3680143–3680147, and / or positions 3679979–3680080, and / or positions 3679912–3679972 of the HPPD gene sequence corresponding to GenBank:AP014958.1 through deletion, mutation, or other means, thereby increasing HPPD gene expression and thus improving plant tolerance to HPPD inhibitors. The method includes gene editing, EMS mutagenesis, radiation mutagenesis, etc. Preferably, the deletion, mutation, or other means alters the HPPD gene sequence specifically corresponding to rice Ge... A base deletion or mutation occurs at positions 3679998–3680011, and / or positions 3679999–3680016, and / or positions 3679995–3680013, and / or positions 3680049–3680058, and / or positions 3679911–3679919, and / or positions 3679914–3679922, and / or positions 3679939–3679948, and / or positions 3679945–3679954, and / or positions 3679946–3679955, and / or positions 3679947–3679956 of the sequence with nBank accession number AP014958.1.

[0028] Preferably, the missing region is a segment of the GenBank:AP014958.1 sequence, specifically bits 3680143–3680147, 3679979–3680080, and 3679912–3679972, with the missing region sequences shown in TCTAA, SEQ ID NO.46, and SEQ ID NO.47, respectively.

[0029] This invention discloses a method for increasing the expression level of the rice HPPD gene through gene editing and the application of the mutant obtained by this method in creating rice resistant to HPPD inhibitor herbicides. Specifically, the method includes the following steps: deleting segments from the rice HPPD gene expression regulatory region, corresponding to positions 3680143–3680147, and / or positions 3679979–3680080, and / or positions 3679912–3679972 of the GenBank:AP014958.1 sequence, thereby increasing the expression level of the rice HPPD gene and thus enhancing the tolerance of the resulting rice mutant to HPPD inhibitor herbicides.

[0030] Furthermore, the method provided by this invention, which involves deleting segments from positions 3680143 to 3680147, and / or positions 3679979 to 3680080, and / or positions 3679912 to 3679972 of the rice HPPD gene expression regulatory region corresponding to the GenBank:AP014958.1 sequence, thereby increasing the expression level of the rice HPPD gene and enhancing its tolerance to HPPD inhibitor herbicides, is achieved by constructing a binary vector CRISPR-HPPDP-1 that targets four sites upstream of the rice HPPD gene translation initiation site and forms double-stranded DNA breaks.

[0031] Specifically, the binary vector CRISPR-HPPDP-1 includes three expression cassettes; the first expression cassette, from upstream to downstream, includes: the rice ribonucleoprotein U3 promoter, the first sgRNA targeting the rice HPPD gene, and the termination sequence of the U3 promoter; the U3 promoter, the second sgRNA targeting the rice HPPD gene, and the termination sequence of the U3 promoter; the rice ribonucleoprotein U6b promoter, the third sgRNA targeting the rice HPPD gene, and the U6b promoter. The termination sequence includes the U6b promoter, the fourth sgRNA targeting the rice HPPD gene, and the termination sequence of the U6b promoter; the second expression cassette, from upstream to downstream, includes the maize Ubiquitin1 gene promoter, the Cas9 protein coding sequence optimized according to rice codon preference, and the Nos terminator; the third expression cassette, from upstream to downstream, includes the cauliflower mosaic virus 35S promoter, the hygromycin resistance gene coding sequence, and the cauliflower mosaic virus 35S terminator.

[0032] Furthermore, the sgRNA sequence corresponding to the first target of the rice HPPD gene is shown in sequence SEQ ID NO.1, the sgRNA sequence corresponding to the second target of the rice HPPD gene is shown in sequence SEQ ID NO.2, the sgRNA sequence corresponding to the third target of the rice HPPD gene is shown in sequence SEQ ID NO.3, and the sgRNA sequence corresponding to the fourth target of the rice HPPD gene is shown in sequence SEQ ID NO.4.

[0033] This invention relates to the application of the binary vector CRISPR-HPPDP-1 in enhancing the expression level of the HPPD gene in rice and creating herbicides resistant to HPPD inhibition in rice.

[0034] The present invention also provides a method for identifying the resistance of rice to HPPD inhibitor herbicides obtained by the method, and for identifying whether the rice possesses the mutant promoter or mutant gene.

[0035] The present invention also provides primers for identifying the rice HPPD gene mutants described above.

[0036] Beneficial Effects: Compared with existing technologies, this invention has the following advantages: The method disclosed in this invention can rapidly obtain rice lines with improved resistance to HPPD inhibitor herbicides, and is expected to improve the efficiency of breeding HPPD inhibitor-resistant rice varieties. This invention also constructs gene-editing vectors with four editing sites, transforms plants, and the transgenic plants show improved tolerance to HPPD herbicides, demonstrating the application value of HPPD gene variants in improving plant tolerance to HPPD herbicides. Attached Figure Description

[0037] Figure 1 This is a structural diagram of the rice HPPD gene; the boxes with arrows represent the two exons of the rice HPPD gene, the short black lines between the exons represent introns, and the four vertical lines upstream of the first exon indicate the locations of the gene editing target sites 1 to 4 selected in this invention.

[0038] Figure 2 This is a T-DNA map of the CRISPR-HPPDP-1 plasmid, the final plant expression vector containing four different sgRNA expression cassettes, a Cas9 gene expression cassette, and a hygromycin resistance gene expression cassette; ZmpUBI: maize Ubiquitin1 gene promoter; SpCas9: Cas9 gene derived from Streptococcus pyogenes, codon-optimized for rice; NOST: NOS terminator; 3: rice U3 promoter; 6: rice U6b promoter; SgRNA: single guide RNA; HPPDP-S1: spacer corresponding to the first target site of the rice HPPD gene selected in this invention; HPPDP-S2: spacer corresponding to the second target site of the rice HPPD gene selected in this invention; HPPDP-S3: spacer corresponding to the third target site of the rice HPPD gene selected in this invention; HPPDP-S4: spacer corresponding to the fourth target site of the rice HPPD gene selected in this invention; 35S Promoter: 35S promoter of cauliflower mosaic virus; HygR: hygromycin resistance gene; 35ST: 35S terminator; LB: left boundary of T-DNA; RB: right boundary of T-DNA;

[0039] Figure 3A: Phenotypic results of wild-type and gene-edited T1 generation mutant lines of vectors 1 to 11 treated with 0.3 μmol / L nicosulfuron; Figure 3 B: Phenotypic results of wild-type and gene-edited T1 generation mutant lines of vectors 12–14 treated with 0.3 μmol / L nicosulfuron; Figure 3 C: Phenotypes of wild-type and T2 generation resistant mutant lines treated with 0.3 μmol / L mesotrione. Figure 3 A and Figure 3 In section B, WT represents the wild type, and 1–14 represent the gene-edited plants corresponding to vectors 1–14. The scale bar in the diagram is 5 cm.

[0040] Figure 4 The figure shows the alignment results of the PCR product of the upstream region of the translation start site of the wild-type HPPD gene and the HPPD gene of the nicosulfuron-resistant mutant in Example 4. The HPPD gene sequence of wild-type rice is the sequence 840 bp upstream of the translation start site (see SEQ ID NO.44), corresponding to positions 3679888 to 3680727 of the GenBank:AP014958.1 sequence. The short black line indicates the sequence corresponding to the 168 bp deletion in the mutant compared to the wild-type rice. The mutant sequence is shown in SEQ ID NO.45.

[0041] Figure 5 The expression levels of the rice HPPD gene in representative nicosulfuron-resistant mutants and wild-type plants are shown. WT: wild-type, Mutant: mutant. The rice Ubiquitin1 gene was used as an internal reference gene. The expression level of the HPPD gene relative to the Ubiquitin1 gene in wild-type rice was set to 1. The error bar represents the standard deviation of three replicates. Detailed Implementation

[0042] The following examples are provided to better understand the present invention, but are not intended to limit the invention. Unless otherwise specified, the experimental methods used in the following examples are conventional methods. Unless otherwise specified, the experimental materials used in the following examples were purchased from conventional biochemical reagent stores.

[0043] Restriction endonucleases Bsa I (Catalog No.: R3733V) and T4 ligase (Catalog No.: M0202V) used for vector construction were purchased from New England Biolabs; KOD-Plus-Neo DNA polymerase (Catalog No.: KOD-401) was purchased from Toyobo (Shanghai) Biotechnology Co., Ltd.; kanamycin (Catalog No.: K8020-10G) and rifampin (Catalog No.: R8010) were purchased from Beijing Solarbio Science & Technology Co., Ltd.; plasmid mini-extraction kit, DNA purification kit, and RNA extraction kit were purchased from Beijing Kangwei Century Biotechnology Co., Ltd.; sucrose (Catalog No.: 10021463) and glucose (Catalog No.: 63005518) were purchased from Sinopharm Chemical Reagent Co., Ltd.; N6 medium (containing vitamins) (Catalog No.: C167- 50L), inositol (Catalog No.: I703-100G), hydrolyzed casein (Catalog No.: C184-100G), proline (Catalog No.: P698-100G), hygromycin (Catalog No.: H397-5G), MS medium (containing vitamins) (Catalog No.: M519-50L), gellan gum (Catalog No.: G434-100G), carbenicillin (Catalog No.: C346-25G), potassium naphthaleneacetate (NAA) (Catalog No.: N610-25G), and kinetin (Catalog No.: K750-5G) were purchased from Phyto Technology Laboratories; 2,4-D (Catalog No.: D7299-100G) was purchased from Sigma-Aldrich (Shanghai) Trading Co., Ltd.; and acetylsuccione (Catalog No.: B21498-100mg) was purchased from Shanghai Yuanye Biotechnology Co., Ltd. The *Escherichia coli* DH5α competent cells (catalog number: C502-02) used for transformation in this invention were purchased from Nanjing Novizan Biotechnology Co., Ltd.; *Agrobacterium tumefaciens* EHA105 competent cells (catalog number: AC1010S) were purchased from Shanghai Weidi Biotechnology Co., Ltd.; DNA Marker DL5000 (TSJ012-500) was purchased from Beijing Qingke Xinyue Biotechnology Co., Ltd.; 2×Taq enzyme premix (catalog number: P222-03), real-time quantitative PCR premix (ChamQSYBR qPCR Master Mix) (catalog number: Q311-02), Phanta high-fidelity DNA polymerase (P505-d1), and homologous recombination kit (catalog number: C112-01, including Exnase) were used. II recombinase was purchased from Nanjing Novizan Biotechnology Co., Ltd.; Hoagland nutrient solution (product number: NSP1020) for soilless rice culture was purchased from Beijing Cooler Technology Co., Ltd.The primers used in this invention were synthesized by Nanjing GenScript Biotech Co., Ltd.; the Sanger sequencing service was provided by Beijing Qingke Xinyue Biotechnology Co., Ltd. The plant expression vector plasmid pMH-Cas9 (17430bp) used for gene editing was provided by Professor Liu Yaoguang's research group at South China Agricultural University. Cas9 mimics the characteristic of high 5' GC content in grass genes and is a designed and synthesized plant optimized codon gene, which is available to the public from South China Agricultural University. The plasmid vectors pYLgRNA-OsU3 and pYLgRNA-OsU6b used to provide the rice polymerase III promoter U3 or U6b were provided by Professor Liu Yaoguang's research group at South China Agricultural University and are available to the public from South China Agricultural University.

[0044] Example 1: Construction of Gene Editing Vector

[0045] Based on the sequence 2498 bp upstream of the translation start site of the rice HPPD gene (corresponding to positions 3679888–3682385 in GenBank: AP014958.1), and combined with the NGG position of the PAM sequence (protospacer adjacent motif) recognized by Cas9, a gene-editing target site specific to the HPPD gene was designed every 100–200 bp within this region. The target site sequences are shown in Table 1. The spacer corresponding to each target site was synthesized by Nanjing GenScript Biotech Co., Ltd. in the form of primers. The 5' ends of the forward and reverse primers contain adapter sequences (Table 2) to construct the sgRNA corresponding to each target site. Specifically, the sequence of target 1 corresponds to bits 3679902 to 3679921 of the GenBank:AP014958.1 sequence, the sequence of target 2 corresponds to bits 3679958 to 3679977 of the GenBank:AP014958.1 sequence, the sequence of target 3 corresponds to bits 3680048 to 3680067 of the GenBank:AP014958.1 sequence, and the sequence of target 4 corresponds to bits 3680127 to 3680146 of the GenBank:AP014958.1 sequence. Using the CRISPR / Cas9 gene editing vector, and referring to the method reported by Ma et al., four sgRNAs were co-constructed on the same gene editing vector using the Golden Gate method (Ma et al., A Robust CRISPR / Cas9 System for Convenient, High-Efficiency Multiplex Genome Editing in Monocot and Dicot Plants. Mol Plant. 2015 8(8):1274-1284.). A total of 14 multi-target gene editing vectors were constructed targeting 43 target sites. The vector combinations are shown in Table 1.

[0046] Table 1. Target site sequences of the HPPD gene transcriptional regulatory region selected in this invention and the vectors to which the target sites belong.

[0047]

[0048]

[0049] Table 2. Forward and reverse primer sequences for the target site Spacer selected in this invention.

[0050]

[0051]

[0052] Taking the first target site's sgRNA (sgRNA 01) as an example, equal volumes of the two Spacer primers corresponding to the target site were mixed: 5 μL of 10 μM Spacer 01 forward primer and 5 μL of 10 μM Spacer 01 reverse primer. The primers were then annealed using a PCR instrument to form double strands under the following conditions: 90℃ for 30 s; ramp to 30℃, 0.1℃ / s. The annealed Spacer was immediately placed on ice for later use. A Golden Gateway-compatible strategy was used to construct a plant expression vector for gene editing (Ma et al., 2015). Template sequences for ligating Spacer to the rice DNA polymerase III promoter OsU3 and sgRNA were constructed using a "cut-and-ligate" strategy. The reaction system consisted of: annealed Spacer 01, 0.5 μL; pYLgRNA-OsU3 plasmid (approximately 20 ng / μL), 1 μL; Bsa I (20,000 U / mL), 0.5 μL; 10×CutSmart buffer, 1 μL; T4 ligase (400,000 U / mL), 0.1 μL; 10×T4 ligase buffer, 1 μL; and deionized water, 5.9 μL. After vortexing and brief centrifugation, the mixture was placed in a PCR instrument for reaction under the following conditions: 37℃ for 5 min; 20℃ for 5 min; for 5 cycles. The template for constructing the U3-sgRNA 01 expression cassette was obtained (the construction method and specific conditions for the templates of the U3-sgRNA 02 to U6b-sgRNA 43 expression cassettes are exactly the same as those for the U3-sgRNA 01 expression cassette, while the selection of the template in the reaction system is based on Table 2, using plasmid pYLgRNA-OsU3 or pYLgRNA-OsU6b).

[0053] Subsequently, the first round of amplification reactions for the U3 / U6b-sgRNA expression cassette was performed. Two sets of PCR reactions were performed simultaneously for each sgRNA. The steps of the first round of amplification reaction are described below using U3-sgRNA 01 as an example (the construction methods for U3-sgRNA 02 to U6b-sgRNA 43 are the same as those for U3-sgRNA 01). PCR reaction system in tube 1: 10mM UF primer (5'-ctccgttttacctgtggaatcg-3'), 0.5μL; Spacer 01 reverse primer, 0.5μL; KOD-Plus-Neo buffer, 2.5μL; 2.5mM dNTP, 2.5μL; MgSO4, 1.5μL; U3-sgRNA 01 template, 1μL; KOD-Plus-Neo DNA polymerase (1000U / mL), 0.5μL; deionized water, 16μL. The second PCR reaction tube contained the following components: 10 mM gRNA-R (5'-cggaggaaaattccatccac-3'), 0.5 μL; Spacer 01 forward primer, 0.5 μL; KOD-Plus-Neo buffer, 2.5 μL; 2.5 mM dNTP, 2.5 μL; MgSO4, 1.5 μL; U3-sgRNA 01 template, 1 μL; KOD-Plus-Neo DNA polymerase (1000 U / mL), 0.5 μL; and deionized water, 16 μL. After vortexing and briefly centrifuging, the mixture was placed in a PCR instrument for reaction under the following conditions: 94℃, 2 min; (98℃, 10 s; 60℃, 15 s; 68℃, 20 s) × 28 cycles.

[0054] PCR products were electrophoresed on a 1% agarose gel, and the size of the PCR bands was observed using a UV gel imaging system. The first tube of PCR product was approximately 500 bp, and the second tube of PCR product was approximately 200 bp, which was in line with the expected size, and was ready for the second round of amplification. The second round of amplification reaction for constructing the U3 / U6b-sgRNA expression cassette was then performed. For each sgRNA, equal volumes of the two amplification products obtained in the first round were mixed and used as templates for the second round of amplification. The second-round PCR amplification reaction system for U3-sgRNA 01 consisted of: 0.75 μL of 10 mM primer B1' (5'-ttcagaggtctctctcgactagtggaatcggcagcaaagg-3'); 0.75 μL of 10 mM primer B2 (5'-agcgtgggtctcgtcagggtccatccactccaagctc-3'); 5 μL of KOD-Plus-Neo buffer; 5 μL of 2.5 mM dNTPs; 3 μL of MgSO4; 2 μL of an equal volume of the first-round amplification product corresponding to sgRNA 01; 1 μL of KOD-Plus-Neo DNA polymerase (1000 U / mL); and 32.5 μL of deionized water. The PCR reaction conditions were: 94 °C for 2 min; (98 °C for 10 s; 58 °C for 15 s; 68 °C for 20 s) × 25 cycles. The primers used for the second round of PCR amplification of U3-sgRNA 02 were B2' (5'-ttcagaggtctctctgacactggaatcggcagcaaagg-3') and B3 (5'-agcgtgggtctcgtcttggtccatccactccaagctc-3'), with the template being an equal volume mixture of the first round amplification products corresponding to sgRNA 02; the primers used for the second round of PCR amplification of U6b-sgRNA 03 were B3' (5'-ttcagaggtctctaagacactggaatcggcagcaaagg-3') and B4 (5'-agcgtgggtctcgagtcggtccatccactccaagctc-3'), with the template being an equal volume mixture of the first round amplification products corresponding to sgRNA 03; U6b-sgRNA The primers used for the second round of PCR amplification in Spacer 04 were B4' (5'-ttcagaggtctctgactcactggaatcggcagcaaagg-3') and BL (5'-agcgtgggtctcgaccgacgcgtccatccactccaagctc-3'), and the template was an equal volume mixture of the first round amplification product corresponding to sgRNA 04. Other PCR components and PCR reaction conditions were the same as those in Spacer 01.The second-round PCR products from the two Spacers were electrophoresed using a 1% agarose gel. The size of the PCR product bands was observed using a UV gel imaging system. A strip containing approximately 500 bp of PCR product was cut off and purified using a kit.

[0055] Finally, the final expression vector for gene editing plants was constructed. First, prepare the following mixture: 1 μL of the second-round PCR amplification product of purified U3-sgRNA01; 1 μL of the second-round PCR amplification product of purified U3-sgRNA02; 1 μL of the second-round PCR amplification product of purified U6b-sgRNA03; 1 μL of the second-round PCR amplification product of purified U6b-sgRNA04; 1.5 μL of Bsa I enzyme buffer; 1 μL of Bsa I (20,000 U / mL); 1 μL of pMH-Cas9 vector plasmid; and 9.5 μL of deionized water. Mix well and place in a PCR instrument, incubating at 37°C for 10 min. Then add 1.5 μL of T4 DNA ligase buffer and 0.1 μL of T4 DNA ligase (400,000 U / mL), mix well, and place in a PCR instrument. The reaction conditions are: (37°C, 2 min; 10°C, 3 min; 20°C, 5 min) × 15 cycles; 37°C, 2 min.

[0056] 5 μL of the PCR ligation product was directly transformed into *E. coli* DH5α competent cells using the heat shock method. The transformed cells were plated on LB agar plates containing 50 μg / mL kanamycin and incubated upside down at 37°C for 12 hours. Seven single colonies were picked using an autoclaved toothpick, and colony PCR was performed using primers Spacer 01 (forward) and Spacer 02 (reverse). Colonies that amplified a PCR product of approximately 500 bp were considered positive colonies. In this invention, positive clone #5 was picked using an inoculating loop sterilized at 121°C and transferred to LB liquid medium containing 50 μg / mL kanamycin. The cells were incubated at 37°C with shaking for 12 hours. Plasmids were extracted using a plasmid mini-extraction kit, and the plasmid concentration was determined to be 255 ng / μL using a Nanodrop microspectrophotometer. Detection primers used: SP-ML: 5'-gcgcggtgtcatctatgttacta-3', SP-R: 5'-cccgacatagatgcaataacttc-3', C121: 5'-acgcctggacttggagagcc-3', C122: 5'-gctcgtccagacctacaacc-3', C123: 5'-acggctctatccctcaccag-3', C124: 5'-cactcttactctcttcgagg-3' C125: 5'-agtctttccttaaggatgac-3', C126: 5'-gagacgaacggtgagactgg-3', and C127: 5'-gcagcacaagcactacctcg-3'. The constructed plasmid vector was sequenced, confirming the Cas9 full-gene sequence and the U3-Spacer-sgRNA element sequence were correct. The plasmid was named CRISPR-HPPDP-1 (i.e., vector 1). The resulting final vector simultaneously contains expression cassettes of sgRNA O1–sgRNA O4 upstream of the HPPD gene translation initiation site, as well as expression cassettes of the Cas9 gene and the hygromycin resistance gene. Figure 2), and used for subsequent rice genetic transformation. When constructing another 13 gene editing vectors, except for the different spacers used for the target sequences (e.g., targets 4, 5, 6, and 7 were used to construct vector 2; targets 7, 8, 9, and 10 were used to construct vector 3; targets 10, 11, 12, and 13 were used to construct vector 4; targets 13, 14, 15, and 16 were used to construct vector 5; targets 16, 17, 18, and 19 were used to construct vector 6; targets 19, 20, 21, and 22 were used to construct vector 7; targets 22, 23, 24, and 25 were used to construct vector 8; targets 25, 26, 27, and 28 were used to construct vector 9; targets 28, 29, and 10 were used to construct vector 2 ...2; targets 28, 29, and 10 were used to construct vector 2; targets 20, 21, and 22 were used to construct vector 2; targets 22, 23, 24, and 25 were used to construct vector 8; targets 25, 26, 27, and 28 were used to construct vector 9; targets 28, 29, and 10 were used to construct vector 2; targets 20, 20, 21, and 22 were used to construct vector 2; targets 22, 23, 24, and 25 were used to construct vector 2; targets 25, 26, 27, and 28 were used to construct vector 2 Vector 10 was constructed using targets 30 and 31; vector 11 was constructed using targets 31, 32, 33, and 34; vector 12 was constructed using targets 34, 35, 36, and 37; vector 13 was constructed using targets 37, 38, 39, and 40; and vector 14 was constructed using targets 40, 41, 42, and 43. The construction method was exactly the same as that used for general-purpose elements. The resulting plasmids were named CRISPR-HPPDP-2 (vector 2) to CRISPR-HPPDP-14 (vector 14). Each final vector simultaneously contains expression cassettes of four different sgRNAs upstream of the translation start site of the target HPPD gene, as well as expression cassettes of the Cas9 gene and the hygromycin resistance gene.

[0057] Take 0.5 μL of plasmids CRISPR-HPPDP-1 to CRISPR-HPPDP-14 and add them to 50 μL of competent cells of Agrobacterium strain EHA105. Place on ice for 5 minutes, freeze in liquid nitrogen for 5 minutes, incubate in water at 37°C for 5 minutes, place on ice for 5 minutes, add 500 μL of antibiotic-free liquid LB medium, and incubate at 28°C for 3 hours. Take 50 μL and spread it on solid LB medium plates containing 50 μg / mL rifampicin and 50 μg / mL kanamycin. Incubate upside down in a 28°C incubator for 48 hours. The Cas9 gene expression cassette-specific detection forward primer ZmUBI-SF: 5'-atgctctaaccttgagtacc-3' and reverse primer C121: 5'-acgcctggacttggagagcc-3' were designed to perform colony PCR on Agrobacterium. Single colonies amplifying the target fragment of approximately 930 bp corresponded to positive strains. These strains were inoculated into a medium containing rifampin and kanamycin and cultured at 28°C with shaking at 220 rpm until OD500. 600 When the concentration is around 0.8, it is used for Agrobacterium-mediated transformation. Example 2: Obtaining and identifying transgenic rice plants with HPPD gene editing.

[0058] 1. Acquisition of callus tissue

[0059] Select plump and healthy seeds of the Nipponbare rice variety and remove the husks; surface sterilize in 70% ethanol for 1 minute, shaking continuously; then sterilize with 3% sodium hypochlorite containing 1 drop of Tween-20 for 30 minutes, shaking on a shaker; wash 5 times with sterile distilled water, about 2 minutes each time, shaking continuously; then use tweezers to remove the washed seeds and place them in a petri dish lined with 5 layers of sterile filter paper, spreading the seeds evenly on the filter paper, and then covering them with 5 more layers of filter paper to allow the seeds to dry; place 15 seeds in callus induction solid medium (N6 salt, 4g; inositol, 0.1g; proline, 2.8g; hydrolyzed casein, 0.3g; sucrose, 30g; 2g / mL). 2,4-D, 1 mL; gellan gum, 4 g; add deionized water to 1 L; autoclave) so that the endosperm is buried in the culture medium and the embryo is exposed on the surface of the culture medium, so that the scutellum is just in contact with the culture medium; place the culture dish in a 28°C incubation room and culture under long-day conditions (16 hours light / 8 hours dark) for about 4 weeks until a large number of firm, light yellow callus masses grow;

[0060] 2. Preparation and co-culture of Agrobacterium

[0061] Agrobacterium, obtained by transforming plasmids CRISPR-HPPDP-1 to CRISPR-HPPDP-14 obtained in Example 1, had three positive colonies collected by sterile toothpicks and inoculated into 20 mL of liquid LB medium containing 50 μg / mL rifampin and 50 μg / mL kanamycin, respectively. The cultures were shaken and cultured for 36 hours. After centrifugation at 5000 rpm for 2 minutes, the cells were resuspended in 1 mL of liquid N6 medium (N6 salt, 4 g; inositol, 0.1 g; hydrolyzed casein, 1 g; glucose, 10 g; sucrose, 30 g; 2 g / mL 2,4-D, 1 mL; deionized water added to 1 L; autoclaved; cooled and then 1 mL of 100 mM acetylsylgenone added). The bacterial concentration was measured using a UV spectrophotometer. The Agrobacterium resuspension was then diluted to OD using liquid N6 medium. 600=0.08; Using tweezers sterilized at 121℃ and cooled, pick up rice callus tissue blocks and immerse them in diluted Agrobacterium solution for 20 minutes; discard the Agrobacterium solution, use sterile tweezers to pick up rice callus blocks, place them in a petri dish lined with 5 layers of sterile filter paper, spread the callus out, and then cover it with another 5 layers of filter paper. Dry the callus for 2 hours; add 500 μL of liquid N6 medium to a co-culture medium plate, place a piece of sterile filter paper with a diameter similar to that of the petri dish on the surface of the medium, ensuring the liquid evenly wets the filter paper, and then place the dried callus on the co-culture medium lined with filter paper (N6 salt, 4g; inositol, 0.1g; hydrolyzed casein, 1g; glucose, 10g; sucrose, 30g; 2g / mL). 2,4-D, 1 mL; plant gel, 4 g; add deionized water to 1 L; autoclave; after cooling, add 100 mM acetylsuccinone, 1 mL; seal the culture dish containing the callus tissue with breathable tape (3M, USA), then wrap the culture medium with aluminum foil, and co-culture at 25°C in the dark for 3 days.

[0062] 3. Screening of positive callus tissue

[0063] Using sterile forceps, wash the co-cultured callus five times with sterile water, then wash it once with sterile distilled water containing 500 μg / mL carbenicillin sodium. Place the callus in a culture dish lined with five layers of sterile filter paper, spread it out, and cover it with another five layers of filter paper to allow it to dry. Transfer the dried callus to recovery culture medium (1L: N6 salt, 4g; inositol, 0.1g; hydrolyzed casein, 1g; glucose, 10g; sucrose, 30g; 2g / mL). 2,4-D, 1 mL; add deionized water to 1 L; autoclave; after cooling, add 250 mg / mL carbenicillin, 1 mL) and place in a 28°C incubator for 3 days under long-day conditions (16 hours light / 8 hours dark); use sterile forceps to transfer the recovered callus to a selection medium containing 50 μg / mL hygromycin (1 L: N6 salt, 4 g; inositol, 0.1 g; hydrolyzed casein, 1 g; glucose, 10 g; sucrose, 30 g; 2 g / mL 2,4-D, 1 mL; add deionized water to 1 L; autoclave; after cooling, add 200 mg / mL carbenicillin sodium, 1 mL; add 50 mg / mL hygromycin B, 1 mL) and incubate under long-day conditions for 2–4 weeks until new, firm, light yellow callus grows.

[0064] 4. Regeneration and culture of transgenic plants

[0065] Using sterile forceps, transfer newly grown hygromycin-resistant callus from the selection medium to regeneration medium (1L volume: MS salt, 4.33g; hydrolyzed casein, 2g; sorbitol, 30g; sucrose, 30g; 2g / mL 2,4-D, 1mL; add deionized water to 1L; autoclave; after cooling, add 200mg / mL carbenicillin sodium, 1mL; add 50mg / mL hygromycin B, 1mL; 1mg / mL NAA, 20μL; 1mg / mL Culture the seedlings on Kinetin (2 mL) under long-day conditions (16 h light / 8 h dark) for 2–4 weeks until green seedlings emerge. Use sterile forceps to remove the green seedlings and place them on solid 1 / 2 MS medium containing 25 μg / mL hygromycin until the seedlings grow to 5–8 cm. Remove the seedlings, wash them, and place them in clean water. In a 28°C incubator, harden the seedlings under long-day conditions for 3–5 days. Transplant the transgenic seedlings into soil and plant them in a 32°C greenhouse under long-day conditions.

[0066] Example 3: Identification of resistance to the herbicide nicosulfuron in transgenic rice lines with gene editing of the upstream sequence of the HPPD gene translation initiation site.

[0067] First, the T1 generation progeny gene-edited lines of all independent T0 generation plants obtained in Example 2 were cultured hydroponically using Hoagland nutrient solution under long-day conditions at 28°C. When the plants reached the 3-4 leaf stage, 0.3 μmol / L of nicosulfuron was added to the culture medium. The wild-type transgenic recipient variety Xiushui 134, under the same growth conditions and herbicide treatment, served as a control. The growth status of the gene-edited plants and the wild-type recipient variety was observed after 7 days. The results showed that the wild-type and all other gene-edited lines (corresponding to vectors 2-14, i.e., CRISPR-HPPDP-2 to CRISPR-HPPDP-14) all exhibited a leaf whitening and death phenotype. However, all 8 test plants of one T1 generation line corresponding to vector 1 containing targets 1-4 did not show whitening, demonstrating tolerance to nicosulfuron. Figure 3 A and 3B show the phenotypes of representative plants; self-pollinated seeds of the T1 generation mutant with nicosulfuron tolerance corresponding to vector 1, namely CRISPR-HPPDP-1, were harvested. Wild-type Xiushui 134 and T2 generation mutants were treated with 0.3 μmol / L nicosulfuron. After 7 days, the growth status of wild-type and mutant plants was observed. The leaves of T2 generation mutant plants remained green and grew normally, while wild-type plants were severely whitish and stunted. Figure 3 C shows the phenotype of a representative plant.

[0068] Example 4: Genotyping and HPPD gene expression level detection of nicosulfuron-tolerant plants

[0069] For all eight T1 progeny lines of the T0 generation corresponding to the T1 generation gene-edited lines exhibiting nicosulfuron tolerance obtained from Example 3, total DNA was extracted from leaves. Genotyping was performed using target site flanking sequence-specific primers HPPDP-SF (5'-ttgagacagctcattctaccgc-3') and HPPDP-SR (5'-tggtccaggtacgtctgtatct-3'). PCR amplification reaction system: 10mM primer HPPDP-SF, 1μL; 10mM primer HPPDP-SR, 1μL; Phanta high-fidelity DNA polymerase buffer, 12.5μL; 2.5mM dNTP, 0.5μL; PCR Enhancer, 2.5μL; DNA, 2μL; Phanta high-fidelity DNA polymerase (1000U / mL), 0.25μL; deionized water, 5.25μL. The PCR reaction conditions were: 95℃, 3 min; (95℃, 15 s; 56℃, 15 s; 72℃, 1 min 30 s) × 35 cycles. The PCR products were sent to Beijing Qingke Xinyue Biotechnology Co., Ltd. for Sanger sequencing.

[0070] Sequencing results showed that the upstream sequences of the HPPD gene translation start site in all eight T1 generation plants exhibiting nicosulfan resistance showed the same homozygous mutation: a 61 bp deletion at positions 25–85 upstream of the ATG position of the wild-type HPPD gene translation start site (ID LOC_Os02g07160 in the Rice Genome Annotation Project, http: / / rice.uga.edu / cgi-bin / gbrowse / rice / ) (corresponding to deletions at positions 3679912–3679972 in GenBank: AP014958.1), a 102 bp deletion at positions 92–193 (corresponding to deletions at positions 3679979–3680080 in GenBank: AP014958.1), and a 5 bp deletion at positions 256–260 (corresponding to deletions at positions 3680143–3680147 in GenBank: AP014958.1). Figure 4 ); while other T1 generation lines that do not have tolerance to nicosulfuron all exhibit the same genotype as the wild type.

[0071] Gene editing of gene promoters or 5'UTR regions can disrupt transcriptional regulatory elements, such as transcriptional enhancement or repression elements. Disruption of repression elements can lead to upregulation of gene transcription levels. To confirm that the upstream transcriptional regulatory sequence of the rice HPPD gene containing the specific sequence deletion obtained in this invention can lead to increased tolerance of gene-edited lines to nicosulfuron, total RNA was extracted from the obtained nicosulfuron-tolerant lines and wild-type varieties in this embodiment. cDNA was obtained through reverse transcription. Rice HPPD gene-specific primers spanning introns were designed: HPPD-TF: 5'-acagggatgaccagggggtgttgc-3' and HPPD-TR: 5'-gcttgcttggcttcaagggatttctca-3'. The rice UBQ gene was used as an internal reference gene (detection primers were UBQ-TF: 5'-gctccgtggcggtatcat-3' and UBQ-TR: 5'-cggcagttgacagccctag-3'). The expression level of the HPPD gene was detected using quantitative PCR. Specifically, a 20 μL reaction system was prepared: 2×ChamQ SYBR qPCR Master Mix, 10 μL; 2.5 μM. HPPD-TF (or UBQ-TF), 1 μL; 2.5 μM HPPD-TR (or UBQ-TR), 1 μL; cDNA, 3 μL; ddH2O to bring the volume to 20 μL; reaction program: 94℃ for 1 min, (94℃ for 20 s, 60℃ for 20 s, 72℃ for 15 s, 82℃ for 5 s reading) × 40 cycles.

[0072] The results showed that the expression level of the HPPD gene in rice lines tolerant to nicosulfuron was significantly higher than that in wild-type varieties, approximately eight times higher. Figure 5 This result indicates that the specific form of sequence deletion upstream of the translation start site of the rice HPPD gene obtained in this invention enhances the rice's tolerance to the HPPD inhibitor herbicide mesotrione by increasing the mRNA expression level of the HPPD gene.

[0073] Example 5: Improving rice tolerance to HPPD inhibitor herbicides by transforming the mutant HPPD gene using a transgenic method.

[0074] The artificially synthesized mutant rice HPPD gene sequence (SEQ ID NO.48) is 5048 bp in length. It includes the sequence upstream of the translation start site ATG (2330 bp), the sequence of two exons and one intron of the HPPD genome (2099 bp), and the sequence downstream of the translation stop codon TAG (619 bp). Specifically, it corresponds to positions 3677170–3682385 of the GenBank:AP014958.1 sequence, and simultaneously deletes the segments corresponding to positions 3680143–3680147, 3679979–3680080, and 3679912–3679972 of the GenBank:AP014958.1 sequence. During synthesis, adapter sequences corresponding to the flanking sites of the Hind III site in the plant expression vector pCambia1300 were added to both ends of the fragment. The 5' adapter sequence was 5'-acctgcaggcatgcaagctt-3', and the 3' adapter sequence was 5'-cgacggccagtgccaagctt-3'. The above mutant rice HPPD gene insert fragment was synthesized by Sangon Biotech (Shanghai) Co., Ltd., and ligated into the HindIII-digested pCambia1300 vector using a homologous recombination kit. The specific reaction system was as follows: Hind III-digested pCambia1300 vector fragment, 200 ng; artificially synthesized mutant rice HPPD gene insert fragment, 200 ng; 5×CE II buffer, 4 μL; Exnase. II. Recombinase, 2 μL; add deionized water to 20 μL, react at 37℃ for 30 min. The ligation product was transformed into Escherichia coli strain DH5α by heat shock. Colony PCR screening was performed using primers pCambia-SF: 5'-ggaaacgacaatctgatccaagc-3' and primers HPPDT-SF: 5'-tgggctgagttacagcgaat-3'. Positive single clones were identified, and plasmids were extracted. The correctly sequenced plasmid was named pC1300-mHPPDP-HPPD-HPPDT-1 and transformed using the Agrobacterium-mediated genetic transformation method described in Example 2. The transgenic rice was transformed into the wild-type rice variety Xiushui 134. Total DNA was extracted from the positive plants obtained from the transgenic rice. PCR amplification was performed using primers 35S-NTF1: 5'-acaatcccactatccttcgcaag-3' and HYG-TR2: 5'-gtacttctacacagccatcggtc-3'. Plants that amplified to obtain a positive band of approximately 1100 bp were transgenic plants containing the mutant rice HPPD gene described in this invention. Their resistance to HPPD inhibitor herbicides was at the same level as that of the mutant obtained by gene editing method described in Example 4 of this invention.

Claims

1. One or more sgRNAs, characterized in that, The sgRNA sequence is selected from one or more of the sequences such as SEQ ID NO.1 to 43.

2. An expression cassette, a recombinant vector, a recombinant cell, or a recombinant bacterial strain, characterized in that: It contains the sgRNA as described in claim 1.

3. An HPPD mutant promoter, characterized in that, Its sequence is shown in SEQ ID NO.

45.

4. An HPPD mutant gene, characterized in that, The HPPD mutant gene is based on a mutation in the expression regulatory region of the wild-type rice HPPD gene sgRNA as described in claim 1. Specifically, it corresponds to a base deletion or mutation at positions 3680143–3680147, and / or positions 3679979–3680080, and / or positions 3679912–3679972 of the rice GenBank accession number AP014958.1 sequence. Preferably, the HPPD mutant gene specifically corresponds to positions 3679998–3679999.1 of the rice GenBank accession number AP014958.1 sequence. Base deletions or mutations occur at positions 3680011, and / or 3679999–3680016, and / or 3679995–3680013, and / or 3680049–3680058, and / or 3679911–3679919, and / or 3679914–3679922, and / or 3679939–3679948, and / or 3679945–3679954, and / or 3679946–3679955, and / or 3679947–3679956; Preferably, the deleted region of the HPPD mutant gene is the fragment at positions 3680143–3680147, 3679979–3680080, and 3679912–3679972 of the GenBank:AP014958.1 sequence, and the sequences of the deleted region are shown in TCTAA, SEQ ID NO.46, and SEQ ID NO.47, respectively.

5. The application of the sgRNA of claim 1, the expression cassette of claim 2, the recombinant vector, the recombinant cell or recombinant strain, the HPPD mutant promoter of claim 3, and the HPPD mutant gene of claim 4 in regulating plant tolerance to HPPD inhibitor herbicides.

6. The application according to claim 5, characterized in that, The application involves selecting one or more editing target sites upstream of the translation start site ATG of the rice HPPD gene, determining each pair of oligo sequences corresponding to each editing target site, introducing adapter sequences at the 5' end of each pair of oligo sequences, designing primer pairs, and constructing one or more gene editing target sites on the same knockout vector.

7. The application according to claim 6, characterized in that, The application involves selecting a first, second, third, and fourth editing target site upstream of the translation initiation site ATG of the rice HPPD gene, determining the first pair of oligo sequences corresponding to the first editing target site, the second pair of oligo sequences corresponding to the second editing target site, the third pair of oligo sequences corresponding to the third editing target site, and the fourth pair of oligo sequences corresponding to the fourth editing target site, respectively, introducing adapter sequences at the 5' end of each pair of oligo sequences, designing four pairs of primer sequences, and constructing the gene editing at the four target sites on the same knockout vector.

8. The application according to claim 7, characterized in that, The first edit site is positions 3679902 to 3679921 of the rice GenBank:AP014958.1 sequence, the second edit site is positions 3679958 to 3679977 of the rice GenBank:AP014958.1 sequence, the third edit site is positions 3680048 to 3680067 of the rice GenBank:AP014958.1 sequence, and the fourth edit site is positions 3680127 to 3680146 of the rice GenBank:AP014958.1 sequence.

9. The application according to claim 7, characterized in that, The HPPD gene promoter in the rice is mutated, and the resulting deletion, mutation site, or binding element is partially or completely located within the sequence segment. Specifically, the HPPD gene promoter corresponds to positions 3680143–3680147, and / or positions 3679979–3680080, and / or positions 3679912–3679972 of the rice GenBank accession number AP014958.1 sequence, where a base deletion or mutation occurs. Preferably, the HPPD mutant gene specifically corresponds to positions 3679998–3680011, and / or positions 3679999–3680016, and / or positions 3679995–3680013 of the rice GenBank accession number AP014958.1 sequence, and / or positions 3679995–3680013, and / or positions 3679998–3680011, and / or positions 3679999–3680016, and / or positions 3679995–3680013, and / or positions 3679998–36800147, and / or positions 3679 ... / or positions 3680049–3680058, and / or positions 3679911–3679919, and / or positions 3679914–3679922, and / or positions 3679939–3679948, and / or positions 3679945–3679954, and / or positions 3679946–3679955, and / or positions 3679947–3679956, where a base deletion or mutation occurs; preferably, the deleted region is a segment corresponding to positions 3680143–3680147, 3679979–3680080, and 3679912–3679972 of the GenBank:AP014958.1 sequence, and the sequences of the deleted region are TCTAA and SEQ, respectively. As shown in ID NO.46 and SEQ ID NO.

47.

10. A method for obtaining plants with improved tolerance to HPPD inhibitor herbicides, characterized in that, The HPPD gene expression level is increased by altering some or all bases at positions 3680143–3680147, and / or positions 3679979–3680080, and / or positions 3679912–3679972 of the GenBank:AP014958.1 sequence through deletion, mutation, or other methods, thereby enhancing plant tolerance to HPPD inhibitor herbicides. The methods include gene editing, EMS mutagenesis, or radiation mutagenesis. Preferably, the deletion, mutation, or other methods specifically alter positions 3679998–3680011, and / or positions 3679999–3680016, and / or positions 3679999 in the rice GenBank accession number AP014958.1 sequence. Bits 5 to 3680013, and / or bits 3680049 to 3680058, and / or bits 3679911 to 3679919, and / or bits 3679914 to 3679922, and / or bits 3679939 to 3679948, and / or bits 3679945 to 3679954, and / or bits 3679946 to 3679955, and / or A base deletion or mutation occurs at positions 3679947 to 3679956; preferably, the deleted region is a segment at positions 3680143 to 3680147, 3679979 to 3680080, and 3679912 to 3679972 of the GenBank:AP014958.1 sequence, and the sequences of the deleted region are shown in TCTAA, SEQ ID NO.46, and SEQ ID NO.47, respectively.

11. A method for identifying rice with increased tolerance to HPPD inhibitor herbicides obtained by the method of claim 10, characterized in that, To determine whether the rice possesses the mutant promoter as described in claim 3 or the mutant gene as described in claim 4.

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