Method for editing osgbssi promoter and intron 1 in rice genome to improve rice quality

Abstract

The application belongs to the field of plant biotechnology, and particularly relates to a method for editing an OsGBSS I promoter and intron 1 in a rice genome to improve rice quality. The technical problem to be solved by the application is to improve the quality of rice. The technical solution to solve the technical problem is to provide a method for improving the amylose content of rice. The method is to direct edit the OsGBSS I promoter and intron 1 based on a gene editing system to reduce the expression of the gene, and then reduce the amylose content to different degrees to obtain rice gene editing materials with excellent rice quality. The method can obtain multiple high-quality rice materials with excellent rice quality. The method steps are simple and easy to operate, and have good prospects in the improvement of rice quality and the research and application of genome function.

Description

Technical Field

[0001] This invention belongs to the field of plant biotechnology, specifically relating to a method for editing the OsGBSSⅠ promoter and intron 1 in the rice genome to improve rice quality. Background Technology

[0002] Rice quality is a comprehensive measure of a series of physicochemical properties during processing, sales, and cooking, generally categorized into four quality types: milling, appearance, nutrition, and cooking taste. The amylose content significantly impacts the taste and quality of rice. Rice varieties with high amylose content produce cooked rice that is less sticky, soft, and glossy. Rice varieties with moderate amylose content produce fluffy, glossy rice that remains moist and soft even after cooling. Rice with an amylose content between 12% and 14% is generally considered to be of excellent quality, but such varieties are very difficult to obtain. Granular starch synthase (GBSS) is a key enzyme in amylose synthesis, maintaining the stable, unbranched state of amylose. The OsGBSSⅠ gene (Wx gene) (LOC_Os06g04200) in rice is located on chromosome 6 of the rice genome, with a CDS length of 1830 bp, encoding a 609-amino acid protein, primarily expressed in the rice endosperm. Studies have found that if Wx expression in rice is blocked, the endosperm contains almost no amylose.

[0003] The emergence of CRISPR / Cas technology has brought new opportunities for Wx gene editing. Currently, novel Wx mutants have been successfully created using CRISPR / Cas technology in crops such as rice, maize, barley, potato, and sweet potato. However, mutations in the coding region of rice result in glutinous rice, which does not meet the standards for high-quality rice. It is difficult to obtain high-quality rice materials with an amylose content between 10% and 14%, especially extremely high-quality rice materials with an amylose content between 12% and 14%. Summary of the Invention

[0004] The technical problem this invention aims to solve is to improve the quality traits of rice. To address this problem, the provided solution is a method for improving the amylose content in rice. This method involves targeted editing of the promoter and intron 1 of the OsGBSSⅠ gene using a CRISPR-Cas12a gene editing system, reducing the gene's expression level and decreasing the amylose content to varying degrees, thereby obtaining high-quality rice gene-edited materials.

[0005] The technical solution of this invention is to provide a method for editing the promoter and intron 1 of the OsGBSSⅠ gene in the rice genome to improve rice quality. This method includes the following steps:

[0006] a. Construct a gene editing expression vector that can express guide RNA targeting the promoter and / or intron 1 of the rice OsGBSSⅠ gene;

[0007] b. Transform rice with the expression vector obtained in step a, and obtain transformed plants using the CRISPR-Cas gene editing system;

[0008] c. Collect seeds from the transformed plants, screen out the gene-edited mutant seeds, and obtain gene-edited rice with improved amylose content.

[0009] The guide RNA mentioned in the above method is at least one of Seq ID No.1, Seq ID No.2, Seq ID No.3, Seq ID No.4, Seq ID No.5, Seq ID No.6, Seq ID No.7, Seq ID No.8, Seq ID No.9, Seq ID No.10, Seq ID No.11, or Seq ID No.12.

[0010] Preferably, the guide RNA described in the above method is at least one of Seq ID No.2, Seq ID No.4, Seq ID No.6, Seq ID No.8, Seq ID No.10, and Seq ID No.12.

[0011] In the above method, the gene editing expression vector capable of expressing the guide RNA is a CRISPR-Cas12a gene editing expression vector or a CRISPR-Cas12b gene editing expression vector. Further, the gene editing expression vector may be pTX377 or pYPQ230.

[0012] Furthermore, the gene editing expression vector described in the above method contains a Cas12a expression unit initiated by the maize ubiquitin promoter ZmUbi1 and a crRNA-scaffold expression unit initiated by the rice ubiquitin promoter OsUbi1.

[0013] Furthermore, the gene editing expression vector described in the above method also contains a hygromycin resistance gene Hyg expression unit initiated by the promoter CaMV35S.

[0014] In this method, the crRNA-scaffold expression unit of the gene editing expression vector expresses at least one of Seq ID No. 2, Seq ID No. 4, Seq ID No. 6, Seq ID No. 8, Seq ID No. 10 or Seq ID No. 12 in tandem.

[0015] Furthermore, the gene editing expression vector described in the above method expresses the crRNA-scaffold expression unit, which includes the fragment shown in Seq ID No. 18.

[0016] In step b of the above method, the rice transformation method uses Agrobacterium-mediated transformation.

[0017] This invention also provides an expression vector. This expression vector can express one or more guide RNAs that edit the promoter of the OsGBSSⅠ gene in the rice genome.

[0018] The guide RNA in the above expression vector is at least one of the guide RNAs shown in Seq ID No.2, Seq ID No.4, Seq ID No.6, Seq ID No.8, Seq ID No.10, or Seq ID No.12.

[0019] The expression vector is at least one of CRISPR-Cas12a or CRISPR-Cas12b gene editing expression vectors.

[0020] The aforementioned expression vector includes a crRNA-scaffold expression unit. Furthermore, the crRNA-scaffold expression unit tandemly expresses at least one guide RNA selected from Seq ID No. 2, Seq ID No. 4, Seq ID No. 6, Seq ID No. 8, Seq ID No. 10, or Seq ID No. 12.

[0021] The crRNA-scaffold expression unit in the above-mentioned expression vector is promoted by the rice ubiquitin promoter OsUbi1; it also includes the Cas12a expression unit promoted by the maize ubiquitin promoter ZmUbi1 and the hygromycin resistance gene Hyg expression unit promoted by the promoter CaMV35S.

[0022] The crRNA-scaffold expression unit in the aforementioned expression vector contains the fragment shown in Seq ID No. 18:

[0023] tgtaaggtgttgggctggaaattTAATTTCTACTAAGTGTAGATgctctgaggcactgacgtgcgggTAATTTCTACTAAGTGTAGATgggtgtgcgtggagcgcgcgcgcTAATTTCTACT AAGTGTAGATtcagcacgtacaagcagccgctaTAATTTCTACTAAGTGTAGATtgtcctacggaatgacgacaagcTAATTTCTACTAAGTGTAGATcacaggcctaatttcaagtccag.

[0024] The present invention also provides a guide RNA or a nucleic acid molecule complementary to it, characterized in that the guide RNA is at least one of Seq ID No.1, Seq ID No.2, Seq ID No.3, Seq ID No.4, Seq ID No.5, Seq ID No.6, Seq ID No.7, Seq ID No.8, Seq ID No.9, Seq ID No.10, Seq ID No.11 or Seq ID No.12.

[0025] Furthermore, the nucleotide sequence of the guide RNA is at least one of Seq ID No. 2, Seq ID No. 4, Seq ID No. 6, Seq ID No. 8, Seq ID No. 10, or Seq ID No. 12.

[0026] Furthermore, this invention also provides the use of the aforementioned guide RNA or its complementary nucleic acid molecule, or the aforementioned expression vector, in editing the promoter of the OsGBSSⅠ gene in the rice genome to improve rice quality. In particular, it provides gene-edited mutants with varying degrees of reduced amylose content in rice for use in improving rice quality.

[0027] The beneficial effects of this invention are as follows: This invention provides a method for improving the quality traits of rice. This method is based on the targeted editing of the promoter elements and intron 1 of the OsGBSSⅠ gene using a gene editing system, thereby regulating the expression of the OsGBSSⅠ gene. This allows for the alteration of amylose content, achieving effective regulation of rice amylose content and improving quality traits related to amylose content in rice. Experiments show that the method of this invention can efficiently edit the promoter elements and intron 1 of the rice OsGBSSⅠ gene. After editing the promoter elements and intron 1 of the rice OsGBSSⅠ gene, new materials with different amylose contents can be obtained, which is very convenient for related research and application in quality improvement. Simultaneously, several high-quality rice materials with excellent rice quality were obtained, including pZJP081-09-1-07 (amylose content 13.3%), pZJP081-06-2-01 (14.0%), pZJP081-18-1-04 (13.6%), pZJP081-13-2-08 (13.5%), and pZJP081-09-2-07 (13.2%). The method of this invention is simple and easy to operate, and shows great promise in rice quality improvement, genome function research, and applications. Attached Figure Description

[0028] Figure 1 The CRISPR-Cas12a multi-site targeted editing system was used to knock out the promoter elements and intron 1 of the rice OsGBSSⅠ gene. A. Schematic diagram of the targeted editing target sites for the rice OsGBSSⅠ gene promoter and intron 1. B. Schematic diagram of the CRISPR-Cas12a multi-site targeted knockout vector design (based on the backbone vector pTX377).

[0029] Figure 2 The T0 identification diagram of CRISPR-Cas12a multisite targeted knockout mutants shows that the light-colored numbers indicate that a large segment deletion has occurred in the individual.

[0030] Figure 3 Determination of amylose (AC) content in seeds of the OsGBSSⅠ directed-edited mutant T2. Boxes indicate the AC content of the highest quality material. OsGBSSⅠ-1 is the control material with the GBSS1 coding region knocked out.

[0031] Figure 4 Determination of the gel consistency (GC) length of seeds of the OsGBSSⅠ directed-edited mutant T2 (the box indicates the GC value for obtaining the best quality material). OsGBSSⅠ-1 is the control material with the GBSS1 coding region knocked out.

[0032] Figure 5. Observation of KI-I2 staining in seeds of the T2 generation of partially edited mutants. The boxes indicate the staining of the high-quality materials obtained (Scale bars = 1cm). OsGBSSⅠ-2 in the figure is the control material with the GBSS1 coding region knocked out.

[0033] Figure 6 Observation of chalkiness in seeds of the T2 generation of partially edited mutants. The boxes indicate the transparency of the obtained high-quality materials (Scale bars = 1 cm). OsGBSSⅠ-2 in the figure is the control material with the GBSS1 coding region knocked out. Detailed Implementation

[0034] Based on extensive prior research on rice quality improvement, this invention has determined that the expression pattern (transcriptional level and endosperm specificity) of the OsGBSSⅠ gene is primarily determined by its promoter (cis-elements). This invention further considers using CRISPR-Cas12a genome editing technology to edit the promoter of the rice OsGBSSⅠ gene, reducing its expression and decreasing amylose content to varying degrees, thereby obtaining high-quality edited material and preferred editing sites. Simultaneously, it also considers designing editing sites targeting intron 1 of the OsGBSSⅠ gene to complement this, aiming to obtain completely non-transcribed material.

[0035] Firstly, based on the analysis of various elements of the promoter and intron 1 of the rice OsGBSSⅠ gene, this invention designs and screens 12 guide RNAs (crRNAs) (such as...). Figure 1(As shown in A). Expression vectors capable of expressing the above-mentioned crRNA were constructed. The backbone vectors for these expression vectors can be pTX377 or pYPQ230 (Tang X, Lowder LG, Zhang T, Malzahn A, Zheng X, Voytas DF, Zhong Z, Chen Y, Ren Q, Li Q, Kirkland ER, Zhang Y, Qi Y. 2017. A CRISPR-Cpf1 system for efficient genome editing and transcriptional repression in plants. Nature Plants, 3:17018; Tang X, Ren Q, Yang L, Bao Y, Zhong Z, He Y, Liu S, Qi C, Liu B, Wang Y, Sretenovic S, Zhang Y, Zheng X, Zhang T, Qi Y, Zhang Y. 2019. Single transcript unit CRISPR 2.0 systems for robust Cas9 and Cas12a mediated plant genome editing. Plant Biotechnology). Journal, 17(7):1431-1445) and other plant Cas12a targeted editing expression vectors. In the embodiments of the present invention, pTX377 was used as a backbone vector, and efficient editing results were achieved. The targeted knockout vector pZJP081 (main expression unit such as Figure 1 (as shown in B).

[0036] The inventors discovered that the above-mentioned gene editing system can efficiently obtain different edited mutants of the rice OsGBSSⅠ gene promoter. Furthermore, nine high-quality edited materials (amylose content between 10% and 14%) were obtained: pZJP081-09-1-07, pZJP081-06-2-01, pZJP081-18-1-04, pZJP081-13-2-08, pZJP081-09-2-07, pZJP080-05-2-09, pZJP081-12-3-10, pZJP081-11-3-05, pZJP081-05-3-03, and pZJP081-11-1-05.

[0037] Therefore, this invention provides the application of the above-mentioned crRNA or expression vector in creating OsGBSSⅠ gene promoter and intron 1 mutants, and improving rice quality. The process includes the following steps:

[0038] a. Design and select crRNA, and construct a CRISPR-Cas12 gene editing expression vector for the OsGBSSⅠ gene promoter and intron 1;

[0039] b. Transform rice with the expression vector obtained in step a to obtain genetically transformed plants;

[0040] c. Screen and identify the transformed plants obtained in b, and screen out the rice OsGBSSⅠ gene-edited mutant.

[0041] d. Agronomic traits were tested on the promoter-edited mutant of the rice OsGBSSⅠ gene to obtain the results of the mutant for rice quality improvement.

[0042] Specifically, the method for creating the promoter and intron 1 mutant of the rice OsGBSSⅠ gene in this invention includes the following steps:

[0043] (1) Selection of crRNA target sites

[0044] The rice OsGBSSⅠ gene (LOC_Os06g04200) is located on chromosome 6 of the genome. Target sites were designed based on the CRISPR-Cas12 system's target site recognition and splicing rules. The target sites were designed at the OsGBSSⅠ gene promoter region (-1400bp-0bp) and the first intron (see...). Figure 1 A). Its crRNA sequence is shown in SEQ ID No. 1 to SEQ ID No. 12, and each crRNA is 23 nt in length. The PAM site is TTTV.

[0045] (2) Construction of OsGBSSⅠ gene promoter-directed editing expression vector

[0046] A vector was constructed using crRNA02, crRNA04, crRNA06, crRNA08, crRNA10, and crRNA12 (see [link to vector]). Figure 1 B). A fragment containing the crRNA to be expressed was synthesized at the biotechnology company, the sequence of which is shown in SEQ ID No. 13.

[0047] SEQ ID No. 13 was assembled into the backbone vector pTX377 (which contains a ZmUbi1-initiated Cas12a expression unit; an OsUbi1-initiated crRNA-scaffold expression unit, a ccdb gene sequence with BsaI restriction sites at both ends; and a CaMV35S-initiated hygromycin resistance gene Hyg expression unit) using the Golden Gate method. The constructed recombinant vector was transformed into *E. coli* DH5α competent cells, and single clones were selected for PCR detection. The resulting vector, pZJP081, was used for targeted editing of the rice OsGBSSⅠ gene promoter and intron 1. Its main expression units are as follows: Figure 1 As shown in B.

[0048] (3) Genetic transformation and genotype identification

[0049] The rice OsGBSSⅠ gene-directed editing expression vector pZJP081 was used for Agrobacterium-mediated genetic transformation, screening, and regenerated transformed plants. Genomic DNA was extracted from the regenerated rice seedlings, and the target fragment was amplified using specific primers Intron-F1 (primer sequence as shown in SEQ ID No. 14) and ZY010-R1 (primer sequence as shown in SEQ ID No. 15) to detect transgenic positivity. After single-plant DNA extraction for positive identification, PCR was performed using designed specific primers OsGBSSⅠ-sscp-F1 (primer sequence as shown in SEQ ID No. 16) and OsGBSSⅠ-sscp-R1 (primer sequence as shown in SEQ ID No. 17) to screen for mutants with large fragment deletions. Sanger sequencing confirmed the presence of directed knockout mutants with large fragment deletions. For mutants without large fragment deletions, homozygous mutants were screened in the T1 generation for sequencing analysis.

[0050] (4) Analysis of quality indicators of mutants

[0051] The quality indicators (amylose content AC and gel consistency value GC) of the OsGBSSⅠ gene promoter and intron 1 were measured, and the starch granules and chalkiness of some mutants were observed.

[0052] The present invention will be described in more detail below through specific examples.

[0053] Example 1: Construction of the rice OsGBSSⅠ promoter and intron 1 editing vector

[0054] (1) crRNA design

[0055] The coding region and (-1400bp-1bp) promoter sequence of the rice OsGBSSⅠ gene were retrieved and downloaded from the NCBI database website (https: / / www.ncbi.nlm.nih.gov / ).

[0056] Then, cis-elements were analyzed using the website (http: / / bioinformatics.psb.ugent.be / webtools / plantcare / html / ). Based on the CRISPR-Cas12 system's target site recognition and cleavage rules, crRNAs were designed for the promoter region and intron 1 region. The mismatch rate and off-target sites of the crRNAs were then predicted online using the CRISPR RGEN Tools website (http: / / www.rgenome.net / cas-offinder / ), and 12 crRNAs were selected. Figure 1 A, SEQ ID No. 1-SEQ ID No. 12). We consider constructing a vector from crRNA02, crRNA04, crRNA06, crRNA08, crRNA10, and crRNA12 (see...). Figure 1 B) Another vector was constructed using crRNA01, crRNA03, crRNA05, crRNA07, crRNA9, and crRNA11. Then, the crRNA fragments to be expressed in the first vector were synthesized at a biotechnology company. Based on the restriction sites of the knockout vector pTX377 used in this invention, BsaI restriction sites were added to both ends, the sequence of which is shown in SEQ ID No. 13. This was then synthesized by Chengdu Qingke Biotechnology Co., Ltd.

[0057] The sequence of SEQ ID No. 13 is as follows (the underlined part is the added BsaI restriction site, and the remaining lowercase letters correspond to crRNA02, crRNA04, crRNA06, crRNA08, crRNA10 and crRNA12 respectively):

[0058] caccggtctcAAGATtgtaaggtgttgggctggaaattTAATTTCTACTAAGTGTAGATgctctgaggcactgacgtgcgggTAATTTCTACTAAGTGTAGATgggtgtgcgtggagcgcgcgcgcTAATTTCTACT AAGTGTAGATtcagcacgtacaagcagccgctaTAATTTCTACTAAGTGTAGATtgtcctacggaatgacgacaagcTAATTTCTACTAAGTGTAGATcacaggcctaatttcaagtccagg gccgGAGACCATCG.

[0059] (2) Connection reaction

[0060] The synthesized fragment SEQ ID No. 13 was assembled into the backbone vector pTX377 via Golden Gate assay. The Golden Gate reaction system consisted of: 1 μL T4 DNA ligase, 2 μL T4 DNA ligase buffer (10x), 1 μL pTX377 backbone vector plasmid (100 ng / μL), 1 μL restriction endonuclease BsaI, 2 μL synthesized fragment, and 13 μL ddH2O. The Golden Gate reaction program was: 15 cycles of 37℃ for 5 min, 16℃ for 10 min, followed by 37℃ for 5 min and 85℃ for 10 min.

[0061] (3) Plasmid transformation of competent Escherichia coli cells

[0062] Add 10 μL of the ligation product to DH5α competent cells, mix gently, place on ice for 20 min, heat shock at 42℃ for 60-90 s, then place on ice for 4 min. Add 350 μL of LB medium and incubate at 37℃ and 200 rpm on a shaker for about 40 min. After incubation, centrifuge at 4000 rpm for 5 min. After centrifugation, resuspend the cells in the remaining supernatant, spread the bacterial culture on LB medium, and incubate overnight at 37℃.

[0063] (4) Colony PCR

[0064] Single colonies were picked from LB agar plates using sterile toothpicks and placed in water containing 50 μL ddH2O. 5 μL of this bacterial culture was used as a template for PCR amplification. A 25 μL system was used, consisting of: 10 μL 2×Taq DNA Polymerase Mix, 0.5 μL Intron-F1 (SEQ ID No. 15), 0.5 μL ZY010-R1 (SEQ ID No. 16), 5 μL bacterial culture template, and 9 μL ddH2O. The PCR program was: 95℃, 3 min → (95℃, 30 s → 58℃, 30 s → 72℃, 30 s) 35 cycles → 72℃, 5 min → 12℃, 10 min (Taq DNA enzyme, dNTPs, etc., were purchased from Tiangen Biotech). After PCR, the samples were detected by electrophoresis on a 1% agarose gel at 130V for 30 min.

[0065] (5) Plasmid extraction and sequencing verification

[0066] For single clones verified by colony PCR, 50 μL of bacterial culture was inoculated into LB broth containing 50 mg / L Kans and shaken for 12-16 hours. Plasmids were then extracted. Plasmid DNA extraction was performed according to the AXYGEN AxyPrep™ Plasmid Miniprep Kit instructions. The extracted plasmids were sent to Qingke Biotechnology Co., Ltd. for sequencing verification. An expression vector targeting the promoter and intron 1 of the rice OsGBSSⅠ gene for directed editing was obtained, named pZJP081. A schematic diagram of its T-DNA region structure is shown below. Figure 1 As shown in B. Using the same method, with vector pTX377 as the backbone, another vector was constructed from crRNA01, crRNA03, crRNA05, crRNA07, crRNA09, and crRNA11 (see Figure B). Figure 1 B), named pZJP080.

[0067] Example 2 Agrobacterium-mediated genetic transformation of rice

[0068] The vector pZJP081 was transformed into competent Agrobacterium rhizogenes EHA105 cells. The specific steps were as follows: Vector pZJP081 was added to competent Agrobacterium rhizogenes EHA105 cells, incubated on ice for 30 min, then flash-frozen in liquid nitrogen for 5 min, placed in a 37°C water bath for 5 min, followed by an ice bath for 5 min. Finally, 1 mL of LB liquid medium was added, and the cells were incubated at 220 rpm and 28°C for 120-150 min. The culture was then spread onto LB solid medium containing 50 mg / L rifampicin and 50 mg / L kanamycin, and incubated at 28°C for 2 days. After incubation, single colonies were picked and subjected to colony PCR for positive verification (method as in Example 1). The Agrobacterium culture was then expanded and stored for later use.

[0069] The experimental method for Agrobacterium-mediated transformation of rice is described in the reference (Tang X, Ren Q, Yang L, Bao Y, Zhong Z, He Y, Liu S, Qi C, Liu B, Wang Y, Sretenovic S, Zhang Y, Zheng X, Zhang T, Qi Y, Zhang Y. 2019. Single transcript unit CRISPR 2.0 systems for robust Cas9 and Cas12a mediated plant genome editing. Plant Biotechnol J 17, 1431-1445.).

[0070] The specific steps of genetic transformation in rice are as follows: Mature rice (Nipponbare) seeds are dehulled and sterilized; the sterilized seeds are inoculated onto N-6-D solid medium containing 0.4% gellan gum and cultured at 32°C under continuous light for 1–5 days; the cultured seeds are transformed into rice using Agrobacterium-mediated transformation, with plasmids pZJP080 and pZJP081 respectively introduced into the rice; the transformed rice seeds are then cultured in induction and selection medium under continuous light at 32°C for 2 weeks; the resulting callus tissue is transferred to RE-III medium; the young plantlets from the callus tissue are transferred to HF medium to induce root development. When the obtained resistant regenerated seedlings reach approximately 15 cm in length, the root culture medium is washed off with water, and the seedlings are transplanted into nutrient soil and cultured in a greenhouse to obtain gene-edited mutant plants.

[0071] Example 3 Identification of the promoter and intron 1 edit mutant of the rice OsGBSSⅠ gene

[0072] (1) Extraction of genomic DNA from rice seedlings

[0073] DNA extraction from rice seedlings was performed using the CTAB method. The specific steps are as follows:

[0074] Take fresh rice leaves, approximately 2-3 cm in length, place them in a 2 mL EP tube, and flash-freeze them with liquid nitrogen. After freezing, shake the foam box vigorously and crush the leaves using the friction of steel balls. Add 600 μL of CTAB, mix well, and heat in a 65°C water bath for 30-45 minutes. After heating, add 600 μL of chloroform and mix well. Centrifuge at 8600 rpm for 10 minutes. Transfer the supernatant to a 1.5 mL EP tube, add 600 μL of dimethyl methanol and mix well. Store at -20°C for 1 hour. Then centrifuge at 12000 rpm for 10 minutes, discard the supernatant, add 500 μL of 75% ethanol, wash twice, let stand and air dry, add 50 μL of ddH2O to dissolve the DNA, and store at -20°C for later use.

[0075] (2) Detection of transgenic rice seedlings

[0076] The target fragment was amplified using the specific primer Intron-F1 (primer sequence as shown in SEQ ID No. 14) and the downstream primer ZY010-R1 (primer sequence as shown in SEQ ID No. 15) to detect transgene positivity. The PCR amplification system and reaction procedure were the same as those for colony PCR in Example 1.

[0077] (3) Genotyping of mutants

[0078] The positive plants obtained from the test were subjected to PCR using specific primers OsGBSSⅠ-sscp-F1 (primer sequence as shown in SEQ ID No. 16) and OsGBSSⅠ-sscp-R1 (primer sequence as shown in SEQ ID No. 17). The PCR products were screened for mutants with large fragment deletions using 1% agarose gel chromatography. Sanger sequencing confirmed the presence of directed-edit mutants with large fragment deletions. The results showed that 13 out of the 32 tested individual plants had large fragment deletions, achieving an efficiency of 40.6%. Figure 2 For mutants without large deletions, homozygous mutants were screened in the T1 generation for sequencing analysis (since the number of plants obtained from the pZJP080 vector transformation was small, detailed analysis will not be conducted in subsequent steps).

[0079] Example 4: Analysis of quality indicators of mutants

[0080] (1) Determination of amylose content (AC)

[0081] The amylose content in homozygous OsGBSSⅠ promoter editing mutant, coding region mutant, and control WT seeds was determined using a dual-wavelength method. First, 100 mg of pure amylose was weighed into a 50 mL volumetric flask, 10 mL of KOH solution was added, and the mixture was gelatinized and dissolved in boiling water. The volume was then adjusted to 50 mL to prepare a 2 mg / mL amylose standard solution. 1 mL of the amylose standard solution was then transferred to a 50 mL volumetric flask, 30 mL of distilled water was added, and the pH was adjusted to approximately 3.5 with 0.1 M HCl solution. 0.5 mL of iodine reagent was added, and the mixture was brought to a final volume with distilled water. The mixture was allowed to stand for 20 min, using distilled water as a blank control, and a full-spectrum visible light scan (400-960 nm) was performed.

[0082] To prepare standard solutions of amylose with different concentration gradients, 0.5, 1.0, 1.5, 2.0, 2.5, and 3.0 mg of each solution were placed into six different 50 mL volumetric flasks. 20–30 mL of ddH₂O was added, and the pH was adjusted to approximately 3.5. Then, 0.5 mL of iodine reagent was added, and the solution was brought to volume with distilled water. The solutions were allowed to stand for 20 min. Using distilled water as a blank, Δλ₁ and Δλ₂ were measured at the reference wavelength λ₁ and the measurement wavelength λ₂, respectively. ΔAs = Aλ₁ - Aλ₁. A standard curve was plotted with the amylose content (mg / mL) on the x-axis and ΔA on the y-axis to fit the regression equation.

[0083] Determination of AC in samples: (1) The seeds of rice mutant plants were dried, crushed and sieved; (2) A reference standard solution was prepared from the starch solution of the mutant plant sample; (3) The absorbance of the mutant plant sample solution was measured and the amylose content in the sample was calculated.

[0084] The calculation formula is as follows: Amylose = Xamyl * 100 * 50 * 100% / 5 * (m * 1000)

[0085] result( Figure 3The results showed that the amylose content in the seeds of the mutant plants changed to varying degrees. Nine high-quality editing materials with amylose content between 10% and 14% were obtained from 47 materials: pZJP081-09-1-07 (13.3%), pZJP081-06-2-01 (14.0%), pZJP081-18-1-04 (13.6%), pZJP081-13-2-08 (13.5%), and pZJP081-09-2-07. (13.2%), pZJP081-12-3-10 (11.8%), pZJP081-11-3-05 (11.4%), pZJP081-05-3-03 (11.3%), pZJP081-11-1-05 (10.8%); meanwhile, the mutant material pZJP081-15-1-07 has an amylose content of 9.9%, and is also a high-quality soft rice. It also includes glutinous rice material pZJP081-03-2-02 (amylose content below 5%), which has no significant difference in AC content from the coding region mutant.

[0086] (2) Gelatin consistency determination (GC)

[0087] The consistency of the gel was determined using the national standard method. 100 mg ± 1 mg of rice seed powder (after grinding and sieving) was weighed into a 15 × 150 mm test tube, and 0.2 mL of 0.025% thymol blue-alcohol solution was added, followed by 2 mL of 0.2 mol / L KOH solution.

[0088] After the sample is thoroughly mixed, heat it in a boiling water bath for 8 minutes. After heating, remove the test tube, let it stand to cool for 10 minutes, and then place it in an ice-water mixture for 20 minutes before removing it.

[0089] Place the removed test tube containing the sample horizontally on a graduated and pre-leveled workbench and let it stand at 25°C for 1 hour. Measure the length of the rice glue flowing in the test tube.

[0090] result( Figure 4 The results showed that the GC length of the mutant materials increased compared to the WT materials. Specifically, the GC length increased as the AC content of the mutant lines decreased, with the mutant lines having the longest GC lengths in those with AC contents below 5%. A negative correlation was found between AC and GC length.

[0091] (3) Observation of starch granules by iodine staining

[0092] The harvested mutant rice seeds were dehulled and soaked in water for 24 hours. The rice endosperm tissue was transversely cut in half, and 200 μL of iodine solution was dropped onto the cut endosperm. After staining for 2 minutes, the residual iodine solution on the endosperm surface was removed with filter paper. The reaction time of each plant material was kept consistent throughout the experiment to facilitate subsequent control and observation of differences. Results ( Figure 5 The results showed that, under the same staining time, the endosperm colors of WT and mutant plant seeds differed significantly after staining. Unlike the bluish-black color of WT seeds, the mutant plant seeds exhibited varying degrees of color change from bluish-black to light reddish-brown as the amylose content decreased.

[0093] (4) Seed chalkiness observation

[0094] Appearance quality is also an important indicator of rice quality. As the amylose content decreases, the transparency of the rice decreases, resulting in a decline in appearance quality. After harvesting mutant rice seed samples and air-drying them, they were dehulled, milled into polished rice using a rice milling machine, and then photographed under natural light to observe changes in seed chalkiness. Results ( Figure 6 The results showed that the chalkiness of seeds from the high-quality (12%–14%) mutant plant pZJP081-09-1-07 (amylose content 13.3%) was not significantly different from that of the wild type; the chalkiness of seeds from the mutant pZJP081-15-1-07 (amylose content 9.9%) increased, and the seeds became translucent; the appearance of seeds from the mutant pZJP081-03-2-02 (amylose content less than 5%) was consistent with that of the mutant seeds in the coding region, and they became completely opaque.

[0095] This invention, through design and screening, obtained specific crRNAs for efficiently editing the OsGBSSⅠ promoter and intron 1 based on the CRISPR-Cas12a system. At the same time, it unexpectedly obtained several mutants with excellent rice quality, such as pZJP081-09-1-07, providing new ideas and selectable target sites for rice quality improvement. sequence list <110> Chengdu Jigu Gene Technology Co., Ltd. <120> Methods for editing the OsGBSSⅠ promoter and intron 1 in the rice genome to improve rice quality <150> 202111246188.4 <151> 2021-10-26 <160> 18 <170> SIPOSequenceListing 1.0 <210> 1 <211> 19 <212> DNA / RNA <213> Artificial Sequence <400> 1 gagccgaggg caaaggaaa 19 <210> 2 <211> twenty three <212> DNA / RNA <213> Artificial Sequence <400> 2 uguaaggugu ugggcuggaa auu 23 <210> 3 <211> twenty three <212> DNA / RNA <213> Artificial Sequence <400> 3 uagcgccguu gcgugaagcc cgc 23 <210> 4 <211> twenty three <212> DNA / RNA <213> Artificial Sequence <400> 4 gcucugaggc acugacgugc ggg 23 <210> 5 <211> twenty three <212> DNA / RNA <213> Artificial Sequence <400> 5 cccuuuugcu uuuccagcuu guu 23 <210> 6 <211> twenty three <212> DNA / RNA <213> Artificial Sequence <400> 6 gggugugcgu ggagcgcgcg cgc 23 <210> 7 <211> twenty three <212> DNA / RNA <213> Artificial Sequence <400> 7 cgcguacgcc gacggcucac aca 23 <210> 8 <211> twenty three <212> DNA / RNA <213> Artificial Sequence <400> 8 ucagcacgua caagcagccg cua 23 <210> 9 <211> twenty three <212> DNA / RNA <213> Artificial Sequence <400> 9 ucgaggcguu aguuggcagg cac 23 <210> 10 <211> twenty three <212> DNA / RNA <213> Artificial Sequence <400> 10 uguccuacgg aaugacgaca agc 23 <210> 11 <211> twenty three <212> DNA / RNA <213> Artificial Sequence <400> 11 auaauucucu cuagcuuauu aca 23 <210> 12 <211> twenty three <212> DNA / RNA <213> Artificial Sequence <400> 12 cacaggccua auuucaaguc cag 23 <210> 13 <211> 273 <212> DNA / RNA <213> Artificial Sequence <400> 13 caccggtctc aagattgtaa ggtgttgggc tggaaattta atttctacta agtgtagatg 60 ctctgaggca ctgacgtgcg ggtaatttct actaagtgta gatgggtgtg cgtggagcgc 120 gcgcgctaat ttctactaag tgtagattca gcacgtacaa gcagccgcta taatttctac 180 taagtgtaga ttgtcctacg gaatgacgac aagctaattt ctactaagtg tagatcacag 240 gcctaatttc aagtccaggg ccggagacca tcg 273 <210> 14 <211> 20 <212> DNA / RNA <213> Artificial Sequence <400> 14 ttctgatcct ctccgttcct 20 <210> 15 <211> 19 <212> DNA / RNA <213> Artificial Sequence <400> 15 aagaccggca acaggattc 19 <210> 16 <211> 21 <212> DNA / RNA <213> Artificial Sequence <400> 16 gacaagtcaa gggaccttag a 21 <210> 17 <211> 22 <212> DNA / RNA <213> Artificial Sequence <400> 17 gagaatctat tccttcacac tg 22 <210> 18 <211> 243 <212> DNA / RNA <213> Artificial Sequence <400> 18 tgtaaggtgt tgggctggaa atttaatttc tactaagtgt agatgctctg aggcactgac 60 gtgcgggtaa tttctactaa gtgtagatgg gtgtgcgtgg agcgcgcgcg ctaatttcta 120 ctaagtgtag attcagcacg tacaagcagc cgctataatt tctactaagt gtagattgtc 180 ctacggaatg acgacaagct aatttctact aagtgtagat cacaggccta atttcaagtc 240 cag 243

Claims

1. A method for editing the promoter and intron 1 of the OsGBSSⅠ gene in the rice genome to improve rice quality, characterized in that... Includes the following steps: a. Construct a Cas gene editing expression vector that can express guide RNA targeting the promoter and intron 1 of the rice OsGBSSⅠ gene; b. Transform rice with the expression vector obtained in step a, and obtain transformed plants using the CRISPR-Cas gene editing system; c. Collect seeds of the transformed plants, screen out the gene-edited mutant seeds, and obtain gene-edited rice with improved amylose content; The Cas gene editing expression vector is a CRISPR-Cas12a gene editing expression vector, comprising a Cas12a expression unit initiated by the maize ubiquitin promoter ZmUbi1 and a crRNA-scaffold expression unit initiated by the rice ubiquitin promoter OsUbi1, as well as a hygromycin resistance gene Hyg expression unit initiated by the promoter CaMV35S; the crRNA-scaffold expression unit tandemly expresses the guide RNAs shown in Seq ID No. 2, Seq ID No. 4, Seq ID No. 6, Seq ID No. 8, Seq ID No. 10, and Seq ID No. 12; the crRNA-scaffold expression unit includes the fragment shown in Seq ID No.

18.

2. The method according to claim 1, characterized in that: The rice transformation described in step b uses Agrobacterium-mediated transformation.

3. An expression vector, characterized in that: The expression vector is a CRISPR-Cas12a gene editing expression vector, capable of expressing multiple guide RNAs that edit the promoter and intron 1 of the OsGBSSⅠ gene in the rice genome; the expression vector includes a crRNA-scaffold expression unit that tandemly expresses the guide RNAs shown in Seq ID No. 2, Seq ID No. 4, Seq ID No. 6, Seq ID No. 8, Seq ID No. 10, and Seq ID No. 12; the crRNA-scaffold expression unit includes the fragment shown in Seq ID No. 18; the crRNA-scaffold expression unit is promoted by the rice ubiquitin promoter OsUbi1; it also includes a Cas12a expression unit promoted by the maize ubiquitin promoter ZmUbi1 and a hygromycin resistance gene Hyg expression unit promoted by the promoter CaMV35S.

4. Use of the expression vector according to claim 3 in editing the promoter and intron 1 of the OsGBSSⅠ gene in the rice genome to improve rice quality.

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