Method for creating osmapk6 weak mutant

Abstract

The invention relates to a method for creating an osmapk6 weak mutant, belongs to the technical field of crop genetic breeding, and particularly relates to a method for creating an osmapk6 weak mutant. The invention aims to solve the bottleneck problem that the functional research of OsMAPK6 and other important protein kinases is limited because the complete knockout of OsMAPK6 and other important protein kinases easily leads to embryo death and fertile seeds are difficult to obtain. According to the present invention, the C-terminal base site of the OsMAPK6 gene is edited so as to obtain the weak mutant encoding the OsMAPK6 protein; according to the invention, key genetic materials and germplasm resources are provided for deep analysis of biological functions of OsMAPK6.

Description

Technical Field

[0001] This invention belongs to the field of crop genetics and breeding technology, and in particular relates to a method for creating a weak mutant of osmapk6. Background Technology

[0002] The MAPK cascade is a highly conserved signal transduction module in eukaryotes, transmitting signals through a stepwise phosphorylation process: MAPKKKs (MAPK kinase kinases) phosphorylate and activate downstream MAPKKs (MAPK kinases), which in turn phosphorylate and activate MAPKs. As an important member of the MAPK family, OsMAPK6 participates extensively in regulating rice growth, development, and stress responses by phosphorylating a variety of substrates.

[0003] In terms of growth and development, OsMAPK6 plays a crucial role through multiple signaling pathways. First, the OsMAPKKK10-OsMAPKK4-OsMAPK6 cascade regulates plant architecture and grain size in rice by phosphorylating the transcription factor OsWRKY53. Additionally, OsMAPK6 can directly phosphorylate serine at position 142 and threonine at position 186 of the GW6a protein, thereby affecting grain development. Second, OsMAPK6 participates in regulating the number of grains per panicle through the ERECTA1-OsMKKK10-OsMKK4-OsMAPK6-DST-OsCKX2 signaling cascade. Third, OsMAPK6 can also activate the gibberellin biosynthesis pathway by phosphorylating and stamen fertility, promoting panicle elongation. Fourth, during anther development, OsMAPK6 participates in regulating tapetum development and stamen fertility through the OsYDA1 / OsYDA2-OsMKK4-OsMPK6 module. It is worth noting that OsMPK6 also plays an important role in the early stages of embryonic development, and its mutations can lead to abnormal initial cell differentiation.

[0004] In stress responses, OsMAPK6 plays a crucial role as a key signaling hub. It participates in immune signaling transduction through its interaction with the bHLH transcription factor RAI1. In chitin-induced immune responses, OsRLCK185 initiates a phosphorylation cascade, activating OsMKK4, which in turn promotes OsMAPK6 phosphorylation, thereby enhancing resistance to rice blast. Under low-temperature stress, OsMAPK6 enhances rice's cold tolerance by phosphorylating and stabilizing OsICE1 and OsIPA1. Faced with salt stress, the OsWRKY53-OsMKK10.2 cascade pathway finely regulates its activity through the OsMAPK6-OsSOS1 module to enhance salt tolerance. Under drought conditions, OsCRK14 activates the OsMKK4-OsMAPK6 cascade, mediating the drought response by stabilizing the transcription factor OsbZIP66.

[0005] Although OsMAPK6 plays a crucial role in plant growth, development, and stress response, its complete knockout leads to embryo lethality, making it difficult to obtain fertile genetic mutants and thus limiting in-depth genetic research. Currently, functional analysis mainly relies on the EMS-induced weak allelic mutant dsg1. Although dsg1 is a sub-effective allelic mutation, it still exhibits severe developmental defects and extremely low seed setting rate. Summary of the Invention

[0006] The purpose of this invention is to provide a method for creating weak mutants of osmapk6 through gene editing, thereby solving the bottleneck problem that the OsMAPK6 gene knockout leads to embryonic lethality and prevents in-depth research on OsMAPK6-related genetics.

[0007] This invention discloses a method for creating a weak mutant of osmapk6, which involves editing the C-terminal base site of the OsMAPK6 gene to obtain a weak mutant encoding the OsMAPK6 protein.

[0008] Furthermore, the editor was guided to target the C-terminal base site of the OsMAPK6 gene.

[0009] Furthermore, based on the deletion of an A base at the 1140bp C-terminus of the OsMAPK6 gene, the target sequence osmapk6(379) was designed; the primers for the osmapk6(379) mutant are FP1 / RP1 and FP2 / RP2;

[0010] The primer FP1 sequence is: 5'-TGCACGAGCAGCATGCATTGTCCGGTTTC-3',

[0011] The primer RP1 sequence is: 5'-CTCTGAAACCGGACAATGCATGCTGCTCG-3';

[0012] The primer FP2 sequence is: 5'-GTGCATCCTTCATTTGTCCTCGGACAATGCATGCATATAAAC-3',

[0013] The primer RP2 sequence is: 5'-TCAAGTTTATATGCATGCATTGTCCGAGGACAAATGAAGGAT-3'.

[0014] Furthermore, based on the insertion of a T base after the 1149th bp of the C-terminus of the OsMAPK6 gene, the target sequence osmapk6(383) was designed; the primers for the osmapk6(383) mutant are FP3 / RP3 and FP4 / RP4;

[0015] The primer FP3 sequence is: 5′-TGCAATTGTCCGAGGAACAAATGAGTTTC-3′.

[0016] The primer RP3 sequence is: 5′-CTCTGAAACTCATTTGTTCCTCGGACAAT-3′.

[0017] The primer FP4 sequence is: 5'-GTGCTAGATTAGATCACTTCATTTGTTCCTCGCTTCCTCT-3',

[0018] The primer RP4 sequence is: 5'-TCAAAGAGGAAGCGAGGAACAAATGAAGTGATCTAATCTA -3'.

[0019] Furthermore, based on the insertion of a G base after the 1159th bp of the C-terminus of the OsMAPK6 gene, a target sequence osmapk6(386) was designed; the primers for the osmapk6(386) mutant are FP5 / RP5 and FP6 / RP6;

[0020] The primer FP5 sequence is: 5′-TGCAGGTTGAACGCAAGGCCTTCTGTTTC-3′,

[0021] The primer RP5 sequence is: 5′-CTCTGAAACAGAAGGCCTTGCGTTCAACC-3′.

[0022] The primer FP6 sequence is: 5'-GTGCAGGATCTAATCTGACCAAGAAGGCCTTGCAAATGAAA-3',

[0023] The primer RP6 sequence is: 5'-TCAATTTCATTTGCAAGGCCTTCTTGGTCAGATTAGATCCT-3'.

[0024] Furthermore, based on the insertion of a T base after the 1164 bp C-terminus of the OsMAPK6 gene, a target sequence osmapk6(388) was designed; the primers for the osmapk6(388) mutant are FP7 / RP7 and FP8 / RP8;

[0025] The primer FP7 sequence is: 5′-TGCAAAGGATCTAATCTACCAAGAGTTTC-3′.

[0026] The primer RP7 sequence is: 5′-CTCTGAAACTCTTGGTAGATTAGATCCTT-3′.

[0027] The primer FP8 sequence is: 5′-GTGCGCAAGGCCTTCATTGGTAGATTAGTAAGGAAT-3′,

[0028] The primer RP8 sequence is: 5′-TCAAATTCCTTACTAATCTACCAATGAAGGCCTTGC-3′.

[0029] Furthermore, based on the insertion of a T base after the 1179 bp C-terminus of the OsMAPK6 gene, a target sequence osmapk6(393) was designed; the primers for the osmapk6(393) mutant are FP9 / RP9 and FP10 / RP10;

[0030] The primer FP9 sequence is: 5′-TGCAACACCAGCTACTGGTAATCAGTTTC-3′,

[0031] The primer RP9 sequence is: 5′-CTCTGAAACTGATTACCAGTAGCTGGTGT-3′.

[0032] The primer FP10 sequence is: 5′-GTGCGGCCTTGCGTTCTAACCCTGATTACCAGTAGCATAATAAA-3′.

[0033] The primer RP10 sequence is: 5′-TCAATTTATTATGCTACTGGTAATCAGGGTTAGAACGCAAGGCC-3′.

[0034] Furthermore, the method is characterized by the following steps:

[0035] I. Vector Construction: Based on the designed target sequence, the corresponding weak mutant primers and universal primers FP / RP and FP' / RP' are annealed into double strands and then ligated into the Epe2 vector through cleavage-ligation to complete the construction of the guide editing vector;

[0036] II. Genetic transformation: The above-mentioned guide editing vector was transformed into Escherichia coli, positive clones were expanded and cultured to extract plasmids, and then the extracted plasmids were used to transform Agrobacterium EHA105 strain. Subsequently, the Agrobacterium strain containing the target plasmid was used to infect rice callus tissue, and differentiation culture was carried out to obtain transgenic rice plants.

[0037] The primer FP sequence is: 5′-AGAGCTATGCTGGAAACAGCATAGCAAGTTGAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGCACCGAGTCG-3′,

[0038] The primer RP sequence is: 5′-GCACCGACTCGGTGCCACTTTTTCAAGTTGATAACGGACTAGCCTTATTTCAACTTGCTATGCTGTTTCCAGCATAG-3′;

[0039] The primer FP' sequence is: 5′-TTGACGCGGTTCTATCTAGTTACGCGTTAAACCAACTAGAAA-3′,

[0040] The primer RP' sequence is: 5′-GGCCTTTCTAGTTGGTTTAACGCGTAACTAGATAGAACCGCG-3′.

[0041] Furthermore, in step one, 20 µl each of the corresponding weak mutant primer and universal primer at a concentration of 100 µM are taken and incubated at 90 °C for 30 seconds, and then cooled to room temperature to anneal the complementary primers to form 4 double-stranded targeting adapters; the 4 double-stranded targeting adapters include 2 double-stranded universal adapters (FP+RP; FP'+FP') and 2 corresponding weak mutant primer double-stranded adapters (e.g., FP1+RP1; FP2+RP2).

[0042] Furthermore, in step one, the four double-stranded targeting adapters are incubated with the Epe2 vector, BsaⅠ, and T4 DNAligase, respectively.

[0043] Incubation of the ligation reaction: 37℃, 5 min; 25℃, 5 min; 25 cycles; 80℃, 10 min.

[0044] Beneficial effects of this invention:

[0045] This invention employs guided editing technology to precisely introduce frameshift mutations at different sites at the C-terminus of the OsMAPK6 gene, prematurely terminating the translation process and thus obtaining a series of weak mutants encoding truncated OsMAPK6 proteins of varying lengths. This method successfully overcomes the research bottleneck of embryo lethality and difficulty in obtaining fertile seeds due to complete OsMAPK6 knockout. The series of osmapk6 mutants (weak mutants) created using this method maintain typical growth and developmental defect phenotypes (such as reduced plant height, smaller leaf angle, smaller grains, and decreased ear elongation) while still achieving normal seed setting. Among them, the seed setting rate of the osmapk6(386) mutant can be restored to approximately 80%. This provides a crucial experimental system and gene resource for in-depth research on the genetic function of OsMAPK6 and the analysis of its molecular function in growth, development, and stress response, and is an indispensable and valuable germplasm resource. Attached Figure Description

[0046] Figure 1This is a schematic diagram of the C-terminal coding sequence of the OsMAPK6 gene and its corresponding protein sequence in Suijing 18 and osmapk6(379), osmapk6(383), osmapk6(386), osmapk6(388) and osmapk6(393);

[0047] Figure 2 The sequencing maps of the OsMAPK6 gene in Suijing 18 and osmapk6(379), osmapk6(383), osmapk6(386), osmapk6(388) and osmapk6(393) are shown.

[0048] Figure 3 The overall morphological diagrams of Suijing 18 and osmapk6(379), osmapk6(383), osmapk6(386), osmapk6(388) and osmapk6(393) at the heading stage;

[0049] Figure 4 The plant height statistics of Suijing 18 and osmapk6(379), osmapk6(383), osmapk6(386), osmapk6(388) and osmapk6(393);

[0050] Figure 5 Statistical results of the sword-shaped leaf angles of Suijing 18 and osmapk6(379), osmapk6(383), osmapk6(386), osmapk6(388) and osmapk6(393);

[0051] Figure 6 The grain morphology diagrams of Suijing 18 and osmapk6(379), osmapk6(383), osmapk6(386), osmapk6(388) and osmapk6(393);

[0052] Figure 7 The statistical results of grain length for Suijing 18 and osmapk6(379), osmapk6(383), osmapk6(386), osmapk6(388) and osmapk6(393) are presented.

[0053] Figure 8 The statistical results of particle width for Suijing 18 and osmapk6(379), osmapk6(383), osmapk6(386), osmapk6(388) and osmapk6(393).

[0054] Figure 9The statistical results of the fruit setting rate of Suijing 18 and osmapk6(379), osmapk6(383), osmapk6(386), osmapk6(388) and osmapk6(393) are presented.

[0055] Figure 10 Ear morphology diagrams of Suijing 18 and osmapk6(379), osmapk6(383), osmapk6(386), osmapk6(388) and osmapk6(393).

[0056] Figure 11 The spike length statistics are for Suijing 18 and osmapk6(379), osmapk6(383), osmapk6(386), osmapk6(388) and osmapk6(393).

[0057] Figure 12 The results of ear elongation are statistically significant for Suijing 18 and osmapk6(379), osmapk6(383), osmapk6(386), osmapk6(388) and osmapk6(393). Detailed Implementation

[0058] The embodiments of the present invention will be described in detail below. The following embodiments are implemented based on the technical solution of the present invention, and detailed implementation schemes and specific operation processes are given. However, the protection scope of the present invention is not limited to the following embodiments.

[0059] Example 1

[0060] Construction of the OsMAPK6 gene editing vector:

[0061] 1. Based on the designed target sequence, the weak mutant primers and universal primers FP / RP and FP' / RP' were annealed into double strands and ligated into the Epe2 vector through cleavage and ligation to complete the construction of the guide editing vector;

[0062] Among them, based on the deletion of an A base at the 1140bp C-terminus of the OsMAPK6 gene, the target sequence osmapk6(379) was designed; the corresponding weak mutant primers for the osmapk6(379) mutant are FP1 / RP1 and FP2 / RP2;

[0063] The primer FP1 sequence is: 5'-TGCACGAGCAGCATGCATTGTCCGGTTTC-3',

[0064] The primer RP1 sequence is: 5'-CTCTGAAACCGGACAATGCATGCTGCTCG-3';

[0065] The primer FP2 sequence is: 5'-GTGCATCCTTCATTTGTCCTCGGACAATGCATGCATATAAAC-3',

[0066] The primer RP2 sequence is: 5'-TCAAGTTTATATGCATGCATTGTCCGAGGACAAATGAAGGAT-3'.

[0067] Based on the insertion of a T base after the 1149th bp of the C-terminus of the OsMAPK6 gene, the target sequence osmapk6(383) was designed; the corresponding weak mutant primers for the osmapk6(383) mutant are FP3 / RP3 and FP4 / RP4;

[0068] The primer FP3 sequence is: 5′-TGCAATTGTCCGAGGAACAAATGAGTTTC-3′.

[0069] The primer RP3 sequence is: 5′-CTCTGAAACTCATTTGTTCCTCGGACAAT-3′.

[0070] The primer FP4 sequence is: 5'-GTGCTAGATTAGATCACTTCATTTGTTCCTCGCTTCCTCT-3',

[0071] The primer RP4 sequence is: 5'-TCAAAGAGGAAGCGAGGAACAAATGAAGTGATCTAATCTA -3'.

[0072] Based on the insertion of a G base after the 1159th bp of the C-terminus of the OsMAPK6 gene, the target sequence osmapk6(386) was designed; the corresponding weak mutant primers for the osmapk6(386) mutant are FP5 / RP5 and FP6 / RP6;

[0073] The primer FP5 sequence is: 5′-TGCAGGTTGAACGCAAGGCCTTCTGTTTC-3′,

[0074] The primer RP5 sequence is: 5′-CTCTGAAACAGAAGGCCTTGCGTTCAACC-3′.

[0075] The primer FP6 sequence is: 5'-GTGCAGGATCTAATCTGACCAAGAAGGCCTTGCAAATGAAA-3',

[0076] The primer RP6 sequence is: 5'-TCAATTTCATTTGCAAGGCCTTCTTGGTCAGATTAGATCCT-3'.

[0077] Based on the insertion of a T base after the 1164 bp C-terminus of the OsMAPK6 gene, the target sequence osmapk6(388) was designed; the corresponding weak mutant primers for the osmapk6(388) mutant are FP7 / RP7 and FP8 / RP8;

[0078] The primer FP7 sequence is: 5′-TGCAAAGGATCTAATCTACCAAGAGTTTC-3′.

[0079] The primer RP7 sequence is: 5′-CTCTGAAACTCTTGGTAGATTAGATCCTT-3′.

[0080] The primer FP8 sequence is: 5′-GTGCGCAAGGCCTTCATTGGTAGATTAGTAAGGAAT-3′,

[0081] The primer RP8 sequence is: 5′-TCAAATTCCTTACTAATCTACCAATGAAGGCCTTGC-3′.

[0082] Based on the insertion of a T base after the 1179 bp C-terminus of the OsMAPK6 gene, the target sequence osmapk6(393) was designed; the corresponding weak mutant primers for the osmapk6(393) mutant are FP9 / RP9 and FP10 / RP10;

[0083] The primer FP9 sequence is: 5′-TGCAACACCAGCTACTGGTAATCAGTTTC-3′,

[0084] The primer RP9 sequence is: 5′-CTCTGAAACTGATTACCAGTAGCTGGTGT-3′.

[0085] The primer FP10 sequence is: 5′-GTGCGGCCTTGCGTTCTAACCCTGATTACCAGTAGCATAATAAA-3′.

[0086] The primer RP10 sequence is: 5′-TCAATTTATTATGCTACTGGTAATCAGGGTTAGAACGCAAGGCC-3′.

[0087] 2. Take 20 µl of the corresponding weak mutant primer and universal primer at a concentration of 100 µM, respectively, incubate at 90 °C for 30 seconds, and then cool to room temperature to allow the complementary primers to anneal and form 4 double-stranded targeting adapters.

[0088] Taking osmapk6(379) as an example, take 20 µl (100 µM concentration) of each of the corresponding weak mutant primers FP1 / RP1 and FP2 / RP2, universal primers FP / RP and FP' / RP', incubate at 90℃ for 30 seconds, and then slowly cool to room temperature to allow the complementary primers to anneal and form a double-stranded targeting adapter.

[0089] 3. The four double-stranded targeting adapters were incubated with the Epe2 vector, BsaⅠ, and T4 DNA ligase, respectively; the reaction systems are shown in Table 1.

[0090] Table 1

[0091] Epe2 vector 1µl Double-chain target connector 1 1µl Double-chain target connector 2 1µl Double-chain target connector 3 1µl Double-chain target connector 4 1µl rcutsmart buffer 1µl T4 DNA Ligase buffer 1µl BsaⅠ 1µl T4 DNA Ligase 1µl <![CDATA[ddH2O]]> 1µl

[0092] The incubation and ligation reaction program was set as follows: 37℃, 5 min; 25℃, 5 min; 25 cycles; 80℃, 10 min.

[0093] 4. Transformation of ligation products into E. coli: (1) Take all the ligation products from step 3 and add them to 100 µL of E. coli Top10 competent cells. Gently tap the bottom of the tube several times with your finger to mix it. Let it stand on ice for 30 minutes. (2) Place the centrifuge tube in a 42℃ water bath for 90 seconds, and then quickly transfer it to ice to cool for 2 minutes. (3) Add 800 µL of antibiotic-free LB liquid medium to the tube and incubate at 37℃ and 120 rpm for 1 hour to allow the cells to recover. (4) Take an appropriate amount of the recovered bacterial solution and spread it evenly on an LB solid plate containing 50 µg / mL kanamycin for positive clone screening.

[0094] 5. Identify positive clones:

[0095] Single colonies were picked, expanded, and plasmids were extracted. Sequencing was performed using ZTJD-F primers. After sequence alignment, positive plasmids were selected for subsequent experiments.

[0096] The ZTJD-F primer sequence is: GTAGTTCAAGGCGGCGAAGTA.

[0097] Example 2

[0098] Transformation of Agrobacterium EHA105:

[0099] 1. Thawing of competent cells

[0100] Take Agrobacterium EHA105 competent cells stored at -80℃ and immediately place them on ice to thaw slowly.

[0101] 2. Plasmid transformation

[0102] Add 0.5–1 µg of the target plasmid (prepared in Example 1) to 100 µL of thawed competent cells, mix gently, and incubate on ice for 30 min.

[0103] 3. Freeze-heat shock treatment

[0104] The mixture was quickly placed in liquid nitrogen and frozen for 5 minutes, and then immediately transferred to a 37°C water bath for heat shock for 5 minutes.

[0105] 4. Bacterial resuscitation

[0106] After heat shock, the sample was quickly placed on ice to cool for 2 min, then 800 µL of antibiotic-free LB liquid medium was added. The sample was then placed in a full-temperature shaker (MKN) and cultured at 28°C and 120 rpm for 4–5 h.

[0107] 5. Resistance plate screening

[0108] After the culture was completed, the bacterial cells were collected by centrifugation, most of the supernatant was discarded, and the bacterial cells were resuspended in the remaining liquid and evenly spread on LB solid plates containing kanamycin (50 µg / mL, Amresco) and rifampin (50 µg / mL, Amresco). The plates were incubated upside down at 28°C for about 3 days.

[0109] 6. Identification and Preservation of Positive Clones

[0110] After single colonies have grown on the plates, positive clones are screened by colony PCR. Verified positive clones are picked and inoculated into LB broth containing the appropriate antibiotic, and cultured at 28°C and 180 rpm for 16 h with shaking. The bacterial culture is then mixed with 30% glycerol at a 1:1 (v / v) ratio and stored at -80°C to obtain the Agrobacterium engineered strain containing the target plasmid. This stored bacterial culture can be directly used for activation in subsequent infection experiments.

[0111] Example 3

[0112] Obtaining the osmapk6 mutant:

[0113] 1. Agrobacterium-mediated infection of rice callus

[0114] (1) Take out the Agrobacterium strain containing the target plasmid preserved in Example 2, and inoculate it into LB liquid medium containing kanamycin (50 µg / mL) and rifampin (50 µg / mL) at a ratio of 1:100, and culture overnight at 28°C and 180 rpm with shaking.

[0115] Wait until the bacterial culture reaches an orange juice-like turbidity (OD).600 When ≈ 1.0), it is used for subsequent infection.

[0116] (2) Take 500 µL of bacterial culture into a 1.5 mL centrifuge tube, centrifuge at 5000 rpm and 28℃ for 3 min, discard the supernatant and collect the bacterial pellet. Gently resuspend the bacterial pellet in 300 µL of co-culture medium containing 20 µg / mL acetylsyringone (Aldrich).

[0117] (3) Select callus tissue of Suijing 18 (SJ18-SJ18-Jing type conventional rice variety) with good growth status and transfer it to a 50 mL centrifuge tube, with the volume about 5 mL.

[0118] (4) Add 20 mL of co-culture medium containing 20 µg / mL acetylsuccinone to a centrifuge tube containing callus tissue, then add the above 300 µL bacterial suspension, mix gently for 2–3 min to complete the infection.

[0119] (5) Discard the co-culture medium, transfer the callus tissue to a culture dish lined with filter paper, and aspirate excess liquid for about 1 minute. Then transfer the callus tissue to a moistened filter paper placed on the surface of the solid co-culture medium and co-culture at 28°C in the dark for 2-3 days.

[0120] 2. Resume culture

[0121] (1) After co-culture, the callus tissue was transferred to a 50 mL centrifuge tube and washed 4-5 times with sterile water containing 400 µg / mL carbenicillin (Amresco) for 1 min each time to remove Agrobacterium.

[0122] (2) Wash the callus tissue 2-3 times with sterile water, dry it, and then transfer it to a recovery culture medium containing 400 µg / mL carbenicillin. Recover the tissue under continuous light at 28°C for 4-5 days.

[0123] 3. Screening and Cultivation

[0124] After recovery culture, the callus tissue was transferred to a selection medium containing 400 µg / mL carbenicillin and 50 µg / mL hygromycin (Roche) and cultured under continuous light at 28°C for about 30 days.

[0125] 4. Differentiation culture

[0126] The resistant callus tissue that survived the screening culture was transferred to the differentiation medium. One cluster of callus tissue was inoculated into each bottle and cultured under continuous light at 28°C for about 30 days to induce the differentiation of transgenic seedlings.

[0127] 5. Molecular identification of transgenic plants

[0128] (1) Take about 20 mg of transgenic seedling leaf sample, place it in a 2 mL centrifuge tube, add sterile steel beads, freeze it with liquid nitrogen, and then shake and grind it into powder.

[0129] (2) Remove the steel ball, add 700 µL of DNA lysis buffer, and incubate at 65°C for 30 min to fully lyse.

[0130] (3) Add an equal volume of chloroform, mix vigorously by inverting, and let stand at room temperature for 10 min.

[0131] (4) Centrifuge at 12000 rpm and 4℃ for 10 min.

[0132] (5) Take the supernatant into a new tube, add an equal volume of isopropanol, invert and mix well, and let stand at room temperature for 10 min.

[0133] (6) Centrifuge at 12000 rpm and 4℃ for 10 min, and discard the supernatant.

[0134] (7) Wash the precipitate with 70% ethanol, centrifuge again and discard the supernatant, then air dry at room temperature.

[0135] (8) Add 50~100 µL of deionized water to dissolve the DNA and obtain a crude DNA solution for subsequent PCR identification.

[0136] (9) Using crude DNA as a template, PCR amplification was performed using primers FP11 (5′-ACTTTTGACCTGATTTTCTCT-3′) and RP11 (5′-ATCCTCTTCCATTGACCACGT-3′) according to the Takara LA Taq DNA Polymerase instructions. The PCR products were sent to Genewiz for sequencing, and plants homozygous for editing different target sites (i.e., weak mutants) were screened to obtain plants.

[0137] Example 4

[0138] Functional identification of osmap6 mutants:

[0139] The Suijing 18 (SJ18) rice variety was used as a wild-type control and planted together with the mutants osmapk6(379), osmapk6(383), osmapk6(386), osmapk6(388), and osmapk6(393) in an experimental field in Harbin, Heilongjiang Province. All materials were managed uniformly using conventional agronomic practices under natural long-day conditions. Key agronomic traits such as plant height, leaf angle, panicle length, number of grains per panicle, grain length, grain width, seed setting rate, and panicle elongation were systematically investigated and statistically analyzed.

[0140] like Figure 1The diagram shows the C-terminal coding sequence of the OsMAPK6 gene and its corresponding protein sequences in Suijing 18 (wild type) and its mutant lines osmapk6(379), osmapk6(383), osmapk6(386), osmapk6(388), and osmapk6(393). An asterisk (*) indicates a stop codon; a hyphen (–) indicates a deleted sequence; and red letters mark inserted nucleotides.

[0141] like Figure 2 The image shows the sequencing maps of the OsMAPK6 gene in the materials SJ18, osmapk6(379), osmapk6(383), osmapk6(386), osmapk6(388), and osmapk6(393).

[0142] By using guided editing technology, an A base was deleted at 1140 bp of the OsMAPK6 gene, resulting in a frameshift mutation and producing a truncated OsMAPK6 protein of 379 amino acids, thus obtaining the osmapk6(379) mutant. Inserting a T base after 1149 bp caused premature translation termination, resulting in a protein of 383 amino acids (osmapk6(383)); inserting a G base after 1159 bp caused premature translation termination, resulting in a protein of 386 amino acids (osmapk6(386)); inserting a T base after 1164 bp caused premature translation termination, resulting in a protein of 388 amino acids (osmapk6(388)); similarly, inserting a T base after 1179 bp caused premature translation termination, resulting in a protein of 393 amino acids (osmapk6(393)). Figure 1 , 2 (As shown).

[0143] like Figures 3-12 The figure shows the statistical results of relevant agronomic traits of Suijing 18 (wild type) and its mutant lines osmapk6(379), osmapk6(383), osmapk6(386), osmapk6(388), and osmapk6(393). The osmapk6(379) mutant exhibits multiple defects similar to those of the previously reported dsg1 mutant, including reduced plant height, erect leaves, smaller grains, decreased seed setting rate, shortened ear length, and ear-clogging phenomenon. Figures 3-12 The osmapk6(383) line exhibited the same severe phenotype: a seed setting rate of only about 17%, a plant height of about half that of the wild type, nearly vertical leaves, significantly reduced ear length and grain size, and severe ear enclosedness ( Figures 3-12In contrast, although osmapk6(386) still exhibited growth and developmental defects, its phenotype was significantly milder than that of osmapk6(379) and osmapk6(383): although its plant height was lower than that of the wild type, it was more than 1.5 times taller than osmapk6(379) and osmapk6(383). Figure 3 , Figure 4 Although the plant type remained compact, the leaf angle was significantly larger than that of osmapk6(379) and osmapk6(383). Figure 5 The grain length and width, ear length and other traits are between those of osmapk6(379) and osmapk6(383) and the wild type, and the seed setting rate has recovered to 80%. Figures 6-11 Although the spikelet neck is still shorter than that of the wild type, the phenomenon of complete panicle envelopment has been completely eliminated. Figure 12 These results indicate that while the deletion of the C-terminal 12 amino acids affects OsMAPK6 function, this effect has been significantly mitigated. Furthermore, osmapk6(388) and osmapk6(393) showed no significant differences from the wild type in any agronomic traits. Figures 3-12 This indicates that removing the 10 amino acids at the C-terminus does not affect the regulatory function of OsMAPK6 in rice growth and development.

[0144] The above experiments fully demonstrate that guided editing can introduce mutations at different sites (e.g., the 379-386 interval) at the C-terminus of the OsMAPK6 gene, thereby obtaining a series of weak mutant osmapk6 variants with different editing types. This solves the bottleneck problem of embryonic lethality caused by traditional methods of OsMAPK6 knockout, which prevents the acquisition of homozygous mutants. Simultaneously, it provides important germplasm resources for the genetic function research of OsMAPK6.

Claims

1. A method for creating a weak mutant of osmapk6, characterized in that, This method edits the C-terminal base site of the OsMAPK6 gene to obtain a weak mutant encoding the OsMAPK6 protein.

2. The method for creating a weak mutant of osmapk6 according to claim 1, characterized in that, The editor was guided to target the C-terminal base site of the OsMAPK6 gene.

3. The method for creating a weak mutant of osmapk6 according to claim 1, characterized in that, Based on the deletion of an A base at the 1140bp C-terminus of the OsMAPK6 gene, the target sequence osmapk6(379) was designed; the primers for the osmapk6(379) mutant are FP1 / RP1 and FP2 / RP2; The primer FP1 sequence is: 5'-TGCACGAGCAGCATGCATTGTCCGGTTTC-3', The primer RP1 sequence is: 5'-CTCTGAAACCGGACAATGCATGCTGCTCG-3'; The primer FP2 sequence is: 5'-GTGCATCCTTCATTTGTCCTCGGACAATGCATGCATATAAAC-3', The primer RP2 sequence is: 5'-TCAAGTTTATATGCATGCATTGTCCGAGGACAAATGAAGGAT-3'.

4. The method for creating a weak mutant of osmapk6 according to claim 1, characterized in that, Based on the insertion of a T base after the 1149th bp of the C-terminus of the OsMAPK6 gene, the target sequence osmapk6(383) was designed; the primers for the osmapk6(383) mutant are FP3 / RP3 and FP4 / RP4; The primer FP3 sequence is: 5′-TGCAATTGTCCGAGGAACAAATGAGTTTC-3′. The primer RP3 sequence is: 5′-CTCTGAAACTCATTTGTTCCTCGGACAAT-3′. The primer FP4 sequence is: 5'-GTGCTAGATTAGATCACTTCATTTGTTCCTCGCTTCCTCT-3', The primer RP4 sequence is: 5'-TCAAAGAGGAAGCGAGGAACAAATGAAGTGATCTAATCTA -3'.

5. The method for creating a weak mutant of osmapk6 according to claim 1, characterized in that, Based on the insertion of a G base after the 1159th bp of the C-terminus of the OsMAPK6 gene, the target sequence osmapk6(386) was designed; the primers for the osmapk6(386) mutant are FP5 / RP5 and FP6 / RP6; The primer FP5 sequence is: 5′-TGCAGGTTGAACGCAAGGCCTTCTGTTTC-3′, The primer RP5 sequence is: 5′-CTCTGAAACAGAAGGCCTTGCGTTCAACC-3′. The primer FP6 sequence is: 5'-GTGCAGGATCTAATCTGACCAAGAAGGCCTTGCAAATGAAA-3', The primer RP6 sequence is: 5'-TCAATTTCATTTGCAAGGCCTTCTTGGTCAGATTAGATCCT-3'.

6. The method for creating a weak mutant of osmapk6 according to claim 1, characterized in that, Based on the insertion of a T base after the 1164 bp C-terminus of the OsMAPK6 gene, the target sequence osmapk6(388) was designed; the primers for the osmapk6(388) mutant are FP7 / RP7 and FP8 / RP8; The primer FP7 sequence is: 5′-TGCAAAGGATCTAATCTACCAAGAGTTTC-3′. The primer RP7 sequence is: 5′-CTCTGAAACTCTTGGTAGATTAGATCCTT-3′. The primer FP8 sequence is: 5′-GTGCGCAAGGCCTTCATTGGTAGATTAGTAAGGAAT-3′, The primer RP8 sequence is: 5′-TCAAATTCCTTACTAATCTACCAATGAAGGCCTTGC-3′.

7. The method for creating a weak mutant of osmapk6 according to claim 1, characterized in that, Based on the insertion of a T base after the 1179 bp C-terminus of the OsMAPK6 gene, the target sequence osmapk6(393) was designed; the primers for the osmapk6(393) mutant are FP9 / RP9 and FP10 / RP10; The primer FP9 sequence is: 5′-TGCAACACCAGCTACTGGTAATCAGTTTC-3′, The primer RP9 sequence is: 5′-CTCTGAAACTGATTACCAGTAGCTGGTGT-3′. The primer FP10 sequence is: 5′-GTGCGGCCTTGCGTTCTAACCCTGATTACCAGTAGCATAATAAA-3′. The primer RP10 sequence is: 5′-TCAATTTATTATGCTACTGGTAATCAGGGTTAGAACGCAAGGCC-3′.

8. The method for creating a weak mutant of osmapk6 according to claim 3, 4, 5, 6 or 7, characterized in that, This method is performed in the following steps: I. Vector Construction: Based on the designed target sequence, the corresponding weak mutant primers and universal primers FP / RP and FP' / RP' are annealed into double strands and then ligated into the Epe2 vector through cleavage-ligation to complete the construction of the guide editing vector; II. Genetic transformation: The above-mentioned guide editing vector was transformed into Escherichia coli, positive clones were expanded and cultured to extract plasmids, and then the extracted plasmids were used to transform Agrobacterium EHA105 strain. Subsequently, the Agrobacterium strain containing the target plasmid was used to infect rice callus tissue, and differentiation culture was carried out to obtain transgenic rice plants. The primer FP sequence is: 5′-AGAGCTATGCTGGAAACAGCATAGCAAGTTGAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGCACCGAGTCG-3′, The primer RP sequence is: 5′-GCACCGACTCGGTGCCACTTTTTCAAGTTGATAACGGACTAGCCTTATTTCAACTTGCTATGCTGTTTCCAGCATAG-3′; The primer FP' sequence is: 5′-TTGACGCGGTTCTATCTAGTTACGCGTTAAACCAACTAGAAA-3′, The primer RP' sequence is: 5′-GGCCTTTCTAGTTGGTTTAACGCGTAACTAGATAGAACCGCG-3′.

9. The method for creating a weak mutant of osmapk6 according to claim 8, characterized in that, In step one, 20 µl each of the corresponding weak mutant primer and the universal primer at a concentration of 100 µM were taken and incubated at 90 °C for 30 seconds, and then cooled to room temperature to allow the complementary primers to anneal and form four double-stranded targeting adapters.

10. The method for creating a weak mutant of osmapk6 according to claim 9, characterized in that, In step one, the four double-stranded targeting adapters are incubated with the Epe2 vector, BsaⅠ, and T4 DNA ligase, respectively. The incubation and ligation reaction were carried out at 37°C for 5 min; 25°C for 5 min; 25 cycles; and 80°C for 10 min.

Images