CN113403321B - Application of OsAKR4C10 in creating non-transgenic glyphosate-resistant rice germplasm resources - Google Patents

Abstract

The invention discloses application of OsAKR4C10 in creating non-transgenic glyphosate-resistant rice germplasm resources. The research of the invention shows that the rice OsAKR4C10 gene is a glyphosate transporter gene of rice, and the rice gene OsAKR4C10 is knocked out to inhibit the absorption and accumulation of the rice on the glyphosate, reduce the glyphosate content in the rice and improve the resistance of the rice on the glyphosate; the glyphosate absorption and accumulation of the rice variety can be adjusted genetically, and the glyphosate-resistant rice variety is cultivated; has wide application prospect.

Description

Role of OsAKR4C10 in creating non-transgenic glyphosate-resistant rice germplasm resources application

technical field

The invention relates to the field of plant genetic engineering, in particular to the application of the rice gene OsAKR4C10 in creating non-transgenic glyphosate-resistant rice germplasm resources by regulating the absorption and accumulation of glyphosate in rice.

Background technique

Rice is the main food for about 2.2 billion people in the world. Proper weed control is the key to ensuring the quality and yield of rice. Manual removal and biological control are efficient and cost-effective. Herbicides can overcome these disadvantages and are still the current The most important means of prevention. At present, there are more than a dozen types of herbicides in paddy fields, with different control targets and different technical requirements for use, which may easily lead to problems such as herbicide injury, excessive pesticide residues, and increased populations of resistant weeds.

Glyphosate is the leading herbicide all year round because of its broad-spectrum, high efficiency, harmlessness to humans and animals, and good environmental compatibility. However, it is also restricted by its non-selective killing of rice. Apply on a large scale in the field. And by cultivating glyphosate-resistant rice is a commonly used coping method at present. For example, Chinese patent CN107129993A discloses a modified glyphosate-resistant gene and a breeding method for glyphosate-resistant rice. Chinese patents CN106497922A, CN106497924A, and CN106497923A disclose A method for constructing borer-resistant and glyphosate-resistant transgenic rice. The above-mentioned methods are all through genetically transforming an exogenous glyphosate-resistant gene into rice, thereby cultivating glyphosate-resistant transgenic rice varieties. However, the problems of long R&D time, low efficiency, and gene contamination in the transfer of resistance genes from exogenous sources can be better solved by excavating the endogenous rice glyphosate transporter gene and inactivating it through plant genetic engineering technology. above question. However, there are few reports on the related genes that regulate the uptake and accumulation of glyphosate in rice.

Contents of the invention

The purpose of the present invention is to overcome the above-mentioned defects and deficiencies in the prior art, and provide the application of the rice OsAKR4C10 gene in regulating the absorption and accumulation of glyphosate in rice.

The second object of the present invention is to provide the application of the rice OsAKR4C10 gene in creating non-transgenic glyphosate-resistant rice germplasm resources.

Above-mentioned purpose of the present invention is given to realize by following technical scheme:

The research of the present invention shows that knocking out the rice OsAKR4C10 gene can inhibit the absorption and accumulation of glyphosate in rice, reduce the glyphosate content in rice, and improve the resistance of rice to glyphosate; Perform gene editing to obtain glyphosate-resistant rice varieties, and use glyphosate-resistant rice varieties obtained through sexual or asexual reproduction. Therefore, the present invention provides the following applications about rice OsAKR4C10 gene and OsAKR4C10 protein:

Application of the rice OsAKR4C10 gene in regulating the absorption and accumulation of glyphosate in rice, the nucleotide sequence of the rice OsAKR4C10 gene is shown in SEQ ID NO: 2; or at least the same as the nucleotide sequence shown in SEQ ID NO: 2 It has 50% homology and encodes the nucleotide sequence of the amino acid shown in SEQ ID NO:1.

The application of the rice OsAKR4C10 protein in regulating the absorption and accumulation of glyphosate in rice, the amino acid sequence of the rice OsAKR4C10 protein is shown in SEQ ID NO: 1; or the amino acid sequence shown in SEQ ID NO: 1 passes through one or several amino acids Substitutions and/or deletions of residues and/or additions still have the same function in the amino acid sequence of the derivative protein.

Amino acid sequence shown in SEQ ID NO: 1:

MAKHFVLNTGAKIPSVGLGTWQSDPGVVGDAVYAAVKAGYRHIDCARMYKNENEVGIALKKLFEEGVVKREDLFITSKLWCDCHAPEDVPESLDKTLSDLQLEYLDLYLIHWPFRVKKGSGISNTEDYIPPDIPSTWGAMEKLYDSGKSRAIGVSNFSSKKLGDLLAVACVPPAVDQVECHPGWQQTKLHNFCQSTGVHLSAYSPLGSPGSTWMNSNVLKESVIISIAEKLGKTPAQVALHWNIQMGHSVLPKSVTEERIKQNIDVYDWSIPEDLLVKFSEIKQVRLLRGDVIVNPHSVYKTHEELWDGEI.

Nucleotide sequence shown in SEQ ID NO: 2:

atggcgaagcatttcgtgctcaacaccggcgccaagatcccctcggtggggctcggcacctggcagtccgacccgggcgtcgtcggcgacgccgtctacgccgctgtcaaggcggggtaccggcacatcgattgcgccagaatgtacaaaaatgaaaatgaggtggggatagctctgaagaagctatttgaagaaggtgttgtcaagcgtgaagatttatttatcacatctaagctatggtgtgattgtcatgccccagaggatgtgcctgagtcactagacaaaactctgagtgacttacagcttgagtacctggatctttaccttattcattggccattcagagtcaagaagggctcaggcattagtaacactgaagactacataccacctgacatcccatctacctggggagcaatggagaagctatatgattctggtaaatctcgtgccattggtgtaagtaacttctcatcaaaaaaactgggtgacctgcttgctgtagcctgtgtacctccagctgttgatcaggtagaatgccatcctggttggcagcaaacgaagctacataacttctgccagtcaactggcgttcatctttctgcatactcgcctctaggttcacctggttcaacatggatgaacagtaacgtccttaaggaatccgtcatcatctcaattgcagagaagctcggcaaaactcctgcacaagtggcactgcactggaacattcagatgggtcacagtgtactcccaaaaagtgtgaccgaagaaaggataaagcagaacatagatgtttatgactggtctattccagaggacttgcttgttaagttctctgagattaagcaggttaggcttctcaggggcgacgtcattgttaatccccacagcgtttataagacccatgaggagctctgggacggcgaaatttag

Application of the rice OsAKR4C10 gene in cultivating glyphosate-resistant rice varieties, the nucleotide sequence of the rice OsAKR4C10 gene is shown in SEQ ID NO: 2; % homology, and the nucleotide sequence encoding the amino acid shown in SEQ ID NO:1.

The application of rice OsAKR4C10 protein in cultivating glyphosate-resistant rice varieties, the amino acid sequence of the rice OsAKR4C10 protein is as shown in SEQ ID NO: 1 or the amino acid sequence shown in SEQ ID NO: 1 after one or several amino acid residues Substitution and/or deletion and/or addition of the amino acid sequence of the derivative protein still having the same function.

Specifically, glyphosate-resistant rice varieties are obtained by inhibiting the expression of the OsAKR4C10 gene in rice, or inhibiting the expression and/or activity of the OsAKR4C10 protein.

Optionally, said suppressing the expression of OsAKR4C10 gene in rice, or suppressing the expression level and/or activity of OsAKR4C10 protein is carried out by conventional methods in the field such as gene editing, RNA interference, homologous recombination or gene knockout.

Optionally, the gene editing is to construct a rice CRISPR-Cas9 system, which contains an sgRNA that recognizes the rice OsAKR4C10 gene target sequence.

Optionally, the target sequence is shown in SEQ ID NO:3.

AGGTGCCGAGCCCCACCGAGGGG (SEQ ID NO: 3).

In the present invention, through the functional identification of the glyphosate transporter gene of rice, the plant gene editing technology is used to inactivate it, and then the glyphosate-resistant rice germplasm resources with the mechanism of low glyphosate absorption are obtained. Its advantages are: taking advantage of glyphosate’s high-efficiency killing characteristics to simplify rice field weeding and prolong the suitable period for weeding; avoiding long research and development time, low efficiency, and genetic pollution caused by exogenous transfer of resistance genes to conventional glyphosate-resistant crops problems; the most important thing is to achieve glyphosate transport selectivity in weeds and crops, and reduce the accumulation and application of glyphosate in rice while killing weeds, which is friendly to the environment and ecology, and can provide " Accelerating the innovation of biological breeding to ensure national food security” provides a new idea.

Compared with the prior art, the present invention has the following beneficial effects:

The invention provides the application of the rice OsAKR4C10 gene in regulating the absorption and accumulation of glyphosate in rice. The research of the invention shows that knocking out the rice OsAKR4C10 gene can inhibit the absorption and accumulation of the herbicide glyphosate in rice and reduce the absorption and accumulation of glyphosate in rice plants. At the same time, knocking out the rice OsAKR4C10 gene can genetically regulate the absorption and accumulation of glyphosate in rice, improve the resistance of rice to glyphosate, and breed glyphosate-resistant rice varieties. The means of combining glyphosate absorption and accumulation with the resistance mechanism of the present invention provides a good choice for the research of herbicide-resistant crops, can be used to create non-transgenic glyphosate-resistant rice germplasm resources, and has great application prospects .

Description of drawings

Fig. 1 is a sequence alignment result of Zhonghua 11 and the mutant of the OsAKR4C10 gene.

Fig. 2 is a diagram showing the real-time fluorescent quantitative PCR detection results of OsAKR4C10 gene at different times after glyphosate spraying treatment of No. 11 Zhonghua. (A) OsAKR4C10 gene expression in shoots of rice; (B) OsAKR4C10 gene expression in shoots of rice.

Figure 3 shows the tolerance of Zhonghua 11 and OsAKR4C10 gene mutants to glyphosate during seed germination. (A) Comparison of seedling shoot length between WT and osakr4c10 in tissue culture media with different concentrations of glyphosate; (B) Comparison of shoot length between WT and osakr4c10 in tissue culture media with different concentrations of glyphosate; (C) Comparison of shoot length between WT and osakr4c10 in tissue culture media with different concentrations of glyphosate Comparison of root length of osakr4c10 seedlings in tissue culture medium with different concentrations of glyphosate.

Figure 4 is a graph showing the results of glyphosate accumulation in different parts of the roots of Zhonghua 11 and the OsAKR4C10 gene mutant after absorbing glyphosate at the seedling stage. (A) Glyphosate accumulation in leaves; (B) Glyphosate accumulation in stems; (C) Glyphosate accumulation in roots; (D) LC-MS/MS detection of glyphosate and its metabolite AMPA peak picture.

Figure 5 shows the tolerance of Zhonghua 11 and the OsAKR4C10 gene mutant after soaking glyphosate in adult plant leaves. (A) Phenotype observation of leaves soaked in 5.75mmol/L glyphosate; (B) Phenotype observation of leaves soaked in 11.5mmol/L glyphosate; (C) Phenotype observation of leaves soaked in 5.75mmol/L glyphosate Fv/Fm of hind leaves; (D) Fv/Fm of leaves soaked in 11.5mmol/L glyphosate.

Figure 6 shows the tolerance of Zhonghua 11 and the OsAKR4C10 gene mutant after spraying glyphosate at the adult plant stage.

Figure 7 shows the changes in chlorophyll content of Zhonghua 11 and OsAKR4C10 gene mutants after spraying glyphosate at the adult plant stage. (A) Changes of chlorophyll content of WT and osakr4c10 rice leaves under 3.6mM glyphosate spraying; (B) Changes of chlorophyll content of WT and osakr4c10 rice leaves under 10.8mM glyphosate spraying.

Figure 8 shows the tolerance of the background OsAKR4C10 gene mutants of Zhonghua No. 1 and Guiyu No. 11 and their offspring after spraying glyphosate at the adult plant stage.

Detailed ways

The present invention will be further described below in conjunction with the accompanying drawings and specific embodiments, but the embodiments do not limit the present invention in any form. Unless otherwise specified, the reagents, methods and equipment used in the present invention are conventional reagents, methods and equipment in the technical field.

Unless otherwise specified, the reagents and materials used in the following examples are commercially available.

In the following examples, Escherichia coli DH5α and Agrobacterium EHA105 are commonly used strains and can be purchased commercially; the rice variety is wild-type Zhonghua 11 (a publicly used rice variety, commercially available). The primers used in the examples were synthesized by Shenzhen Huada Gene Company, and the sequencing was performed at Shenzhen Huada Gene Company.

Quantitative experiments in the following examples were all set up to repeat the experiments three times, and the results were averaged.

Example 1 CRISPR Knockout Construction of OsAKR4C10 Mutant Plants

1. Use the CRISPR/Cas9 system to select the target sequence according to the exon sequence of OsAKR4C10

Using the simple and efficient CRISPR/Cas9 system, select the specific target sequence according to the OsAKR4C10 exon sequence, the target sequence: 5'-AGGTGCCGAGCCCCACCGAGGGG-3'. The target sequence is directed against the OsAKR4C10 gene and specifically inactivates the OsAKR4C10 protein.

2. Construct the pCRISPR/Cas9 recombinant vector containing the above target sequence fragment

1) According to the target sequence, design adapter primers with sticky ends

Add the designed target sequence to the pCRISPR/Cas9 system's specific sticky end adapter, and synthesize the complete adapter primer.

F1: 5'-TGTGTGGGTGCCGAGCCCCACCGAG-3';

R1: 5'-AAACCTCGGTGGGGCTCGGCACCCA-3'.

2) Annealing the adapter primer with sticky ends to complement each other to form small double-stranded fragments with sticky ends

Dilute the F1 primer and R1 primer to a solution with a concentration of 10 μM, take 10 μL each and mix well, perform annealing reaction in a PCR machine, and lower it from 98°C to 22°C, so that the F1 primer and R1 primer are complementary to form a double strand with sticky ends small snippet.

3) Digest the original vector pOs-sgRNA containing sg-RNA (TAKARA Cat#632640)

The original vector pOs-sgRNA containing sg-RNA was digested with restriction endonuclease BsaI to generate cohesive ends complementary to the cohesive ends of the target sequence. The system for digesting the original pOs-sgRNA vector with BsaⅠ is: 2 μL of 10×buffer, 1 μL of BsaⅠ enzyme, 4 μg of pOs-sgRNA vector, supplemented with ddH ₂ O to 20 μL, and digested at 37°C for 12 hours. After the digested product was electrophoresed on 1% agarose gel to check the size of the band, the kit (OMEGA Cat#D2500-02) was used to recover and purify the digested product to obtain the digested pOs-sgRNA vector, and add sterilized ddH ₂ O was dissolved and the concentration was measured for use.

4) Ligate the small double-stranded fragment with cohesive ends to the digested pOs-sgRNA vector to form a recombinant vector containing the target sequence and sg-RNA

Use T4 ligase to connect the double-stranded small fragment in step 2) and the pOs-sgRNA vector digested in step 3) to form a complete recombinant vector containing the target sequence for OsAKR4C10 protein and sg-RNA. The 15 μL ligation system is: 1.5 μL of 10×T4 ligation buffer, 4 μL of small double-stranded fragments, 3 μL of digested pOs-sgRNA vector, 1 μL of T4 DNA ligase, supplemented with ddH ₂ O to 15 μL, and ligated at 16°C for 12 hours. The ligation product was transformed into Escherichia coli DH5α, cultivated overnight on a kana-resistant LB plate (containing 10 mg/L kanamycin), and the positive strains were selected for sequencing to obtain a recombinant vector containing the target sequence and sg-RNA with correct sequencing.

5) Use LR mix to perform LR reaction recombination of the recombinant vector containing the target sequence and sg-RNA and the vector pH-Ubicas9-7 containing Cas9 to form a complete recombinant vector containing the target sequence-sg-RNA+Cas9

Use LR mix (Shanghai Beinuo Biotechnology Co., Ltd.) to perform LR reaction recombination on the recombinant vector obtained in step 4) and the vector pH-Ubi-cas9-7 containing Cas9 (provided by Baige Gene Technology Co., Ltd.). LR reaction system: recombinant vector containing target sequence and sg-RNA 25-50ng, pH-Ubi-cas9-7 vector 75ng, 5×LR Clonase TM buffer 1μL, TE Buffer (pH8.0) supplemented to 4.5μL, LR ClonaseTM 0.5 μL. Incubate the system at 25°C for 2 hours, add 2 μL of 2 μg/μL Proteinase K after the reaction, treat at 37°C for 10 minutes, then transfer 2 μL of the reaction product into E. coli DH5α, and gentamicin-resistant LB plate overnight at 37°C Cultivate, select positive strains for sequencing, and obtain a complete pCRISPR/Cas9-OsAKR4C10 recombinant expression vector containing the OsAKR4C10 protein target sequence-sg-RNA+Cas9 with correct sequencing.

3. Introduce the obtained complete recombinant vector containing OsAKR4C10 protein target sequence-sg-RNA+Cas9 into rice callus to obtain transgenic plants

1) The recombinant expression vector pCRISPR/Cas9-OsAKR4C10 obtained in step 2 was electroporated into Agrobacterium EHA105 (Olivia C.D, 2019) to obtain the recombinant strain AGL1/pCRISPR/Cas9-OsAKR4C10.

2) The recombinant strain AGL1/pCRISPR/Cas9-OsAKR4C10 was transformed into the rice callus of Zhonghua No. 11 by the method mediated by Agrobacterium, as follows:

Pick a single colony of AGL1/pCRISPR/Cas9-OsAKR4C10 and inoculate it in 10 mL of Agrobacterium medium (containing 50 mg/L kanamycin and 50 mg/L rifampicin), and culture it on a shaker at 180 rpm at 28°C for 2-3 days . Take 4mL of bacterial liquid, centrifuge at 4000rpm for 3min, discard the supernatant, add a small amount of AAM medium to resuspend the cells, then add 20mL of AAM medium (containing 0.1mM acetosyringone As), and incubate in the dark at 28°C and 150rpm After 1-2 hours, cultivate to about OD ₆₀₀ =0.4. Select the rice callus of Zhonghua No. 11 (hereinafter also referred to as wild-type rice) in good growth state, and immerse it in the Agrobacterium culture solution (YEP without agar), shake it at 28°C and 150-200rpm for 20min, and shake the callus The tissue was poured out, and the excess bacterial solution was blotted with sterile filter paper, and the callus was spread flat on a sterile plate containing multiple layers of filter paper, and dried on an ultra-clean table (the callus was scattered and not agglomerated), and then the callus was Tissues were transferred to co-cultivation medium and cultured in the dark for 2-3 days. The callus was transferred to the NB minimal medium containing 100 mg/L hygromycin and 400 mg/L cephalosporin for selection for 3-4 weeks (one screening). The surviving calli were transferred to the second screening medium (NB basic medium containing 100 mg/L hygromycin and 200 mg/L cephalosporin) for selection for 3 weeks. Transfer the resistant callus to the differentiation medium (containing 100mg/L hygromycin) for differentiation, and transfer the regenerated plants after rooting on the strong seedling medium containing 100mg/L hygromycin (about 3-4 weeks) In the greenhouse, transgenic plants in which the OsAKR4C10 protein is completely inactivated can be obtained from the T0 generation plants.

The media used in the above transformations were as follows:

Co-cultivation medium (Beijing Huayueyang Biotechnology Co., Ltd.): callus induction and subculture medium + As (0.1mmol/L) + glucose (10g/L), pH 5.2.

Agrobacterium-infected rice callus medium (AAM medium, Beijing Huayueyang Biotechnology Co., Ltd.): AA macroelements + AA trace elements + AA amino acids + MS vitamins + hydrolyzed casein (500 mg/L) + sucrose ( 68.5g/L) + glucose (36g/L) + As (0.1mM), pH 5.2.

NB basic medium (Beijing Huayueyang Biotechnology Co., Ltd.): N6 macroelement + B5 trace element + B5 organic component + iron salt + hydrolyzed casein (300mg/L) + proline (500mg/L) + sucrose ( 30g/L)+agar (8g/L), pH 5.8.

Callus induction and subculture medium: NB basic medium + 2,4-D (2mg/L).

Differentiation medium: NB basic medium + 6-BA (3mg/L) + NAA (1mg/L).

Strong seedling medium: 1/2MS medium + NAA (0.5mg/L) + MET (0.25mg/L).

Agrobacterium medium (YEP): 10g/L tryptone + 10g/L yeast extract + 5g/L sodium chloride + 15g/L agar.

4. Screen transgenic positive plants among transgenic plants

The DNA (OMEGA Cat#D3485-02) was extracted from the transgenic plants (T ₀ generation) transplanted in step 3, and the target sequence site was detected, and a total of 12 positive plants were detected.

5. Using transgenic positive plants to obtain mutant plants

1) Identification of mutation sites

Extract DNA (OMEGA Cat#D3485-02) from the positive plants transplanted in step 4, design specific primers F2 and R2 for the DNA fragment within 500bp containing the target site, and amplify the DNA fragment containing the target site, The amplified 289bp PCR product was purified and then sent to the company for sequencing. The sequencing results were compared with the sequence of the wild-type plant, and mutant plants were screened out.

F2: 5'-GGCCGCTGCCTACAGTAAAG-3';

R2: 5'-AGAGGAGAGGAGGAGACGC-3'.

2) The mutant plants were propagated, and the seeds were harvested from individual plants that did not contain transgenic elements such as hygromycin and Cas9 in the T1 transgenic segregation population, and a functional loss mutant was obtained, named osakr4c10. The results of mutational analysis of loss-of-function mutants and wild-type plants are shown in Figure 1.

Real-time fluorescence quantitative test after embodiment 2 glyphosate treats rice

A 10.8 mmol/L glyphosate solution was prepared, and the plants of the wild type variety Zhonghua No. 11 grown for 25 days were sprayed, and the water treatment was used as a control. RNA (OMEGA Cat#R6827-02) was extracted from the aerial part (stem and leaf) and underground part 5, 72, and 120 h after treatment, and was reverse-transcribed (Takara, PrimeScript RT reagent Kit with gDNAEraser) for real-time fluorescent quantitative PCR. The expression of OsAKR4C10 gene was detected. The experiment was repeated three times, and the results were averaged.

Real-time fluorescent quantitative PCR was performed using Bio-Rad CFX96. The PCR reaction system (20 μL) was carried out according to the product instruction manual SYBR Green Real-Time PCR Master Mix reagent (Takara), and the specific system was as follows: 10 μL SYBR Green Real-Time PCR Master Mix, 2 μL upstream and downstream primer mixture (concentration of both upstream and downstream primers was 10 μM ), 7 μL RNase-free water, 1 μL cDNA template. The specific reaction procedure is as follows: enzyme heat activation at 95°C for 30s, 1 cycle; denaturation at 95°C for 5s, extension at 60°C for 30s, a total of 40 cycles.

The primer sequences for amplifying the OsAKR4C10 gene are:

F3: 5'-AACACTGAAGACTACATACCACCT-3';

R3: 5'-ACTTACACCAATGGCACGAGA-3'.

Using UBQ2 as the internal reference gene, the primer sequence for amplifying the internal reference UBQ2 is:

F4: 5'-TGCTATGTACGTCGCCATCCAG-3';

R4: 5'-AATGAGTAACCACGCTCCGTCA-3'.

Data processing adopts the method of Comparative Ct, that is, the Ct value is the number of cycles experienced when the fluorescent signal in the PCR tube reaches the set threshold value, ΔCt=Ct(OsAKR4C10)-Ct(UBQ2), and the value of ^2-△△Ct The value measures the gene transcription level, and analyzes and compares the expression of the OsAKR4C10 gene in the samples.

The results are shown in Figure 2. After glyphosate-induced treatment for 5 h, the expression of OsAKR4C10 in the shoot of rice was up-regulated by 2.62 times compared with the control group treated with water; There was no difference between the control group. After 72 hours, there was no statistically significant difference in the expression of OsAKR4C10 gene in the aboveground or underground parts of rice between the treatment group and the clean water control group, and the expression of OsAKR4C10 gene in the aboveground part of rice in the glyphosate treatment group decreased. After 120 hours, compared with the clean water control group, there was no statistically significant difference in the expression of OsAKR4C10 gene in the aboveground part of rice; while in the underground part of rice, the induced expression of OsAKR4C10 gene in the treatment group was significantly increased by 9.71 times compared with the control group.

Example 3 Sensitivity test of rice at germination stage to glyphosate

The susceptibility test of osakr4c10 mutant and wild type rice to glyphosate at germination stage was carried out by planting tissue culture seedlings. Seeds of osakr4c10 mutant rice and Zhonghua 11 wild-type rice as a control were baked at 49°C for 4 days to recover seed vigor. The pre-planted rice seeds are first washed with 75% alcohol (prepared with absolute ethanol and sterilized water), then with 50% NaClO solution, and finally rinsed with sterile water to complete the thorough disinfection of the seeds. When planting, heat the tweezers under an alcohol lamp, and after cooling, clamp the seeds and place them in MS medium (containing 0, 10, 25, and 50 μmol/L glyphosate respectively). Germinate at 28°C in the dark, and after emergence, switch to a light-dark cycle of 12/12h, with a temperature of 30°C in light and 28°C in darkness for 15 days. Observe the phenotype and take pictures to record and measure the length of aboveground and underground parts.

The results showed that at the concentration of 10 μmol/L glyphosate, the ratio of root length to shoot length of mutant plants was significantly higher than that of wild type. At the concentration of 25 μmol/L glyphosate, the growth of osakr4c10 mutant rice seedlings was only slightly inhibited compared with the growth without glyphosate, while the wild-type rice seeds could hardly grow after germination. At a concentration of 50 μmol/L glyphosate, the seeds of osakr4c10 mutant rice seedlings could still germinate, but the further vegetative growth stage of seedlings was significantly inhibited, and the wild-type rice seeds completely turned black and shrunk to death (Fig. 3). The results showed that the deletion of OsAKR4C10 gene significantly improved the tolerance of rice seeds to glyphosate during germination and was beneficial to the growth of plants.

Embodiment 4 Seedling stage rice root absorbs glyphosate test

The osakr4c10 mutant and Zhonghua 11 rice seedlings with uniform growth after 21 days of hydroponics were transferred to 50mL centrifuge tubes, with 6 plants in each group as a replicate, and 3 replicates per treatment. Before spraying, place the rice roots in 0.5mmol/LCaCl ₂ (pH=5.8) for 1 hour pre-cultivation, then replace with 0.5mM CaCl ₂ (pH=5.8) containing 0.5mmol/L glyphosate liquid for cultivation Three days later, the roots, stems, and leaves of rice were collected and weighed for storage. The sample pretreatment method is as follows: grind the sample to powder with liquid nitrogen, extract each 0.2g sample with 1mL sterilized water, transfer the extract mixture to a 10mL centrifuge tube, vortex for 5min, sonicate for 30min, and centrifuge at 6000rpm for 5min. Take 1mL of the supernatant, add 0.4mL CH ₂ Cl ₂ to remove impurities, vortex mix for 3min, centrifuge at 6000rpm for 5min, take 1mL of the supernatant aqueous phase, add 50mg C18 for purification, vortex for 3min, centrifuge at the highest speed for 10min, take 0.8mL Add 0.4mL of 5% sodium borate buffer solution and 0.4mL of 1.0g/L FMOC-Cl acetone solution to the centrifuged supernatant, vortex and mix well, and place it at 37°C overnight for derivatization treatment. μm organic filter membrane, LC-MS/MS to determine the amount of glyphosate and AMPA.

The results showed that the glyphosate content of the osakr4c10 mutant was lower than that of wild-type rice in roots, stems, and leaves after 24 h, indicating that the glyphosate absorption ability of OsAKR4C10 was reduced after 72 h; The glyphosate content in roots and roots was lower than that of wild-type rice, but there was no significant difference between the glyphosate content in stems of osakr4c10 mutants and wild-type, which may be the result of the saturation of the upward transport capacity of rice roots, while all The glyphosate metabolite AMPA was not detected in the samples (Figure 4). The results indicated that the OsAKR4C10 gene was involved in the upward transport of glyphosate from rice roots.

Example 5 Glyphosate Resistance Test of Detached Rice Leaves at Adult Plant Stage

Select the plants grown in the rice incubator for 35 days, cut the leaves of the same length and soak them in 20mL 0.1% (v/v) Silwet L-77 (Beijing Biotuoda Technology Co., Ltd.) solution (containing 5.75 or 11.5mmol/L glyphosate), placed in a light-dark cycle of 12/12h, and the temperature was 30°C in the light and 28°C in the dark for cultivation. The development process of the phytotoxicity was observed at different time periods, and the maximum quantum yield (Fv/Fm) of each leaf PS II was measured at 5h, 24h, and 48h by using a chlorophyll fluorescence imager. After 4 days, the phytotoxicity of the leaves of the osakr4c10 mutant and wild-type rice were inspected and recorded by taking pictures.

The results showed that under the treatment of low concentration of glyphosate, the leaves of wild-type (Zhonghua 11) and osakr4c10 mutant rice were not seriously damaged, and Fv/Fm decreased with time, but there was no significant difference (Figure 5A and C) ; while at high concentrations, the wild-type rice leaves were more phytotoxic than the osakr4c10 mutant, with more macules and severe chlorosis. Combining with the photosynthesis index Fv/Fm, it was consistent that the osakr4c10 mutant was more effective than the wild-type pair. Glyphosate was more tolerated (Figure 5B and D).

Embodiment 6 adult plant stage rice plant resistance test to glyphosate

The osakr4c10 mutant rice seeds and the wild-type Zhonghua 11 rice seeds were sterilized and germinated, sown in potted pots, and placed in a light-dark cycle of 12/12h, and the temperature was 30°C in the light and 28°C in the dark. After 55 days, a glyphosate spray experiment (concentration of 3.6 or 10.8mmol/L) was carried out. The commercial glyphosate product Roundup (30% glyphosate effective content) was selected as the agent, and the growth and morphological characteristics of the plants were continuously recorded.

At the same time, the time points of 0, 1, 3, 5, 7, and 9 days after spraying were selected to measure the chlorophyll content of the leaves of each treatment. The experimental conditions and methods were in accordance with the literature (Zhou Yong, Fan Xiaolei, Lin Yongjun, Chen Hao. (2018). Rice chlorophyll Determination of content. Bio-101e1010147.).

The results showed that the growth state of the osakr4c10 mutant rice and the wild type Zhonghua 11 were comparable under the condition of no glyphosate application; After 10.8mmol/L glyphosate treatment, WT plants showed yellowing and curly leaves after 4 days, and withered and died after 9 days. However, the growth of osakr4c10 mutant rice had no obvious phytotoxicity characteristics after 4 days or 9 days under the two concentrations of treatment, which was comparable to the growth status of the plants without glyphosate spraying ( FIG. 6 ). At the same time, the chlorophyll content of leaves after different treatments was detected, and it was found that the chlorophyll content of WT leaves continued to decrease over time, and the chlorophyll content of osakr4c10 mutant rice leaves decreased with glyphosate application, but not significantly (Figure 7). It shows that the loss of OsAKR4C10 gene function endows the adult rice plants with resistance to glyphosate.

Example 7 Determination of glyphosate resistance in the offspring of OsAKR4C10 gene-edited plants crossed with rice varieties in the background of Guiyu No. 11

The OsAKR4C10 gene-edited plants in the background of Guiyu 11, the plants that can flower on the same day after the ears of rice have been fully or partially pulled out, were used as the male plants; the plants that can flower on the same day were used as the male plants; Zhonghua 1 was selected, and the ears of rice had been completely or partially pulled out, but there were still some plants. Plants with many grains and not yet flowered were used as female parents. Use scissors to cut off the fully flowered branches on the upper part of the ear of the female plant and the undeveloped branches on the lower part of the ear, and then cut off the upper 1/2 to 2/3 of the grains that are opaque in the middle and have anthers inside. The chaff is specifically based on cutting the anthers in the grain and not harming the female organs in the grain. Spray the above-mentioned ears of rice with a watering can filled with 70% medicinal alcohol subsequently, and emasculate and bag them. Artificial pollination at noon of the same day or at noon of the next day. Use scissors to gently cut off the rice ears with high pollen content at the neck of the ear at the flowering stage of the male parent’s rice ear, put the rice ear down and gently put it into the kraft paper hybridization bag of the female parent plant prepared in advance, and shake it off to make the father’s ear grow. The pollen fully falls on the rice ear of the female plant to achieve the purpose of pollination. To obtain F1, identify the mutation of the OsAKR4C10 gene locus according to the method in Example 1, select a homozygous mutant, and perform self-crossing for 3 consecutive generations, and identify the mutation of the target site in each generation according to the method in Example 1. Obtain a purified strain of the OsAKR4C10 gene site mutation, and use Zhonghua No. 1 as a control to identify the glyphosate resistance of the plant according to the method in Example 6. It was found that the growth status of the mutant rice lines GY11-1, GY11-2 and GY11-3 and Zhonghua No. 1 were comparable under the condition of no application of glyphosate; Under the treatment, the leaves of the Zhonghua No. 1 plant showed severe yellowing and curling after 4 days, and withered and died after 9 days. However, the mutant rice lines GY11-1, GY11-2 and GY11-3 had no obvious phytotoxicity characteristics, and their growth status was comparable to that of plants not sprayed with glyphosate ( FIG. 8 ). It shows that the loss of OsAKR4C10 gene function endows adult rice plants with resistance to glyphosate, and can be used to create glyphosate-resistant germplasm resources through hybridization.

The above-mentioned embodiment is a preferred embodiment of the present invention, but the embodiment of the present invention is not limited by the above-mentioned embodiment, and any other changes, modifications, substitutions, combinations, Simplifications should be equivalent replacement methods, and all are included in the protection scope of the present invention.

sequence listing

<110> South China Agricultural University

<120> Application of OsAKR4C10 in the creation of non-transgenic glyphosate-resistant rice germplasm resources

<141> 2021-05-08

<160> 3

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Tyr Lys Asn Glu Asn Glu Val Gly Ile Ala Leu Lys Lys Leu Phe Glu

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Pro Phe Arg Val Lys Lys Gly Ser Gly Ile Ser Asn Thr Glu Asp Tyr

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Asp Ser Gly Lys Ser Arg Ala Ile Gly Val Ser Asn Phe Ser Ser Lys

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Lys Leu Gly Asp Leu Leu Ala Val Ala Cys Val Pro Pro Ala Val Asp

165 170 175

Gln Val Glu Cys His Pro Gly Trp Gln Gln Thr Lys Leu His Asn Phe

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Cys Gln Ser Thr Gly Val His Leu Ser Ala Tyr Ser Pro Leu Gly Ser

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aaactgggtg acctgcttgc tgtagcctgt gtacctccag ctgttgatca ggtagaatgc 540

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Claims (2)

1. the application of the reagent of knocking out rice OsAKR4C10 gene in suppressing the absorption and accumulation of rice leaves and roots to glyphosate, it is characterized in that, said reagent suppresses the absorption and accumulation of rice to glyphosate by knocking out rice OsAKR4C10 gene, Reduce glyphosate content in rice, improve rice resistance to glyphosate, the nucleotide sequence of the rice OsAKR4C10 gene is shown in SEQID NO: 2; the knockout rice OsAKR4C10 gene is constructed by constructing rice CRISPR-Cas9 The system, the system contains sgRNA that recognizes the rice OsAKR4C10 gene target sequence; the target sequence is shown in SEQ ID NO:3. 2. The application of the reagent for knocking out the rice OsAKR4C10 gene in cultivating glyphosate-resistant rice varieties, characterized in that the reagent obtains the glyphosate-resistant rice variety by inhibiting the expression of the OsAKR4C10 gene in rice, and the rice OsAKR4C10 gene The nucleotide sequence is shown in SEQID NO: 2; the expression of the rice OsAKR4C10 gene is suppressed by constructing the rice CRISPR-Cas9 system, which contains the sgRNA that recognizes the rice OsAKR4C10 gene target sequence; the target sequence is as SEQ ID NO: 3 shown.

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