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

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

Technical Field

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

Background

The rice is the main grain of about 22 hundred million people all over the world, wherein proper weed control is the key for ensuring the quality and the yield of the rice, the manual removal and biological control modes have low efficiency and high cost, and the herbicide can well overcome the defects and is still the most important control means at present. At present, the types of herbicides in rice fields are more than ten, the control objects are different, the use technical requirements are different, and the problems of herbicide phytotoxicity, excessive pesticide residues, increase of tolerant weed groups and the like are easily caused.

Due to the characteristics of broad spectrum, high efficiency, no harm to human and livestock and good environmental compatibility, the glyphosate is in the leading position of herbicide all the year round, but is also limited by the non-selective inactivation of the glyphosate on rice, and the glyphosate cannot be applied to large areas in rice fields. However, the cultivation of glyphosate-resistant rice is a commonly used method, for example, chinese patent CN107129993A discloses a modified glyphosate-resistant gene and a cultivation method of glyphosate-resistant rice, chinese patents CN106497922A, CN106497924A, CN106497923A, etc. disclose the construction method of transgenic rice resistant to snout moth's larva and glyphosate, and all of the above methods are to genetically transform an exogenous glyphosate-resistant gene into rice, thereby cultivating a glyphosate-resistant transgenic rice variety. However, the exogenous transfer of resistance genes has the problems of long research and development time, low efficiency and gene pollution; the problems can be better solved by exploring the endogenous glyphosate transporter gene of the rice and inactivating the glyphosate transporter gene by a plant genetic engineering technology. However, related genes which play a role in regulating the glyphosate absorption and accumulation of rice are rarely reported in rice at present.

Disclosure of Invention

The invention aims to overcome the defects 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 purpose of the invention is to provide the application of the rice OsAKR4C10 gene in the establishment of non-transgenic glyphosate-resistant rice germplasm resources.

The above object of the present invention is achieved by the following technical solutions:

the research of the invention shows that the rice OsAKR4C10 gene knockout can 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 gene editing technology is utilized to carry out gene editing on OsAKR4C10 gene of a main production cultivar to obtain a glyphosate-resistant rice cultivar, and the glyphosate-resistant rice cultivar is obtained by sexual or asexual propagation. Therefore, the present invention provides the following applications of the rice OsAKR4C10 gene and OsAKR4C10 protein:

the application of the rice OsAKR4C10 gene in regulating the absorption and accumulation of glyphosate in rice is disclosed, wherein the nucleotide sequence of the rice OsAKR4C10 gene is shown as SEQ ID NO:2 is shown in the specification; or with SEQ ID NO:2, and encodes a nucleotide sequence shown as SEQ ID NO:1, or a nucleotide sequence of the amino acid shown in the specification.

The application of the rice OsAKR4C10 protein in regulating the absorption and accumulation of glyphosate in rice is disclosed, wherein the amino acid sequence of the rice OsAKR4C10 protein is shown as SEQ ID NO:1 is shown in the specification; or SEQ ID NO:1 by substitution and/or deletion and/or addition of one or more amino acid residues, and/or the like.

SEQ ID NO: 1:

MAKHFVLNTGAKIPSVGLGTWQSDPGVVGDAVYAAVKAGYRHIDCARMYKNENEVGIALKKLFEEGVVKREDLFITSKLWCDCHAPEDVPESLDKTLSDLQLEYLDLYLIHWPFRVKKGSGISNTEDYIPPDIPSTWGAMEKLYDSGKSRAIGVSNFSSKKLGDLLAVACVPPAVDQVECHPGWQQTKLHNFCQSTGVHLSAYSPLGSPGSTWMNSNVLKESVIISIAEKLGKTPAQVALHWNIQMGHSVLPKSVTEERIKQNIDVYDWSIPEDLLVKFSEIKQVRLLRGDVIVNPHSVYKTHEELWDGEI.

SEQ ID NO: 2:

atggcgaagcatttcgtgctcaacaccggcgccaagatcccctcggtggggctcggcacctggcagtccgacccgggcgtcgtcggcgacgccgtctacgccgctgtcaaggcggggtaccggcacatcgattgcgccagaatgtacaaaaatgaaaatgaggtggggatagctctgaagaagctatttgaagaaggtgttgtcaagcgtgaagatttatttatcacatctaagctatggtgtgattgtcatgccccagaggatgtgcctgagtcactagacaaaactctgagtgacttacagcttgagtacctggatctttaccttattcattggccattcagagtcaagaagggctcaggcattagtaacactgaagactacataccacctgacatcccatctacctggggagcaatggagaagctatatgattctggtaaatctcgtgccattggtgtaagtaacttctcatcaaaaaaactgggtgacctgcttgctgtagcctgtgtacctccagctgttgatcaggtagaatgccatcctggttggcagcaaacgaagctacataacttctgccagtcaactggcgttcatctttctgcatactcgcctctaggttcacctggttcaacatggatgaacagtaacgtccttaaggaatccgtcatcatctcaattgcagagaagctcggcaaaactcctgcacaagtggcactgcactggaacattcagatgggtcacagtgtactcccaaaaagtgtgaccgaagaaaggataaagcagaacatagatgtttatgactggtctattccagaggacttgcttgttaagttctctgagattaagcaggttaggcttctcaggggcgacgtcattgttaatccccacagcgtttataagacccatgaggagctctgggacggcgaaatttag

the application of the rice OsAKR4C10 gene in culturing glyphosate-resistant rice varieties is characterized in that the nucleotide sequence of the rice OsAKR4C10 gene is shown as SEQ ID NO:2 is shown in the specification; or with SEQ ID NO:2, and encodes a nucleotide sequence shown as SEQ ID NO:1, or a nucleotide sequence of the amino acid shown in the specification.

The application of the rice OsAKR4C10 protein in culturing glyphosate-resistant rice varieties is characterized in that the amino acid sequence of the rice OsAKR4C10 protein is shown as SEQ ID NO:1 or SEQ ID NO:1 by substitution and/or deletion and/or addition of one or more amino acid residues, and/or the like.

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

Optionally, the inhibition of the expression of the OsAKR4C10 gene in rice, or the inhibition of the expression level and/or activity of the OsAKR4C10 protein is performed by a conventional method 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, and the system contains sgRNA recognizing a rice OsAKR4C10 gene target sequence.

Optionally, the sequence of the target is as set forth in SEQ ID NO:3, respectively.

AGGTGCCGAGCCCCACCGAGGGG(SEQ ID NO：3)。

The invention obtains the glyphosate resistant rice germplasm resource by taking low-absorption glyphosate as a mechanism through the functional identification of the glyphosate transporter gene of the rice and the inactivation of the glyphosate transporter gene by utilizing a plant gene editing technology. The advantages are that: the characteristics of efficient killing of glyphosate are exerted to simplify weeding in the rice field and prolong the suitable weeding period; the problems of long research and development time, low efficiency and gene pollution caused by exogenous transfer of resistance genes into conventional glyphosate-resistant crops are solved; most importantly, the glyphosate conductance selectivity is realized in weeds and crops, the cumulant and the application amount of glyphosate in rice are reduced while weeds are killed, the method is environment-friendly and ecological, and a new thought can be provided for accelerating the innovation of biological breeding and ensuring the national food safety.

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

the invention provides application of a rice OsAKR4C10 gene in regulation of glyphosate absorption and accumulation of rice, and research of the invention shows that the rice OsAKR4C10 gene knockout can inhibit the absorption and accumulation of the rice on herbicide glyphosate, reduce the content of glyphosate in rice plants and reduce the health risk to human bodies; meanwhile, the rice OsAKR4C10 gene is knocked out, so that the absorption and accumulation of the rice on the glyphosate can be genetically adjusted, the resistance of the rice on the glyphosate is improved, and a glyphosate-resistant rice variety is bred. The method combines the glyphosate absorption accumulation and resistance mechanism, provides a good choice for the research of herbicide-resistant crops, can be used for creating non-transgenic glyphosate-resistant rice germplasm resources, and has a great application prospect.

Drawings

FIG. 1 is a diagram showing the results of sequence alignment of mutants of Zhonghua No. 11 and OsAKR4C10 genes.

FIG. 2 is a graph showing the real-time fluorescent quantitative PCR detection results of OsAKR4C10 gene at different times after glyphosate spray treatment of flower No. 11. (A) OsAKR4C10 gene expression condition of the overground part of rice; (B) the expression condition of OsAKR4C10 gene in the underground part of the rice.

FIG. 3 shows the glyphosate tolerance of the Zhonghua No. 11 and OsAKR4C10 gene mutants in the seed germination stage. (A) Comparing the seedling bud length of WT and osakr4c10 in the glyphosate tissue culture medium with different concentrations; (B) Comparing the lengths of the overground parts of the seedlings of WT and osakr4c10 in the glyphosate tissue culture mediums with different concentrations; (C) WT was compared to osakr4c10 for root length in different concentrations of glyphosate tissue culture media.

FIG. 4 is a graph showing the results of detecting the accumulation of different parts of the Zhonghua No. 11 and OsAKR4C10 gene mutants after glyphosate is absorbed by roots in seedling stage. (A) leaf glyphosate accumulation; (B) a glyphosate accumulation amount of the stem; (C) the accumulated amount of glyphosate at the root; (D) And detecting the peak patterns of the glyphosate and a metabolite AMPA thereof by LC-MS/MS.

FIG. 5 shows the tolerance of the Zhonghua No. 11 and OsAKR4C10 gene mutant after glyphosate soaking of leaves in an adult plant. (A) Observing the phenotype of the leaves after being soaked in glyphosate of 5.75 mmol/L; (B) Observing the phenotype of the leaves after being soaked in 11.5mmol/L glyphosate; (C) soaking in 5.75mmol/L glyphosate to obtain Fv/Fm of the leaf; (D) soaking in 11.5mmol/L glyphosate, and then soaking the Fv/Fm of the leaf blade.

FIG. 6 shows the tolerance of the Zhonghua No. 11 and OsAKR4C10 gene mutants after spraying glyphosate in the adult stage.

FIG. 7 shows the change of chlorophyll content of the Zhonghua No. 11 and OsAKR4C10 gene mutants after spraying glyphosate in the adult stage. (A) Chlorophyll content of WT and osakr4c10 rice leaves is changed after 3.6mM glyphosate is sprayed; (B) The chlorophyll content of rice leaves under the condition that 10.8mM glyphosate is sprayed on WT and osakr4c10 is changed.

FIG. 8 shows the tolerance of the OsAKR4C10 gene mutant and its filial generation after spraying glyphosate in the adult stage, both of which are the Chinese No. 1 and Guiyu No. 11 background.

Detailed Description

The invention is further described with reference to the drawings and the following detailed description, which are not intended to limit the invention in any way. Reagents, methods and apparatus used in the present invention are conventional in the art unless otherwise indicated.

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

Coli DH 5. Alpha. And Agrobacterium EHA105 in the following examples are commonly used strains and commercially available; the rice variety is wild type Zhonghua No. 11 (publicly used rice variety, commercially available). The primers used in the examples were synthesized by Shenzhen Huamao Gene Co, and the sequencing was performed by Shenzhen Huamao Gene Co.

The quantitative tests in the following examples, all set up three replicates and the results averaged.

Example 1CRISPR knockout construction of OsAKR4C10 mutant plants

1. Selection of target sequences based on the exon sequences of OsAKR4C10 using the CRISPR/Cas9 system

Selecting a specific target sequence according to an OsAKR4C10 exon sequence by utilizing a simple and efficient CRISPR/Cas9 system, wherein the target sequence comprises the following steps: 5 'AGGTGCCGAGCCCCACCGAGGGG-activated 3'. The target sequence is specific to OsAKR4C10 gene, and specifically inactivates OsAKR4C10 protein.

2. Construction of pCRISPR/Cas9 recombinant vector containing the target sequence fragment

1) Design of adaptor primer with cohesive end according to target sequence

The designed target sequence is added to the specific sticky-end linker of the pCRISPR/Cas9 system and the complete linker primer is synthesized.

F1：5’-TGTGTGGGTGCCGAGCCCCACCGAG-3’；

R1：5’-AAACCTCGGTGGGGCTCGGCACCCA-3’。

2) Annealing and complementing the adaptor primer with the cohesive end to form a double-stranded small fragment with the cohesive end

Diluting the F1 primer and the R1 primer into a solution with the concentration of 10 mu M, uniformly mixing 10 mu L of each solution, carrying out annealing reaction in a PCR instrument, and reducing the temperature from 98 ℃ to 22 ℃ so that the F1 primer and the R1 primer are complemented to form a double-stranded small fragment with a sticky end.

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

The original vector pOs-sgRNA containing the sg-RNA was digested with the restriction enzyme Bsa I, resulting in cohesive ends which could be complementary to the cohesive ends of the target sequence. The system of cutting the pOs-sgRNA original vector by Bsa I enzyme is as follows: 10 XBuffer 2. Mu.L, bsa I enzyme 1. Mu.L, pOs-sgRNA vector 4. Mu.g, ddH ₂ The amount of O is up to 20. Mu.L, and the enzyme is cleaved at 37 ℃ for 12h. Checking the size of the cut enzyme product with 1% agarose gel electrophoresis, passing the product through a column of a kit (OMEGA Cat # D2500-02), recovering and purifying the cut enzyme product to obtain cut enzyme pOs-sgRNA vector, and adding sterilized ddH ₂ Dissolving O, and measuring the concentration for later use.

4) Ligating double-stranded small fragments with cohesive ends to the digested pOs-sgRNA vector to form a recombinant vector comprising the target sequence and sg-RNA

Connecting the double-stranded small fragment in the step 2) with the cut pOs-sgRNA vector in the step 3) by using T4 ligase to form a complete recombinant vector containing a target sequence aiming at the OsAKR4C10 protein and sg-RNA. The 15 μ L linker is: 10 XT 4 ligation buffer 1.5. Mu.L, double-stranded small fragment 4. Mu.L, digested pOs-sgRNA vector 3. Mu.L, T4 DNA ligation 1. Mu.L, ddH ₂ O to 15. Mu.L, and ligation was performed at 16 ℃ for 12 hours. The ligation product was transformed into E.coli DH 5. Alpha. And kanamycin-resistant LB plates were cultured overnight (containing 10mg/L kanamycin), and positive strains were selected for sequencing to obtain the assayA correctly sequenced recombinant vector comprising a target sequence and sg-RNA.

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

LR mix (North Noro Biotech Co., ltd, shanghai) was used to recombine the recombinant vector obtained in step 4) with the vector pH-Ubi-Cas9-7 (supplied by Baige Gene technology Co., ltd) containing Cas9 by LR reaction. LR reaction system: 25-50ng of recombinant vector containing the target sequence and sg-RNA, 75ng of pH-Ubi-cas9-7 vector, 1. Mu.L of 5 XR Clonase TM Buffer, TE Buffer (pH 8.0) were supplemented to 4.5. Mu.L, LR Clonase TM 0.5. Mu.L. Incubating the system at 25 ℃ for 2h, adding 2 mu L of 2 mu g/mu L of protease K after reaction, treating at 37 ℃ for 10min, transferring 2 mu L of reaction product into escherichia coli DH5 alpha, overnight culturing at 37 ℃ of gentamicin resistant LB plate, selecting positive strains for sequencing, and obtaining the complete pCRISPR/Cas9-OsAKR4C10 recombinant expression vector containing OsAKR4C10 protein target sequence-sg-RNA + Cas9 with correct sequencing.

3. The obtained complete recombinant vector containing the OsAKR4C10 protein target sequence-sg-RNA + Cas9 is introduced into rice callus to obtain a transgenic plant

1) And (3) transferring the recombinant expression vector pCRISPR/Cas9-OsAKR4C10 obtained in the step (2) into agrobacterium EHA105 (Olivia C.D, 2019) by electric shock to obtain a recombinant strain AGL1/pCRISPR/Cas9-OsAKR4C10.

2) The recombinant strain AGL1/pCRISPR/Cas9-OsAKR4C10 is used for transforming medium-flowering No. 11 rice callus by an agrobacterium-mediated method, and the method specifically comprises the following steps:

AGL1/pCRISPR/Cas9-OsAKR4C10 single colony is picked up, inoculated into 10mL of agrobacterium culture medium (containing 50mg/L kanamycin and 50mg/L rifampicin), and shake-cultured at 28 ℃ and 180rpm for 2-3 days. Centrifuging 4mL of bacterial solution at 4000rpm for 3min, pouring out the supernatant, adding a small amount of AAM culture medium to reconstitute the suspended cells, adding 20mL of AAM culture medium (containing 0.1mM acetosyringone As), performing shake cultivation at 28 deg.C and 150rpm in the dark for 1-2h, and culturing to OD ₆₀₀ And about = 0.4. Selecting callus of granular Zhonghua No. 11 (hereinafter, also referred to as wild-type rice) rice having good growth statusImmersing in Agrobacterium culture solution (YEP without agar), shaking at 150-200rpm for 20min at 28 deg.C, pouring out callus, sucking off excess bacteria solution with sterile filter paper, spreading the callus in sterile plate containing multiple layers of filter paper, blow drying on ultra-clean bench (callus is dispersed without agglomeration), transferring the callus onto co-culture medium, and culturing in dark for 2-3 days. The calli were transferred to NB minimal medium containing 100mg/L hygromycin and 400mg/L cefamycin for 3-4 weeks (one screen). The surviving calli were transferred to a secondary screening medium (NB minimal medium containing 100mg/L hygromycin and 200mg/L cefuroxime) for 3 weeks. Transferring the resistant callus to a differentiation culture medium (containing 100mg/L hygromycin) for differentiation, transferring a regenerated plant to a greenhouse after rooting on a strong seedling culture medium (about 3-4 weeks) containing 100mg/L hygromycin, and obtaining a transgenic plant with the OsAKR4C10 protein completely inactivated in a T0 generation plant.

The media used in the above transformation were as follows:

coculture medium (beijing waryo biotechnology limited): the callus and subculture medium was induced + As (0.1 mmol/L) + glucose (10 g/L), pH 5.2.

Agrobacterium infection rice callus culture medium (AAM medium, beijing Huayuyang Biotech Co., ltd.): AA macroelement, AA microelement, AA amino acid, MS vitamin, hydrolyzed casein (500 mg/L), sucrose (68.5 g/L), glucose (36 g/L), as (0.1 mM) and pH 5.2.

NB minimal medium (bio-technologies ltd, waryo, beijing): macroelement N6 + trace element B5 + organic component B5 + iron salt + hydrolyzed casein (300 mg/L) + proline (500 mg/L) + sucrose (30 g/L) + agar (8 g/L), pH 5.8.

Induction of callus and subculture medium: NB minimal medium +2,4-D (2 mg/L).

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

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

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

4. Screening transgenic positive plants in transgenic plants

The transgenic plant (T) transplanted in the step 3 ₀ Generation) to extract DNA (OMEGA Cat # D3485-02), and detecting target sequence locus to obtain 12 positive plants.

5. Obtaining mutant plants using transgenic positive plants

1) Identification of the site of mutation

Extracting DNA (OMEGA Cat # D3485-02) from the transplanted positive plants in the step 4, designing specific primers F2 and R2 aiming at DNA fragments within 500bp containing target sites, amplifying the DNA fragments containing the target sites, purifying and then sending 289bp PCR products to a company for sequencing, and comparing sequencing results with wild plant sequences to screen out mutant plants.

F2:5’-GGCCGCTGCCTACAGTAAAG-3’；

R2:5’-AGAGGAGAGGAGGAGACGC-3’。

2) And (3) breeding the mutant plant, detecting a plant single plant without hygromycin, cas9 and other transgenic elements in a T1 generation transgenic segregation population, and harvesting seeds to obtain a function deletion mutant, which is named as osakr4c10. The results of mutation analysis of the loss-of-function mutants and wild-type plants are shown in FIG. 1.

Example 2 real-time fluorescent quantitation assay after Glyphosate treatment of Rice

10.8mmol/L glyphosate solution is prepared, spray treatment is carried out on No. 11 plants growing in 25-day wild type varieties, and clear water treatment is used as a control. After treatment, 5h, 72h and 120h of RNA (OMEGA Cat # R6827-02) of the overground part (stem and leaf) and the underground part (stem and leaf) are respectively extracted, and after reverse transcription (Takara, primeScript RT reagent Kit with gDNA Eraser), real-time fluorescence quantitative PCR is carried out to detect the expression of the OsAKR4C10 gene. The experiment was repeated three times and the results averaged.

Real-time fluorescent quantitative PCR was performed using Bio-Rad CFX 96. The PCR reaction system (20. Mu.L) was carried out according to the product instruction SYBR Green Real-Time PCR Master Mix reagent (Takara) as follows: 10 μ L SYBR Green Real-Time PCR Master Mix, 2 μ L upstream and downstream primer Mix (10 μ M for both upstream and downstream primer concentrations), 7 μ L RNase-free water, 1 μ L cDNA template. The specific reaction procedure is as follows: enzymatic heat activation is carried out at 95 ℃ for 30s for 1 cycle; denaturation at 95 deg.C for 5s, extension at 60 deg.C for 30s, for 40 cycles.

The primer sequence for amplifying the OsAKR4C10 gene is as follows:

F3：5’-AACACTGAAGACTACATACCACCT-3’；

R3：5’-ACTTACACCAATGGCACGAGA-3’。

the primer sequence for amplifying the internal reference UBQ2 by taking the UBQ2 as an internal reference gene is as follows:

F4：5’-TGCTATGTACGTCGCCATCCAG-3’；

R4：5’-AATGAGTAACCACGCTCCGTCA-3’。

the data are processed by using a Comparative Ct method, i.e., the Ct value is the number of cycles that the fluorescence signal in the PCR tube passes through when reaching a set threshold value, and the delta Ct = Ct (OsAKR 4C 10) -Ct (UBQ 2) is calculated by 2 ^－△△Ct The expression of OsAKR4C10 gene in the sample is analyzed and compared.

The results are shown in fig. 2, and after glyphosate induction treatment for 5 hours, the expression level of OsAKR4C10 in the upper part of the rice is up-regulated by 2.62 times compared with the control group treated by clear water; in the underground part of rice, the expression level of OsAKR4C10 gene was not different between the treated group and the control group. After 72h, compared with the clear water control group, the treatment group has no statistical difference in the expression level of the OsAKR4C10 gene of the rice overground part or the underground part, and the expression level of the OsAKR4C10 gene of the rice overground part of the glyphosate treatment group falls back. After 120h, compared with a clear water control group, the expression quantity of the OsAKR4C10 gene on the overground part of the rice has no statistical significance difference; compared with the control group, the induction expression level of the OsAKR4C10 gene of the treatment group in the underground part of the rice is obviously increased by 9.71 times.

Example 3 susceptibility test of Rice in germinating stage to Glyphosate

And carrying out a sensitivity test of the osakr4c10 mutant and wild rice in the germination period on glyphosate by adopting a planting mode of tissue culture seedlings. The osakr4c10 mutant rice and the wild type rice seed of Zhonghua No. 11 as a control were baked at 49 ℃ for 4 days, and the seed vigor was recovered. The pre-seeded rice seeds were washed with 75% alcohol (prepared with absolute ethanol and sterile water), then with 50% naclo solution, and finally rinsed clean with sterile water to complete the thorough disinfection of the seeds. When in seeding, the tweezers are heated under an alcohol lamp, the cooled tweezers are clamped and placed in an MS culture medium (containing 0, 10, 25 and 50 mu mol/L glyphosate respectively), after seeding, the mouth of a tissue culture bottle is sealed, the seedling is placed in a dark place at 28 ℃ for sprouting at the early stage, after seedling emergence, the seedling is transferred to a light-dark period for 12/12h, the temperature is 30 ℃ under the light, and the seedling is continuously cultured for 15 days under the dark environment condition at 28 ℃. Watch phenotypic photographic records and measure above and below ground section lengths.

The results show that at a glyphosate concentration of 10. Mu. Mol/L, the root length and shoot length of the mutant plants are significantly higher than that of the wild type. At a glyphosate concentration of 25 μmol/L, growth of osakr4c10 mutant rice seedlings was only slightly inhibited compared to growth in the absence of glyphosate, whereas wild type rice seeds were barely able to grow after germination. At a glyphosate concentration of 50. Mu. Mol/L, seeds of osakr4c10 mutant rice seedlings still germinated but further vegetative growth stages of the seedlings were significantly inhibited, and wild type rice seeds completely darkened and shrivelled to die (FIG. 3). The result shows that the deficiency of the OsAKR4C10 gene obviously improves the tolerance of the rice seeds to glyphosate in the germination period and is beneficial to the growth of plants.

Example 4 test for Glyphosate uptake by roots of rice at seedling stage

The osakr4c10 mutant and middle-flowering No. 11 rice seedlings which grow uniformly in 21 days of water culture are transferred to a 50mL centrifuge tube, 6 plants in each group are one repeat, and 3 groups are repeated in each treatment. Before application, rice roots were placed at 0.5mmol/LCaCl ₂ (pH = 5.8) after 1h of preculture, 0.5mM CaCl was used instead ₂ (pH = 5.8) after culturing with 0.5mmol/L glyphosate solution for 3 days, the roots, stems and leaves of rice were collected and weighed for storage. The sample pretreatment method comprises the following steps: the sample was ground to a powder with liquid nitrogen, 1mL of sterile water was used per 0.2g of sample, and the extract mixture was transferred to a10 mL centrifuge tube, vortexed for 5min, then centrifuged for 5min with ultrasound 30min, 6000rpm. 1mL of the supernatant was taken, and 0.4mL of CH was added ₂ Cl ₂ Removing impurities, mixing uniformly by vortex for 3min, centrifuging at 6000rpm for 5min, collecting supernatant 1mL, adding 50mg C18 for purification, vortex for 3min, centrifuging at the highest speed for 10min,and (3) taking 0.8mL of centrifuged supernatant, adding 0.4mL of 5% sodium borate buffer solution and 0.4mL of 1.0g/L FMOC-Cl acetone solution, uniformly mixing by vortex, placing at 37 ℃ for overnight derivatization treatment, passing through a 0.45-micrometer organic filter membrane after derivatization is finished, and determining the amounts of glyphosate and AMPA by LC-MS/MS.

The result shows that the glyphosate content of the OsAKR4C10 mutant is lower than that of the wild rice in roots, stems and leaves after 24 hours, which indicates that the glyphosate absorption capacity is reduced after the function of the OsAKR4C10 is lost; the glyphosate content in the leaf and root of the osakr4c10 mutant was lower than that of the wild type rice after 72h, but there was no significant difference between the glyphosate content in the leaf of the osakr4c10 mutant and the wild type, which is probably the result of saturation of the up-transport capacity of the rice root, while no glyphosate metabolite AMPA was detected in all samples (fig. 4). The results show that the OsAKR4C10 gene is involved in the transport of glyphosate from the root of rice to the upper part.

Example 5 resistance test of Ex vivo leaves of adult-stage Rice against Glyphosate

Selecting plants growing in a rice incubator for 35 days, cutting leaves with the same length, soaking the leaves in 20mL of 0.1% (v/v) Silwet L-77 (Beijing Bootouda technologies Co., ltd.) solution (containing 5.75 or 11.5mmol/L glyphosate), standing in a dark and light cycle of 12/12h, and culturing at the temperature of 30 ℃ in the light and 28 ℃ in the dark. Observing the development process of the phytotoxicity at different time periods, and measuring the PS II maximum quantum yield (Fv/Fm) value of each leaf by using a chlorophyll fluorescence imager at 5h, 24h and 48h time periods respectively. After 4 days, the osakr4c10 mutant and wild-type rice leaf sections were examined for phytotoxicity and recorded by photography.

The results show that wild type (middle flower No. 11) and osakr4C10 mutant rice leaves were not severely damaged under low glyphosate treatment, fv/Fm decreased with time but no significant difference (fig. 5A and C); whereas at high concentrations, the phytotoxicity was more pronounced in leaves of wild-type rice compared to the osakr4c10 mutant, with more yellow spots and severe chlorosis, consistent with the photosynthesis index Fv/Fm indicating that the osakr4c10 mutant was more tolerant to glyphosate than the wild-type (fig. 5B and D).

Example 6 resistance test of Rice plants at adult stage to Glyphosate

The osakr4c10 mutant rice seeds and the wild type Zhonghua No. 11 rice seeds are sterilized, germinated and sowed in pot pots, the pot pots are placed in a light-dark period of 12/12h, cultured for 55 days under the environmental conditions that the temperature is 30 ℃ under the light and 28 ℃ under the dark, glyphosate spraying experiments (the concentration is 3.6 or 10.8 mmol/L) are carried out, glyphosate commercial products are selected as agents, namely, the agricultural product is achieved (the effective content of glyphosate is 30%), and the growth and morphological characteristics of plants are continuously recorded.

The chlorophyll content of each treated leaf is measured at 0, 1, 3, 5, 7 and 9 days after spraying, and the experimental conditions and the method are according to the literature (Zhouyong, severe dawn, lin champion, chen Hao. (2018). Measuring the chlorophyll content of rice. Bio-101e 1010147.).

The results show that the growth status of flower 11 in osakr4c10 mutant rice and wild type was comparable without application of glyphosate; under the treatment of 3.6mmol/L glyphosate, growth retardation appears after 4 days on wild type Zhonghua No. 11 plants, and the leaves are yellowed and curled after 9 days because of water loss and withering; the WT plants were yellow-curled after 4 days and withered and dead after 9 days under 10.8mmol/L glyphosate treatment. The growth of the osakr4c10 mutant rice showed no obvious phytotoxicity characteristics after 4 days or 9 days at both concentrations, comparable to the growth of plants without sprayed glyphosate (fig. 6). The chlorophyll content of the leaves after different treatments was simultaneously detected, and it was found that the chlorophyll content of the WT leaves was continuously reduced with time, and the chlorophyll content of the osakr4c10 mutant rice leaves was reduced but not significant following glyphosate application (FIG. 7). The loss of the function of the OsAKR4C10 gene is proved to endow the rice adult plant with glyphosate resistance.

Example 7 determination of Glyphosate resistance of hybrid progeny of OsAKR4C10 Gene-edited plants and Rice varieties with Guiyu Yuyu 11 background

OsAKR4C10 gene editing plants with the Guiyu No. 11 background are completely or partially extracted from rice ears, and plants capable of flowering on the same day are used as male parent plants; the Chinese No. 1 plant is selected as the female parent, the ear of rice is completely or partially extracted, but more grains are not bloomed yet. Cutting off the branches of the rice ear of the female parent plant which are completely bloomed and the branches of the lower part of the rice ear which are not developed by scissors, then cutting off the glumes of 1/2-2/3 of the upper part of the grains which are opaque and contain anther in the middle, and specifically taking cutting off the anther in the grains and not damaging the female organs in the grains as the standard. The ears of rice thus treated were then sprayed with a spray can filled with 70% medicinal alcohol to remove the ears and bagging. Artificial pollination is carried out at noon of the day or at noon of the next day. And slightly shearing the rice ears with large pollen amount at the ear necks by using scissors in the full bloom stage of the male parent rice ears, slightly putting the rice ears downwards into a female parent plant kraft paper hybridization bag prepared in advance, shaking off to ensure that the male parent pollen fully falls on the rice ears of the female parent plant, and achieving the aim of pollination. F1 was obtained by identifying the mutation of the OsAKR4C10 gene locus according to the method of example 1, selecting homozygous mutants, and subjecting them to successive selfing for 3 generations, each of which identified the mutation of the target locus according to the method of example 1. A OsAKR4C10 gene locus mutation purification strain is obtained, and the glyphosate resistance of the plant is identified according to the method of the embodiment 6 by taking the Chinese No. 1 as a contrast. As a result, it was found that the growth states of the mutant rice lines of the strains GY11-1, GY11-2 and GY11-3 and Zhonghua No. 1 were comparable without application of glyphosate; under the treatment of 10.8mmol/L glyphosate, chinese No. 1 plants have severe yellowing and curling of leaves after 4 days, and die after 9 days. The GY11-1, GY11-2 and GY11-3 mutant rice lines had no obvious phytotoxicity characteristics, and the growth situation was comparable to that of the plants not sprayed with glyphosate (FIG. 8). The loss of the OsAKR4C10 gene function endows the rice plant with glyphosate resistance, and can be used for creating glyphosate resistance germplasm resources in a hybridization mode.

The above embodiments are preferred embodiments of the present invention, but the present invention is not limited to the above embodiments, and any other changes, modifications, substitutions, combinations, and simplifications which do not depart from the spirit and principle of the present invention should be construed as equivalents thereof, and all such changes, modifications, substitutions, combinations, and simplifications are intended to be included in the scope of the present invention.

Sequence listing

<110> southern China university of agriculture

Application of OsAKR4C10 in establishment of non-transgenic glyphosate-resistant rice germplasm resources

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Claims (2)

1. Knock-out riceOsAKR4C10Use of a genetic reagent for inhibiting glyphosate absorption and accumulation in rice leaves and roots by knocking out riceOsAKR4C10The gene inhibits the absorption and accumulation of the glyphosate in the rice, reduces the glyphosate content in the rice, and improves the glyphosate resistance of the riceOsAKR4C10The nucleotide sequence of the gene is shown as SEQID NO:2 is shown in the specification; the knock-out riceOsAKR4C10The gene is obtained by constructing a rice CRISPR-Cas9 system which contains recognized riceOsAKR4C10sgRNA of a gene target sequence; the sequence of the target is shown as SEQ ID NO:3, respectively.

2. Knock-out riceOsAKR4C10Application of gene reagent in culturing glyphosate-resistant rice variety, characterized in that the reagent inhibits the glyphosate in riceOsAKR4C10Expressing the gene to obtain glyphosate-resistant rice variety, and the rice isOsAKR4C10The nucleotide sequence of the gene is shown as SEQID NO:2 is shown in the specification; the inhibition of riceOsAKR4C10The expression of the gene is realized by constructing a rice CRISPR-Cas9 system which contains recognized riceOsAKR4C10sgRNA of a gene target sequence; the sequence of the target is shown as SEQ ID NO:3, respectively.

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