Plant anti-paraquat herbicide PUT mutant protein and application thereof

Abstract

The invention belongs to the field of plant anti-herbicides, and discloses a plant anti-paraquat herbicide PUT mutant protein and application thereof. According to the invention, transmembrane structural domain analysis is carried out on proteins PUT1, PUT2 and PUT3 of paddy rice, so that potential mutation sites PUT1G458, PUT2G359 and PUT3G444 of the paddy rice OsPUT1 / 2 / 3 gene are predicted. According to the present invention, the OsPUT1 / 2 / 3 gene is subjected to precise mutation by using the guide editing system method, the conserved site is mutated into the positively charged PUT1G458R, PUT2G359R or PUT3G444R so as to successfully obtain the target mutant plant, and the paraquat resistance identification is performed to finally create the paraquat-resistant rice germplasm, such that the important significance is provided for the diversity of the rice germplasm resource.

Description

Technical Field

[0001] This invention belongs to the field of plant herbicide resistance, specifically relating to a plant-resistant paraquat-resistant PUT mutant protein and its application. Background Technology

[0002] Weed control is a crucial step in ensuring high and stable rice yields, especially with simplified rice cultivation becoming the mainstream technology, highlighting its increasing importance. Rice paddies are home to a wide variety of weeds, and large-scale use of various herbicides can easily lead to weed resistance, soil pollution, and pesticide residues. In contrast, non-selective herbicides are widely used due to their high efficiency, low dosage, and simple application methods, significantly reducing herbicide usage and labor costs. However, non-selective herbicides lack selectivity for both weeds and rice. Breeding herbicide-resistant crops is an effective way to improve herbicide application efficiency, and altering the sensitive sites of herbicide effector proteins is a common strategy for cultivating herbicide-resistant crops.

[0003] Paraquat (PQ), also known as methyl viologen (MV), is a highly effective, broad-spectrum, contact herbicide. It can rapidly and effectively kill hundreds of weed species and is quickly deactivated upon contact with soil, leaving no residue. Paraquat kills plants rapidly by disrupting the electron transport chain I of the photosynthetic system, generating superoxide radicals that interrupt photosynthesis and destroy chlorophyll synthesis. Therefore, it is widely used in agricultural production.

[0004] As a model plant, there is considerable research on paraquat-resistant mutants and resistance mechanisms in Arabidopsis thaliana, but very little research on them in major crops. The LAT1 protein encoded by the Arabidopsis gene PUT3 (POLYAMINE UPTAKE TRANSPORTER 3), At5g05630, also known as RMV1 (RESISTANT TO METHYL VIOLOGEN 1) (Miki Fujitaa et al., Natural variation in a polyamine transporter determines paraquat tolerance in Arabidopsis PNAS, 2012, 109(16), 6343-6347), belongs to the L-type amino acid (LAT) transporter family within the Arabidopsis amino acid, polyamine, and organic cation superfamily. This protein is located on the cell membrane and mediates the transport of extracellular polyamines / paraquat into the cell via a proton concentration gradient. Its polymorphism and natural variation can regulate the uptake and tolerance of polyamines / paraquat in Arabidopsis thaliana. The Arabidopsis thaliana paraquat-resistant mutant put3 exhibits high resistance to paraquat by reducing its uptake activity. Simultaneous knockout of three homologous genes in rice (OsPUT1, OsPUT2, and OsPUT3) using CRISPR / Cas9 gene editing technology also yielded homozygous mutant rice lines that effectively enhance paraquat resistance (Yu-Shu Lyu1 et al., Disruption of three polyamine uptaketransporter genes in rice by CRISPR / Cas9 gene editing confers tolerance to herbicide paraquat, aBIOTECH, published online on June 25, 2022).

[0005] The development of precision gene editing technology has provided technical support for precisely altering the sensitive sites of herbicide effector proteins. Among these technologies, Prime Editing (PE), with its superior precision gene editing capabilities combined with protein structure prediction technology, can accurately analyze the acidity / basicity of different amino acids and their binding ability to paraquat molecules, thereby precisely targeting the paraquat resistance site in the rice homolog OsPUT gene. This technology enables the efficient and precise creation of paraquat-resistant rice germplasm resources, providing strong technical support for herbicide-resistant rice breeding.

[0006] Currently, research on herbicide-resistant rice breeding is relatively limited. Although paraquat has been banned by the state due to its high toxicity and potential risks to human health, it has the characteristics of rapidly killing weeds in the field and quickly binding to the soil and becoming inactive. Therefore, creating paraquat-resistant rice germplasm resources is still of great significance. Summary of the Invention

[0007] The purpose of this invention is to provide a plant-resistant PUT mutant protein resistant to paraquat herbicide and its application.

[0008] This invention predicts potential mutation sites in the rice OsPUT1 / 2 / 3 gene by analyzing the transmembrane domains of the rice PUT1, PUT2, and PUT3 proteins. Furthermore, the Prime Editing (PE) technology is used to precisely mutate the OsPUT1 / 2 / 3 gene, successfully obtaining target mutant plants. Paraquat resistance is then identified, ultimately creating paraquat-resistant rice germplasm.

[0009] First, this invention identified homologous genes of AtPUT3 in Arabidopsis thaliana in the rice genome through protein sequence homology: OsPUT1 (Os02g47210), OsPUT2 (Os12g39080), and OsPUT3 (Os03g37984). Further protein structure prediction was performed to identify conserved amino acids PUT1G458, PUT2G359, and PUT3G444, which may affect paraquat transport. Paraquat is a positively charged dicationic molecule that disrupts the redox homeostasis of the photosynthetic system, competitively binding electrons delivered by ferricrene in the light-dependent reactions, generating large amounts of ROS that lead to plant death. Studies in Arabidopsis thaliana have shown that the transporter protein encoded by the PUT3 gene mediates the directional transport of paraquat from the cytoplasm to the chloroplast by regulating the membrane vesicle transport pathway between the Golgi apparatus and chloroplasts. Therefore, mutating the conserved sites to positively charged PUT1G458R, PUT2G359R, and PUT3G444R can reduce the absorption of paraquat molecules by plant leaves, thereby producing paraquat resistance.

[0010] Secondly, this invention utilizes the plant precision editing system ePE2 to precisely edit the potential paraquat resistance sites in the aforementioned rice, obtaining precise mutants PUT1G458R, PUT2G359R, and PUT3G444R after genetic transformation and passaging screening. Simultaneously, the OsPUT1, OsPUT2, and OsPUT3 genes were knocked out using the CRISPR / Cas9 system to obtain the put1 / put2 / put3 triple knockout mutant, which served as a control.

[0011] Specifically, the present invention provides the following technical solutions:

[0012] This invention provides a plant PUT mutant protein, which has a substitution mutation at the G458, G359 or G444 site relative to the wild-type PUT proteins PUT1, PUT2 or PUT3 of the plant, respectively. The substitution mutation is a mutation from G to a basic amino acid K, R or H, wherein the amino acid sequences of the wild-type PUT proteins PUT1, PUT2 or PUT3 are as shown in SEQ ID NO.19-21, respectively.

[0013] Preferably, the above mutation is a mutation from G to R, and the plant is rice.

[0014] The present invention provides a nucleic acid, which is the nucleic acid encoding the PUT mutant protein as described above.

[0015] The present invention provides a biological material, which is an expression cassette, vector, microbial cell, plant cell or plant cell line containing the nucleic acid described above.

[0016] This invention provides applications of the aforementioned PUT mutant proteins, nucleic acids, or biological materials:

[0017] (1) Application in equipping plants with resistance to paraquat herbicides or improving plant resistance to paraquat herbicides;

[0018] (2) Application in reducing the phytotoxicity of paraquat-type herbicides;

[0019] (3) Application in weed control;

[0020] (4) Application in the genetic breeding of plants resistant to paraquat herbicides; and / or

[0021] (5) Application in the improvement of plant germplasm resources resistant to paraquat herbicides.

[0022] This invention provides a method for preparing herbicide-resistant plants, wherein the plants contain the nucleic acids as described above or express one, two, or three of the PUT mutant proteins as described above; preferably, the plants contain the nucleic acids as described above or express one, two, or three of the PUT mutant proteins as described above by gene editing, hybridization, backcrossing, self-pollination, or asexual reproduction.

[0023] The present invention provides a reagent or kit for detecting or screening PUT mutations, the reagent or kit being used to detect PUT mutant proteins as described above or the nucleic acids encoding PUT mutant proteins as described above.

[0024] This invention provides a method for detecting or identifying whether a plant has paraquat resistance, and for detecting or identifying whether the plant has the PUT mutant protein or the nucleic acid as described above, preferably using the reagents or kits described above.

[0025] Paraquat resistance testing revealed that the three precise mutants of this invention—PUT1G458R, PUT2G359R, and PUT3G444R—exhibited superior resistance compared to the put1 / put2 / put3 triple knockout mutants. Therefore, this invention, through transmembrane domain analysis of rice PUT1, PUT2, and PUT3 proteins, predicted potential mutation sites PUT1G458, PUT2G359, and PUT3G444 in the rice OsPUT1 / 2 / 3 gene. Furthermore, guided editing was used to precisely mutate the OsPUT1 / 2 / 3 gene, converting conserved sites to positively charged PUT1G458R, PUT2G359R, or PUT3G444R, successfully obtaining target mutant plants. Paraquat resistance identification was then conducted, ultimately creating paraquat-resistant rice germplasm, which is of significant importance to the diversity of rice germplasm resources. Attached Figure Description

[0026] Figure 1 The amino acid sequence homology between rice and Arabidopsis thaliana was compared, and potential resistance target sites in rice, PUT1G458, PUT2G359, and PUT3G444, were identified.

[0027] Figure 2 Analysis of protein domains in rice.

[0028] Figure 3 Rice plant phenotypes for resistance testing of various rice PUT mutants.

[0029] Figure 4 Statistics on plant height for resistance testing of various mutant rice strains. Compared with the wild type, there was a statistically significant difference (p<0.05). Compared with the wild type, there was a statistically significant difference (p<0.001). Detailed Implementation

[0030] The present invention will be described below through specific embodiments to better understand the present invention, but this does not constitute a limitation thereof.

[0031] Example 1: PUT homology sequence alignment and protein structure analysis

[0032] Three rice homologous genes with high homology to Arabidopsis PUT3 were identified in the rice gene database through amino acid sequence homology comparison: OsPUT1 (gene accession number LOC_Os02g47210, encoded amino acid sequence as shown in SEQ ID NO. 19), OsPUT2 (gene accession number LOC_Os12g39080, encoded amino acid sequence as shown in SEQ ID NO. 20), and OsPUT3 (gene accession number LOC_Os03g37984, encoded amino acid sequence as shown in SEQ ID NO. 21). Figure 1 The amino acid sequence homology of the protein it encodes with that of Arabidopsis thaliana PUT3 is 57.62%, 43.96%, and 63.65%, respectively.

[0033] Protein domain analysis showed that ( Figure 2 OsPUT1 is located on chromosome 2 of rice, encoding 565 amino acid residues and possessing 12 transmembrane helical domains; OsPUT2 is located on chromosome 12, encoding 497 amino acids and possessing 10 transmembrane helical domains; OsPUT3 is located on chromosome 3, encoding 551 amino acids and possessing 9 transmembrane helical domains. Among these, the OsPUT1 / 2 / 3 proteins are more conserved in the 9th and 10th transmembrane domains. Therefore, the highly conserved amino acids PUT1G458, PUT2G359, and PUT3G444 were selected as potential resistance target sites. Figure 1 ).

[0034] Example 2: Obtaining precisely gene-edited plants using PUT1G458R, PUT2G359R, and PUT3G444R.

[0035] Construction of precise editing carrier

[0036] The precise editing process is described in the reference: Prime editing-mediated precise knockin of proteintag sequences in the rice genome. Plant Commun. 2023 May 8;4(3):100572. doi:10.1016 / j.xplc.2023.100572.

[0037] A specific 18-20 bp sequence from the genomic sequence of the target gene was selected using the target sequence design website (http: / / skl.scau.edu.cn / targetdesign), with an NGG 3' end and a GC content between 40% and 70%. The template sequence was input into the PlantPegDesigner website (www.plantgenomeediting.net), and the target site and RTT-PBS sequence were designed based on the target sequence. Then, an 8-nt linker sequence between PBS and evopreQ1 was designed using the pegLIT website (https: / / peglit.liugroup.us / ). GTGC was added before the forward target sequence, followed by the 8-nt linker GAACATTT. The complementary sequence TCAAAAAATGTTC of the TCAA-8-nt linker GAACATTT was added before the reverse complementary target sequence (Table 1).

[0038] Table 1. Names and sequences of primers designed for pegRNA

[0039]

[0040] Primer design for target sites: The single strand of DNA containing NGG is usually called the forward target sequence (g++), and the opposite strand is called the reverse target sequence (g--). TGCA is added to the front end of the forward target sequence, followed by GTTTC, and CTCTGAAAC is added to the front end of the reverse target sequence to synthesize primers (Table 2).

[0041] Table 2. Primer names and sequences designed for target sites

[0042]

[0043] After annealing the target primers (RT, PBS, sg2.0, and evopreQ1), the ligation products were ligated into the BsaⅠ-HFv2 pre-digested ePE2 expression vector using the Golden Gate cloning method. The ligation product was then transformed into *E. coli* Dh5. Clones containing the correct insert fragment were selected, and the correctness of the vector was verified by Sanger sequencing. Finally, the ePE2-PUT1G458R, ePE2-PUT2G359R, and ePE2-PUT3G444R vectors were successfully constructed.

[0044] Obtaining transgenic materials through Agrobacterium-mediated genetic transformation:

[0045] The ePE2-PUT1G458R, ePE2-PUT2G359R, and ePE2-PUT3G444R vectors were transformed into the Kitaake rice variety using the EHA105 Agrobacterium-mediated transformation method. After obtaining transgenic material, the editing sites of the transformed T0 generation plants were detected by Hi-TOM sequencing, and correctly edited rice plants were selected. These plants were then propagated through planting to obtain precisely edited plants with homozygous mutations of PUT1G458R, PUT2G359R, and PUT3G444R.

[0046] Example 3: Obtaining OsPUT1, OsPUT2, and OsPUT3 multi-gene knockout plants

[0047] OsPUT1, OsPUT2, and OsPUT3 were knocked out using the CRISPR / Cas9 system, and T0 generation mutant materials of the put1 / put2 / put3 triple knockout mutant were successfully obtained through genetic transformation. The put1 / put2 / put3 triple knockout mutant was used as a control for subsequent screening of resistant plants (refer to Chinese Patent CN114480482A).

[0048] Construction of the put1 / put2 / put3 three-knockout carrier (refer to Chinese Patent CN201510485573.2).

[0049] (1) Target sequence design: Select a specific sequence of 18-20 bp from the genome sequence of the target gene through the target sequence design website (http: / / skl.scau.edu.cn / targetdesign), with NGG at the 3' end and GC content between 40% and 70%.

[0050] (2) Primer design: The single strand of DNA containing NGG is usually called the forward target sequence (g++), and the opposite strand is called the reverse target sequence (g--). Add GGCA to the front of the forward target sequence and add AAAC to the front of the reverse target sequence to synthesize primers (Table 3).

[0051] Table 3. Primer names and sequences for the design of the put1 / put2 / put3 triple knockout vector.

[0052]

[0053] (3) Construction of multi-target knockout vectors

[0054] Sticky ends were generated by digesting the vector SK-5G with Aar I enzyme. The annealed product was then ligated using T4 ligase. The mixture was then processed using E. coli DH5α. Genetic transformation and screening yielded intermediate vectors SK-5GOsPUT1, SK-5GOsPUT2, and SK-5GOsPUT3. The pC1300-Ubi-Cas9 vector was ligated into the intermediate vectors using three pairs of isosigmazydase. The mixture was then processed using *E. coli* DH5α... Transformation screening was performed, and the correctness of the vector was verified by Sanger sequencing. Finally, the pC1300-Ubi-Cas9-gOsPUT1-gOsPUT2-gOsPUT3 vector was successfully constructed.

[0055] The vector was transformed into rice kitaake material using the EHA105 Agrobacterium transformation method. To identify positive materials among the mutants, the T0 generation plants obtained from the transformation were analyzed by Hi-TOM sequencing to detect the editing sites, and triple knockout mutants put1 / put2 / put3, in which OsPUT1, OsPUT2, and OsPUT3 were knocked out, were screened. Seeds of homozygous triple knockout mutants put1 / put2 / put3 were obtained and used as controls in the paraquat resistance experiment.

[0056] Example 4: Paraquat Resistance Test

[0057] Based on preliminary experiments, 0.5 μM was determined as the tolerance concentration for paraquat resistance testing. The relative growth index (RGI, =ΔH / H0×100%) represents the difference in growth and development status and plant height of seedlings in response to paraquat.

[0058] Growth status of rice put mutants: The seedling morphology of put mutants was not significantly different from that of WT. Quantitative parameter analysis of the growth and development of put mutants through statistical analysis of seedling height phenotypic characteristics revealed that none of the put mutants showed abnormal growth and development under the absence of paraquat stress.

[0059] Paraquat resistance detection in rice put mutants: Resistance of various rice put mutants was detected at a paraquat concentration of 0.5 μM. Results Figure 3 and Figure 4 As shown, the results indicate that after 72 hours of treatment with 0.5 μM paraquat, the relative growth coefficient of plant height of the three knockout mutants put1 / put2 / put3 increased by 1.48 times compared to WT, demonstrating a significant effect. The relative growth coefficients of plant height of the three point mutants PUT1G458R, PUT2G359R, and PUT3G444R increased by 2.12 times, 1.87 times, and 2.45 times, respectively, showing extremely significant effects.

[0060] The above description is merely a preferred embodiment of the present invention and is not intended to limit the invention. Various modifications and variations can be made to the present invention by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. A plant PUT mutant protein, characterized in that, The wild-type PUT proteins PUT1, PUT2, or PUT3 of the plant have substitution mutations at sites G458, G359, or G444, respectively. The substitution mutations are mutations from G to basic amino acids K, R, or H. The amino acid sequences of the wild-type PUT proteins PUT1, PUT2, or PUT3 are shown in SEQ ID NO.19-21, respectively.

2. A nucleic acid, characterized in that, The nucleic acid is the encoding nucleic acid of the PUT mutant protein as described in claim 1.

3. A biomaterial, characterized in that, It is an expression cassette, expression vector, microbial cell, or plant cell containing the nucleic acid described in claim 2.

4. The application of the PUT mutant protein as described in claim 1, the nucleic acid as described in claim 2, or the biomaterial as described in claim 3, characterized in that, The application is: (1) Application in equipping plants with resistance to paraquat herbicides or improving plant resistance to paraquat herbicides; (2) Application in reducing the phytotoxicity of paraquat-type herbicides; (3) Application in weed control; (4) Application in the genetic breeding of plants resistant to paraquat herbicides; and / or (5) Application in the improvement of germplasm resources of plants resistant to paraquat herbicides.

5. The application as described in claim 4, characterized in that, The plant in question is either rice or Arabidopsis thaliana.

6. A method for preparing a herbicide-resistant plant, characterized in that, The plant is made to contain the nucleic acid of claim 2 or to express one, two, or three of the PUT mutant proteins of claim 1.

7. The preparation method according to claim 6, characterized in that, The plant is made to contain the nucleic acid of claim 2 or to express one, two or three of the PUT mutant proteins of claim 1 by means of gene editing, hybridization, backcrossing, self-pollination or asexual reproduction.

8. The preparation method according to claim 6, characterized in that, The plant in question is either rice or Arabidopsis thaliana.

9. A reagent or kit for detecting or screening PUT mutations, characterized in that, The reagent or kit is used to detect the PUT mutant protein of claim 1 or the nucleic acid of claim 2.

10. A method for detecting or identifying whether a plant has resistance to paraquat herbicide, characterized in that, Detect or identify whether the plant has the PUT mutant protein of claim 1 or the nucleic acid of claim 2.

11. The method as described in claim 10, characterized in that, Use the reagent or kit described in claim 9.

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