Plant paraquat-resistant herbicide put mutant protein and application thereof

Abstract

The application belongs to the field of plant herbicide resistance, and discloses a plant paraquat herbicide-resistant PUT mutant protein and application thereof. Through transmembrane domain analysis on rice PUT1, PUT2 and PUT3 proteins, potential mutation sites PUT1G458, PUT2G359 and PUT3G444 of the rice OsPUT1 / 2 / 3 gene are predicted. The OsPUT1 / 2 / 3 gene is precisely mutated by using a guide editing system method, the conserved sites are mutated into positive charges PUT1G458R, PUT2G359R or PUT3G444R, the target mutant plants are successfully obtained, and the paraquat resistance is identified, and finally, the paraquat-resistant rice germplasm is created, which has important significance for rice germplasm resource diversity.

Description

TECHNICAL FIELD

[0001] The application belongs to the field of plant resistance to herbicides, and particularly relates to a plant resistance to paraquat herbicide PUT mutant protein and application thereof. BACKGROUND

[0002] Weed control is a key link to ensure high and stable yield of rice, especially under the background of light and simple planting technology becoming the mainstream technology. There are various types of weeds in rice fields, and large-scale use of various herbicides for control can easily cause weed resistance, soil pollution and pesticide residues. In contrast, non-selective herbicides can significantly reduce herbicide use and labor costs due to their high efficiency, low use amount and simple application method, and are widely used. However, non-selective herbicides lack selectivity for weeds and rice, and cultivating herbicide-resistant crops is an effective means to improve the efficiency of herbicide application, and changing the sensitive site of herbicide effect protein is a common strategy for cultivating herbicide-resistant crops.

[0003] Paraquat (abbreviation PQ), also known as methyl viologen (MV), is a highly efficient and broad-spectrum contact-type non-selective herbicide. It can quickly and effectively kill hundreds of weeds, and will be quickly inactivated after contacting with soil, without residue. Paraquat can quickly kill plants by destroying the photosystem I electron transport chain, generating superoxide free radicals to interrupt photosynthesis and destroy chlorophyll synthesis, and is therefore widely used in agricultural production.

[0004] As a model plant, more studies on paraquat-resistant mutants and resistance mechanisms in Arabidopsis have been conducted, while few studies have been conducted in major crops. The Arabidopsis gene PUT3 (POLYAMINE UPTAKE TRANSPORTER 3), At5g05630, also known as RMV1 (RESISTANT TO METHYL VIOLIGEN 1) (Miki Fujitaa et al., Natural variation in a polyamine transporter determines paraquat tolerance in Arabidopsis PNAS, 2012, 109(16), 6343-6347), encodes the LAT1 protein, which belongs to the L-type amino acid (LAT) transporter family in the amino acid, polyamine, and organic cation superfamily of Arabidopsis. The protein is located on the cell membrane and can mediate the transport of polyamine / paraquat from the outside of the Arabidopsis cell to the inside of the cell driven by the proton concentration gradient. Its polymorphic natural variation can regulate the uptake activity and tolerance of paraquat / polyamine in Arabidopsis. The paraquat-resistant mutant put3 of Arabidopsis shows high resistance to paraquat by reducing the uptake activity of paraquat. Using CRISPR / Cas9 gene editing technology, three homologous genes OsPUT1, OsPUT2, and OsPUT3 in rice were simultaneously knocked out, and a rice homozygous mutant strain that can effectively improve paraquat resistance was also obtained (Yu-Shu Lyu1 et al., Disruption of three polyamine uptake transporter genes in rice by CRISPR / Cas9 gene editing confers tolerance to herbicide paraquat, a BIOTECH, June 25, 2022, online publication).

[0005] With the development of precise gene editing technology, it provides technical support for precisely changing the sensitive site of herbicide effect protein. Among them, the Prime Editing (PE) technology, with its excellent precision gene editing ability, combined with protein structure prediction technology, can accurately analyze the acid-base properties of different amino acids and their binding ability with paraquat molecules, and then accurately target the paraquat resistance site in the rice homologous gene OsPUT. This technical means realizes the efficient and precise creation of rice paraquat-resistant germplasm resources, and provides strong technical support for rice herbicide-resistant breeding.

[0006] At present, the research on rice herbicide-resistant breeding is relatively less. Although paraquat has been banned by the state due to its high toxicity and potential risk to human health, it has the characteristics of quickly killing weeds in the field and being quickly combined with soil to passivate, so it is still of great significance to create paraquat-resistant rice germplasm resources. SUMMARY

[0007] The present application aims to provide a plant paraquat-resistant herbicide PUT mutant protein and its application.

[0008] The present application predicts the potential mutation sites of the rice OsPUT1 / 2 / 3 gene by analyzing the transmembrane domain of the rice PUT1, PUT2 and PUT3 proteins. Further, the Prime Editing (PE) technology is used to precisely mutate the OsPUT1 / 2 / 3 gene, and the target mutant plants are successfully obtained. Through paraquat resistance identification, paraquat-resistant rice germplasm is finally created.

[0009] Firstly, the present application finds that the homologous genes of Arabidopsis thaliana AtPUT3 in the rice genome are OsPUT1 (Os02g47210), OsPUT2 (Os12g39080) and OsPUT3 (Os03g37984) through protein sequence homologous alignment. Further protein structure prediction is performed to find the conserved amino acids PUT1G458, PUT2G359 and PUT3G444 in the transmembrane domain region that may affect the transport of paraquat. Paraquat is a positively charged dicationic molecule that interferes with the redox state of the photosynthetic system, competitively binds to the electrons delivered by ferredoxin in the light reaction, and generates a large amount of ROS to cause plant death. Studies in Arabidopsis thaliana have shown that the transport 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 and the chloroplast. Therefore, mutating the conserved sites to PUT1G458R, PUT2G359R and PUT3G444R can reduce the absorption of paraquat molecules by plant leaves, thereby producing paraquat resistance.

[0010] Secondly, the present application uses the plant precise editing system ePE2 to precisely edit the potential paraquat resistance sites in the above rice. After genetic transformation and passage screening, PUT1G458R, PUT2G359R and PUT3G444R precise mutants are obtained. At the same time, the OsPUT1, OsPUT2 and OsPUT3 genes are simultaneously knocked out by the CRISPR / Cas9 system to obtain put1 / put2 / put3 triple knockout mutants as controls.

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

[0012] The present application provides a plant PUT mutant protein, which has a substitution mutation at G458, G359 or G444 site of wild type PUT protein PUT1, PUT2 or PUT3 of the plant, respectively, the substitution mutation is a mutation from G to basic amino acid K, R or H, wherein the amino acid sequence of wild type PUT protein PUT1, PUT2 or PUT3 is 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 application provides a nucleic acid, which is a coding nucleic acid of the PUT mutant protein as described above.

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

[0016] The present application provides the use of the PUT mutant protein, the nucleic acid or the biological material as described above:

[0017] (1) in imparting or improving the resistance of a plant to centaurea herbicides;

[0018] (2) in reducing the phytotoxicity of centaurea herbicides to a plant;

[0019] (3) in weed control;

[0020] (4) in the genetic breeding of a plant with resistance to centaurea herbicides; and / or

[0021] (5) in the improvement of a plant germplasm resource with resistance to centaurea herbicides.

[0022] The present application provides a method for preparing a herbicide-resistant plant, which contains one, two or three of the nucleic acid as described above or expresses one, two or three of the PUT mutant protein as described above; preferably, the plant contains one, two or three of the nucleic acid as described above or expresses one, two or three of the PUT mutant protein as described above by gene editing, hybridization, backcrossing, selfing or asexual reproduction.

[0023] The present application provides a reagent or kit for detecting or screening a PUT mutation, which is used for detecting the PUT mutant protein as described above or the coding nucleic acid of the PUT mutant protein as described above.

[0024] The application provides a method for detecting or identifying whether a plant has paraquat herbicide resistance, detecting or identifying whether the plant has a PUT mutant protein as described above or a nucleic acid as described above, preferably using a reagent or kit as described above.

[0025] Through paraquat resistance detection, it is found that the resistance of the three precise mutants PUT1G458R, PUT2G359R and PUT3G444R of the application is better than that of the put1 / put2 / put3 triple knockout mutant. Therefore, by analyzing the transmembrane domain of the rice PUT1, PUT2 and PUT3 proteins, the application predicts the potential mutation sites PUT1G458, PUT2G359 and PUT3G444 of the rice OsPUT1 / 2 / 3 gene. Further, the guide editing system method is used to precisely mutate the OsPUT1 / 2 / 3 gene, and the conserved sites are mutated into positive charge PUT1G458R, PUT2G359R or PUT3G444R, and the target mutant plant is successfully obtained, and the paraquat resistance is identified, and finally the paraquat-resistant rice germplasm is created, which has important significance for the diversity of rice germplasm resources. BRIEF DESCRIPTION OF DRAWINGS

[0026] Figure 1 Rice and Arabidopsis amino acid sequence homology alignment and potential resistance target sites PUT1G458, PUT2G359 and PUT3G444 of rice.

[0027] Figure 2 Rice protein domain analysis.

[0028] Figure 3 Rice plant phenotype of resistance detection of each mutant of rice put.

[0029] Figure 4 Plant height statistics of resistance detection of each mutant of rice put. : Statistically significantly different from wild type (p<0.05); : Statistically extremely significantly different from wild type (p<0.001). DETAILED DESCRIPTION

[0030] The application will be described in the following specific embodiments, so that the application can be better understood, but it does not constitute a limitation on the application.

[0031] Example 1, PUT homologous 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 sequence of 18-20 bp in the genomic sequence of the target gene was selected 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%. The template sequence was input on the PlantPegDesigner website (www.plantgenomeediting.net), and the target site, RTT-PBS sequence, and 8-nt Linker sequence between PBS and evopreQ1 were designed based on the Target sequence. GTGC was added at the front of the forward target sequence, and 8-nt Linker GAACATTT was added at the back. TCAA-8-nt Linker GAACATTT complementary sequence TCAAAAATGTTC was added at the front of the reverse complementary target sequence (Table 1).

[0038] Table 1, primer name and sequence of pegRNA design

[0039]

[0040] Target site primer design: the DNA single strand containing NGG is usually referred to as the forward target sequence (g++), and the opposite strand is referred to as the reverse target sequence (g--). TGCA is added at the front of the forward target sequence, and GTTTC is added at the back. CTCTGAAAC is added at the front of the reverse target sequence, and a primer is synthesized (Table 2).

[0041] Table 2, primer name and sequence of target site design

[0042]

[0043] After the RT, PBS, sg2.0 and evopreQ1 primers were annealed, they were connected to the ePE2 expression vector pre-cut by BsaⅠ-HFv2 enzyme in a Golden Gate cloning manner. Subsequently, the ligation product was transformed into E. coli Dh5 , and clones containing correct insertion fragments were screened, and the correctness of the vector was verified by Sanger sequencing. Finally, ePE2-PUT1G458R, ePE2-PUT2G359R and ePE2-PUT3G444R vectors were successfully constructed.

[0044] Transgenic materials were obtained by genetic transformation through Agrobacterium infection:

[0045] The ePE2-PUT1G458R, ePE2-PUT2G359R and ePE2-PUT3G444R vectors were transformed into the rice kitaake variety by using the EHA105 Agrobacterium transformation method. After obtaining the transgenic material, the editing sites of the T0 generation plants obtained by transformation were detected by Hi-TOM sequencing, and correct edited rice plants were screened. The plants were planted and expanded to obtain PUT1G458R, PUT2G359R and PUT3G444R homozygous mutation precise editing plants.

[0046] Example 3, obtaining of OsPUT1, OsPUT2, OsPUT3 multi-gene knockout plants

[0047] OsPUT1, OsPUT2 and OsPUT3 were triple-targeted by the CRISPR / Cas9 system, and the put1 / put2 / put3 triple-knockout mutant T0 generation mutant material was successfully obtained by genetic transformation. The put1 / put2 / put3 triple-knockout mutant was used as a control for subsequent resistance plant screening (reference Chinese patent CN114480482A).

[0048] Construction of put1 / put2 / put3 triple-knockout vector (reference Chinese patent CN201510485573.2).

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

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

[0051] Table 3, primer name and sequence of put1 / put2 / put3 triple-knockout vector design

[0052]

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

[0054] The vector SK-5G was cut by Aar I to produce sticky ends. The annealing product was connected by T4 ligase. The ligation product was transformed into E. coli DH5 Genetic transformation, screening to obtain SK-5GOsPUT1, SK-5GOsPUT2, SK-5GOsPUT3 intermediate vectors. pC1300-Ubi-Cas9 vector is connected to the intermediate vector with three pairs of same tail enzymes. After being transformed into E. coli DH5 Transformation screening, and the correctness of the vector is verified by Sanger sequencing. Finally, the pC1300-Ubi-Cas9-gOsPUT1-gOsPUT2-gOsPUT3 vector is successfully constructed.

[0055] The vector is transformed into rice kitaake material by EHA105 agrobacterium transformation method. To determine the positive material in the above mutant, the editing site of the T0 generation plant obtained by transformation is detected by Hi-TOM sequencing, and the triple knockout mutant put1 / put2 / put3 in which OsPUT1, OsPUT2, and OsPUT3 are all knocked out is screened. Planting for expansion, obtain triple knockout mutant put1 / put2 / put3 homozygous mutant seeds as a control for paraquat resistance experiment.

[0056] Example 4, paraquat resistance detection

[0057] According to the pre-experiment, 0.5 μM is determined as the tolerance concentration for paraquat resistance detection. Among them, the relative growth index (RGI, Relative Growth Index = ΔH / H0 x 100%) is the difference in growth and development state and plant height of seedlings to paraquat.

[0058] Growth state of rice put mutant: there is no significant difference in plant morphology of put mutant seedlings and WT. Through quantitative parameter analysis of growth and development of put mutant by seedling height phenotype statistical analysis, it is found that put mutant does not appear abnormal growth and development under the condition of no paraquat stress.

[0059] Paraquat resistance detection of rice put mutant: the resistance of rice put mutant is detected under the concentration of 0.5 μM paraquat. The results Figure 3 and Figure 4 As shown in the table, the results show that after 72 hours of 0.5 μM paraquat treatment, the relative growth index of plant height of triple knockout mutant put1 / put2 / put3 is increased by 1.48 times compared with WT, and the effect is significant. The relative growth index of plant height of 3 point mutant plants PUT1G458R, PUT2G359R, and PUT3G444R is increased by 2.12 times, 1.87 times, and 2.45 times, respectively, and the effect is extremely significant.

[0060] The above merely provides the preferred embodiments of the present application, and is not used to limit the present application. For those skilled in the art, the present application can have various modifications and changes. Any modifications, equivalent replacements, improvements, etc. made within the principles and technical scope of the present application shall fall into the scope of the present application.

Claims

1. A plant PUT mutant protein, characterized in that, The plant PUT mutant protein is a substitution mutation at site G458 in the amino acid sequence of wild-type PUT protein PUT1; or the plant PUT mutant protein is a substitution mutation at site G359 in the amino acid sequence of wild-type PUT protein PUT2; or the plant PUT mutant protein is a substitution mutation at site G444 in the amino acid sequence of wild-type PUT protein PUT3; the substitution mutation is a mutation from G to basic amino acid R; the amino acid sequences of the wild-type PUT protein PUT1, the wild-type PUT protein PUT2, and the wild-type PUT protein PUT3 are shown in SEQ ID NOs. 19-21, respectively.

2. A nucleic acid, characterized in that, The nucleic acid is a coding nucleic acid of the PUT mutant protein of claim 1.

3. A biomaterial, characterized by, It is an expression cassette, an expression vector, a microbial cell, or a plant cell comprising the nucleic acid of claim 2.

4. Use of a PUT mutant protein according to claim 1, a nucleic acid according to claim 2 or a biological material according to claim 3, characterized in that, The application is: (1) application in conferring or improving the resistance of rice to bentazon herbicides; (2) application in reducing the phytotoxicity of bentazon herbicides to rice; (3) application in the genetic breeding of rice with resistance to bentazon herbicides; and / or (4) application in the improvement of rice germplasm resources with resistance to bentazon herbicides.

5. A method for making a herbicide-resistant plant, characterized by, The plant contains one of the nucleic acids of claim 2 or expresses one of the PUT mutant proteins of claim 1; the plant is rice.

6. The production method according to claim 5, wherein The plant contains one of the nucleic acids of claim 2 or expresses one of the PUT mutant proteins of claim 1 by gene editing, hybridization, backcrossing, selfing, or asexual reproduction.

7. A reagent or kit for detecting or screening for a PUT mutation, characterized in that, The reagent or kit is used for detecting the PUT mutant protein of claim 1 or the nucleic acid of claim 2.

8. A method for detecting or identifying whether a plant has resistance to paraquat herbicide, characterized in that, Detecting or identifying whether the plant has the PUT mutant protein of claim 1 or the nucleic acid of claim 2; the plant is rice.

9. The method of claim 8, wherein, Using the reagent or kit of claim 7.

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