Amaranthus retroflexus ArCYP81Q32 gene and application thereof

Abstract

The invention belongs to the technical field of plant biology, and particularly relates to an amaranthus retroflexus ArCYP81Q32 gene and application thereof. The invention discovers that amaranthus retroflexus ArCYP81Q32 participates in the degradation of a metabolic detoxification protoporphyrinogen oxidase (PPO) inhibitor herbicide fluoroglycofen-ethyl in a plant body. The relationship between the amaranthus retroflexus ArCYP81Q32 gene and fluoroglycofen-ethyl resistance is disclosed for the first time, whether a plant material generates metabolic resistance to herbicides or not can be identified by detecting the expression quantity of the amaranthus retroflexus ArCYP81Q32 gene, and the method has guiding significance on timely discovery of resistance, guidance of scientific pesticide application in farmland and alleviation of further spreading of pesticide resistance. The amaranthus retroflexus ArCYP81Q32 gene detoxifies protoporphyrinogen oxidase inhibitor herbicides through metabolism, and also provides a new gene resource for developing transgenic crops resisting the herbicides.

Description

Technical Field

[0001] This invention belongs to the field of plant biotechnology. Specifically, this invention relates to an ArCYP81Q32 gene of Amaranthus retroflexus and its application. Background Technology

[0002] Amaranthus retroflexus L. is the most frequent and widely distributed species in the genus Amaranthus, and is listed as a noxious weed in many parts of the world. Its seeds produce a large quantity of fruit, have a long lifespan, and a wide germination period, easily forming a persistent soil seed bank. As a C4 plant, it is highly competitive for light, water, and nutrients, causing severe damage in soybean-producing areas, leading to yield reductions of 20%-80%.

[0003] Fluoroglycofen-ethyl, a diphenyl ether herbicide developed by Rohm and Haas, has good weed control efficacy and is suitable for controlling broadleaf weeds in wheat, peanut, and soybean fields. However, due to long-term, large-scale, and singular use, resistant weed populations have been detected in soybean fields in Northeast China. Weed resistance to herbicides mainly occurs through mutations at target sites, preventing the herbicide from binding effectively and thus losing its weed-controlling activity. In weeds resistant to acetolactate synthase (ALS) inhibitors, the ALS gene is mutated, altering the enzyme structure and preventing binding to the herbicide. In weeds resistant to acetyl-CoA carboxylase (ACCase) inhibitors, ACCase gene mutations significantly reduce the affinity of the target enzyme for the herbicide. Some weeds also increase the activity or content of their internal detoxification enzyme systems, enabling them to break down herbicides into non-toxic or low-toxic metabolites. Cytochrome P450s (CYP450s) catalyze hydroxylation (alkyl and aryl hydroxylation) and dealkylation (N-, O-, S-demethylation, ethylation, etc.) reactions, which are one of the main pathways for weed degradation and metabolism of herbicides. Increased expression of certain genes in the P450s family can lead to herbicide resistance in previously sensitive weed populations. The reactions catalyzed by CYP450s are the initiating steps of most herbicide metabolism and play a role in the formation of herbicide resistance in weeds. Therefore, identifying the key nodes and metabolic pathways of herbicide degradation in various weeds is an important means of developing new herbicides or mitigating weed resistance to relevant herbicides. Summary of the Invention

[0004] To overcome the shortcomings in existing technologies regarding the resistance of Amaranthus retroflexus to herbicides such as ethoxysulfuron, and the lack of new methods for controlling Amaranthus retroflexus, this invention, through extensive research, discovered an Amaranthus retroflexus ArCYP81Q32 gene. This ArCYP81Q32 gene is the CYP450 gene for Amaranthus retroflexus to metabolize protoporphyrinogen oxidase inhibitors, and can metabolize and detoxify protoporphyrinogen oxidase inhibitors in plants, thus completing this invention.

[0005] In one aspect, the present invention discloses a retroflex amaranth ArCYP81Q32 gene, the nucleotide sequence of which is shown in SEQ ID NO:1 and the encoded amino acid sequence is shown in SEQ ID NO:2.

[0006] In one aspect, the present invention discloses an application of the ArCYP81Q32 gene of Amaranthus retroflexus, which identifies the resistance of different Amaranthus retroflexus materials to protoporphyrinogen oxidase inhibitor herbicides by detecting the relative expression level of the ArCYP81Q32 gene in plants.

[0007] In some embodiments, the present invention discloses a method for identifying a protrude amaranth material with high resistance to porphyrinogen oxidase inhibitor herbicides. The method includes detecting the expression of the ArCYP81Q32 gene in the protrude amaranth material. If the ArCYP81Q32 gene is highly expressed, then the protrude amaranth material has strong resistance to porphyrinogen oxidase inhibitor herbicides.

[0008] In some embodiments, the protoporphyrinogen oxidase inhibitor herbicide is ethoxysulfuron.

[0009] In some embodiments, the detection of the relative expression level of the ArCYP81Q32 gene in plants includes the following steps: S1. Extract total RNA from the target plant *Amaranthus retroflexus* and reverse transcribe it into cDNA; S2. Using the cDNA obtained in step S1 as a template and β-actin as an internal reference gene, perform qPCR reaction, establish a standard curve, and calculate the relative expression level of the ArCYP81Q32 gene of the reverse-branch amaranth in the test reverse-branch amaranth. In one embodiment, the qPCR reaction system consisted of: 1 μL of cDNA obtained in S1, 10 μL of TransStart® TopGreen qPCR SuperMix (+Dye II), 0.4 μL of forward primer, 0.4 μL of reverse primer, and 8.2 μL of ddH2O. In one embodiment, the qPCR reaction procedure is as follows: 94°C pre-denaturation for 30 s; 94°C denaturation for 5 s, 60°C annealing for 34 s, with simultaneous fluorescence acquisition, for a total of 40 cycles; In this invention, the nucleotide sequence of the forward primer of the ArCYP81Q32 gene of Amaranthus retroflexus is shown in SEQ ID NO:3, and the nucleotide sequence of the reverse primer of the ArCYP81Q32 gene of Amaranthus retroflexus is shown in SEQ ID NO:4. In this invention, the nucleotide sequence of the forward primer of β-actin is shown in SEQ ID NO:5, and the nucleotide sequence of the reverse primer of β-actin is shown in SEQ ID NO:6.

[0010] In practical applications, this identification can help agricultural scientists, agricultural producers, and environmental protection personnel to identify highly resistant Amaranthus repens materials in a timely manner, thereby enabling them to select other types of herbicides or adopt other weeding methods to improve the efficiency of herbicide use.

[0011] In one aspect, the present invention discloses an application of the ArCYP81Q32 gene of Amaranthus retroflexus, which is used to improve the plant's tolerance to ethoxysulfuron by overexpressing the ArCYP81Q32 gene in plants.

[0012] In some embodiments, the plant is a crop, and by overexpressing the ArCYP81Q32 gene of Amaranthus retroflexus in the crop, the crop's tolerance to ethoxysulfuron can be improved, thereby avoiding interference with the crop when the herbicide is applied. Beneficial effects

[0013] This invention is the first to clone a CYP450 gene, ArCYP81Q32, from *Amaranthus retroflexus*. The protein encoded by ArCYP81Q32 shows high homology in amino acid sequence with the CYP81Q32 proteins of amaranth, beet, and spinach. Furthermore, its expression level in a population of *Amaranthus retroflexus* resistant to ethoxysulfuron was significantly higher than in a susceptible population (P < 0.01). This invention also reveals for the first time the relationship between the *Amaranthus retroflexus* ArCYP81Q32 gene and resistance to protoporphyrinogen oxidase inhibitor herbicides. The expression level of the *Amaranthus retroflexus* ArCYP81Q32 gene can be used to identify whether *Amaranthus retroflexus* has developed metabolic resistance to protoporphyrinogen oxidase inhibitor herbicides.

[0014] 2. In this invention, the ArCYP81Q32 gene of *Amaranthus retroflexus* was overexpressed in *Arabidopsis thaliana*, and the sensitivity of transgenic *Arabidopsis thaliana* to ethoxysulfuron was determined. The results showed that the transgenic *Arabidopsis thaliana* had significantly higher tolerance to ethoxysulfuron than wild-type *Arabidopsis thaliana*, proving that ArCYP81Q32 is one of the reasons for conferring resistance to ethoxysulfuron in *Amaranthus retroflexus*, and also providing a new gene resource for developing transgenic crops resistant to this type of herbicide.

[0015] 3. This invention provides qPCR primers for detecting the expression level of the ArCYP81Q32 gene in *Amaranthus retroflexus*, and a method based on these primers for detecting the ArCYP81Q32 gene expression level in *Amaranthus retroflexus*, thereby determining the resistance of *Amaranthus retroflexus* to protoporphyrinogen oxidase inhibitor herbicides (especially ethoxysulfuron). This method has the advantages of simplicity, high sensitivity, and strong specificity, and has important application prospects in the detection and research of non-target resistance of *Amaranthus retroflexus* to ethoxysulfuron. Attached Figure Description

[0016] Figure 1 1. Structural domain analysis of ArCYP81Q32 of reverse-branched amaranth in Embodiment 2 of the present invention.

[0017] Figure 2 Phylogenetic tree of ArCYP81Q32 of the reverse branch amaranth in Embodiment 2 of the present invention.

[0018] Figure 3 The standard curves and melting curves of the qPCR primers for the target gene ArCYP81Q32 and the internal reference gene β-actin in Example 3 of this invention are shown. (The first row shows the standard curves and melting curves of the qPCR primers for the target gene ArCYP81Q32, and the second row shows the standard curves and melting curves of the qPCR primers for the internal reference gene β-actin).

[0019] Figure 4 The qPCR gene expression levels of the ArCYP81Q32 gene in the susceptible (S) and resistant (R) populations of *Amaranthus retroflexus* in Example 3 of this invention were verified before and 3 and 6 h after treatment with ethoxysulfuron. (** in the figure indicates a significant difference of P < 0.01).

[0020] Figure 5 Agarose gel electrophoresis image of the transgenic Arabidopsis marker gene HPT in Example 4 of this invention.

[0021] Figure 6 The resistance phenotype of Arabidopsis thaliana transgenic with the ArCYP81Q32 gene after treatment with ethoxysulfuron in Example 4 of this invention. Detailed Implementation

[0022] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to embodiments. Unless otherwise specified, the equipment and reagents used in the embodiments and experimental examples are commercially available. Unless otherwise stated, all reagents used in this invention are analytical grade reagents. The specific embodiments described herein are for illustrative purposes only and are not intended to limit the invention. Example

[0023] When *Amaranthus retroflexus* reached the 3-4 leaf stage, it was treated with 1 / 4 of the field recommended dose (26.1 g ai ha−1) of ethoxysulfuron, with water spraying serving as a control. After 3 and 6 hours of treatment, aboveground tissues were collected, rapidly frozen in liquid nitrogen, and sent to the company for parameter-free transcriptome sequencing. Three biological replicates were set up for each treatment.

[0024] Based on the transcriptome sequencing results, differentially expressed genes were screened using DESeq2 software with |log2 FoldChange|≥1 and q<0.05 as criteria. Gene information and sequences were obtained from the transcriptome sequencing results. Finally, a significantly differentially expressed gene was identified: the nucleotide sequence of the *Amaranthus retroflexus* ArCYP81Q32 gene is shown in SEQ ID NO.1, and the amino acid sequence of the encoded protein is shown in SEQ ID NO.2. Example

[0025] 2.1 The ArCYP81Q32 gene sequence (SEQ ID NO.1) of *Amaranthus retroflexus* was uploaded to NCBI. Conserved domain analysis was performed using the Conserved Domains Database tool. The results are as follows: Figure 1 As shown, the results indicate that the ArCYP81Q32 gene of Amaranthus retroflexus belongs to the CYP81 subfamily of the CYP450 superfamily, possesses the conserved core domain of CYP450, and contains multiple heme binding sites, suggesting that it may be a cytochrome P450 monooxygenase with redox catalytic activity.

[0026] 2.2 The amino acid sequence (SEQ ID NO.2) of the protein encoded by the ArCYP81Q32 gene of *Amaranthus retroflexus* was uploaded to NCBI and compared using the BLASTP tool. The top 50 sequences with the highest sequence identity were selected and a phylogenetic tree was constructed using MEGA. The results are as follows: Figure 2 As shown, the protein encoded by this gene is highly consistent with the CYP81Q32 protein of amaranth, beet, and spinach, with identity rates of 94.49%, 74.69%, and 72.09%, respectively, and the query coverage rate is 100% for all of them. Example

[0027] The resistant population R and the susceptible population S of *Amaranthus retroflexus* were cultured to the 3-4 leaf stage and then treated with the field-recommended dose (26.1 g aiha−1) of ethoxysulfuron, with water as a control. Aboveground tissues were collected 3 and 6 h after treatment, flash-frozen in liquid nitrogen, and stored at -80°C. Four replicates were set up for each sampling point.

[0028] Total RNA was extracted from the target plant, *Amaranthus retroflexus*, according to the kit instructions. RNA integrity was assessed using 1% agarose gel electrophoresis, and RNA concentration and purity were determined using a spectrophotometer. RNA bands that were clear, undegraded, and at a concentration greater than 200 ng / μL, with A260 / 280 and A260 / 230 values ​​meeting the requirements, were suitable for reverse transcription into cDNA.

[0029] Reverse transcription system: 1000 ng total RNA, 4 μL TransScript® All-in-One SuperMix for qPCR, 1 μL gDNA Remover, ddH2O to a final volume of 20 μL. Reverse transcription program: Incubate at 42°C for 15 min, then heat at 85°C for 5 s.

[0030] Based on the ArCYP81Q32 gene sequence SEQ ID NO.1, qPCR primers ArCYP81Q32-F1 and ArCYP81Q32-R1 were designed using NCBI Primer-BLAST; and β-actin gene was used as internal reference gene, with primers β-actin-F and β-actin-R.

[0031] The nucleotide sequence of primer ArCYP81Q32-F1 is shown in SEQ ID NO.3; The nucleotide sequence of primer ArCYP81Q32-R1 is shown in SEQ ID NO.4; The nucleotide sequence of primer β-actin-F is shown in SEQ ID NO.5; The nucleotide sequence of primer β-actin-R is shown in SEQ ID NO.6.

[0032] The cDNA stock solution was used as a standard and diluted using a serial dilution method: it was diluted to five levels: 1, 1 / 3, 1 / 9, 1 / 27, and 1 / 81, which served as templates for the preparation of the standard curve.

[0033] The 20 μL qPCR reaction system includes: 1 μL template cDNA, 10 μL TransStart® Top Green qPCRSuperMix (+Dye II), 0.4 μL forward primer, 0.4 μL reverse primer, and 8.2 μL ddH2O.

[0034] The qPCR reaction program is as follows: 94°C pre-denaturation for 30 s; 94°C denaturation for 5 s, 60°C annealing for 34 s, with fluorescence acquisition performed simultaneously, for a total of 40 cycles.

[0035] The results are as follows Figure 3 As shown: Figure 3 The following are standard curves and melting curves of qPCR primers for the internal reference gene β-actin and the target gene ArCYP81Q32 of *Amaranthus retroflexus* (the first row shows the standard curve and melting curve of qPCR primers for the target gene ArCYP81Q32, and the second row shows the standard curve and melting curve of qPCR primers for the internal reference gene β-actin). The results show that the amplification efficiency of the primers for both the internal and target genes is between 90% and 110%, the correlation coefficient R² of the standard curves is greater than 0.99, and the melting curves are single-peaked with no primer dimers, indicating good primer specificity and suitability for qPCR gene expression measurement.

[0036] The relative expression level of the ArCYP81Q32 gene was calculated using the 2-ΔΔCT method. ΔCT = CT(ArCYP81Q32) - CT(β-actin); ΔΔCT = ΔCT (experimental sample) - ΔCT (control sample). The experimental data were analyzed using SPSS with a T-test, and plotted using Prism software.

[0037] The qPCR gene expression levels of the ArCYP81Q32 gene in susceptible (S) and resistant (R) populations of *Amaranthus retroflexus* before and 3 and 6 h after treatment with ethoxysulfuron are shown in [reference needed]. Figure 4 At 3 h and 6 h after treatment with ethoxysulfuron, the expression level of the ArCYP81Q32 gene in the resistant population (R) was 1.61 and 6.01 times that in the susceptible population (S), respectively. Example

[0038] Primers ArCYP81Q32-F2 (SEQ ID NO.7) and ArCYP81Q32-R2 (SEQ ID NO.8) containing a full-length CDS region with homologous arms were designed based on the multiple cloning site of the PHG expression vector and SEQ ID NO.1. The CDS region of the ArCYP81Q32 gene was amplified, and the target fragment was recovered by gel electrophoresis. The PHG vector was double-digested with restriction endonucleases BamHI and PstI, and the linearized vector was recovered by gel electrophoresis. The recovered target gene fragment and the digested vector were ligated using a seamless cloning kit to construct the PHG-ArCYP81Q32 recombinant plasmid. The 10 μL seamless cloning reaction system consisted of: 5 μL 2×Basic AssemblyMix, 2 μL PHG linearized vector, 1 μL ArCYP81Q32 gene fragment, and 2 μL ddH2O. After gentle mixing, the mixture was incubated at 50°C for 10 min.

[0039] The ligation reaction product was transformed into *E. coli* Top10 competent cells and plated on LB (LB / Kan) plates containing kanamycin, then incubated overnight at 37°C. Single colonies were picked and cultured overnight at 37°C and 220 rpm in LB / Kan liquid medium before sequencing. After successful sequencing, the plasmid was extracted and stored at -20°C. The PHG-ArCYP81Q32 recombinant plasmid was transformed into *Agrobacterium* GV3101 competent cells using a freeze-thaw method.

[0040] The ArCYP81Q32 gene was transferred into wild-type Arabidopsis thaliana using an Agrobacterium-mediated flower-dip method. The T0 generation was then... After sterilization by inverting and mixing with 70% ethanol for 1 min, inverting and mixing with 7% sodium hypochlorite for 10 min, and rinsing five times with sterile water, the plants were cultured on 1 / 2 MS medium plates (containing 30 μg•mL⁻¹ hygromycin). Sixteen positive plants were selected. DNA was extracted from the leaves of the positive plants, and the marker gene HPT was amplified using primers HPT-F (nucleotide sequence shown in SEQ ID NO. 9) and HPT-R (nucleotide sequence shown in SEQ ID NO. 10). Figure 5 The results of agarose gel electrophoresis of the marker gene HPT in transgenic Arabidopsis thaliana show that all 16 plants were positive for successful transgenic growth.

[0041] Sixteen positive plants were further cultured, and seeds were collected after maturity to obtain T1 generation seeds. After sterilization, T1 generation seeds were screened on MS medium plates containing 30 μg·mL⁻¹ hygromycin. Positive plants were then transplanted into small square pots, three plants per pot. At the 6-true-leaf stage, wild-type and transgenic Arabidopsis were sprayed with 26.1 g aiha-1 of ethoxysulfuron (1 / 4X, where X is the field recommended dose). The growth status of wild-type and transgenic Arabidopsis was observed 14 days after application. See [link to relevant documentation]. Figure 6 This is the validation result of transgenic Arabidopsis thaliana with the resistance gene ArCYP81Q32. In the figure, WT represents wild-type Arabidopsis thaliana, ArCYP81Q32 represents transgenic Arabidopsis thaliana, and CK represents water treatment. A total of 7 transgenic lines with significant resistance to ethoxysulfuron were obtained, further proving that ArCYP81Q32 is one of the reasons why Amaranthus retroflexus acquired resistance to ethoxysulfuron.

[0042] In summary, the ArCYP81Q32 gene of *Amaranthus retroflexus* of this invention participates in the resistance of plants to herbicides (ethoxyflufenican), which are inhibitors of the metabolic detoxification protoporphyrinogen oxidase (PPO). Overexpression of the ArCYP81Q32 gene of *Amaranthus retroflexus* in plants can improve the tolerance of plants to ethoxyflufenican.

[0043] The above description, in conjunction with specific embodiments, provides a further detailed explanation of the present invention. It should not be construed that the specific implementation of the present invention is limited to these descriptions. For those skilled in the art, several simple deductions or substitutions can be made without departing from the concept of the present invention, and all such deductions or substitutions should be considered to fall within the scope of protection defined by the claims submitted herein.

Claims

1. A reverse-branch amaranth ArCYP81Q32 gene, characterized in that, The nucleotide sequence of the ArCYP81Q32 gene of *Amaranthus retroflexus* is shown in SEQ ID NO:1, and the encoded amino acid sequence is shown in SEQ ID NO:

2.

2. An application of the ArCYP81Q32 gene of *Amaranthus retroflexus* according to claim 1, characterized in that, By detecting the relative expression level of the ArCYP81Q32 gene in Amaranthus retroflexus, the resistance of different plant materials to protoporphyrinogen oxidase inhibitor herbicides was identified.

3. The application according to claim 2, characterized in that, The detection of the relative expression level of the ArCYP81Q32 gene in *Amaranthus retroflexus* in plants includes the following steps: S1. Extract total RNA from the plant material to be tested and reverse transcribe it into cDNA; S2. Using the cDNA obtained in step S1 as a template and β-actin as an internal reference gene, perform qPCR reaction, establish a standard curve, and calculate the relative expression level of the ArCYP81Q32 gene of Amaranthus retroflexus in the test plant.

4. The application according to claim 2, characterized in that, The plant material is Amaranthus retroflexus.

5. The application according to claim 2, characterized in that, The nucleotide sequence of the forward primer of the ArCYP81Q32 gene of Amaranthus retroflexus is shown in SEQ ID NO:3, and the nucleotide sequence of the reverse primer of the ArCYP81Q32 gene of Amaranthus retroflexus is shown in SEQ ID NO:

4.

6. A method for identifying amaranth resistant to herbicides containing high porphyrinogen oxidase inhibitors, characterized in that, The method includes detecting the expression of the ArCYP81Q32 gene in the reverse-branch amaranth material. If the ArCYP81Q32 gene is highly expressed, then this reverse-branch amaranth material is highly resistant to porphyrinogen oxidase inhibitor herbicides. The nucleotide sequence of the reverse-branch amaranth ArCYP81Q32 gene is shown in SEQ ID NO:

1.

7. The identification method according to claim 6, characterized in that, The protoporphyrinogen oxidase inhibitor herbicide is ethoxysulfuron.

8. An application of the ArCYP81Q32 gene in Amaranthus retroflexus, characterized in that, The ArCYP81Q32 gene of *Amaranthus retroflexus* was overexpressed in plants to improve their tolerance to ethoxysulfuron; the nucleotide sequence of the ArCYP81Q32 gene is shown in SEQ ID NO:

1.

9. The application of the ArCYP81Q32 gene of *Amaranthus retroflexus* according to claim 8, characterized in that, The plant in question is an agricultural crop. By overexpressing the ArCYP81Q32 gene of Amaranthus retroflexus in the crop, the crop's tolerance to ethoxysulfuron can be improved, thereby avoiding interference with the crop when applying herbicides.

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