An Apa2aAg03750 gene resistant to amaranth herbicide and its application

Abstract

This invention relates to the field of genetic engineering technology, and proposes an Apa2aAg03750 gene resistant to the herbicide *Amaranthus pungens* and its application. The nucleotide sequence of the Apa2aAg03750 gene is shown in SEQ ID NO.1, and the amino acid sequence encoded by the Apa2aAg03750 gene is shown in SEQ ID NO.2. This technical solution solves the problems of high resistance to *Amaranthus pungens* and low crop resistance to this herbicide in related technologies.

Description

Technical Field

[0001] This invention relates to the field of genetic engineering technology, specifically to an Apa2aAg03750 gene resistant to the herbicide Amaranthus praecox and its application. Background Technology

[0002] Herbicide resistance is a global problem threatening agricultural production. The extensive use of herbicides leads to the rapid development of herbicide resistance in weeds, exacerbating crop yield losses and increasing agricultural production costs. (Example: Long-awned amaranth) Amaranthus palmeri It is a major invasive weed worldwide, with strong environmental adaptability and high resistance to herbicides, which exacerbates its invasion and spread.

[0003] However, research on herbicide resistance in Amaranthus longicornis is relatively weak. In particular, previous studies on non-selective glyphosate resistance in Amaranthus longicornis mainly focused on target resistance, namely the increase in copy number and overexpression of the target gene EPSPS, but only on EPSPS target resistance.

[0004] Therefore, exploring the herbicide resistance mechanism beyond EPSPS target resistance and identifying the key sites of herbicide tolerance in resistant populations will help in the development of new herbicides and the cultivation of herbicide-resistant crops. Summary of the Invention

[0005] This invention proposes an Apa2aAg03750 gene resistant to the herbicide Amaranthus lanceolata and its application, which solves the problems of high resistance to Amaranthus lanceolata and low crop resistance to herbicide Amaranthus lanceolata in related technologies.

[0006] The technical solution of the present invention is as follows: This invention proposes an Apa2aAg03750 gene resistant to the herbicide Amaranthus longifolia. The nucleotide sequence of the Apa2aAg03750 gene is shown in SEQ ID NO.1, and the amino acid sequence encoded by the Apa2aAg03750 gene is shown in SEQ ID NO.2.

[0007] The present invention also proposes a recombinant expression vector containing the Apa2aAg03750 gene as described in claim 1.

[0008] As a further technical solution, the herbicide is glyphosate.

[0009] As a further technical solution, the vector backbone of the recombinant expression vector is pEASY-Blunt E1.

[0010] This invention also proposes a method for constructing a recombinant expression vector, comprising the following steps: S1. Using Amaranthus longicornis cDNA as a template, PCR amplification was performed to obtain the PCR amplification product; S2. The PCR amplification product is ligated with pEASY-Blunt E1 to obtain the ligation product. S3. The ligation product was then introduced into competent E. coli cells, and the results confirmed that the target gene was successfully inserted into the recombinant expression vector pEASY-Blunt E1.

[0011] As a further technical solution, in step S1, the PCR amplification reaction program is as follows: pre-denaturation at 95℃ for 2 min; then 40 cycles of denaturation at 95℃ for 20 s, annealing at 55℃ for 20 s, and extension at 72℃ for 15 s; and further extension at 72℃ for 5 min; finally, maintaining at 4℃.

[0012] As a further technical solution, in step S1, the PCR reaction system is: 2 μL cDNA, 1 μL upstream primer, 1 μL downstream primer, 10 μL 5×TransStart FastPfu Buffer, 4 μL dNTPs, 1 μL DNA polymerase, 31 μL ddH2O, totaling 50 μL.

[0013] As a further technical solution, the upstream primer is: ATGTCATCAAAAGAAGATGATATAGTGC (SEQ ID NO.3).

[0014] As a further technical solution, the downstream primer is: TTAATCTATTCCATACTTTTTCCTTAATTCC (SEQ ID NO.4).

[0015] As a further technical solution, the concentration of the upstream primer is 10µM.

[0016] As a further technical solution, the concentration of the downstream primer is 10µM.

[0017] As a further technical solution, in step S2, the connection temperature is 25°C and the connection time is 5 minutes.

[0018] As a further technical solution, in step S2, during the ligation process, the volume ratio of PCR amplification product to pEASY-BluntE1 is 1:4.

[0019] The present invention also proposes the application of the Apa2aAg03750 gene, which is used to resist the herbicide Amaranthus lanceolata, in the development of new target Amaranthus lanceolata herbicides.

[0020] This invention also proposes the application of the Apa2aAg03750 gene, which is resistant to the herbicide Amaranthus praecox, in the breeding of herbicide-resistant crops.

[0021] The working principle and beneficial effects of this invention are as follows: In this invention, the Apa2aAg03750 gene was used to construct a recombinant expression strain. Experiments were conducted to investigate the effects of different concentrations of glyphosate on the absorbance of the recombinant plasmid bacterial solution and the empty vector bacterial solution, as well as the effects of different time points on the absorbance of the recombinant plasmid bacterial solution and the empty vector bacterial solution. These two experiments verified that the recombinant expression strain exhibits resistance to the herbicide *Amaranthus longicornis*. Therefore, the development of the Apa2aAg03750 gene provides a reference for genetic improvement of crops using genetic engineering technology and offers a new target for overcoming herbicide resistance in weeds such as *Amaranthus longicornis*. Attached Figure Description

[0022] The present invention will now be described in further detail with reference to the accompanying drawings and specific embodiments.

[0023] Figure 1 This is a graph showing the change in the relative expression level of the Apa2aAg03750 gene before and after glyphosate treatment in the long-awned amaranth resistant (GR) population in Example 1 of the present invention; In the figure, GR6hck is the 6h blank group, GR6h is the 6h treatment group, GR24hck is the 24h blank group, GR24h is the 24h treatment group, GR72hck is the 72h blank group, GR72h is the 72h treatment group, and * indicates that the 6h treatment group is significantly different from the 6h blank group (P < 0.05). Figure 2 This is a graph showing the absorbance results of recombinant plasmid bacterial suspension and empty bacterial suspension treated with different concentrations of glyphosate in Example 3 of the present invention; In the figure, pEASY-BL21 is the empty vector bacterial culture, and pEASY-GST-BL21 is the recombinant plasmid bacterial culture; Figure 3 The graph shows the absorbance results of the recombinant plasmid bacterial solution and the empty vector bacterial solution in Example 3 of the present invention at 1h, 2h, 3h, 4h, 5h, and 6h. In the figure, pEASY-BL21 is the empty bacterial culture, and pEASY-GST-BL21 is the recombinant plasmid bacterial culture. Detailed Implementation

[0024] The technical solutions of the present invention will be clearly and completely described below with reference to the embodiments of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative effort are within the scope of protection of the present invention.

[0025] Example 1: Obtaining the target gene Test samples: The long-awned amaranth resistant (GR) population was collected from Xingyang City, Henan Province; the long-awned amaranth sensitive (GS) population was collected from Xi'an City, Shaanxi Province; Both resistant (GR) and susceptible (GS) populations of *Amaranthus longicornis* at the 10-12 leaf stage were randomly divided into control and treatment groups. The treatment group was sprayed with half the recommended field dose of glyphosate (effective spray dose: 450 g ai / h, spray volume: 900 μL / plant), while the control group was sprayed with an equal volume of sterile water. Aboveground tissues were collected from both groups at 6 h, 24 h, and 72 h post-treatment (6 h control group, 6 h treatment group, 24 h control group, 24 h treatment group, 72 h control group, and 72 h treatment group, respectively). After cleaning with sterile water, the tissues were placed in 15 mL centrifuge tubes and flash-frozen in liquid nitrogen. Each sample contained three biological replicates. The collected *Amaranthus longicornis* tissue samples were then sent to the company for transcriptome sequencing using the Illumina NovaSeq platform.

[0026] Raw sequencing data were cleaned by removing adapter sequences and low-quality sequences using Trimmomatic software. Then, Hisat2 software was used to map the sequencing data of each sample onto the *Amaranthus longicornis* reference genome (reference genome number GWHGENU00000000.1), and StringTie2 software was used to estimate the read counts and expression levels of genes in each sample. Next, differential expression analysis was performed using the Bioconductor software package DESeq2, with the following selection thresholds for differentially expressed genes: q-value < 0.05, log2(Fold change) ≥ 1.

[0027] Focusing on genes specifically upregulated in the long-awned amaranth resistant (GR) population, this invention found that the Apa2aAg03750 gene (SEQ ID NO.1) was significantly upregulated in the early stage after glyphosate treatment in the long-awned amaranth resistant (GR) population.

[0028] SEQ ID NO.1: ATGTCATCAAAAGAAGATGATATAGTGCTATTAGACTTTTGGGCAAGTCCATTTGTAGCAAGAGTCAAATTAGCCTAGAAGAGAAAGGTATTACTAACTATGAATGTCTCCATGAAGATGATATAATGGTTTCCAAAAGTTCTCTTCTTATGGAGATGAACCCGGTTCA CAAAAAAGTCCCGGTTCTAATACATAATGGTAAACCGGTTTGTGAATCTTTACTTATAGTGGAGTATATTGATGAGGTATGGAAGCATAAGTCCCCTTCTTTGATGCCTCTTCATCCTTATGATGTAGCTCGGGCTCGGTTTTGGGCCTACTCTTTTGATAAAAAGTTTC CCGAGTTTTCCTCAAGTTATGGACACAAAAAACCGTAATTCAACAAGAAGAGAAGAATGATTTCATGAACTTCCTTAAAATGGTGGAAGAAGAGCTTGGAGACAAGCCTTATTTTGGAGGAAATACTTTTGGATATTGTGATATTGCACTAATTTCAATTTATAGTTGG TTTTATACCTATCAAAAATTTACTAAACTAGACATAGAATATGAGTGCCCAAAACTTATTGCATGGGGTAAGAGATGTATGGATAGAGATAGTGTCAAAAAAGTTGTTCTTAATGAGGTTCAAATTTATAATTATGCATTGGAATTAAGGAAAAAGTATGGAATAGATTAA The nucleotide sequence of the Apa2aAg03750 gene was translated into an amino acid sequence (SEQ ID NO.2) using the online software tool Translate (https: / / web.expasy.org / translate / ) on the Expasy website. Then, the sequence was compared with the Pfam database (E value = 1e-2) using HMMER v3.1b2 software. This confirmed that the Apa2aAg03750 gene contains a conserved domain of glutathione S-transferase (GST), and therefore belongs to the GST gene family.

[0029] SEQ ID NO.2: MSSKEDDIVLLDFWASPFVARVKLALEEKGITNYECLHEDDIMVSKSSLLMEMNPVHKKVPVLIHNGKPVCESLLIVEYIDEVWKHKSPSLMPLHPYDVARARFWAYSFDKKF PEFLLKLWTQKTVIQQEEKNDFMNFLKMVEEELGDKPYFGGNTFGYCDIALISIYSWFYTYQKFTKLDIEYECPKLIAWGKRCMDRDSVKKVVLNEVQIYNYALELRKKYGID Finally, the relative expression levels of the Apa2aAg03750 gene before and after glyphosate treatment in the long-awned amaranth resistant (GR) population were verified by real-time quantitative PCR (with Actin gene as the internal reference). Figure 1 The results showed that the Apa2aAg03750 gene exhibited specific expression characteristics in the long-awned amaranth resistant (GR) population, and its expression level was significantly upregulated after 6 h of glyphosate treatment, indicating that the Apa2aAg03750 gene is resistant to glyphosate.

[0030] Example 2: Construction of Recombinant Expression Vector 2.1 RNA extraction and cDNA synthesis: Long-awned amaranth plants were cultivated at 28℃ under 14 hours of light / 10 hours of darkness. Leaves of long-awned amaranth at the 6-8 leaf stage were harvested, placed in cryovials, and then flash-frozen in liquid nitrogen and stored at -80℃ for subsequent experiments.

[0031] RNA was extracted from the leaves of *Amaranthus longicornis* using an ultrapure RNA extraction kit (CW0851S, Kangwei Century Biotechnology Co., Ltd.). cDNA synthesis was performed using the FastSensi one-step method for synthesizing the first strand of genomic cDNA using a premixed reagent (KR38-02, Tiangen Biotech (Beijing) Co., Ltd.). The specific steps were carried out according to the instructions in the corresponding kit.

[0032] 2.2 Obtaining Recombinant Expression Vectors Using the cDNA library of *Amaranthus longicornis* (2.1) as a template to amplify the target gene, the primer sequences were designed as follows: primer-F:ATGTCATCAAAAGAAGATGATATAGTGC; (SEQ ID NO.3); primer-R:TTAATCTATTCCATACTTTTTCCTTAATTCC; (SEQ ID NO. 4).

[0033] The pEASY-Blunt E1::Apa2aAg03750 vector was constructed using directed cloning. The specific construction method was as follows: the target gene was amplified by PCR using TransStart FastPfu DNA Polymerase (hot-start high-fidelity DNA polymerase). The PCR reaction system is shown in Table 1. The PCR reaction program was: 95℃ pre-denaturation for 2 min; then 40 cycles of 95℃ denaturation for 20 s, 55℃ annealing for 20 s, and 72℃ extension for 15 s; followed by a further extension at 72℃ for 5 min; finally, the temperature was maintained at 4℃ to obtain the target gene PCR product. Then, the target gene PCR product was mixed with pEASY-Blunt E1 Expression Vector (CE111, Beijing TransGen Biotech Co., Ltd.) at a volume ratio of 1:4, reacted at 25℃ for 5 min, and then ligated to obtain the ligation product. The ligation product was added to 50 µL of Trans1-T1 E. coli competent cells and placed in a 1.5 mL centrifuge tube. The mixture was mixed, incubated on ice for 30 min, and then heat-shocked in a 42℃ water bath for 30 s. Then, the mixture was transferred to ice and allowed to stand for 2 min. 500 µL of LLB liquid medium (1 L L L LB liquid medium contains: 10 g sodium chloride, 10 g tryptone, 5 g yeast extract, and the remainder is water; the LLB liquid medium described below is the same as here) was added to the centrifuge tube and cultured at 200 rpm and 37℃ for 45 min with shaking to obtain the transformed competent cells. 200 µL of transformed competent cells were plated and cultured for 16 h. Single colonies were then picked and cultured in 1 mL of LB broth containing 100 mg / L ampicillin at 37 °C for 6 h at 200 rpm. Colony PCR amplification was performed using primers for the target gene (PCR procedure and system as above). PCR products were obtained and detected by electrophoresis on a 1% agarose gel. Bacterial cultures showing the corresponding target band on the agarose gel were sent to Shanghai Sangon Biotech for first-generation sequencing verification. Verification showed that the target gene fragment was successfully inserted into the vector.

[0034]

[0035] Note: TransStart FastPfu The buffer contained 100 mM Tris-SO4 (pH 9.2), 200 mM KCl, 50 mM (NH4)2SO4, 10 mM MgSO4, and 10% Glycerol; the final concentration of cDNA in the PCR system was 20 ng. TransStart FastPfu DNA Polymerase (hot-start high-fidelity DNA polymerase), model AP221-01, was purchased from Beijing TransGen Biotech Co., Ltd.

[0036] 2.3 Obtaining Recombinant Expression Strains Single colonies with sequencing results consistent with the target gene sequence were amplified, and recombinant plasmids were extracted using a plasmid extraction kit (DP103, Tiangen Biotech Co., Ltd.). 1 µL of the recombinant plasmid and the pEASY-Blunt E1 empty vector plasmid were added to 100 µL of BL21(DE3) competent cells, respectively. The cells were incubated on ice for 30 min, then in a 42°C water bath for 60 s, followed by 2 min on ice. 1 mL of LB liquid medium was added, and the cells were cultured at 37°C with shaking at 200 rpm for 45 min to obtain transformed competent cells. 100 µL of the transformed competent cells were plated and cultured at 37°C for 16 h. Single colonies were then picked and cultured in 1 mL of LB liquid medium containing 100 mg / L ampicillin, at 37°C for approximately 6 h to obtain recombinant plasmid colonies and empty vector colonies. Colony PCR amplification was performed on recombinant plasmid colonies and empty vector colonies using the target gene primers (same as in Experiment 2.2 above) and T7 universal primers (T7 Forward: TAATACGACTCACTATAGGG (SEQ ID NO. 5); T7 Terminator: GCTAGTTATTGCTCAGCGG (SEQ ID NO. 6)). The PCR amplification system and procedure were the same as described in 2.2 above. The PCR products were detected by electrophoresis on a 1% agarose gel. Bacterial solutions corresponding to the target band size were stored in 50% glycerol at -80°C to obtain recombinant plasmid colonies and pEASY-Blunt E1 empty vector colonies.

[0037] Example 3: Identification of herbicide resistance phenotypes in Amaranth arvensis 3.1 Take 2µL of the recombinant plasmid colonies and pEASY-Blunt E1 empty vector colonies preserved in Example 2.3 above into a solid medium containing 100mg / L Amp (ampicillin) + LB (1L solid medium contains: 10g tryptone, 5g yeast extract, 10g sodium chloride, 15g agar powder, and the remainder is water), streak in a zigzag pattern using an inoculation loop, and incubate at 37℃ for 20h. Single colonies were picked and inoculated into 10 mL of LB liquid medium. The culture was incubated at 37°C and 200 rpm until OD600 = 0.6, completing the culture phase. 400 µL of the culture was then added to LB liquid medium containing 100 mg / L ampicillin, 1 mM IPTG (isopropyl-β-D-thiogalactopyranoside), and glyphosate at final concentrations of 0, 5, 10, 15, 20, and 25 mM, with a final volume of 5 mL. The medium was incubated at 37°C and 200 rpm for 10 h to obtain recombinant plasmid and empty vector cultures. The absorbance changes of the recombinant plasmid and empty vector cultures under different glyphosate gradients were recorded. Three replicates were set for each glyphosate concentration, and the absorbance was measured three times for each replicate, with the average value taken. The growth of the recombinant plasmid and empty vector cultures after adding different concentrations of glyphosate is shown below. Figure 2 As shown.

[0038] like Figure 2 As shown, the recombinant plasmid bacterial culture exhibited strong resistance to glyphosate, indicating that the protein encoded by the Apa2aAg03750 gene is highly resistant to glyphosate and also resistant to the herbicide Amaranthus longicornis.

[0039] 3.2. The bacterial culture was cultured until OD600 = 0.7 (the culture stage was the same as in 3.1 above, the only difference being the OD value). 400 µL of the bacterial culture was added to LB liquid medium containing 100 mg / L ampicillin, 1 mM IPTG (isopropyl-β-D-thiogalactopyranoside), and a final concentration of 20 mM glyphosate, resulting in a final volume of 5 mL. The medium was incubated at 37°C with shaking at 200 rpm. The absorbance of the recombinant plasmid and empty vector bacterial cultures was recorded every 1 hour after drug addition (1 h, 2 h, 3 h, 4 h, 5 h, and 6 h). Three replicates were set up for each time point. The absorbance was measured three times for each replicate, and the average value was taken. The growth of the recombinant plasmid and empty vector bacterial cultures at 1 h, 2 h, 3 h, 4 h, 5 h, and 6 h after adding 20 mM glyphosate is shown below. Figure 3 As shown.

[0040] Depend on Figure 3It can be seen that the growth of the empty vector bacterial solution was significantly inhibited, but the recombinant plasmid bacterial solution could still maintain a stable reproduction rate, which further indicates that the protein encoded by the Apa2aAg03750 gene has strong resistance to glyphosate, thus conferring resistance to high concentrations of glyphosate to the recombinant plasmid bacterial solution, i.e., resistance to the herbicide Amaranthus longicornis.

[0041] Therefore, this invention constructs a recombinant expression strain to evaluate the herbicide resistance (glyphosate) of the Apa2aAg03750 gene, and determines that its recombinant expression can significantly enhance the herbicide resistance of the recombinant expression strain; it provides a reference for using genetic engineering technology to cultivate herbicide-resistant crops and provides a potential target for overcoming herbicide resistance in weeds such as amaranth.

[0042] The above are merely preferred embodiments of the present invention and are not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. An Apa2aAg03750 gene resistant to the herbicide *Amaranthus longicornis*, characterized in that, The nucleotide sequence of the Apa2aAg03750 gene is shown in SEQ ID NO.1, and the amino acid sequence encoded by the Apa2aAg03750 gene is shown in SEQ ID NO.

2.

2. A recombinant expression vector, characterized in that, The recombinant expression vector contains the Apa2aAg03750 gene as described in claim 1.

3. A recombinant expression vector according to claim 2, characterized in that, The recombinant expression vector has a vector backbone of pEASY-Blunt E1.

4. A method for constructing a recombinant expression vector, used to construct the recombinant expression vector according to any one of claims 2 to 3, characterized in that, Includes the following steps: S1. Using Amaranthus longicornis cDNA as a template, PCR amplification was performed to obtain the PCR amplification product; S2. The PCR amplification product is ligated with pEASY-Blunt E1 to obtain the ligation product. S3. The ligation product was then introduced into competent E. coli cells, and the results were verified by PCR amplification and gel electrophoresis, confirming that the target gene was successfully inserted into the recombinant expression vector pEASY-Blunt E1.

5. The method for constructing a recombinant expression vector according to claim 4, characterized in that, In step S1, the PCR amplification reaction program is as follows: pre-denaturation at 95°C for 2 min; then 40 cycles of denaturation at 95°C for 20 s, annealing at 55°C for 20 s, and extension at 72°C for 15 s; and further extension at 72°C for 5 min; finally, maintaining at 4°C.

6. A method for constructing a recombinant expression vector according to claim 4, characterized in that, In step S1, the PCR reaction system consists of: 2 μL cDNA, 1 μL upstream primer, 1 μL downstream primer, 10 μL 5×TransStart FastPfuBuffer, 4 μL dNTPs, 1 μL DNA polymerase, and 31 μL ddH2O, for a total of 50 μL.

7. The method for constructing a recombinant expression vector according to claim 4, characterized in that, In step S2, the connection temperature is 25°C and the connection time is 5 minutes.

8. A method for constructing a recombinant expression vector according to claim 4, characterized in that, In step S2, during the ligation process, the volume ratio of the PCR amplification product to pEASY-Blunt E1 is 1:

4.

9. The application of the Apa2aAg03750 gene, which is a herbicide resistant to amaranth as described in claim 1, in the development of amaranth herbicides targeting new herbicides.

10. The application of the Apa2aAg03750 gene, as described in claim 1, resistant to the herbicide *Amaranthus longicornis*, in the breeding of herbicide-resistant crops.

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