Method for visually and rapidly detecting Ile-1781-Leu mutation of ACCase gene of moleplant seeds

Abstract

The invention discloses a method for visually and rapidly detecting Ile-1781-Leu mutation of a moleplant seed ACCase gene, and relates to the technical field of herbicide-resistant weed detection. Specific primers (SEQ ID NO.1 and SEQ ID NO.2) and a probe (SEQ ID NO.3) for RAA reaction are designed on the basis of a 1781st amino acid resistance mutation region of a moleplant seed ACCase gene, a CRISPR / Cas12a fluorescent color development system is combined, an RAA-CRISPR / Cas12a visual detection method is established, a sample with Ile-1781-Leu mutation shows fluorescence under a blue light instrument, and the detection result shows that the detection result shows that the detection result shows that the detection result shows that the detection result shows that the detection result shows that the detection result shows that the detection result shows that the detection result shows that the detection result shows that the detection result shows that the detection result shows that the detection result shows that the detection result shows that the detection result shows that Compared with a traditional PCR method, the method is easy to operate, high in sensitivity and short in reaction time, supports visual rapid detection, and provides technical support for rapid diagnosis and scientific prevention and treatment of resistant moleplant seed target mutation.

Description

Technical Field

[0001] The present invention relates to the field of herbicide-resistant weed detection, and in particular to a method for rapid and visual detection of the Ile-1781-Leu mutation in the ACCase gene of resistant Leptochloa chinensis based on RAA-CRISPR / Cas12a. Background Art

[0002] Leptochloa chinensis (L.) Nees is one of the common grass weeds in rice fields and can cause a serious reduction in the yield of early, mid- and late rice. Cyhalofop-butyl is currently the most widely used acetyl-CoA carboxylase (ACCase) inhibitor in rice fields. It controls annual grass weeds in rice fields, especially Leptochloa chinensis. However, the continuous use of cyhalofop-butyl for many years has inevitably led to the rapid development of cyhalofop-butyl resistance in Leptochloa chinensis. The emergence of this resistant Leptochloa chinensis has made cyhalofop-butyl lose its effectiveness against such weeds, and the herbicides famoxadone and fenoxaprop-butyl, which have the same mechanism of action, are also greatly reduced in their effectiveness against resistant Leptochloa chinensis. Blindly increasing the amount of herbicide used not only increases the cost of the drug, but also affects rice yield. Therefore, there is an urgent need for a method that can quickly and easily detect cyhalofop-butyl resistance in Leptochloa chinensis.

[0003] The target of cyhalofop-butyl is acetyl-CoA carboxylase in plants. It inhibits the activity of ACCase in plastids, thereby affecting the normal fatty acid synthesis in plants and causing plant death. The main reason for the resistance of Leptochloa chinensis to cyhalofop-butyl is the occurrence of point mutations in the ACCase gene, which results in the obstruction or weakening of the binding of the herbicide to the target, thereby reducing the effect of the herbicide. Traditional target gene resistance mutation detection methods, including PCR, LAMP and other technologies (such as Chinese patent CN103820551 A), although these methods have certain applicability, have problems such as long detection cycle and high equipment dependence.

[0004] Recombinase-aided amplification (RAA) is a method that rapidly amplifies DNA at a constant temperature (37-42°C) using recombinase, single-strand binding protein, and DNA polymerase, without the need for thermostable enzymes or precision thermal cyclers. Currently, no RAA-based method for detecting ACCase gene mutations has been reported. Summary of the Invention

[0005] To address the above issues, this application combines RAA and CRISPR / Cas12a for the first time, first using RAA for rapid, constant-temperature nucleic acid amplification, and then using the highly specific recognition and cutting capabilities of the CRISPR / Cas12a system to detect specific DNA sequences, so that the results can be directly judged by fluorescent colorimetric reactions. Compared with traditional PCR methods, this method is simple to operate, highly sensitive, and has a short reaction time. It supports rapid visual detection, providing a new technical means for the rapid detection and scientific prevention of target mutations in resistant Leptospermum chinense.

[0006] Specifically, the above invention object is achieved through the following solutions:

[0007] First, this application has adopted a visual rapid detection method for the Ile-1781-Leu mutation of the ACCase gene in the resistant Leptochloa chinensis. The specific steps of the RAA-CRISPR / Cas12a detection are as follows:

[0008] (1) Using a conventional DNA extraction kit to extract genomic DNA from the leaves of the Leptochloa chinensis plant to be tested;

[0009] (2) Using the genomic DNA extracted in step (1) as a template, perform RAA amplification:

[0010] The RAA reaction system (50 μL) consists of: 25 μL buffer, 2.5 μL magnesium acetate (280 mmol / L), 2 μL each of the upstream primer RAA-F (10 μM) and the downstream primer RAA-R2 (10 μM) with nucleotide sequences as shown in SEQ ID NO. 1 and SEQ ID NO. 2, 2 μL DNA template (concentration approximately 20-200 ng / μL), and 16.5 μL ddH2O.

[0011] Reaction conditions: 37°C, 30 min; after the reaction is completed, 50 μL of DNA extraction reagent (volume ratio: Tris-saturated phenol: chloroform: isoamyl alcohol = 25:24:1) is added to each reaction tube, and then centrifuged at 12,000 rpm for 5 min. The supernatant is the RAA amplification product.

[0012] (3) CRISPR / Cas12a color development reaction using RAA amplification products

[0013] The CRISPR / Cas12a reaction system is 25 μL: 2.5 μL of 10×NEB buffer, 0.5 μL of RNase inhibitor, 0.5 μL of Cas12a (Cpf1) nuclease, 2 μL of ssDNA (10 μM), 2.5 μL of crRNA (10 μM) with the nucleotide sequence shown in SEQ ID NO. 3, 2 μL of the RAA amplification product obtained in step (2), and 15 μL of ddH2O;

[0014] Reaction conditions: 37°C, 30 min, to obtain CRISPR / Cas12a amplification products;

[0015] The CRISPR / Cas12a amplification products were detected by fluorescence using a blue light gel imager. Samples showing fluorescent reactions were determined to have an Ile-1781-Leu mutation in the ACCase gene.

[0016] Secondly, the present application provides a kit for detecting the Ile-1781-Leu mutation in the ACCase gene of resistant Leptochloa chinensis, including an RAA detection kit and a CRISPR / Cas12a detection kit:

[0017] The RAA detection kit includes: 25 μL of buffer, 2.5 μL of magnesium acetate (280 mmol / L), 2 μL of 10 μM upstream primer RAA-F (SEQ ID NO. 1), 2 μL of 10 μM downstream primer RAA-R2 (SEQ ID NO. 2), and 16.5 μL of ddH2O.

[0018] The CRISPR / Cas12a detection kit includes: 2.5 μL of 10×NEB buffer, 0.5 μL of RNase inhibitor, 0.5 μL of Cas12a nuclease, 2 μL of ssDNA (10 μM), 2.5 μL of crRNA (SEQ ID NO.3) with a concentration of 10 μM, and 15 μL of ddH2O; wherein the ssDNA molecular sequence is

[0019] 6-FAM-TTATT-BHQI.

[0020] Third, the present application provides a pair of primers for detecting the Ile-1781-Leu mutation of the ACCase gene of Leptochloa chinensis, and the nucleotide sequences of the primers are shown in SEQ ID NO.1 and SEQ ID NO.2, respectively.

[0021] In this application, the term "Ile-1781-Leu mutation" refers to the mutation of the amino acid codon at position 1781 of the ACCase gene in Leptochloa chinensis (GeneBank accession numbers: MW266986 and MW266987) from ATA to TTA, and the corresponding amino acid from Ile (isoleucine) to Leu (leucine). The mutant Leptochloa chinensis plants are resistant to the herbicide cyhalofop-butyl. The term "visualization" refers to the appearance of fluorescence under blue light.

[0022] Based on the published ACCase gene sequence of Leptochloa chinensis in the GenBank database (GeneBank accession numbers: MW266986 and MW266987), and based on the amino acid mutation region at position 1781 of the ACCase gene, this application designs specific RAA primers and CRISPR probes for detecting the Ile-1781-Leu mutation type of the ACCase gene of resistant Leptochloa chinensis. Combined with the CRISPR / Cas12a fluorescence colorimetric system, Leptochloa chinensis samples with the Ile-1781-Leu mutation of the ACCase gene can exhibit a fluorescent colorimetric reaction under blue light ( Figure 6 ).

[0023] This detection method can amplify the target gene under constant temperature conditions. By combining RAA and CRISPR / Cas12a, the reaction conditions are optimized. Amplification detection can be performed after crude gene extraction of samples in the field, significantly shortening the time required for the detection cycle. The detection method only requires a metal bath or water bath for amplification, and the results can be directly judged by observing fluorescence under blue light, eliminating the complex temperature change process and getting rid of the dependence on complex instruments and equipment such as centrifuges and PCR machines. It not only has the advantages of high sensitivity and simple operation, but also has a short reaction time (only 1 hour, while the conventional PCR reaction time is generally about 2.5 hours). It can achieve rapid detection of large quantities of samples and can provide strong technical support for the scientific prevention and control of resistant Leptospermum chinense. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 Whole plant dose-response bioassays for sensitive and resistant Leptochloa chinensis populations 1 and 2.

[0025] Figure 2 The base sequences of the ACCase gene at position 1781 of sensitive and resistant Leptochloa chinensis; (1) is the sensitive Leptochloa chinensis, and (2) is the resistant Leptochloa chinensis population 1.

[0026] Figure 3 Sequence of the RAA amplified product of the ACCase gene from Leptochloa chinensis. Green represents the RAA primer.

[0027] Figure 4 Results of RAA amplification of the ACCase gene in sensitive and resistant Leptochloa chinensis. M: DNA relative molecular mass standard; S: sensitive population; R: resistant population; NC: negative control.

[0028] Figure 5 Fluorescence intensity graph of the RAA amplified products of the sensitive and resistant Leptochloa chinensis ACCase genes after CRISPR / Cas12a reaction; NC: negative control.

[0029] Figure 6Validation experiments were performed on multiple samples of P. japonica with known genotypes. DETAILED DESCRIPTION

[0030] Example 1 PCR, RAA, crRNA, and ssDNA Primer Synthesis of Leptochloa chinensis ACCase Gene

[0031] According to the GenBank database, combined with the two published ACCase gene sequences of Leptochloa chinensis (GeneBank accession numbers: MW266986 and MW266987), PCR primers, RAA primers and CRISPR guide RNA (crRNA) probes were designed targeting the Ile-1781-Leu mutation site of the ACCase gene of Leptochloa chinensis.

[0032] The RAA forward primer (RAA-F) was 30 bases before the target site (towards the 5′ direction), and three reverse primers RAA-R1, RAA-R2, and RAA-R3 were designed based on the RAA-F sequence.

[0033] The single-stranded DNA (ssDNA) reporter sequence is 6-FAM-TTATT-BHQI, modified at the 5' end with 6-carboxyfluorescein (6-FAM) and at the 3' end with black hole quencher-I (BHQI). The primers used in this example were synthesized by Sangon Biotech (Shanghai) Co., Ltd. See Table 1 for details.

[0034] Table 1 Sequence information of primers for amplifying the ACCase gene in Leptochloa chinensis

[0035]

[0036] Example 2 Extraction of genomic DNA from sensitive and resistant Leptochloa chinensis and PCR amplification of the ACCase gene

[0037] Experimental materials: A known sensitive Leptochloa chinensis (wild type) population was collected from Yangzhou, Jiangsu Province, with no history of herbicide use. A resistant Leptochloa chinensis population 1 was collected from a rice field in Yangzhou, Jiangsu Province. The whole-plant bioassay method was used to detect the resistance levels of sensitive and resistant Leptochloa chinensis populations to cyhalofop-butyl, respectively, according to the method published in the reference "BURGOS N R. Whole-plant and seed bioassays for resistance confirmation [J]. Weed Science, 2015, 63 (SP1): 152-165.". The sensitive group showed no resistance to cyhalofop-butyl (e.g. Figure 1 ), while the resistant Leptochloa chinensis population 1 developed a high level of resistance to cyhalofop-butyl (as shown in Figure 1(shown in B).

[0038] Experimental method: About 100 mg of leaves of Leptochloa chinensis were cut and the sample DNA was extracted using a high-efficiency plant genomic DNA extraction kit (Tiangen Biochemical Technology (Beijing) Co., Ltd., DP350) and then PCR amplification was performed.

[0039] PCR reaction system: 2×Taq PCRMix 12.5 μL, upstream and downstream primers (SEQ ID NO. 4, SEQ ID NO. 5, both 10 μM) 1 μL each, DNA template 1 μL, ddH2O 11.5 μL.

[0040] PCR amplification protocol: 94°C for 5 min, 34 cycles of (94°C for 30 s, 58°C for 30 s, 72°C for 60 s), and 72°C for 5 min. After completion of the reaction, 5 μL of the amplified product was analyzed by 1% agarose gel electrophoresis (120 V, 30 min) and sent to Sangon Biotech (Shanghai) Co., Ltd. for bidirectional sequencing.

[0041] Experimental results: Sequencing results showed that the amino acid codon at position 1781 of the ACCase gene of the resistant Leptochloa chinensis mutated from ATA (sensitive) to TTA (resistant), resulting in the substitution of the encoded amino acid from isoleucine (Ile) to leucine (Leu), as shown in Figure 2. Figure 2 As shown in (1) and (2).

[0042] Example 3 RAA amplification primer screening

[0043] Experimental materials: Same as Example 2.

[0044] Experimental method: 50 μL RAA reaction system, including: buffer 25 μL, magnesium acetate 2.5 μL (280 mmol / L), upstream primer RAA-F (SEQ ID NO. 1) with a concentration of 10 μM 2 μL, downstream primer (10 μM) 2 μL, DNA template 2 μL, ddH2O 16.5 μL.

[0045] The buffer used in this embodiment is Abuffer in the DNA Constant Temperature Rapid Amplification Kit (WLB8201KIT) of Anpu Future (Changzhou) Biotechnology Co., Ltd., and magnesium acetate is the B buffer in the kit; in specific implementations, other conventional recombinase-mediated isothermal nucleic acid amplification (RAA) reaction buffers can also be used.

[0046] Using Leptochloa chinensis genomic DNA as a template (the extraction method was the same as in Example 2) and ddH2O as a negative control, the entire reaction system was amplified at 37°C for 30 min and set aside. After the reaction was completed, 50 μL of DNA extraction reagent (Tris-saturated phenol: chloroform: isoamyl alcohol = 25:24:1 by volume) was added to each reaction tube, and then centrifuged at 12000 rpm for 5 min. The supernatant was the RAA amplification product. 5 μL of the RAA amplification product was detected by 1% agarose gel electrophoresis (120 V, 30 min).

[0047] The above RAA reactions used RAA-R1, RAA-R2, and RAA-R3 as downstream primers for comparative detection.

[0048] Experimental results: Using the RAA method, the RAA-F / R2 primers successfully amplified the target DNA fragment after 1% agarose gel electrophoresis analysis. This primer can be used for subsequent RAA amplification of the ACCase gene in Leptochloa chinensis. The amplified fragment sequence of the RAA-F / R2 primer set is as follows: Figure 3 shown.

[0049] Example 4 RAA Amplification of Sensitive and Resistant Leptochloa chinensis ACCase Genes

[0050] Experimental materials: Same as Example 2.

[0051] Experimental method: Same as Example 3.

[0052] The RAA amplification products of the sensitive and resistant Leptochloa chinensis ACCase genes were obtained respectively and set aside.

[0053] Experimental results: Figure 4 As shown: using primers RAA-F and RAA-R2 with nucleotide sequences shown in SEQ ID NO.1 and SEQ ID NO.2 respectively, DNA fragments of the sensitive and resistant Leptochloa chinensis ACCase genes were successfully amplified, and the amplified product band size was 353 bp.

[0054] Example 5 RAA amplification product undergoes CRISPR / Cas12a fluorescence detection reaction

[0055] Experimental materials: Same as Example 2.

[0056] Experimental method: A total of 25 μL of the RAA-CRISPR / Cas12a detection system included: 2.5 μL of 10×NEB buffer (New England Biolabs, B6002S), 0.5 μL of RNase inhibitor (Tiangen Biochemical Technology (Beijing) Co., Ltd., NG209), 0.5 μL of Cas12a (Cpf1) nuclease (New England Biolabs, M0653S), 2 μL of ssDNA (10 μM), 2.5 μL of crRNA (10 μM), 2 μL of the RAA amplification product obtained in Example 3, and 15 μL of ddH2O.

[0057] At the same time, ddH2O was used as a negative control, and the detection system was placed in an enzyme-free centrifuge tube at 37°C for reaction for 30 min. After the reaction, it was placed under a blue light gel imager (Hangzhou Miou Instrument Co., Ltd., DUT-48) for fluorescence intensity detection.

[0058] Experimental results: The RAA amplification product was detected by fluorescence using the CRISPR / Cas12a detection system. Figure 5 As shown: Only the resistant Leptochloa chinensis sample with the Ile-1781-Leu mutation in the ACCase gene emitted fluorescence under a blue light gel imager and was therefore labeled positive. However, the sensitive Leptochloa chinensis sample without the ACCase gene mutation and the negative control failed to capture fluorescence and were therefore considered negative.

[0059] The above test results show that the RAA-CRISPR / Cas12a visualization system detection method established in the present invention is specific to the Ile-1781-Leu mutation type of the ACCase gene in Leptochloa chinensis.

[0060] Example 6: Accuracy Verification of RAA-CRISPR / Cas12a Detection Method

[0061] Experimental materials: Population 2 of Leptochloa chinensis with known ACCase genotypes was collected from rice fields in Yangzhou, Jiangsu Province, and its sensitivity to cyhalofop-butyl was determined by whole-plant bioassay (e.g. Figure 1 (as shown in C).

[0062] Experimental method: A commercially available DNA extraction kit was used to extract genomic DNA of known samples (the extraction method was the same as in Example 2), and the ACCase genotype of 24 individuals of the resistant Leptochloa chinensis population 2 was identified using the RAA-CRISPR / Cas12a detection method established in Example 5 above and the conventional PCR method (Example 1).

[0063] Table 2 Sensitivity of Radix Angelicae Dahuricae samples and PCR detection results of ACCase gene mutation

[0064]

[0065]

[0066] Experimental results: The genotypes of 24 individuals from the resistant Leptochloa chinensis population 2 with known genotypes were detected using the established RAA-CRISPR / Cas12a and traditional PCR methods.

[0067] The results showed that all plant samples with Ile-1781-Leu mutation in the ACCase gene emitted fluorescence under blue light, while sensitive plant samples did not show any fluorescence reaction (Table 2, Figure 6 (As shown in Figures AD). This indicates that the RAA-CRISPR / Cas12a detection method can accurately and quickly detect the Ile-1781-Leu mutation type of the ACCase gene in Leptochloa chinensis.

[0068] Compared with traditional PCR detection methods, the RAA-CRISPR / Cas12a detection system has a short reaction time, with two reactions taking only 1 hour in total. It has low equipment requirements and can perform reaction operations using a metal bath, water bath, etc. It also supports visual detection, providing a new technical means for the rapid detection of target mutations in resistant Leptochloa chinensis.

Claims

1. A method for rapid visual detection of the Ile-1781-Leu mutation in the ACCase gene of Leptochloa chinensis, characterized in that: Here are the steps: 1) Extract genomic DNA from the Leptochloa chinensis plants to be tested; 2) Using the extracted genomic DNA as a template, perform RAA amplification: The RAA reaction system is as follows: 25 µL of buffer, 2.5 µL of magnesium acetate, 2 µL of 10 µM upstream primer, 2 µL of 10 µM downstream primer, and ddH2O to 50 µL. The nucleotide sequences of the upstream primer and downstream primer are shown in SEQ ID NO. 1 and SEQ ID NO. 2, respectively. RAA reaction conditions: 37°C, 30 min. After the reaction is complete, add 50 µL of DNA extraction reagent to each reaction tube and centrifuge. The supernatant obtained is the RAA amplification product and is reserved for future use. The DNA extraction reagent is obtained by mixing Tris-saturated phenol, chloroform, and isoamyl alcohol in a volume ratio of 25:24:1; 3) CRISPR / Cas12a colorimetric reaction using RAA amplification products CRISPR / Cas12a reaction system: 2.5 μL of 10 × NEB buffer, 0.5 μL of RNase inhibitor, 0.5 μL of Cas12a nuclease, 2 μL of 10 μM ssDNA, 2.5 μL of 10 μM crRNA, 2 μL of RAA amplification product, and ddH2O to 25 μL; the nucleotide sequence of the crRNA is shown in SEQ ID NO. 3; the nucleotide sequence of the ssDNA is TTATT, and its 5' end is modified with 6-FAM and its 3' end is modified with BHQI; Reaction conditions: 37°C, 30 min, to obtain CRISPR / Cas12a amplification products; The obtained CRISPR / Cas12a amplification products were detected by fluorescence using a blue light gel imager. Samples showing fluorescent reactions were determined to have an Ile-1781-Leu mutation in the ACCase gene.

2. A kit for detecting the Ile-1781-Leu mutation in the ACCase gene of Leptochloa chinensis, characterized in that: The kit consists of an RAA detection kit and a CRISPR / Cas12a detection kit; The RAA detection kit comprises: an upstream primer and a downstream primer whose nucleotide sequences are shown as SEQ ID NO.1 and SEQ ID NO.2 respectively; The CRISPR / Cas12a detection kit includes: crRNA and ssDNA having a nucleotide sequence as shown in SEQ ID NO.3; the ssDNA nucleotide sequence is TTATT, and its 5' end is modified with 6-FAM and its 3' end is modified with BHQI.

3. The kit according to claim 2, wherein The RAA detection kit further comprises a buffer solution and magnesium acetate.

4. The kit according to claim 2, wherein The CRISPR / Cas12a detection kit also includes 10× NEB buffer, RNase inhibitor, and Cas12a nuclease.

5. A pair of primers for detecting the Ile-1781-Leu mutation in the ACCase gene of Leptochloa chinensis, characterized in that: The nucleotide sequences of the primer pair are shown as SEQ ID NO.1 and SEQ ID NO.2 respectively.

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