CN116218957B - A method for detecting herbicide-resistant AHAS genes in millet - Google Patents

Abstract

The present invention provides a method for detecting the herbicide-resistant AHAS gene of millet, which comprises the steps of detecting by using gRNA, Cas protein and single-stranded nucleic acid detector. The present invention improves the detection efficiency by mining and screening SNP sites and screening and optimizing gRNA, and has broad application prospects.

Description

A method for detecting herbicide-resistant AHAS genes in millet

Technical Field

The present invention relates to the field of agricultural biotechnology, and in particular to a method for detecting millet AHAS genes, and in particular to a method, system and kit for detecting millet herbicide-resistant AHAS genes based on CRISPR technology.

Background technique

Millet (Latin name: Setaria italica) is a plant of the Poaceae family. It was called millet, millet, and sorghum in ancient times. In my country, millet is a special drought-resistant crop. The germination of seeds only requires 26% of its weight in water, while the germination of sorghum, wheat, and corn seeds requires 40%, 45%, and 48% of their weight in water, respectively. At the same time, millet has a higher tolerance to low soil fertility, but the current millet planting area is shrinking. Millet herbicide-resistant varieties are rarely used in production, and millet planting requires manual weeding, which has always been one of the main factors hindering the development of millet industrialization.

Acetohydroxyacid synthase (AHAS), as a key enzyme in the common pathway for the synthesis of branched-chain amino acids in plants, is the target enzyme of highly effective and low-toxic chemical herbicides such as imidazolinones and sulfonylureas. AHAS inhibitors inhibit the AHAS activity of plants, thereby preventing the synthesis of branched-chain amino acids, causing damage to protein synthesis and hindering DNA synthesis during cell division, thereby stopping the mitosis of plant cells in the S phase (DNA synthesis phase) of the G1 phase and the M phase of the G2 phase. As a result, cells cannot complete mitosis, which in turn stops the growth of plants and eventually leads to the death of individual plants. Important progress has also been made in the detailed mechanism of action of AHAS inhibitors.

Imidazolin is an important monocotyledon herbicide. Imidazolin is a typical representative of imidazolinone herbicides and belongs to the AHAS inhibitor herbicide. It is one of the most important herbicide varieties in the world and one of the most widely used herbicides. It is widely used for weed control in crops such as soybeans and wheat. Compared with domestic commonly used soybean herbicides such as fomesafen, Imidazolin has a broader spectrum, lower cost, better weed control effect, can be used in the early or late seedling stage, high activity and low quantity, etc. At present, millet varieties resistant to herbicides are rarely used in production. Breeding new millet varieties resistant to herbicides is an important basis for realizing large-scale and mechanized millet planting.

The present invention provides a novel method for detecting the herbicide-resistant AHAS gene in millet. The method is based on CRISPR technology, especially based on the trans activity of Cas12i protein, and provides a rapid detection method with high specificity and high detection sensitivity.

Summary of the invention

The present invention provides a method, system and kit for detecting the herbicide-resistant AHAS gene of millet based on CRISPR technology.

On the one hand, the present invention provides a method for detecting the millet imidacloprid herbicide-resistant gene AHAS gene, the method comprising contacting a nucleic acid to be tested with a Cas12i protein, the above-mentioned gRNA and a single-stranded nucleic acid detector; the gRNA comprises a region binding to the Cas12i protein and a guide sequence hybridizing with the target nucleic acid, and the guide sequence hybridizing with the target nucleic acid is shown in any one of SEQ ID No.3-4; and detecting a detectable signal generated by the Cas protein cutting the single-stranded nucleic acid detector, thereby detecting the millet herbicide-resistant AHAS gene.

In the present invention, the region that binds to the CRISPR/CAS effector protein is also called a direct repeat sequence, a backbone region or a spacer sequence, and this region interacts with the Cas protein, thereby binding to the Cas protein.

In one embodiment, the gRNA includes, from the 5' end to the 3' end, a region that binds to the Cas protein and a guide sequence that hybridizes with the target nucleic acid.

In one embodiment, the guide sequence that hybridizes with the target nucleic acid contains 20-30 bases and hybridizes with the sequence shown in SEQ ID No. 9 or its reverse complementary sequence, and the guide sequence comprises the sequence shown in any one of SEQ ID No. 3-4.

In a preferred embodiment, the guide sequence that hybridizes to the target nucleic acid contains 20-30 bases, for example, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30 bases.

The hybridization with the sequence shown in SEQ ID No. 9 or its reverse complementary sequence means that the above-mentioned guide sequence can be continuously complementary to a continuous section of SEQ ID No. 9 or the reverse complementary sequence of SEQ ID No. 9. For example, if the guide sequence hybridized with the target nucleic acid contains 30 bases, then the 30 bases of the guide sequence need to be complementary to 30 continuous bases of SEQ ID No. 9 or the complementary sequence of SEQ ID No. 9.

Preferably, the sequence of the region that binds to the Cas protein is shown as SEQ ID No.7.

In one embodiment, the Cas12i protein is selected from any one of the following:

1. The amino acid sequence of the Cas12i protein is shown in SEQ ID No.2;

II. The amino acid sequence of the Cas12i protein is compared with SEQ ID No.2, and there is a mutation in the 944th amino acid corresponding to the amino acid sequence shown in SEQ ID No.2.

In one embodiment, the amino acid position 944 mutates to a non-S amino acid, for example, A, D, G, L, Q, F, W, Y, V, N, E, K, M, T, C, P, H, R, I; preferably, the amino acid position 944 mutates to R.

It should be recognized that proteins can be changed in various ways, including amino acid substitutions, deletions, truncations and insertions, and methods for such operations are generally known in the art. For example, amino acid sequence variants of the above-mentioned proteins can be prepared by mutations to DNA. It can also be accomplished by other forms of mutagenesis and/or by directed evolution, for example, using known mutagenesis, recombination and/or shuffling methods, combined with related screening methods, to perform single or multiple amino acid substitutions, deletions and/or insertions. Those skilled in the art will understand that these minor amino acid changes in the Cas protein of the present invention can occur (e.g., naturally occurring mutations) or be produced (e.g., using r-DNA technology) without loss of protein function or activity. If these mutations occur in the catalytic domain, active site or other functional domain of the protein, the properties of the polypeptide may change, but the polypeptide may maintain its activity. If the mutations present are not close to the catalytic domain, active site or other functional domain, a smaller effect can be expected.

Those skilled in the art can identify the essential amino acids of the Cas mutant protein of the present invention according to methods known in the art, such as site-directed mutagenesis or protein evolution or analysis of a bioinformatics system. The catalytic domain, active site or other functional domain of the protein can also be determined by physical analysis of the structure, such as by the following techniques: such as nuclear magnetic resonance, crystallography, electron diffraction or photoaffinity labeling, combined with mutations of putative key site amino acids.

In the present invention, amino acid residues can be represented by single letters or three letters, for example: alanine (Ala, A), valine (Val, V), glycine (Gly, G), leucine (Leu, L), glutamine (Gln, Q), phenylalanine (Phe, F), tryptophan (Trp, W), tyrosine (Tyr, Y), aspartic acid (Asp, D), asparagine (Asn, N), glutamic acid (Glu, E), lysine (Lys, K), methionine (Met, M), serine (Ser, S), threonine (Thr, T), cysteine (Cys, C), proline (Pro, P), isoleucine (Ile, I), histidine (His, H), arginine (Arg, R).

The term "AxxB" means that the amino acid A at position xx is changed to amino acid B, for example S944R means that S at position 944 is mutated to R.

The specific amino acid position (number) within the protein of the present invention is determined by aligning the amino acid sequence of the target protein with SEQ ID No. 2 using standard sequence alignment tools, such as the Smith-Waterman algorithm or the CLUSTALW2 algorithm, wherein the sequences are considered aligned when the alignment score is the highest. The alignment score can be calculated according to the method described in Wilbur, W.J. and Lipman, D.J. (1983) Rapid similarity searches of nucleic acid and protein data banks. Proc. Natl. Acad. Sci. USA, 80: 726-730. The default parameters are preferably used in the ClustalW2 (1.82) algorithm: protein gap opening penalty = 10.0; protein gap extension penalty = 0.2; protein matrix = Gonnet; protein/DNA end gap = -1; protein/DNAGAPDIST = 4. Preferably, the AlignX program (part of the vectorNTI group) is used to determine the position of specific amino acids in the protein of the present invention by comparing the amino acid sequence of the protein with SEQ ID No. 2 with default parameters suitable for multiple alignment (gap opening penalty: 10og gap extension penalty 0.05).

In one embodiment, the nucleic acid sequence of the herbicide-resistant AHAS gene is shown as SEQ ID No.9, and the amino acid sequence is shown as SEQ ID No.1.

Furthermore, the method also includes the step of obtaining the nucleic acid to be tested from the sample to be tested, for example, the step of performing PCR amplification on the sample to be tested.

Furthermore, the method further comprises the step of using a primer pair to amplify and obtain the nucleic acid to be tested, wherein the upstream primer of the primer pair is shown as SEQ ID NO:5, and the specific downstream primer is shown as SEQ ID NO:6.

In the present invention, the nucleic acid to be detected may be a double-stranded nucleic acid or a single-stranded nucleic acid.

The amplification of the present invention is selected from one or any combination of PCR, nucleic acid sequencing-based amplification (NASBA), recombinase polymerase amplification (RPA), loop-mediated isothermal amplification (LAMP), strand displacement amplification (SDA), helicase-dependent amplification (HDA), or nickase amplification reaction (NEAR), multiple displacement amplification (MDA), rolling circle amplification (RCA), ligase chain reaction (LCR), or derivative amplification method (RAM).

In the present invention, the sample can be a sample from a plant, for example, millet, potato, Brazilian morning glory, Japanese morning glory, common morning glory; in other embodiments, the sample can also come from other plants, for example, tobacco, amaranth, beet, Brassica, fennel, cucumber, gourd, Datura, globe amaranth, tomato, heart-leaf tobacco, kidney bean, radish, cowpea.

On the other hand, the present invention also provides a system, composition or kit for detecting or diagnosing the above-mentioned herbicide-resistant AHAS gene, the system, composition or kit comprising Cas12i protein, the above-mentioned gRNA and a single-stranded nucleic acid detector. Further, the system, composition or kit also includes an amplification primer; preferably, the amplification primer includes the above-mentioned primer.

On the other hand, the present invention also provides the use of the above system, composition or kit in detecting herbicide-resistant AHAS genes.

On the other hand, the present invention also provides the use of the above-mentioned system and composition in detecting millet resistance to imazethapyr herbicide, or in preparing a reagent or kit for detecting millet resistance to imazethapyr herbicide.

On the other hand, the present invention also provides use of the above composition in millet breeding.

In a preferred embodiment, the amino acid sequence of the Cas12i protein is selected from the group consisting of:

(1) the protein shown in SEQ ID NO: 2;

(2) A derivative protein having substantially the same function formed by replacing, deleting or adding one or more (e.g., 2, 3, 4, 5, 6, 7, 8, 9 or 10) amino acid residues of the amino acid sequence shown in SEQ ID NO: 2 or an active fragment thereof;

(3) A protein having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% sequence identity to SEQ ID NO: 2 and having trans activity.

In one embodiment, the Cas protein mutant comprises an amino acid substitution, deletion or replacement, and the mutant at least retains its trans cleavage activity. Preferably, the mutant has Cis and trans cleavage activity.

In the present invention, "Cas mutant protein" can also be referred to as a mutated Cas protein, or a Cas protein variant.

In the present invention, the single-stranded nucleic acid detector includes single-stranded DNA, single-stranded RNA or single-stranded DNA-RNA hybrid. In other embodiments, the single-stranded nucleic acid detector includes a mixture of any two or three of single-stranded DNA, single-stranded RNA or single-stranded DNA-RNA hybrid, for example, a combination of single-stranded DNA and single-stranded RNA, a combination of single-stranded DNA and single-stranded DNA-RNA hybrid, and a combination of single-stranded RNA and single-stranded DNA-RNA. In other embodiments, the single-stranded nucleic acid detector also includes a modification of the base.

In a preferred embodiment, the single-stranded nucleic acid detector is a single-stranded oligonucleic acid detector.

The single-stranded nucleic acid detector does not hybridize with the gRNA.

In the present invention, the detectable signal is realized by the following means: vision-based detection, sensor-based detection, color detection, gold nanoparticle-based detection, fluorescence polarization, fluorescence signal, colloidal phase transition/dispersion, electrochemical detection and semiconductor-based detection.

In some embodiments, the method of the present invention further comprises the step of measuring a detectable signal generated by a CRISPR/CAS effector protein (Cas protein). The Cas protein can stimulate the cleavage activity of any single-stranded nucleic acid after recognizing the target nucleic acid or hybridizing with the target nucleic acid, thereby cleaving the single-stranded nucleic acid detector and generating a detectable signal.

In the present invention, the detectable signal can be any signal generated when the single-stranded nucleic acid detector is cut. For example, detection based on gold nanoparticles, fluorescence polarization, fluorescence signal, colloidal phase change/dispersion, electrochemical detection, semiconductor-based sensing. The detectable signal can be read out by any suitable means, including but not limited to: measurement of detectable fluorescence signals, gel electrophoresis detection (by detecting changes in bands on the gel), detection of the presence or absence of color based on vision or sensors, or differences in the presence of colors (e.g., based on gold nanoparticles) and differences in electrical signals.

In a preferred embodiment, the detectable signal is achieved in the following manner: different reporter groups are respectively set at the 5' end and the 3' end of the single-stranded nucleic acid detector, and when the single-stranded nucleic acid detector is cut, a detectable reporter signal can be exhibited; for example, a fluorescent group and a quenching group are respectively set at the two ends of the single-stranded nucleic acid detector, and when the single-stranded nucleic acid detector is cut, a detectable fluorescent signal can be exhibited.

In one embodiment, the fluorescent group is selected from one or any several of FAM, FITC, VIC, JOE, TET, CY3, CY5, ROX, Texas Red or LC RED460; the quenching group is selected from one or any several of BHQ1, BHQ2, BHQ3, Dabcy1 or Tamra.

In other embodiments, the detectable signal can also be achieved in the following manner: different labeling molecules are respectively set at the 5' end and the 3' end of the single-stranded nucleic acid detector, and the reaction signal is detected by colloidal gold detection.

In some embodiments, the measurement of the detectable signal can be quantitative, and in other embodiments, the measurement of the detectable signal can be qualitative.

Preferably, the single-stranded nucleic acid detector generates a first detectable signal before being cleaved by the Cas protein, and generates a second detectable signal different from the first detectable signal after being cleaved.

In other embodiments, the single-stranded nucleic acid detector includes one or more modifications, such as base modifications, backbone modifications, sugar modifications, etc., to provide new or enhanced features (such as improved stability) to the nucleic acid. Examples of suitable modifications include modified nucleic acid backbones and non-natural internucleoside linkages, and nucleic acids with modified backbones include those that retain phosphorus atoms in the backbone and those that do not have phosphorus atoms in the backbone. Suitable modified oligonucleotide backbones containing phosphorus atoms include phosphorothioates, chiral phosphorothioates, phosphorodithioates, phosphotriesters, aminoalkylphosphotriesters, methyl and other alkylphosphonates. In some embodiments, the single-stranded nucleic acid detector contains one or more phosphorothioates and/or heteroatom nucleoside bonds. In other embodiments, the single-stranded nucleic acid detector can be a nucleic acid mimetic; in certain embodiments, the nucleic acid mimetic is a peptide nucleic acid (PNA), another type of nucleic acid mimetic is based on a linked morpholinyl unit (morpholinyl nucleic acid) having a heterocyclic base attached to a morpholine ring, and other nucleic acid mimics also include cyclohexenyl nucleic acids (CENA), and also include ribose or deoxyribose chains.

In one embodiment, the molar ratio of the Cas protein to the gRNA is (0.8-1.2):1.

In one embodiment, the final concentration of the Cas protein is 20-200 nM, preferably, 30-100 nM, more preferably, 40-80 nM, more preferably, 50 nM.

In one embodiment, the final concentration of the gRNA is 20-200 nM, preferably, 30-100 nM, more preferably, 40-80 nM, more preferably, 50 nM.

In one embodiment, the final concentration of the nucleic acid to be tested is 5-100 nM, preferably, 10-50 nM.

In one embodiment, the single-stranded nucleic acid detector is used at a final concentration of 100-1000 nM, preferably 150-800 nM, preferably 200-800 nM, preferably 200-500 nM, preferably 200-300 nM.

In one embodiment, the single-stranded nucleic acid detector has 2-300 nucleotides, preferably 3-200 nucleotides, preferably 3-100 nucleotides, preferably 3-30 nucleotides, preferably 4-20 nucleotides, more preferably 5-15 nucleotides.

The term "hybridization" or "complementary" or "substantially complementary" refers to a nucleic acid (e.g., RNA, DNA) comprising a nucleotide sequence that enables it to non-covalently bind, i.e., to form base pairs and/or G/U base pairs, "anneal" or "hybridize" with another nucleic acid in a sequence-specific, antiparallel manner (i.e., nucleic acid-specific binding to complementary nucleic acids). Hybridization requires that the two nucleic acids contain complementary sequences, although there may be mismatches between the bases. Suitable conditions for hybridization between two nucleic acids depend on the length of the nucleic acids and the degree of complementarity, which are variables well known in the art. Typically, the length of a hybridizable nucleic acid is 8 nucleotides or more (e.g., 10 nucleotides or more, 12 nucleotides or more, 15 nucleotides or more, 20 nucleotides or more, 22 nucleotides or more, 25 nucleotides or more, or 30 nucleotides or more).

It should be understood that the sequence of a polynucleotide need not be 100% complementary to the sequence of its target nucleic acid to specifically hybridize. A polynucleotide may comprise 60% or more, 65% or more, 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, 95% or more, 98% or more, 99% or more, 99.5% or more, or 100% sequence complementarity to the target region in the target nucleic acid sequence with which it hybridizes.

General Definition:

Unless defined otherwise, technical and scientific terms used herein have the same meanings as commonly understood by one of ordinary skill in the art.

The term "amino acid" refers to a carboxylic acid containing an amino group. Various proteins in living organisms are composed of 20 basic amino acids.

The terms "polynucleotide", "nucleotide sequence", "nucleic acid sequence", "nucleic acid molecule" and "nucleic acid" are used interchangeably and include DNA, RNA or hybrids thereof, which may be double-stranded or single-stranded.

The term "oligonucleotide" refers to a sequence containing 3-100 nucleotides, preferably, 3-30 nucleotides, preferably, 4-20 nucleotides, more preferably, 5-15 nucleotides.

The terms "homology" or "identity" are used to refer to the matching of sequences between two polypeptides or between two nucleic acids. When a position in the two sequences being compared is occupied by the same base or amino acid monomer subunit (e.g., a position in each of the two DNA molecules is occupied by adenine, or a position in each of the two polypeptides is occupied by lysine), then the molecules are identical at that position. Between the two sequences. Typically, the comparison is made when the two sequences are aligned to produce maximum identity. Such comparison can be by using, for example, the identity of amino acid sequence can be by conventional methods, reference such as Smith and Waterman, 1981, Adv.Appl.Math.2:482Pearson & Lipman, 1988, Proc.Natl Acad.Sci.USA 85:2444, Thompson et al., 1994, Nucleic Acids Res 22:467380 etc., by computerized operation algorithm (GAP, BESTFIT, FASTA, and TFASTA in Wisconsin Genetics software package, Genetics Computer Group) determine. Also can use the BLAST algorithm that can be obtained from the National Center for Biotechnology Information (NCBI www.ncbi.nlm.nih.gov/), use default parameters to determine.

As used herein, the "CRISPR" refers to Clustered regularly interspaced short palindromic repeats, which are derived from the immune system of microorganisms.

As used herein, "biotin", also known as vitamin H, is a small molecule vitamin with a molecular weight of 244 Da. "Avidin", also known as anti-biotin, is an alkaline glycoprotein with 4 binding sites with extremely high affinity to biotin. Commonly used avidins include streptavidin. The extremely strong affinity of biotin and avidin can be used to amplify or enhance the detection signal in the detection system. For example, biotin can easily bind to proteins (such as antibodies, etc.) with covalent bonds, and the avidin molecules bound to the enzyme react with the biotin molecules bound to the specific antibody, which not only plays a multi-stage amplification role, but also produces color due to the catalytic effect of the enzyme when it encounters the corresponding substrate, thereby achieving the purpose of detecting unknown antigen (or antibody) molecules.

SNP

Single nucleotide polymorphism (SNP) mainly refers to the DNA sequence polymorphism caused by the variation of a single nucleotide at the genomic level. SNP is a dimorphic marker caused by the conversion or transversion of a single base, or by the insertion or deletion of a base. SNP may be within the gene sequence or in the non-coding sequence outside the gene. The polymorphism expressed by SNP only involves the variation of a single base, which can be caused by the conversion or transversion of a single base, or by the insertion or deletion of a base.

The generation of imazethapyr resistance in millet described herein is based on the generation of SNP sites in the AHAS gene.

gRNA

As used herein, the "gRNA" is also referred to as guide RNA or guide RNA, and has the meaning generally understood by those skilled in the art. In general, the guide RNA may include a direct repeat sequence and a guide sequence, or may consist essentially of or consist of a direct repeat sequence and a guide sequence (also referred to as a spacer in the context of an endogenous CRISPR system). In different CRISPR systems, gRNA may include crRNA and tracrRNA, or may only contain crRNA, depending on the different Cas proteins it depends on. crRNA and tracrRNA may be artificially modified and fused to form a single guide RNA (sgRNA). In some cases, the guide sequence is any polynucleotide sequence that has sufficient complementarity with the target sequence (the characteristic sequence described in the present invention) to hybridize with the target sequence and guide the CRISPR/Cas complex to specifically bind to the target sequence, typically having a sequence length of 12-25nt. The direct repeat sequence may be folded to form a specific structure (such as a stem-loop structure) for recognition by the Cas protein to form a complex. The guide sequence does not need to be 100% complementary to the characteristic sequence (target sequence). The guide sequence is not complementary to the single-stranded nucleic acid detector.

In certain embodiments, when optimally aligned, the degree of complementarity (match) between a guide sequence and its corresponding target sequence is at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, or at least 99%. Determining optimal alignment is within the capabilities of one of ordinary skill in the art. For example, there are publicly available and commercially available alignment algorithms and programs, such as, but not limited to, ClustalW, Smith-Waterman in matlab, Bowtie, Geneious, Biopython, and SeqMan.

The gRNA described in the present invention may be natural, or artificially modified or designed and synthesized.

Single-stranded nucleic acid detector

The single-stranded nucleic acid detector of the present invention refers to a sequence containing 2-200 nucleotides, preferably 2-150 nucleotides, preferably 3-100 nucleotides, preferably 3-30 nucleotides, preferably 4-20 nucleotides, more preferably 5-15 nucleotides, preferably a single-stranded DNA molecule, a single-stranded RNA molecule or a single-stranded DNA-RNA hybrid.

The single-stranded nucleic acid detector is used in the detection method or system to report whether the target nucleic acid exists in the sample. The single-stranded nucleic acid detector includes different reporting groups or labeling molecules at both ends. When it is in the initial state (i.e., uncleaved state), it does not present a reporting signal. When the single-stranded nucleic acid detector is cleaved, it presents a detectable signal, i.e., a detectable difference is shown after cleavage and before cleavage. In the present invention, if a detectable difference can be detected, it reflects that the target nucleic acid can be detected; or, if the detectable difference cannot be detected, it reflects that the target nucleic acid cannot be detected.

In one embodiment, the reporter group or labeling molecule includes a fluorescent group and a quencher group, the fluorescent group is selected from one or any several of FAM, FITC, VIC, JOE, TET, CY3, CY5, ROX, Texas Red or LC RED460; the quencher group is selected from one or any several of BHQ1, BHQ2, BHQ3, Dabcy1 or Tamra.

In other embodiments, the single-stranded nucleic acid detector has a first molecule (such as FAM or FITC) connected to one end and a second molecule (such as biotin) connected to the other end. The reaction system containing the single-stranded nucleic acid detector is used to detect the target nucleic acid in conjunction with the flow strip (preferably, colloidal gold detection method). The flow strip is designed to have two capture lines, with an antibody that binds to the first molecule (i.e., the first molecule antibody) at the sample contact end (colloidal gold), an antibody that binds to the first molecule antibody at the first line (control line), and an antibody that binds to the second molecule (i.e., the second molecule antibody, such as avidin) at the second line (test line). When the reaction flows along the strip, the first molecule antibody binds to the first molecule and carries the cut or uncut oligonucleotide to the capture line, and the cut reporter will bind to the antibody of the first molecule antibody at the first capture line, and the uncut reporter will bind to the second molecule antibody at the second capture line. The binding of the reporter group to each line will result in a strong readout/signal (e.g., color). As more reporters are cut, more signals will accumulate at the first capture line, and less signals will appear at the second line. In some aspects, the present invention relates to the use of a flow strip as described herein for detecting nucleic acids. In some aspects, the present invention relates to a method for detecting nucleic acids using a flow strip as defined herein, such as a (lateral) flow test or a (lateral) flow immunochromatographic assay. In some aspects, the molecules in the single-stranded nucleic acid detector can be replaced with each other, or the position of the molecules can be changed, as long as the reporting principle is the same or similar to the present invention, the improved method is also included in the present invention.

The detection method of the present invention can be used for quantitative detection of target nucleic acid. The quantitative detection index can be quantified according to the signal strength of the reporter group, such as according to the luminescence intensity of the fluorescent group, or according to the width of the color band.

Sequence information

The partial sequence information involved in the present invention is provided as follows:

BRIEF DESCRIPTION OF THE DRAWINGS

In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

Figure 1. Phenotypes of Huagu 11 and Yugu 1 after being treated with the herbicide imidacloprid. The upper left is the control group CK of untreated Yugu 1, the upper right is the experimental group of Yugu 1 treated with 0.25% of the herbicide imidacloprid for 7 days, the lower right is the control group CK of untreated Huagu 11, and the lower right is the experimental group of Huagu 11 treated with 0.25% of the herbicide imidacloprid for 7 days.

Figure 2. SNPs and indel distribution of Huagu11 based on Yugu1. The window was selected as 50kb, cyan indicates the number of SNPs in each window, and orange indicates the total insertion length (bp) of each window (including insertions and deletions). Purple below the x-axis indicates SNPs enrichment area greater than 450kb. The arrow is the AHAS locus, which explains the herbicide resistance trait of Huagu11.

Figure 3. Alignment of AHAS protein sequences from Huagu 11 and Yugu 1. The letters at the arrow positions represent different amino acids.

Figure 4. Nucleotide sequence alignment of AHAS genes from Huagu 11 and Yugu 1.

Figure 5. Amino acid sequence alignment of AHAS proteins from different varieties with different resistance to the herbicide imazethapyr. Four millet varieties sensitive to the herbicide imazethapyr, TT8, zhanggu, Yugu1, Yugu18, Setaria viridis, and resistant varieties Huagu11 and Y6492.

Figure 6. Detection results of different gRNAs for dsDNA target nucleic acid. Among them, line 1 is the experimental group, line 2 is the control group without adding target nucleic acid, Figure 6A shows that when gRNA-1 and dsDNA target nucleic acid react, the fluorescence signal reaches the peak value (reaches the plateau) at 18 minutes, and Figure 6B shows that when gRNA-2 reacts with dsDNA target nucleic acid, the fluorescence signal reaches the peak value (reaches the plateau) at about 32 minutes. Figure 6C shows that the target nucleic acid was detected by gRNA-1 for the susceptible variety Yugu 18, and the results show that no peak was detected.

Figure 7. Verification results of Cas mutant protein editing efficiency.

Detailed ways

The present invention is further described below in conjunction with the embodiments. The following description is only a preferred embodiment of the present invention, and does not limit the present invention in other forms. Any technician familiar with the profession may use the above disclosed technical content to change it into an equivalent embodiment with equivalent changes. Any simple modification or equivalent change made to the following embodiments based on the technical essence of the present invention without departing from the content of the present invention falls within the protection scope of the present invention.

The technical solution of the present invention is based on the following principles. According to the results of whole gene sequencing, single nucleotide polymorphism (SNP) sites related to the imidazole acetyl nicotinic acid resistance gene of millet are excavated, and PCR amplification is combined with CRISPR technology, which has the characteristics of fast, sensitive, specific and efficient. Under the premise that there is a specific nucleic acid marked by the target SNP in the sample, the specific primer binds to the target sequence, and the target sequence is enriched by PCR amplification. The Cas enzyme (Cas protein) binds to the amplified product under the guidance of gRNA, activates the trans-cleavage (trans) activity of the Cas protein, and the Reporter in the shearing system (Reporter is connected to a fluorescent group at one end and a quenching group at the other end). After being sheared by the Cas protein, the Reporter will release fluorescence to present the test results. In other embodiments, the two ends of the single-stranded nucleic acid detector (Reporter) can also be set as a marker that can be detected by colloidal gold.

Example 1: SNP site mining and primer screening

1.1 Materials and methods

Material acquisition: Yugu 18 was used as the female parent and Y6492 as the male parent. Bag hybridization was carried out at the experimental base of Huada Agricultural Research Institute in Dapeng New District, Shenzhen, and the hybrid seeds were harvested as F1 generation. The F1 generation was sown, and 400 F2 generation seeds were harvested by bagging and self-pollination. The best single plant was selected from the F2 generation, and the first generation backcross (BC1) was carried out with Yugu 18 as the recurrent parent. The best single plant among the offspring of the backcross was continued to be selected as the donor parent for the second generation backcross (BC2). Then the best single plant was selected for the second generation of self-pollination purification, and the pure and single plant with stable resistance to imidacloprid was finally obtained, and the target single plant was named Huagu 11.

Phenotypic identification of imidazolin resistance: The purchased imidazolin herbicide product (purchased from Shandong Xianda Agrochemical Co., Ltd., an aqueous formulation with an active ingredient content of 5%) was added to 40 kg of clean water at a dosage of 133 mL/mu to prepare a herbicide spray solution. Six days after the millet seedlings emerged, the group experimental materials were sprayed with the herbicide spray solution. The leaf changes of the group experimental materials were observed 3-5 days after spraying to identify the imidazolin resistance of each generation of group materials. Among them, the normal leaves are imidazolin resistance type, the faded ones are partially imidazolin resistance type, and the dead ones are not imidazolin resistance type.

As shown in Figure 1, Yugu No. 1 and Huagu No. 11 have completely different resistance to the herbicide imazethapyr. After being treated with 0.25% of the herbicide imazethapyr for 7 days, the leaves of Yugu No. 1 wilt and die, while Huagu No. 11 grows normally. The genomes of Yugu No. 1 and Huagu No. 11 were sequenced and analyzed.

Acquisition of parental and population genotype data: The extracted DNA was subjected to second-generation sequencing to build a library, in which the parents were subjected to 10X resequencing and the F2 generation population was subjected to RAD simplified genome sequencing to obtain genotype data.

Imidazole acetylcholine resistance gene/QTL positioning: Through bioinformatics analysis, the obtained genotype data and phenotypic data are associated to perform gene/QTL positioning. Through genetic positioning of high-density SNP molecular markers in the collection population, the imazole acetylcholine herbicide resistance gene is accurately located within a length range of 1931bp (Chr1: 24269962-24271893).

1.2 Mining and screening of SNP sites

The genome of Huagu 11 was aligned to the genome of Yugu 1 to identify genomic variation information. The results are shown in Figure 2. After analysis, a total of 969,596 SNPs and 156,282 indels (a total of 617,674 bp) were identified, with an average of 2.42 SNPs and 0.39 indels (1.54 bp) per kilobase. The distribution of SNPs and indels was positively correlated (correlation R = 0.9859, P < 0.001). In addition, this study found that the distribution of SNPs was not uniform, but clustered on the genome. Using windows to calculate the distribution of SNPs, 1,593 windows were found to be SNP-enriched regions, which contained 19.78% of the genome, including 89.55% of SNPs. Windows close to 100 kb were merged, and only windows over 450 kb were displayed. These enriched SNPs may be the reason for the differences in genetic resources between Huagu 11 and Yugu 1, and thus the phenotypic diversity of the two.

The AHAS gene is located at 24623024–24624955bp on chromosome 1 of Huagu 11, which happens to be an area rich in SNPs (as shown in Figure 2). The length of the contig containing the AHAS gene is 6760944bp, of which 3606289bp is before the translation start codon and 3152723bp is after the stop codon, which means that the genome sequence of Huagu 11 can provide all the information about AHAS. The sequence of AHAS was extracted from the genomes of the other three varieties: Yugu 1, Zhanggu and TT8, and it was found that the contig length corresponding to this gene in Yugu 1 was 207997, which is much smaller than that of Huagu 11. The AHAS gene sequence in Zhanggu is incomplete, and the AHAS gene sequence in the TT8 genome is missing. This shows that the quality of the Huagu 11 genome is high, and it also implies that it has greater application potential than other genomes. Figure 4 shows that compared with Yugu No. 1, the AHAS gene of Huagu 11 has four single nucleotide mutations, but Figure 3 shows that the four single nucleotide mutations only caused one amino acid mutation, and the serine at position 626 of the AHAS protein of Yugu No. 1 mutated to asparagine in Huagu 11.

In order to verify the single nucleotide mutation, the nucleotide sequence of the AHAS gene (SEQ ID No. 9) was amplified and sequenced from four other varieties with different resistance to imidazopyrine, Setaria viridis and these two millet varieties (Huagu 11 and Yugu 1), the amplification primers are shown in Table 1, the amplification system is shown in Table 2, the amplification program is shown in Table 3, and the product length is 1932bp. The PCR product was extracted from 1.0% agarose gel and cloned into pGEM-T EasyVector plasmid. The recombinant plasmid was then transformed into Escherichia coli DH5α cells, and the positive clones were sent to Shanghai Biotech Company for sequencing.

Table 1. AHAS gene amplification primer information

AHAS-F ATGGCCACGACGACCGCCGC AHAS-R TCAATACACGGTCCTGCCAT

Table 2. Amplification system

Table 3. Amplification procedures

The results are shown in Figure 5. The single nucleotide mutation does exist in Huagu 11 and Y6492. The other varieties are the same as Yugu 1, without this mutation and are sensitive to imidazole acetylcholine. The entire coding of AHAS and the SNPs site of s626n can be found in the transcriptome data of Huagu 11 leaves. The above results show that s626n is the reason why Huagu 11 is resistant to imidazole acetylcholine. The nucleic acid sequence of AHAS of Huagu 11 is as follows (compared with Yugu 1, the 1877th nucleotide mutated from G to A):

atggccacgacgaccgccgccaccgcggccgccgcgctcaccggtgccatcaccaccgcc

tcgcccaggccgaggcgccgcgcgcacctcccgtccgccgcccggcgcgccgcgcccatc

aggtgctcggcggcgtcgcccgccgcgccgacggcgacctcggctcccccggccaccccg

ctccggccgtggggccccaccgagccccgcaagggcgccgacatcctcgtcgaggccctc

gagcgctgcggcgtcagcgacgtcttcgcctaccccggcggcgcgtccatggagatccac

caggcgctcacccgttcccccgtcatcgcgaaccacctattccgccacgagcaaggggag

gccttcgccgcctccgggtacgcgcgctcgtccggccgcgtcggcgtctgcgtcgccacc

tcgggccccggcgccaccaacctcgtctccgcgctcgccgacgcgctgctcgactccgtc

cccatggtcgccataacaggccaggtgccccgacgcatgatcggcaccgacgccttccag

gagacgccaatcgtcgaggtcacccgctccatcaccaagcacaactacctggtcctcgac

gtcgaagacatcccccgcgttgtgcaggaggcgttcttcctcgcctcctccggtcgcccg

gggccggtgctcgttgacatccccaaggatatccagcagcagatggcagtgccggtctgg

gacacgcacatgtgtctgcctgggtacattgcgcgcctgcccaagcctcctgcaactgaa

ctccttgagcaggtgctgcgtcttgttggtgagtcacggcgccctgttctttatgttggt

ggtggctgcgctgcatccggtgaggagctgcgccgctttgttgagatgaccggaatccca

gttacaactactctgatgggccttggcaacttccccagtgacgacccactgtctctgcgc

atgcttggtatgcatggtaccgtatatgcaaattatgctgtggataaggccgacctgttg

cttgcatttggtgtgcggttcgatgatcgtgtgacagggaaaattgaggcttttgcaagc

agggctaagattgtgcacattgatattgatccggctgagattggcaagaacaagcagcca

catgtgtccatctgtgcagatgtcaagcttgctctgcagggcatgaacactcttctggaa

ggaatcacatcaaagaagagctttgactttggctcatggcatgatgagttggatcagcag

aagaggggattccccctggggtacaaaacttttgatgaggagatccagccacagtatgct

atccaggttctggatgagctgacgaaaggagaggccatcattgccacaggtgttgggcag

caccagatgtgggcggcacagtactacacctacaagcgtccaaggcaatggttgtcttca

gctggtcttggggctatgggatttggtttgccggctgctgctggtgctgctgtggccaac

ccaggtgtcacagttgttgacatcgatggggatggtagcttccaaatgaacattcaggag

ttggctatgatccgcattgagaacctcccagtgaaggtctttgtgctaaacaaccagcac

ctggggatggtggtgcagtgggaggacaggttctacaaggccaaccgggcacacacatac

ttggggaacccagacaatgagagcgagatatatccagattttgtgacgattgccaaagga

ttcaacattccagcagcccgtgtgacaaagaagagcgaggtccgtgcagcaatcaagaag

atgctcgagactccagggccatacctgttggatatcattgtcccgcaccaggagcatgtg

ttgcctatgatcccgaacggtggcgctttcaaggacatgatcctggatggtgatggcagg

accgtgtattga

Example 2: Detection of SNP sites based on CRISPR technology

CRISPR-based nucleic acid detection technology is a new type of gene detection technology developed in recent years, which has the characteristics of simplicity, high efficiency, and easy operation. The use of CRISPR-based nucleic acid detection technology can be combined with molecular breeding to improve the detection efficiency of molecular markers. This embodiment provides a method for detecting millet SNPs based on CRISPR. The details are as follows:

Using the SNP site of the AHAS gene of millet resistance to imidazopyrine herbicide screened in Example 1, AHAS-F/R primers were used to amplify the DNA group of millet, and gRNA (gRNA-1, gRNA-2) was designed at the SNP site according to the specific sequence (SEQ ID No.9). The present embodiment is based on Cas12i (SEQ ID No.2) designed to bind to Cas12i gRNA, and the first 3 bases at the 5' end of each gRNA are TTN (PAM sequence).

The sequences of the designed gRNAs are as follows:

In order to verify the detection efficiency of the above-mentioned different gRNAs and Cas12i proteins, the activity of different gRNAs was verified in this embodiment.

A double-stranded target sequence (dsDNA, SEQ ID No. 9) was used as the double-stranded target nucleic acid.

The double-stranded target nucleic acid is obtained by the PCR reaction in Example 1 (amplified using primers AHAS-F/R). After the PCR reaction is completed, the PCR product is added to the Cas enzyme cutting system.

The sequence of the single-stranded nucleic acid detector is FAM-TTATT-BHQ1;

The following detection system was used: Cas12i final concentration was 50 nM, gRNA final concentration was 50 nM, target nucleic acid (dsDNA obtained by PCR amplification in Example 1) was 2 μl, and single-stranded nucleic acid detector final concentration was 200 nM. Incubate at 37 °C and read FAM fluorescence/20 sec. The control group did not add target nucleic acid, and 1 was the experimental group.

As shown in Figure 6, the results of gRNA-1 and gRNA-2 reacting with the target nucleic acid of dsDNA, as shown in Figures 6A-6B, the time for gRNA-1 to detect the target nucleic acid to reach the plateau is 18 minutes, and the time for gRNA-2 to reach the plateau is 32 minutes. Among them, gRNA-1 has a good sensitivity for detecting dsDNA, and it is feasible to use gRNA for molecular marker detection, and the detection is more convenient and the sensitivity is higher.

The screened gRNA-1 was used to detect the susceptible variety Yugu 18 to verify the specificity of the gRNA. The results are shown in Figure 6C. The target fragment was not detected for the susceptible variety Yugu 18, and the peak of the fluorescence signal could not be detected in experimental group 1, indicating that gRNA-1 has good specificity and can efficiently and conveniently distinguish between resistant varieties and susceptible varieties.

The results showed that when the test sample contained the single nucleotide polymorphism point of the AHAS gene that was resistant to the imazethapyr herbicide in millet (that is, the nucleotide at position 1877 of the AHAS gene was A, resulting in the change of the amino acid at position 626 from serine to aspartic acid), the Cas protein bound to the gRNA and targeted the target segment, stimulating Trans activity and cutting the single-stranded nucleic acid detector to emit identifiable fluorescence, thereby proving that the test sample contained a mutation in the resistance site. If the target fragment was not detected, the single-stranded nucleic acid detector would not be cut and would not emit detectable fluorescence, thereby proving that the test sample did not contain the corresponding resistance mutation site (that is, the nucleotide at position 1877 of the AHAS gene was G), thereby further identifying the resistance of the millet variety to the imazethapyr herbicide.

Example 3: Further improving detection efficiency by mutating Cas protein

In order to further improve the detection efficiency of the mutation site of the AHAS gene for millet resistance to imidazole acetyl nicotinic acid herbicide, the present embodiment performs mutation optimization on the Cas protein to improve its editing and detection activity. The applicant predicts the key amino acid sites that may affect its biological function through bioinformatics, and mutates the amino acid sites to obtain a Cas mutant protein with improved editing activity. Specifically, the amino acids that the potential Cas12i binds to the target sequence are subjected to site-directed mutagenesis by bioinformatics methods. In this embodiment, the 944th amino acid site from the N-terminus shown in SEQ ID No.2 was predicted by bioinformatics methods, and it was subjected to site-directed mutagenesis, and the nucleotide sequence of the wild-type Cas12i is shown in SEQ ID No.8.

Variants of Cas protein were generated by PCR-based site-directed mutagenesis. The specific method is to divide the DNA sequence of Cas12i protein into two parts with the mutation site as the center, design two pairs of primers to amplify the two parts of DNA sequence respectively, introduce the sequence to be mutated on the primers, and finally load the two fragments into pcDNA3.3-eGFP vector by Gibson cloning. The combination of mutants is constructed by splitting the DNA of Cas12i protein into multiple segments and using PCR and Gibson clone. Fragment amplification kit: TransStart FastPfu DNAPolymerase (containing 2.5mM dNTPs), please refer to the instructions for the specific experimental process. Gel recovery kit: Gel DNA Extraction Mini Kit, please refer to the instruction manual for the specific experimental process. Kit used for vector construction: pEASY-Basic Seamless Cloning and Assembly Kit (CU201-03), please refer to the instruction manual for the specific experimental process. The involved mutated amino acid sites and primer sequences are shown in the following table:

A Cas protein in which the amino acid at position 944 of SEQ ID No. 2 was mutated from S to R was obtained through experiments, and the activity of the mutant protein was then experimentally verified.

The mutant protein S944R of Cas12i was obtained to verify its gene editing activity in animal cells, and the target was designed for the FUT8 gene of Chinese hamster ovary cells (CHO). The vector pcDNA3.3 was modified to carry EGFP fluorescent protein and PuroR resistance gene. The SV40NLS-Cas-XX fusion protein was inserted through the restriction sites XbaI and PstI; the U6 promoter and gRNA sequence were inserted through the restriction site Mfe1. The CMV promoter drives the expression of the fusion protein SV40NLS-Cas-XX-NLS-GFP. The protein Cas-XX-NLS is connected to the protein GFP with the connecting peptide T2A. The promoter EF-1α drives the expression of the puromycin resistance gene. Plating: CHO cells were plated when the confluence was 70-80%, and the number of cells inoculated in a 12-well plate was 8*10^4 cells/well. Transfection: 24 hours after plating, transfection was performed. 6.25 μl HieffTrans ^TM liposome nucleic acid transfection reagent was added to 100 μl opti-MEM and mixed. 2.5 ug plasmid was added to 100 μl opti-MEM and mixed. The diluted HieffTrans ^TM liposome nucleic acid transfection reagent was mixed evenly with the diluted plasmid and incubated at room temperature for 20 minutes. The incubated mixture was added to the culture medium with cells for transfection. Puromycin screening: Puromycin was added 24 hours after transfection, with a final concentration of 10 μg/ml. After 24 hours of puromycin treatment, the culture medium was replaced with normal medium and cultured for another 24 hours. 48 hours after transfection, the cells were digested with trypsin-EDTA (0.05%) and cells with GFP signals were sorted using a flow cytometer (FACS).

Extract DNA, PCR amplify the editing area, and send to hiTOM sequencing: The cells were collected after trypsin digestion, and genomic DNA was extracted using the Cell/Tissue Genomic DNA Extraction Kit (Biotech). The genomic DNA was amplified near the target site. The PCR product was sequenced by hiTOM. Sequencing data analysis was performed to count the types and proportions of sequences within 15nt upstream and 10nt downstream of the target site, and to count the sequences with SNV frequencies greater than/equal to 1% or non-SNV mutation frequencies greater than/equal to 0.06% in the sequence, and the editing efficiency of the Cas-XX protein at the target site was obtained.

The results are shown in Figure 7. Compared with the wild-type Cas12i protein (WT), the editing efficiency of the Cas12i protein after the S944R amino acid site mutation is significantly improved, which is about twice the editing efficiency of the wild type, indicating that the 944th amino acid site from the N-terminus of SEQ ID No.2 is the key site for Cas12i to exert its activity.

Using the method of Example 2, the gRNA-1 with good effect on detecting double-stranded target sequences in Example 2 was further verified to be efficient in detecting double-stranded target sequences (dsDNA) in combination with mutant protein Cas12i-S944R. A double-stranded target sequence (dsDNA, SEQ ID No. 9) was used as a double-stranded target nucleic acid, dsDNA was the dsDNA targeted by the corresponding gRNA, the single-stranded nucleic acid detector sequence was FAM-TTATT-BHQ1, the double-stranded target nucleic acid amplification system was referenced to Example 1, and the target nucleic acid detection method and system were referenced to Example 2.

The experimental results show that the combination of the optimized Cas12i mutant protein (S944R) and the gRNA-1 screened in Example 2 can significantly improve the detection efficiency; when detecting dsDNA, the peak value of the fluorescence signal can be reached in about 11 minutes; compared with the combination of wild-type Cas12i and gRNA, the detection time of the Cas12i mutant protein is shorter, the sensitivity is higher, and the effect is more significant.

Although the specific embodiments of the present invention have been described in detail, it will be understood by those skilled in the art that various modifications and changes may be made to the details according to all the teachings that have been published, and these changes are within the scope of protection of the present invention. The entire invention is given by the attached claims and any equivalents thereof.

Claims (7)

1. A method for detecting the millet herbicide-resistant AHAS gene, the method comprising contacting a nucleic acid to be tested with a Cas12i protein, a gRNA and a single-stranded nucleic acid detector; the gRNA comprises a region that binds to the Cas12i protein and a guide sequence that hybridizes with the target nucleic acid, and the guide sequence that hybridizes with the target nucleic acid is shown in SEQ ID No. 3; detecting a detectable signal generated by cleavage of the single-stranded nucleic acid detector by the Cas protein, thereby detecting the millet herbicide-resistant AHAS gene; Compared with SEQ ID No.2, the amino acid sequence of the Cas12i protein has a mutation in the 944th amino acid corresponding to the amino acid sequence shown in SEQ ID No.2, and the 944th amino acid mutates to R; the nucleic acid sequence of the herbicide-resistant AHAS gene is shown in SEQ ID No.9. 2. The method according to claim 1 is characterized in that the method also includes the step of performing PCR amplification on the nucleic acid to be tested. 3. The method according to claim 2, characterized in that the nucleotide sequence of the upstream primer for PCR amplification is as shown in SEQ ID No.5, and the nucleotide sequence of the downstream primer for PCR amplification is as shown in SEQ ID No.6. 4. A system, composition or kit for detecting the herbicide-resistant AHAS gene in millet, the system, composition or kit comprising the Cas12i protein, gRNA and single-stranded nucleic acid detector described in claim 1. 5. The system, composition or kit according to claim 4, characterized in that the system, composition or kit also includes the primer of claim 3. 6. Use of the composition according to any one of claims 4-5 in detecting the herbicide-resistant AHAS gene in millet. 7. The use according to claim 6, characterized in that the nucleotide sequence of the herbicide resistance gene AHAS is shown as SEQ ID No.9.