CN117965565B - Tribulus alfalfa MtPAIR gene, gene editing vector and application thereof - Google Patents

Abstract

The invention discloses a medicago sativa MtPAIR gene, a gene editing vector and application thereof. The nucleotide sequence of the alfalfa MtPAIR gene in the caltrop is shown as SEQ ID No.1, and the amino acid sequence of the encoded protein is shown as SEQ ID No. 2; the invention also provides a target gene fragment for MtPAIR gene editing, a gene editing vector for editing MtPAIR gene and application of the target gene fragment in regulating and controlling fertility development of medicago truncatula or fixation of heterosis of medicago truncatula. The invention utilizes the gene editing technology and the reverse screening Tnt mutant library to obtain the mutant for controlling meiosis PAIR1 gene in alfalfa, verifies whether the gene function is conservative, lays a foundation for creating alfalfa MiMe materials and realizing the hybrid vigor fixing system, and lays a foundation for realizing the fixation of alfalfa hybrid vigor.

Description

Tribulus alfalfa MtPAIR gene, gene editing vector and application thereof

Technical Field

The invention relates to a gene separated from medicago truncatula (Medicagotruncatula) and application thereof, in particular to a gene separated from medicago truncatula and related to fertility, a target gene fragment and a gene editing vector and application thereof, belonging to the field of plant fertility genes and application thereof.

Background

Plant fertility and reproductive development are not only closely related to plant reproduction offspring, but also are genetic bases of crop heterosis utilization technologies. However, since genetic information is recombined during sexual reproduction and heterosis cannot be maintained in offspring due to segregation, breeders and seed companies must spend a lot of manpower, material resources and land resources for seed production. If the fixation of the heterosis of crops can be realized, the seed selection production process is greatly simplified, and the method has important economic and social benefits. By artificially creating apomixis in plants, it is expected to be a possible way to achieve heterosis fixation, providing a very useful tool for breeders.

Currently, the institute of agricultural sciences, wang Kejian, found that knocking out OsMTL gene by CRISPR CAS in rice could induce haploid material formation and simultaneously edit OsREC, osPAIR1, osOSD1 (three genes mutation at the same time could transform meiosis into similar mitosis, i.e. MiMe) and OsMTL (induce parthenocarpy) four genes, and that apomixis characteristics could be introduced into hybrid rice, thus achieving fixation （Wang, C., et al.Clonal seeds from hybrid rice bysimultaneous genome engineering of meiosis and fertilization genes.Nature biotechnology. 37,283-286(2019)）. of heterozygous genotypes and thus better understanding the genetic mechanism of apomixis is important for developmental and evolutionary studies and design of apomixis characteristics into crop plants, and could enable the possibility of propagating heterosis in a range of progeny.

The medicago sativa (Medicagotruncatula) is annual herbaceous plant, grazing pasture, and is an ideal mode plant for research of leguminous plant genetics and genomics due to the characteristics of short growth cycle, small ploidy, small genome, self-fertilization, nitrogen fixation and the like.

Disclosure of Invention

One of the purposes of the present invention is to provide the alfalfa MtPAIR gene and the encoded protein thereof;

another object of the present invention is to provide a target gene fragment for editing the gene MtPAIR of alfalfa;

The third object of the present invention is to provide a gene editing vector for performing a loss-of-function mutation on the alfalfa MtPAIR gene;

the fourth object of the present invention is to apply the gene MtPAIR gene of Tribulus terrestris, its target gene fragment or gene editing carrier of Tribulus terrestris MtPAIR gene to construct MtPAIR gene mutant, fix alfalfa heterosis or create alfalfa MiMe material.

In order to achieve the above purpose, the main technical scheme adopted by the invention comprises the following steps:

In still another aspect of the present invention, there is provided a alfalfa MtPAIR gene having a nucleotide sequence selected from any one of the nucleotide sequences set forth in the following (a) - (e):

(a) A polynucleotide sequence shown in SEQ ID No. 1;

(b) A polynucleotide sequence encoding the amino acid sequence shown in SEQ ID No. 2;

(c) A polynucleotide sequence capable of hybridizing under stringent hybridization conditions to the polynucleotide sequence of (a) or (b), the polynucleotide sequence being associated with regulatory fertility;

(d) A polynucleotide sequence having at least 75% or more identity to a polynucleotide sequence set forth in any one of (a) - (c), said polynucleotide sequence being associated with regulatory fertility; preferably, a polynucleotide sequence having at least 85% or more identity to a polynucleotide sequence set forth in any one of (a) - (c), said polynucleotide sequence being associated with regulatory fertility; more preferably, a polynucleotide sequence having at least 90% or more identity to a polynucleotide sequence set forth in any one of (a) - (c), said polynucleotide sequence being associated with regulatory fertility; most preferably, a polynucleotide sequence having at least 95% or more identity to a polynucleotide sequence set forth in any one of (a) - (c), said polynucleotide sequence being associated with regulatory fertility;

(e) A polynucleotide sequence complementary to the polynucleotide sequence of any one of (a) - (d), which polynucleotide sequence is associated with regulatory fertility.

The percentage of sequence identity described in the present invention may be obtained by well known bioinformatics algorithms, including Myers and Miller algorithms, needleman-Wunsch global alignment, smith-Waterman local alignment, pearson and Lipman similarity search, karlin and Altschul algorithms, as is well known to those skilled in the art.

In addition, the nucleotide shown in SEQ ID NO.1 can be optimized by one skilled in the art to enhance the expression efficiency in plants.

In another aspect of the present invention, there is provided a protein encoded by the alfalfa MtPAIR gene (PAIR 1 protein) having an amino acid sequence selected from the amino acid sequences shown in any one of the following (a) to (d):

(a) An amino acid sequence shown in SEQ ID NO. 2;

(b) A protein variant obtained by deleting or replacing one or more amino acid residues in the amino acid sequence shown in SEQ ID NO.2, wherein the protein variant still has the function or activity of regulating plant fertility;

(c) A protein variant obtained by inserting one or more amino acid residues into the amino acid sequence shown in SEQ ID NO.2, wherein the protein variant still has the function or activity of regulating plant fertility;

(d) A protein which has 80% or more identity with the amino acid sequence shown in SEQ ID No.2, and has the function or activity of regulating plant fertility.

In order to facilitate purification or detection of the PAIR1 protein provided by the invention, a tag may be attached to the amino-or carboxy-terminus of the protein, and a person skilled in the art may attach a corresponding tag to the amino-or carboxy-terminus of the protein, as required for purification or detection, all of which are known to a person skilled in the art.

The PAIR1 protein can be synthesized artificially or obtained by synthesizing the encoding gene and then biologically expressing.

The nucleotide sequence encoding the PAIR1 protein of the present invention can be easily mutated by a person skilled in the art using known methods, such as directed evolution or point mutation. Those artificially modified nucleotides having 75% or more identity to the nucleotide sequence of the PAIR1 protein isolated according to the present invention are derived from the nucleotide sequence of the present invention and are equivalent to the sequence of the present invention as long as the PAIR1 protein is encoded.

The coding gene of the PAIR1 protein can be obtained by deleting one or a plurality of codons in the nucleotide sequence shown in SEQ ID No.1, and/or carrying out missense mutation of one or a plurality of base PAIRs, and/or connecting a coding sequence of a tag at the 5 '-end and/or the 3' -end of the mutant.

In addition, the nucleotide according to the present invention may be DNA such as cDNA, genomic DNA or recombinant DNA; RNA, such as mRNA or hnRNA, may also be used.

In another aspect of the present invention, there is provided a target gene fragment for editing the gene of alfalfa MtPAIR, the nucleotide sequences of which are shown as SEQ ID No.3 and SEQ ID No.4, respectively.

In yet another aspect, the present invention further provides an expression cassette, recombinant vector or recombinant host cell comprising the MtPAIR gene; preferably, the expression cassette, recombinant vector or recombinant host cell is a recombinant eukaryotic expression cassette, recombinant eukaryotic expression vector or recombinant plant cell; more preferably, the recombinant eukaryotic expression cassette or recombinant eukaryotic expression vector is a recombinant plant expression cassette or recombinant plant expression vector.

Operably linking the MtPAIR gene with an expression regulatory element to obtain a recombinant plant expression vector; the recombinant plant expression vector can consist of a 5 '-end non-coding region, a nucleotide shown as SEQ ID NO.1 and a 3' -non-coding region; wherein, the 5' non-coding region can comprise a promoter sequence, an enhancer sequence or/and a translation enhancing sequence; the promoter may be a constitutive promoter, an inducible promoter, a tissue or organ specific promoter; the 3' non-coding region may comprise a terminator sequence, an mRNA cleavage sequence, and the like. Suitable terminator sequences can be taken from the Ti-plasmid of Agrobacterium tumefaciens, such as the octopine synthase and nopaline synthase termination regions.

The recombinant plant expression vector may also contain a selectable marker gene for selection of transformed cells, for selection of transformed cells or tissues. The marker gene includes: genes encoding antibiotic resistance, genes conferring resistance to herbicidal compounds, and the like. In addition, the marker gene also includes phenotypic markers such as beta-galactosidase and fluorescent protein.

Transformation protocols and protocols for introducing the polynucleotide or polypeptide into a plant may vary depending on the type of plant or plant cell used for transformation. Suitable methods for introducing the polynucleotide into a plant cell include: microinjection, electroporation, agrobacterium-mediated transformation, direct gene transfer, high-velocity ballistic bombardment, and the like. In certain embodiments, the alfalfa MtMET gene may be provided to plants using a variety of transient transformation methods. The transformed cells can be regenerated into stably transformed plants using conventional methods (McCormick et al PLANT CELL reports 1986.5:81-84).

The MtPAIR gene in alfalfa can be subjected to the function deletion mutation by a person skilled in the art by adopting a conventional method such as a conventional gene knockout or gene editing technology, for example, a MtPAIR gene knockout vector is constructed or a CRISPR/Cas9 gene editing vector of MtPAIR gene is constructed by adopting a gene editing technology, and the MtPAIR gene in plants can be subjected to the function deletion mutation by the person skilled in the art.

It is known to those skilled in the art that the main principle of CRISPR/Cas gene editing systems or gene editing methods is to find the location where gene editing is to be performed, i.e. to target DNA sequences, in the host genome by means of a nucleic acid fragment called guide-RNA (gRNA), and then cleave the DNA by means of Cas proteins. In the present application, the Cas protein includes, but is not limited to, cas9, cas12a, cas12j, cas12e, cas13, and/or Cas14, among others.

As a referenced embodiment, the present invention provides CRISPR/Cas9 gene editing vectors of MtPAIR genes comprising a sgRNA expression cassette and a Cas9 nuclease expression cassette, wherein the sgRNA expression cassette comprises the target gene fragment of the MtPAIR gene shown in SEQ ID No.3 and SEQ ID No. 4.

Another aspect of the present invention is to apply the MtPAIR gene, its coding protein (PAIR 1 protein) or MtPAIR gene editing vector to regulate and control alfalfa fertility; wherein, the regulation of alfalfa fertility is sterility or pollen inactivity of the medicago truncatula.

As a reference embodiment, the sterility or pollen inactivity of alfalfa in Tribulus terrestris may be achieved by subjecting the MtPAIR gene in alfalfa to a loss-of-function mutation; wherein, the deletion mutation refers to the occurrence of a terminator or a reading frame shift after the target site of the normal MtPAIR coding sequence.

In still another aspect, the present invention provides a method for constructing a gene function deletion mutant of alfalfa MtPAIR, comprising:

(1) Constructing a CRISPR/Cas9 gene editing vector of MtPAIR gene;

(2) Transforming the CRISPR/Cas9 gene editing vector of the MtPAIR gene into the tissue or cell of alfalfa to make MtPAIR gene function deletion mutation, screening to obtain the alfalfa mutant with MtPAIR gene function deletion mutation; the alfalfa mutant is sterile or has reduced pollen activity.

In addition, the invention also provides the application of MtPAIR gene, its coding protein (PAIR 1 protein) or MtPAIR gene editing vector and the like in the fixation of hybrid vigor of medicago sativa, including creation of medicago sativa MiMe material, such as deletion of MtPAIR gene through CRISPR CAS to block homologous recombination in meiosis process, and editing other genes capable of converting meiosis into similar mitosis and inducing parthenogenesis to realize fixation of heterozygous genotype.

The invention utilizes the gene editing technology and the reverse screening Tnt mutant library to obtain the mutant for controlling meiosis PAIR1 gene in alfalfa, and verifies whether the gene function is conserved, thus laying a foundation for creating alfalfa MiMe materials and realizing a heterosis fixing system. The invention has important significance for the creation of MiMe materials of leguminous plants, and can lay a foundation for realizing the fixation of alfalfa heterosis.

Definition of terms in connection with the present invention

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

The term "polynucleotide" or "nucleotide" means deoxyribonucleotides, deoxyribonucleosides, ribonucleosides, or ribonucleotides and polymers thereof in either single-or double-stranded form. Unless specifically limited, the term encompasses nucleic acids containing known analogs of natural nucleotides that have binding properties similar to reference nucleic acids and are metabolized in a manner similar to naturally occurring nucleotides. Unless otherwise specifically limited, the term also means oligonucleotide analogs, which include PNAs (peptide nucleic acids), DNA analogs used in antisense technology (phosphorothioates, phosphoroamidites, etc.). Unless otherwise specified, a particular nucleic acid sequence also implicitly encompasses conservatively modified variants thereof (including, but not limited to, degenerate codon substitutions) and complementary sequences as well as the sequence explicitly indicated. In particular, degenerate codon substitutions may be achieved by generating sequences in which the 3 rd position of one or more selected (or all) codons is substituted with mixed bases and/or deoxyinosine residues.

The terms "polypeptide", "peptide" and "protein" are used interchangeably herein to mean a polymer of amino acid residues. That is, the description for polypeptides applies equally to the description of peptides and to the description of proteins, and vice versa. The term applies to naturally occurring amino acid polymers and to amino acid polymers in which one or more amino acid residues are non-naturally encoded amino acids. As used herein, the term encompasses amino acid chains of any length, including full-length proteins (i.e., antigens) in which the amino acid residues are linked via covalent peptide bonds.

The term "stringent hybridization conditions" as used herein means conditions of low ionic strength and high temperature known in the art. In general, the probe hybridizes to its target sequence to a greater degree of detectability (e.g., at least 2-fold over background) under stringent conditions than to other sequences, stringent hybridization conditions are sequence-dependent (because the target sequence is present in excess and 50% of the probe is occupied under the equilibrium conditions) longer sequences can be specifically hybridized at higher temperatures by controlling the stringency or wash conditions of hybridization to identify a target sequence 100% complementary to the probe, more specifically, the stringent conditions are typically selected to be about 5-10℃below the thermal melting point (T _m) of the specific sequence at a defined ionic strength pH, T _m is the temperature at which 50% of the probe complementary to the target sequence hybridizes to the target sequence at the equilibrium (at a specified ionic strength, pH and nucleic acid concentration) (stringent conditions can be those in which 50% of the probe is occupied at the equilibrium at T _m) at a pH of 7.0 to 8.3, typically about 1.0M sodium ion, about 0. 1.0M ℃below sodium ion, and 5-10℃or less than 5% of the other nucleotide (5X 24) can be used as a stable hybridization signal (e.g., 5X 30% of the nucleotide) is included at the temperature or at least about 5X 30% of the time of the hybridization) under conditions, such as described below the temperature (e.g., 5X 60% of the nucleotide) and 5X nucleotide (30% of the nucleotide) is not shown) under conditions for hybridization or at the background conditions, 1% SDS, at 65℃in 0.2 XSSC washing and at 65℃in 0.1% SDS. The washing may be performed for 5, 15, 30, 60, 120 minutes or more.

The term "recombinant host cell strain" or "host cell" means a cell comprising a polynucleotide of the invention, regardless of the method used to insert to produce a recombinant host cell, such as direct uptake, transduction, f-pairing, or other methods known in the art. The exogenous polynucleotide may remain as a non-integrating vector, such as a plasmid, or may integrate into the host genome. The host cell may be a prokaryotic cell or a eukaryotic cell, and the host cell may also be a monocotyledonous or dicotyledonous plant cell.

The term "operably linked" refers to a functional linkage between two or more elements that may be contiguous or non-contiguous.

The term "recombinant plant expression vector" means one or more DNA vectors for effecting transformation of a plant; these vectors are often referred to in the art as binary vectors. Binary vectors, together with vectors with helper plasmids, are most commonly used for agrobacterium-mediated transformation. Binary vectors typically include: cis-acting sequences required for T-DNA transfer, selectable markers engineered to be capable of expression in plant cells, heterologous DNA sequences to be transcribed, and the like.

The term "transformation" refers to a method of introducing a heterologous DNA sequence into a host cell or organism.

The term "expression" refers to the transcription and/or translation of an endogenous gene or transgene in a plant cell.

The term "expression" refers to the transcription and/or translation of an endogenous gene or transgene in a plant cell.

Drawings

FIG. 1 shows the sterile phenotype of alfalfa R108, mutant mtpair1-1 and mutant mtpair-2 plants; a: the phenotype of the whole plant of the mutant pair1-1, the mutant pair1-2 and the alfalfa R108; b: is the branching phenotype of mutant pair1-1, mutant pair1-2 and medicago truncatula R108.

FIG. 2 shows the phenotype of alfalfa R108, mutant mtpair-1 and mutant mtpair-2 male gametophytes.

FIG. 3 shows the phenotype of alfalfa R108, mutant mtpair-1 and mutant mtpair-2 female gametophytes.

FIG. 4 shows the phenotype of sterility and pollen inactivity of the alfalfa R108, knockout material mtpair1-CR plants.

Detailed Description

The invention will be further described with reference to specific embodiments, and advantages and features of the invention will become apparent from the description. These examples are merely exemplary and do not limit the scope of the invention in any way. It will be understood by those skilled in the art that various changes and substitutions can be made in the details and form of the invention without departing from the spirit and scope of the invention, but these modifications and substitutions are intended to be within the scope of the invention.

The experimental methods in the following examples are conventional methods unless otherwise specified.

The test materials used in the examples described below, unless otherwise specified, were purchased from conventional biochemical reagent stores.

The quantitative tests in the following examples were all set up in triplicate and the results averaged.

Test materials

Tribulus alfalfa R108 is provided by The Nobel Foundation (website: https:// www.nobelprize.org/the-nobel-prize-organization/the-nobel-foundation /).

PDIREC-22C plasmid was purchased from Beijing Zhongyuan polymer biotechnology Co., ltd; agrobacterium tumefaciens AGL1 is provided by biological technology at the national academy of agricultural sciences.

YEP liquid medium: 10g of peptone, 10g of yeast extract and 5g of sodium chloride were dissolved in an appropriate amount of distilled water, and then autoclaved at 121℃for 20min with distilled water to a volume of 1L.

Callus induction liquid medium: 100mL of macroelement mother liquor, 1mL of microelement mother liquor, 1mL of organic element mother liquor, 20mL of ferric salt mother liquor, 100mg of inositol, 30g of sucrose, 4mg of auxin and 0.5mg of cytokinin are dissolved by a proper amount of distilled water, then distilled water is used for constant volume to 1L, the pH value is regulated to 5.8, and the autoclave is sterilized at 121 ℃ for 20min.

Callus induction solid medium: 100mL of macroelement mother liquor, 1mL of microelement mother liquor, 1mL of organic element mother liquor, 20mL of ferric salt mother liquor, 100mg of inositol, 30g of sucrose, 4mg of auxin, 0.5mg of cytokinin, 200mg of cephalosporin, 250mg of timentin, 50mg of kanamycin and 3.2g of Phytagel are dissolved by a proper amount of distilled water, then distilled water is used for constant volume to 1L, the pH value is adjusted to 5.8, and the mixture is autoclaved for 20min at 121 ℃.

Differentiation medium: 100mL of macroelement mother liquor, 1mL of microelement mother liquor, 1mL of organic element mother liquor, 20mL of ferric salt mother liquor, 100mg of inositol, 20g of sucrose, 200mg of cephalosporin, 250mg of timentin, 50mg of kanamycin and 3.2g of Phytagel are dissolved by a proper amount of distilled water, then distilled water is used for volume fixation to 1L, the pH value is adjusted to 5.8, and the autoclave is sterilized at 121 ℃ for 20min.

Rooting medium: 2.215g Murashige&Skoog Basal Medium with Vitamins (product of PhytoTechnologyLaboratories company, product No. 16B 0519138A) was dissolved in a suitable amount of distilled water, and then distilled water was used to fix the volume to 1L, pH was adjusted to 5.8, and the mixture was autoclaved at 121℃for 20 minutes.

Ferric salt mother liquor: disodium ethylenediamine tetraacetate 37.3mg and ferrous sulfate heptahydrate 27.8mg were dissolved with an appropriate amount of distilled water, and then the volume was fixed to 1L with distilled water.

Macroelement mother liquor: 1.85g of magnesium sulfate heptahydrate, 28.3g of potassium nitrate, 4.63g of ammonium sulfate, 1.66g of calcium chloride dihydrate and 4g of potassium dihydrogen phosphate are dissolved in a proper amount of distilled water, and then distilled water is used for volume fixation to 1L.

Trace element mother liquor: 1g of manganese sulfate monohydrate, 500mg of boric acid, 100mg of zinc sulfate heptahydrate, 100mg of potassium iodide, 10mg of sodium molybdate dihydrate, 20mg of copper sulfate pentahydrate and 10mg of cobalt chloride hexahydrate are dissolved in a proper amount of distilled water, and then distilled water is used for constant volume to 1L.

Organic element mother liquor: 500mg of nicotinic acid, 500mg of thiamine hydrochloride and 500mg of pyridoxine hydrochloride are dissolved with an appropriate amount of distilled water, and then the volume is fixed to 1L with distilled water.

Alexander dye liquor formula: 95% ethanol 5mL, 1% malachite green 500. Mu.L, 1% acid fuchsin 2.5mL, 1% orange G250. Mu.L, glycerol 12.5mL, glacial acetic acid 2mL, and distilled water to a volume of 50mL.

EXAMPLE 1 cloning of the alfalfa MtPAIR Gene

1. Overall phenotype analysis of alfalfa mutant pair1-1 and mutant pair1-2 seedlings

The mutant pair1-1 and the mutant pair1-2 are provided by inserting the alfalfa Tnt1 into a mutant library of The Nobel Foundation (website: https:// www.nobelprize.org/the-nobel-prize-organization/the-nobel-foundation /), and the numbers of the mutant pair1-1 and the mutant pair1-2 in the alfalfa Tnt1 insertion mutant library are N19538 and NF1382 in sequence. According to the information recorded in the insertion mutant library of the medicago truncatula Tnt, the mutant PAIR1-1 is a medicago truncatula mutant obtained by inserting Tnt into the second exon of the PAIR1 gene, and the mutant PAIR1-2 is a medicago truncatula mutant obtained by inserting Tnt1 into the first exon of the PAIR1 gene.

Respectively planting a mutant pair1-1, a mutant pair1-2 and medicago truncatula R108, and observing the fruiting condition of the whole seedling and the fruiting condition of branches; the whole seedling phenotype is shown in FIG. 1A, and the branching phenotype is shown in FIG. 1B. The results indicate that R108 can be normally set, but the pair1-1 and pair1-2 mutants are almost sterile.

2. Phenotypic analysis of mutant pair1-1 and mutant pair1-2 male gametophytes

Pollen Alexander staining procedure:

(1) The alfalfa anther is taken and placed in a proper amount of Carnot's fixative (absolute ethyl alcohol: glacial acetic acid=3:1), and is fixed at room temperature for 3-4 h hours, if necessary, overnight.

(2) The fixed liquid is sucked away in a ventilation kitchen, then an appropriate volume of Alexander staining liquid is added, and the mixture is placed in a 37 ℃ incubator for dark staining for an appropriate time.

(3) Transferring the dyed anther to a centrifuge tube containing 10% glycerol for decolorizing at room temperature for 45min on the next day, poking the anther under a microscope to make pollen flow out, and observing the dyeing condition of pollen.

As a result, as shown in FIG. 2 (A, B, C), R108 pollen was active normally, but pollen of the pair1-1 and pair1-2 mutants was inactive.

The pollen scanning electron microscope observation step:

And (3) taking the anther of the medicago tribulus powder just scattered on the dry paper, fixing the anther, spraying gold, and observing the pollen morphology under a scanning electron microscope.

As a result, R108 pollen was full, but pollen morphology of pair1-1 and pair1-2 mutants was abnormal, showing shrinkage and shrinkage, as shown in FIG. 2 (D, E, F).

3. Phenotype observations of mutant pair1-1 and mutant pair1-2 mature ovules

The mature ovule transparent-interference observation method comprises the following steps:

(1) Mature ovules of alfalfa were placed in a fixing solution of FAA fixing solution (formalin: ethanol: acetic acid: water=1:0.5:5:3.5) overnight.

(2) The ovule was then placed on a slide containing a clear solution (chloral hydrate: glycerol: water=8:1:2), covered with a coverslip, and the morphology of the ovule was then observed on a olympus microscope.

As a result, as shown in FIG. 3, the structure of seven-cell eight-nucleus embryo sac was observed in the R108 ovule, but embryo sacs of the pair1-1 and pair1-2 mutants did not develop.

4. Cloning of MtPAIR Gene

1. Extracting total RNA of flowers on the alfalfa R108 plants, and then carrying out reverse transcription to obtain cDNA of the alfalfa R108 plants.

2. After the step 1 is completed, the cDNA of R108 is used as a template, and the primers MtPAIR to attB1-F and MtPAIR to attB2-R are adopted for amplification to obtain PCR amplification products of about 1371BP, and the PCR products are recovered and then subjected to BP reaction with the vector pDONR207 to obtain an intermediate vector.

MtPAIR1-attB1-F：

5’gtggggacaagtttgtacaaaaaagcaggcttcATGAAGATCAACAAAGCCTCCGA-3’ (SEQ ID No.5)

MtPAIR1-attB2-R：

5’gtggggaccactttgtacaagaaagctgggtcCAGATCAAAGTCAAACATGC-3’ (SEQ ID No.6)

3. Sequencing the intermediate vector obtained in the step 2.

Sequencing results show that the intermediate vector for PAIR1 gene contains the DNA molecule shown in SEQ ID No. 1. The DNA molecule shown in SEQ ID No.1 is the alfalfa MtPAIR gene of Tribulus terrestris, and the amino acid sequence of the encoded PAIR1 protein is shown in SEQ ID No. 2.

Nucleotide sequence of alfalfa MtPAIR gene in caltrop ：ATGAAGATCAACAAAGCCTCCGATCTCTCCTCCATCTCCGTCTTCCCTCCTCCTCACGTTTATTCCAGGAAGATGAACAATGCATCCAATGGATTTCAAGCCTCGCAACATCGATCGCAACCATCGCATCGAGATATAGAGAGGCAGAACAATGTATCGAATGGATTGCATGCGATGTCGCAACATCGATCACAACCGTCTCAGCAGTCATTTTCTCAGGGACTTTCATCTCAGCAAGGAATTTTGTCTCATTTCTCTCAGAGTTCTCTTGATGAAGCTATCACAACAAATGACCAGAGAGCTGCTTCTCAAGAACTTGAGAACTCTTCAAGGAGGTTTTCTAGTTTGCCCCGCCTTACTTATTCAAAGGACGAGAGTCAACCGCACAACTCAAGATCTTCATCAAATCTCCTGGTCAAATGGAACTCTGCAGATAATAAAAATCAGTTAAGTGAAGGACTCGAAAACAGAATTGGCATAATGGAAACCTCATTGAGCAGGTTTGCAATGATCATGGATTCTGTTCAAAGTGACGTAATGCAAGTAAACAAAGGAACAAAGGAAATGCACTTGGAGATGGAGTGCATAAGACAGAAGTTGATTGCTCAGGATAACACACTTCAGTTAATGATGAAAGGACAAGAAGAAATTAAAGCAAGCATCAATGGGAGTTTGAAATCTTTATCTGAACAAATGAGCCGTGTTACAGACATAGAGAAGTTACAGGAAGTATATATGTTGGTTTCTTCCATGCCTCAGCTAATTGAAGGCTCTCTGCGAAATTTGCAAAATGATCTCCAGAACACCACCAAGGAAATGAAGGAAATCTCTTGCAGTTTAAAACATTCTAACCAAAAGGATCTGGCACAGCCTATTCTATCTCCAAAGTGTGTTAGCAAACAAGTTATCACACCGAAAATGCGTCAGACTCCAGCCGTTGAAGCGAAGAAGAACACTCCAGTTATTGTAGCTTCAGAAGCAGACACAGGAGGCTGGAGGCCAGTAAAAAGAGAAAGAGTCACTTTTTCAGATAGAATATCTGAGAAGGTTCAAAAGCGAATTCCACCAAAAGTGGAGAAGGGTACAAGGGGAGGAAGAGACTATGCAATTGTCATTGAATCTGACGAGGAGTCTGATTGCTTTGAAGTAAAATCAGCAGGAAAGTGGGAAGGGAAGAAAAAAGAAAAAATTACAAATGACGCAAGTAACATCAGCCAAATGGAGGGAGCTAAGTTGGTTACAACAGATGGACATGCAGCGGTTAGGAGGAGTGCAAGGAAGAGACTCCCTAACCAAATGATGGATGATTTTGTGTGCAAAGGGTATAGGAAAAGGAATAGGAGAAAAAAGAGCATGTTTGACTTTGATCTGTAA（SEQ ID No.1）.

Amino acid sequence of medicago truncatula PAIR1 protein:

MKINKASDLSSISVFPPPHVYSRKMNNASNGFQASQHRSQPSHRDIERQNNVSNGLHAMSQHRSQPSQQSFSQGLSSQQGILSHFSQSSLDEAITTNDQRAASQELENSSRRFSSLPRLTYSKDESQPHNSRSSSNLLVKWNSADNKNQLSEGLENRIGIMETSLSRFAMIMDSVQSDVMQVNKGTKEMHLEMECIRQKLIAQDNTLQLMMKGQEEIKASINGSLKSLSEQMSRVTDIEKLQEVYMLVSSMPQLIEGSLRNLQNDLQNTTKEMKEISCSLKHSNQKDLAQPILSPKCVSKQVITPKMRQTPAVEAKKNTPVIVASEADTGGWRPVKRERVTFSDRISEKVQKRIPPKVEKGTRGGRDYAIVIESDEESDCFEVKSAGKWEGKKKEKITNDASNISQMEGAKLVTTDGHAAVRRSARKRLPNQMMDDFVCKGYRKRNRRKKSMFDFDL*（SEQ ID No.2）.

example 2, acquisition and phenotypic analysis of T ₀ generation MtPAIR1 Gene knockout alfalfa mutant

1. MtCRISPR/Cas9: : construction of MtPAIR1 binary vectors

1. And selecting target sequences of target genes, and respectively designing two targets for each gene.

Wherein, two targets for MtPAIR gene are:

5’-AGGAGGAGGGAAGACGGAGATGG-3’（SEQ ID No.3）；

5’-GGATTGACGTGAAGTGAACCTGG-3’（SEQ ID No.4）。

2. The following fragments were amplified using KOD Hi-Fidelity enzyme using pDIRECTT-22C vector (Beijing Zhongyuan Polymer biosciences Co., ltd., product No. 91135-ADG) as a template:

segment 1: adopting a primer combination of CmYLCV + MtPAIR-B_gRNA 1;

Fragment 2: adopting a primer combination of MtPAIR < 1 > -C_gRNA < 1 > + MtPAIR-D_gRNA <2 >;

Fragment 3: adopting a primer combination of MtPAIR <1 > -C_gRNA < 2+ > oCsy-E;

The information on each primer is as follows:

CmYLCV：

5’-TGCTCTTCGCGCTGGCAGACATACTGTCCCAC-3’；(SEQ ID No.7)

Mt-PAIR1-B-gRNA1：

5’-TCGTCTCCTTCCCTCCTCCTCTGCCTATACGGCAGTGAACCTG-3’；(SEQ ID No.8)

Mt-PAIR1-C-gRNA1：

5’-TCGTCTCAGGAAGACGGAGAGTTTTAGAGCTAGAAATAGC-3’ (SEQ ID No.9)

Mt-PAIR1-D-gRNA2：

5’-TCGTCTCATCACGTCAATCCCTGCCTATACGGCAGTGAACCTG-3’；(SEQ ID No.10)

Mt-PAIR1-C-gRNA2：

5’-TCGTCTCAGTGAAGTGAACCGTTTTAGAGCTAGAAATAGC-3’；(SEQ ID No.11)

oCsy-E：

5’-TGCTCTTCTGACCTGCCTATACGGCAGTGAAC-3’；(SEQ ID No.12)

3. The 3 fragments were recovered by a recovery kit, and the concentration was detected and measured by electrophoresis. Each fragment was added 5-7ng, pDIRECT-22C vector was added 50ng, sapI enzyme was added 0.5. Mu.L, esp I enzyme was added 0.5. Mu.L, T7 DNA LIGASE enzyme was added 1. Mu.L, 2 XT 7 DNA LIGASE buffer was added 10. Mu.L, and finally dd H ₂ O was added to 20. Mu.L.

4. The reaction procedure is: 20× (37 ℃/5 min+25 ℃/10 min) +4 ℃ and the number of cycles of connection can be increased if necessary.

The vector after sequencing verification to be correct is named MtCRISPR/Cas9: : mtPAIR 1A 1.

2. Acquisition of recombinant Agrobacterium

MtCRISPR/Cas9: : mtPAIR1 binary vector is introduced into agrobacterium tumefaciens AGL1 to obtain recombinant agrobacterium, and the recombinant agrobacterium is named AGL1/MtCRISPR/Cas9: : mtPAIR 1A 1.

3. Acquisition of T ₀ generation MtPAIR1 Gene knockout mutant

1. Preparation of the dyeing liquor

(1) AGL1/MtCRISPR/Cas9: : mtPAIR 1A single colony was inoculated into YEP liquid medium containing 50mg/mL rifampicin and 50mg/mL kanamycin, respectively, and cultured overnight at 28℃under shaking at 200rpm to give culture broth 1.

(2) After the completion of the step (1), 500. Mu.L of the culture broth 1 was inoculated into 5mL of YEP liquid medium, and then 5. Mu.L of 100mg/mL acetosyringone aqueous solution was added thereto, followed by shaking culture at 28℃and 200rpm, to obtain a culture broth 2 having an OD _600nm of 0.8.

(3) After the step (2) is completed, the culture solution 2 is taken and centrifuged at 3800rpm for 15min, and the bacterial cells are collected.

(4) After the step (3) is completed, the thalli are taken and resuspended by using a callus induction liquid culture medium containing 100mg/L acetosyringone, and the invasion solution with the OD _600nm value of 0.2 is obtained.

2. Obtaining of T ₀ generation MtPAIR1 gene knockout medicago sativa

(1) Multiple leaves of alfalfa R108 plants grown to 4 weeks were taken and the leaves were cut with a razor blade for 4-5 cuts.

(2) After the step (1) is completed, the small blade blocks are placed in the dyeing liquid obtained in the step (1) and are shaken in darkness for 30min.

(3) After the step (2) is completed, the leaf small pieces are transferred to a callus induction solid culture medium, and are subjected to dark culture at 24 ℃ for 4 weeks (the culture medium is replaced every 2 weeks), so that white embryogenic callus is obtained.

(4) After the step (3) is completed, the white embryogenic callus is transferred to a differentiation medium, and is alternately cultured for 4 weeks at 24 ℃ in a light-dark mode (the medium is replaced every 2 weeks), so that green embryoids are differentiated.

(5) After the step (4) is completed, transferring the green embryoid to a rooting culture medium, alternately culturing at 24 ℃ in a light-dark mode (changing the culture medium every 2 weeks), and transferring the rooted long leaves to vermiculite until seedlings are grown.

(6) And extracting the obtained genome DNA of the transgenic alfalfa plant containing the recombinant vector targeted by the gene CRISPR/Cas9 of the alfalfa MtPAIR by using a CTAB method. The DNA was used as a template, and the sequence containing the target region was amplified with 2X RAPID TAQ MASTER MixPCR and sequenced.

After sequencing, it was confirmed that single mutant mtpair-CR (due to nucleotide insertions or deletions, resulting in frame shifts and premature termination of the translated protein) was obtained.

4. Phenotypic analysis of MtPAIR1 knockout mutants

And (3) taking a picture of the whole of mtpair-CR mutant and wild (medicago truncatula R108) seedlings obtained in the step three, taking a picture of branches and staining Alexander pollen.

Pollen Alexander staining procedure:

(1) The anther of mature alfalfa pollen but not scattered pollen is taken and placed in a proper amount of Carnot's fixative (absolute ethyl alcohol: glacial acetic acid=3:1), and the mixture is fixed for 3-4 hours at room temperature, and if necessary, can be fixed overnight.

(2) The fixed liquid is sucked away in a ventilation kitchen, then an appropriate volume of Alexander staining liquid is added, and the mixture is placed in a 37 ℃ incubator for dark staining for an appropriate time.

(3) The next day the stained anthers were transferred to centrifuge tubes containing 10% glycerol for 45min at room temperature and finally the staining of the pollen was observed under a microscope.

The detection result is shown in fig. 4; from FIG. 4A, B, C and D, it can be seen that the mtpair1-CR mutant is not firm, exhibiting a sterile phenotype; mtpair1-CR pollen was found to be inactive based on the result of Alexander activity staining (E, F of FIG. 4).

Claims (3)

1. Application of the gene MtPAIR of medicago sativa in regulating fertility development of medicago sativa; the regulation and control of fertility development of the medicago sativa is that the gene MtPAIR of the medicago sativa is subjected to function deletion mutation so as to cause sterility of the medicago sativa;

The nucleotide sequence of the alfalfa MtPAIR gene in Tribulus terrestris is the polynucleotide sequence shown as SEQ ID No. 1.

2. Application of CRISPR/Cas9 gene editing vector of Tribulus alnicosus MtPAIR gene in regulating and controlling fertility development of Tribulus alnicosus; the fertility development of the medicago truncatula is regulated to ensure that the medicago truncatula is sterile by carrying out function deletion mutation on MtPAIR genes; the CRISPR/Cas9 gene editing vector comprises an sgRNA expression frame and a Cas9 nuclease expression frame, wherein the sgRNA expression frame comprises a nucleotide sequence shown in SEQ ID No.3, and the nucleotide sequence of the alfalfa MtPAIR gene in Tribulus terrestris is a polynucleotide sequence shown in SEQ ID No. 1.

3. A method for constructing a gene loss-of-function mutant of medicago truncatula MtPAIR, comprising:

(1) Constructing a CRISPR/Cas9 gene editing vector of the alfalfa MtPAIR gene; the nucleotide sequence of the alfalfa MtPAIR gene in Tribulus terrestris is the polynucleotide sequence shown in SEQ ID No. 1;

the CRISPR/Cas9 gene editing vector comprises an sgRNA expression frame and a Cas9 nuclease expression frame, wherein the sgRNA expression frame comprises a nucleotide sequence shown in SEQ ID No. 3;

(2) Transforming the constructed CRISPR/Cas9 gene editing vector of the gene MtPAIR of the medicago truncatula into tissues or cells of the medicago truncatula to cause the function deletion mutation of the MtPAIR gene, and screening to obtain the medicago truncatula mutant with the function deletion mutation of the MtPAIR gene; the alfalfa mutant of the tribulus terrestris is sterile.