WO2020007331A1 - 一种利用CRISPR/Cas9***对紫花苜蓿基因定点突变的方法 - Google Patents

一种利用CRISPR/Cas9***对紫花苜蓿基因定点突变的方法 Download PDF

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WO2020007331A1
WO2020007331A1 PCT/CN2019/094630 CN2019094630W WO2020007331A1 WO 2020007331 A1 WO2020007331 A1 WO 2020007331A1 CN 2019094630 W CN2019094630 W CN 2019094630W WO 2020007331 A1 WO2020007331 A1 WO 2020007331A1
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alfalfa
medium
crispr
cas9
gene
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French (fr)
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陈海涛
王文
谢雄平
邱强
尚占环
苏克先
何辉
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广东三杰牧草生物科技有限公司
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Priority to CA3105396A priority Critical patent/CA3105396A1/en
Priority to US17/257,617 priority patent/US20210292777A1/en
Priority to EP19830148.3A priority patent/EP3848464A4/en
Priority to AU2019299296A priority patent/AU2019299296B2/en
Publication of WO2020007331A1 publication Critical patent/WO2020007331A1/zh
Priority to US17/141,354 priority patent/US20210147844A1/en

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Definitions

  • the invention belongs to the technical field of gene editing, and particularly relates to a method for site-directed mutation of an alfalfa gene by using a CRISPR / Cas9 system.
  • Alfalfa (Medicago sativa.) Is a widely cultivated legume forage, rich in high-quality dietary fiber, edible protein, multivitamins (including vitamin B, vitamin C, vitamin E, etc.), a variety of beneficial minerals, and saponins, flavones Bioactive ingredients such as carotenoids, phenolic acids, etc. have high protein content and good palatability, which is of great significance for raising the level of feed for livestock.
  • the well-developed root system of alfalfa plays an important role in preventing soil erosion and environmental governance.
  • alfalfa has a large number of nitrogen-fixing microorganisms symbiosis
  • alfalfa is also of great significance in improving soil fertility.
  • the research on alfalfa gene function is helpful to improve the alfalfa traits and assist in breeding new alfalfa varieties with better traits.
  • the use of gene silencing technology in alfalfa to suppress the expression of target genes is a very effective means to study gene function and breed new varieties.
  • spontaneous mutations often occur in nature, the frequency of mutations is extremely low.
  • the mutation rate of spontaneous mutations at most gene loci is about 10 -5 or 10 -6 per generation, and only mutations that occur in gametes can be transmitted to the next. generation.
  • Chemical mutations, physical mutagenesis, and DNA insertion mutations can be used to treat plant materials to obtain useful mutants in a short period of time. Mutagenic factors cause a higher frequency of mutations, hundreds of times or even thousands of times higher than the frequency of spontaneous mutations, and a wide range of mutations and types of induced mutations, which can sometimes induce new or rare new mutations in nature.
  • mutations introduced by mutagenesis are random, and it is difficult to obtain homozygous mutants for a certain site.
  • RNAi RNA interference
  • gene editing technologies such as ZFN, TALLEN, and CRISPR / Cas9 have been developed for targeted gene silencing.
  • RNAi technology mainly degrades the mRNA sequence transcribed by the target gene and down-regulates the translation level of the mRNA, thereby achieving the effect of inhibiting the expression of the target gene.
  • the gene sequence of the target gene is not mutated, and the target gene may still be expressed to a certain extent.
  • CRISPR / Cas9 technology was first applied to gene editing in 2013, it has developed rapidly and widely.
  • the CRISPR / Cas9 system is an acquired immune system from the genus Streptococcus, which can resist the invasion of foreign genes. It consists mainly of CRISPR and a specific Cas9 protein.
  • CRISPR is a cluster of regularly spaced short palindromic repeats. It is a new type of DNA repeats. It consists of a series of short highly conserved forward repeats and spacers of similar length.
  • Cas9 protein is a multi-domain protein composed of 1409 amino acids and contains two nuclease domains RuvC-like and HNH.
  • the CRISPR / Cas9-based genome editing system includes two parts: sgRNA and the enzyme Cas9. This system can specifically recognize the target sequence adjacent to the PAM (NGG) motif and cut it 3nt upstream to form a DSB (double strains break) .
  • the body uses its own repair mechanisms such as homologous recombination (HR), non-homogeneous Source end ligation (NHEJ) to repair damaged DNA.
  • the NHEJ repair process can generate Indels (including deletions and insertions), which in turn lead to frameshift mutations in the DNA coding sequence.
  • Alfalfa is a strictly cross-pollinated autotetraploid plant with four alleles per locus. If you need to obtain a homozygous mutant line that can be stably inherited for a certain locus, the ideal is to obtain simultaneous mutations. Multiple strains of four alleles can achieve hybridization and stable inheritance among multiple strains; the second is to obtain stable pure genetics through repeated crosses between naturally occurring or mutagenesis hybrid mutants. Combining mutant strains, but this is a time-consuming, labor-intensive, high-input, and inefficient process. Therefore, in general, it is particularly difficult to obtain homozygous mutants that can be stably inherited by using naturally occurring or mutagenesis mutants in alfalfa.
  • RNAi RNAi technology to reduce the expression of alfalfa genes.
  • No gene editing technology such as ZFN, TALEN, CRISPR / Cas9 has been used for gene editing to obtain homozygous mutants.
  • RNAi has limited in silencing and unstable characteristics, which limits its application.
  • a method for site-directed mutation of alfalfa genes using the CRISPR / Cas9 system characterized in that the method includes the following steps:
  • Step (1) Construct a universal binary expression vector Ms CRISPR / Cas9 transformed by Agrobacterium tumefaciens in alfalfa;
  • Step (2) Design a CRISPR / Cas9-based sgRNA targeting sequence for the target gene in alfalfa, connect the DNA fragment containing the sgRNA targeting sequence to a common vector Ms CRISPR / Cas9 in alfalfa to construct the vector Ms CRISPR / Cas9 :: target;
  • Step (3) Agrobacterium tumefaciens-mediated transformation of alfalfa to achieve site-directed mutation of a specific gene in alfalfa.
  • alfalfa includes wild varieties, local varieties, bred varieties, and introduced varieties.
  • the target gene includes any biologically functional gene or DNA sequence in the alfalfa genome.
  • This technology relies on the transformation of alfalfa by Agrobacterium tumefaciens.
  • the T-DNA sequence containing the selection gene Hpt expression frame, sgRNA expression frame and Cas9 protein expression frame is integrated into the alfalfa genome to obtain transgenic plants through transformation.
  • the exogenous expression elements are expressed in alfalfa, thus functioning and obtaining gene knockout plants.
  • an expression vector MsCRISPR / Cas9 was constructed.
  • the binary expression vector Ms CRISPR / Cas9 backbone vector for expressing the CRISPR / Cas9 system in alfalfa is pCambia1300 vector.
  • the pCambia1300 backbone vector has a T-DNA region for transforming plants.
  • the T-DNA region includes a left border repeat (LB) repeat, a right border repeat (RB) repeat, and a sequence used for transformation inside the left and right border sequences.
  • the T-DNA region contains left and right borders for transformation
  • the sequence contains the Hpt gene expression element expressing a protein that is resistant to the plant screening agent hygromycin, the Cas9 protein expression element, the sgRNA expression element, and the target sequence between the two Aar1 digestion sites inside the sgRNA expression element
  • the expression element of the Hpt gene contains a Camv 35S promoter (enhanced) promoter sequence that initiates the transcription of the Hpt gene, a CDS sequence that expresses the Hpt gene, and a Camv poly (A) termination sequence that terminates the transcription of the Hpt gene.
  • the expression element contains a 2xCaMV 35S promoter sequence that initiates Cas9 sequence transcription, a DNA sequence that expresses Cas9, and a Nos that terminates Cas9 transcription Terminator termination sequence, the sgRNA-expressing expression element internally contains the Tribulus truncatula MtU6 promoter sequence that initiates transcription of the sgRNA sequence and a DNA sequence containing the sgRNA expression.
  • LB T-DNA repeat / RB T-DNA repeat represents the left and right border repeat sequences of T-DNA; CaMV 35s promoter / MtU6 promoter / 2x CaMV 35s promoter represents the promoter element; Hpt / sgRNA / Cas9 Represents the gene sequence; CaMV poly (A) / NosT represents the termination sequence; AarI represents the insertion site of the sgRNA targeting sequence.
  • using the alfalfa U6 promoter to promote sgRNA expression 2XCaMV35S promoter to promote Cas9 gene expression; CaMV35S (Enhanced) promoter to promote the expression of the selection marker gene Hpt.
  • sequence of the MtU6 promoter is shown in SEQ ID NO.2.
  • the sgRNA-expressing DNA sequence is as shown in SEQ ID No. 3, and the sequence of positions 1 to 30 in the sgRNA-expressing DNA sequence contains two AarI restriction endonuclease recognition sites, To construct a site-directed mutation expression vector for the target gene.
  • Step (1) Design a pair of oligo primers (18-24bp) according to the target sequence to form a DNA fragment with a linker at both ends and synthesize:
  • Step (2) annealing the synthesized primer pair to form a complementary DNA double-stranded fragment with a sticky end
  • Step (3) Use AarI restriction enzyme to cut the binary expression vector MsCRISPR / Cas9; after dephosphorylation by CIP reaction, perform electrophoretic separation in a 0.8-1.2% agarose gel to recover and purify the linear vector;
  • Step (4) The annealed complementary DNA double-stranded fragment DNA with sticky ends and the recovered and purified MsCRISPR / Cas9 linear vector are ligated with a T4 DNA ligase in a 16 ° C water bath for 10-16 hours;
  • Step (5) The ligation product is transformed into E. coli DH5 ⁇ competent cells by a heat shock method, and after resuscitation, it is spread on an LB plate medium containing 50 mg / L kanamycin and cultured in a 37 ° C incubator for 12-16 h;
  • Step (6) Pick a single colony and clone it in 1.5 ml of LB liquid medium containing 50 mg / L kanamycin, and shake it at 37 ° C for 12-16 h;
  • Step (7) Sequencing the monoclonal bacterial solution with the sequencing primer MtU6-TF, selecting the correct target and cloning the clone in 50-100ml LB liquid medium containing 50mg / L kanamycin, shaking at 37 ° C with a shaker for 12 -16h, extract the plasmid.
  • sequence of the sequencing primer MtU6-T-F is shown in SEQ ID NO.4.
  • the method for obtaining mutant plants using Agrobacterium tumefaciens-mediated transformation of alfalfa comprises the following steps:
  • Step (1) Recovery of Agrobacterium tumefaciens strains; Agrobacterium strains transformed by electric shock are coated in YM solid plate culture medium, cultured at 25-29 ° C for 24h-48h; single clones are picked and inoculated in 30-50mLYM liquid In the medium, shake at 100-220r / min for 24h-48h at 25-29 ° C on a shaker; both the YM solid medium and YM liquid medium contain 30-60mg / L kanamycin and 200-450mg / L Fuping
  • Step (2) Obtaining alfalfa callus: The process of obtaining alfalfa callus is as follows:
  • the components of the callus induction medium are: SH basic medium + 1.0-3.0mg / L 2,4-D (2,4-difluorophenoxyacetic acid) + 0.1-0.3mg / L KT (kinin) + 0.1-0.5mg / L hydrolyzed complex protein + 15-40g / L sucrose + 5.8-9g / L agar; preferably, the components of the callus induction medium are: SH basic medium + 2mg / L 2,4-D + 0.2mg / L KT + 0.3mg / L hydrolyzed complex protein + 30g / L sucrose + 8g / L agar;
  • Step (3) Infection of alfalfa callus by Agrobacterium tumefaciens The process of infecting alfalfa callus by said Agrobacterium strain is as follows: inoculate the Agrobacterium tumefaciens strain recovered in step (1) to 1-2 days in advance In 50-100mL of YM liquid medium (Sigma), culture at 100-220r / min on a 25-29 ° C shaker to an OD value of 260/280 between 0.5-0.8; transfer the bacterial solution to a 50mL sterile centrifuge tube Centrifuge in a centrifuge at 3000-4500 r / min for 10-15 minutes at 0-8 ° C.
  • YM liquid medium Sigma
  • the resuspension is MS liquid culture medium;
  • the callus induced in step (2) is collected into a sterile triangle bottle with a breathable plastic sealing film, and poured into the resuspended agricultural
  • the bacillus liquid is sealed with the sealing film that comes with the triangular flask, and the vacuum pump is evacuated to 0.5kpa for a total of 0.5-1.5h. During the period, it is gently shaken every 10-15min. Remove the triangular flask and shake at 25-29 ° C. Shake at 100-150r / min for 0.5-1.5h on the bed, pour out the bacterial solution, and spread the material to dry in a sterile petri dish covered with filter paper;
  • Step (4) Co-culture of alfalfa callus infected with Agrobacterium tumefaciens:
  • the co-cultivation process is as follows: the dried infected material is inoculated into a co-culture medium Sterile filter paper), dark culture in an incubator at 25 ⁇ 1 °C for 2-5 days; the components of the co-culture medium are: MS basic medium + 1-3mg / L 2,4-D + 0.1-0.3mg / L KT + 15-40g / L sucrose + 5.8-9g / L agar + 50-150umol / L acetylsyringone; preferably, the co-culture medium component is: MS basic medium + 2mg / L 2,4-D + 0.2mg / L KT + 30g / L sucrose + 8g / L agar + 100umol / L acetylsyringone;
  • Step (5) Screening and cultivation of alfalfa callus The screening and culture process is as follows: inoculate the material obtained from the co-cultivation into a screening medium, and culture in the light in a 25 ⁇ 1 ° C incubator or incubation room for 30-60 days;
  • the components of the screening medium are: SH basic medium + 1-3mg / L 2,4-D + 0.1-0.3mg / L KT + 15-40g / L sucrose + 5.8-9g / L agar + 150-450mg / L cephalosporin Thiokine + 150-450mg / L Carbenicillin + 10-50mg / L Hygromycin;
  • the components of the screening medium are: SH basic medium + 2mg / L2,4-D + 0.2mg / L KT + 30g / L sucrose + 8g / L agar + 250mg / L Cefotaxime + 250mg / L Carbenicillin + 15mg / L Hygromycin;
  • Step (6) Alfalfa callus differentiation culture: transfer the screened culture material to the differentiation medium, and culture in the light in a 25 ⁇ 1 ° C incubator or incubation room for 15-30 days;
  • the components of the differentiation medium are: UM basic medium + 0.5-5g / L hydrolyzed complex protein + 0.1-2mg / L kinetin + 15-40g / L sucrose + 5.8-9g / L agar + 150-450mg / L Cefotaxime + 4-10mg / L Hygromycin;
  • the components of the differentiation medium are: UM basal medium + 2g / L hydrolyzed complex protein + 0.4mg / L kinetin + 30g / L sucrose + 8g / L agar + 250mg / L cefotaxime + 5mg / L hygromycin;
  • Step (7) Alfalfa regenerating bud rooting culture: transfer the differentiated 1-3cm buds to the rooting medium, the medium components are: MS basic medium + 0.5-2mg / L indolebutyric acid + 15-40g / L sucrose + 5.8-9g / L agar + 150-450mg / L cefotaxime; preferably, the rooting medium composition is: MS basic medium + 1mg / L indolebutyric acid + 30g / L sucrose + 8g / L agar + 250mg / L cefotaxime;
  • Step (8) Screening and detection of alfalfa mutant strains.
  • the method for screening and detection of alfalfa mutant strains includes PCR-RE, T7E1 enzyme digestion detection and target deep sequencing.
  • the detection methods are as follows:
  • PCR-RE detection DNA samples were extracted from the regenerated plants obtained above, and specific amplification primers were designed for the target gene sequence for amplification. If the amplified fragment has a specific endonuclease cleavage site on the Cas9 protein cleavage site, the mutated sequence cannot be cleaved by restriction enzymes due to the loss of its cleavage site.
  • the specific operations are: a. Using specific primers designed for the target gene to amplify the target gene fragments of the regenerated plant and the control plant; b. Recovering the amplified target fragment, and using the target-specific restriction enzyme to perform the target fragment Digestion; c.
  • the control sequence has the same size band and no other bands, and the restriction enzyme digested control sequence is cut into two smaller bands, indicating that the target gene is knocked out and the regenerated shoots are homozygous mutation type; After restriction endonuclease digestion of the amplified plant regeneration product, there are bands in the electrophoresis results that are the same as those of the control sequence without digestion, and the bands are the same as those after restriction enzyme digestion.
  • the two smaller bands of the control sequence are the same size, indicating that the target gene has been knocked out and the regenerating shoots are of the type of heterozygous mutation; if the amplified products of the regenerating shoots are digested with restriction enzymes, there are no bands in the electrophoretic bands.
  • the size of the control sequence is the same, and the size of the digested band is the same as that of the two smaller bands of the control sequence after restriction digestion, indicating that the target gene has not been knocked out; d.
  • the above uncut bands were recovered and ligated to pMD19-T by TA cloning. After transforming E. coli, a batch of monoclonals were selected for sequencing and the types of mutations on the target were analyzed.
  • T7E1 detection DNA samples were extracted from the regenerated plants obtained above, and specific amplification primers were designed for the target gene sequence for amplification. If the amplified target gene fragment does not have a restriction enzyme restriction site at the Cas9 protein cleavage site, the mutant plant is detected by T7E1 restriction digestion.
  • the specific operations are: a. Designing primers to amplify the target gene sequence; b. Mixing the wild type and the regenerated plant target gene amplification products; c. Denaturing and renaturing the mixed products; d. Using the T7E1 enzyme (NEB The company) digested the refolded product and detected it on agarose gel electrophoresis.
  • Target deep sequencing detection DNA samples are extracted from the regenerated plants obtained above, and specific amplification primers are designed for the target gene sequence for amplification. The PCR products were TA cloned and ligated to the pMD19-T vector. After transforming E. coli, a batch of monoclonals was selected for deep sequencing of the target, and whether there were mutations at the target site and the type of mutation were analyzed.
  • the invention also protects the application of the method of site-directed mutation of alfalfa genes using the CRISPR / Cas9 system in alfalfa breeding and gene editing alfalfa breeding.
  • the invention successfully establishes the gene editing technology system based on CRISPR / Cas9 for the first time in alfalfa, successfully achieves the knockout of the target gene and obtains a homozygous mutant plant with an obvious phenotype.
  • the present invention adopts an Agrobacterium transformation method to contain expression resistance to anti-screening agents on the basis of an existing platform for transforming alfalfa by Agrobacterium tumefaciens to obtain transgenic plants.
  • the Hpt gene expression frame, sgRNA expression frame and Cas9 protein expression frame of the mycin protein are integrated into the alfalfa genome.
  • the key point of introducing the CRISPR / Cas9 system into alfalfa by transgenic method is to find a suitable promoter to start the expression of various elements, including the CaMV 35S (Enhanced) promoter that starts the transcription of the Hpt gene and the 2xCaMV 35S that starts the transcription of the Cas9 gene. Promoters have been shown to initiate transcription and function of related genes in alfalfa; the promoters that initiate sgRNA transcription are generally U6 or U3 promoters, but there have been no reports of U6 / U3 promoters in alfalfa. The sgRNA transcription is efficiently initiated in the order of Asteraceae. Through literature surveys, the more studied MtU6 promoter in the same species of alfalfa, Tribulus alfalfa, was selected to initiate sgRNA transcription.
  • the invention uses CRISPR / Cas9 technology for the first time in cross-pollination of autotetraploid plant alfalfa and obtains homozygous mutant plants; it uses the MtU6 promoter to start sgRNA transcription in alfalfa for the first time, and the MtU6 promoter is derived from alfalfa
  • This promoter a close-knit species of Tribulus terrestris, has been shown to be able to initiate the transcription of snRNA (small nuclear clear RNA) in alfalfa's relative species, Tribulus terrestris, and achieve gene silencing in Tribulus terrestris via RNAi and CRISPR / Cas9.
  • snRNA small nuclear clear RNA
  • the present invention can achieve cross pollination in autotetraploid alfalfa obtained within one generation
  • Homozygous mutants have important scientific research and application value and can accelerate the breeding of alfalfa.
  • the invention Compared with natural mutations and traditional artificially induced mutations, the invention accurately introduces mutations with higher mutation efficiency, and can reach a mutation rate of 52% (of which the homozygous mutation rate reaches 12%).
  • This technology opens up a new field for gene editing in alfalfa, laying a technical foundation for subsequent simultaneous simultaneous silence of multiple genes, deletion of large chromosomal fragments, and precise insertion of foreign genes into targets; meanwhile, the use of CRISPR /
  • the site-directed function of Cas9 we can inactivate Cas9 protein mutations into dCas9.
  • By coupling protein domains with other functions on dCas9 we can achieve target gene expression up / down regulation, methylation / demethylation, and base substitution. Wait.
  • Figure 1 is a structural diagram of the Ms CRISPR / Cas9 expression vector
  • FIG. 2 is a wild type and mutant regenerated plant obtained by infecting alfalfa with Agrobacterium tumefaciens by using a CRISPR / Cas9 expression vector Ms CRISPR / Cas9 :: PDS containing a PDS gene target;
  • Figure 3 shows the sequencing results of the PDS gene PCR products of alfalfa wild-type and mutant plants
  • 5 is a leaf phenotype of wild type and mutant plants obtained by infecting alfalfa with the CRISPR / Cas9 expression vector MsCRISPR / Cas9 :: PALM1 containing the target of the PALM1 gene;
  • Figure 6 shows the sequencing results of the alfalfa wild-type and mutant plant PALM1 gene PCR products.
  • Phytoene desaturase is the main rate-limiting enzyme in the carotenoid pigment synthesis pathway. It can catalyze the colorless C40 octahydrolycopene to produce ⁇ -carotene, streptavidin, and tomato. Lycopene, 3,4-dehydrolycopene, 3,4,3'4'-dehydrolycopene or 3,4-dehydrostreptavidin. Mutant plants of this gene exhibit an albino phenotype for easy observation. Using this gene as a target gene in plants is a convenient method to detect whether the gene knockout system works and evaluate the efficiency of the work.
  • Aar1 to digest the carrier 1300DM-MtU6 (Aar1) -Cas9; after the CIP reaction, separate in a 0.8-1.2% agarose gel, and tap the gel to recover the linear carrier;
  • the ligation product is transformed into E. coli DH5 ⁇ competent cells by heat shock, and after resuscitation, they are spread on a LB plate medium containing 50 mg / L kanamycin and cultured in a 37 ° C incubator for 12-16 hours;
  • sequencing primers are as follows:
  • MtU6-T-F GGCATGCAAGCTTATCGATAC.
  • the above expression vector was transformed into competent cells of Agrobacterium tumefaciens strain EHA105 by electric shock, recovered and spread in YM medium (addition of 50 mg / L kanamycin, 250 mg / L rifampicin), and cultured at 28 ° C for 24 h -48h; Pick a single clone and inoculate it in 30-50ml YM liquid medium (add 50mg / L kanamycin, 250mg / L rifampicin), shake at 200r / min on a shaker at 28 ° C for 24h-48h.
  • Agrobacterium transformation was used to integrate the DNA sequence expressing the CRISPR / Cas9 system and the plant-resistant Hpt protein into the alfalfa genome, to make the expression products of these elements function, and to obtain positive plants in which the target gene was silenced by screening.
  • the specific conversion method is as follows:
  • Alfalfa seed germination Select full and round alfalfa seeds, soak in 75% alcohol for 2 minutes, and wash twice in sterile water for 1 minute each time; soak in 0.1% liter of mercury and shake for 10 minutes in hands, and wash in sterile water for 5 minutes. Times. Spread the sterilized seeds in a large sterile petri dish containing filter paper and air-dry. Inoculate on MS solid medium, germinate in light incubator or culture room for 7-14 days;
  • callus induction take the cotyledons and hypocotyls of the above-mentioned germinated seedlings, cut them into small pieces with a sterile scalpel, inoculate them in callus induction medium, and incubate in the dark at 25 ⁇ 1 °C incubator or culture room for 3 days .
  • the components of callus induction medium are: SH basic medium + 2mg / L 2,4-D + 0.2mg / L KT + 0.3mg / L hydrolyzed complex protein + 30g / L sucrose + 8g / L agar;
  • Agrobacterium tumefaciens strains prepared above were inoculated in 50-100ml of YM liquid medium 1-2 days in advance, and cultured at 28 ° C on a shaker at 200r / min to an OD value of 260/280 between 0.5-0.8 Transfer the bacterial solution to a 50ml sterile centrifuge tube, centrifuge at 4000r / min for 12 minutes at 4 ° C in the centrifuge, remove the centrifuge tube, discard the supernatant, add the resuspension and resuspend to OD value 260/280.
  • Co-cultivation Inoculate the dry and infected material above into the co-cultivation medium (lay a piece of sterilized filter paper in the medium).
  • the components of the co-cultivation medium are: MS basic medium + 2 mg / L2, 4-D + 0.2mg / L KT + 30g / L sucrose + 8g / L agar + 100umol / L acetylsyringone. Dark culture in a 25 ⁇ 1 ° C incubator or incubator for 3 days.
  • the above co-culture material is inoculated into the screening medium, and the components of the screening medium are: SH basic medium + 2 mg / L 2, 4-D + 0.2 mg / L KT + 30 g / L sucrose + 8 g / L agar + 250mg / L Cefotaxime + 250mg / L Carbenicillin + 15mg / L Hygromycin; light culture in 25 ⁇ 1 °C incubator or culture room for 30-60 days;
  • Differentiation Transfer the recovered material to the differentiation medium.
  • the components of the differentiation medium are: UM basic medium + 2g / L hydrolyzed complex protein + 0.4mg / L kinetin + 30g / L sucrose + 8g / L agar + 250mg / L Cefotaxime + 5mg / L Hygromycin;
  • Rooting Transfer the above-mentioned differentiated 1-3cm buds to the rooting medium.
  • the components of the rooting medium are: MS basic medium + 1mg / L indolebutyric acid + 30g / L sucrose + 8g / L agar + 250mg / L Ceftioxine.
  • Target deep sequencing detection DNA samples were extracted from the regenerated plants and amplified using specific amplification primers for the PDS sequence of the target gene. The PCR products were TA cloned and ligated to the pMD19-T vector. After transforming E. coli strain DH5 ⁇ , a batch of monoclonals were selected for deep sequencing of the target, and whether there were mutations on the target site and the type of mutation were analyzed.
  • Fig. 2 An albino seedling was obtained (Fig. 2).
  • a DNA sample was extracted from the albino seedling and the PDS gene sequence was amplified.
  • the sequencing result (Fig. 3) showed that the plant had a 2bp base deletion at the target site.
  • the base deletion site is the cleavage site of the Cas9 protein.
  • Figure 2 shows: wild type (PDS-wt); mutant type (PDS-mt). Wild-type plants exhibit a normal green phenotype; mutant plants exhibit a white phenotype. This result is consistent with the phenotype of plants obtained by CRISPR / Cas9-mediated PDS gene knockout in poplar, apple and other species. This indicates that the obtained albino vaccine is probably caused by the mutation of PDS gene mediated by the introduced CRISPR / Cas9 system.
  • Figure 3 shows that from the sequencing results, compared with the wild type, the PDS mutant plant had a 2 bp base deletion on the target and the mutation occurred at the cleavage site of the Cas9 protein. It is explained that the alfalfa albino seedlings in Fig. 2 are caused by CRISPR / Cas9 cutting the PDS gene target site, resulting in a 2bp base deletion during gene repair.
  • Embodiment 1 The difference from Embodiment 1 lies in:
  • Primers were designed based on the PALM1 sequence in the alfalfa near-source species Medicago truncatula, and primers were designed to amplify and sequence the alfalfa MsPALM1 gene sequence.
  • a target site was designed based on the obtained MsPALM1 gene sequence; the target site was ligated to the vector Ms CRISPR / Cas9 site to obtain The MsPALM1 gene knockout vector Ms CRISPR / Cas9 :: PALM1 was used to transform the alfalfa explants through the above-mentioned Agrobacterium tumefaciens transformation method and obtain regenerated plants.
  • the detection method was PCR-RE.
  • the genomic DNA samples were extracted from the plants, amplified using specific amplification primers targeting the MsPALM1 gene containing the target sequence, and the mutants were screened by BstU I restriction endonuclease digestion PCR products (Figure 4); and observed by phenotype ( Figure 5) Mutation was confirmed by deep sequencing with the target ( Figure 6).
  • Figure 4 shows that, by PCR-RE detection, among the 25 alfalfa regeneration plants sampled, a total of 13 mutant plants (there are bands that cannot be cut by the restriction enzyme BstUI at 768bp), of which 10 Heterozygous mutant strains (in addition to the band that cannot be cut by the restriction enzyme BstUI at 768bp, there are two bands generated by the restriction enzyme BstUI, which are located at 442bp and 326bp, respectively) There are 3 homozygous mutant plants (there is a band that cannot be cut by the restriction enzyme BstUI only at 768bp); the mutation rate is 52%, of which the homozygous mutation rate is 12%.
  • Figure 5 shows that among the 25 alfalfa regenerated plants sampled, three of the compound leaves (second, third, and fourth from left to right) exhibited obvious phenotypic changes, which were in contrast to the control (left to right In comparison, the number of leaflets in the compound leaves of the mutant plants changed from three to five. Among them, the mutant No. 3 (that is, No. 20 in Fig. 4) showed a weak band that could be cut by the BstUI restriction enzyme in the PCR-RE result, which may be because the TO plant was a chimera. s reason.
  • FIG. 6 shows that a part of the plants selected from the screened in FIG. 4 was subjected to deep target sequencing of the MsPALM1 gene, and mutations were successfully introduced at the predetermined target of CRISPR / Cas9 on the MsPALM1 gene.

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Abstract

一种利用CRISPR/Cas9***对紫花苜蓿基因定点突变的方法。首先构建紫花苜蓿中通用的由根癌农杆菌介导转化的双元表达载体MsCRISPR/Cas9；再针对紫花苜蓿中的目标基因设计基于CRISPR/Cas9的sgRNA序列，将含有编码所述sgRNA序列的DNA片段连接到MsCRISPR/Cas9中构建载体MsCRISPR/Cas9::target；然后使用根癌农杆菌介导转化紫花苜蓿，通过筛选获得目的基因被敲除的突变体转化植株。该方法使用MtU6启动子在紫花苜蓿中启动sgRNA转录。

Description

一种利用CRISPR/Cas9***对紫花苜蓿基因定点突变的方法 技术领域
本发明属于基因编辑技术领域，尤其涉及一种利用CRISPR/Cas9***对紫花苜蓿基因定点突变的方法。
背景技术
紫花苜蓿(Medicago sativa.)是广泛种植的豆科牧草，富含优质膳食纤维、食用蛋白、多种维生素(包括维生素B、维生素C、维生素E等)、多种有益的矿物质以及皂苷、黄酮类、类胡萝卜素、酚醛酸等生物活性成分，蛋白含量高、适口性好，对于提升牲畜的饲料水平具有重要意义。除了作为饲草料作物，紫花苜蓿发达的根系在防治水土流失、环境治理方面发挥重要作用。此外，因为紫花苜蓿根部有大量的固氮微生物共生，紫花苜蓿在改善土壤肥力上也具有重要的意义。针对紫花苜蓿基因功能的研究有助于改善紫花苜蓿的性状，辅助培育具备更优性状的紫花苜蓿新品种。在紫花苜蓿中使用基因沉默技术抑制目的基因的表达是研究基因功能与培育新品种的一种非常有效的手段。
自然界中虽经常发生自发突变，但突变频率极低，大多数基因位点每代自发突变的突变率约为10 -5或10 -6，且只有发生上在配子中的突变才可以传到下一代。通过化学诱变、物理诱变以及DNA***突变等诱变方法处理植物材料，可以在短时间内获得有利用价值的突变体。诱变因素引发的突变频率较高，比自发突变频率高几百倍，甚至上千倍，而且诱发突变的变异范围广泛，类型多样，有时能够诱发产生自然界稀有的或未曾有过的新突变。但是通过诱变方式引入的突变是随机的，很难针对某一位点得到纯合突变体，同样的，使用诱变方法产生的突变只有发生在配子中的突变才可以传到下一代。靶向基因沉默技术是研究特定基因功能的有效手段。目前，针对靶向基因沉默已经开发出RNAi(RNA干扰)，以及ZFN、TALLEN、CRISPR/Cas9等基因编辑技术。RNAi技术主要降解目的基因转录出来的mRNA序列，下调mRNA的翻译水平，从而实现抑制目的基因表达的作用。但是，通过RNAi技术降低目的基因表达时，目的基因的基因序列没有被突变，在一定程度上仍可能有目的基因表达。相反，使用ZFN、TALLEN、CRISPR/Cas9技术实现的基因沉默，在DNA水平上突变基因序列，使基因彻底 丧失功能。与RNAi相比，这三种技术更加稳定、高效。在TALEN、ZFN、CRISPR/Cas9三种技术中，TALEN与ZFN需要针对每一个靶位点将识别不同碱基的氨基酸序列元件按靶点序列拼装起来，每一个靶位点都有自己独特的蛋白质序列，这就导致该过程耗时、耗力、成本高昂。与TALLEN以及ZFN不同的是，CRISPR/Cas9技术是通过sgRNA上的靶向序列识别靶位点，靶向序列与靶点之间通过碱基互补配对的方式互相识别，这种方式更加简便、廉价、操作方便。所以，自2013年CRISPR/Cas9技术被首次应用于基因编辑以来，得到快速、广泛的发展。CRISPR/Cas9***是来自链球菌属的一种获得性免疫***，可以抵御外源基因的入侵。它主要由CRISPR和特异的Cas9蛋白组成。CRISPR是一成簇的规律间隔的短回文重复序列，是一类新型的DNA重复序列，它由一系列短的高度保守的正向重复序列和长度相似的间隔序列间隔排列组成。Cas9蛋白是一种由1409个氨基酸组成的多结构域蛋白，含有两个核酸酶结构域RuvC-like和HNH。基于CRISPR/Cas9的基因组编辑体系包括2部分：sgRNA及酶Cas9。这种***能够特异的识别毗邻PAM(NGG)基序的靶序列，并在其上游3nt处进行切割形成DSB(double strains break),随后机体通过自身的修复机制如同源重组(HR)、非同源末端连接(NHEJ)来修复损伤的DNA。NHEJ方式的修复过程能够产生Indel(包括缺失和***)，进而导致DNA编码序列移码突变。
紫花苜蓿是严格异花授粉的同源四倍体植物，每个座位有四个等位基因，如果需要针对某一座位获得可稳定遗传的纯合突变体品系，最理想的就是获得同时突变了四个等位基因的多个株系，能够实现多个株系间的杂交与稳定遗传；其次就是通过自然发生的或诱变产生的杂合突变体之间的反复杂交获得可稳定遗传的纯合突变体品系，但这是一个耗时、耗力、高投入、低效率的过程。因此综合而言，在紫花苜蓿中利用自然发生的或者诱变产生的突变体通过杂交获得能够稳定遗传的纯合突变体尤为困难，这严重阻碍了紫花苜蓿的研究与育种，尤其针对只有获得纯合体才能表现出性状变化的隐性基因突变而言更加困难。在紫花苜蓿中开发高效的靶向突变技术实现在一个世代之内获得稳定遗传的纯合突变体，对于紫花苜蓿研究与育种意义重大。目前在紫花苜蓿中已知的是使用RNAi技术降低紫花苜蓿基因的表达，尚无使用ZFN、TALEN、CRISPR/Cas9等基因编辑技术进行基因编辑获得纯合突变体的报道。如上文所述，与ZFN、TALEN、 CRISPR/Cas9等基因编辑技术相比，RNAi因其沉默不彻底以及不稳定的特性，限制了该技术的应用。在水稻、拟南芥、番茄、杨树、白杨、苹果、小麦、玉米、大豆等物种中通过CRISPR/Cas9技术已经成功获得可稳定遗传的纯合突变体，但在紫花苜蓿中还没有使用CRISPR/Cas9等靶向基因编辑技术进行基因编辑的报道。
发明内容
为了在同源四倍体植物紫花苜蓿中实现基因编辑，并运用于生产，我们针对紫花苜蓿开发了一项利用CRISPR/Cas9***实现定点突变的方法，实现了紫花苜蓿中的精准定点基因突变，突变率高达52％，并获得了具备理想表型的同时沉默了四个等位基因的纯合突变植株(纯合突变率达12％)，率先在紫花苜蓿中建立了高效的靶向基因编辑体系，对紫花苜蓿研究与育种具有重大意义。因此，本发明拟提供一种利用CRISPR/Cas9***对紫花苜蓿基因定点突变的方法。
本发明的技术方案如下：
一种利用CRISPR/Cas9***对紫花苜蓿基因定点突变的方法，其特征在于，所述方法包括以下步骤：
步骤(1)构建紫花苜蓿中通用的由根癌农杆菌介导转化的双元表达载体MsCRISPR/Cas9；
步骤(2)针对紫花苜蓿中的目标基因设计基于CRISPR/Cas9的sgRNA靶向序列，将含有编码所述sgRNA靶向序列的DNA片段连接到紫花苜蓿中通用的载体MsCRISPR/Cas9中构建载体MsCRISPR/Cas9::target；
步骤(3)使用根癌农杆菌介导转化紫花苜蓿，实现对紫花苜蓿中特定基因的定点突变。
进一步地，所述紫花苜蓿包括野生品种、地方品种、育成品种、引进品种。
进一步地，所述目标基因包括紫花苜蓿基因组中任何有生物学功能的基因或DNA序列。
进一步地，所述的在紫花苜蓿中表达CRISPR/Cas9***的双元表达载体MsCRISPR/Cas9的全序列如SEQ ID NO.1所示。
该技术依托的是通过根癌农杆菌对紫花苜蓿进行转化，通过转化将含有筛选 基因Hpt表达框、sgRNA表达框以及Cas9蛋白表达框的T-DNA序列整合到紫花苜蓿基因组中获得转基因植株，转化的外源表达元件在紫花苜蓿中表达，从而发挥功能，获得基因敲除的植株。针对农杆菌转化的方式，构建了一个表达载体MsCRISPR/Cas9。
优选地，所述的在紫花苜蓿中表达CRISPR/Cas9***的双元表达载体MsCRISPR/Cas9的骨架载体是pCambia1300载体，所述的pCambia1300骨架载体上有一段用于转化植物的T-DNA区域，所述T-DNA区域包含一个左边界重复序列(LB repeat)、一个右边界重复序列(RB repeat)以及左右边界序列内部的用于转化的序列，所述的T-DNA区域左右边界内部用于转化的序列中包含表达抗植物筛选剂潮霉素的蛋白的Hpt基因表达元件、表达Cas9蛋白的表达元件、表达sgRNA的表达元件以及sgRNA表达元件内部两个Aar1酶切位点之间的靶点序列，所述Hpt基因表达元件内部包含启动Hpt基因转录的Camv 35S promoter(enhanced)启动子序列、表达Hpt基因的CDS序列和终止Hpt基因转录的Camv poly(A)终止序列，所述表达Cas9蛋白的表达元件内部包含启动Cas9序列转录的2xCaMV 35S promoter启动子序列、表达Cas9的DNA序列和终止Cas9转录的Nos terminator终止序列，所述表达sgRNA的表达元件内部包含启动sgRNA序列转录的蒺藜苜蓿MtU6启动子序列和包含有表达sgRNA的DNA序列。
在该载体(图1)中，LB T-DNA repeat/RB T-DNA repeat表示T-DNA左右边界重复序列；CaMV 35s promoter/MtU6 promoter/2x CaMV 35s promoter表示启动子元件；Hpt/sgRNA/Cas9表示基因序列；CaMV poly(A)/NosT代表终止序列；AarI代表sgRNA靶向序列***位点。如图1所示：使用蒺藜苜蓿U6启动子启动sgRNA表达；2XCaMV35S启动子启动Cas9基因表达；CaMV35S(Enhanced)启动子启动筛选标记基因Hpt表达。
进一步地，所述的MtU6启动子的序列SEQ ID NO.2所示。
优选地，所述的表达sgRNA的DNA序列如SEQ ID NO.3所示，表达sgRNA的DNA序列中第1位到第30位的序列内部包含两个AarI限制性内切酶识别位点，用以针对目标基因构建定点突变的表达载体。
进一步地，所述表达载体MsCRISPR/Cas9::target构建方法操作如下：
步骤(1)根据靶点序列设计一对oligo引物(18-24bp),形成如下两端带有接头的DNA片段,并合成：
5’-T T T G N 16-23-3’
3’-C N 16-23 C A A A-5’；
步骤(2)将上述合成的引物对进行退火，形成带粘性末端的互补DNA双链片段；
步骤(3)使用AarI限制性内切酶酶切双元表达载体MsCRISPR/Cas9；经CIP反应去磷酸化后在0.8-1.2％的琼脂糖凝胶中进行电泳分离，回收纯化线性载体；
步骤(4)将退火化后带粘性末端的互补DNA双链片段DNA与回收纯化的MsCRISPR/Cas9线性载体使用T4 DNA连接酶在16℃水浴锅中连接10-16h；
步骤(5)将连接产物通过热击法转化大肠杆菌DH5ɑ感受态细胞，复苏后涂布于含50mg/L卡那霉素的LB平板培养基上，37℃培养箱培养12-16h；
步骤(6)挑取单菌落克隆于1.5ml含50mg/L卡那霉素的LB液体培养基中，37℃摇床摇12-16h；
步骤(7)使用测序引物MtU6-T-F对单克隆菌液进行测序，选择靶点连接正确的克隆于50-100ml含50mg/L卡那霉素的LB液体培养基中，37℃摇床摇12-16h，提取质粒。
进一步地，所述测序引物MtU6-T-F的序列如SEQ ID NO.4所示。
优选地，所述使用根癌农杆菌介导转化紫花苜蓿获得突变植株的方法包含以下步骤：
步骤(1)根癌农杆菌菌种复苏；将电击转化后的农杆菌菌株涂布于YM固体平板培养基中，25-29℃培养24h-48h；挑取单克隆，接种于30-50mLYM液体培养基中，25-29℃摇床上100-220r/min摇24h-48h；所述YM固体培养基和YM液体培养基中均包含30-60mg/L卡那霉素和200-450mg/L利福平；
步骤(2)紫花苜蓿愈伤组织的获得:所述紫花苜蓿愈伤组织获得过程如下：
挑选饱满、色泽圆润的紫花苜蓿种子，65-80％酒精浸泡1-3分钟，无菌水洗2-4次，每次0.5-2分钟；0.05-0.15％升汞浸泡并手摇6-12分钟，无菌水洗3～5次，接种于MS固体培养基上，光照培养箱或培养室中萌发7-16天；
取萌发后的子叶和下胚轴，切成小块接种于愈伤诱导培养基中，25±1℃培 养箱或培养室中暗培养2～4天；所述愈伤诱导培养基成分为：SH基础培养基+1.0-3.0mg/L 2,4-D(2,4-二氟苯氧乙酸)+0.1-0.3mg/L KT(激动素)+0.1-0.5mg/L水解络蛋白+15-40g/L蔗糖+5.8-9g/L琼脂；优选地，所述愈伤诱导培养基成分为：SH基础培养基+2mg/L 2,4-D+0.2mg/L KT+0.3mg/L水解络蛋白+30g/L蔗糖+8g/L琼脂；
步骤(3)根癌农杆菌侵染紫花苜蓿愈伤组织:所述农杆菌菌株侵染紫花苜蓿愈伤组织过程如下：提前1-2天将步骤(1)复苏的根癌农杆菌菌株接种于50-100mL的YM液体培养基(Sigma公司)中，25-29℃摇床上100-220r/min培养至OD值260/280在0.5-0.8之间；将菌液转移到50mL无菌离心管中，离心机中0-8℃条件下3000-4500r/min离心10-15分钟，取出离心管，弃上清液，加入重悬液重悬至OD值260/280在0.5-0.8之间，加入50-150umol/L乙酰丁香酮，重悬液为MS液体培养基；将步骤(2)中诱导的愈伤组织收集到带透气塑料封口膜的无菌三角瓶中，倒入重悬后的农杆菌菌液，用三角瓶自带的封口膜封口，真空泵中抽真空到0.5kpa，一共抽0.5-1.5h，期间每10-15min的时候轻轻摇晃一下；取出三角瓶，25-29℃摇床上100-150r/min摇0.5-1.5h，倒干菌液，将材料摊开在铺了滤纸的无菌培养皿中晾干；
步骤(4)被根癌农杆菌侵染过的紫花苜蓿愈伤组织共培养:所述共培养过程如下：将晾干的侵染过的材料接种于共培养培养基(培养基中铺一张灭菌滤纸)，25±1℃培养箱中暗培养2-5天；所述共培养培养基成分为：MS基础培养基+1-3mg/L 2,4-D+0.1-0.3mg/L KT+15-40g/L蔗糖+5.8-9g/L琼脂+50-150umol/L乙酰丁香酮；优选地，所述共培养培养基成分为：MS基础培养基+2mg/L 2,4-D+0.2mg/L KT+30g/L蔗糖+8g/L琼脂+100umol/L乙酰丁香酮；
步骤(5)紫花苜蓿愈伤组织筛选培养:所述筛选培养过程如下：将共培养得到的材料接种于筛选培养基，25±1℃培养箱或培养室中光照培养30-60天；所述筛选培养基成分为：SH基础培养基+1-3mg/L 2,4-D+0.1-0.3mg/L KT+15-40g/L蔗糖+5.8-9g/L琼脂+150-450mg/L头孢噻亏+150-450mg/L羧苄青霉素+10-50mg/L潮霉素；优选地，所述筛选培养基成分为：SH基础培养基+2mg/L2,4-D+0.2mg/L KT+30g/L蔗糖+8g/L琼脂+250mg/L头孢噻亏+250mg/L羧苄青霉素+15mg/L潮霉素；
步骤(6)紫花苜蓿愈伤组织分化培养:将筛选培养后的材料转接至分化培养基，25±1℃培养箱或培养室中光照培养15-30天；所述分化培养基成分为：UM基础培养基+0.5-5g/L水解络蛋白+0.1-2mg/L激动素+15-40g/L蔗糖+5.8-9g/L琼脂+150-450mg/L头孢噻亏+4-10mg/L潮霉素；优选地，所述分化培养基成分为：UM基础培养基+2g/L水解络蛋白+0.4mg/L激动素+30g/L蔗糖+8g/L琼脂+250mg/L头孢噻亏+5mg/L潮霉素；
步骤(7)紫花苜蓿再生芽生根培养:将分化出的1-3cm的芽转接至生根培养基，培养基成分为：MS基础培养基+0.5-2mg/L吲哚丁酸+15-40g/L蔗糖+5.8-9g/L琼脂+150-450mg/L头孢噻肟；优选地，所述生根培养基成分为：MS基础培养基+1mg/L吲哚丁酸+30g/L蔗糖+8g/L琼脂+250mg/L头孢噻亏；
步骤(8)紫花苜蓿突变株的筛选与检测，所述紫花苜蓿突变株的筛选与检测的方法包括PCR-RE、T7E1酶切检测与靶点深度测序。检测方法具体如下：
PCR-RE检测：将上述获得的再生植株提取DNA样品，针对靶基因序列设计特异性的扩增引物进行扩增。若扩增所得的片段中，在Cas9蛋白切割位点上有特异性内切酶酶切位点，则发生突变的序列内因其酶切位点丢失不能被限制性内切酶切割。具体操作为：a.利用针对靶基因设计的特异性引物扩增再生植株与对照植株靶基因片段；b.将扩增所得目标片段回收，利用靶点特异的限制性内切酶对目标片段进行酶切；c.将酶切产物通过琼脂糖凝胶电泳检测，电泳结果分析：若再生苗扩增产物进行限制性内切酶酶切后，电泳条带中有条带与未进行酶切的对照序列条带大小一致且无其他条带，而进行限制性内切酶酶切的对照序列被切割为两条较小条带，则说明靶基因被敲除且再生苗为纯合突变类型；若再生苗扩增产物进行限制性内切酶酶切后，电泳结果中有条带与未进行酶切的对照序列条带大小一致，且有条带与进行限制性内切酶酶切后的对照序列的两条较小条带大小一致，则说明靶基因被敲除且再生苗为杂合突变类型；若再生苗扩增产物进行限制性内切酶酶切后，电泳条带中没有条带与未进行酶切的对照序列条带大小一致，且酶切后的条带与且进行限制性内切酶酶切后的对照序列的两条较小条带大小一致，则说明靶基因没有被敲除；d.将上述未被切割的条带回收，并通过TA克隆连接到pMD19-T上，转化大肠杆菌以后挑选一批单克隆进行测序，分析靶点上的突变类型。
T7E1检测：将上述获得的再生植株提取DNA样品，针对靶基因序列设计特异性的扩增引物进行扩增。若扩增所得的靶基因片段中，在Cas9蛋白切割位点处没有限制性内切酶酶切位点，则通过T7E1酶切方式检测突变植株。具体操作为：a.设计引物扩增靶基因序列；b.将野生型与再生植株的靶基因扩增产物进行混合；c.将混合的产物进行变性、复性；d.使用T7E1酶(NEB公司)对复性后的产物进行酶切，在琼脂糖凝胶电泳进行检测。结果分析：若是再生植株中的靶基因存在突变，则经过变性与复性后，会与野生型植株的扩增产物形成异源双链核酸分子，该异源双链核酸分子能够被T7E1酶切割得到两条理想大小的条带；e.将能被T7E1酶酶切的再生苗的PCR产物进行TA克隆连接到pMD19-T载体(Takara公司)上，转化大肠杆菌以后挑选一批单克隆进行测序，分析在靶位点上的突变类型以及再生苗是否为纯合敲除。
靶点深度测序检测：将上述获得的再生植株提取DNA样品，针对靶基因序列设计特异性的扩增引物进行扩增。将PCR产物进行TA克隆连接到pMD19-T载体上，转化大肠杆菌以后挑选一批单克隆进行靶点深度测序，分析在靶位点上是否有突变、分析突变类型。
同时，本发明还保护利用CRISPR/Cas9***对紫花苜蓿基因定点突变的方法在紫花苜蓿育种和基因编辑紫花苜蓿育种中的应用。
本发明在紫花苜蓿中首次成功建立了基于CRISPR/Cas9的基因编辑技术体系，成功实现了对目的基因的敲除并获得了具有明显表型的纯合突变植株。本发明为了在同源四倍体植物紫花苜蓿中实现基因编辑，在基于已有的根癌农杆菌转化紫花苜蓿获得转基因植株的平台基础上，采用农杆菌转化的方法将含有表达抗筛选剂潮霉素的蛋白的Hpt基因的表达框、sgRNA表达框、Cas9蛋白表达框的T-DNA区域整合到紫花苜蓿基因组上，这些元件在紫花苜蓿细胞内转录、翻译并发挥功能，通过筛选获得目的基因被敲除的突变体转化植株。通过转基因方式将CRISPR/Cas9***导入紫花苜蓿中进行表达的关键点在于寻找合适的启动子启动各个元件的表达，其中启动Hpt基因转录的CaMV 35S(Enhanced)启动子与启动Cas9基因转录的2xCaMV 35S启动子已经被证明可以在紫花苜蓿中启动相关基因转录并发挥功能；启动sgRNA转录的启动子一般为U6或者U3启动子，但在紫花苜蓿中尚无研究U6/U3启动子的报道，为了在紫花目中高效启动sgRNA 的转录，通过文献资料调研，选择紫花苜蓿的同属近源种蒺藜苜蓿中研究较多的MtU6启动子启动sgRNA的转录。
与现有的在紫花苜蓿中使用RNAi进行基因表达下调的技术相比，本发明的优点在于：
本发明首次在异花授粉的同源四倍体植物紫花苜蓿中使用CRISPR/Cas9技术，并获得纯合突变植株；首次使用MtU6启动子在紫花苜蓿中启动sgRNA转录，MtU6启动子来源于紫花苜蓿的近源物种蒺藜苜蓿，该启动子已经被证明能够在紫花苜蓿近缘种蒺藜苜蓿中启动snRNA(small nuclear RNA)的转录，通过RNAi与CRISPR/Cas9实现蒺藜苜蓿中的基因表达沉默。在紫花苜蓿中使用蒺藜苜蓿MtU6启动sgRNA转录比使用其他亲缘关系较远的物种效率会更高。
1.本发明能够实现异花授粉的同源四倍体紫花苜蓿中在一个世代之内获得
纯合突变体，具有重要的科研与应用价值，可以加速紫花苜蓿育种。
2.首次在紫花苜蓿中建立了基于根癌农杆菌转化的紫花苜蓿CRISPR/Cas9基因敲除平台，为紫花苜蓿及其它植物基因编辑奠定基础，可以直接在基因编码序列上进行突变，使目的基因沉默、丧失功能，该技术更加彻底、稳定。
3.与自然突变以及传统人工诱导突变相比，本发明以更高的突变效率精准地引入突变，能达到突变率52％(其中纯合突变率达12％)。
4.该技术为在紫花苜蓿中进行基因编辑开辟了一个新的领域，为后续同时沉默多个基因、染色体大片段缺失、外源基因精准***靶点等奠定了技术基础；同时，利用CRISPR/Cas9的定点功能，我们可以将Cas9蛋白突变失活成为dCas9，通过在dCas9上偶联具备其他功能的蛋白结构域实现靶基因的表达上调/下调、甲基化/去甲基化、碱基替换等。
附图说明
图1为MsCRISPR/Cas9表达载体结构图；
图2为将含有PDS基因靶点的CRISPR/Cas9表达载体MsCRISPR/Cas9::PDS通过根癌农杆菌侵染紫花苜蓿获得的野生型与突变型再生植株；
图3为紫花苜蓿野生型与突变型植株PDS基因PCR产物测序结果；
图4为将含有PALM1基因靶点的CRISPR/Cas9表达载体的根癌农杆菌侵染紫花苜蓿获得的再生植株，经PCR-RE检测筛选突变植株的琼脂糖凝胶电泳结果；
图5为将含有PALM1基因靶点的CRISPR/Cas9表达载体 MsCRISPR/Cas9::PALM1的根癌农杆菌侵染紫花苜蓿获得的野生型与突变型植株的叶片表型；
图6为紫花苜蓿野生型与突变型植株PALM1基因PCR产物测序结果。
具体实施方式
下面结合具体实施例对本发明的技术方案做进一步详细说明，但本发明并不局限于以下技术方案。
实施例1利用CRISPR/Cas9***对花苜蓿中八氢番茄红素脱氢酶编码基因PDS突变
八氢番茄红素脱氢酶(phytoene desaturase,PDS)，是类胡萝卜色素合成途径中的主要限速酶，可催化无色C40八氢番茄红素生成ζ-胡萝卜素、链孢红素、番茄红素、3,4-脱氢番茄红素、3,4,3‘4’-脱氢番茄红素或3,4-脱氢链孢红素等。该基因的突变植株表现出白化表型，便于观察。在植物中以该基因作为靶基因是检测基因敲除体系是否工作以及评估工作效率的一种便捷地方法。
依据紫花苜蓿近源种蒺藜苜蓿中的PDS序列，设计引物扩增紫花苜蓿PDS基因序列并测序，依据所得PDS基因序列并参考蒺藜苜蓿中的案例，设计靶位点；将靶位点连接到载体1300DM-MtU6(Aar1)-Cas9的位点上获得针对PDS基因的敲除载体，将该载体通过上述根癌农杆菌转化的方式转化紫花苜蓿外殖体，并获得再生植株(图2)。其中表达载体构建、农杆菌菌株的制备、使用农杆菌进行转化、突变体的筛选与检测过程如下：
一、表达载体构建过程如下：
1.根据靶点序列设计一对oligo引物(18-24bp),形成如下两端带有接头的DNA片段,并合成：
5’-T T T G N16-23-3’
3’-C N16-23C A A A-5’；
2.将上述合成的成对引物进行退火；
3.使用Aar1酶酶切载体1300DM-MtU6(Aar1)-Cas9；CIP反应后在0.8-1.2％的琼脂糖凝胶中进行分离，割胶回收线性载体；
4.将退火磷酸化的DNA片段与胶回收的1300DM-MtU6(Aar1)-Cas9线性载 体使用T4 DNA连接酶在16℃水浴锅中连接10-16h；
5.将连接产物通过热击法转化大肠杆菌DH5ɑ感受态细胞，复苏后涂布于含50mg/L卡那霉素的LB平板培养基上37℃培养箱培养12-16h；
6.挑取单克隆于1.5ml含50mg/L卡那霉素的LB液体培养基中，37℃摇床摇12-16h；
7.使用测序引物对单克隆菌液进行测序，选择靶点连接正确的克隆于50-100ml含50mg/L卡那霉素的LB液体培养基中，37℃摇床摇12-16h,提取质粒。测序引物如下：
MtU6-T-F:GGCATGCAAGCTTATCGATAC。
二、农杆菌菌株的制备
将上述表达载体通过电击法转化根癌农杆菌菌株EHA105感受态细胞中，复苏并涂布于YM培养基(附加50mg/L卡那霉素，250mg/L利福平)中，28℃培养24h-48h；挑取单克隆，接种于30-50mlYM液体培养基中(附加50mg/L卡那霉素，250mg/L利福平)，28℃摇床上200r/min摇24h-48h。
三、使用农杆菌进行转化
使用农杆菌转化的方式将表达CRISPR/Cas9***以及抗植物筛选剂的Hpt蛋白的DNA序列整合到紫花苜蓿基因组中，使这些元件的表达产物发挥功能，通过筛选获得目的基因被沉默的阳性植株。具体的转化方式如下：
1.紫花苜蓿种子萌发：挑选饱满、色泽圆润的紫花苜蓿种子，75％酒精浸泡2分钟，无菌水洗两次，每次1分钟；0.1％升汞浸泡并手摇10分钟，无菌水洗五次。将灭菌种子摊开于含滤纸的无菌大培养皿中，风干。接种于MS固体培养基上，光照培养箱或培养室中萌发7-14天；
2.愈伤组织诱导：取上述萌发苗的子叶和下胚轴，无菌手术刀切成小块，接种于愈伤诱导培养基中，25±1℃培养箱或培养室中暗培养3天。愈伤诱导培养基成分为：SH基础培养基+2mg/L 2,4-D+0.2mg/L KT+0.3mg/L水解络蛋白+30g/L蔗糖+8g/L琼脂；；
3.侵染：提前1-2天将上述制备的根癌农杆菌菌株接种于50-100ml的YM液 体培养基中，28℃摇床上200r/min培养至OD值260/280在0.5-0.8之间；将菌液转移到50ml无菌离心管中，离心机中4℃条件下4000r/min离心12分钟，取出离心管，弃上清液，加入重悬液重悬至OD值260/280在0.5-0.8之间，依据每升加入100umol乙酰丁香酮加入对应体积的100umol/ml的乙酰丁香酮，重悬液为MS液体培养基(MS+30g/L蔗糖)；将步骤2中诱导的外殖体收集到带透气塑料封口膜的100ml无菌三角瓶，倒入适量重悬后的菌液(能没过材料即可)，用三角瓶自带的封口膜封口，真空泵中抽真空到0.5kpa，一共抽1h，期间每15min的时候轻轻摇晃一下；取出三角瓶，28℃摇床上120r/min摇1h，倒干菌液，将材料摊开在铺了滤纸的无菌培养皿中晾干；
4.共培养：将上述晾干的侵染过的材料接种于共培养培养基(培养基中铺一张灭菌滤纸)，共培养培养基成分为：MS基础培养基+2mg/L 2,4-D+0.2mg/L KT+30g/L蔗糖+8g/L琼脂+100umol/L乙酰丁香酮。25±1℃培养箱或培养室中暗培养3天。
5.筛选：将上述共培养材料接种于筛选培养基,筛选培养基成分为：SH基础培养基+2mg/L 2,4-D+0.2mg/L KT+30g/L蔗糖+8g/L琼脂+250mg/L头孢噻亏+250mg/L羧苄青霉素+15mg/L潮霉素；25±1℃培养箱或培养室中光照培养30-60天；
6.分化：将上述恢复的材料转接至分化培养基，分化培养基成分为：UM基础培养基+2g/L水解络蛋白+0.4mg/L激动素+30g/L蔗糖+8g/L琼脂+250mg/L头孢噻亏+5mg/L潮霉素；
7.生根：将上述分化出的1-3cm的芽转接至生根培养基，生根培养基成分为：MS基础培养基+1mg/L吲哚丁酸+30g/L蔗糖+8g/L琼脂+250mg/L头孢噻亏。
四、突变体的筛选与检测
靶点深度测序检测：将获得的再生植株提取DNA样品，用针对靶基因PDS序列的特异性扩增引物进行扩增。将PCR产物进行TA克隆连接到pMD19-T载体上，转化大肠杆菌菌株DH5ɑ以后挑选一批单克隆进行靶点深度测序，分析在靶位点上是否有突变、分析突变类型。
通过实验与检测，我们发现：
通过表型观察，获得一株白化苗(图2)，将白化苗提取DNA样品并扩增PDS基因序列，测序结果(图3)表明：该植株在靶位点上有2bp碱基缺失，碱基缺失位点为Cas9蛋白的切割位点。
图2表明：野生型(PDS-wt)；突变型(PDS-mt)。野生型植株表现出正常的绿色表型；突变植株表现出白色表型。该结果与白杨、苹果等物种中通过CRISPR/Cas9介导的PDS基因敲除所获得的植株表型一致。说明所得的白化苗很可能是由导入的CRISPR/Cas9***介导的PDS基因突变引起。
图3表明：从测序结果看，PDS突变植株与野生型相比，在靶点上有2bp的碱基缺失且该突变发生在Cas9蛋白的切割位点上。说明图2中的紫花苜蓿白化苗是由CRISPR/Cas9切割PDS基因靶位点，导致基因修复过程中有2bp碱基缺失所引起的。
实施例2紫花苜蓿中PALM1基因突变
与实施例1不同之处在于：
依据紫花苜蓿近源种蒺藜苜蓿中的PALM1序列，设计引物扩增紫花苜蓿MsPALM1基因序列并测序，依据所得MsPALM1基因序列设计靶位点；将靶位点连接到载体MsCRISPR/Cas9的位点上获得针对MsPALM1基因的敲除载体MsCRISPR/Cas9::PALM1，将该载体通过上述根癌农杆菌转化的方式转化紫花苜蓿外殖体，并获得再生植株；检测方式为PCR-RE，将所获得的再生植株提取基因组DNA样品，用针对MsPALM1基因包含靶点序列的特异性扩增引物进行扩增，并通过BstU I限制性内切酶酶切PCR产物筛选突变株(图4)；并通过表型观察(图5)与靶点深度测序(图6)确认突变。
图4表明：通过PCR-RE检测，在所抽检的25株紫花苜蓿再生植株中，共有13株突变植株(在768bp处有不可被限制性内切酶BstUI所切割的条带)，其中有10株杂合突变株(除了在768bp处有不可被限制性内切酶BstUI所切割的条带外，还有被限制性内切酶BstUI切割后生成的两条带，分别位于442bp与326bp处)，有3株纯合突变植株(只在768bp处有不可被限制性内切酶BstUI所切割的条带)；突变率达52％，其中纯合突变率达12％。
图5表明，在所抽检的25株紫花苜蓿再生植株中，有三株的复叶(从左往右第二、三、四号)表现出明显的表型变化，与对照(从左往右第一号)相比，突变植株的复叶中小叶数由三个变为五个。其中，第3号突变株(也即图4中的第20号)在PCR-RE结果中出现微弱的可被BstUI限制性内切酶切割的条带，这或许是因为T0代植株为嵌合体的原因。
图6表明，从图4所筛选得到的植株中抽取一部分针对MsPALM1基因进行靶点深度测序，在MsPALM1基因上的CRISPR/Cas9的预定靶点处成功引入了突变。
以上结果表明：使用CRISPR/Cas9技术在紫花苜蓿中成功实现定点突变。
Figure PCTCN2019094630-appb-000001
Figure PCTCN2019094630-appb-000002
Figure PCTCN2019094630-appb-000003
Figure PCTCN2019094630-appb-000004
Figure PCTCN2019094630-appb-000005
Figure PCTCN2019094630-appb-000006
Figure PCTCN2019094630-appb-000007
Figure PCTCN2019094630-appb-000008
Figure PCTCN2019094630-appb-000009
Figure PCTCN2019094630-appb-000010
Figure PCTCN2019094630-appb-000011
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Figure PCTCN2019094630-appb-000013
Figure PCTCN2019094630-appb-000014

Claims (11)

  1. 一种利用CRISPR/Cas9***对紫花苜蓿基因定点突变的方法，其特征在于，所述方法包括以下步骤：
    步骤(1)构建紫花苜蓿中通用的由根癌农杆菌介导转化的双元表达载体MsCRISPR/Cas9；
    步骤(2)针对紫花苜蓿中的目标基因设计基于CRISPR/Cas9的靶标序列，将含有编码所述靶标序列的DNA片段连接到紫花苜蓿中通用的载体MsCRISPR/Cas9中构建载体MsCRISPR/Cas9::target；
    步骤(3)使用根癌农杆菌介导转化紫花苜蓿，实现对紫花苜蓿中特定基因的定点突变。
  2. 根据权利要求1所述的利用CRISPR/Cas9***对紫花苜蓿基因定点突变的方法，其特征在于，所述紫花苜蓿包括野生品种、地方品种、育成品种、引进品种。
  3. 根据权利要求1所述的利用CRISPR/Cas9***对紫花苜蓿基因定点突变的方法，其特征在于，所述目标基因包括紫花苜蓿基因组中任何有生物学功能的基因或DNA序列。
  4. 根据权利要求1所述的利用CRISPR/Cas9***对紫花苜蓿基因定点突变的方法，其特征在于，所述的在紫花苜蓿中表达CRISPR/Cas9***的双元表达载体MsCRISPR/Cas9的全序列如SEQ ID NO.1所示。
  5. 根据权利要求1所述的利用CRISPR/Cas9***对紫花苜蓿基因定点突变的方法，其特征在于，所述的在紫花苜蓿中表达CRISPR/Cas9***的双元表达载体MsCRISPR/Cas9的骨架载体是pCambia1300载体，所述的pCambia1300骨架载体上有一段用于转化植物的T-DNA区域，所述T-DNA区域包含一个左边界重复序列LB repeat、一个右边界重复序列RB repeat以及左右边界序列内部的用于转化的序列，所述的T-DNA区域左右边界内部用于转化的序列中包含表达抗植物筛选剂潮霉素的蛋白的Hpt基因表达元件、表达Cas9蛋白的表达元件、表达sgRNA的表达元件以及sgRNA表达元件内部两个Aar1酶切位点之间的靶点序列，所述Hpt基因表达元件内部包含启动Hpt基因转录的Camv 35S promoter enhanced启动子序列、表达Hpt基因的CDS序列和终止Hpt基因转录的Camv poly A终止序列，所述表达Cas9蛋白的表达元件内部包含启动Cas9序列转录 的2xCaMV 35S promoter启动子序列、表达Cas9的DNA序列和终止Cas9转录的Nos terminator终止序列，所述表达sgRNA的表达元件内部包含启动sgRNA序列转录的蒺藜苜蓿MtU6启动子序列和包含有表达sgRNA的DNA序列。
  6. 如权利要求5所述的利用CRISPR/Cas9***对紫花苜蓿基因定点突变的方法，其特征在于，所述的MtU6启动子的序列如SEQ ID NO.2所示。
  7. 如权利要求5所述的利用CRISPR/Cas9***对紫花苜蓿基因定点突变的方法，其特征在于，所述的表达sgRNA的DNA序列如SEQ ID NO.3所示，第1位到第30位的DNA序列中包含两个AarI限制性内切酶识别位点，用以针对目标基因构建定点突变的表达载体。
  8. 如权利要求1-7任一所述的利用CRISPR/Cas9***对紫花苜蓿基因定点突变的方法，其特征在于，所述表达载体MsCRISPR/Cas9::target构建方法操作如下：
    步骤(1)根据靶点序列设计一对oligo引物18-24bp,能形成如下两端带有接头的DNA片段,并合成：
    5’-T T T G N 16-23-3’
    3’-C N 16-23C A A A-5’；
    步骤(2)将上述合成的引物对进行退火反应，形成带粘性末端的互补oligo引物二聚体双链片段；
    步骤(3)使用AarI限制性内切酶酶切双元表达载体MsCRISPR/Cas9；经小牛碱性磷酸酶CIP去磷酸化反应后在0.8-1.2％的琼脂糖凝胶中进行电泳分离，回收纯化线性载体；
    步骤(4)将退火磷酸化后带粘性末端的互补DNA双链片段DNA与回收纯化的MsCRISPR/Cas9线性载体使用T4 DNA连接酶在16℃水浴锅中连接10-16h；
    步骤(5)将连接产物通过热击法转化大肠杆菌DH5ɑ感受态细胞，复苏后涂布于含50mg/L卡那霉素的LB平板培养基上，37℃培养箱培养12-16h；
    步骤(6)挑取单菌落克隆于1.5ml含50mg/L卡那霉素的LB液体培养基中，37℃摇床摇12-16h；
    步骤(7)使用测序引物MtU6-T-F对单克隆菌液进行测序，选择靶点连接正确的单克隆于50-100ml含50mg/L卡那霉素的LB液体培养基中，37℃摇床摇 12-16h，提取质粒。
  9. 如权利要求6所述的利用CRISPR/Cas9***对紫花苜蓿基因定点突变的方法，其特征在于，所述测序引物MtU6-T-F的序列如SEQ ID NO.4所示。
  10. 如权利要求1所述的利用CRISPR/Cas9***对紫花苜蓿基因定点突变的方法，其特征在于，所述使用根癌农杆菌介导转化紫花苜蓿获得突变植株的方法包含以下步骤：
    步骤(1)根癌农杆菌菌种复苏：将电击转化后的农杆菌菌株涂布于YM固体平板培养基中，25-29℃培养24h-48h；挑取单克隆，接种于30-50mLYM液体培养基中，25-29℃摇床上100-220r/min摇24h-48h；所述YM固体培养基和YM液体培养基中均包含30-60mg/L卡那霉素和200-450mg/L利福平；
    步骤(2)紫花苜蓿愈伤组织的获得：所述紫花苜蓿愈伤组织获得过程如下：
    挑选饱满、色泽圆润的紫花苜蓿种子，65-80％酒精浸泡1-3分钟，无菌水洗2-4次，每次0.5-2分钟；0.05-0.15％升汞浸泡并手摇6-12分钟，无菌水洗3～5次，接种于MS固体培养基上，光照培养箱或培养室中萌发7-16天；
    取萌发后的子叶和下胚轴，切成小块接种于愈伤诱导培养基中，25±1℃培养箱或培养室中暗培养2～4天；所述愈伤诱导培养基成分为：SH基础培养基+1.0-3.0mg/L 2,4-二氟苯氧乙酸+0.1-0.3mg/L激动素+0.1-0.5mg/L水解络蛋白+15-40g/L蔗糖+5.8-9g/L琼脂；优选地，所述愈伤诱导培养基成分为：SH基础培养基+2mg/L 2,4-二氟苯氧乙酸+0.2mg/L激动素+0.3mg/L水解络蛋白+30g/L蔗糖+8g/L琼脂；
    步骤(3)根癌农杆菌侵染紫花苜蓿愈伤组织：所述农杆菌菌株侵染紫花苜蓿愈伤组织过程如下：提前1-2天将步骤(1)复苏的根癌农杆菌菌株接种于50-100mL的YM液体培养基中，25-29℃摇床上100-220r/min培养至OD值260/280在0.5-0.8之间；将菌液转移到50mL无菌离心管中，离心机中0-8℃条件下3000-4500r/min离心10-15分钟，弃上清液，加入重悬液重悬至OD值260/280在0.5-0.8之间，加入50-150umol/L乙酰丁香酮；所述重悬液为MS基础培养基+30g/L蔗糖；将步骤(2)中诱导的愈伤组织收集到带透气塑料封口膜的无菌三角瓶中，倒入重悬后的农杆菌菌液，用三角瓶自带的封口膜封口，真空泵中抽真空到0.5kpa，一共抽0.5-1.5h；取出三角瓶，25-29℃摇床上100-150 r/min摇0.5-1.5h，倒干菌液，晾干；
    步骤(4)将被根癌农杆菌侵染过的紫花苜蓿愈伤组织进行共培养：所述共培养过程如下：将晾干的侵染过的材料接种于共培养培养基，培养基中铺一张灭菌后的滤纸，25±1℃培养箱中暗培养2-5天；所述共培养培养基成分为：MS基础培养基+1-3mg/L 2,4-二氟苯氧乙酸+0.1-0.3mg/L激动素+15-40g/L蔗糖+5.8-9g/L琼脂+50-150umol/L乙酰丁香酮；优选地，所述共培养培养基成分为：MS基础培养基+2mg/L 2,4-二氟苯氧乙酸+0.2mg/L激动素+30g/L蔗糖+8g/L琼脂+100umol/L乙酰丁香酮；
    步骤(5)紫花苜蓿愈伤组织筛选培养：所述筛选培养过程如下：将共培养得到的材料接种于筛选培养基，25±1℃培养箱或培养室中光照培养30-60天；所述筛选培养基成分为：SH基础培养基+1-3mg/L 2,4-二氟苯氧乙酸+0.1-0.3mg/L激动素+15-40g/L蔗糖+5.8-9g/L琼脂+150-450mg/L头孢噻亏+150-450mg/L羧苄青霉素+10-50mg/L潮霉素；优选地，所述筛选培养基成分为SH基础培养基+2mg/L2,4-二氟苯氧乙酸+0.2mg/L激动素+30g/L蔗糖+8g/L琼脂+250mg/L头孢噻亏+250mg/L羧苄青霉素+15mg/L潮霉素；
    步骤(6)紫花苜蓿愈伤组织分化培养：将筛选培养后的材料转接至分化培养基，25±1℃培养箱或培养室中光照培养15-30天；所述分化培养基成分为：UM基础培养基+0.5-5g/L水解络蛋白+0.1-2mg/L激动素+15-40g/L蔗糖+5.8-9g/L琼脂+150-450mg/L头孢噻亏+4-10mg/L潮霉素；优选地，所述分化培养基成分为：UM基础培养基+2g/L水解络蛋白+0.4mg/L激动素+30g/L蔗糖+8g/L琼脂+250mg/L头孢噻亏+5mg/L潮霉素；
    步骤(7)紫花苜蓿再生芽生根培养：将分化出的1-3cm的芽转接至生根培养基，培养基成分为：MS基础培养基+0.5-2mg/L吲哚丁酸+15-40g/L蔗糖+5.8-9g/L琼脂+150-450mg/L头孢噻肟；优选地，所述生根培养基成分为：MS基础培养基+1mg/L吲哚丁酸+30g/L蔗糖+8g/L琼脂+250mg/L头孢噻亏；
    步骤(8)紫花苜蓿突变株的筛选与检测，所述紫花苜蓿突变株的筛选与检测的方法包括PCR-RE检测、T7E1酶切检测与靶点深度测序。
  11. 如权利要求1-10任一权利要求所述的利用CRISPR/Cas9***对紫花苜蓿基因定点突变的方法在紫花苜蓿育种和基因编辑紫花苜蓿育种中的应用。
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