CN116716317B - Application of PSK3 gene in promotion of genetic transformation efficiency of alfalfa - Google Patents

Abstract

The invention relates to application of a PSK3 gene in promoting genetic transformation efficiency of alfalfa, belongs to the technical field of plant genetic engineering and gene editing, utilizes the advantage that the PSK3 gene can promote callus proliferation and regeneration of alfalfa, combines a PSK3-CRISPR_2.0-pRGEB31R gene editing efficient and simple carrier system, successfully carries out homozygous editing on a alfalfa PALM1 gene within 4-6 months by taking alfalfa of Gongnong No. 1 as a genetic background, and obtains multi-leaf alfalfa. The positive efficiency of the identified transgenes can reach more than 63%, wherein 79% of positive transgenic lines of PALM1 genes are edited.

Description

Application of PSK3 gene in promoting genetic transformation efficiency of alfalfa

Technical field

The invention belongs to the technical fields of plant genetic engineering and gene editing, and particularly relates to the application of PSK3 gene in promoting the genetic transformation efficiency of alfalfa.

Background technique

Alfalfa is known as the "King of Forage" and is the forage crop with the largest planting area in the world. The leaf-to-stem ratio is one of the key indicators that determines important agronomic traits such as alfalfa biomass and protein content. Cultivating varieties with more leaves can increase the leaf-to-stem ratio of alfalfa, thereby increasing its biomass and nutritional value. Cys(2)His(2) zinc finger transcription factor PALMate-likePentafoliata1 (PALM1) is involved in the initiation of alfalfa lateral leaflets and the establishment of primary leaf morphology. The loss-of-function PALM1 mutant of alfalfa exhibits a split leaf phenotype, with leaf tips gathering. 4-5 leaflets can significantly increase the leaf-to-stem ratio of alfalfa.

In addition to using conventional breeding techniques to innovate and breed alfalfa germplasm, biotechnology has also been continuously introduced into alfalfa breeding work, and important breakthroughs have been made. Among them, gene editing technology provides a new technical approach for cultivating excellent new alfalfa germplasm. However, currently most plant gene editing relies on genetic transformation technology. Therefore, how to improve the efficiency of genetic transformation has become one of the bottlenecks restricting plant gene editing. The low callus generation and regeneration efficiency of alfalfa results in its genetic transformation system being heavily dependent on varieties and genotypes. In addition, the genetic background of alfalfa is complex, the genome is large, and the autopolyploid genomes are highly similar, resulting in low or no gene editing efficiency and complex vector construction, which seriously hinders the promotion and application of gene editing technology in alfalfa genetic breeding. .

Plant callus formation and regeneration mainly involve the transformation of cell fate and the establishment of cell totipotency at the cellular level. This process is cooperatively controlled by a series of core transcription factors that regulate plant embryonic development. Heterologous overexpression of the above-mentioned core transcription factors, such as BabyBoom-WUS2, GRF4-GIF1, TaWOX5, etc., can be used as molecular auxiliary tools to promote rapid dedifferentiation of explants to form calluses or accelerate callus redifferentiation to form organs, thereby significantly reducing Requirements for explant source and genotype for gene transformation. However, the above-mentioned molecular auxiliary tools cannot achieve rapid proliferation and efficient regeneration of callus at the same time, and many molecular auxiliary tools are prone to deformity, resulting in limited application. The PSK3 gene encodes a sulfonated pentapeptide growth factor (sulfopeptin), which can simultaneously promote rapid proliferation and efficient regeneration of callus. However, previous reports mostly involved the exogenous application of sulfonated pentapeptide growth factors. This substance has not been commercialized, and its application amount fluctuates greatly with different plant species and varieties. It is unstable and has poor repeatability), and this has not been seen so far. The use of genes in rapid propagation and genetic transformation of plant tissue culture. Therefore, the patented PSK3 simultaneously promotes alfalfa callus proliferation and regeneration, and constructs a new genetic transformation auxiliary molecular tool for efficient callus proliferation and regeneration, which can be used for alfalfa gene editing and the creation of new germplasm.

Contents of the invention

In view of the above technical problems, the present invention provides an application of PSK3 gene in promoting the genetic transformation efficiency of alfalfa. The vector of the present invention is composed of a strong promoter of Arabidopsis Ubiquitin10, a constitutive strong promoter, to simultaneously activate the Cas9 module and the sgRNA module to synergistically and efficiently expression to achieve efficient editing effects, and the tRNA tandem system (using tRNA to express multiple sgRNAs in tandem) can achieve simultaneous editing of multiple target sites on the same gene or different genes. In addition, this vector also contains the Phytosulfokine3precursor (PSK3) encoding gene and the plant resistance screening gene (hph/bar/NPTⅡ). The PSK3 gene can significantly promote alfalfa callus proliferation and regeneration.

The present invention is achieved through the following technical solutions:

Application of PSK3 gene in promoting callus proliferation and regeneration of alfalfa.

The present invention also provides the application of the alfalfa gene editing vector system, which encodes the corresponding PSK3 protein and improves the efficiency of alfalfa callus occurrence and regeneration. The vector system is PSK3-CRISPR_2.0-pRGEB31R, wherein Cas9 and sgRNA is expressed from the strong promoter of Arabidopsis Ubiquitin10.

The invention also provides a gene editing vector system PSK3-CRISPR_2.0-pRGEB31R-KOPALM1, which can be used for MsPALM1 gene editing.

Furthermore, the PALM1 gene in the gene editing vector system is another gene that controls traits.

The present invention also provides a method for obtaining multi-leaf alfalfa using the PSK3-CRISPR_2.0-pRGEB31R gene editing vector system. The method includes the following steps:

(1) Obtain the alfalfa PSK3 gene sequence and design primers;

(2) Based on the obtained PSK3 gene sequence, design primers as follows:

PSK3-F:CATTTCATTTGGAGAGGACAATGAGGCTAAGTTTTATCTT

PSK3-R: TCAAGGCTTATGATGTTGTG

(3) Amplify the CaMV35Spromoter sequence from the PEG100 vector and design the primers as follows:

35S-PSK3-F:TCCCGCCTTCAGTTTTGAGACTTTTCAACAAAGGA

35S-PSK3-R：TGTCCTTCCCAAATGAAATGA

(4) Amplify the OCSterminator sequence from the PEG100 vector and design the primers as follows: OCS-PSK3-F: ACATCATAAGCCTTGACTGCTTTAATGAGATATGCG OCS-PSK3-R: AAACACTGATAGTTTCTGCTGAGCCTCGACAGTT

(5) Use high-fidelity enzymes to perform PCR amplification of three fragments of PSK3, CaMV35Spromoter and OCSterminator. The fragment sizes are 369bp, 346bp and 708bp respectively. Use the three PCR reaction products as templates to perform a second round of PCR reaction and use bridging method to The three PCR reaction products are connected into a complete PSK3 expression cassette. The connection primer sequence is as follows:

35S-PSK3-F:TCCCGCCTTCAGTTTTGAGACTTTTCAACAAAGGA OCS-PSK3-R:AAACACTGATAGTTTCTGCTGAGCCTCGACAGTT

(6) Use PmeⅠ restriction endonuclease to perform a single enzyme digestion on the CRISPR_2.0-pRGEB31R vector and cut the gel to recover it. Connect the target fragment recovered in 5) into the digested CRISPR_2.0-pRGEB31R vector through a seamless connection. , the ligation reaction product was transferred into DH5α, positive clones were identified and sequenced, the sequencing results were compared with the reported sequences, the correctly sequenced colonies were cultured and the PSK3-CRISPR_2.0-pRGEB31R vector plasmid was extracted and stored at -20°C;

(7) Obtain the alfalfa MsPALM1 gene sequence and design primers;

(8) Based on the obtained alfalfa MsPALM1 gene sequence and the CRISPR (Protospacer-adjacentMotif (PAM) is NGG) vector design principles, the four selected target sites are:

‘GGAATATTATGAACACAACA’, ‘CGGTCAGCTCAAGCTCTTGG’, ‘GGAAACGAGCACGGTCGCGG’, ‘TTTACCATTTCTTCCGTGTT’;

(9) After selecting the target site, use a high-fidelity enzyme to perform PCR amplification of the target site, tRNA and sgRNA fragments; design corresponding primers for different target sites, using the pGTR vector containing tRNA and sgRNA sequences as a template; PCR amplification and gel recovery were performed to obtain a 200bp fragment, and then the PCR reaction product was removed for gel recovery; the primer sequence is as follows:

MsPALM1-tRNA-CRISPRCas92.0-1:

TAGGTCTCAATCGAACAAAGCACCAGTG MsPALM1-tRNA-CRISPRCas92.0-2:

GCGGTCTCATCATAATATTCCTGCACCAGCCGGGAA MsPALM1-tRNA-CRISPRCas92.0-3:

TAGGTCTCAATGAACACAACAGTTTTAGAGCTAGAAA MsPALM1-tRNA-CRISPRCas92.0-4:

GCGGTCTCATTGAGCTGACCGTGCACCAGCCGGGAA MsPALM1-tRNA-CRISPRCas92.0-5:

TAGGTCTCATCAAGCTCTTGGGTTTTAGAGCTAGAAA MsPALM1-tRNA-CRISPRCas92.0-6:

GCGGTCTCAGTGCTCGTTTCCTGCACCAGCCGGGAA MsPALM1-tRNA-CRISPRCas92.0-7:

TAGGTCTCAGCACGGTCGCGGGTTTTAGAGCTAGAAA MsPALM1-tRNA-CRISPRCas92.0-8:

GCGGTCTCAAGAAATGGTAAATGCACCAGCCGGGAA MsPALM1-tRNA-CRISPRCas92.0-9:

TAGGTCTCATTCTTCCGTGTTGTTTTAGAGCTAGAAA MsPALM1-tRNA-CRISPRCas92.0-10:

TAGGTCTCAATATAAAAAAAGCACCGACTCGGTGCC;

(10) The recovered target fragment is ligated into the PSK3-CRISPR_2.0-pRGEB31R backbone vector using BsaⅠ restriction endonuclease and T4 ligase through a one-step ligation reaction. The ligation reaction product is transferred into DH5α. Positive clones are identified and sequenced. The results were compared with the reported sequences. The correctly sequenced colonies were cultured and the PSK3-CRISPR_2.0-pRGEB31R-KOPALM1 vector plasmid was extracted and stored at -20°C.

(11) Prepare the Agrobacterium infection solution, soak the alfalfa leaf pieces in the Agrobacterium infection solution to induce callus, transfer the grown callus to the differentiation medium to induce regenerative shoots, and transfer the regenerated shoots to rooting The culture medium induces the shoots to take root. The rooted seedlings grow to a height of 6-8cm and are transferred to breathable and moist soil until the alfalfa grows new leaves.

Further, step (8) CRISPR/Cas9 vector design target design principles: the target site is 20 bases and must end with -NGG-; the GC content of the target site should be between 40% and 70%, and avoid It has 4 consecutive T bases; 2-4 target sites are designed for the alfalfa PALM1 gene to improve the success rate of gene editing.

Further, the reaction system of PCR in the steps (2) (3) (4) (5) (9) is: 25 μL Phanta (2×), cDNA, 1 μL each of forward/reverse primer (10 μM), 2 μL pGTR vector, 21 μL ddH ₂ O; PCR reaction conditions are: 95℃ for 3min; 95℃ for 15s, 56℃ for 15s; 72℃ for 45s, 34 cycles; 72℃ for 10min.

Further, the ligation reaction system of step (6): 4 μL of 5×CEII buffer, 2 μL of Express II, 300 ng of carrier, 60 ng of target gel recovery fragment, and deionized water to complete the total reaction system of 20 μL. Ligation PCR program: ligation at 37°C for 30 minutes. After the program is completed, store in the refrigerator at 4°C for later use.

Further, the ligation reaction system of step (10): BsaⅠ15U, 10×BasⅠbuffer 1.5μL, T4 ligase 50U, carrier 0.5μg, target gel recovery fragment 30ng, deionized water to complete the total reaction system of 15μL; connect the PCR program : Digest at 37°C for 10 minutes, ligate at 10°C for 5 minutes, extend at 20°C for 10 minutes, 3 cycles, digest at 37°C for 10 minutes, connect at 10°C for 5 minutes, extend at 20°C for 10 minutes, 10 cycles, store in the refrigerator at 4°C for later use. .

Further, in step (10), the constructed plasmid is added to the melted Agrobacterium competent cells in an ice bath for 30 minutes, then quickly frozen in liquid nitrogen for 3 minutes, heat shocked at 37°C for 90 seconds, bathed in ice for 2 minutes, then added with LB liquid culture medium, and shaken at 28°C Incubate at 160rpm for 2-3h.

The beneficial effects of the present invention compared with the prior art:

The present invention takes advantage of the PSK3 gene that can simultaneously promote the proliferation and regeneration of alfalfa callus, combines it with the PSK3-CRISPR_2.0-pRGEB31R gene editing efficient and simple vector system, and uses Gongnong No. 1 alfalfa as the genetic background. Within 4-6 months, The alfalfa PALM1 gene was successfully edited homozygously, and multi-leaf alfalfa was obtained. It was determined that the transgenic positive efficiency could reach over 63%, and 79% of the positive transgenic lines had their PALM1 genes edited.

Description of the drawings

Figure 1 is a simplified structural diagram of the alfalfa PSK3-CRISPR_2.0-pRGEB31R vector;

Figure 2 is a phenotypic diagram of PSK3 promoting the rapid occurrence of callus in alfalfa Gongnong No. 1;

A. Phenotype of callus growth of CRISPR_2.0-pRGEB31R-KOPALM1 transgenic Gongnong No. 1 alfalfa at 30 days. B. Phenotype of callus growth of PSK3-CRISPR_2.0-pRGEB31R-KOPALM1 transgenic Gongnong No. 1 alfalfa at 30 days. Figure, C, CRISPR_2.0-pRGEB31R-KOPALM1 transgenic Gongnong No. 1 alfalfa callus growth for 45 days details, D, PSK3-CRISPR_2.0-pRGEB31R-KOPALM1 transgenic Gongnong No. 1 callus growth details for 45 days picture.

Figure 3 shows that PSK3 promotes callus regeneration of Gongnong No. 1 alfalfa;

A. CRISPR_2.0-pRGEB31R-KOPALM1 transgenic Gongnong No. 1 alfalfa callus regeneration phenotype chart, B. PSK3-CRISPR_2.0-pRGEB31R-KOPALM1 transgenic Gongnong No. 1 alfalfa callus regeneration phenotype chart, C. Statistical chart of PSK3 promoting callus regeneration efficiency of Gongnong No. 1 alfalfa.

Figure 4 is a schematic diagram of the MsPALM1 target site location and gene editing status;

A. Schematic diagram of the location of the MsPALM1 target site, B. Statistical diagram of the MsPALM1 gene editing type mutation efficiency of two homozygous edited plants;

Figure 5 shows the phenotype of MsPALM1 homozygous edited alfalfa Gongnong 1;

A. Overall picture of Gongnong No. 1 alfalfa and PALM1-KO Gongnong No. 1 alfalfa, B. Gongnong No. 1 alfalfa and PALM1-KO Gongnong No. 1 alfalfa leaves.

Detailed ways

The technical solutions of the present invention are further explained below through examples, but the protection scope of the present invention is not limited in any way by the examples.

Example 1: PSK3-CRISPR_2.0-pRGEB31R-KOPALM1 vector construction process is as follows:

(1) Obtain the PSK3 gene sequence from the whole genome sequence of alfalfa, design primers, and use alfalfa Gongnong No. 1 cDNA as a template to amplify the PSK3 sequence. The sequence information is as follows: ATGAGGCTAAGTTTTATCTTTGGAGCTCAAATCTTTTTCCTTTTCTTTCTACTCTCCTCCTCAATACTCTCTGCCAGACAACTTACCAATGAACAAGGTACGCATGCAATACTTAACTCTTCATTAGGGCCAAACTTTGTTT TGGAGTTGGAAGGAGATGAATCTTTCAAGGTGTCAGGAATGGAAGAGTGTAATATTGAAGATGAAGATTGTATGCAAAGAAGAATGACTTTAGAAGCTCACCTAGACTACATCTACACACAACATCATAAGCCTTGA;

(2) Based on the obtained PSK3 gene sequence, design primers as follows:

PSK3-F:CATTTCATTTGGAGAGGACAATGAGGCTAAGTTTTATCTT

PSK3-R: TCAAGGCTTATGATGTTGTG

(3) Amplify the CaMV35Spromoter sequence from the PEG100 vector and design the primers as follows: 35S-PSK3-F: TCCCGCCTTCAGTTTTGAGACTTTTCAACAAAGGA

35S-PSK3-R：TGTCCTCTCCAAATGAAATGA

(4) Amplify the OCSterminator sequence from the PEG100 vector and design the primers as follows: OCS-PSK3-F: ACATCATAAGCCTTGACTGCTTTAATGAGATATGCG OCS-PSK3-R: AAACACTGATAGTTTCTGCTGAGCCTCGACAGTT

(5) Use high-fidelity enzymes to perform PCR amplification of the above three fragments of PSK3, CaMV35Spromoter and OCSterminator. The reaction system is: 25μL Phanta (2×), cDNA, 1μL of forward/reverse primer (10μM), 2μL of pGTR vector, 21μL of ddH ₂ O . PCR reaction conditions were: 95°C for 3 min; 95°C for 15 s, 56°C for 15 s; 72°C for 45 s, 34 cycles; and 72°C for 10 min. PCR amplification and gel recovery were performed. The sizes of the above three fragments were 369bp, 346bp, and 708bp respectively. Then the PCR reaction products were taken out for a second round of PCR reaction. The three amplification products were connected into one fragment using bridging method. The primer sequences are as follows :

35S-PSK3-F:TCCCGCCTTCAGTTTTGAGACTTTTCAACAAAGGA OCS-PSK3-R:AAACACTGATAGTTTCTGCTGAGCCTCGACAGTT

(6) Use PmeⅠ restriction endonuclease to perform a single enzyme digestion on the CRISPR_2.0-pRGEB31R vector and cut the gel to recover it. Connect the target fragment recovered in 5) into the digested CRISPR_2.0-pRGEB31R vector through a seamless connection. , the ligation reaction product was transferred into DH5α, positive clones were identified and sequenced, the sequencing results were compared with the reported sequences, the correctly sequenced colonies were cultured and the PSK3-CRISPR_2.0-pRGEB31R vector plasmid was extracted and stored at -20°C, as shown in the figure 1 shown;

The PSK3 gene can significantly promote alfalfa callus proliferation: 30 days after screening, PSK3-CRISPR2.0-pRGEB31R-KOPALM1 transgenic calli increased significantly compared with CRISPR2.0-pRGEB31R-KOPALM1 transgenic calli. The specific phenotype is shown in Figure 2. The PSK3 gene can significantly promote alfalfa callus regeneration: the number of PSK3-CRISPR2.0-pRGEB31R-KOPALM1 transgenic calli that differentiates into bud points is significantly increased compared to that of CRISPR2.0-pRGEB31R-KOPALM1 transgenic calli. The specific phenotype is shown in Figure 3. .

Example 2 Obtaining the PSK3-CRISPR_2.0-pRGEB31R-KOPALM1 vector plasmid: Based on the obtained alfalfa MsPALM1 gene sequence and the CRISPR vector design principles, the four target sites selected were ‘GGAATATTATGAACACAACA’,

‘CGGTCAGCTCAAGCTCTTGG’, ‘GGAAACGAGCACGGTCGCGG’,

‘TTTACCATTTCTTCCGTGTT’,

It was identified that MsPALM1 was edited in 79% of the positive transgenic lines. The specific data are shown in Figure 4.

CRISPR/Cas9 vector design target design principles: the target site is 20 bases and must end with "NGG"; the GC content of the target site should be between 40% and 70%; and avoid having 4 consecutive T bases; for The alfalfa PALM1 gene is designed with four target sites to improve the success rate of gene editing.

After selecting the target site, use a high-fidelity enzyme (Phanta) to PCR amplify the fragment, design corresponding primers for different target sites (primer sequences are as follows), and use the pGTR vector containing tRNA and sgRNA sequences as a template. The reaction system is: 25 μL Phanta (2×), cDNA, 1 μL each of forward/reverse primer (10 μM), 2 μL pGTR vector, and 21 μL ddH ₂ O. PCR reaction conditions were: 95°C for 3 min; 95°C for 15 s, 56°C for 15 s; 72°C for 45 s, 34 cycles; and 72°C for 10 min. PCR amplification and gel recovery were performed to obtain a fragment of approximately 200 bp, and then the PCR reaction product was removed for gel recovery. The primer sequence is as follows: MsPALM1-tRNA-CRISPRCas92.0-1:

TAGGTCTCAATCGAACAAAGCACCAGTG MsPALM1-tRNA-CRISPRCas92.0-2:

GCGGTCTCATCATAATATTCCTGCACCAGCCGGGAA MsPALM1-tRNA-CRISPRCas92.0-3:

TAGGTCTCAATGAACACAACAGTTTTAGAGCTAGAAA MsPALM1-tRNA-CRISPRCas92.0-4:

GCGGTCTCATTGAGCTGACCGTGCACCAGCCGGGAA MsPALM1-tRNA-CRISPRCas92.0-5:

TAGGTCTCATCAAGCTCTTGGGTTTTAGAGCTAGAAA MsPALM1-tRNA-CRISPRCas92.0-6:

GCGGTCTCAGTGCTCGTTTCCTGCACCAGCCGGGAA MsPALM1-tRNA-CRISPRCas92.0-7:

TAGGTCTCAGCACGGTCGCGGGTTTTAGAGCTAGAAA MsPALM1-tRNA-CRISPRCas92.0-8:

GCGGTCTCAAGAAATGGTAAATGCACCAGCCGGGAA MsPALM1-tRNA-CRISPRCas92.0-9:

TAGGTCTCATTCTTCCGTGTTGTTTTAGAGCTAGAAA MsPALM1-tRNA-CRISPRCas92.0-10:

TAGGTCTCAATATAAAAAAAGCACCGACTCGGTGCC

(8) One-step ligation reaction system: 15 U of BsaⅠ (purchased from NEB Company), 1.5 μL of 10×BasⅠ buffer, 50 U of T4 ligase, 0.5 μg of carrier, 30 ng of target gel recovery fragment, and deionized water to complete the total reaction system of 15 μL. One-step ligation PCR program: digest at 37°C for 10 minutes, ligate at 10°C for 5 minutes, extend at 20°C for 10 minutes, 3 cycles, digest at 37°C for 10 minutes, ligate at 10°C for 5 minutes, extend at 20°C for 10 minutes, 10 cycles, 4 °C and store in the refrigerator for later use.

(9) The ligation reaction product is transferred into DH5α, and positive clones are identified and sent for sequencing.

(10) Compare the sequencing results with the reported sequences, culture the correctly sequenced colonies, extract the PSK3-CRISPR_2.0-pRGEB31R-KOPALM1 vector plasmid and store it at -20°C for subsequent experiments.

Example 3: Obtaining the homozygous edited line of alfalfa PALM1 Gongnong No. 1

(1) Collect the leaves of Jianzhuang Gongnong No. 1 alfalfa. The size of the leaves is about 5cm. If the leaves are too large or too small, they will be affected by the disinfectant. Disinfect with 7.5% sodium hypochlorite for 10 minutes. Wash with sterile water until there is no disinfectant residue. dry moisture.

(2) Prepare the infection solution in advance: transfer the PSK3-CRISPR_2.0-pRGEB31R-KOPALM1 plasmid obtained in Example 1 to EHA105 Agrobacterium under thermal stimulation, screen out positive colonies on kanamycin and rifampicin LB plates, and place them Store in -80℃ refrigerator for later use. Take out the bacterial solution and place it in Kan(50mg/L)+Rif(50mg/L) LB and shake the bacteria. Shake the bacterial solution overnight until OD ₆₀₀ = 0.6-0.8. Centrifuge at 3500rpm at 18℃ for 15min. Discard the liquid and add an equal amount of SH3a. Transformation solution, 0.1% AS (100mM), continue shaking for 2-3h. Then dilute the infection solution to 0.2-0.3, and the preparation of the infection solution is completed.

(3) Cut the leaves treated in step (1) into 1cm pieces and soak them in the prepared infection solution, while ultrasonicating for 15 minutes. Blot the bacterial solution with sterilized filter paper. Placed in co-culture SH3a+AS (SH basic medium containing final concentration of 30g·L ^-1 sucrose, 4mg·L ^-1 2,4-D, 0.5mg·L ^-1 6-BA, 100μmol·L ^-1 acetosyringone, pH 5.95) for two days.

(4) Transfer the leaves without mold and bacteria to the screening medium SH3a medium (SH basic medium, 30g·L-1 sucrose, pH value 5.95, 4g·L ^-1 plant gel, 4mg·L ^{– 1} 2,4-D, 0.5mg·L ^–1 6-BA, 400mg·L ^–1 Gentamicin, 2mg·L ^–1 bialaphos/7.5mg·L ^–1 hyg-B/15mg·L ^–1 Gentamicin, Induce callus for 1.5-2 months.

(5) Transfer the grown calli to differentiation medium MSBK (MS basic medium, 30g·L-1 sucrose, pH value 5.955, 7.8g·L ^-1 agar, 0.5mg·L ^–1 6- BA, 1mg·L ^–1 KT, 300mg·L ^–1 Gentamicin, 1.5mg·L ^{– 1} bialaphos/5mg·L ^–1 hyg-B/10mg·L ^–1 Gentamicin, induce 1.5-2 regenerated buds moon.

(6) Transfer the induced regenerated shoots to rooting medium MS ₀ (MS basic medium, 15g·L ^-1 sucrose, pH value 6, 7.8g·L ^-1 agar, 2mg·L ^-1 NAA, 300mg ·L ^-1 Temetin, induces shoots to take root for 2-3 weeks.

(7) When the rooted seedlings grow to 6-8cm high, add sterile water and open the lid to cultivate the seedlings for 7 days. Then wash the rooting medium, soak it in water with rooting powder for 30 minutes, and transfer it to a medium mixed with vermiculite. Place in moist, breathable soil, cover with plastic wrap, and wait until the alfalfa grows new leaves for 2-3 weeks. Remove the plastic wrap. The PALM1 knockout homozygous transgenic line will show a multi-leaf phenotype, with 4 clusters at the leaf tips. -5 leaflets. As shown in Figure 5.

(8) PCR identification of transgenic alfalfa, the alfalfa DNA extracted in step (7) is used as a PCR template, the reaction system is: 10 μL TapMix (2×), cDNA, 1 μL of forward/reverse primer (10 μM), 2 μL of alfalfa DNA, 6 μL of ddH ₂ O . PCR reaction conditions were: 95°C for 3 min; 95°C for 15 s, 56°C for 15 s; 72°C for 20 s, 28 cycles; and 72°C for 10 min. The PCR identification product was subjected to agarose gel electrophoresis. The hygromycin fragment was approximately 398 bp and the Cas9 fragment was approximately 547 bp. The two positive target fragments at the same time were considered transgenic plants. The primer sequences are as follows:

hph-F: AAGGAATCGGTCAATACACTACATGG

hph-R:AAGACCAATGCGGAGCATATACG

Cas9-F: AAGAACCGGATTCTGCTATCTGC

Cas9-R: GGTCGAAGTTGCTCTTGAAGTTG

(9) Select the DNA of positive alfalfa as the PCR template, and the reaction system is: 25 μL Phanta

(2×), cDNA, 1 μL each of forward/reverse primer (10 μM), 2 μL alfalfa DNA, 21 μL ddH ₂ O. PCR reaction conditions were: 95°C for 3 min; 95°C for 15 s, 56°C for 15 s; 72°C for 45 s, 34 cycles; and 72°C for 10 min. A fragment of approximately 611 bp was obtained through PCR amplification gel recovery, and then the PCR reaction product was removed for gel recovery. The primer sequences are as follows:

MsPALM1 sequencing-F:ACTTGGATGTGGAACCCTA,

MsPALM1 Sequencing-R: AGCTCAAGATCAAGCTCTTC

(10) PCR identification was performed on plants with multi-leaf phenotypes. The gel recovery products were connected to T vectors and transformed into DH5α by heat shock method. If positive identification was performed, single clone sequencing was performed. Alfalfa was an autotetraploid, and all DNA chains were edited into pure Co-editor. Three of the plants were randomly selected, numbered KO1 and KO2 respectively, and primers were designed within 100-150 bp near the editing site, and primer adapters were added. The DNA of the above three homozygous alfalfa plants was used as a template for PCR amplification, and Hi- TOM deep sequencing verification results: As shown in B in Figure 4, the proportion of editing types that insert 1 base in KO1 is 17.6%, and the proportion of editing types that delete 3 bases and insert 1 base is 52.9 %, the editing type that deletes 2 bases accounts for 6%, and the editing type that deletes 8 bases accounts for 23.5%; the editing type that deletes 4 bases and inserts 1 base accounts for KO2 17%, the editing type that deletes 9 bases and inserts 1 base accounts for 22%, the editing type that deletes 9 bases and inserts 2 bases accounts for 11%, and the editing type that deletes more than 9 bases accounts for 17%. The editing type accounts for 50%.

Claims (2)

1.PSK3Use of a gene for promoting callus proliferation and regeneration of alfalfa, characterized in that the gene comprisesPSK3The sequence information of the gene cDNA is shown as SEQ ID NO. 23; the application is thatPSK3The gene is transferred into alfalfa and is used for promoting the proliferation and regeneration of the callus.

2. The application of alfalfa genome editing carrier system is characterized in that the system encodes corresponding PSK3 protein to improve alfalfa callus generation and regeneration efficiency, and the carrier system is PSK3-CRISPR_2.0-pRGEB31R, as describedPSK3The sequence information of the gene cDNA is shown as SEQ ID NO.23, and the application is thatPSK3The gene is transferred into alfalfa for promoting the proliferation and regeneration of the callus,Cas9the sgRNA is prepared from Arabidopsis thalianaUbiquitin 10Is used for promoting expression.