Efficient transgenic method for potato tetraploid variety

Abstract

The invention discloses an efficient transgenic method for a potato tetraploid variety. The method comprises the following steps: pre-culture, agrobacterium infection and co-culture, callus induction and differentiation, and two rounds of rooting screening. According to the method, by optimizing a culture medium formula, agrobacterium tumefaciens-mediated transformation conditions and a CRISPR / Cas9 gene editing strategy, the genetic transformation efficiency and the gene editing accuracy are improved. Experimental results show that the editing efficiency of about 63% can be achieved by adopting the method, and the screening accuracy can reach 100%. The method not only accelerates the breeding process of new potato varieties, but also provides a powerful tool for functional gene research, and has wide application prospects and important economic values.

Description

Technical Field

[0001] The invention belongs to the field of plant genetic engineering and relates to a highly efficient genetic transformation and gene editing method for potato tetraploid varieties. Background Art

[0002] As the world's fourth most important food crop, potato (Solanum tuberosum L.) plays a pivotal role in global food security and agricultural economy. With the growth of population and changes in consumption patterns, the requirements for potato yield and quality are increasing. Traditional potato breeding methods mainly rely on hybridization and selection. Although these methods have improved the traits of potatoes to a certain extent, due to the genetic complexity and long life cycle of potatoes, the breeding cycle of new varieties is long and the efficiency is low.

[0003] In recent years, with the development of biotechnology, genetic engineering breeding technology has provided a new approach for potato breeding. In particular, the emergence of CRISPR / Cas9 gene editing technology has made it possible to precisely modify plant genes. This technology uses guide RNA (gRNA) and Cas9 nuclease to accurately target and edit specific genes in the plant genome, thereby achieving precise regulation of plant traits.

[0004] Although CRISPR / Cas9 technology has achieved remarkable research results in model plants, its application in potato still faces challenges. Potato is an autologous tetraploid plant with a large and complex genome, which brings additional difficulties to genetic transformation and gene editing. In addition, existing genetic transformation methods are inefficient, the transformation process is complicated, and it is often accompanied by non-target effects, leading to problems with the phenotypic and genetic stability of transgenic plants. Summary of the invention

[0005] In view of this, the present invention aims to provide a method for efficient transgenic potato tetraploid varieties. The method improves the efficiency of genetic transformation and the accuracy of gene editing by optimizing the culture medium formula, Agrobacterium-mediated genetic transformation conditions and gene editing strategy. The present invention can not only accelerate the breeding process of new potato varieties, but also provide a powerful tool for the study of potato functional genes, and is expected to play an important role in potato molecular breeding.

[0006] The inventors have continuously innovated and reformed through long-term exploration and attempts, as well as multiple experiments and efforts, to solve the above technical problems. The technical solution provided by the present invention is to provide a method for efficient transgenic potato tetraploid varieties, comprising the following steps:

[0007] S1. Pre-culture: Take sterile potato seedlings with a growth period of 2 to 4 weeks, cut stem segments with a length of 0.8 to 1.5 cm without axillary buds and healthy and fully developed leaves without petioles as explants, inoculate them in pre-culture / co-culture medium and pre-culture them in the dark for 1 to 3 days;

[0008] S2. Agrobacterium infection and co-cultivation: place the stem segments and leaves pre-cultured for 1 to 3 days in a centrifuge tube containing Agrobacterium culture solution of OD600=0.5 and infect for 6 to 10 minutes, slowly turning it upside down during the infection. After the infection is completed, pour out the culture solution, dry the excess culture solution on the explants, transfer the explants to the pre-culture / co-culture medium, and co-cultivate at 22°C in the dark for 1 to 3 days;

[0009] S3. Callus induction: transfer the explants co-cultured for 1 to 3 days to callus induction medium and culture them in the dark at 22°C for 10 to 15 days;

[0010] S4. Callus differentiation: transfer the explants after callus induction for 10 to 15 days to callus differentiation medium to induce callus differentiation, and replace the medium every 10 to 15 days;

[0011] S5. After two rounds of rooting screening, the 1.5-2 cm shoots differentiated from the callus tissue are cut off and inserted into the rooting screening medium. After the taproot grows for the first time, the terminal bud is cut again and inserted into a new rooting screening medium to induce rooting. If taproots grow in two rounds of rooting screening, it is a transgenic positive plant.

[0012] Preferably, the method comprises the following steps:

[0013] S1. Pre-culture: Take sterile potato seedlings with a growth period of 4 weeks, cut 1 cm long stem segments without axillary buds and healthy and fully developed leaves without petioles as explants and inoculate them in pre-culture / co-culture medium for pre-culture in the dark for 2 days;

[0014] S2. Agrobacterium infection and co-cultivation: The stem segments and leaves pre-cultured for 2 days were placed in a centrifuge tube containing Agrobacterium culture solution with OD600 = 0.5 and infected for 8 minutes, and slowly inverted during the infection. After the infection was completed, the culture solution was poured out, the excess culture solution on the explants was dried, and the explants were transferred to the pre-culture / co-culture medium and co-cultured for 2 days at 22°C in the dark;

[0015] S3. Callus induction: the explants co-cultured for 2 days were transferred to callus induction medium and cultured in the dark at 22°C for 12 days;

[0016] S4. Callus differentiation: transfer the explants after callus induction for 12 days to callus differentiation medium to induce callus differentiation, and replace the medium every 14 days;

[0017] S5. After two rounds of rooting screening, the 1.5-2 cm shoots differentiated from the callus tissue are cut off and inserted into the rooting screening medium. After the taproot grows for the first time, the terminal bud is cut again and inserted into a new rooting screening medium to induce rooting. If taproots grow in two rounds of rooting screening, it is a transgenic positive plant.

[0018] Compared with the prior art, the present invention has the following beneficial effects:

[0019] The present invention shows significant improvements in improving transgenic efficiency, reducing costs, enhancing screening accuracy and environmental friendliness, and provides a new and efficient technical solution for the genetic improvement of potatoes and other crops. The present invention significantly improves the genetic transformation efficiency of potato tetraploid varieties by optimizing conditions such as pre-culture, Agrobacterium infection and co-culture, shortens the acquisition cycle of transgenic plants, and thus improves the efficiency of breeding work. The two-round rooting screening method adopted by the present invention can effectively identify transgenic positive plants. This method is simple, efficient and accurate, and the screening accuracy rate can reach 100%, which greatly reduces the generation of false positives and improves the reliability of screening. In addition, the method of the present invention reduces the experimental cost, saves experimental resources by reducing the amount of culture medium used and simplifying the operating steps, and reduces the use of reagents that may cause harm to the human body during the experiment.

[0020] On the basis of the above technical solution, the present invention can also be improved as follows:

[0021] Further:

[0022] The pre / co-culture medium consisted of the following components: MS basal salts, 20 g / L sucrose, 1 mL / L 1000x N&N vitamins, 1.0 mg / L TZR, 0.0278 mg / L GA3, 0.02 mg / L NAA, and 2.0 g / L Phytagel.

[0023] Compared with the prior art, the beneficial effects of adopting the above further technical solution are:

[0024] By accurately proportioning ingredients such as MS basal salts, sucrose, N&N vitamins, TZR, GA3, NAA and Phytagel, the pre- / co-culture medium can better meet the nutritional and growth requirements of potato sterile seedlings in the pre-culture stage, thereby improving the survival rate and growth quality of explants. This optimized culture medium formula can promote the rapid growth of explants and the efficient induction of callus tissue, laying a good foundation for subsequent Agrobacterium infection and transgenic processes. In addition, the precise ratio of culture medium components also helps to reduce the variability between experiments, improve the repeatability of experiments and the reliability of results. Therefore, this improvement not only improves the efficiency of genetic transformation and the quality of transgenic plants, but also helps to shorten the breeding cycle, reduce costs, and further promote the development of potato genetic improvement and gene editing technology.

[0025] On the basis of the above technical solution, the present invention can also be improved as follows:

[0026] Further:

[0027] The callus induction medium consists of the following components: MS basal salts, 20 g / L sucrose, 1 mL / L 1000x N&N vitamins, 1.0 mg / L TZR, 0.0278 mg / L GA3, 0.02 mg / L NAA, 2.0 g / L Phytagel and 200 mg / L Tim.

[0028] Compared with the prior art, the beneficial effects of adopting the above further technical solution are:

[0029] By adding 200 mg / L of Tim (Timentin) to MS basic salts, sucrose, N&N vitamins, TZR, GA3, NAA and Phytagel, this improved culture medium can more effectively promote the formation of callus tissue of potato explants. As a broad-spectrum antibiotic, Timentin can effectively inhibit bacterial contamination and improve the success rate of callus induction. In addition, the optimized formula of this culture medium helps to improve the regeneration ability of explants and reduce the risk of contamination during the culture process, thereby improving the efficiency of genetic transformation and the stability of transgenic plants. Therefore, this improvement not only improves the efficiency and quality of the callus induction stage, but also helps to optimize the entire genetic transformation process, providing a more efficient and reliable technical solution for potato gene editing and genetic improvement.

[0030] On the basis of the above technical solution, the present invention can also be improved as follows:

[0031] Further:

[0032] The callus differentiation medium consists of the following components: MS basal salts, 20 g / L sucrose, 1 mL / L 1000x N&N vitamins, 2.0 mg / L TZR, 10 mg / L GA3, 2.0 g / L Phytagel, 50 mg / L Kan, 200 mg / L Tim and 200 mg / L Cef.

[0033] Compared with the prior art, the beneficial effects of adopting the above further technical solution are:

[0034] By accurately proportioning MS basic salts, sucrose, N&N vitamins, TZR, GA3, Phytagel, and antibiotics Kan, Tim, and Cef, the culture medium not only provides a suitable nutrition and hormone environment, promotes the differentiation of callus tissue into regenerated plants, but also effectively inhibits bacterial contamination, improves the stability of the culture process and the success rate of transgenic plants. In particular, the use of Kan (kanamycin) provides an effective means for screening transgenic plants containing resistance genes. The optimized formula of this culture medium helps to improve differentiation efficiency and reduce variation during the culture process, thereby improving the efficiency of genetic transformation and the quality of transgenic plants, and provides a more efficient and reliable technical solution for potato genetic improvement and gene function research.

[0035] On the basis of the above technical solution, the present invention can also be improved as follows:

[0036] Further:

[0037] The rooting screening medium consists of the following components: 4.74 g / L MS medium without agar and sucrose, 30 g / L sucrose, 2.0 g / L Phytagel, 50 mg / L Kan and 200 mg / L Tim.

[0038] Compared with the prior art, the beneficial effects of adopting the above further technical solution are:

[0039] The culture medium can provide a more suitable environment for the rooting and screening of transgenic positive plants. This optimized culture medium not only promotes the rooting process of the plants, but also effectively screens out transgenic positive plants through the selection pressure of the antibiotic Kan. At the same time, the use of Tim further inhibits possible bacterial contamination. Such improvements improve the rooting efficiency and the accuracy of transgenic plant screening, reduce the false positive rate, thereby improving the efficiency and reliability of the entire genetic transformation process, and providing a more accurate and efficient workflow for potato genetic improvement and gene editing research.

[0040] On the basis of the above technical solution, the present invention can also be improved as follows:

[0041] Further:

[0042] The method includes using the gRNA sequences specified by SEQ ID NO.1 and SEQ ID NO.2 to guide the Cas9 protease to recognize and cut the upstream cis-regulatory element T#01 of the potato StCDF1 gene.

[0043] Compared with the prior art, the beneficial effects of adopting the above further technical solution are:

[0044] By using a specific gRNA sequence, the present invention can accurately guide the Cas9 protease to recognize and cut the cis-regulatory element T#01 upstream of the potato StCDF1 gene. The selection of this specific target site helps to achieve fine regulation of the expression of potato tuberization-related genes. This precise gene editing method not only improves the success rate of gene editing, but also reduces the risk of off-target effects, thereby achieving effective improvement of specific traits while maintaining the stability of other traits of the plant. In addition, the application of this method can accelerate the breeding process of new potato varieties, improve the accuracy and efficiency of breeding, and has important practical significance for the genetic improvement and agricultural production of potatoes.

[0045] On the basis of the above technical solution, the present invention can also be improved as follows:

[0046] Further:

[0047] The method further comprises constructing a CRISPR / Cas9 knockout vector before step S1, wherein the vector comprises the gRNA sequences specified by SEQ ID NO.1 and SEQ ID NO.2.

[0048] Compared with the prior art, the beneficial effects of adopting the above further technical solution are:

[0049] Construct a CRISPR / Cas9 knockout vector containing a specific gRNA sequence in advance to improve the targeting and efficiency of gene editing and ensure the accuracy and success rate of subsequent genetic transformation steps.

[0050] On the basis of the above technical solution, the present invention can also be improved as follows:

[0051] Further:

[0052] The method further comprises propagating sterile test tube potato seedlings before step S1, cutting sterile virus-free potato stem segments with axillary buds as explants, and inserting the explants into MS solid culture medium.

[0053] Compared with the prior art, the beneficial effects of adopting the above further technical solution are:

[0054] By pre-propagating sterile test tube seedlings and using stem segments with axillary buds as explants, the quality and consistency of the explants can be ensured, thereby improving the success rate and efficiency of genetic transformation.

[0055] On the basis of the above technical solution, the present invention can also be improved as follows:

[0056] Further:

[0057] The method further comprises preparing Agrobacterium bacterial liquid before step S2, taking Agrobacterium EHA105 bacterial liquid containing plant knockout vector pJCV55-StU6-200-StUBI10-T#01, and streaking the liquid on a LB solid medium containing 50 mg / L Spec and 20 mg / L Rif.

[0058] Compared with the prior art, the beneficial effects of adopting the above further technical solution are:

[0059] By preparing the Agrobacterium culture containing a specific knockout vector in advance and streaking it on a medium containing antibiotics, the stability and transformation ability of the Agrobacterium can be ensured, thereby improving the efficiency of subsequent infection of explants. In addition, this method helps to screen out Agrobacterium containing the target gene editing vector, reduce the interference of non-target strains, and further improve the accuracy of genetic transformation and the quality of transgenic plants.

[0060] On the basis of the above technical solution, the present invention can also be improved as follows:

[0061] Further:

[0062] The method further includes detecting mutations by Sanger sequencing and Hi-TOM sequencing after step S5, extracting genomic DNA of transgenic positive seedlings by CTAB method, and performing PCR amplification and sequencing, wherein the primer sequence of PCR amplification target fragment T#01 in Sanger sequencing is:

[0063] The forward primer T01-F is shown in SEQ ID NO.7, and the reverse primer T01-R is shown in SEQ ID NO.8;

[0064] The primer sequence for PCR amplification of target fragment T#01 in Hi-TOM sequencing is:

[0065] The forward primer Hi-F is shown as SEQ ID NO.9, and the reverse primer Hi-R is shown as SEQ ID NO.10.

[0066] Compared with the prior art, the beneficial effects of adopting the above further technical solution are:

[0067] By combining Sanger sequencing and Hi-TOM sequencing technology, accurate mutation detection can be performed on the genomic DNA of transgenic positive seedlings. This combined method uses the high accuracy of Sanger sequencing to preliminarily determine the mutation type and site, and then obtains more detailed mutation information through the high resolution of Hi-TOM sequencing, thereby ensuring the accuracy and comprehensiveness of mutation detection. The CTAB method is a fast and economical method for extracting genomic DNA, which is suitable for processing large-scale samples. In addition, by PCR amplification of the target fragment T#01 and using specific primer sequences, the CRISPR / Cas9 editing effect can be detected in a targeted manner, improving the efficiency and reliability of mutation detection. The improvement of this method not only enhances the confirmation of the gene editing status of transgenic plants, but also provides a solid molecular biological foundation for subsequent genetic analysis and breeding decisions. BRIEF DESCRIPTION OF THE DRAWINGS

[0068] In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings required for use in the embodiments will be briefly introduced below. It should be understood that the following drawings only show certain embodiments of the present invention and therefore should not be regarded as limiting the scope. For ordinary technicians in this field, other related drawings can be obtained based on these drawings without paying creative work.

[0069] Figure 1 This is the pre-culture status of explants of 'Chuanyu 50'.

[0070] Figure 2 This is the callus induction of 'Chuanyu 50' explants.

[0071] Figure 3 This is the callus differentiation of 'Chuanyu 50' explants.

[0072] Figure 4 This is a schematic diagram of the target sequence T#01 position, gRNA position, and CRISPR / Cas9 vector construction.

[0073] Figure 5 This is the PCR test result of the regenerated seedlings.

[0074] Figure 6 This is the peak diagram of the first generation sequencing of transgenic positive plants.

[0075] Figure 7 This is a schematic diagram of target site editing in mutant plants. DETAILED DESCRIPTION

[0076] The following describes the invention in conjunction with specific embodiments.

[0077] In order to make the purpose, technical solutions and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below in combination with the embodiments of the present invention. Obviously, the described embodiments are part of the embodiments of the present invention, rather than all the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention. Therefore, the following detailed description of the embodiments of the present invention is not intended to limit the scope of the invention claimed for protection, but merely represents selected embodiments of the present invention.

[0078] In the present invention, unless otherwise specified, all equipment and raw materials can be purchased from the market or are commonly used in the industry. The methods in the following embodiments, unless otherwise specified, are conventional methods in the art.

[0079] In this example, the sterile test tube seedlings of potato variety 'Chuanyu 50' provided by the Crop Research Institute of Sichuan Academy of Agricultural Sciences and propagated and preserved by the Plant Epigenome and Bioinformatics Laboratory of the School of Life Sciences of Sichuan Normal University were used as materials.

[0080] Potato sterile test tube seedling propagation:

[0081] Cut the stem segment (about 1 cm long) of sterile virus-free potato 'Chuanyu 50' with one axillary bud as explant, insert its morphological lower end into MS solid culture medium (11.74 g / L MS culture medium containing agar but not sucrose + 30 g / L sucrose, pH 5.8), and inoculate 7 stem segments in each culture bottle. Place the culture bottle in the tissue culture room for culture, and the culture conditions are 2000 lx light intensity, 22 ° C temperature, and 16h / 8h (light / dark) light cycle. After the seedlings are 4 weeks old, carry out propagation culture in the same way as above.

[0082] 1. Screening the optimal culture medium system for potato genetic transformation and gene editing

[0083] The propagated and preserved sterile tissue culture seedlings were subjected to potato explant regeneration and transformation under four culture medium systems with different hormone ratios, namely A, B, C, and D (see Table 1), and the callus induction rate and callus differentiation rate were compared to select the optimal culture medium system.

[0084] The pH value of the culture medium in Table 1 is 5.7.

[0085] Table 1 Culture medium

[0086]

[0087] Take sterile potato seedlings with a growth period of 4 weeks, cut a 1 cm long stem segment without axillary buds and a healthy and fully developed leaf without petiole (cut perpendicular to the main vein to form an incision, and the leaf is inverted) as explants and inoculate them in the pre- / co-culture medium for pre-culture in the dark for 2 days. Figure 1 As shown in the figure, except for the browning of some stem segments and leaves in A1, most of the stem segments and leaves in A1 and BS / BL pre-culture mediums have begun to swell and differentiate into callus tissue. However, less than 20% of the stem segments and leaves in C1 and D culture mediums differentiated into callus tissue, and a large number of leaves turned brown. Therefore, among all the pre-culture mediums tested, BS / BL is the best pre-culture medium for 'Chuanyu 50'. It should be noted that in this example, BS is the culture medium for stem segment culture, and BL is the culture medium for leaf culture.

[0088] The explants co-cultured for 2 days were transferred to A1, BS / BL, C1 and D+200 mg / L Tim medium and cultured in the dark at 22°C for 12 days to induce callus formation. The callus induction rate was calculated after 12 days.

[0089] Callus induction rate = (number of callus formation / total number of explants) × 100%.

[0090] Depend on Figure 2 As shown in Table 2, in the A1+200mg / L Tim callus induction medium, the callus induction rates of stem segments and leaves were 90% and 80%, respectively (Table 2). In the BS / BL+200mg / L Tim callus induction medium, the callus induction rates of stem segments and leaves were 93% and 88%, respectively (Table 2); in the C1+200mg / L Tim and D+200mg / L Tim callus induction medium, the callus differentiation rates of stem segments were 65% and 43%, respectively, and the callus differentiation rates of leaves were 58% and 62%, respectively. The comprehensive results showed that the best callus induction medium for 'Chuanyu 50' was BS / BL.

[0091] The explants after callus induction for 12 days were transferred to callus differentiation medium to induce callus differentiation. The medium was replaced every 14 days, and the callus differentiation rate was counted.

[0092] Callus differentiation rate = (number of differentiated calli / number of calli) × 100%.

[0093] In the A2+50mg / L Kan+200mg / L Tim+200mg / L Cef differentiation medium, the differentiation rate of stem callus was 9% (Table 2), while no differentiated buds were observed in leaf callus ( Figure 3); In the differentiation medium of B2+50mg / L Kan+200mg / L Tim+200mg / L Cef, both stem segment and leaf callus differentiated into shoots ( Figure 3 ), the callus differentiation rates were 25% and 22%, respectively (Table 2). Since most of the stem segments and leaves turned brown during the callus induction stage, the calli in the differentiation mediums C2+50mg / L Kan+200mg / L Tim+200mg / L Cef and D+50mg / L Kan+200mg / L Tim+200mg / L Cef did not differentiate ( Figure 3 , Table 2). Therefore, B2+50mg / L Kan+200mg / L Tim+200mg / L Cef differentiation medium was selected as the best differentiation medium for 'Chuanyu 50'.

[0094] Table 2 Effects of different culture media on callus formation and differentiation in stem segments and leaves

[0095]

[0096]

[0097] Experimental data show that the components of the culture medium are important factors affecting the induction rate and differentiation rate of stem segments and leaves. Different hormone ratios and additional ingredients (such as TZR, GA3, NAA, IAA, etc.) have a significant effect on the growth and differentiation of plant tissues. For example, TZR (thiadiazuron), as a highly efficient cytokinin, can promote the induction of callus tissue; while auxins such as IAA (indoleacetic acid) and NAA (naphthaleneacetic acid) affect tissue differentiation. The combination of different culture medium components leads to different induction and differentiation rates, showing the direct influence of culture medium components on the induction and differentiation process of plant tissues.

[0098] Changes in hormone concentrations had a significant effect on the induction and differentiation rates of stem segments and leaves. Adjustment of the concentrations of hormones such as TZR and GA3 played a key role in the induction and differentiation of callus tissue. For example, when a higher concentration of TZR (such as 4.78 mg / L) was combined with a lower concentration of GA3 (0.2 mg / L), the induction rate of stem segments decreased (0.43), while the differentiation rate was also affected. This shows that the optimization of hormone concentrations is crucial to improving the induction and differentiation rates.

[0099] The physiological state and hereditary factors of explant are also important factors affecting induction rate and differentiation rate. In the present embodiment, the growth cycle of use is the sterile seedling of 4 weeks as explant, and its physiological state is relatively consistent, which helps the stability of experimental result. Different types of plants react differently to medium components and hormone concentrations because of the difference of their hereditary factors, which also explains why it is necessary to optimize the culture medium system for specific plants and their varieties.

[0100] By optimizing these factors, a targeted nutritional and hormonal environment is provided according to the biological characteristics and needs of stem segments and leaves, which helps to balance cell division and differentiation, thereby increasing the induction rate and differentiation rate, and can significantly improve the genetic transformation and gene editing efficiency of potato tetraploid varieties.

[0101] 2. Vector construction for potato genetic transformation and gene editing

[0102] The upstream cis-regulatory element T#01 of the potato StCDF1 gene was used as the target sequence, and gRNA was designed using the CRISPR-GE website (http: / / skl.scau.edu.cn / ). The gRNA fragment was inserted into the pJCV55-StU6-200-StUBI10 vector containing Cas9 by PCR and Golden Gate cloning ( Figure 4 ), transformed into Escherichia coli Trans 5α, screened and obtained Escherichia coli containing the recombinant vector pJCV55-StU6-200-StUBI10-T#01, extracted the plasmid of Escherichia coli, transformed into Agrobacterium EHA105, used Agrobacterium-mediated method to transform the target plant, screened the transgenic positive plants, and identified the mutants.

[0103] 2.1 Target fragment acquisition

[0104] Based on gRNA1 (as shown in SEQ ID NO.1) and gRNA2 (as shown in SEQ ID NO.2), adapters are added to both ends to obtain forward primer gRNA1-F (as shown in SEQ ID NO.3) and reverse primer gRNA2-R (as shown in SEQ ID NO.4). gRNA1 and gRNA2 play a vital role in potato genetic transformation and gene editing. As guide RNAs of the CRISPR / Cas9 system, they can accurately guide the Cas9 enzyme to a specific target sequence in the potato genome to achieve precise editing of the target gene. The complementary pairing regions of gRNA1 and gRNA2 specifically bind to the target DNA sequence, triggering the nucleic acid cleavage activity of the Cas9 enzyme, thereby generating double-strand breaks at the predetermined gene site. These breaks can then be used by the cell's repair mechanism to introduce insertion or deletion mutations, or replace specific gene sequences through non-homologous end joining (NHEJ) or homology-directed repair (HDR) pathways. Since the complementary pairing region of each gRNA must completely match the specific sequence of the target gene to ensure the accuracy of editing, gRNA1 and gRNA2 are highly specific and irreplaceable, and their design and selection are crucial to achieving effective gene editing.

[0105] PCR amplification was performed using GTR plasmid as a template and gRNA1-F and gRNA2-R as primers. The amplification reaction system was: 10xEx buffer 2μL, dNTP 1.6μL, Ex Taq 0.1μL (negligible), DNA 1μL, T01-F 1μL (10μM), T01-R 1μL (10μM), and ddH2O 13.4μL.

[0106] The reaction conditions were as follows: pre-denaturation at 95°C for 3 min; denaturation at 95°C for 30 s, annealing at 66°C for 30 s, extension at 72°C for 14 s, 35 cycles; extension at 72°C for 5 min, and storage at 4°C.

[0107] The product obtained by PCR amplification was detected by agarose gel electrophoresis. If it met the target size, the target fragment was cut out and recovered according to the steps of the agarose gel kit and stored at -20°C for future use.

[0108] 2.2 Carrier connection

[0109] The obtained target fragment was mixed with the plasmid containing the pJCV55-StU6-200-StUBI10 vector in a certain ratio, and the restriction enzyme digestion and ligation were performed according to the following reaction system: 2 μL of plasmid, 2 μL of target fragment, 1.5 μL of rCutSmart buffer, 1 μL of BsaⅠ, 1 μL of T4 Ligase, 7.5 μL of ddH2O, and gently mixed. The reaction was carried out at 37°C for 30 minutes, 50°C for 5 minutes, and 80°C for 10 minutes. After the reaction, it was placed on ice to cool for a few seconds, and then the restriction enzyme digestion and ligation products could be stored at -20°C for later use or directly used for transformation.

[0110] 2.3 E. coli transformation and colony PCR verification

[0111] Take out the competent E. coli cells stored at -80°C, place them on ice to thaw, dispense 25 μL of competent cells into each tube, add 1-2 μL of the recombinant product obtained according to (2), mix well, place on ice for 30 min, heat shock at 42°C for 45 s, place on ice for 2 min, add 250 μL of pure LB medium, culture at 37°C, 200 rpm, shake for 1 h, draw the bacterial solution (10-100 μL) and spread it on the LB medium containing spectinomycin, culture at 37°C overnight. Pick a single colony and perform colony PCR verification. The reaction system is as follows: 2×PCR mix 5 μL, forward primer (as shown in SEQ ID NO.5) 0.5 μL, reverse primer (as shown in SEQ ID NO.6) 0.5 μL, template volume is not counted, ddH 2 O was filled to 10 μL, and the PCR reaction program was set according to the reaction program for obtaining the target fragment. After the amplification was completed, agarose gel electrophoresis was performed to observe the results; the colonies verified by colony PCR were picked into liquid LB medium containing spectinomycin, and cultured overnight at 37°C and 200 rpm. After the bacteria were expanded, they were sent to the company for sequencing for further verification; the bacterial solution verified by sequencing was used to extract plasmids according to the steps of the plasmid extraction kit of OMEGA Biological Reagent Company in the United States. The plasmids can be stored at -20°C for standby use or directly used for transformation.

[0112] 2.4 Agrobacterium transformation

[0113] Take out the competent Agrobacterium cells stored at -80°C, place them on ice to melt, and dispense 25 μL competent cells into each tube, add 1-2 μL of the plasmid constructed in step (3), mix well, place in an ice bath for 5 minutes, place in liquid nitrogen for 5 minutes, place in a 28°C water bath for 5 minutes, place in an ice bath for 5 minutes, add 700 μL pure LB liquid culture medium, and culture in a temperature-controlled shaker at 28°C and 200 rpm for 2-3 hours. Collect the bacterial solution at 6000 rpm for 1 minute, draw 10-100 μL of the bacterial solution and spread it on the LB culture medium containing the corresponding antibiotics, culture at 28°C for 2 days, pick a single colony for colony PCR verification (the reaction system and procedure are the same as those for Escherichia coli PCR verification). Place the colony verified by colony PCR into the liquid LB culture medium containing the corresponding antibiotics, and culture overnight at 28°C and 200 rpm. Mix the bacterial solution with glycerol in a volume ratio of 1:1 and store at -80°C for later use.

[0114] 3. Potato genetic transformation and gene editing

[0115] The constructed CRISPR / Cas9 vector was introduced into potato 'Chuanyu 50' via Agrobacterium, and transgenic positive plants were screened and mutants were identified.

[0116] 3.1 Take 1 μL of Agrobacterium EHA105 culture medium containing the plant knockout vector pJCV55-StU6-200-StUBI10-T#01 (targeting the T#01 sequence in the core promoter region upstream of the potato StCDF1 gene, driven by the potato U6 snoRNA promoter, and using Kan as a selection marker), streak it on a solid LB medium containing 50 mg / L Spec and 20 mg / L Rif, and culture it in the dark at 28°C for 2 days. Pick a single colony and inoculate it in 10 mL of liquid LB medium containing 50 mg / L Spec and 20 mg / L Rif, at 28°C, 200 rpm, overnight until the logarithmic phase of growth, and take 0.5 mL of the culture medium and further culture it in 20 mL of liquid LB medium containing 50 mg / L Spec and 20 mg / L Rif at 28°C until OD600 = 0.5. The cultured Agrobacterium culture liquid was centrifuged at 4800 rpm for 6 min, the bacterial precipitate was collected, and the bacterial cells were resuspended in 15 mL MS liquid culture medium (4.33 g / L MS basal salt (PhytoNO.524) + 30 g / L sucrose) for subsequent explant infection.

[0117] 3.2 Take sterile potato seedlings with a growth period of 4 weeks, cut 1 cm long stem segments without axillary buds and healthy and fully developed leaves without petioles as explants and inoculate them in pre / co-culture medium for pre-culture in the dark for 2 days.

[0118] The pre / co-culture medium consisted of the following components: MS basal salts, 20 g / L sucrose, 1 mL / L 1000xN&N vitamins, 1.0 mg / L TZR, 0.0278 mg / L GA3, 0.02 mg / L NAA, and 2.0 g / L Hytagel.

[0119] 3.3 Place the stem segments and leaves pre-cultured for 2 days in a 50 mL centrifuge tube containing OD600 = 0.5 Agrobacterium culture solution and infect for 8 minutes, slowly turning it upside down during the infection. After the infection is completed, pour out the culture solution and place the explants on sterilized filter paper to absorb the excess culture solution. Transfer the explants to the pre-culture / co-culture medium and co-culture for 2 days at 22°C in the dark.

[0120] 3.4 Transfer the explants after 2 days of co-culture to callus induction medium and culture them in the dark at 22°C for 12 days.

[0121] Callus induction medium consisted of the following components: MS basal salts, 20 g / L sucrose, 1 mL / L 1000x N&N vitamins, 1.0 mg / L TZR, 0.0278 mg / L GA3, 0.02 mg / L NAA, 2.0 g / L Phytagel, and 200 mg / L Tim.

[0122] 3.5 Transfer the explants after callus induction for 12 days to callus differentiation medium to induce callus differentiation, and replace the medium every 14 days.

[0123] Callus differentiation medium consisted of the following components: MS basal salts, 20 g / L sucrose, 1 mL / L 1000x N&N vitamins, 2.0 mg / L TZR, 10 mg / L GA3, 2.0 g / L Phytagel, 50 mg / L Kan, 200 mg / L Tim, and 200 mg / L Cef.

[0124] 3.6 When the buds grew to about 1.5-2 cm, they were cut off and transferred to the rooting screening medium with the corresponding screening resistance for two rounds of rooting screening. A total of 40 second-round regenerated seedlings were screened from 63 regenerated seedlings.

[0125] The rooting screening medium consisted of the following components: 4.74 g / L MS medium without agar and sucrose, 30 g / L sucrose, 2.0 g / L Phytagel, 50 mg / L Kan and 200 mg / L Tim.

[0126] 3.7 Extract the DNA of the second-round potato regeneration seedlings and perform PCR verification. Figure 5 As shown, the gel electrophoresis results showed that the band size was correct, indicating that all 40 second-round regenerated seedlings were transgenic positive, proving that the secondary rooting screening method is accurate and effective.

[0127] 3.8 Two groups of PCR amplifications were performed using the DNA extracted in step 3.7 as a template: the first group performed PCR amplification on 40 transgenic positive plants of 'Chuanyu 50' and the wild type of 'Chuanyu 50', and then purified the PCR products by agarose gel electrophoresis as samples for Sanger sequencing to preliminarily detect the mutation type and site; the second group used #1, #12 and #19 and the wild type of 'Chuanyu 50' as examples, and the PCR products were directly sent for Hi-TOM sequencing without purification after the PCR amplification reaction to obtain more detailed mutation information.

[0128] The primer sequences for PCR amplification of target fragment T#01 in Sanger sequencing are: forward primer T01-F (as shown in SEQ ID NO.7), reverse primer T01-R (as shown in SEQ ID NO.8). The PCR amplification reaction system is: 10x Ex buffer 2μL, dNTP 1.6μL, Ex Taq 0.1μL, DNA 1μL, T01-F 1μL (10μM), T01-R 1μL (10μM), ddH2O 13.4μL. The reaction conditions are: 95℃ pre-denaturation for 3min; 95℃ denaturation for 30s, 55℃ annealing for 30s, 72℃ extension for 50s, 35 cycles; 72℃ extension for 5min, 4℃ storage.

[0129] like Figure 6 As shown, compared with the wild-type sequencing peak graph of 'Chuanyu 50', all plants showed chaotic peaks near the two target sites, indicating that the target DNA sequences of these plants have changed in these areas. The appearance of chaotic peaks is usually due to the presence of polymorphism or variation in the DNA sequence, which causes the sequencing results to appear in these areas with a standard peak pattern different from that of the wild type, which means that the CRISPR / Cas9 system has successfully introduced mutations at the target site. Among the 63 regenerated seedlings, 40 showed editing positive, that is, mutations occurred at the target site, and the editing efficiency was about 63%. The ability to edit specific genes with high efficiency is of great significance for crop improvement and gene function research. In addition, this also verifies the accuracy and efficiency of the secondary rooting screening method in screening transgenic positive plants.

[0130] The primer sequences for PCR amplification of target fragment T#01 in Hi-TOM sequencing are: forward primer Hi-F (as shown in SEQ ID NO.9), reverse primer Hi-R (as shown in SEQ ID NO.10). The PCR amplification reaction system is: 10x Ex buffer 2μL, dNTP 1.6μL, Ex Taq 0.1μL, DNA 1μL, Hi-F 1μL (10μM), Hi-R 1μL (10μM), ddH2O 13.4μL. The reaction conditions are: 95℃ pre-denaturation for 3min; 95℃ denaturation for 30s, 62℃ annealing for 30s, 72℃ extension for 12s, 35 cycles; 72℃ extension for 5min, 4℃ storage.

[0131] like Figure 7 As shown, in #1, the wild type accounted for 68.34%, the mutation accounted for 31.66%, and the mutation type was mainly deletion, of which -2bp / no mutation (gRNA1 mutation type / gRNA2 mutation type) mutation type accounted for 28.95%. Insertion and deletion mutations appeared in #12, accounting for 100% in total, of which -33bp / +1bp mutation type accounted for the highest proportion, at 32.68%. In #19, insertion and deletion accounted for 97.8% in total, the wild type accounted for only 2.2%, and the no mutation / -1bp mutation type accounted for as high as 40.4%. These results show that the mutation types and frequencies caused by the CRISPR / Cas9 system in different plants in this embodiment are different, which is related to multiple factors such as the design of gRNA, the delivery efficiency of the CRISPR / Cas9 system, and the repair mechanism of the plant. Hi-TOM sequencing provides detailed mutation information, which is helpful for researchers to understand the specific effects of gene editing and optimize gene editing strategies. Through these data, researchers can evaluate the accuracy and efficiency of gene editing and further study gene function and crop improvement.

[0132] In the description of the present invention, it should be understood that "-" and "~" represent the range between two values, and the range includes the endpoints. For example: "AB" represents a range greater than or equal to A and less than or equal to B. "A~B" represents a range greater than or equal to A and less than or equal to B.

[0133] In the description of the present invention, the term "and / or" herein is merely a description of the association relationship of associated objects, indicating that three relationships may exist. For example, A and / or B may represent three situations: A exists alone, A and B exist at the same time, and B exists alone.

[0134] In the description of the invention, the numerical values ​​of time, temperature, ratio, mass, etc. involved may be based on actual measurements, standard parameters of equipment, simplified rounding results, or within an acceptable error range, ensuring the practicability and repeatability of the invention.

[0135] In the description of the present invention, the term "about" or "approximately" is used to express the approximate value of a numerical value or range, allowing a certain error to ensure the flexibility and practicality of the description while remaining within an acceptable error range, with the maximum error range not exceeding 10% of the corresponding numerical value or numerical range.

[0136] The above are only preferred embodiments of the present invention. It should be noted that the above preferred embodiments should not be regarded as limiting the present invention, and the protection scope of the present invention should be based on the scope defined by the claims. For ordinary technicians in this technical field, several improvements and modifications can be made without departing from the spirit and scope of the present invention, and these improvements and modifications should also be regarded as the protection scope of the present invention.

Claims

1. A method for efficient transgenic potato tetraploid varieties, characterized in that: The following steps are involved: S1. Pre-culture: Take sterile potato seedlings with a growth period of 2 to 4 weeks, cut stem segments with a length of 0.8 to 1.5 cm without axillary buds and healthy and fully developed leaves without petioles as explants, inoculate them in pre-culture / co-culture medium and pre-culture them in the dark for 1 to 3 days; S2. Agrobacterium infection and co-cultivation: place the stem segments and leaves pre-cultured for 1 to 3 days in a centrifuge tube containing Agrobacterium culture solution of OD600=0.5 and infect for 6 to 10 minutes, slowly turning it upside down during the infection. After the infection is completed, pour out the culture solution, dry the excess culture solution on the explants, transfer the explants to the pre-culture / co-culture medium, and co-cultivate at 22°C in the dark for 1 to 3 days; S3. Callus induction: transfer the explants co-cultured for 1 to 3 days to callus induction medium and culture them in the dark at 22°C for 10 to 15 days; S4. Callus differentiation: transfer the explants after callus induction for 10 to 15 days to callus differentiation medium to induce callus differentiation, and replace the medium every 10 to 15 days; S5. After two rounds of rooting screening, the 1.5-2 cm shoots differentiated from the callus tissue are cut off and inserted into the rooting screening medium. After the taproot grows for the first time, the terminal bud is cut again and inserted into a new rooting screening medium to induce rooting. If taproots grow in two rounds of rooting screening, it is a transgenic positive plant.

2. The method according to claim 1, characterized in that The pre / co-culture medium consisted of the following components: MS basal salts, 20 g / L sucrose, 1 mL / L 1000x N&N vitamins, 1.0 mg / L TZR, 0.0278 mg / L GA3, 0.02 mg / L NAA, and 2.0 g / L Phytagel.

3. The method according to claim 1, characterized in that The callus induction medium consists of the following components: MS basal salts, 20 g / L sucrose, 1 mL / L 1000x N&N vitamins, 1.0 mg / L TZR, 0.0278 mg / L GA3, 0.02 mg / L NAA, 2.0 g / L Phytagel and 200 mg / L Tim.

4. The method according to claim 1, characterized in that: The callus differentiation medium consists of the following components: MS basal salts, 20 g / L sucrose, 1 mL / L 1000x N&N vitamins, 2.0 mg / L TZR, 10 mg / L GA3, 2.0 g / L Phytagel, 50 mg / L Kan, 200 mg / L Tim and 200 mg / L Cef.

5. The method according to claim 1, characterized in that The rooting screening medium consists of the following components: 4.74 g / L MS medium without agar and sucrose, 30 g / L sucrose, 2.0 g / L Hytagel, 50 mg / L Kan and 200 mg / L Tim.

6. The method according to claim 1, characterized in that The method includes using the gRNA sequences specified by SEQ ID NO.1 and SEQ ID NO.2 to guide the Cas9 protease to recognize and cut the upstream cis-regulatory element T#01 of the potato StCDF1 gene.

7. The method according to claim 1, characterized in that The method further comprises constructing a CRISPR / Cas9 knockout vector before step S1, wherein the vector comprises the gRNA sequences specified by SEQ ID NO.1 and SEQ ID NO.

2.

8. The method according to claim 1, characterized in that The method further comprises propagating sterile test tube potato seedlings before step S1, cutting sterile virus-free potato stem segments with axillary buds as explants, and inserting the explants into MS solid culture medium.

9. The method according to claim 1, characterized in that: The method further comprises preparing Agrobacterium bacterial liquid before step S2, taking Agrobacterium EHA105 bacterial liquid containing the plant knockout vector pJCV55-StU6-200-StUBI10-T#01, and streaking the plate on an LB solid medium containing 50 mg / L Spec and 20 mg / L Rif.

10. The method according to claim 1, characterized in that The method further includes detecting mutations by Sanger sequencing and Hi-TOM sequencing after step S5, extracting genomic DNA of transgenic positive seedlings by CTAB method, and performing PCR amplification and sequencing, wherein the primer sequence of PCR amplification target fragment T#01 in Sanger sequencing is: The forward primer T01-F is shown in SEQ ID NO.7, and the reverse primer T01-R is shown in SEQ ID NO.8; The primer sequence for PCR amplification of target fragment T#01 in Hi-TOM sequencing is: The forward primer Hi-F is shown as SEQ ID NO.9, and the reverse primer Hi-R is shown as SEQ ID NO.10.

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