Application and method of knocking out LrTPP3 gene in inhibiting the occurrence of Lycium ruthenicum branch thorn

Abstract

This invention relates to the field of genetic engineering technology, and in particular to the application and method of knocking out the LrTPP3 gene in suppressing the occurrence of thorns on branches of black wolfberry. This invention provides the application of knocking out the LrTPP3 gene in suppressing the occurrence of thorns on branches of black wolfberry, and the suppression of thorn occurrence after knocking out the LrTPP3 gene.

Description

Technical Field

[0001] This invention relates to the field of genetic engineering technology, and in particular to the application and method of knocking out the LrTPP3 gene in inhibiting the occurrence of thorns on branches of black wolfberry. Background Technology

[0002] Black goji berry (Lycium barbarum) is a perennial halophytic medicinal shrub belonging to the Solanaceae family. Rich in natural water-soluble anthocyanins and flavonoids, it possesses outstanding antioxidant effects and has high development value in the food, medicinal, and health care fields. Its salt and drought tolerance also plays a vital role in vegetation restoration in ecologically fragile areas, making it a rare plant resource with both economic and ecological value. However, black goji berry plants generally exhibit a dense network of thorns, a biological characteristic that, while providing self-protection against grazing by herbivores in the natural environment, poses significant challenges to artificial cultivation, fruit harvesting, and large-scale production.

[0003] Currently, the harvesting of black goji berries is still mainly done manually. The dense thorns easily scratch the hands of harvesters and injure the soft berries, reducing harvesting efficiency. Furthermore, due to the inconvenience of harvesting, some harvesters resort to a predatory method of cutting off fruiting branches, which not only severely affects the plant's fruit production the following year but also causes serious damage to black goji berry resources, contradicting the principles of ecological protection and sustainable utilization. In addition, the presence of thorns increases the difficulty and cost of field management (such as pruning, fertilization, and pest and disease control), hindering the large-scale and standardized development of the black goji berry industry and becoming a key bottleneck in the current industrialization process of black goji berries.

[0004] To address this industry challenge, producers can employ strategies such as physical pruning, chemical regulation, traditional breeding, and mechanized harvesting. Physical pruning typically involves manually or mechanically removing existing thorns from the plant. Because black goji berry thorns are dense and point outwards in all directions, this requires significant manual labor, making it too costly. Furthermore, pruning inevitably damages the plant, making it highly susceptible to pathogen infection. Chemical regulation involves spraying growth regulators such as IAA to suppress thorns; however, these methods often negatively impact other aspects of plant growth and contradict the principles of green agriculture and organic farming. Traditional breeding methods, such as distant hybridization combined with grafting, can indeed significantly reduce thorn formation, as exemplified by the Ministry of Agriculture and Rural Affairs' new variety "Yunqichang 9." However, this method is unlikely to yield new varieties with nutritional and health benefits equivalent to or similar to black goji berries, and the breeding cycle is often lengthy. Mechanized harvesting with vibration can avoid hand injuries, but black goji berry branches flower sequentially, and even fruits on the same branch mature at different times, so mechanized harvesting would result in an extreme waste of fruit resources.

[0005] Suppressing thorn development in black goji berries through gene manipulation is a relatively ideal method, yielding materials that, while still belonging to the black goji berry species, possess advantages such as stable traits. For example, knocking out key genes for thorn development using CRISPR-Cas9 gene editing technology holds promise for cultivating superior black goji berry lines with no or few thorns. This invention establishes a novel gene editing technology for black goji berries: whole black goji berry plants infected with Agrobacterium are obtained through traditional transient genetic transformation; leaf sections are used as explants for tissue culture; and stable target gene-edited plants are obtained through direct organogenesis. Thorn development in these plants is significantly suppressed, resulting in completely thornless black goji berry plants. This method does not require kanamycin to screen tissue-cultured plants and can even obtain stably knocked-out lines when the exogenous vector gene (including screening resistance genes) is not integrated, creating favorable conditions for new variety approval and promotion; it also does not require the addition of any plant growth regulators (PGRs) to the culture medium, belonging to a PGR-free direct organogenesis system. Summary of the Invention

[0006] To address the aforementioned issues, this invention provides the application and method of knocking out the LrTPP3 gene in suppressing the occurrence of thorns on branches of black wolfberry. Knocking out the LrTPP3 gene suppresses the occurrence of thorns on branches of black wolfberry.

[0007] To achieve the above objectives, the present invention provides the following technical solution:

[0008] This invention provides the application of knocking out the LrTPP3 gene in inhibiting the occurrence of thorns on black wolfberry branches.

[0009] Preferably, the nucleotide sequence of the LrTPP3 gene is shown in SEQ ID No. 1.

[0010] This invention also provides a method for permanently knocking out the LrTPP3 gene based on transient genetic transformation, comprising the following steps:

[0011] 1) Agrobacterium containing the pTarget2-LrTPP vector was mixed with MS liquid medium containing acetylsuccinone to obtain the infection solution;

[0012] The pTarget2-LrTPP vector was obtained by inserting a sequence fragment into the original vector pEgP237, and the nucleotide sequence of the sequence fragment is shown in SEQ ID No. 2.

[0013] 2) After infecting the black wolfberry seedlings with the infection solution described in step 1), they were co-cultured to obtain co-cultured seedlings;

[0014] 3) The co-cultured seedlings described in step 2) are subjected to heat treatment, and then tissue culture and regeneration line propagation are carried out in sequence to obtain the line with the LrTPP3 gene knocked out.

[0015] Preferably, in step 1), the volume ratio of Agrobacterium containing the pTarget2-LrTPP vector to MS liquid culture medium containing acetylsuccinone is 1:1.

[0016] The concentration of acetylsuccinone in the MS liquid culture medium was 1 µmol / L.

[0017] Preferably, the conditions for immersion in step 2) include: immersion at a rotation speed of 90 rpm and a temperature of 25°C for 5 hours;

[0018] The volume ratio of the inoculum to the number of black goji berry seedlings is 33 mL: 1 seedling. The black goji berry seedlings are then immersed in the inoculum.

[0019] Preferably, the co-culture conditions in step 2) include: co-culturing in MS solid medium of 100 µmol / L AS at 28°C in the dark for 2 days.

[0020] Preferably, the heat treatment conditions in step 3) include: culturing in an environment with a temperature of 37°C and a photoperiod of 16 h light / 8 h darkness for 24 h, followed by recovery culturing in an environment of 25°C for 24 h, and then culturing again in an environment of 37°C for 24 h.

[0021] Preferably, after the heat treatment described in step 3), the tissue culture and regenerated line propagation are carried out after culturing at 23~27℃ for 2 days.

[0022] Preferably, the tissue culture conditions in step 3) include: taking the leaf tips of black wolfberry seedlings (3-7 mm) as explants, culturing them on MS solid medium containing 300 mg / L Cef and 5 mg / L AgNO3 until the seedlings reach a height of 6-8 cm, and replacing the MS solid medium every 7 days.

[0023] Preferably, the conditions for propagating the regenerated line in step 3) include: inoculating 0.8-1.2 cm of stems with apical buds into 1 / 2 MS solid medium to induce rooting and form a line.

[0024] The beneficial effects of this invention are:

[0025] Knocking out the LrTPP3 gene inhibited the occurrence of thorns on black wolfberry branches. Attached Figure Description

[0026] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the accompanying drawings used in the embodiments will be briefly described below.

[0027] Figure 1 This is a technical roadmap of the present invention;

[0028] Figure 2 PCR electrophoresis images of wild-type and transiently transformed tissue culture lines of black wolfberry; WT, wild-type; 2, 4, tissue culture lines after transient transformation; M: DL5,000 DNA Marker;

[0029] Figure 3 CDS sequence alignment of the LrTPP3 gene between wild-type (WT) and gene-edited strain of black goji berry;

[0030] Figure 4 Prediction of protein function in wild-type (WT) black goji berries and gene-edited black goji berries;

[0031] Figure 5 PCR detection diagram of LrTPP3 gene-edited line insertion in black goji berries. M: DL5,000 DNAMaker; +: positive vector; 2, 4: gene-edited transgenic lines; WT: wild type.

[0032] Figure 6 Representative plants (A), leaves (B), stems (C), and leafless stems (D) of wild-type (WT) and LrTPP3 knockout lines (Cas2-2 and Cas2-4) of black goji berries. Detailed Implementation

[0033] This invention provides the application of LrTPP3 gene knockout in inhibiting thorn formation in black wolfberry branches. In this invention, the nucleotide sequence of the CDS of the LrTPP3 gene is shown in SEQ ID No. 1, and is as follows:

[0034]

[0035] This invention also provides a method for permanently knocking out the LrTPP3 gene based on transient genetic transformation, comprising the following steps:

[0036] 1) Agrobacterium containing the pTarget2-LrTPP vector was mixed with MS liquid medium containing acetylsuccinone to obtain the infection solution;

[0037] The pTarget2-LrTPP vector was obtained by inserting a sequence fragment into the original vector pEgP237, and the nucleotide sequence of the sequence fragment is shown in SEQ ID No. 2.

[0038] 2) After infecting the black wolfberry seedlings with the infection solution described in step 1), they were co-cultured to obtain co-cultured seedlings;

[0039] 3) The co-cultured seedlings described in step 2) are subjected to heat treatment, and then tissue culture and regeneration line propagation are carried out in sequence to obtain the line with the LrTPP3 gene knocked out.

[0040] In this invention, Agrobacterium containing the pTarget2-LrTPP vector is mixed with MS liquid medium containing acetylsuccinone to obtain an infection solution; the pTarget2-LrTPP vector is obtained by inserting a sequence fragment into the original vector pEgP237, and the nucleotide sequence of the sequence fragment is shown in SEQ ID No. 2.

[0041] In this invention, the volume ratio of Agrobacterium containing the pTarget2-LrTPP vector to MS liquid culture medium containing acetylsuccinone is preferably 1:1, and the concentration of acetylsuccinone in the MS liquid culture medium is preferably 100 µmol / L.

[0042] SEQ ID No. 2:

[0043] 5'-CTTTCACGCGAATGAGCCGG-3'.

[0044] In this invention, the method for obtaining the pTarget2-LrTPP vector preferably includes the following steps:

[0045] 1) Design of target sites and single-strand renaturation of LrTPP3 gene editing vector in black goji berries

[0046] Using the CRISPR-GE online website (http: / / skl.scau.edu.cn), the targetDesign subroutine was invoked to design gRNAs targeting the LrTPP3 gene of black goji berries. After screening for high-scoring gRNA sequences, the corresponding single-stranded DNA sequences were synthesized using a primer synthesis method. The specific sequence information is as follows: LrTPP-cas-F (5'-GATTCGTCTTTCACGCGAATGAGC-3') (SEQ ID No. 3), LrTPP-cas-R (5'-AAACGCTCATTCGCGTGAAAGACG-3') (SEQ ID No. 4).

[0047] The synthesized single-stranded DNA sequence was subjected to annealing. The annealing PCR reaction program was set as follows: 95℃ for 5 min; 85℃ for 5 min; 75℃ for 5 min; 65℃ for 5 min; 55℃ for 5 min; 45℃ for 5 min; 35℃ for 5 min; 25℃ for 5 min, and finally stored at 4℃. The annealing PCR reaction system was prepared as follows (total volume 20 μL): 10×Ex Taq Buffer (20 mM) 2 μL, LrTPP-cas-F (100 μM) 9 μL, LrTPP-cas-F (100 μM) 9 μL.

[0048] 2) Extraction and enzyme digestion of the gene editing vector pEgP237 plasmid

[0049] The pEgP237 plasmid was extracted using the Zhongke Ruitai standard plasmid mini-prep kit. After determining the concentration and purity of the plasmid using a spectrophotometer, it was digested with the restriction endonuclease BsaⅠ. The digestion reaction system was prepared as follows (total volume 20 μL): ddH2O to 20 μL, 10×LabFD™ Buffer 2 μL, pEgP237 plasmid 1 μg, and LabFD™ BsaⅠ 1 μL. The digestion reaction was incubated at 37℃ for 4 h.

[0050] After confirming successful plasmid digestion by agarose gel electrophoresis, the digested pEgP237 vector was purified and recovered using a Kangwei Century purification and recovery kit. The recovered product was stored at -20°C for later use.

[0051] 3) Ligation and transformation of the LrTPP3 gene target site of black wolfberry with pEgP237 vector

[0052] Using T4 DNA ligase, the renatured product of the target site was ligated overnight at 16°C with the digested pEgP237 vector. The ligation reaction system was prepared as follows (total volume 10 μL): ddH2O to 10 μL, Ligase 10× Buffer 1 μL, BsaⅠ digested vector (200 ng / μL) 1 μL, renatured fragment 2 μL, and T4 DNA Ligase 1 μL. The constructed LrTPP3 gene-editing recombinant plasmid was named pTarget 2-LrTPP and subsequently transformed into E. coli TOP10 competent cells. The specific steps are as follows:

[0053] (1) Take 5 μL of overnight ligation product and add it to 50 μL of freshly thawed E. coli TOP10 competent cells. Gently tap the cells to mix them evenly.

[0054] (2) Let it stand in an ice bath environment for 30 minutes;

[0055] (3) Place in a 42℃ water bath for 90 s, and immediately after taking it out, perform an ice bath treatment for 2-3 min. Avoid shaking the centrifuge tube during this period.

[0056] (4) Add 500 μL of non-resistant LB liquid medium to the centrifuge tube and shake at 220 rpm and 37°C for 1 h to activate the cells;

[0057] (5) Take 100 μL of activated bacterial solution and spread it evenly on LB resistant solid medium with 50 mg / L Kan added. Incubate at 37℃ for 12-16 h.

[0058] After plating and culturing, single colonies were picked and incubated with shaking at 37°C. PCR detection of the bacterial culture was performed using vector primers M13-F (sequence: 5'-GTTGTAAAACGACGGCCAG-3') (SEQ ID No. 5) and LrTPP-cas-R. The target band size was approximately 500 bp. Positive colonies were selected for sequencing. After successful sequencing verification, plasmids were extracted using the Zhongke Ruitai standard plasmid mini-prep kit. The specific steps are as follows:

[0059] (1) Take 1-5 mL of overnight cultured Escherichia coli culture and add it in batches to 2 mL centrifuge tubes. Centrifuge at 10,000 g for 1 min, discard the supernatant and keep the bacterial cells;

[0060] (2) Add all of the RNase A from the Zhongke Ruitai plasmid miniprep kit to solution P1 in advance (store at 4℃), add 250 μL of solution P1 and 5 μL of Lysis Dye to the bacterial precipitate, and resuspend the precipitate thoroughly with a pipette until the solution turns turbid red.

[0061] (3) Add 250 μL of solution P2, gently invert the centrifuge tube 6-8 times to fully lyse the bacteria, and the solution will turn into a clear red color;

[0062] (4) Add 350 μL of solution P3, immediately and gently invert the container to mix thoroughly until the solution is completely transparent yellow. Centrifuge at 10,000 g for 10 min;

[0063] (5) Place the adsorption column CP into the collection tube, carefully transfer the supernatant into the adsorption column, centrifuge at 10,000 g for 30-60 s, discard the waste liquid, and put the adsorption column CP back into the collection tube.

[0064] (6) Add anhydrous ethanol to the washing solution PW in advance and mix well. Add 700 μL of washing solution PW to the adsorption column CP, centrifuge at 10,000 g for 30-60 s, discard the waste liquid, and put the adsorption column CP back into the collection tube.

[0065] (7) Add 500 μL of washing solution PW to the adsorption column CP again, centrifuge at 10,000 g for 30-60 s, and discard the waste liquid;

[0066] (8) Put the adsorption column CP back into the collection tube and centrifuge at 10,000 g for 2 min to ensure that the residual washing solution in the adsorption column is completely removed.

[0067] (9) Place the adsorption column CP in a clean 1.5 mL centrifuge tube, add 40 μL of sterile ddH2O to the center of the adsorption membrane, place at room temperature for 2-3 min, and centrifuge at 10,000 g for 2 min.

[0068] (10) Take the collected plasmid solution and resuspend it in the center of the adsorption membrane. Let it stand at room temperature for 2-3 min, centrifuge at 10,000 g for 2 min, determine the concentration, and store the plasmid solution at -20 ℃.

[0069] The pTarget 2-LrTPP plasmid was transformed into GV3101 Agrobacterium competent cells using the freeze-thaw method. The specific steps are as follows:

[0070] (1) Place 100 μL of Agrobacterium competent cells GV3101 on ice to thaw beforehand;

[0071] (2) Add pTarget 2-LrTPP recombinant plasmid to 100 μL of GV3101 Agrobacterium competent cells and gently mix.

[0072] (3) Ice bath for 5 min, liquid nitrogen quick freeze for 5 min, 37℃ water bath for 5 min, ice bath for 5 min, avoid shaking during the process;

[0073] (4) Add 700 μL of YEP liquid medium without resistance and incubate at 28℃ and 180 rpm for 3-4 h with shaking.

[0074] (5) Centrifuge at 5000 rpm for 5 min, discard 400-500 μL of supernatant, and resuspend the bacterial cells in the remaining supernatant;

[0075] (6) Take 100 mL of resuspended bacterial solution and spread it evenly on YEP solid medium containing 50 mg / L Rif + 50 mg / L Kan, and incubate it upside down at 28℃ for 48 h;

[0076] (7) Select single clones of bacteria and culture them in YEP liquid medium containing resistance for 12 h. Perform bacterial PCR. The PCR system is as follows: 1 μL of bacterial culture; 1 μL of 10 μM M13F; 1 μL of 10 μM LrTPP-cas2-R; 0.5 μL of Max enzyme; 12.5 μL of 2×Buffer; 0.5 μL of 10 mM dNTP; 8.5 μL of ddH2O. The PCR reaction program is as follows: 95℃ for 15 min; 95℃ for 30 s, 59.2℃ for 30 s, 72℃ for 3 min 15 s, 35 cycles; 72℃ for 5 min, and store at 4℃. After the PCR products are verified by agarose gel electrophoresis, select the positive bacterial species with the correct bands for sequencing. Mix sterile glycerol with the bacterial culture at a ratio of 1:1. Store the transformed Agrobacterium culture at -80℃ for later use.

[0077] This invention involves infecting black goji berry seedlings with the aforementioned infection solution and then co-culturing them to obtain co-cultured seedlings. In this invention, the preferred infection conditions include: immersion at 90 rpm and 25°C for 5 hours. In this invention, the preferred ratio of the volume of the infection solution to the number of black goji berry seedlings is 33 mL:1 seedling. In this invention, the preferred co-culturing conditions include: co-culturing in 100 µmol / L AS MS solid medium at 28°C in the dark for 2 days.

[0078] This invention involves heat-treating the co-cultured seedlings, followed by tissue culture and propagation of regenerated lines to obtain lines with the LrTPP3 gene knocked out. Preferably, the heat-treating conditions include: culturing at 37°C with a photoperiod of 16 h light / 8 h dark for 24 h, followed by recovery culture at 25°C for 24 h, and then culturing again at 37°C for 24 h. Preferably, after heat treatment, the seedlings are cultured at 23-27°C for 2 days before tissue culture and propagation of regenerated lines. Preferably, the tissue culture conditions include: using leaf tips (3-7 mm) from black goji berry seedlings as explants, culturing on MS solid medium containing 300 mg / L Cef and 5 mg / L AgNO3 until the seedlings reach a height of 6-8 cm, changing the MS solid medium every 7 days. In this invention, the preferred conditions for propagating the regenerated lineage include: inoculating 0.8-1.2 cm stems with apical buds into 1 / 2 MS solid medium to induce rooting and form a lineage.

[0079] WT black goji berry LrTPP3 gene CDS sequence (5'-3') (SEQ ID No. 1):

[0080] ATGGTGTCAAGATCCTATTCCAATCTTTTAGAGCTAGCTTCAGGCGAGGCTCCTTCGCCGTCTTTCACGCGAATGAGCCGGAGGATTCCACGTATTATGACCGTTGC C GGGATAATGTCTGATCTGGATGATGATGGATCGGG T AGCGCTTGCTCTGATCCATCGTCCTCAGCTCAGAAAGATAGGATAATTGTTGTAGCTAATCAGCTGCCAATTAGAGTACAAAGAAAAACTGATGGTAGTAAAGGATGGTTATTCAGCTGGGACGAGAATTCGCTTCTCCTCCAGCTGAAAGATGGTTTAGGTGATGATGAAATTGAGGTTATATATGTTGGTTGCCTTAAGGAAGAAATCCACCCGAATGAGC AA

[0081] The bold text indicates Cas2-2 mutation sites, the underlined text indicates Cas2-4 mutation sites, and GA is inserted between the two AAs.

[0082] The amino acid sequence corresponding to the CDS sequence of the LrTPP3 gene in WT black goji berries (SEQ ID No. 6):

[0083] MVSRSYSNLLELASGEAPSPSFTRMSRRIPRIMTVAGIMSDLDDDGSGSACSDPSSSSAQKDRIIVVANQLPIRVQRKTDGSKGWLFSWDENSLLLQLKDGLGDDEIEVIYVGCLKEEIHPNEQ DEVSQILLETFKCVPTFVPPDLFTKYYHGFCKQQLWPLFHYMLPLSPDLGGRFNRLLWQAYVSVNKIFADRIMEVINPEDDFVWVHDYHLMVLPTFLRKRFNRVKLGFFLHSPFPSSEIYKTLP IREEILRALLNSDLIGFHTFDYARHFLSCCSRMLGISYESKRGYIGLEYYGRTVSIKILPVGIHMGQLQQVLSLPETEAKVAELVEQFNHQGRTLLLGVDDMDIFKGISLKLLAMEQLLLQHPE KQGKVVLVQIANPPRGKGKDVEEVREETNSTVKRINEVYGRPGYQPVILIDKPLKFYERIAYYVVAECCLVTAVRDGMNLIPYEYIISRQGNEKLDKVLKLDSSTQKKSMLVVSEFIGCSPSLS.

[0084] The bolded areas represent Cas2-2 mutation sites.

[0085] CDS sequence (5'-3') of the LrTPP3 gene in the Cas2-2 strain (SEQ ID No. 7):

[0086]

[0087] The bolded areas indicate abrupt changes.

[0088] The amino acid sequence corresponding to the CDS sequence of the LrTPP3 gene in the Cas2-2 strain (SEQ ID No. 8):

[0089] MVSRSYSNLLKLASGEAPSPPFTLMSRRIPRIMTVAGIMSDLDDDGSGSACSDPSSSSSAQKDRIIVVANQLPIRVQRKTDGSKGWLFSWDENSLLLQLKDGLGDDEIEVIYVGCLKEEIHPNEQ DEVSQILLETFKCVPTFVPPDLFTKYYHGFCKQQLWPLFHYMLPLSPDLGGRFNRLLWQAYVSVNKIFADRIMEVINPEDDFVWVHDYHLMVLPTFLRKRFNRVKLGFFLHSPFPSSEIYKTLP IREEILRALLNSDLIGFHTFDYARHFLSCCSRMLGISYESKRGYIGLEYYGRTVSIKILPVGIHMGQLQQVLSLPETEAKVAELVEQFNHQGRTLLLGVDDMDIFKGISLKLLAMEQLLLQHPE KQGKVVLVQIANPPRGKGKDVEEVREETNSTVKRINEVYGRPGYQPVILIDKPLKFYERIAYYVVAECCLVTAVRDGMNLIPYEYIISRQGNEKLDKVLKLDSSTQKKSMLVVSEFIGCSPSLS.

[0090] The bolded areas indicate abrupt changes.

[0091] CDS sequence (5'-3') of LrTPP3 gene in Cas2-4 strain (SEQ ID NO.9):

[0092]

[0093] Bold text indicates mutations or base insertions.

[0094] The amino acid sequence corresponding to the CDS sequence of the LrTPP3 gene in the Cas2-4 strain (SEQ ID NO.10):

[0095] MVSRSYSNLLELASGEAPSPSFTRMSRRIPRIMTVAGIMSDLDDDGSGSACSDPSSSSAQKDRIIVVANQLPIRVQRKTDGSKGWLFSWDENSLLLQLKDGLGDDEIEVIYVGCLKEEIHPNEQKMKCHKYSLRHLSVFPLLFRLICLLSTIMDFVNNNFGLCSTICCHFRPILVVDSIDYYGRLMFL*.

[0096] * indicates early termination.

[0097] To further illustrate the present invention, the following detailed description is provided in conjunction with embodiments, but these should not be construed as limiting the scope of protection of the present invention.

[0098] Example 1

[0099] 1. Experimental materials, reagents and culture media

[0100] 1.1 Plant materials

[0101] The sterile black goji berry tissue culture seedlings were preserved in the tissue culture room of the Liaoning Provincial Key Laboratory of Forest Genetics, Breeding and Cultivation (College of Forestry, Shenyang Agricultural University).

[0102] 1.2 Preparation of reagents and culture media

[0103] 50 mg / mL kanamycin (Kana): Weigh 1 g of Kana powder and dissolve it in sterile water to a final volume of 20 ml. Use a filter and a disposable syringe to filter the Kana solution in a laminar flow hood. Aliquot the filtered solution into 2 mL and 200 μmol / L centrifuge tubes that have been autoclaved and store them in a refrigerator at -20°C for later use.

[0104] 100 mmol / L Acetyleugenone (AS): Dissolve 1.962 g of AS powder in 100 mL of DMSO. Filter the AS solution in a clean bench using a filter and a disposable syringe. Aliquot the filtered solution into 2 mL autoclaved centrifuge tubes and store at -20 ℃ for later use. Rifampin was purchased from Zhongke Ruitai Biotechnology Co., Ltd. Cefotaxime sodium was purchased from Sangon Biotech Co., Ltd. 5 U / μL Real Taq DNA Polymerase, 10x Real Taq Buffer, 10 mmol / L dNTP (Zhongke Ruitai), Kana, Tritonx-100, agar, Na2EDTA, MS dry powder (Solarbio), yeast extract, beef extract (AOBOX), syringe filter, DMSO (Sangon), AS (MERYER), glacial acetic acid, anhydrous ethanol, sodium chloride, sucrose, and domestically produced analytical grade AR and Tween 20 reagents were used.

[0105] The culture medium used in this embodiment and its composition are shown in Table 1.

[0106] Table 1 Culture medium formulation

[0107] culture medium formula YEP liquid culture medium Sodium chloride 5g / L + beef extract powder 10g / L + yeast extract powder 10g / L (pH=7.0) YEP solid culture medium Sodium chloride 5g / L + beef extract powder 10g / L + yeast extract powder 10g / L + agar 1.5g / L (pH=7.0) 1 / 2MS liquid culture medium Sucrose 20g / L + MS dry powder 2.37g / L (pH=5.8) 1 / 2MS solid culture medium Sucrose 20g / L + MS dry powder 2.37g / L + agar 4.7g / L (pH=5.8) MS solid culture medium Sucrose 40g / L + MS dry powder 4.74g / L + agar 4.7g / L (pH=5.8)

[0108] 1.3 Carriers and strains

[0109] The LrTPP3 gene editing vector of black goji berries used in this embodiment was obtained by inserting a 5'-CTTTCACGCGAATGAGCCGG-3' (SEQ ID No. 2) fragment into the original vector pEgP237 using an enzyme digestion method. The constructed vector was named pTarget2-LrTPP. The Agrobacterium engineered strain used was LBA4404.

[0110] 2. Test Methods

[0111] 2.1 Transient genetic transformation of black goji berries based on gene editing vectors

[0112] 2.1.1 Transient conversion material cultivation of black wolfberry

[0113] Select black goji berry tissue culture seedlings with good growth status that have been growing for 1-2 months. In a clean bench, use sterile scissors to cut off 3-4 cm of the stem with the top bud of the black goji berry tissue culture seedling. Inoculate the seedlings into 1 / 2 MS medium and culture for about 14 days until the stem is about 10 cm long and the root is about 2-3 cm long. Use the seedlings for transient transformation experiments.

[0114] 2.1.2 Preparation of Agrobacterium infection solution

[0115] Agrobacterium containing pTarget2-LrTPP, stored at -80 ℃, was thawed on ice. Inoculum was streaked onto YEP plates containing 100 mg / mL Rif and 100 mg / L Kana. After 48 h of incubation in the dark, single colonies were picked and inoculated into 1 mL of YEP liquid medium (containing 100 mg / L Kan and 100 mg / L Rif). The culture was then incubated at 28 ℃ with shaking at 200 rpm for 24 h. The bacterial culture after 24 h of shaking was then added to 100 mL of YEP liquid medium (containing 100 mg / L Kan and 100 mg / L Rif) for further culture. The culture was then incubated at 28 ℃ with shaking at 200 rpm until OD reached. 600 Approximately 0.6~0.8; centrifuge at 4000 rpm for 10 min at 4 ℃, discard the supernatant, resuspend the cells in an equal volume of MS liquid medium with 1 μmol / L AS added, and incubate the infection solution in a 4 ℃ refrigerator for 30 min.

[0116] 2.1.2 Ultrasonic treatment

[0117] The cultured black goji berry tissue culture plants were ultrasonically treated in a sterile environment (24 W, 46 kHz) (ultrasonic treatment liquid: 600 mL purified water + 600 μL between 20 s) for 10 s.

[0118] 2.1.3 Co-cultivation

[0119] The infection solution was poured into a sterile 100 ml Erlenmeyer flask, and the ultrasonically treated black goji berry plants were placed inside. The flask was then placed in a shaker at 90 r / min and inoculated at 25 ℃ for 5 h to infect the sterile black goji berry seedlings. Afterward, the bacterial solution on the surface of the black goji berry plants was blotted dry with sterilized filter paper, and the plants were inoculated into MS solid medium supplemented with 100 μmol / L AS. The plants were co-cultured at 28 ℃ in the dark for 2 days.

[0120] 2.1.4 Heat Treatment

[0121] After co-culturing, the black goji berries were placed in an environment with a temperature of 37 °C and a photoperiod of 16 h light / 8 h darkness for 24 h, followed by a recovery culture at 25 °C for 24 h. They were then placed in a 37 °C environment for another 24 h, thus completing two rounds of heat treatment. After the heat treatment, they were cultured at 25 ± 2 °C for 2 days.

[0122] 2.1.5 Tissue Culture

[0123] First, the bacterial solution on the surface of the heat-treated black goji berries was washed using MS liquid culture (containing 100 μmol / L As and 300 mg / L Cef). Then, the tips of the cleaned black goji berry leaves, approximately 3-7 mm in diameter, were cut and used as explants for tissue culture. The leaf tip explants were placed adaxially downwards on the MS solid medium to induce direct organogenesis (rooting followed by budding). The MS solid medium was supplemented with 300 mg / L Cef and 5 mg / L AgNO3, but no plant growth regulators (PGRs) were added. Rooting began in the leaf tip explants after 7-14 days of culture, and budding started from the new roots around 30 days later. The MS solid medium was replaced every 7 days during this period.

[0124] 2.1.6 Propagation of regenerated lines

[0125] When the explants from the leaf tips after transient transformation reach a height of approximately 6-8 cm, a 0.8-1.2 cm section of the stem with the terminal bud is cut and re-inoculated onto 1 / 2 MS solid medium without PGR to induce rooting and form a line, which takes approximately 7-14 days. Once the plants reach a suitable size, molecular identification is performed to determine whether the LrTPP3 gene in the obtained line has been successfully edited or knocked out. The above tissue culture line system will be referred to below as the transiently transformed tissue culture line.

[0126] 2.2 Molecular detection of tissue culture lines after transient transformation of black wolfberry

[0127] 2.2.1 Determination of LrTPP3 gene target region editing status

[0128] Total genomic DNA was extracted from wild-type and transiently transformed tissue culture lines of *Lycium barbarum* using the RealPure Plant Genomic DNA Extraction Kit (RTG2404-01). DNA quality and concentration were determined by 1% gel electrophoresis and a NanoDropone spectrophotometer (Thermo). The qualified DNA was stored at -20 °C for later use. Using the extracted qualified DNA as a template, PCR was performed using primers LrTPP-Cas2-F (5'-TTGCTTGGTTTCACTCA-3') (SEQ ID No. 11) and LrTPP-Cas2-R (5'-CACTTCATCTTGCTCAT-3') (SEQ ID No. 12) to amplify the target fragment of the LrTPP3 gene. DNA amplification was assessed by agarose gel electrophoresis. PCR reaction system: DNA 1 μL; primer LrTPP-Cas2-F 0.5 μL; primer LrTPP-Cas2-R 0.5 μL; Taq 0.25 μL; Taq Buffer 2.5 μL; dNTP 0.3 μL; ddH2O 19.95 μL; total volume 25 μL. PCR reaction program: 95 ℃ for 15 min; 35 cycles (95 ℃ for 30 s; 60 ℃ for 30 s; 72 ℃ for 1 min); 72 ℃ for 5 min, store at 12 ℃.

[0129] The target PCR product was sent to Suzhou Genewise Technology Co., Ltd. for DNA sequencing to verify whether the LrTPP3 gene sequence was successfully edited and the edited sequence.

[0130] 2.2.2 Protein function prediction of LrTPP3 edited lines of black wolfberry

[0131] The CDS sequences of the LrTPP3 gene in wild-type black goji berries and the successfully edited LrTPP3 gene lines Cas2-2 and Cas2-4 were converted into amino acid sequences. The protein function was predicted using the InterProScan website (https: / / www.ebi.ac.uk / interpro / ) to determine whether the major function of the LrTPP3 protein in the successfully edited lines was lost. If it was lost, it was preliminarily considered that the gene was successfully knocked out.

[0132] 2.2.3 Detection of DNA insertion into the LrTPP3 gene-edited strain vector

[0133] Total genomic DNA was extracted from wild-type black wolfberry and LrTPP3 knockout lines. Using primers M13F (5'-GTTGTAAAACGACGGCCAG-3') (SEQ ID No. 5) and LrTPP-R (5'-AATCCGAGGAGGT TTCC-3') (SEQ ID No. 13), PCR was used to verify whether the above infection operation permanently inserted the corresponding region of the vector into the black wolfberry genome. The pEgP237 vector was used as a positive control during PCR. The PCR products were detected by agarose gel electrophoresis.

[0134] 2.3 Propagation and Transplantation of LrTPP3 Gene-Edited Lines of Black Goji Berry

[0135] Since no visible thorns were observed in the tissue culture bottles of black goji berries, propagation, hardening-off, and transplanting were carried out on wild-type and gene-edited black goji berry lines to observe changes in the thorn phenotype of the gene-edited lines. Tender stems with terminal buds from vigorous gene-edited lines were cut in a clean bench and inoculated into 1 / 2 MS solid medium supplemented with 300 mg / L Cef for rooting and propagation. After approximately 45 days of growth on the medium (until complete rooted plants had formed), the stems were placed under natural light for acclimatization for 7-15 days until the plants showed good growth (mainly characterized by thick stems and dark green leaves). At this point, the well-grown plants were removed from the tissue culture bottles, and the roots were rinsed under a gentle stream of water. After washing, the roots were soaked in a 0.33% carbendazim solution for 20 minutes to prevent fungal diseases during plant growth. The seedling substrate was sterilized at 121 °C for 40 minutes, followed by cooling, in preparation for transplanting the tissue culture seedlings. The seedling nutrient substrate was purchased from Shenyang Fanyu Horticulture Technology Co., Ltd. The substrate, officially named soilless seedling nutrient substrate, has a moisture content ≤40% and an organic matter content ≥40%. This seedling nutrient substrate was mixed with perlite and vermiculite in a 3:1:1 ratio. The transplanted plants were covered with perforated disposable plastic cups to maintain humidity. After approximately 14 days, when the plants were growing well and sprouting new leaves, the plastic cups were removed. The transplanted plants were placed in a soil cultivation room with a light intensity of 48 μmol / m² / s and a temperature of 25±2 ℃.

[0136] 2.4 Phenotypic determination and data statistics of wild-type and gene-edited lines

[0137] After transplanting and cultivation for approximately 40 days, newly sprouted stems from healthy wild-type and gene-edited lines with similar developmental stages were used as experimental materials for phenotypic data determination. Thorny and thornless branches were identified visually. Branch length was measured using a measuring tape (2 m range), and branch diameter, main leaf length, and leaf width were measured using vernier calipers. These data were then used to calculate the average main leaf length (total main leaf length / number of main leaves) and main leaf width (total main leaf width / number of main leaves) for both thorny and thornless branches. The thorn emergence rate of thorny branches was statistically analyzed (number of thorny leaf axils ÷ total number of leaf axils × 100%), and thorn length and width were measured using vernier calipers. The experiment was conducted in triplicate.

[0138] One-way ANOVA analysis was performed on plant phenotypic data using SPSS 20.0 (P < 0.05).

[0139] 2.5 Technical Route

[0140] The technical route of this embodiment is described below. Figure 1 .

[0141] 3 Results and Analysis

[0142] 3.1 Identification of LrTPP3 gene-edited lines in black wolfberry

[0143] 3.1.1 Identification of LrTPP3 gene DNA sequence in black goji berry gene-edited strain

[0144] DNA was extracted from wild-type and transiently transformed tissue culture lines, and PCR was performed using primers LrTPP-Cas2-F and LrTPP-Cas2-R to amplify the target fragment of the LrTPP3 gene. The results are as follows: Figure 2 As shown, bands were detected in both wild-type and transiently transformed tissue culture lines, with the bands accurately located and approximately 705 bp in length.

[0145] 3.1.2 DNA sequence changes in tissue culture lines of black goji berries after transient transformation

[0146] The LrTPP3 gene sequence was compared between the obtained transiently transformed tissue culture lines and the wild type. The comparison results are as follows: Figure 2 As shown, in the CDS sequence of the LrTPP3 gene of the Cas2-2 strain, G→A at 31nt, T→C at 61nt, G→T at 71nt, and C→T at 108nt; in the Cas2-4 strain, C→T at 108nt, T→G at 143nt, and two additional GA bases are added at 372nt. Figure 3 This indicates that two LrTPP3 gene-edited lines of black goji berries were successfully obtained in this embodiment. Sequencing results showed no overlapping peaks, proving that the obtained gene-edited lines are homozygous mutant lines.

[0147] 3.1.3 Functional prediction of LrTPP3 protein in gene-edited black goji berries

[0148] Mutations in the LrTPP3 gene in two Cas2 strains resulted in different alterations to their protein products: Cas2-2 underwent a two-amino acid substitution (the total length of 496 aa remained unchanged, SEQ ID No. 9); while Cas2-4, due to an insertion mutation, resulted in a frameshift and premature termination of translation, producing a truncated protein (only 188 aa, SEQ ID No. 10). InterProScan bioinformatics functional prediction showed that both mutants lost the key functional domain (GT20_TPS domain) of wild-type LrTPP3. Figure 4 Among them, the truncated protein of Cas2-4, lacking a complete functional domain, cannot perform the normal biological function of LrTPP3 and is a knockout mutant with complete loss of function. Cas2-2 was predicted to lose a key functional domain, and in this embodiment, it was used as a candidate loss-of-function mutant along with Cas2-4 for subsequent experiments. In summary, this embodiment obtained two loss-of-function mutants of the LrTPP3 gene (including one knockout mutant and one candidate loss-of-function mutant).

[0149] 3.1.4 Detection of DNA Insertion into LrTPP3 Gene-Edited Lines

[0150] PCR results using primers M13F and LrTPP-R showed that no band was detected in the wild-type (WT) strain, while the transgenic line showed a band at an accurate location of approximately 751 bp, consistent with the length of the positive control plasmid band. Figure 5 This indicates that the T-DNA sequence of the LrTPP3 gene-editing vector has been successfully inserted into the genome of *Lycium chinense*. This demonstrates that although this example used *Agrobacterium* infection of the entire tissue culture plant of *Lycium chinense*, a method conventionally defined as transient transformation, after transformation, leaf tips were explanted for tissue culture. Even though plants formed via direct organogenesis without callus formation, the corresponding vector fragment could still stably insert into the genome of the resulting plant, a result similar to stable genetic transformation. Of course, in actual experiments, there are also cases where the vector fragment does not insert into the genome, yet stable edited plants are still obtained.

[0151] 3.2 LrTPP3 gene knockout causes disappearance of thorns on black wolfberry branches

[0152] In the comparative analysis of branches and thorns, it was found that, compared with the wild type (WT), the LrTPP3 gene knockout lines (Cas2-2 and Cas2-4) showed an upward trend in the number of newly developed lateral branches. Figure 6(Table A) but no significant difference was observed (Table 2). The knockout lines did not exhibit thorny lateral branches, which was significantly different from the 22.67% thorny lateral branch rate of the WT line (Table 2), indicating that LrTPP3 gene knockout significantly inhibited thorn formation in black wolfberry, resulting in completely thornless lines. Figure 6 (C and D). Furthermore, in the comparative analysis of thornless lateral branches, no significant differences were found between lateral branch length and width and WT (Table 2), while the number of leaves per cluster, leaf length, and leaf width all increased (Table 2 and WT). Figure 6 (B) but no significant difference was found. In summary, knocking out the LrTPP3 gene in black goji berry completely inhibits the occurrence of thorns in its potted plants, but has no significant negative impact on stem and leaf growth.

[0153] Table 2. Effects of LrTPP3 gene knockout on the phenotype of new shoots in transplanted black goji berries

[0154] series WT Cas2-2 Cas2-4 Number of new lateral branches per plant <![CDATA[7.33±0.57 b ]]> <![CDATA[8.50±0.50 a ]]> <![CDATA[7.34±0.58 b ]]> Number of new lateral branches per branch <![CDATA[6.34±0.58 b ]]> <![CDATA[7.50±0.0.5 a ]]> <![CDATA[6.34±0.58 b ]]> Percentage of thorny lateral branches (%) <![CDATA[22.67±4.48 a ]]> <![CDATA[0.00±0.00 b ]]> <![CDATA[0.00±0.00 b ]]> Thorn-bearing lateral branches thorn production rate (%) <![CDATA[10.67±1.86 a ]]> —— —— Thorny lateral branch thorn length (mm) 4.02±0.21 —— —— Thorny lateral branches, thorn width (mm) 0.97±0.04 —— —— Length of thornless lateral branches (cm) <![CDATA[137.47±21.79 a ]]> <![CDATA[114.15±7.75 a ]]> <![CDATA[137.55±11.35 a ]]> Width of thornless lateral branches (mm) <![CDATA[1.31±0.14 a ]]> <![CDATA[1.38±0.08 a ]]> <![CDATA[1.29±0.14 a ]]> Number of leaves per cluster of thornless lateral branches <![CDATA[3.47±0.25 a ]]> <![CDATA[4.16±0.48 a ]]> <![CDATA[4.02±0.17 a ]]> Length of thornless lateral branch leaves (cm) <![CDATA[30.63±4.58 a ]]> <![CDATA[31.93±3.91 a ]]> <![CDATA[36.45±2.54 a ]]> Width of thornless lateral branches (mm) <![CDATA[2.48±0.50 a ]]> <![CDATA[2.93±0.14 a ]]> <![CDATA[3.35±0.02 a ]]>

[0155] Note: The data in the table are the mean ± standard deviation of three replicate experiments. Data in the same column marked with different lowercase letters are significantly different (P < 0.05), indicating that no such data exists. Number of new lateral branches per plant: the average number of new lateral branches per plant after pruning; Number of new lateral branches per branch: the average number of new lateral branches on a single pruned branch.

[0156] Although the above embodiments have provided a detailed description of the present invention, they are only some embodiments of the present invention, and not all embodiments. People can obtain other embodiments based on these embodiments without creative effort, and these embodiments all fall within the protection scope of the present invention.

Claims

1. Application of LrTPP3 gene knockout in inhibiting thorn formation in black wolfberry branches.

2. The application according to claim 1, characterized in that, The nucleotide sequence of the LrTPP3 gene is shown in SEQ ID No.

1.

3. A method for permanently knocking out the LrTPP3 gene based on transient genetic transformation, characterized in that, Includes the following steps: 1) Agrobacterium containing the pTarget2-LrTPP vector was mixed with MS liquid medium containing acetylsuccinone to obtain the infection solution; The pTarget2-LrTPP vector was obtained by inserting a sequence fragment into the original vector pEgP237, and the nucleotide sequence of the sequence fragment is shown in SEQ ID No.

2. 2) After infecting the black wolfberry seedlings with the infection solution described in step 1), they were co-cultured to obtain co-cultured seedlings; 3) The co-cultured seedlings described in step 2) are subjected to heat treatment, and then tissue culture and regeneration line propagation are carried out in sequence to obtain the line with the LrTPP3 gene knocked out.

4. The method according to claim 3, characterized in that, Step 1) The volume ratio of Agrobacterium containing the pTarget2-LrTPP vector to MS liquid culture medium containing acetylsuccinone is 1:1; The concentration of acetylsuccinone in the MS liquid culture medium was 100 µmol / L.

5. The method according to claim 3, characterized in that, Step 2) The conditions for immersion include: immersion at a rotation speed of 90 rpm and a temperature of 25°C for 5 hours; The volume ratio of the inoculum to the number of black goji berry seedlings is 33 mL: 1 seedling. The black goji berry seedlings are then immersed in the inoculum.

6. The method according to claim 3, characterized in that, Step 2) The co-culture conditions include: co-culturing in MS solid medium of 100 µmol / L AS at 28°C in the dark for 2 days.

7. The method according to claim 3, characterized in that, Step 3) The heat treatment conditions include: incubating for 24 h in an environment with a temperature of 37°C and a photoperiod of 16 h light / 8 h darkness, followed by recovery incubation at 25°C for 24 h, and then incubating again at 37°C for 24 h.

8. The method according to claim 3, characterized in that, After the heat treatment described in step 3), the tissue culture and regenerated line propagation are carried out after culturing at 23~27℃ for 2 days.

9. The method according to claim 3, characterized in that, Step 3) The tissue culture conditions include: taking the leaf tips of black wolfberry seedlings (3-7 mm) as explants, culturing them on MS solid medium containing 300 mg / L Cef and 5 mg / L AgNO3 until the seedlings reach a height of 6-8 cm, and changing the MS solid medium every 7 days.

10. The method according to claim 3, characterized in that, Step 3) The conditions for propagating the regenerated line include: inoculating 0.8-1.2 cm stems with apical buds into 1 / 2 MS solid medium to induce rooting and form a line.

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