A gene editing system targeting a pds gene of a celtis sinensis and application thereof

Abstract

The present application relates to the technical field of plant genetic engineering, and provides a gene editing system targeting Lagerstroemia indica PDS gene and application thereof. The CRISPR / Cas9 gene editing technology system is successfully constructed and applied in Lagerstroemia indica for the first time, which fills the technical gap in the field and opens a new way for Lagerstroemia indica functional genomics research. The gene editing strategy of the present application is efficient and accurate, preferably adopts a double-target editing design, and the effect is significantly better than that of the traditional single-point mutation strategy. The Lagerstroemia indica sterile seedling with axillary bud stem segment is innovatively selected as the explant, and the transformation system is stable and reliable. The present application has important practical value for Lagerstroemia indica molecular breeding or gene function research, and has a wide application prospect.

Description

Technical Field

[0001] This invention relates to the field of plant genetic engineering technology, and more specifically, to a gene editing system targeting the PDS gene of Lagerstroemia indica and its application. Background Technology

[0002] Crape myrtle ( Lagerstroemia indica Lagerstroemia indica (also known as Chinese crape myrtle) is a deciduous flowering shrub or small tree belonging to the genus Lagerstroemia indica of the family Lythraceae. As a landscaping plant, Lagerstroemia indica boasts a rich variety of flower colors, a long flowering period, and strong adaptability. Furthermore, it is easy to manage and maintain, and has environmental purification functions, making it an ideal choice for urban landscaping. Lagerstroemia indica originated in China and has a cultivation history of over 1600 years. my country has abundant Lagerstroemia indica germplasm resources. Common flower colors in the genus Lagerstroemia in nature include red, pink, purple, and white, but blue and yellow flowers have not been observed, and there are no double-flowered varieties.

[0003] Breeding research on Lagerstroemia indica in my country started relatively late, mainly employing traditional breeding methods to cultivate new varieties. However, due to the long growth cycle and complex genetic regulation of woody plants, as well as factors such as incompatibility in distant hybridization, it is extremely difficult to cultivate yellow, blue-flowered, or double-petaled varieties using traditional breeding techniques. Current technologies mostly integrate exogenous genes into the plant genome through genetic transformation to improve specific traits. Building upon this, gene editing technology can achieve trait changes without introducing exogenous genes, resulting in homozygous mutants. This method effectively reduces the ethical controversies associated with conventional transgenic plants. Lagerstroemia indica is a woody plant with high polyphenol content, making callus induction difficult and genetic transformation systems highly unstable. Therefore, establishing a suitable and efficient transformation and editing system for Lagerstroemia indica is a pressing technical challenge in this field. Gene editing technology based on the CRISPR / Cas9 system has attracted significant attention due to its advantages of high efficiency, flexibility, and avoidance of exogenous gene integration. This technology uses sgRNA to guide the Cas9 nuclease to target and cleave specific gene sequences, creating DNA double-strand breaks. The cell's own homologous recombination or non-homologous end joining repair mechanisms then allow for precise insertion or deletion of the target gene. CRISPR / Cas9 technology has been successfully applied to various ornamental plants such as petunias, chrysanthemums, lilies, and tea trees, significantly accelerating the plant breeding process. Summary of the Invention

[0004] The purpose of this invention is to provide a gene editing system that targets the PDS gene of Crape Myrtle and its application.

[0005] In order to achieve the objective of the present invention, in a first aspect, the present invention provides an sgRNA that targets the PDS gene of Lagerstroemia indica, which is sgRNA1 and / or sgRNA2. The sgRNA1 targets the nucleotide sequence or its complementary sequence shown in SEQ ID NO:1 in the Crape Myrtle PDS gene; The sgRNA2 targets the nucleotide sequence or its complementary sequence shown in SEQ ID NO:2 in the crape myrtle PDS gene.

[0006] The crape myrtle PDS gene is the nucleotide sequence shown in SEQ ID NO:3 or its complementary sequence.

[0007] Secondly, the present invention provides a gene editing system targeting the PDS gene of Lagerstroemia indica, the system comprising: a) a CRISPR / Cas9 protein or nucleic acid encoding the protein; and, b) The sgRNA or the nucleic acid encoding the sgRNA.

[0008] Furthermore, the system is constructed in one or more plant expression vectors.

[0009] Preferably, in the plant expression vector, the nucleic acid encoding the CRISPR / Cas9 protein is operatively linked to a 35S promoter (formed by tandem sequences shown in SEQ ID NO:4 and SEQ ID NO:5).

[0010] Preferably, the nucleic acid encoding the sgRNA1 is driven by the AtU3d promoter (SEQ ID NO:6); and the nucleic acid encoding the sgRNA2 is driven by the AtU3b promoter (SEQ ID NO:7).

[0011] Thirdly, the present invention provides the application of the gene editing system in the preparation of gene-edited crape myrtle plants.

[0012] Fourthly, this invention provides a method for constructing gene-edited crape myrtle plants, comprising the following steps: (1) Provide stem segments with axillary buds from Lagerstroemia indica as explants; (2) Using Agrobacterium-mediated transformation, the explants were transformed with Agrobacterium carrying the gene editing system; (3) The transformed explants were cultured on a selection medium to obtain resistant shoots; (4) The resistant buds were transferred to a rooting medium to obtain the Lagerstroemia indica gene-edited plant.

[0013] Further, the transformation in step (2) includes: infecting the explants with Agrobacterium suspension under vacuum conditions. For example, treatment under a vacuum of 0.1 MPa for 10-15 minutes.

[0014] Further, the screening medium in step (3) is a WPM medium containing hygromycin. For example, the screening medium contains hygromycin antibiotic at a concentration of 2.5 mg / L and is supplemented with 200 mg / L termethin (TMT) for antibacterial activity.

[0015] Preferably, the screening medium further contains 0.3-0.8 mg / L of 6-BA (6-benzyladenine) and 0.03-0.08 mg / L of IBA (indolebutyric acid), more preferably it contains 0.5 mg / L of 6-BA and 0.05 mg / L of IBA.

[0016] Further, step (4) includes inoculating the resistant buds onto a rooting medium to induce rooting; wherein the rooting medium is 1 / 2 MS medium.

[0017] Preferably, the rooting medium further contains 0.3-0.5 mg / L of IBA, more preferably 0.3 mg / L of IBA.

[0018] Fifthly, the present invention provides the application of the crape myrtle gene-edited plants constructed according to the method in crape myrtle molecular breeding (such as breeding for the purpose of obtaining the crape myrtle albino phenotype) or in crape myrtle gene function research.

[0019] By employing the above technical solution, the present invention has at least the following advantages and beneficial effects: (i) This invention is the first to successfully construct and apply the CRISPR / Cas9 gene editing technology system in Lagerstroemia indica, filling the technological gap in this field and opening up a new avenue for functional genomics research in Lagerstroemia indica.

[0020] (ii) A dual-target editing design is preferred. Multi-target deletion is easier to detect than single-point mutation and completely destroys protein function. By rationally selecting targets, the knockout of target gene fragments can be achieved in a more thorough manner, with significantly better results than traditional single-point mutation strategies.

[0021] (III) The conversion system of this invention is stable and reliable: 1. Innovatively, aseptic seedlings of Lagerstroemia indica with axillary buds were selected as explants. Compared with leaves, axillary bud stem segments not only showed strong resistance to browning but also directly produced seedlings, avoiding the long callus induction period and greatly shortening the breeding cycle. They exhibit strong regeneration ability and high proliferation efficiency, fundamentally overcoming the problem of difficult regeneration of materials such as leaves.

[0022] 2. The optimized Agrobacterium infection process and culture medium formula effectively inhibited explant browning, ensuring the activity and subsequent regeneration capacity of transformed tissues.

[0023] 3. The clearly defined antibiotic screening concentrations and phased culture procedures ensure good reproducibility of the system and establish reliable technical standards.

[0024] (iv) This invention provides a complete solution from target design, vector construction, genetic transformation to mutant identification. This system can not only be used to create specific crape myrtle mutants, but also provides a core tool platform for the future targeted improvement of the ornamental traits, growth characteristics and stress resistance of crape myrtle, which is expected to significantly shorten the crape myrtle breeding cycle and has important scientific research and industrial application value. Attached Figure Description

[0025] Figure 1 The image shows the pYLCRISPRCas9P35s-N-PDS vector in Example 1.

[0026] Figure 2 This is the process of obtaining crape myrtle stem segment genetic transformation and gene-edited plants in Example 2 of the present invention; A is the crape myrtle stem segment explant in the co-culture stage (pre-cultured for 2 days and then infected with Agrobacterium); B is the crape myrtle explant in the callus induction and adventitious bud differentiation stage; C is the crape myrtle tissue culture seedling control; D is the crape myrtle tissue culture seedling exhibiting the albino phenotype.

[0027] Figure 3 This is the sequencing result of the PCR amplification product of the edited plant in Example 2 of this invention. The red highlighted areas indicate the target mutation results.

[0028] Figure 4 This is an electrophoresis diagram of the PCR amplification product of the transgenic positive seedling in Example 3 of the present invention. Detailed Implementation

[0029] This invention aims to provide an sgRNA (guide RNA) targeting the PDS gene of Crape Myrtle, a gene editing system, and its applications.

[0030] The present invention adopts the following technical solution: This invention provides an sgRNA that targets the PDS gene of Crape Myrtle, wherein the targeting sequence of the sgRNA is selected from the nucleotide sequences shown in SEQ ID NO:1 and / or SEQ ID NO:2.

[0031] Preferably, the system comprises two guide RNAs targeting different sites on the Lagerstroemia indica PDS gene, thereby achieving dual-target editing. SEQ ID NO:1 and SEQ ID NO:2 are spaced 460 bp apart on the Lagerstroemia indica PDS gene. This design facilitates targeted deletion of a specific segment of the gene through cell repair mechanisms triggered by dual-target cleavage (to facilitate deletion of the gene segment through dual-target cleavage).

[0032] The present invention also provides a crape myrtle gene editing system, the system comprising a CRISPR / Cas gene editing tool and at least one guide RNA targeting the crape myrtle PDS gene.

[0033] In one specific implementation, the CRISPR / Cas gene editing tool is the CRISPR / Cas9 system.

[0034] Preferably, the system comprises two guide RNAs that target different sites of the crape myrtle PDS gene, forming a dual-target editing system to achieve more efficient gene manipulation.

[0035] Furthermore, the system is constructed in a plant expression vector comprising: a Cas9 protein-coding sequence driven by a constitutive 35S promoter; and an expression unit of one or both guide RNAs driven by a Pol III-type U3 promoter.

[0036] Preferably, the plant expression vector is a pYLCRISPRCas9P35s-N recombinant vector.

[0037] This invention also provides a method for obtaining crape myrtle gene-edited plants using the above-described gene-editing system. The method includes the following steps: Step 1: Provide stem segments with axillary buds from aseptic seedlings of Crape Myrtle as explants; Step 2: Using the gene editing system described above, the explants described in Step 1 are infected by Agrobacterium tumefaciens-mediated infection, with the axillary buds of the stem segments inserted into the co-culture medium with the stem segments facing upwards. Step 3: The explants co-cultured in Step 2 are transferred to a selection medium for resistance screening and preliminary culture. The selection medium is a WPM medium supplemented with plant hormones, containing hygromycin at a concentration of 2.5 mg / L. Step four: The resistant material obtained in step three is sequentially transferred to the selection medium and the rooting medium for culture to induce shoot differentiation and root formation, ultimately obtaining complete gene-edited positive seedlings; wherein, the selection medium is based on WPM medium supplemented with 0.3-0.8 mg / L of 6-BA and 0.03-0.08 mg / L of IBA (preferably with 0.5 mg / L of 6-BA and 0.05 mg / L of IBA), and the rooting medium is based on 1 / 2 MS medium supplemented with 0.3-0.5 mg / L of IBA (preferably with 0.3 mg / L of IBA).

[0038] Further, the method includes the following steps: (1) providing axillary bud stem segments of sterile crape myrtle seedlings as explants; (2) constructing a CRISPR / Cas9 expression vector containing the target sequences of SEQ ID NO:1 and / or SEQ ID NO:2, and transforming it into Agrobacterium; (3) immersing the explants in Agrobacterium infection solution and treating them under a vacuum of 0.1 MPa for 10-15 minutes; (4) placing the transformed explants on a selection medium for culture, wherein the selection medium contains hygromycin antibiotic at a concentration of 2.5 mg / L; (5) sequentially transferring the obtained resistant material into the selection medium for culture to obtain complete gene-edited positive seedlings; wherein the selection medium is based on WPM medium and the rooting medium is based on 1 / 2 MS medium.

[0039] The present invention also provides a crape myrtle plant cell containing the aforementioned vector, wherein the PDS gene in the cell genome is edited.

[0040] This invention also provides the uses of the aforementioned crape myrtle gene editing system. By transferring the crape myrtle CRISPR / Cas9 gene editing vector into crape myrtle, gene-edited crape myrtle plants (crape myrtle gene mutant materials) can be obtained. The system can be used for crape myrtle gene function research, gene expression regulation analysis, or applied in crape myrtle molecular design breeding to accelerate the improvement of traits such as flower color, plant type, and resistance. The obtained crape myrtle gene mutant materials can be used for crape myrtle gene function research or crape myrtle molecular breeding.

[0041] The following embodiments provide an sgRNA gene editing system targeting the PDS gene of Lagerstroemia indica, which is used to generate gene-edited plants of Lagerstroemia indica, thereby achieving targeted editing of the PDS gene of Lagerstroemia indica and helping to improve the progress of research on gene function of Lagerstroemia indica.

[0042] The following examples use Lagerstroemia indica ( Lagerstroemia indica The variety is 'Binfen Jiaren'. Healthy, semi-lignified young shoots were taken, disinfected, and inoculated onto 1 / 2 MS medium. They were cultured at a temperature of 24-27 ℃, a light intensity of 1580-4290 Lux, and a light intensity of 14 h / d to obtain sterile tissue culture seedlings with uniform growth, which served as the source of explants.

[0043] The target gene involved in the following examples is the phytoene dehydrogenase gene of Lagerstroemia indica (LiPDS gene, nucleotide sequence as shown in SEQ ID NO:3). Two specific sgRNA target sites—sgRNA1 and sgRNA2—were designed and synthesized, having sequences as shown in SEQ ID NO:1 and SEQ ID NO:2, or their complementary sequences, respectively. This invention uses the plant binary expression vector pYLCRISPRCas9P35s-N based on the CRISPR / Cas9 system (vector map shown in...). Figure 1 For reference, see Ma X, Zhang Q, Zhu Q, et al. A Robust CRISPR / Cas9 System for Convenient, High-Efficiency Multiplex Genome Editing in Monocot and Dicot Plants[J].Molecular Plant,2015,8(8):1274-1284.DOI:10.1016 / j.molp.2015.04.007), provided by Professor Liu Yaoguang's research group at South China Agricultural University. It includes the coding sequence of the guide RNA targeting the PDS gene of chestnut plants, the Cas9 gene expression cassette driven by a constitutive promoter (35S promoter, whose nucleotide sequence is composed of the sequences shown in SEQ ID NO:4 and SEQ ID NO:5 in tandem), and the sgRNA expression unit driven by a Pol III promoter (U3 promoter, for example, the nucleotide sequence of the AtU3d promoter is shown in SEQ ID NO:6, and the nucleotide sequence of the AtU3b promoter is shown in SEQ ID NO:7).

[0044] After transforming the constructed pYLCRISPRCas9P35s-N-PDS gene editing vector into Agrobacterium, Lagerstroemia indica was transformed into Lagerstroemia indica using stem segments as explants. Positive gene editing results were obtained after screening and culture.

[0045] The WPM culture medium used in the following examples was purchased from CAISSON LABS, model WPP03.

[0046] The following examples are used to illustrate the present invention, but are not intended to limit the scope of the invention. Unless otherwise specified, the technical means used in the examples are conventional means well known to those skilled in the art, and the raw materials used are all commercially available products.

[0047] Example 1: Construction of a dual-target CRISPR / Cas9 plant expression vector exist LiPDSOne target was designed for the ORF 5' region and one for the functional domain of the gene sequence, ensuring that mutations at either target would result in loss of function, or that the sequence between the two targets would be knocked out. The GC percentage of the target should ideally not be lower than 40%. There should be no more than four consecutive T's within the target to prevent RNA Pol III from using them as transcription termination signals. An online tool (CRISPR Design Tool) was used to design sgRNA sequences targeting the LiPDS gene, synthesize the sgRNA, and verify its sequence correctness. The website for target screening and primer design is http: / / skl.scau.edu.cn / targetdesign / .

[0048] Design and synthesize target primer adapters complementary to the BsaI enzyme cleavage site, and anneal to obtain well-bound dual-linked heads (wherein, the annealing products of the sequences shown in SEQ ID NO:8 and 9 encode sgRNA1, and the annealing products of the sequences shown in SEQ ID NO:10 and 11 encode sgRNA2). The pYLgRNA-AtU3b and pYLgRNA-AtU3d plasmids (from the research group of Liu Yaoguang at South China Agricultural University, see reference Ma X, Zhang Q, Zhu Q, et al. A Robust CRISPR / Cas9 System for Convenient, High-Efficiency Multiplex Genome Editing in Monocot and DicotPlants[J].Molecular Plant,2015,8(8):1274-1284.DOI:10.1016 / j.molp.2015.04.007) were digested with enzymes, and the digested pYLgRNA-AtU3b and pYLgRNA-AtU3d plasmids were ligated with their corresponding adapters to obtain circular plasmids. The ligation product was linearized and amplified in large quantities through two rounds of PCR amplification. Next, based on the BsaI restriction site, a "cleansing-while-ligating" process was performed to ligate the dual-target site and sgRNA expression cassette into the final vector pYLCRISPRCas9P35s-N (containing the kanamycin resistance gene). The pYLCRISPRCas9P35s-N-PDS gene editing vector map was successfully constructed as shown below. Figure 1 As shown.

[0049] Example 2: Genetic transformation method of crape myrtle stem segments and acquisition of gene-edited plants 1. The pYLCRISPRCas9P35s-N-PDS vector constructed in Example 1 was transformed into Agrobacterium EHA105. A single colony was picked and placed in 2 mL of LB liquid medium and cultured in a shaking incubator at 200 rpm and 28 ℃ for about 12 h. The colonies were then detected by PCR to detect single positive colonies. The PCR product length was 875 bp.

[0050] 2. Transfer the positive bacterial culture to 50 mL of LB liquid medium and incubate in a shaking incubator at 200 rpm and 28 ℃ for about 12 h, followed by centrifugation at 5000 rpm for 10 min. Discard the supernatant in a clean bench and resuspend the collected bacterial cells in a solution containing sterile water, 10 mmol / L 2-(N-morpholino)ethanesulfonic acid (MES), 10 mmol / L magnesium chloride hexahydrate (MgCl2·6H2O), and 200 μmol / L acetylsyleugenone (AS). Use the resuspended solution as a control. Measure the bacterial concentration using a UV spectrophotometer and adjust the concentration to 1.0-1.5. Incubate at 28 ℃ in the dark for 2 h to obtain the infection solution.

[0051] 3. Using stem segments with axillary buds from aseptic 'Binfen Jiaren' seedlings as explants, inoculated into WPM medium and pre-cultured in the dark for 2 days. Then, under aseptic conditions, the explants and bacterial solution were placed together in the infection solution, ensuring full contact between the stem segments and the bacterial solution, and sealed with sealing film. Infection was carried out under a vacuum of 0.1 MPa for 10-15 minutes, with vacuuming performed intermittently during the process to alleviate the stress of prolonged negative pressure on the material. The infection temperature was 28 ℃. After completion, the vacuum pump was turned off, and the pressure was slowly released to prevent damage to the stem segments from sudden decompression.

[0052] 4. After infection, remove the bacterial solution attached to the explants and inoculate them onto WPM co-culture medium supplemented with 0.5 mg / L 6-BA and 0.05 mg / L IBA. Co-culture is carried out in the dark. After co-culture, the explants are washed with sterile water containing 100 mg / L termetidine (TMT) for 3 min under sterile conditions, then washed three times with sterile water. After drying, they are inoculated onto selection medium and selected under light conditions. The selection medium used is WPM medium containing 0.5 mg / L 6-BA, 0.05 mg / L IBA, and 2.5 mg / L hygromycin, with 200 mg / L TMT added for antibacterial effect. The medium is changed every 14 days.

[0053] The differentiation medium used to induce shoot differentiation was the same as the screening medium mentioned above; the rooting medium used was 1 / 2 MS medium containing 0.3 mg / L IBA.

[0054] 5. After 60 days of screening and culture, using uninfected sterile seedlings as controls, DNA was extracted from the leaves of positive plants using the Vazyme Plant Genomic DNA Extraction Kit (DC104-01). PCR detection was performed using specific primers for the target gene. PCR amplification was performed using vector-specific primers (product length 875 bp). Transgenic positive plants were identified, and the positive rate was calculated. Figure 2 It can be seen that the edited plants exhibited an albino phenotype approximately 45 days after the transformation.

[0055] 6. For the DNA of positive seedlings, target site sequence amplification was performed using target detection primers. The PCR amplification products were then subjected to first-generation Sanger sequencing. The results showed that gene editing occurred in one of the 72 transformed plants. Near target site 1 (01), there were two base mismatches and one base deletion; near target site 2 (02), there was one base mismatch. Figure 3 As shown, mutants with PDS-deficient phenotypes were obtained using the CRISPR / Cas9 system. Sequencing revealed that the mutant had a mutation and deletion near the expected target site, with a gene editing efficiency of 1.39%.

[0056] The LB liquid medium contains: 25 g / L Luria-Bertani (LB) medium, 50 mg / L kanamycin, 25 mg / L rifampin, and the culture temperature is 27-29 ℃.

[0057] The pre-culture and co-culture media were: WPM medium + 0.5 mg / L 6-BA, 0.05 mg / L IBA + 30 g / L sucrose + 7 g / L agar, pH 5.8-6.0.

[0058] The screening medium includes: WPM medium, 0.5 mg / L 6-BA, 0.05 mg / L IBA, 2.5 mg / L Hygromycin, and 200 mg / L TMT for antibacterial effect; The colony PCR primers are vector-specific primers with the following sequences: 5'-GTCGTGCTCCACATGTTGACCGG-3' and 5'-CCCGACATAGATGCAATAACTTC-3'.

[0059] The target detection primers have the following sequences: target 1 detection primer sequences are 5'-TGTAGTCTCAAGTTGGGATC-3' and 5'-TGGGAATACATTTTTTTTAT-3'; target 2 detection primer sequences are 5'-CTTGGAAAACAATATAAAGT-3' and 5'-ATCACATCTGTATTATAGGC-3'.

[0060] Example 3: Effects of different explant types on genetic transformation and regeneration of Lagerstroemia indica 1. Using aseptic tissue culture seedlings of the crape myrtle variety 'Binfen Jiaren' as material, aseptic leaves and aseptic stem segments with axillary buds were used as explants to compare the effects of different explants on callus induction, adventitious bud differentiation, and genetic transformation efficiency. Two to three wounds were made on the aseptic leaves, and the aseptic stem segments with axillary buds were cut to a length of 1.0-1.5 cm, retaining 1-2 pairs of axillary buds.

[0061] 2. Using leaves as explants, Agrobacterium-mediated transformation of the pCAMBIA1300super vector was performed (see reference Lan-Ying L, EMK, Burgund B, et al. Novel plant transformation vectors containing the superpromoter[J]. Plant physiology, 2007, 145 (4): 1294-300). Pre-culture time (0 d, 1 d, 2 d, 3 d) and bacterial concentration OD were set. 600 A four-factor, four-level orthogonal experiment was conducted with 16 combinations, including (0.4, 0.6, 0.8, 1.0), infection time (5 min, 10 min, 15 min, 20 min), and co-culture time (1 d, 2 d, 3 d, 4 d). Each group treated 30 explants, with 3 replicates, and the results were statistically analyzed after 35 days of culture.

[0062] 3. Using stem segments with axillary buds as explants, Agrobacterium-mediated transformation of pHDE-35S::RUBY vector was performed. Pre-culture for 1 day and co-culture for 2 days were conducted, with different bacterial concentrations (OD) set. 600 Fifteen combinations were used, including explant concentrations of 0.4, 0.6, 0.8, 1.2, and 1.5, and infection times of 5, 10, and 15 minutes. Ten explants were treated in each group, with three replicates. Results were collected after 60 days of culture.

[0063] 4. Explants were pre-cultured in WPM medium supplemented with 0.5 mg / L 6-BA and 0.05 mg / L IBA. Leaves were infected using a shaker, while stem segments were infected under vacuum at 0.1–0.5 MPa. After infection, excess bacterial solution was aspirated, and the explants were transferred to the same culture medium for co-culture in the dark.

[0064] 5. After co-culture, wash the explants with sterile water containing 100 mg / L termethin, rinse three times with sterile water, and transfer them to WPM medium containing 0.5 mg / L 6-BA and 0.05 mg / L IBA. Add 200 mg / L termethin to inhibit bacterial growth. Change the medium every 14 days.

[0065] 6. The results showed that when leaves were used as explants, the callus induction rate was the highest, only 53.3±1.9%. All combinations failed to differentiate adventitious buds, exhibited severe browning, and all PCR tests were negative, resulting in no positive plants. When stem segments with axillary buds were used as explants, the callus induction rate and adventitious bud induction rate were both 100% in all combinations, with low browning and good growth. (OD...) 600 Under conditions of 1.2-1.5, PCR amplification yielded the following results: Figure 4 The positive band results in the sample indicate that transgenic positive plants have been successfully obtained.

[0066] This indicates that stem segments with axillary buds have significantly better regeneration capacity, browning resistance, and genetic transformation efficiency than leaves, making them the most suitable explants for the gene editing system of this invention.

[0067] Although the present invention has been described in detail above with general descriptions and specific embodiments, modifications or improvements can be made to it, which will be obvious to those skilled in the art. Therefore, all such modifications or improvements made without departing from the spirit of the present invention fall within the scope of protection claimed by the present invention.

Claims

1. An sgRNA targeting the crape myrtle PDS gene, characterized in that, They are sgRNA1 and sgRNA2; The sgRNA1 targets the nucleotide sequence shown in SEQ ID NO:1 in the Crape Myrtle PDS gene; The sgRNA2 targets the nucleotide sequence shown in SEQ ID NO:2 in the crape myrtle PDS gene.

2. A gene editing system targeting the PDS gene of Lagerstroemia indica, characterized in that, The system comprises: a) a CRISPR / Cas9 protein or nucleic acid encoding the protein; and, b) The sgRNA of claim 1 or the nucleic acid encoding the sgRNA.

3. The gene editing system according to claim 2, characterized in that, The system is constructed in one or more plant expression vectors.

4. The gene editing system according to claim 3, characterized in that, In the plant expression vector, the nucleic acid encoding the CRISPR / Cas9 protein is operatively linked to a 35S promoter; The nucleic acid encoding sgRNA1 is driven by the AtU3d promoter; the nucleic acid encoding sgRNA2 is driven by the AtU3b promoter.

5. The application of the gene editing system according to any one of claims 2-4 in the preparation of gene-edited plants of Lagerstroemia indica.

6. A method for constructing gene-edited plants of Lagerstroemia indica, characterized in that, Includes the following steps: (1) Provide stem segments with axillary buds from Lagerstroemia indica as explants; (2) Using Agrobacterium-mediated transformation, the explants are transformed with Agrobacterium carrying the gene editing system of any one of claims 2-4; (3) The transformed explants were cultured on a selection medium to obtain resistant shoots; (4) The resistant buds were transferred to a rooting medium to obtain the Lagerstroemia indica gene-edited plant.

7. The method according to claim 6, characterized in that, The transformation in step (2) includes: infecting the explants in an Agrobacterium suspension under vacuum conditions.

8. The method according to claim 6 or 7, characterized in that, The screening medium mentioned in step (3) is WPM medium containing hygromycin.

9. The method according to claim 8, characterized in that, The screening medium also contains 0.3-0.8 mg / L of 6-BA and 0.03-0.08 mg / L of IBA.

10. The method according to claim 6 or 7, characterized in that, Step (4) includes inoculating the resistant buds onto a rooting medium to induce rooting; wherein the rooting medium is 1 / 2 MS medium.

11. The method according to claim 10, characterized in that, The rooting medium also contains 0.3-0.5 mg / L of IBA.

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