Electric shock transformation method of rubber tree leaf protoplast large plasmids

Abstract

The invention discloses a rubber tree leaf protoplast large plasmid electric shock transformation method. The method comprises the following steps: constructing a gene editing vector; selecting leaves in the bronze stage, soaking the leaves in mannitol, cutting off main leaf veins, cutting the leaves into long and thin strips, and performing enzymolysis treatment; terminating enzymolysis when the volume is equal to W5; adding plasmid DNA into a centrifugal tube, adding the protoplast suspension, transferring into an electric shock cup for ice bath and electric shock transformation, adding WI for resuspension, and culturing; genome DNA is extracted, and PCR and high-throughput sequencing are carried out; and identifying whether the test-tube seedlings of the rubber trees are edited or not by utilizing the reference genome sequence of the rubber trees and combining high-throughput sequencing. The method for completing large plasmid (more than 15 k) transformation on the protoplast cells of the rubber tree through an electric shock transformation mode is established for the first time, recipient bacteria do not need to be transformed in the process, toxicity of plasmids to the recipient bacteria is prevented, and technical support is provided for rapid identification of large plasmid gene functions such as rubber tree transgenosis and gene editing.

Description

Technical Field

[0001] This invention relates to the field of DNA transformation technology, specifically to a method for electroporation transformation of large plasmids from protoplasts of rubber tree leaves. Background Technology

[0002] Transient transformation of protoplasts is currently the most widely used transformation method for rubber trees. However, existing transient transformation methods using rubber tree cells as transformation recipients can only use small DNA fragments or small vectors (below 8k) as transformation donors. As the size of the transformation donor DNA increases, the transformation efficiency decreases exponentially. Furthermore, PEG is a chemical reagent that is poorly soluble in water, which is time-consuming to prepare and carries the risk of deterioration leading to performance degradation. The entire transformation process is also quite cumbersome.

[0003] Electroporation conversion does not require chemical reagents and uses physical equipment such as an electroporator. After parameter settings, the conversion conditions can be kept consistent. It is simple to operate, fast and efficient. However, there are currently no reports of successful electroporation conversion of large DNA fragments (over 15k) from rubber tree protoplasts.

[0004] However, the gene-editing vectors for rubber trees often exceed 15 kJ, or even reach around 20 kJ. Furthermore, stable genetic transformation of rubber trees is complicated, labor-intensive, costly, and time-consuming. To ensure the effectiveness of stable transformation, it is necessary to conduct preliminary verification of gene (vector) function through transient transformation before stable transformation. Therefore, it is urgent to establish a transformation method for large plasmid DNA from protoplasts of rubber tree leaves.

[0005] However, existing technologies have the following drawbacks;

[0006] (1) Limited information carried: Small fragments or small plasmids have a small capacity and may not be able to provide enough functional gene elements, including upstream and downstream regulatory sequences, multi-gene functional complexes, and long fragment gene combinations. For the study of the complex biological functions or genetic improvement of rubber trees, it is difficult to quickly, comprehensively and accurately identify the function and regulatory mechanism of genes in rubber trees because they cannot provide enough complete functional gene modules.

[0007] (2) Genetic transformation of rubber trees is time-consuming, labor-intensive, and inefficient. In order to assess the transformation effect before transformation and reduce the risk of ineffective transformation, it is necessary to conduct preliminary identification and evaluation of vector function through transient transformation before stable transformation. The donor vector size of all reported transient transformations of rubber trees is less than 8k, and the transformation efficiency decreases exponentially with the increase of vector size. However, the size of transgenic or gene-editing plasmids for stable transformation of rubber trees exceeds 10k, especially gene-editing vectors, which are all above 15k and even reach about 20k. There is an urgent need to develop new methods for transient transformation of large plasmid DNA for preliminary functional identification of transgenic and gene-editing plasmids (large fragment DNA). Summary of the Invention

[0008] To address the shortcomings of existing technologies, this invention provides a method for electroporation transformation of large plasmids from rubber tree leaf protoplasts. This method solves the problems of existing transient transformation methods that use rubber tree cells as transformation recipients, which can only use small DNA fragments or small vectors (below 8k) as transformation donors. These methods are time-consuming to prepare, carry the risk of deterioration leading to performance degradation, and are cumbersome throughout the entire transformation process. Furthermore, there are currently no reports of successful electroporation transformation of large DNA fragments (above 15k) from rubber tree protoplasts.

[0009] To achieve the above objectives, the present invention provides the following technical solution: a method for electroporation transformation of large plasmids in protoplasts of rubber tree leaves, comprising the following steps:

[0010] Step 1: Vector Construction

[0011] The gene editing vector of this invention was constructed using the pCAMBIA1300-2×35SCas9-HbU6.2 rubber tree gene editing vector as a backbone.

[0012] Step 2: Genetic transformation

[0013] Step 2.1: Perform plasmid extraction and concentration detection;

[0014] Step 2.2: Extraction of protoplasts

[0015] Leaves at the bronze stage were selected, soaked in 0.6 M mannitol for 10 min, the main vein was removed with a scalpel, and the leaves were cut into thin strips 0.5–1 mm wide. The strips were then transferred to enzyme solution E2 and enzymatically digested in a dark incubator at 28 °C on a shaker at 60 r / min for 5 h. The digestion was terminated with an equal volume of W5, filtered, collected in 50 ml round-bottom centrifuge tubes, centrifuged at 800 rpm for 3 min and the supernatant was discarded. The leaves were rinsed once with W5, centrifuged again and the supernatant was discarded. The leaves were then resuspended in PBS buffer.

[0016] Step 2.3: Electroporation of protoplasts

[0017] Add 20 μl of plasmid DNA to a 2 ml round-bottom centrifuge tube, add 300 μl of the above protoplast suspension, mix well, transfer to an electroporation cuvette, incubate on ice for 5 min, electroporate, transfer to a 1.5 ml centrifuge tube, incubate at room temperature for 15 min, centrifuge at 800 rpm for 3 min and discard the supernatant, rinse once with WI, centrifuge and discard the supernatant, add 1 ml of WI to resuspend, transfer to a 12-well cell culture plate and incubate in a dark room at 26℃ for 48 h;

[0018] Step 3: Gene Editing Identification

[0019] Step 3.1: DNA extraction and PCR

[0020] Genomic DNA was extracted from protoplasts that had been transformed and cultured in a dark chamber at 26°C for 48 hours. Using the genomic DNA as a template, fragments containing the target region were amplified using primer pairs T10-msF1: ggagtgagtacggtgtgcCAGTCTATGCTGGAGTTAG and T10-msR1: gagttggatgctggatggGCTTTGCTCTGATCTGCAG, and then high-throughput sequencing was performed.

[0021] Step 3.2: Editing and Appraisal

[0022] Using a reference genome sequence of a rubber tree, combined with high-throughput sequencing, we can identify whether the transformed protoplasts have undergone editing.

[0023] Preferably, in step 1, the method for preparing the gene editing vector includes:

[0024] 1) Anneal the PDS-T10F forward primer and the PDS-T10R reverse primer to obtain the double-stranded target sequence;

[0025] The nucleotide sequence of the forward primer of PDS-T10F is 5'-ATTGATGAGATCCATTCTTCTGC-3', and the nucleotide sequence of the reverse primer of PDS-T10R is 3'-TACTCTAGGTAAGAAGACGCAAA-5'.

[0026] 2) The vector is digested with AarI enzyme and then ligated with the double-stranded target sequence;

[0027] 3) Take 10 μL of the ligation product obtained in step 2) and add it to 100 μL of DH5α competent cells. After gently mixing, place it on ice and immediately heat shock it in a metal bath for 45 seconds. Then immediately place it on ice and let it stand. Transfer it to 500 µL of antibiotic-free LB liquid medium, mix it by inverting, and place it on a shaker to revive the cells. Then take 100 μL of the bacterial culture and spread it evenly on an LB solid medium plate containing kanamycin resistance. After drying, invert it and incubate it in an incubator.

[0028] The next day, after bacterial colonies grew on the plates, colony PCR was performed using primers PDS-T10F+GFP-seq-R (5'AGCTCACTCATTAGGCACCC-3'). Clones that could amplify a specific 273 bp were selected for sequencing identification.

[0029] Preferably, in step 1), the annealing method includes: rinsing the dry powders of the PDS-T10F forward primer and the PDS-T10R reverse primer using dd... Dilute with water to 100 μM, then take 20 μL of each and put them into PCR tubes to mix. Then place them in a PCR instrument for annealing, and cool to room temperature after the process is complete.

[0030] The annealing conditions include a temperature of 100℃ and a time of 5 minutes.

[0031] Preferably, in step 2), the enzyme digestion system comprises: 5 μL of 10x AarI buffer, 1 μL of 50x oligonucleotide, 1 μL of AarI, 1 μg of pCAMBIA1300-2×35SCas9-HbU 6.2, and dd... Bring the volume to 50 μL;

[0032] The enzyme digestion conditions include: a temperature of 37°C and a time of 5 hours.

[0033] Preferably, in step 2), the ligation system comprises: 50 ng of purified vector fragment, 1 μL of 10x T4 ligase buffer, 7 μL of annealed double-stranded target sequence, 0.5 μL of T4 ligase, and supplemented with dd... Up to 10 μL;

[0034] The conditions for the connection include: room temperature and a time of 1 hour.

[0035] Preferably, in step 2.1, the identified positive clones are sterilized, plasmids are extracted, and the plasmid concentration is detected.

[0036] Preferably, in step 2.2, E2 comprises 20 mM potassium chloride, 10 mM calcium chloride, 0.6 M mannitol, 10 mM MES, 1 g / L BSA, 15 g / L cellulase, 7.5 g / L ionase, and pH 5.7.

[0037] Preferably, in step 2.2, W5 comprises 154 mM sodium chloride, 5 mM potassium chloride, 125 mM calcium chloride, 5 mM glucose, and 2 mM MES.

[0038] Preferably, in step 2.3, the electrocup is preheated in an ice bath for 30 minutes, the electrocup parameters of the electroconverter are set to 400V, the LV electrocup is dried with a paper towel before electrocup is electrocuted, and then the electrocup is placed in the electrocup tank for electrocup electrocution.

[0039] Preferably, in step 2.3, WI includes 0.5M mannitol, 4mM MES, and 20mM potassium chloride.

[0040] This invention provides a method for the electroporation transformation of large plasmids in rubber tree leaf protoplasts. It has the following beneficial effects:

[0041] (1) Carrying more genes: Large plasmids have a large capacity and can carry multiple target genes, regulatory elements and marker genes. In the genetic improvement of rubber trees, multiple genes related to excellent traits (such as high rubber yield, strong resistance, etc.) can be introduced at the same time to achieve multi-gene synergistic regulation and accelerate the process of rubber tree variety improvement.

[0042] (2) Convenient and fast: There is no need to transfer plasmids into intermediate strains. Just add protoplasts directly into electroporation cuvettes, put them into electroporation apparatus, press the switch, and the transformation is completed in seconds. The process is convenient, time-saving and fast.

[0043] (3) Complete gene environment: Large plasmids can contain complete upstream and downstream regulatory sequences of the target gene, providing a gene environment closer to the natural state for studying the function of rubber tree genes; it helps to study the gene expression regulation mechanism in rubber trees more accurately and gain a deeper understanding of the growth, development, latex production and other physiological processes of rubber trees.

[0044] (4) Facilitates screening and identification: Large plasmids usually carry multiple screening marker genes (such as antibiotic resistance genes, fluorescent protein genes, etc.), which can more conveniently and accurately screen out positive transformants during the screening process after transformation; by using corresponding screening reagents or detection methods, rubber tree cells or plants containing foreign genes can be quickly identified, thereby improving screening efficiency.

[0045] (5) Avoid incompatibility between plasmid and intermediate host Agrobacterium: Traditional transient and stable transformations require plasmid to be transferred into Agrobacterium first, which requires purchasing suitable Agrobacterium competent cells. The transformation steps are relatively cumbersome and complicated. More importantly, the transformation plasmid may be toxic to Agrobacterium, leading to transformation failure, or the immune mechanism of Agrobacterium may modify the plasmid, affecting replication and plasmid function. The transformation efficiency is not 100%, and positive transformation strains must be screened and identified.

[0046] This invention uses gene-editing vectors as transformation donors, which can classify the proportion of transgenic cells and the type of editing in positive protoplasts. Attached Figure Description

[0047] Figure 1 This is a diagram of the gene editing vector used for transformation in this invention;

[0048] Figure 2 This is a schematic diagram of the HbPDS gene structure of the present invention;

[0049] Figure 3 This is a PCR (T10-msF1+R1) detection image of high-throughput sequencing fragments after DNA extraction according to the present invention;

[0050] Figure 4 This is a diagram showing the mutation sequence and proportions of the present invention.

[0051] Figure 5 This is a complete experimental flowchart of the present invention. Detailed Implementation

[0052] The technical solution of the present invention will now be clearly and completely described with reference to the accompanying drawings. Obviously, the described embodiments are only a part of the embodiments of the present invention, and not all of them. All other embodiments obtained by those skilled in the art based on the embodiments of the present invention without creative effort are within the scope of protection of the present invention.

[0053] Please see the appendix Figure 1 -Appendix Figure 5 This invention provides a method for electroporation transformation of large plasmids in protoplasts of rubber tree leaves, comprising the following steps:

[0054] Step 1: Vector Construction

[0055] A gene editing vector was constructed using the pCAMBIA1300-2×35SCas9-HbU6.2 rubber tree gene editing vector as the backbone;

[0056] Furthermore, in step 1, the method for preparing the gene editing vector includes:

[0057] 1) Anneal the PDS-T10F forward primer and the PDS-T10R reverse primer to obtain the double-stranded target sequence;

[0058] The nucleotide sequence of the forward primer of PDS-T10F is 5'-ATTGATGAGATCCATTCTTCTGC-3', and the nucleotide sequence of the reverse primer of PDS-T10R is 3'-TACTCTAGGTAAGAAGACGCAAA-5'.

[0059] 2) The vector is digested with AarI enzyme and then ligated with the double-stranded target sequence;

[0060] 3) Take 10 μL of the ligation product obtained in step 2) and add it to 100 μL of DH5α competent cells. After gently mixing, place it on ice and immediately heat shock it in a metal bath for 45 seconds. Then immediately place it on ice and let it stand. Transfer it to 500 µL of antibiotic-free LB liquid medium, mix it by inverting, and place it on a shaker to revive the cells. Then take 100 μL of the bacterial culture and spread it evenly on an LB solid medium plate containing kanamycin resistance. After drying, invert it and incubate it in an incubator.

[0061] The next day, after bacterial colonies grew on the plates, colony PCR was performed using primers PDS-T10F+GFP-seq-R (5'AGCTCACTCATTAGGCACCC-3'). Clones that could amplify a specific 273 bp were selected for sequencing identification.

[0062] Further, in step 1), the annealing method includes: annealing the dry powders of the PDS-T10F forward primer and the PDS-T10R reverse primer using dd... Dilute with water to 100 μM, then take 20 μL of each and put them into PCR tubes to mix. Then place them in a PCR instrument for annealing. After the annealing is complete, cool to room temperature to obtain the final product.

[0063] The annealing conditions include a temperature of 100℃ and a time of 5 minutes.

[0064] Further, in step 2), the enzyme digestion system comprises: 5 μL of 10x AarI buffer, 1 μL of 50x oligonucleotide, 1 μL of AarI, 1 μg of pCAMBIA1300-2×35SCas9-HbU 6.2, and dd... Bring the volume to 50 μL;

[0065] The enzyme digestion conditions include: a temperature of 37°C and a time of 5 hours.

[0066] Further, in step 2), the ligation system includes: 50 ng of purified vector fragment, 1 μL of 10x T4 ligase buffer, 7 μL of annealed double-stranded target sequence, 0.5 μL of T4 ligase, and supplemented with dd... Up to 10 μL;

[0067] The conditions for the connection include: room temperature and a time of 1 hour.

[0068] Further, in step 3), the ice bath time is 30 min, the metal bath at 42 ℃ is heat-shocked for 45 sec, the static time is 2 min, the shaker at 37 ℃ is placed on a shaker at 180 rpm for 1 h, the culture temperature in the incubator is 37 ℃, and the culture time is 16 h.

[0069] Step 2: Genetic transformation

[0070] Step 2.1: Perform plasmid extraction and concentration detection;

[0071] Further, in step 2.1, the identified positive clones are sterilized, plasmids are extracted, and the plasmid concentration is detected.

[0072] Step 2.2: Extraction of protoplasts

[0073] Leaves at the bronze stage were selected, soaked in 0.6 M mannitol for 10 min, the main vein was removed with a scalpel, and the leaves were cut into thin strips 0.5–1 mm wide. The strips were then transferred to enzyme solution E2 and enzymatically digested in a dark incubator at 28 °C on a shaker at 60 r / min for 5 h. The digestion was terminated with an equal volume of W5, filtered, collected in 50 ml round-bottom centrifuge tubes, centrifuged at 800 rpm for 3 min and the supernatant was discarded. The leaves were rinsed once with W5, centrifuged again and the supernatant was discarded. The leaves were then resuspended in PBS buffer.

[0074] Further, in step 2.2, E2 includes 20mM potassium chloride, 10mM calcium chloride, 0.6M mannitol, 10mM MES, 1g / L BSA, 15g / L cellulase, 7.5g / L ionase, and pH 5.7;

[0075] Further, in step 2.2, W5 includes 154 mM sodium chloride, 5 mM potassium chloride, 125 mM calcium chloride, 5 mM glucose, and 2 mM MES.

[0076] Step 2.3: Electroporation of protoplasts

[0077] Add 20 μl of plasmid DNA to a 2 ml round-bottom centrifuge tube, add 300 μl of the above protoplast suspension, mix well, transfer to an electroporation cuvette, incubate on ice for 5 min, electroporate, transfer to a 1.5 ml centrifuge tube, incubate at room temperature for 15 min, centrifuge at 800 rpm for 3 min and discard the supernatant, rinse once with WI, centrifuge and discard the supernatant, add 1 ml of WI to resuspend, transfer to a 12-well cell culture plate and incubate in a dark room at 26℃ for 48 h;

[0078] Furthermore, in step 2.3, the electrocup is preheated in an ice bath for 30 minutes, the electroconversion device is set to 400V, the electrocup is dried with a paper towel before electrocution, and then the electrocup is placed in the electrocution tank for electrocution.

[0079] Further, in step 2.3, WI includes 0.5M mannitol, 4mM MES, and 20mM potassium chloride.

[0080] Step 3: Gene Editing Identification

[0081] Step 3.1: DNA extraction and PCR

[0082] Genomic DNA was extracted from protoplasts that had been transformed and cultured in a dark chamber at 26°C for 48 hours. Using the genomic DNA as a template, fragments containing the target region were amplified using primer pairs T10-msF1: ggagtgagtacggtgtgcCAGTCTATGCTGGAGTTAG and T10-msR1: gagttggatgctggatggGCTTTGCTCTGATCTGCAG, and then high-throughput sequencing was performed.

[0083] Step 3.2: Editing and Appraisal

[0084] Using a reference genome sequence of a rubber tree, combined with high-throughput sequencing, we can identify whether the transformed protoplasts have undergone editing.

[0085] The results of protoplast editing were identified, as shown in Table 1.

[0086] Table 1:

[0087] Voltage strength (low voltage) Plasmid concentration (ng / μl) Total number of samples Edit sample number 400V 400+ 8 5 400V 1000+ 15 7

[0088] The sgRNA target of the HbPDS gene designed in this invention is located on the 12th exon, and its sequence is as follows:

[0089] PDS-T10: -5'ATGAGATCCATTCTTCTGC-3';

[0090] Although embodiments of the invention have been shown and described, it will be understood by those skilled in the art that various changes, modifications, substitutions and alterations can be made to these embodiments without departing from the principles and spirit of the invention, the scope of which is defined by the appended claims and their equivalents.

Claims

1. A method for electroporation transformation of large plasmids in protoplasts of rubber tree leaves, characterized in that, Includes the following steps: Step 1: Vector Construction The gene editing vector of this invention was constructed using the pCAMBIA1300-2×35SCas9-HbU6.2 rubber tree gene editing vector as a backbone. Step 2: Genetic transformation Step 2.1: Perform plasmid extraction and concentration detection; Step 2.2: Extraction of protoplasts Leaves at the bronze stage were selected, soaked in 0.6 M mannitol for 10 min, the main vein was removed with a scalpel, and the leaves were cut into thin strips 0.5–1 mm wide. The strips were then transferred to enzyme solution E2 and enzymatically digested in a dark incubator at 28 °C on a shaker at 60 r / min for 5 h. The digestion was terminated with an equal volume of W5, filtered, collected in 50 ml round-bottom centrifuge tubes, centrifuged at 800 rpm for 3 min and the supernatant was discarded. The leaves were rinsed once with W5, centrifuged again and the supernatant was discarded. The leaves were then resuspended in PBS buffer. Step 2.3: Electroporation of protoplasts Add 20 μl of plasmid DNA to a 2 ml round-bottom centrifuge tube, add 300 μl of the above protoplast suspension, mix well, transfer to an electroporation cuvette, incubate on ice for 5 min, electroporate, transfer to a 1.5 ml centrifuge tube, incubate at room temperature for 15 min, centrifuge at 800 rpm for 3 min and discard the supernatant, rinse once with WI, centrifuge and discard the supernatant, add 1 ml of WI to resuspend, transfer to a 12-well cell culture plate and incubate in a dark room at 26℃ for 48 h; Step 3: Gene Editing Identification Step 3.1: DNA extraction and PCR Genomic DNA was extracted from protoplasts that had been transformed and cultured in a dark chamber at 26°C for 48 hours. Using the genomic DNA as a template, fragments containing the target region were amplified using primer pairs T10-msF1: ggagtgagtacggtgtgcCAGTCTATGCTGGAGTTAG and T10-msR1: gagttggatgctggatggGCTTTGCTCTGATCTGCAG, and then high-throughput sequencing was performed. Step 3.2: Editing and Appraisal Using a reference genome sequence of a rubber tree, combined with high-throughput sequencing, we can identify whether the transformed protoplasts have undergone editing.

2. The method for electroporation transformation of large plasmids in rubber tree leaf protoplasts according to claim 1, characterized in that, In step 1, the method for preparing the gene editing vector includes: 1) Anneal the PDS-T10F forward primer and the PDS-T10R reverse primer to obtain the double-stranded target sequence; The nucleotide sequence of the forward primer of PDS-T10F is 5'-ATTGATGAGATCCATTCTTCTGC-3', and the nucleotide sequence of the reverse primer of PDS-T10R is 3'-TACTCTAGGTAAGAAGACGCAAA-5'. 2) The vector is digested with AarI enzyme and then ligated with the double-stranded target sequence; 3) Take 10 μL of the ligation product obtained in step 2) and add it to 100 μL of DH5α competent cells. After gently mixing, place it on ice and immediately heat shock it in a metal bath for 45 seconds. Then immediately place it on ice and let it stand. Transfer it to 500 µL of antibiotic-free LB liquid medium, mix it by inverting, and place it on a shaker to revive the cells. Then take 100 μL of the bacterial culture and spread it evenly on an LB solid medium plate containing kanamycin resistance. After drying, invert it and incubate it in an incubator. The next day, after bacterial colonies grew on the plates, colony PCR was performed using primers PDS-T10F+GFP-seq-R (5'AGCTCACTCATTAGGCACCC-3'). Clones that could amplify a specific 273 bp were selected for sequencing identification.

3. The method for electroporation transformation of large plasmids in rubber tree leaf protoplasts according to claim 2, characterized in that, In step 1), the annealing method includes: rinsing the dry powders of the PDS-T10F forward primer and the PDS-T10R reverse primer with dd... Dilute with water to 100 μM, then take 20 μL of each and put them into PCR tubes to mix. Then place them in a PCR instrument for annealing, and cool to room temperature after the process is complete. The annealing conditions include a temperature of 100℃ and a time of 5 minutes.

4. The method for electroporation transformation of large plasmids in rubber tree leaf protoplasts according to claim 2, characterized in that, In step 2), the enzyme digestion system comprises: 5 μL of 10x AarI buffer, 1 μL of 50x oligonucleotide, 1 μL of AarI, 1 μg of pCAMBIA1300-2×35SCas9-HbU 6.2, and dd... Bring the volume to 50 μL; The enzyme digestion conditions include: a temperature of 37°C and a time of 5 hours.

5. The method for electroporation transformation of large plasmids in rubber tree leaf protoplasts according to claim 2, characterized in that, In step 2), the ligation system includes: 50 ng of purified vector fragment, 1 μL of 10x T4 ligase buffer, 7 μL of annealed double-stranded target sequence, 0.5 μL of T4 ligase, and supplemented with dd... Up to 10 μL; The conditions for the connection include: room temperature and a time of 1 hour.

6. The method for electroporation transformation of large plasmids in rubber tree leaf protoplasts according to claim 1, characterized in that, In step 2.1, the identified positive clones are sterilized, plasmids are extracted, and the plasmid concentration is detected.

7. The method for electroporation transformation of large plasmids in rubber tree leaf protoplasts according to claim 1, characterized in that, In step 2.2, E2 includes 20mM potassium chloride, 10mM calcium chloride, 0.6M mannitol, 10mM MES, 1g / L BSA, 15g / L cellulase, 7.5g / L ionase, and pH 5.

7.

8. The method for electroporation transformation of large plasmids in rubber tree leaf protoplasts according to claim 1, characterized in that, In step 2.2, W5 includes 154 mM sodium chloride, 5 mM potassium chloride, 125 mM calcium chloride, 5 mM glucose, and 2 mM MES.

9. The method for electroporation transformation of large plasmids in rubber tree leaf protoplasts according to claim 1, characterized in that, In step 2.3, the electrocup is preheated in an ice bath for 30 minutes, the electrocup parameters of the electroconversion device are set to 400V, the LV electrocup is dried with a paper towel before electrocup is electrocuted, and then the electrocup is placed in the electrocup tank for electrocution.

10. The method for electroporation transformation of large plasmids in rubber tree leaf protoplasts according to claim 1, characterized in that, In step 2.3, WI includes 0.5M mannitol, 4mM MES, and 20mM potassium chloride.

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