CN117487847A - Method for obtaining homozygous gene editing plant of rubber tree - Google Patents
Method for obtaining homozygous gene editing plant of rubber tree Download PDFInfo
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- CN117487847A CN117487847A CN202311421131.2A CN202311421131A CN117487847A CN 117487847 A CN117487847 A CN 117487847A CN 202311421131 A CN202311421131 A CN 202311421131A CN 117487847 A CN117487847 A CN 117487847A
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Abstract
The invention provides a method for obtaining a homozygous gene editing plant of a rubber tree, and relates to the technical field of genetic engineering. And (3) carrying out enzyme digestion on the gene editing vector to obtain an enzyme digestion vector, connecting the enzyme digestion vector with a target sequence to obtain a connecting product, converting the connecting product into escherichia coli and agrobacterium to obtain a conversion bacterium, infecting a rubber tree somatic embryo and the like to obtain a rubber tree homozygous gene editing plant. The invention uses the gene editing carrier as transformation donor, and judges whether the positive resistance somatic embryo is chimeric or not by high-throughput sequencing of the positive resistance somatic embryo, and the T0 generation resistance somatic embryo is chimeric. The T0 generation positive chimeric embryo is subjected to embryo cutting and purification and then is subjected to continuous subculture, so that a T1 generation resistant somatic embryo is obtained, the T1 generation resistant somatic embryo is homozygous at a proportion of about 50%, and the T1 generation homozygous editing somatic embryos are selected for plant regeneration.
Description
Technical Field
The invention relates to the technical field of genetic engineering, in particular to a method for obtaining a homozygous gene editing plant of a rubber tree.
Background
The existing genetic transformation with rubber tree embryo as transformation receptor, after transformation, T is obtained through the process of resistance callus induction-resistance embryo formation 0 The generation of resistant somatic embryos is determined to be positive by detecting the transformation gene or the resistance gene, but the method only can detect whether the resistant somatic embryos contain transgenic cells, cannot judge the proportion of the transgenic cells to non-transformed wild type cells in the somatic embryos, and obtains T 0 The positive somatic embryos are chimeric somatic embryos and are formed by T 0 The regenerated plants obtained from the generation positive somatic embryos are chimeric plants, so that the transgenic function is invalid, and the accuracy of transgenic function judgment is seriously affected.
Disclosure of Invention
In order to solve the problems, the invention provides a method for obtaining a homozygous gene editing plant of a rubber tree, which takes a pCAMBIA 1300-2X 35SCas9-HbU6.2 vector (gene editing vector) as a transformation donor, and is different from a common functional gene over-expression donor, so that whether the transgenic plant is a chimeric body can be judged by high-throughput sequencing of positive resistant somatic embryos, and the result proves that T0-generation resistant somatic embryos are all chimeric bodies. The most critical is that the T0 generation positive chimeric embryo is subjected to embryo cutting and purification and then is subjected to successive transfer, so that the T1 generation resistant embryo is obtained, and the T1 generation resistant embryo is detected to be homozygous at about 50 percent, and the T1 generation homozygous edited embryo is selected for plant regeneration.
In order to achieve the above object, the present invention provides the following technical solutions:
the invention provides a method for obtaining a homozygous gene editing plant of a rubber tree, which comprises the following steps:
1) Enzyme cutting is carried out on the pCAMBIA1300-2 multiplied by 35SCas9-HbU6.2 carrier to obtain an enzyme-cut carrier;
the nucleotide sequence of the pCAMBIA 1300-2X 35SCas9-HbU6.2 vector is shown as SEQ ID No. 9;
2) Connecting the enzyme-cleaved vector obtained in the step 1) with a target sequence to obtain a connection product;
3) Converting the connection product obtained in the step 2) into escherichia coli, extracting plasmids, and transferring the plasmids into agrobacterium to obtain transformed bacteria;
4) Infecting the rubber tree embryo with the transformed bacteria obtained in the step 3) to obtain an infected embryo;
5) Cutting the infected somatic embryo obtained in the step 4) to obtain an embryo block, performing callus induction culture and somatic embryo formation culture on the embryo block, and screening positive transformant embryos to obtain T0 generation somatic embryos;
6) Cutting the T0 generation somatic embryo obtained in the step 5) to obtain embryo blocks, carrying out callus induction culture and somatic embryo formation culture on the embryo blocks to obtain T1 generation somatic embryo, and selecting homozygous gene editing somatic embryo to carry out plant regeneration to obtain the homozygous gene editing plant of the rubber tree.
Preferably, the target sequence in the step 2) is obtained by annealing a primer pair, the nucleotide sequence of an upstream primer of the primer pair is shown as SEQ ID No.1, and the nucleotide sequence of a downstream primer of the primer pair is shown as SEQ ID No. 2.
Preferably, the system connected in step 2) comprises: 50ng of enzyme digestion vector, 1. Mu.L of 10 xT 4 ligase buffer, 0.5. Mu.L of target sequence 7. Mu. L, T4 ligase, and ddH 2 O is added to 10 mu L;
the conditions of the connection include: connecting for 1h at 20-30 ℃.
Preferably, the system for enzyme digestion in the step 1) comprises: 10 XAarI buffer 5. Mu.L, 50 Xoligonucleotide 1. Mu. L, aarI enzyme 1. Mu. L, pCAMBIA 1300-2X 35SCas9HbU6.2 vector 1. Mu.g, ddH 2 O is added to 50 mu L;
the conditions for the cleavage include: the enzyme was cleaved at 37℃for 5h.
Preferably, the sizes of the embryo blocks in the steps 5) and 6) are 2-3 mm multiplied by 2-3 mm.
Preferably, said step 5) uses a specific primer pair to screen positive transformant embryos;
the nucleotide sequence of the upstream primer of the specific primer pair is shown as SEQ ID No.3, and the nucleotide sequence of the downstream primer of the specific primer pair is shown as SEQ ID No. 4.
Preferably, the genome DNA of the somatic embryo obtained by the formation and culture of the somatic embryo is used as a template, PCR amplification is carried out through the specific primer pair, and when a 433bp band is amplified, the somatic embryo is a positive transformant embryo.
Preferably, the agrobacterium of step 3) is agrobacterium EHA105;
the conditions of the step 4) infection comprise: after acetosyringone is added into bacterial liquid containing the transformed bacteria, the rubber tree embryo is placed into the bacterial liquid for static incubation for 8min, and after ultrasonic treatment, the rubber tree embryo is subjected to static incubation for 10min.
Preferably, the conditions of the ultrasonic treatment include: treatment was carried out at 40kHz for 50s.
Preferably, the conditions of the callus induction culture in steps 5) and 6) comprise: dark culture on MS callus induction medium containing 3.0mM calcium chloride, 7.0. Mu.M KT, 8.1. Mu.M naphthalene acetic acid, 6.8. Mu.M 2,4-D, 204.5mM sucrose, 2.2g/L phytase, 10mg/L hygromycin and 500mg/L timentin for 30D at 24 ℃;
the conditions of the embryo formation culture in steps 5) and 6) both include: the germinated calli were transferred to embryogenic medium containing 10mg/L hygromycin for 2 months.
The beneficial effects of the invention are as follows:
the invention uses the gene editing carrier as transformation donor, which is different from the common functional gene donor, so that the positive resistance somatic embryo can be judged whether to be a chimeric body by high-throughput sequencing, and the result proves that the T0 generation resistance somatic embryo is all chimeric body. The most critical is that the T0 generation positive chimeric embryo is subjected to embryo cutting and purification and then is subjected to successive transfer, so that the T1 generation resistant embryo is obtained, and the T1 generation resistant embryo is detected to be homozygous at about 50 percent, and the T1 generation homozygous edited embryo is selected for plant regeneration.
The invention uses the gene editing vector as a transformation donor, and can classify the specific alignment and types of the transgenic cells in the positive somatic embryos. Because the gene editing vector edits the target gene HbPDS gene, positive somatic embryos can be classified into the following 4 types by high-throughput sequencing of sequences at the target: firstly, the non-mutated type contains only wild-type cells; secondly, chimeric bodies of wild type and mutant type exist simultaneously; thirdly, a chimeric body only comprising a plurality of mutation types; fourth, homozygous gene editing transformant.
After the T0 generation positive transformant embryo is obtained, the method does not enter a plant regeneration program like conventional transformation, but carries out a round of embryo cutting-resistance callus induction-resistance subculture screening process on the T0 generation positive transformant embryo, so that 50% of homozygous embryos exist in the obtained T1 generation positive transformant embryo, and the homozygous gene editing embryo is selected for plant regeneration, thereby obtaining the homozygous gene editing plant of the rubber tree for the first time.
Drawings
In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings that are required to be used in the embodiments will be briefly described below.
FIG. 1 is a diagram of a gene editing vector used for transformation in the present invention;
FIG. 2 is a schematic view of HbPDS gene structure;
FIG. 3 is a PCR detection of a resistant somatic embryo Cas9 gene fragment;
FIG. 4 shows the mutant sequence and the proportion of T0 generation editing embryo A29;
FIG. 5 is a phenotype of T1 generation edited somatic embryo regenerated plants;
FIG. 6 is a genotype of 4 homozygous gene editing plants;
FIG. 7 is a flow chart of the present invention for obtaining homozygous gene-edited plants (wherein the lower row is the more part of the invention than the prior art).
Detailed Description
The invention provides a method for obtaining a homozygous gene editing plant of a rubber tree, which comprises the following steps:
1) Enzyme cutting is carried out on the pCAMBIA1300-2 multiplied by 35SCas9-HbU6.2 carrier to obtain an enzyme-cut carrier;
the nucleotide sequence of the pCAMBIA 1300-2X 35SCas9-HbU6.2 vector is shown as SEQ ID No. 9;
2) Connecting the enzyme-cleaved vector obtained in the step 1) with a target sequence to obtain a connection product;
3) Converting the connection product obtained in the step 2) into escherichia coli, extracting plasmids, and transferring the plasmids into agrobacterium to obtain transformed bacteria;
4) Infecting the rubber tree embryo with the transformed bacteria obtained in the step 3) to obtain an infected embryo;
5) Cutting the infected somatic embryo obtained in the step 4) to obtain an embryo block, performing callus induction culture and somatic embryo formation culture on the embryo block, and screening positive transformant embryos to obtain T0 generation somatic embryos;
6) Cutting the T0 generation somatic embryo obtained in the step 5) to obtain embryo blocks, carrying out callus induction culture and somatic embryo formation culture on the embryo blocks to obtain T1 generation somatic embryo, and selecting homozygous gene editing somatic embryo to carry out plant regeneration to obtain the homozygous gene editing plant of the rubber tree.
The invention carries out enzyme digestion on the pCAMBIA 1300-2X 35SCas9-HbU6.2 vector to obtain an enzyme digestion vector.
In the present invention, the cleavage system preferably comprises: 10 XAarI buffer 5. Mu.L, 50 Xoligonucleotide 1. Mu. L, aarI enzyme 1. Mu. L, pCAMBIA 1300-2X 35SCas9-HbU6.2 vector 1. Mu.g, ddH 2 O was made up to 50. Mu.L. In the present invention, the conditions for the cleavage preferably include: the enzyme was cleaved at 37℃for 5h.
In the invention, the nucleotide sequence of the pCAMBIA 1300-2X 35SCas9-HbU6.2 vector is shown as SEQ ID No.9, and the nucleotide sequence is specifically as follows:
1. the double underlined sequence is a gRNA sequence;
2. underlined 19bp is HbPDS specific target sequence;
3. the 342bp sequence of the bolded part is the promoter of HbU6.2 gene;
4. 737bp of the wavy line is a 2X 35S promoter sequence
5. The 4206bp in italics is Cas9 gene;
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the invention connects the enzyme cutting carrier with the target sequence to obtain the connection product.
In the invention, the target sequence is obtained by annealing a primer pair, the nucleotide sequence of an upstream primer of the primer pair is shown as SEQ ID No.1, and the nucleotide sequence of a downstream primer of the primer pair is shown as SEQ ID No. 2. In the present invention, the annealing conditions include: run at 100℃for 5min.
SEQ ID No.1：5’-ATTGATGAGATCCATTCTTCTGC-3’；
SEQ ID No.2：3’-TACTCTAGGTAAGAAGACGCAAA-5’。
The sgRNA target of HbPDS gene designed in the invention is positioned on the 12 th exon, and has the following sequence:
PDS-T10 SEQ ID No.5：-5'CCTGCAGAAGAATGGATCTCAT-3'。
in the present invention, the linked system preferably comprises: 50ng of enzyme digestion vector, 1. Mu.L of 10 xT 4 ligase buffer, 0.5. Mu.L of target sequence 7. Mu. L, T4 ligase, and ddH 2 O is added to 10 mu L; the conditions of the connection preferably include: connecting for 1h at 20-30 ℃.
The obtained connection product is transformed into escherichia coli, plasmids are extracted, and the plasmids are transferred into agrobacterium to obtain transformed bacteria. In the present invention, the agrobacterium is preferably agrobacterium EHA105. The method of the present invention for transforming the ligation product into E.coli, extracting the plasmid and transforming the plasmid into Agrobacterium is not particularly limited, and those skilled in the art can adopt the conventional method.
The obtained transformant is used for infecting rubber tree somatic embryos to obtain infected somatic embryos.
In the present invention, the conditions of the infestation preferably include: after acetosyringone is added into bacterial liquid containing the transformed bacteria, the rubber tree embryo is placed into the bacterial liquid for static incubation for 8min, and after ultrasonic treatment, the rubber tree embryo is subjected to static incubation for 10min. In the present invention, the conditions of the ultrasonic treatment preferably include: treatment was carried out at 40kHz for 50s.
The method comprises the steps of cutting the obtained infected somatic embryo to obtain an embryo block, carrying out callus induction culture and somatic embryo formation culture on the embryo block, and screening positive transformant embryos to obtain T0 generation somatic embryos.
The method comprises the steps of cutting the obtained T0 generation somatic embryo to obtain embryo blocks, carrying out callus induction culture and somatic embryo formation culture on the embryo blocks to obtain T1 generation somatic embryo, and selecting homozygous gene editing somatic embryo for plant regeneration to obtain the homozygous gene editing plant of the rubber tree.
In the present invention, the size of the embryo is preferably 2 to 3mm×2 to 3mm. The invention preferably uses a specific primer pair to screen positive transformant embryos; the nucleotide sequence of the upstream primer of the specific primer pair is shown as SEQ ID No.3, and the nucleotide sequence of the downstream primer of the specific primer pair is shown as SEQ ID No. 4.
SEQ ID No.3：5’-ccgatcggcacagcatcaag-3’；
SEQ ID No.4：5’-tccacatcgctattgtccgg-3’。
The invention preferably uses genome DNA of somatic embryo obtained by forming and culturing the somatic embryo as a template, and carries out PCR amplification by the specific primer pair, and when 433bp band is amplified, the somatic embryo is positive transformant embryo.
In the present invention, the conditions for callus induction culture preferably include: dark culture was performed on MS callus induction medium containing 3.0mM calcium chloride, 7.0. Mu.M KT, 8.1. Mu.M naphthalene acetic acid, 6.8. Mu.M 2,4-D, 204.5mM sucrose, 2.2g/L phytase, 10mg/L hygromycin and 500mg/L timentin for 30D at 24 ℃. In the present invention, the conditions of the embryo-forming culture preferably include: the germinated calli were transferred to embryogenic medium containing 10mg/L hygromycin for 2 months. The embryo-forming medium is not particularly limited, and those skilled in the art can adopt a conventional method.
The present invention will be described in detail with reference to examples for further illustration of the invention, but they should not be construed as limiting the scope of the invention.
Example 1
1 vector construction
The constructed pCAMBIA 1300-2X 35SCas9-HbU6.2 rubber tree gene editing vector is taken as a framework, and the specific flow is as follows:
(1) synthesizing a pair of complementary targets with joints, wherein the sequences are as follows;
PDS-T10F SEQ ID No.1:5’-ATTGATGAGATCCATTCTTCTGC-3’；
PDS-T10R SEQ ID No.2:3’-TACTCTAGGTAAGAAGACGCAAA-5’。
(2) primer annealing to double strand
The PDS-T10F forward primer and the PDS-T10R reverse primer were subjected to ddH 2 O-water was diluted to 100. Mu.M, and 20. Mu.L each was mixed in a PCR tube, followed by running at 100℃for 5min and cooling at room temperature to form a double-stranded target sequence having a sticky end.
(3) Enzyme cutting of carrier
And (3) placing the prepared enzyme digestion system at 37 ℃ for enzyme digestion for 5 hours, and purifying and recovering enzyme digestion vectors by using a large amount of DNA product purification kit (DP 205).
(4) Target fragment-editing vector ligation
(5) Coli transformation and cloning identification
Adding 10 mu L of the connection product into 100 mu L of DH5 alpha competent cells, slightly mixing, ice-bathing for 30min, immediately placing in a metal bath at 42 ℃ for heat shock for 45sec, immediately placing on ice for standing for 2min, transferring into 500 mu L of LB liquid medium without antibiotics, standing upside down, mixing uniformly, placing on a 180rpm shaking table for shaking culture at 37 ℃ for 1h to revive the cells, then uniformly smearing 100 mu L of bacterial liquid on a plate of LB solid medium with kana resistance, drying, and then culturing in a 37 ℃ incubator for 16h in an inverted manner.
After bacterial plaque grows on the flat plate on the next day, a primer PDS-T10F+GFP-SEQ-R (SEQ ID No.6: 5'-AGCTCACTCATTAGGCACCC-3') is used for colony PCR identification, and clones which can expand specific 273bp are selected and sent to a company for sequencing identification.
2 genetic transformation
Extracting plasmids after the positive cloning identified above, transferring into an agrobacterium EHA105 strain, shaking the bacterial solution to OD 600=0.45 at 28 ℃, adding 100 mu M acetosyringone, then placing rubber tree embryo into the bacterial solution for incubation for 8min, then treating for 50s at 40kHz ultrasonic intensity, then standing for incubation for 10min, then taking out the somatic embryo from the bacterial solution, washing 3 times with sterile water, placing the bacterial embryo on sterile dry filter paper, placing the bacterial embryo in an ultra clean bench for blow drying, placing the bacterial embryo on a PCM culture medium, and co-culturing the bacterial embryo for 84h at 22 ℃ under dark condition.
3 resistance selection and plant regeneration
(1) Induction and editing identification of T0 generation somatic embryo
The infected rubber tree embryos were placed on MS medium without antibiotics for 2 days, then cut into 3X 3mM pieces, then placed on MS-based callus induction medium (3.0 mM calcium chloride, 7.0. Mu.M KT, 8.1. Mu.M naphthalene acetic acid, 6.8. Mu.M 2,4-D, 204.5mM sucrose and 2.2g/L phytase), 10mg/L hygromycin and 500mg/L timentin were added, and cultured in dark at 24 ℃. After one month, the newly germinated calli were transferred to embryogenic medium containing 10mg/L hygromycin for 2 months to form T0-generation somatic embryos, at which time 2mm square pieces of small embryos were excised to extract genomic DNA, and PCR was performed using Cas9 gene specific primers (FSEQ ID No.3:5'-ccgatcggcacagcatcaag-3', RSEQ ID No.4: 5'-tccacatcgctattgtccgg-3') to initially screen positive transformant embryos (capable of expanding 433bp specific band, FIG. 3, marker was DL 2000).
Genomic DNA from PCR positive resistant embryos was selected as template, fragments containing targets were amplified with T10-ms primers (FSEQ ID No.7: ggagtgagtacggtgtgcCAGTCTATGCTGGAGTTAG; R SEQ ID No.8: gagttggatgctggatggGCTTTGCTCTGATCTGCAG, where lowercase letters are high throughput sequencing linker sequences) and sent to the company for high throughput sequencing (Hi-TOM, http:// 121.40.237.174/Hi-TOM/logic. Php), and positive embryo pieces were detected that were edited and all positive embryo pieces were chimeric. The ratio of the mutant sequences of the chimeric editing embryo A29 was 9.7%, and the specific mutant sequences were as follows (FIG. 4).
(2) Induction and editing identification of T2 generation somatic embryo
In the past, the genetic transformation of the rubber tree embryo is carried out, after the T0 generation embryo is identified to have transgene insertion, the plant regeneration procedure is carried out under the condition that whether the positive embryo is chimeric or not is not known. The invention utilizes a gene editing vector and combines high-throughput sequencing to find that the T0 generation embryo blocks are all chimeric. Thus, the T0-generation embryo was innovatively not regenerated, but the A29 embryo identified as the occurrence of editing was cut into a plurality of pieces (33 pieces in total) of 2X 2mm, and one round of the above was performed3-(1) As a result, 33T 1-generation resistant embryos were obtained, and high-throughput detection was performed by the method described in 3- (1) above, and it was found that the 29 embryo pieces were all subjected to gene editing, the lowest editing ratio was 50%, the 17 embryo pieces were 100%, and 16 individual embryos were homozygous embryos of single cell origin, accounting for 48.5% of all 33 embryos (Table 1).
TABLE 1 editing efficiency of 33A 29T 1 generation embryos and genotypes thereof
(3) Phenotype and molecular characterization of regenerated plants
The 29T 2 generation embryo blocks edited in 3- (2) are selected for plant regeneration, and 8 regenerated plants are obtained as a result, wherein 4 plants are chimeric plants (A-D in fig. 5) and 4 plants are homozygous plants (E-F in fig. 5).
Sequencing the chlorosis, green and white parts of the chimeric plant respectively, and finding that the green and yellow parts of the chimeric plant are edited, wherein the editing proportion is between 66 and 69 percent, and the editing proportion of the white part is between 86 and 100 percent.
Subsequently, high throughput sequencing of E-H showed that homozygous gene editing occurred in all 4 plants, where E was a homozygote editing plant and F-H was a homozygote editing plant with the genotype shown in FIG. 6.
Although the foregoing embodiments have been described in some, but not all, embodiments of the invention, it should be understood that other embodiments may be devised in accordance with the present embodiments without departing from the spirit and scope of the invention.
Claims (10)
1. A method for obtaining a homozygous gene editing plant of a hevea brasiliensis plant, comprising the steps of:
1) Enzyme cutting is carried out on the pCAMBIA1300-2 multiplied by 35SCas9-HbU6.2 carrier to obtain an enzyme-cut carrier;
the nucleotide sequence of the pCAMBIA 1300-2X 35SCas9-HbU6.2 vector is shown as SEQ ID No. 9;
2) Connecting the enzyme-cleaved vector obtained in the step 1) with a target sequence to obtain a connection product;
3) Converting the connection product obtained in the step 2) into escherichia coli, extracting plasmids, and transferring the plasmids into agrobacterium to obtain transformed bacteria;
4) Infecting the rubber tree embryo with the transformed bacteria obtained in the step 3) to obtain an infected embryo;
5) Cutting the infected somatic embryo obtained in the step 4) to obtain an embryo block, performing callus induction culture and somatic embryo formation culture on the embryo block, and screening positive transformant embryos to obtain T0 generation somatic embryos;
6) Cutting the T0 generation somatic embryo obtained in the step 5) to obtain embryo blocks, carrying out callus induction culture and somatic embryo formation culture on the embryo blocks to obtain T1 generation somatic embryo, and selecting homozygous gene editing somatic embryo to carry out plant regeneration to obtain the homozygous gene editing plant of the rubber tree.
2. The method according to claim 1, wherein the target sequence in step 2) is obtained by annealing a primer pair, the nucleotide sequence of the upstream primer of the primer pair is shown in SEQ ID No.1, and the nucleotide sequence of the downstream primer of the primer pair is shown in SEQ ID No. 2.
3. The method according to claim 1, wherein the system of step 2) connections comprises: 50ng of enzyme digestion vector, 1. Mu.L of 10 xT 4 ligase buffer, 0.5. Mu.L of target sequence 7. Mu. L, T4 ligase, and ddH 2 O is added to 10 mu L;
the conditions of the connection include: connecting for 1h at 20-30 ℃.
4. The method according to claim 1, wherein the system for cleavage of step 1) comprises: 10 XAarI buffer 5. Mu.L, 50 Xoligonucleotide 1. Mu. L, aarI enzyme 1. Mu. L, pCAMBIA 1300-2X 35SCas9-HbU6.2 vector 1. Mu.g, ddH 2 O is added to 50 mu L;
the conditions for the cleavage include: the enzyme was cleaved at 37℃for 5h.
5. The method of claim 1, wherein the size of each of the embryo pieces of steps 5) and 6) is 2-3 mm x 2-3 mm.
6. The method of claim 1, wherein step 5) uses a specific primer pair to screen positive transformant embryos;
the nucleotide sequence of the upstream primer of the specific primer pair is shown as SEQ ID No.3, and the nucleotide sequence of the downstream primer of the specific primer pair is shown as SEQ ID No. 4.
7. The method according to claim 6, wherein the PCR amplification is performed using genomic DNA of the embryo obtained by the embryo-forming culture as a template, and the embryo is a positive transformant embryo when a 433bp band is amplified.
8. The method according to claim 1, wherein the agrobacterium of step 3) is agrobacterium EHA105;
the conditions of the step 4) infection comprise: after acetosyringone is added into bacterial liquid containing the transformed bacteria, the rubber tree embryo is placed into the bacterial liquid for static incubation for 8min, and after ultrasonic treatment, the rubber tree embryo is subjected to static incubation for 10min.
9. The method of claim 8, wherein the conditions of the ultrasonic treatment comprise: treatment was carried out at 40kHz for 50s.
10. The method according to claim 1, wherein the conditions of the callus induction culture of steps 5) and 6) each comprise: dark culture on MS callus induction medium containing 3.0mM calcium chloride, 7.0. Mu.M KT, 8.1. Mu.M naphthalene acetic acid, 6.8. Mu.M 2,4-D, 204.5mM sucrose, 2.2g/L phytase, 10mg/L hygromycin and 500mg/L timentin for 30D, said dark culture having a temperature of 24 ℃;
the conditions of the embryo formation culture in steps 5) and 6) both include: the germinated calli were transferred to embryogenic medium containing 10mg/L hygromycin for 2 months.
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