Genetic transformation method of erigeron breviscapus chloroplast

Abstract

The invention discloses a genetic transformation method of erigeron breviscapus chloroplast. According to the method, plasmids containing target genes are introduced into explants prepared from erigeron breviscapus leaves through a gene gun method, and erigeron breviscapus transfection plants are obtained; wherein the parameters of the gene gun bombardment are as follows: the plasmid DNA concentration is 1-1.5 [mu] g / [mu] l, the number of bombardment times is 1-2, the target distance is 3-9 cm, the bombardment pressure is 900-1100 psi, and the vacuum degree is 26-28 mmHg. By adopting the method established by the invention, the homogenized erigeron breviscapus transfection plant can be efficiently obtained. The method has an important application value.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology, and in particular relates to a method for genetic transformation of chloroplasts of Erigeron breviscapus. Background Art

[0002] Brevifolia is the dried whole herb of Erigeron breviscapus (Vant.) Hand.-Mazz., a plant of the Asteraceae family. It is a perennial herbal medicinal plant with a history of more than 1,000 years of medicinal use. Brevifolia scutellarin is the medicinal active ingredient of Brevifolia scutellariae, which has the effects of dilating blood vessels, inhibiting platelet aggregation, promoting blood circulation, alleviating myocardial ischemia-reperfusion injury and reducing oxidative damage, and has become an important drug for the clinical treatment of cardiovascular and cerebrovascular diseases. However, traditional nuclear genetic engineering has failed to significantly increase the yield of Brevifolia scutellariae. Although the total synthesis of scutellarin has been achieved through engineered yeast, there are still problems such as high production cost and low yield. Therefore, there is an urgent need to establish an efficient and low-cost method to increase the yield or medicinal ingredient content of Brevifolia scutellariae to meet market demand.

[0003] Plastid genetic engineering is an effective means of genetic improvement, and it is easier to obtain ideal traits than traditional methods. In addition, increasing the carbon assimilation rate and strengthening the fixation of carbon dioxide by photosynthetic enzymes in chloroplasts through plastid genetic engineering has become one of the effective strategies to improve metabolism (improve the photosynthetic efficiency of transgenic varieties) and increase yield. However, problems such as low conversion rate of transplastomic plants, difficulty in homogenization, and sterility of transplastomic plants have further limited the widespread application of plastid genetic engineering, especially in the field of medicinal plants. These problems have seriously restricted the further development of genetic improvement of medicinal plants. So far, only Artemisia annua has established a mature and stable plastid genetic transformation system. Summary of the invention

[0004] The purpose of the present invention is to establish a method for genetic transformation of chloroplasts of Erigeron breviscapus and obtain Erigeron breviscapus transplastomic plants.

[0005] The present invention firstly protects a method for genetic transformation of chloroplasts of Erigeron breviscapus, which can be a method of using gene gun method to introduce a plasmid containing a target gene into an explant prepared by Erigeron breviscapus leaves to obtain an Erigeron breviscapus transplastomic plant.

[0006] In the above method, the parameters of the gene gun bombardment can be as follows: the plasmid DNA concentration can be 1-1.5 μg / μl (such as 1-1.25 μg / μl, 1.25-1.5 μg / μl, 1 μg / μl, 1.25 μg / μl or 1.5 μg / μl); the number of bombardments can be 1-2 times (such as 1 time or 2 times), the target distance can be 3-9 cm (such as 3-6 cm, 6-9 cm, 3 cm, 6 cm or 9 cm), the bombardment pressure can be 900-1100 psi (such as 900-1000 psi, 1000-1100 psi, 900 psi, 1000 psi or 1100 psi), and the vacuum degree can be 26-28 mmHg (such as 26-27 mmHg, 27-28 mmHg, 26 mmHg, 27 mmHg or 28 mmHg).

[0007] In the above method, the parameters of the gene gun bombardment can be specifically as follows: bombardment pressure 1100 psi, target distance 9 cm, vacuum degree 26 mmHg, bombardment number 1 time, plasmid DNA concentration 1 μg / μL.

[0008] In the above method, the parameters of the gene gun bombardment can be specifically as follows: bombardment pressure 1100 psi, target distance 3 cm, vacuum degree 28 mmHg, bombardment number 1 time, plasmid DNA concentration 1.5 μg / μL.

[0009] In the above method, the steps of "using a gene gun method to introduce a plasmid containing a target gene into an explant prepared by using leaves of Erigeron breviscapus to obtain an Erigeron breviscapus transplastomic plant" can be as follows:

[0010] (1) inoculating leaves of Erigeron breviscapus on differentiation medium 1 and culturing;

[0011] (2) placing leaves of Erigeron breviscapus in a hypertonic medium for hypertonic treatment;

[0012] (3) introducing a plasmid containing the target gene by gene gun bombardment, and then placing it in differentiation medium 1 and culturing;

[0013] (4) placing the callus on a screening medium, culturing it, and obtaining resistant callus;

[0014] (5) placing the resistant callus containing the target gene on differentiation medium 1 and culturing;

[0015] (6) placing in differentiation medium 2 and culturing;

[0016] (7) placing in differentiation medium 3 and culturing to obtain clustered shoots;

[0017] (8) placing the plant on a rooting medium and culturing it to obtain a transplastomic plant of Erigeron breviscapus.

[0018] In the above step (2), the purpose of performing the hypertonic treatment is to reduce the damage to the recipient cells during the bombardment process.

[0019] The solutes and their concentrations in any of the above-mentioned differentiation medium 1 may be 4.20-4.60 g / L of MS powder (e.g., 4.20-4.43 g / L, 4.43-4.60 g / L, 4.20 g / L, 4.43 g / L or 4.60 g / L), 28-30 g / L of sucrose (e.g., 28-29 g / L, 29-30 g / L, 28 g / L, 29 g / L or 30 g / L), 4-6 g / L of plant gel (e.g., 4-5 g / L, 5-6 g / L, 4 g / L, 5 g / L or 6 g / L), 6-BA 1-2 mg / L (such as 1-1.5 mg / L, 1.5-2 mg / L, 1 mg / L, 1.5 mg / L or 2 mg / L) and NAA 0.1-0.2 mg / L (such as 0.1-0.15 mg / L, 0.15-0.2 mg / L, 0.1 mg / L, 0.15 mg / L or 0.2 mg / L); the solvent is water; the pH value is 5.8-6.0 (5.8-5.9, 5.9-6.0, 5.8, 5.9 or 6.0).

[0020] Any of the above-mentioned differentiation medium 1 can be prepared by dissolving 4.43g MS powder, 30g sucrose and 4g plant gel in an appropriate amount of deionized water, then making the volume up to 1L with deionized water, adjusting the pH value to 5.8; sterilizing at 121°C and high temperature and high pressure for 15min; when cooled to about 40-50°C, adding sterilized 6-BA and NAA and making their concentrations in the system 1mg / L and 0.1mg / L respectively.

[0021] The solutes and their concentrations in any of the above-mentioned hypertonic culture media may be 4.20-4.60 g / L (e.g., 4.20-4.43 g / L, 4.43-4.60 g / L, 4.20 g / L, 4.43 g / L or 4.60 g / L) of MS powder, 28-30 g / L (e.g., 28-29 g / L, 29-30 g / L, 28 g / L, 29 g / L or 30 g / L) of sucrose, Mannitol 68-72 g / L (such as 68-70 g / L, 70-72 g / L, 68 g / L, 70 g / L or 72 g / L) and plant gel 4-6 g / L (such as 4-5 g / L, 5-6 g / L, 4 g / L, 5 g / L or 6 g / L); the solvent is water; the pH value is 5.8-6.0 (5.8-5.9, 5.9-6.0, 5.8, 5.9 or 6.0).

[0022] Any of the above-mentioned hypertonic culture media can be specifically prepared by dissolving 4.43g MS powder, 30g sucrose, 72g mannitol and 4g plant gel in an appropriate amount of deionized water, then making up to 1L with deionized water, adjusting the pH value to 5.8, and sterilizing at 121°C for 15min.

[0023] The solutes and their concentrations in any of the above-mentioned screening medium may be 4.20-4.60 g / L of MS powder (e.g., 4.20-4.43 g / L, 4.43-4.60 g / L, 4.20 g / L, 4.43 g / L or 4.60 g / L), 28-30 g / L of sucrose (e.g., 28-29 g / L, 29-30 g / L, 28 g / L, 29 g / L or 30 g / L), 4-6 g / L of plant gel (e.g., 4-5 g / L, 5-6 g / L, 4 g / L, 5 g / L or 6 g / L), 6-BA 1-2 mg / L (such as 1-1.5 mg / L, 1.5-2 mg / L, 1 mg / L, 1.5 mg / L or 2 mg / L), NAA 0.1-0.2 mg / L (such as 0.1-0.15 mg / L, 0.15-0.2 mg / L, 0.1 mg / L, 0.15 mg / L or 0.2 mg / L) and spectinomycin (such as 50-53 mg / L, 53-55 mg / L, 50 mg / L, 53 mg / L or 55 mg / L); the solvent is water; the pH value is 5.8-6.0 (5.8-5.9, 5.9-6.0, 5.8, 5.9 or 6.0).

[0024] Any of the above-mentioned screening culture media can specifically be prepared by dissolving 4.43g MS powder, 30g sucrose and 4g plant gel in an appropriate amount of deionized water, then making the volume up to 1L with deionized water, adjusting the pH value to 5.8; sterilizing at 121°C and high temperature and high pressure for 15min; when cooled to about 40-50°C, adding sterilized 6-BA, NAA and spectinomycin (Spe) and making their concentrations in the system 1mg / L, 0.1mg / L and 50mg / L, respectively.

[0025] The solutes and their concentrations in any of the above-mentioned differentiation medium 2 may be 4.20-4.60 g / L of MS powder (e.g., 4.20-4.43 g / L, 4.43-4.60 g / L, 4.20 g / L, 4.43 g / L or 4.60 g / L), 28-30 g / L of sucrose (e.g., 28-29 g / L, 29-30 g / L, 28 g / L, 29 g / L or 30 g / L), 4-6 g / L of plant gel (e.g., 4-5 g / L, 5-6 g / L, 4 g / L, 5 g / L or 6 g / L), 6-BA 1-2 mg / L (such as 1-1.5 mg / L, 1.5-2 mg / L, 1 mg / L, 1.5 mg / L or 2 mg / L), NAA 0.1-0.2 mg / L (such as 0.1-0.15 mg / L, 0.15-0.2 mg / L, 0.1 mg / L, 0.15 mg / L or 0.2 mg / L) and spectinomycin 5-8 mg / L (such as 5-6 mg / L, 6-8 mg / L, 5 mg / L, 6 mg / L or 8 mg / L); the solvent is water; the pH value is 5.8-6.0 (5.8-5.9, 5.9-6.0, 5.8, 5.9 or 6.0).

[0026] Any of the above-mentioned differentiation culture media 2 can specifically be prepared by dissolving 4.43g MS powder, 30g sucrose and 4g plant gel in an appropriate amount of deionized water, then making the volume up to 1L with deionized water, adjusting the pH value to 5.8; sterilizing at 121°C and high temperature and high pressure for 15min; when cooled to about 40-50°C, adding sterilized 6-BA, NAA and spectinomycin and making their concentrations in the system 1mg / L, 0.1mg / L and 5mg / L respectively.

[0027] The solutes and their concentrations in any of the above-mentioned differentiation medium 3 may be 4.20-4.60 g / L of MS powder (e.g., 4.20-4.43 g / L, 4.43-4.60 g / L, 4.20 g / L, 4.43 g / L or 4.60 g / L), 28-30 g / L of sucrose (e.g., 28-29 g / L, 29-30 g / L, 28 g / L, 29 g / L or 30 g / L), 4-6 g / L of plant gel (e.g., 4-5 g / L, 5-6 g / L, 4 g / L, 5 g / L or 6 g / L), 6-BA 1-2 mg / L (such as 1-1.5 mg / L, 1.5-2 mg / L, 1 mg / L, 1.5 mg / L or 2 mg / L), NAA 0.1-0.2 mg / L (such as 0.1-0.15 mg / L, 0.15-0.2 mg / L, 0.1 mg / L, 0.15 mg / L or 0.2 mg / L) and spectinomycin 10-12 mg / L (10-11 mg / L, 11-12 mg / L, 10 mg / L, 11 mg / L or 12 mg / L); the solvent is water; the pH value is 5.8-6.0 (5.8-5.9, 5.9-6.0, 5.8, 5.9 or 6.0).

[0028] Any of the above-mentioned differentiation medium 3 can be specifically prepared by dissolving 4.43g MS powder, 30g sucrose and 4g plant gel in an appropriate amount of deionized water, then making the volume up to 1L with deionized water, adjusting the pH value to 5.8; sterilizing at 121°C and high temperature and high pressure for 15min; when cooled to about 40-50°C, adding sterilized 6-BA, NAA and spectinomycin and making their concentrations in the system 1mg / L, 0.1mg / L and 10mg / L respectively.

[0029] The solutes and their concentrations in any of the above-mentioned rooting medium may be 2.00-2.40 g / L of MS powder (e.g., 2.00-2.22 g / L, 2.22-2.40 g / L, 2.00 g / L, 2.22 g / L or 2.40 g / L), 28-30 g / L of sucrose (e.g., 28-29 g / L, 29-30 g / L, 28 g / L, 29 g / L or 30 g / L), 4-6 g / L of plant gel (e.g., 4-5 g / L, 5-6 g / L, 4 g / L, 5 g / L), L or 6g / L), IBA 0.5-0.8mg / L (such as 0.5-0.6mg / L, 0.6-0.8mg / L, 0.5mg / L, 0.6mg / L or 0.8mg / L) and NAA 0.3-0.5mg (such as 0.3-0.4mg, 0.4-0.5mg, 0.3mg, 0.4mg or 0.5mg); the solvent is water; the pH value is 5.8-6.0 (5.8-5.9, 5.9-6.0, 5.8, 5.9 or 6.0).

[0030] Any of the above-mentioned rooting culture media can specifically be prepared by dissolving 2.22g of MS powder, 30g of sucrose and 4g of plant gel in an appropriate amount of deionized water, then making the volume up to 1L with deionized water, adjusting the pH value to 5.8; sterilizing at 121°C and high temperature and high pressure for 15min; when cooled to about 40-50°C, adding sterilized IBA and NAA and making their concentrations in the system 0.5mg / L and 0.3mg / L respectively.

[0031] In the above method, in step (1), the culture can be dark cultured at 23-27°C (such as 23-25°C, 25-27°C, 23°C, 25°C or 27°C) for 5-9 days (such as 5-7 days, 7-9 days, 5 days, 6 days, 7 days, 8 days or 9 days).

[0032] In the above method, in step (2), the hypertonic treatment method can be dark culturing at 23-27°C (such as 23-25°C, 25-27°C, 23°C, 25°C or 27°C) for 5 to 6 hours (such as 5 days or 6 days).

[0033] In the above method, in step (3) and step (5), the culture can be carried out at 23-27°C (such as 23-25°C, 25-27°C, 23°C, 25°C or 27°C) with alternating light and dark for 1-3 weeks (such as 1-2 weeks, 2-3 weeks, 1 week, 2 weeks or 3 weeks).

[0034] In the above method, in step (4), the culture may be carried out in the dark at 23-27°C (such as 23-25°C, 25-27°C, 23°C, 25°C or 27°C).

[0035] In the above method, in step (6), the culture can be carried out at 23-27°C (such as 23-25°C, 25-27°C, 23°C, 25°C or 27°C) with alternating light and dark for 3-5 weeks (such as 3-4 weeks, 4-5 weeks, 3 weeks, 4 weeks or 5 weeks).

[0036] In the above method, in step (7) and step (8), the culture can be carried out at 23-27°C (such as 23-25°C, 25-27°C, 23°C, 25°C or 27°C) with alternating light and dark.

[0037] Any of the above-mentioned light-dark alternating cultures can specifically be 16h light / 8h dark, wherein the light intensity is 1900lux.

[0038] The present invention also protects a kit for genetic transformation of chloroplasts of Erigeron breviscapus, which may include at least one of the above-mentioned differentiation medium 1, the above-mentioned hypertonic medium, the above-mentioned screening medium, the above-mentioned differentiation medium 2, the above-mentioned differentiation medium 3 and the above-mentioned rooting medium.

[0039] Any of the above-mentioned kits for genetic transformation of chloroplasts of Erigeron breviscapus can specifically be composed of at least one of the above-mentioned differentiation medium 1, the above-mentioned hypertonic medium, the above-mentioned screening medium, the above-mentioned differentiation medium 2, the above-mentioned differentiation medium 3 and the above-mentioned rooting medium.

[0040] The use of at least one of the above-mentioned differentiation medium 1, any of the above-mentioned hypertonic medium, any of the above-mentioned screening medium, any of the above-mentioned differentiation medium 2, any of the above-mentioned differentiation medium 3 and any of the above-mentioned rooting medium in the genetic transformation of chloroplasts of Erigeron breviscapus also falls within the protection scope of the present invention.

[0041] Any of the plasmids containing the target gene described above can specifically be the recombinant plasmid pBtEat3 mentioned in the embodiment. The recombinant plasmid pBtEat3 is a recombinant plasmid obtained by inserting the Eb-trnA fragment into the recognition site of the restriction endonuclease Sac I of the recombinant plasmid pBtEa3. The target gene can be the spectinomycin resistance selection marker gene aadA and / or the reporter gene eGFP.

[0042] After a large number of experiments, the inventors of the present application have established a method for genetic transformation of chloroplasts of Erigeron breviscapus. The method is to clarify the critical concentration of antibiotics for screening and culturing of Erigeron breviscapus plastid transformation, optimize the conditions for gene gun transformation of Erigeron breviscapus plastid transformation, and develop a tissue culture scheme suitable for the entire Erigeron breviscapus plastid transformation process, and finally obtain homogenized Erigeron breviscapus chloroplast transgenic plants (i.e., Erigeron breviscapus plastid plants). It can be seen that the present invention provides a vector and method for the genetic transformation of chloroplasts of Erigeron breviscapus. In addition, the inventors of the present application have established an efficient plastid transformation method by orthogonal method to reduce the number of experiments and the complexity of experimental analysis methods, thereby improving efficiency and experimental accuracy. This also provides a reference for the establishment of plastid genetic transformation systems of other medicinal plants. The present invention has important application value. BRIEF DESCRIPTION OF THE DRAWINGS

[0043] Figure 1 Schematic diagram of the structure of the chloroplast operon of the recombinant plasmid pBtEat3.

[0044] Figure 2 The effect of different concentrations of spectinomycin on callus induction of Erigeron breviscapus.

[0045] Figure 3 This is the screening and regeneration process of transplastomic plants of Erigeron breviscapus.

[0046] Figure 4 This is the PCR analysis of eGFP gene and aadA gene in the chloroplast genome of Erigeron breviscapus transplastomic plants.

[0047] Figure 5 This is the PCR analysis of the sequences LF-eGFP and RF-aadA in the chloroplast genome of the transplastomic plants of Erigeron breviscapus.

[0048] Figure 6 To detect the expression of eGFP gene in transplastomic plants of Erigeron breviscapus. DETAILED DESCRIPTION

[0049] The present invention is further described in detail below in conjunction with specific embodiments, and the examples provided are only for illustrating the present invention, rather than for limiting the scope of the present invention. The examples provided below can be used as a guide for further improvements by those of ordinary skill in the art, and do not constitute a limitation of the present invention in any way.

[0050] The experimental methods in the following examples, unless otherwise specified, are all conventional methods, and are performed according to the techniques or conditions described in the literature in the field or according to the product instructions. The materials, reagents, etc. used in the following examples, unless otherwise specified, can all be obtained from commercial channels.

[0051] Phusion high-fidelity DNA polymerase, restriction endonuclease Not I, restriction endonuclease HindIII, restriction endonuclease Sal I, restriction endonuclease Kpn I and restriction endonuclease Sac I are all products of NEB. EasyTaq DNA polymerase is a product of Beijing Quanshijin Biotechnology Co., Ltd. pEASY-Uni Seamless Cloning and Assembly Kit and EasyPure Quick Gel Extraction Kit are both products of Beijing Quanshijin Biotechnology Co., Ltd. HiPure Plasmid Mini Kit and Plasmid MaxiPrep Kit are both products of Shanghai Promega Biological Products Co., Ltd. High-efficiency plant genomic DNA extraction kit is a product of Tiangen Biochemical Technology (Beijing) Co., Ltd.

[0052] The names of the culture media and their compositions involved in the following examples are as follows:

[0053] Hypertonic culture medium: Dissolve 4.43 g MS powder, 30 g sucrose, 72 g mannitol and 4 g plant gel in an appropriate amount of deionized water, then dilute to 1 L with deionized water, adjust the pH value to 5.8; sterilize at 121°C for 15 min.

[0054] Differentiation medium 1: Dissolve 4.43 g MS powder, 30 g sucrose and 4 g plant gel in an appropriate amount of deionized water, then dilute to 1 L with deionized water, and adjust the pH value to 5.8; sterilize at 121°C for 15 min; when cooled to about 40-50°C, add sterilized 6-BA and NAA and make their concentrations in the system 1 mg / L and 0.1 mg / L, respectively.

[0055] Rooting medium: Dissolve 2.22g MS powder, 30g sucrose and 4g plant gel in an appropriate amount of deionized water, then dilute to 1L with deionized water, and adjust the pH value to 5.8; sterilize at 121℃ for 15min; when cooled to about 40-50℃, add sterilized IBA and NAA and make their concentrations in the system 0.5mg / L and 0.3mg / L respectively.

[0056] Example

[0057] 1. Experimental Methods

[0058] 1. Effect of spectinomycin on callus induction of Erigeron breviscapus

[0059] (1) Preparation of screening medium with different concentration gradients of spectinomycin

[0060] 4.43g MS powder, 30g sucrose and 4g plant gel were dissolved in an appropriate amount of deionized water, and then the volume was adjusted to 1L with deionized water, and the pH value was adjusted to 5.8; sterilized at 121°C for 15min; when cooled to about 40-50°C, sterilized 6-BA and NAA were added and their concentrations in the system were 1mg / L and 0.1mg / L respectively, and spectinomycin (Spe) was added and its concentration in the system was 0mg / L, 10mg / L, 20mg / L, 30mg / L, 40mg / L, 50mg / L, 100mg / L, 150mg / L, 200mg / L, 250mg / L, 300mg / L, 350mg / L, 400mg / L, 450mg / L or 500mg / L to obtain a screening medium.

[0061] (2) In order to ensure the differentiation and proliferation ability of transplastomic cells, young leaves of 4-8-week-old sterile seedlings of Erigeron breviscapus (i.e., young leaves of Erigeron breviscapus) were selected as explants. The sterile seedlings of Erigeron breviscapus were provided by the National Key Laboratory of Quality Assurance and Sustainable Utilization of Authentic Medicinal Materials, China Academy of Chinese Medical Sciences.

[0062] (3) Each treatment was repeated three times. The method of each treatment was as follows: In a clean bench, the young leaves of Erigeron breviscapus were cut into pieces with an area of ​​about 1 cm. 2The explants were divided into small pieces, and then placed in the screening medium prepared in step (1), and cultured at 25° C. with alternating light and dark conditions (16 h light / 8 h dark, with a light intensity of 1900 lux) for 30 days.

[0063] The state of the explants and the induction of callus were observed and recorded, and the effects of different concentrations of spectinomycin on callus induction and growth were statistically analyzed.

[0064] 2. Construction of chloroplast expression vectors (i.e., recombinant plasmid pBa3, recombinant plasmid pBEa3, recombinant plasmid pBtEa3, and recombinant plasmid pBtEat3)

[0065] Recombinant plasmids pBa3, pBEa3, pBtEa3 and pBtEat3 are all described in the following document: Yu Y, Ouyang Z, Guo J, Zeng W, Zhao Y, Huang L. Complete Chloroplast Genome Sequence of Erigeron breviscapus and Characterization of Chloroplast Regulatory Elements. Front Plant Sci. 2021; 12: 758-290. The names in the document are pBa3, pBEa3, pBtEa3 and pBtEat3, respectively. The construction steps are as follows:

[0066] (1) Chloroplast DNA (cpDNA) was extracted from young leaves of Erigeron breviscapus and used as a template for PCR amplification using a primer pair consisting of primer Trps16-F: 5'-CCCCTATTTCTATGTTATCCAATCAAA-3' and primer Trps16-R: 5'-CAAACAAAATTAAATTAAGGAAATAAAAAAAG-3' and Phusion high-fidelity polymerase. The 3'UTR sequence Trps16 of about 300 bp was recovered using the EasyPure Quick Gel Extraction Kit. Using the 3'UTR sequence Trps16 as a template, PCR amplification was performed using a primer pair consisting of primer pBaTrps16-F: 5'-CCAAGGTAGTCGGCAAATAATCAAACAAAATTAAATTAAGGAAATAAAAAAAG-3' and primer pBaTrps16-R: 5'-GAGCCCGCGGTGGCGGCCGCATGGAATCCATATAGAATAAAGAACTTCTATTC-3' to recover an Eb-Trps16a fragment of about 350 bp.

[0067] (2) Chloroplast DNA (cpDNA) was extracted from young leaves of Erigeron breviscapus and used as a template. The primer pair consisting of primer Prrn-5F: 5'-AACCCAATGTGAGTTTTTCATTTTG and primer Prrn-5R: 5'-TTCATAGTTGCATTACTTATAGCTTCCTT and Phusion super-fidelity polymerase were used for PCR amplification, and the promoter sequence Prrn of about 100 bp was recovered using the EasyPure Quick Gel Extraction Kit. The promoter sequence Prrn was used as a template, and the primer pair consisting of primer pBaPrrn-F: 5'-TCGACGGTATCGATAAGCTTTGCTACCCCGCCGTGAT-3' and primer pBaPrrn-R: 5'-GCGATCACCGCTTCCCCCATAAATCCCTCCCTACAACGTATCCATGCGCTTCATATTCG-3' was used for PCR amplification, and an Eb-Prrna fragment of about 170 bp was recovered.

[0068] (3) Chloroplast DNA (cpDNA) was extracted from young leaves of Erigeron breviscapus and used as a template for PCR amplification using a primer pair consisting of primer trnI-F: 5'-GGGCTATTAGCTCAGTGGTAGAG-3' and primer trnI-R: 5'-TGGGCCATCCTGGACTTG-3' and Phusion high-fidelity polymerase. The homologous recombinant sequence trnI of about 1300 bp was recovered using the EasyPure Quick Gel Extraction Kit. Using the homologous recombination sequence trnI as a template, PCR amplification was performed using a primer pair consisting of primer pBtrnI-recom-F: 5'-TATAGGGCGAATTGGGTACCGGGCCTTGTACACACCGC-3' and primer pBtrnI-recom-R: 5'-TCGAGGGGGGGCCCGGTACCATTCTTTTCCCTGGCGCA-3' to recover an Eb-trnI fragment of about 1300-1400 bp.

[0069] (4) Chloroplast DNA (cpDNA) was extracted from young leaves of Erigeron breviscapus and used as a template for PCR amplification using a primer pair consisting of primer trnA-F: 5'-GGGGATATAGCTCAGTTGGTAGAG-3' and primer trnA-R: 5'-TGGAGATAAGCGGACTCGAAC-3' and Phusion high-fidelity polymerase. The homologous recombinant sequence trnA of about 900 bp was recovered using the EasyPure Quick Gel Extraction Kit. Using the homologous recombination sequence trnA as a template, PCR amplification was performed using a primer pair consisting of primer pBtrnA-recom-F: 5'-TAGAGCGGCCGCCACCGCGGGCTCCACTTGGCTCGGGG-3' and primer pBtrnA-recom-R: 5'-AAGCTGGAGCTCCACCGCGGAGCTTTGTATCGGCTAAGTTCACG-3' to recover an Eb-trnA fragment of about 900-1000 bp.

[0070] (5) The plasmid pBluescriptSK(+) (a product of Beijing Huayueyang Biotechnology Co., Ltd., catalog number VECT4780) was double-digested with restriction endonucleases Not I and Hind III to recover the vector backbone of about 2900 bp. The vector backbone, aadA fragment, Eb-Prrna fragment and Eb-Trps16a fragment were then recombined using the pEASY-UniSeamless Cloning and Assembly Kit to obtain the recombinant plasmid pBa3.

[0071] The aadA fragment is a DNA fragment amplified using plasmid pH7WG2D (described in the following literature: Zhao Y, Zhang Y, Su P, Yang J, Huang L, Gao W. Genetic Transformation System for Woody Plant Tripterygium wilfordii and Its Application to Product Natural Celastrol. Front Plant Sci. 2018; 8: 2221.) as a template and a primer pair consisting of aadA-F: ​​5'-ATGACATGTTTTTTTGGGGTACAGTC-3' and aadA-R: 5'-TTATTTGCCGACTACCTTGGTGAT-3'. The aadA fragment is the spectinomycin resistance selection marker gene aadA.

[0072] (6) The recombinant plasmid pBa3 was digested with restriction endonuclease SalI to recover the linearized recombinant plasmid pBa3. Then, the linearized recombinant plasmid pBa3, eGFP, Eb-Prrna fragment and Eb-Trps16a fragment were recombined using the pEASY-Uni Seamless Cloning and Assembly Kit to obtain the recombinant plasmid pBEa3.

[0073] The eGFP fragment is a DNA fragment amplified using the PBI-1300 vector (described in the following literature: Zhao Y, Zhang Y, Su P, Yang J, Huang L, Gao W. Genetic Transformation System for Woody Plant Tripterygium wilfordii and Its Application to Product Natural Celastrol. Front Plant Sci. 2018; 8: 2221) as a template and a primer pair consisting of eGFP-F: 5'-ATGGTGAGCAAGGGCGAGGA-3' and eGFP-R: 5'-TTAAAGATCTTCTTCAGAAATCAACTTTTGTTC-3'. The eGFP fragment is the reporter gene eGFP.

[0074] The recombinant plasmid pBEa3 was submitted to Beijing Liuhe BGI Co., Ltd. for sequencing. The sequencing results showed that the recombinant plasmid pBEa3 was obtained by inserting the reporter gene eGFP into the recognition site of the restriction endonuclease Sal I of the recombinant plasmid pBa3. The recombinant plasmid pBEa3 also contained the spectinomycin resistance selection marker gene aadA and the reporter gene eGFP.

[0075] (7) The recombinant plasmid pBEa3 was digested with restriction endonuclease Kpn I to recover the linearized recombinant plasmid pBEa3. The linearized recombinant plasmid pBEa3 and the Eb-trnI fragment were then recombined using the pEASY-Uni Seamless Cloning and Assembly Kit to obtain the recombinant plasmid pBtEa3.

[0076] The recombinant plasmid pBtEa3 was submitted to Beijing Liuhe BGI Co., Ltd. for sequencing. The sequencing results showed that the recombinant plasmid pBtEa3 was obtained by inserting the Eb-trnI fragment into the recognition site of the restriction endonuclease Kpn I of the recombinant plasmid pBEa3. The recombinant plasmid pBtEa3 contained the spectinomycin resistance selection marker gene aadA, the reporter gene eGFP and the homologous recombination sequence trnI.

[0077] (8) The recombinant plasmid pBtEa3 was digested with restriction endonuclease Sac I to recover the linearized recombinant plasmid pBtEa3. The linearized recombinant plasmid pBtEa3 and the Eb-trnA fragment were then recombined using the pEASY-Uni Seamless Cloning and Assembly Kit to obtain the recombinant plasmid pBtEat3.

[0078] The recombinant plasmid pBtEat3 was submitted to Beijing Liuhe BGI Co., Ltd. for sequencing. The sequencing results showed that the recombinant plasmid pBtEat3 was obtained by inserting the Eb-trnA fragment into the recognition site of the restriction endonuclease Sac I of the recombinant plasmid pBtEa3. The recombinant plasmid pBtEat3 contains the spectinomycin resistance selection marker gene aadA, the reporter gene eGFP, the homologous recombination sequence trnI and the homologous recombination sequence trnA.

[0079] 3. Establishment and optimization of chloroplast transformation method

[0080] Chloroplast transformation was performed according to the Daniell method (described in the following literature: Verma D, Samson NP, Koya V, Daniell H. A protocol for expression of offoreign genes in chloroplasts. Nat Protoc. 2008; 3(4): 739-758.). The details are as follows:

[0081] (1) Optimization of transformation conditions

[0082] The optimization factors included bombardment pressure, target distance, bombardment number, vacuum level and plasmid DNA concentration. These factors and the variable levels studied are shown in Table 1.

[0083] Table 1

[0084]

[0085] Based on the above five factors (bombardment pressure, target distance, bombardment times, vacuum degree and plasmid DNA concentration) and different levels of each factor (3 levels in total), a mixed orthogonal table L was used. 18 (3 7 ) The conditions for gene gun transformation were optimized. Each factor was repeated three times, and the whole set of experiments was repeated twice.

[0086] Table 2 is the orthogonal experiment table.

[0087] Table 2

[0088]

[0089]

[0090] (2) Explant preparation

[0091] In a clean bench, cut the tender leaves (about 1 cm × 2 cm, as explants) of sterile seedlings of Erigeron breviscapus with good growth conditions and 4 to 8 weeks old, and then inoculate them on differentiation medium 1, with 5-6 explants placed in each culture dish. Finally, place the culture dish in an incubator for dark culture at 25°C. After 1 week of culture, transfer the explants to a hypertonic culture medium (for the purpose of hypertonicity) and culture them at 25°C in the dark for 5 to 6 hours.

[0092] (3) Microcarrier preparation and embedding

[0093] (3-1) Add 1 ml of 70% (v / v) ethanol aqueous solution to 30 mg of gold powder (gold powder particle diameter is 0.6 μm), vortex for 3-5 minutes, let stand at room temperature for 15 minutes, centrifuge at 10,000 rpm for 5 minutes, and collect the precipitate. Subsequently, wash the gold powder twice with sterile water. The steps of each washing are as follows: add 1 ml of sterile water to resuspend for 1 minute, centrifuge at 10,000 rpm for 1 minute, and collect the precipitate. Finally, add 500 μl of 50% (v / v) sterile glycerol aqueous solution to the precipitate, suspend it for 2-3 seconds, and obtain a microcarrier with a gold powder concentration of 60 mg / ml. The microcarrier can be stored at room temperature for 2 weeks.

[0094] (3-2) Obtaining embedded microcarriers

[0095] 5 μl of recombinant plasmid pBtEat3 aqueous solution (concentration of 1.0 μg / μL, 1.5 μg / μL or 2.0 μg / μL), 50 μl of 2.5M calcium chloride aqueous solution and 20 μl of 0.1M sterilized spermidine solution (Beijing Coolbo Technology Co., Ltd., item number PPT9520) were added to the 50 μl microcarrier in sequence, vortexed for 2-3 minutes, allowed to stand for 1 minute, centrifuged at 10000 rpm for 1 minute, and precipitate 1 was collected. Then 140 μl of 70% (v / v) ethanol aqueous solution was added to precipitate 1, slightly mixed, centrifuged at 10000 rpm for 1 minute, and precipitate 2 was collected. Then 140 μl of ethanol was added to precipitate 2 (for the purpose of washing the precipitate), centrifuged at 10000 rpm for 1 minute, and precipitate 3 was collected. Finally, 48 μl of ethanol was added to precipitate 3, and lightly shaken for 2-3 seconds to obtain embedded microcarriers. The embedded microcarriers obtained were sufficient for 6 bombardments.

[0096] (4) Gene gun bombardment

[0097] Use tweezers to transfer the explants completed in step (2) from the hypertonic culture medium to an area of ​​about 2.5 cm in diameter in the middle of a culture dish containing differentiation medium 1. The explants are placed closely to each other, leaving as little space as possible. Then, use a gene gun (PDS1000 / He, Bio-Rad) to bombard the leaves of Erigeron breviscapus with the embedded microcarriers obtained in step (3). For specific operation methods, please refer to the gene gun operation guide. Bombardment was performed according to 16 groups of different transformation conditions listed in Table 2, and each group of experiments was repeated twice.

[0098] 4. Chloroplast transformation, homogenization screening and acquisition of Erigeron breviscapus transplastomic plants

[0099] (1) After completing step 3, place the explant in differentiation medium 1 and culture at 25°C for 2 to 3 days, then divide the explant into approximately 0.8 to 1 cm 2 The cells were divided into small pieces and transferred to differentiation medium 1 and cultured at 25°C with alternating light and dark conditions (16 h light / 8 h dark, light intensity of 1900 lux) for 2 weeks.

[0100] (2) After completing step (1), the divided explants were transferred to a screening medium (with a spectinomycin concentration of 50 mg / L) (for resistance screening) and cultured in the dark at 25° C. until resistant callus was obtained; during this period, fresh screening medium was replaced every 2 weeks.

[0101] The preparation method of the screening medium (the concentration of spectinomycin is 50 mg / L) is as follows: dissolve 4.43 g MS powder, 30 g sucrose and 4 g plant gel in an appropriate amount of deionized water, then make up to 1 L with deionized water, and adjust the pH value to 5.8; sterilize at 121°C and high temperature and high pressure for 15 min; when cooled to about 40-50°C, add sterilized 6-BA, NAA and spectinomycin (Spe) and make their concentrations in the system 1 mg / L, 0.1 mg / L and 50 mg / L, respectively.

[0102] (3) After completing step (2), a small amount of newly generated resistant callus tissue was clamped, and total DNA was extracted using the CTAB method. Then, PCR amplification was used to detect the eGFP gene and the aadA gene. The primers used to detect the eGFP gene were primer PeGFP-F: 5'-ATGGTGAGCAAGGGCGAGGA-3' and primer PeGFP-R: 5'-TTAAAGATCTTCTTCAGAAATCAACTTTTGTTC-3'. The primers used to detect the aadA gene were primer PaadA-F: ​​5'-ATGGGGGAAGCGGTGATC-3' and primer PaadA-R: 5'-TTATTTGCCGACTACCTTGGTGAT-3'. The results were determined as follows:

[0103] If primers PeGFP-F and primer PeGFP-R are used for PCR amplification, a DNA fragment of 762 bp can be obtained, indicating that the resistant callus contains the eGFP gene; if primers PaadA-F and primer PaadA-R are used for PCR amplification, a DNA fragment of 792 bp can be obtained, indicating that the resistant callus contains the aadA gene.

[0104] (4) After completing step (3), select resistant callus tissue containing eGFP gene and aadA gene identified by PCR, cut it into 5 mm × 5 mm size with tweezers, and place it in differentiation medium 1, 25°C, light and dark alternating culture (16 h light / 8 h dark, light intensity of 1900 lux) for 2 weeks (first round of differentiation); then place it in differentiation medium 2, 25°C, light and dark alternating culture (16 h light / 8 h dark, light intensity of 1900 lux) for 4 weeks, during which time it is subcultured once every 2 weeks (second round of differentiation); then place it in differentiation medium 3, 25°C, light and dark alternating culture (16 h light / 8 h dark, light intensity of 1900 lux), subcultured once every 2 weeks (third round of differentiation), until resistant clustered shoots growing to 2 to 3 cm are obtained.

[0105] Differentiation medium 2: Dissolve 4.43 g MS powder, 30 g sucrose and 4 g plant gel in an appropriate amount of deionized water, then dilute to 1 L with deionized water, adjust the pH to 5.8; sterilize at 121°C for 15 min; when cooled to about 40-50°C, add sterilized 6-BA, NAA and spectinomycin and adjust their concentrations in the system to 1 mg / L, 0.1 mg / L and 5 mg / L, respectively.

[0106] Differentiation medium 3: Dissolve 4.43 g MS powder, 30 g sucrose and 4 g plant gel in an appropriate amount of deionized water, then dilute to 1 L with deionized water, adjust the pH to 5.8; sterilize at 121°C for 15 min; when cooled to about 40-50°C, add sterilized 6-BA, NAA and spectinomycin and adjust their concentrations in the system to 1 mg / L, 0.1 mg / L and 10 mg / L, respectively.

[0107] (4) After completing step (4), the resistant clustered buds that have grown to 2 to 3 cm are transferred to a rooting medium for rooting culture (25°C, alternating light and dark culture (16 h light / 8 h dark, light intensity of 1900 lux)) to obtain Erigeron breviscapus transplastomic plants.

[0108] 5. Molecular detection of transplastomic plants of Erigeron breviscapus

[0109] (1) PCR detection of target genes and selectable marker genes in transplastomic plants of Erigeron breviscapus

[0110] The genomic DNA of the transplastomic plants of Erigeron breviscapus was extracted using the Hi-DNAsecure Plant Kit, and then the eGFP gene and aadA gene were detected by PCR amplification. The primers used to detect the eGFP gene were primer PeGFP-F: 5'-ATGGTGAGCAAGGGCGAGGA-3' and primer PeGFP-R: 5'-TTAAAGATCTTCTTCAGAAATCAACTTTTGTTC-3'. The primers used to detect the selective marker gene aadA were primer PaadA-F: ​​5'-ATGGGGGAAGCGGTGATC-3' and primer PaadA-R: 5'-TTATTTGCCGACTACCTTGGTGAT-3'. The results were determined as follows:

[0111] If primers PeGFP-F and primers PeGFP-R are used for PCR amplification, a DNA fragment of 762 bp in size can be obtained, indicating that the transplastomic plants of Erigeron breviscapus contain the eGFP gene; if primers PaadA-F and primers PaadA-R are used for PCR amplification, a DNA fragment of 792 bp in size can be obtained, indicating that the transplastomic plants of Erigeron breviscapus contain the aadA gene.

[0112] (2) PCR detection of whether eGFP and aadA genes are integrated into the chloroplast genome of Erigeron breviscapus

[0113] The genomic DNA of the transplastomic plants of Erigeron breviscapus was extracted using the Hi-DNAsecure Plant Kit, and then the LF-eGFP and RF-aadA sequences were detected by PCR amplification. The LF-eGFP sequence was about 1.8 kb in size and contained part of the left homologous recombination sequence and part of the eGFP gene expression cassette sequence. The RF-aadA sequence was about 2.1 kb in size and contained part of the right homologous recombination sequence and part of the aadA gene expression cassette sequence (see Figure 1 ). The primers used to detect whether the sequence LF-eGFP is integrated into the chloroplast genome of Erigeron breviscapus are primer PtrnI-LF: 5'-GGTAGCCGTACTGGAAGGTGC-3' and primer PeGFP-R2: 5'-TCGGCCATGATATAGACGTTGTG-3'. The primers used to detect whether the aadA gene is integrated into the chloroplast genome of Erigeron breviscapus are primer PaadA-F: ​​5'-ATGGGGGAAGCGGTGATC-3' and primer PtrnA-RF: 5'-AGCTTTGTATCGGCTAAGTTCAC-3'. The results are determined as follows:

[0114] If primers PtrnI-LF and primers PeGFP-R2 are used for PCR amplification, a DNA fragment of 1.8 kb can be obtained, indicating that the eGFP gene is integrated into the chloroplast genome of the transplastomic plant of Erigeron breviscapus; if primers PaadA-F and primers PtrnA-RF are used for PCR amplification, a DNA fragment of 2.1 kb can be obtained, indicating that the aadA gene is integrated into the chloroplast genome of the transplastomic plant of Erigeron breviscapus; the integration of the eGFP gene into the chloroplast genome of the transplastomic plant of Erigeron breviscapus and the integration of the aadA gene into the chloroplast genome of the transplastomic plant of Erigeron breviscapus Figure 1 The chloroplast operon of the recombinant plasmid pBtEat3 shown was integrated into the chloroplast genome of the Erigeron breviscapus transplastomic plant.

[0115] 6. Fluorescence detection

[0116] The chloroplast morphology and fluorescence expression of leaves of the transplastomic plants of Erigeron breviscapus were observed using a laser confocal scanning microscope. The excitation light wavelength was set to 488 nm, the detectable green fluorescent protein emission wavelength was 516 nm, and the detectable chlorophyll fluorescence emission wavelength was 650-682 nm.

[0117] 2. Experimental Results

[0118] 1. Effect of spectinomycin on callus induction of Erigeron breviscapus

[0119] Effects of spectinomycin on callus induction in Erigeron breviscapus Figure 2 : Spectinomycin has a significant inhibitory effect on callus induction in the range of 250-500 mg / L. The leaf explants of Erigeron breviscapus show a slight swelling, thickening of the corners and curling inwards. Only a small amount of light yellow granular protrusions are produced at the incision of individual Erigeron breviscapus leaf explants, and callus formation is basically not induced; Spectinomycin is in the range of 100-200 mg / L. A thin layer of callus with white granular shape is formed on the edge of the leaf explants of Erigeron breviscapus after 2 weeks of culture. After 30 days of culture, the callus shows obvious browning phenomenon and growth stagnation; Spectinomycin in the range of 10-40 mg / L cannot effectively inhibit the growth of Erigeron breviscapus callus. If it is used for the screening of resistant callus, it will lead to the generation of a large number of non-transformed cells; and when spectinomycin is 50 mg / L, it can effectively inhibit the growth of callus without causing a large number of cell death. Therefore, 50 mg / L of spectinomycin is a more ideal concentration for screening Erigeron breviscapus plastid transformation resistant callus.

[0120] 2. Screening and regeneration of transplastomic plants of Erigeron breviscapus

[0121] The screening and regeneration process of transplastomic plants of Erigeron breviscapus is shown in Figure 3 .

[0122] The bombarded leaves of Erigeron breviscapus ( Figure 3 Middle A) Recover in culture at 25°C for 2-3 days on differentiation medium 1, then divide the explants to approximately 0.8-1 cm 2 Small pieces ( Figure 3 B), and transferred to differentiation medium 1 and cultured in alternating light and dark at 25°C for 2 weeks; then the Erigeron breviscapus explants were transferred to screening medium (spectinomycin concentration was 50 mg / L) and cultured in the dark at 25°C to induce resistant callus ( Figure 3 C), during which fresh screening medium was replaced every 2 weeks. During the induction culture of resistant callus, callus was observed on the edge of the leaves after about 5 to 8 weeks. The screening concentration of 50 mg / L spectinomycin caused most of the calli to turn brown and die, while another part of the calli neither browned nor differentiated during the long screening process, but were able to proliferate continuously, indicating that this screening concentration can obtain resistant calli but is not conducive to callus differentiation. In the first round of differentiation culture, the screening pressure was cancelled, and after 2 weeks of culture, the spectinomycin concentration was gradually increased, with subculture every 2 weeks until the screening pressure reached 10 mg / L. Green resistant callus can be induced in the 8th to 11th week of differentiation culture ( Figure 3 D), the callus is clearly different from the non-transformed callus (albino, Figure 3 Middle C).

[0123] The statistics of the number of green resistant calli during the screening culture are shown in Table 3. The leaves of Experimental Group 1 (gene gun transformation conditions: bombardment pressure 1100psi, target distance 6cm, vacuum degree 27mmHg, bombardment times 2 times, plasmid DNA concentration 1μg / μL) produced green resistant calli the latest, and green resistant calli were not observed until the 11th week of screening culture. A total of 4 green resistant calli were produced during the screening culture; the leaves of Experimental Groups 4, 7 and 11 produced calli from the 10th to 11th week, but the calli were in a white state during the entire screening culture, and no green resistant calli were observed; Experimental Group 10 (gene gun transformation conditions: The leaves of experimental group 12 (bombardment pressure 1100psi, target distance 9cm, vacuum 26mmHg, bombardment number 1, plasmid DNA concentration 1μg / μL) produced green resistant callus earliest, and green callus was observed in the 8th week. During the screening culture, 9 green resistant callus were produced. The leaves of experimental group 12 (gene gun transformation conditions: bombardment pressure 1100psi, target distance 3cm, vacuum 28mmHg, bombardment number 1, plasmid DNA concentration 1.5μg / μL) produced green resistant callus in the 9th to 10th week, and 8 green resistant callus were produced during the screening culture.

[0124] Table 3. Statistics of green resistant callus during screening and cultivation

[0125]

[0126] After 2 to 4 weeks of differentiation screening, multiple green budding points can be seen ( Figure 3 E), when the clustered buds obtained by screening and culture grow to 2-3 cm ( Figure 3 After about 2 to 3 weeks of rooting culture, only a few clustered buds can take root ( Figure 3 Among all the experimental groups, only experimental groups 10 and 12 eventually obtained transplastomic plants with strong root systems. Although experimental group 1 produced green callus, it did not differentiate into green resistant clustered buds.

[0127] 3. Molecular identification of transplastomic plants of Erigeron breviscapus

[0128] Using gDNA of Erigeron breviscapus transplastomic plants, recombinant plasmid pBtEat3 or genomic DNA of non-transgenic Erigeron breviscapus sterile seedlings (i.e., wild-type Erigeron breviscapus sterile seedlings) as templates, primers PeGFP-F and PeGFP-R were used to amplify the eGFP gene, and primers PaadA-F and PaadA-R were used to amplify the aadA gene. The PCR amplification products were detected by 1.0% agarose gel electrophoresis. The results are as follows: Figure 4 As shown in the figure (M: DL 2000DNA Maker; pBtEat3 is the recombinant plasmid pBtEat3; WT is the genomic DNA of the non-transgenic Erigeron breviscapus sterile seedlings, G10 is the Erigeron breviscapus transplastomic plant obtained from experimental group 10, and G12 is the Erigeron breviscapus transplastomic plant obtained from experimental group 12). The results showed that the Erigeron breviscapus transplastomic plant obtained from experimental group 10 and the Erigeron breviscapus transplastomic plant obtained from experimental group 12, as well as the recombinant plasmid pBtEat3, could amplify the target bands that were consistent with the size of the eGFP gene (about 760 bp) and the aadA gene (about 800 bp), while the non-transgenic Erigeron breviscapus sterile seedlings did not amplify the target bands that were consistent with the size of the eGFP gene and the aadA gene.

[0129] In order to further verify whether the eGFP gene and aadA gene were integrated into the chloroplast genome of Erigeron breviscapus, gDNA of Erigeron breviscapus transplastomic plants, recombinant plasmid pBtEat3 or genomic DNA of sterile seedlings of non-transgenic Erigeron breviscapus was used as templates, and the sequence LF-eGFP was amplified by primers PtrnI-LF and PeGFP-R2, and the sequence RF-aadA was amplified by primers PaadA-F and PtrnA-RF. The PCR amplification products were detected by 1.0% agarose gel electrophoresis. The results are shown in Figure 2. Figure 5As shown (M: Trans2K PlusDNAMaker; pBtEat3 is the recombinant plasmid pBtEat3; WT is the genomic DNA of the non-transgenic Erigeron breviscapus sterile seedlings, the one containing "G10" is the Erigeron breviscapus transplastomic plant obtained from experimental group 10, and the one containing "G12" is the Erigeron breviscapus transplastomic plant obtained from experimental group 12). The results showed that the Erigeron breviscapus transplastomic plants obtained from experimental group 10 and experimental group 12, as well as the recombinant plasmid pBtEat3, could amplify DNA bands consistent with the size of the sequences LF-eGFP (1.8kb) and RF-aadA (2.1kb), while the non-transgenic Erigeron breviscapus sterile seedlings did not amplify DNA bands consistent with the size of the sequences LF-eGFP and RF-aadA. Explanation Figure 1 The chloroplast operon of the vector pBtEat3 shown has been targeted and integrated into the chloroplast genome of the Erigeron breviscapus transplastomic plants.

[0130] 4. The coefficients of the transplastomic plants of Erigeron breviscapus obtained in experimental groups 10 and 12 are shown in Table 4. A total of 7 transplastomic plant lines of Erigeron breviscapus were obtained in experimental group 10, of which 2 were mutants; a total of 2 transplastomic plant lines of Erigeron breviscapus were obtained in experimental group 12, and no mutant lines were obtained. Finally, the optimal transformation conditions of Erigeron breviscapus plastid transformation gene gun were determined as follows: bombardment pressure 1100psi, target distance 9cm, vacuum degree 26mmHg, bombardment times 1 time, and plasmid DNA concentration 1μg / μL.

[0131] Table 4. Statistics of transplastomic Erigeron breviscapus

[0132]

[0133] 5. Fluorescence detection of transplastomic plants of Erigeron breviscapus

[0134] The mesophyll cells of transplastomic plants and sterile seedlings of non-transgenic Erigeron breviscapus were observed using a laser confocal microscope.

[0135] Results Figure 6 (EbTp is a transplastomic plant of Erigeron breviscapus, and WT is a non-transgenic sterile seedling of Erigeron breviscapus). The results showed that under the bright field of view, gray chloroplasts were visible in the mesophyll cells of both the transplastomic plants of Erigeron breviscapus and the sterile seedlings of wild-type Erigeron breviscapus, and the chloroplasts were flat and complete spherical or ellipsoidal in shape; when the wavelength of the excitation light was adjusted to 488nm, chlorophyll autofluorescence (red) was visible in the mesophyll cells of both the transplastomic plants of Erigeron breviscapus and the sterile seedlings of wild-type Erigeron breviscapus, and green fluorescence produced by eGFP under the action of the excitation light could be observed in the transplastomic plants of Erigeron breviscapus, and it appeared yellow after being superimposed with the red fluorescence of chlorophyll, indicating that the eGFP gene was expressed in the chloroplasts; while no green fluorescence was observed in the sterile seedlings of wild-type Erigeron breviscapus, and no yellow fluorescence appeared when it was superimposed with the red fluorescence.

[0136] The above results show that the present invention provides a method for genetic transformation of chloroplasts of Erigeron breviscapus mediated by gene gun, comprising the following steps: using gene gun method to introduce a plasmid containing a target gene into sterile leaf explants of Erigeron breviscapus seedlings; in the gene gun method, the parameters of gene gun bombardment are set as follows: DNA concentration: 1 μg / μl; bombardment times: 1 time; target distance: 9 cm; bombardment pressure: 1100 psi; vacuum degree: 26 mmHg; the critical concentration of antibiotics for screening culture of Erigeron breviscapus plastid transformation is 50 mg / L, and through PCR verification and microscopic detection, it is determined that the target gene is integrated into the Erigeron breviscapus chloroplast genome, and finally the Erigeron breviscapus transplastomic plant is obtained. The present invention obtains homogenized Erigeron breviscapus chloroplast transgenic plant materials (i.e., Erigeron breviscapus transplastomic plants) for the first time, and obtains molecular evidence.

[0137] The present invention has been described in detail above. For those skilled in the art, without departing from the purpose and scope of the present invention, and without the need to carry out unnecessary experimental conditions, the present invention can be implemented in a wide range under equivalent parameters, concentrations and conditions. Although the present invention provides specific embodiments, it should be understood that the present invention can be further improved. In a word, according to the principles of the present invention, the application is intended to include any changes, uses or improvements to the present invention, including departure from the disclosed scope in the application, and changes made with conventional techniques known in the art.

Claims

1. A method for genetic transformation of chloroplasts of Erigeron breviscapus, comprising using a gene gun method to introduce a plasmid containing a target gene into an explant prepared from leaves of Erigeron breviscapus to obtain a transplastomic plant of Erigeron breviscapus; Features: The parameters of gene gun bombardment are as follows: plasmid DNA concentration is 1-1.5 μg / μl, bombardment times are 1-2 times, target distance is 3-9 cm, bombardment pressure is 900-1100 psi, and vacuum degree is 26-28 mmHg.

2. The method according to claim 1, Features: The steps of "using a gene gun method to introduce a plasmid containing a target gene into an explant prepared from leaves of Erigeron breviscapus to obtain an Erigeron breviscapus transplastomic plant" are as follows: (1) inoculating leaves of Erigeron breviscapus on differentiation medium 1 and culturing; The solutes and concentrations of differentiation medium 1 are 4.20-4.60 g / L of MS powder, 28-30 g / L of sucrose, 4-6 g / L of phytagel, 1-2 mg / L of 6-BA, and 0.1-0.2 mg / L of NAA; the solvent is water; the pH value is 5.8-6.0; (2) placing leaves of Erigeron breviscapus in a hypertonic medium for hypertonic treatment; The solutes and concentrations of the hypertonic culture medium are 4.20-4.60 g / L of MS powder, 28-30 g / L of sucrose, 68-72 g / L of mannitol, and 4-6 g / L of plant gel; the solvent is water; the pH value is 5.8-6.0; (3) introducing a plasmid containing the target gene by gene gun bombardment, and then placing it in differentiation medium 1 and culturing; (4) placing the callus on a screening medium and culturing it to obtain resistant callus; The solutes and concentrations of the screening culture medium are 4.20-4.60 g / L of MS powder, 28-30 g / L of sucrose, 4-6 g / L of phytagel, 1-2 mg / L of 6-BA, 0.1-0.2 mg / L of NAA and 50-55 mg / L of spectinomycin; the solvent is water; the pH value is 5.8-6.0; (5) placing the resistant callus containing the target gene on differentiation medium 1 and culturing; (6) placing in differentiation medium 2 and culturing; The solutes and concentrations of differentiation medium 2 are 4.20-4.60 g / L MS powder, 28-30 g / L sucrose, 4-6 g / L phytagel, 1-2 mg / L 6-BA, 0.1-0.2 mg / L NAA and 5-8 mg / L spectinomycin; the solvent is water; the pH value is 5.8-6.0; (7) placing in differentiation medium 3 and culturing to obtain resistant clustered shoots; The solutes and concentrations of differentiation medium 3 are 4.20-4.60 g / L MS powder, 28-30 g / L sucrose, 4-6 g / L phytagel, 1-2 mg / L 6-BA, 0.1-0.2 mg / L NAA and 10-12 mg / L spectinomycin; the solvent is water; the pH value is 5.8-6.0; (8) placing the plant on a rooting medium and culturing the plant to obtain a transplastomic plant of Erigeron breviscapus; The solutes and concentrations of the rooting medium are 2.00-2.40 g / L of MS powder, 28-30 g / L of sucrose, 4-6 g / L of plant gel, 0.5-0.8 mg / L of IBA and 0.3-0.5 mg / L of NAA; the solvent is water; and the pH value is 5.8-6.

0.

3. The method according to claim 2, Features: In the step (1), the culture is carried out in the dark at 23-27° C. for 5-9 days.

4. The method according to claim 2, Features: In the step (2), the method of hypertonic treatment is dark culturing at 23-27° C. for 5-6 hours.

5. The method according to claim 2, Features: In the steps (3) and (5), the culture is carried out at 23-27° C. with alternating light and dark for 1-3 weeks.

6. The method according to claim 2, Features: In the step (4), the culture is carried out in the dark at 23-27°C.

7. The method according to claim 2, Features: In the step (6), the culture is carried out at 23-27° C. with alternating light and dark for 3-5 weeks.

8. The method according to claim 2, Features: In the steps (7) and (8), the culture is carried out at 23-27° C. with alternating light and dark.

9. A kit for genetic transformation of chloroplasts of Erigeron breviscapus, comprising at least one of the differentiation medium 1, the hypertonic medium, the screening medium, the differentiation medium 2, the differentiation medium 3 and the rooting medium in claim 2.

10. Use of at least one of the differentiation medium 1, the hypertonic medium, the screening medium, the differentiation medium 2, the differentiation medium 3 and the rooting medium in claim 2 in genetic transformation of chloroplasts of Erigeron breviscapus.

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