Cultivation method of isatis tinctoria plant overexpressing EVM0003075 gene

Abstract

The invention relates to a cultivation method of an overexpressed EVM0003075 gene isatis tinctoria plant, and belongs to the technical field of plant cultivation. The method comprises the following steps: firstly, transforming an agrobacterium strain through a constructed agrobacterium transformation vector for expressing the isatis tinctoria EVM0003075 gene, then transforming an explant of the isatis tinctoria by using a screened recombinant agrobacterium strain, regenerating a seedling of the isatis tinctoria, and screening and culturing the seedling of the isatis tinctoria with stable overexpression of the EVM0003075 gene, so as to obtain an isatis tinctoria plant with increased isatin content in the isatis tinctoria. The expression level of the EVM0003075 gene of the transgenic isatis tinctoria is improved to 5-9 times, the content of the isatis tinctoria is remarkably improved, and the character that the accumulation period of the isatis tinctoria is short is shown. By adopting the method disclosed by the invention, the content of the isatin in the isatis tinctoria can be increased to 10-66 times. The specific information content of the patent of the method for improving the content of the isatin in the isatis tinctoria through overexpression of the EVM0003075 gene is shown in the specification.

Description

Technical Field

[0001] This invention relates to the field of plant cultivation technology, specifically to an overexpression method. EVM0003075 Methods for cultivating genetically modified Isatis indigotica plants. Background Technology

[0002] Isatis indigotica ( Isatis tinctoria L. Isatis tinctoria is a biennial herb of the Brassicaceae family with at least 2,000 years of cultivation history. It is an important industrial raw material for medicinal, dyeing, food, and cosmetic purposes. However, through long-term cultivation and artificial breeding, different varieties or farm-cultivated germplasm have been formed, and their quality often varies greatly, which seriously restricts the production and application of Isatis tinctoria.

[0003] Studies have shown that indigo and indirubin are the main medicinal components of Isatis indigotica. Indigo has pharmacological effects such as anti-inflammatory, hepatoprotective, and immune-enhancing properties. It is also a natural organic pigment widely used in printing and dyeing, food, and cosmetics industries. Indirubin is an anti-tumor drug, clinically effective in treating chronic myeloid leukemia. However, in the early stages of cultivation, Isatis indigotica breeding primarily used conventional methods to select high-yielding and insect-resistant varieties, such as "Jilan No. 1," "Dinglan No. 1," and tetraploid Isatis indigotica. Varieties or strains specifically targeting indigo and indirubin content are currently lacking. Therefore, the indigo and indirubin content in Isatis indigotica is currently low, at only 3.4% and 0.6%, respectively.

[0004] Therefore, in order to solve the above problems, there is an urgent need for a method to cultivate Isatis indigotica plants that can improve the quality of Isatis indigotica and increase the content of indigo and indirubin in Isatis indigotica, which is of great significance to the production and application of Isatis indigotica. Summary of the Invention

[0005] To solve or partially solve the problems existing in related technologies, the present invention provides a method for cultivating Isatis indigotica plants that not only results in a high survival rate of positive seedlings and good seedling growth, but also significantly increases the indigo glycoside content and shortens the accumulation period.

[0006] This application provides an overexpression EVM0003075 A method for cultivating Isatis indigotica plants using genetically modified styrax, the method comprising the following steps: (1) Constructing an expression of Isatis indigotica EVM0003075 Agrobacterium-mediated transformation vector for genes; (2) The Agrobacterium transformation vector constructed in step (1) was transformed into Agrobacterium GV3101 competent cells by freezing and thawing to obtain the transformed Agrobacterium strain; (3) Using Isatis indigotica as the recipient material, the Agrobacterium strain obtained in step (2) was genetically transformed to obtain a stable overexpression strain. EVM0003075 Isatis seedlings with genetically modified citronella; (4) The Isatis indigotica seedlings obtained in step (3) are screened and then cultivated to obtain overexpression EVM0003075 Isatis indigotica plants with genetic defects.

[0007] Furthermore, in step (1), the pRI101-GFP vector is used to construct an expression vector for Isatis indigotica. EVM0003075 Agrobacterium-mediated transformation vector for genes.

[0008] Furthermore, the specific operation of the freeze-thaw method in step (2) is as follows: 1) Thaw Agrobacterium GV3101 competent cells stored at -80℃ on ice; 2) Pipette 10 μL of pRI101-GFP- from step 1). EVM0003075 Plasmids were added to Agrobacterium GV3101 competent cells, and the cells were gently mixed and then incubated on ice for 5 min, in liquid nitrogen for 5 min, in a 37°C water bath for 5 min, and in an ice bath for 5 min. 3) Add 700 μL of antibiotic-free LB medium to 2), mix well, and revive at 28°C and 200 rpm for 2 h; take 200 μL of the revived bacterial solution and spread it on a solid culture medium containing 50 μg / mL kanamycin and 20 μg / mL rifampin using a spreader. Invert the plate and place it in a 28°C incubator for 2-3 days.

[0009] Furthermore, the genetic transformation in step (3) is carried out using Agrobacterium-mediated genetic transformation with Isatis tinctoria as the recipient material.

[0010] Furthermore, the cultivation in step (4) involves directly transplanting the rooted Isatis indigotica seedlings into the soil after hardening them off.

[0011] Beneficial effects 1. The method for cultivating Isatis indigotica plants provided in this application can directionally induce an increase in indigo glycoside content and shorten the accumulation period of indigo glycosides, thereby enabling transgenic overexpression. EVM0003075 In the gene-derived Isatis indigotica, the content of indoside is increased by 10 to 66 times or more.

[0012] 2. It was determined that when culturing Isatis tinctoria using the method provided in this application, the accumulation of indoside was significantly accelerated (the transgenic plants had accumulated indoside on the 120th day after transplanting into the soil, but it was not detected in the control group).

[0013] 3. The method provided in this application is not affected by climate or seasonal restrictions; it increases production rapidly, has significant economic benefits, and has a promising application prospect.

[0014] It should be understood that the above general description and the following detailed description are exemplary and explanatory only, and do not limit this application. Attached Figure Description

[0015] The above and other objects, features and advantages of this application will become more apparent from the more detailed description of exemplary embodiments thereof in conjunction with the accompanying drawings.

[0016] Figure 1 Overexpression EVM0003075 Diagram of the tissue culture process of genes; a) Regeneration of transgenic shoots, b) Elongation of transgenic regenerated shoots, c) Regenerated shoots divided into individual individuals, d) Rooting of transgenic plants, d) Growth of rooted plants.

[0017] Figure 2 Overexpression EVM0003075 PCR positive identification of transgenic plants of the gene: M in lanes 1 and 16 is the Trans 15K DNA marker; lanes 2-8, numbered 1-1 to 1-7, are the identification results of overexpression materials; lanes 9 and 10 are the negative control of non-transgenic Isatis indigotica material and negative control with added double distilled water, respectively; lanes 11-15 are the identification results of the control group.

[0018] Figure 3 Overexpression EVM0003075 qRT-PCR gene expression level diagram; EV represents the average expression level of the control group, and 1-1 to 1-6 represent positive transgenic plants.

[0019] Figure 4 Overexpression EVM0003075 The indoside content of the gene was measured in μg / g, with EV-1 to EV-3 representing the control group and 1-1 to 1-6 representing the positive transgenic plants.

[0020] Figure 5 Overexpression EVM0003075 Genes were transplanted into soil and cultivated for 120 days to produce plants that were growing well. Detailed Implementation

[0021] Embodiments of this application will now be described in more detail with reference to the accompanying drawings. While embodiments of this application are shown in the drawings, it should be understood that this application may be implemented in various forms and should not be limited to the embodiments set forth herein. Rather, these embodiments are provided to make this application more thorough and complete, and to fully convey the scope of this application to those skilled in the art.

[0022] Within the plant, indigo and indirubin are stored in vacuoles as precursors—indican. In mature leaves or after physical damage to the plant, the colorless indican in the vacuoles comes into contact with β-D-glucosidase (GLU) in the chloroplasts, catalyzing the cleavage of its glycosidic bonds and reducing it to unstable indoxyl. Most of the indoxyl then further condenses to form indigo and indirubin. Additionally, a small portion of indoxyl combines with isatitin to also form indirubin. Therefore, increasing the content of the precursor indican can further increase the content of indigo and indirubin in Isatis tinctoria.

[0023] Previous research by our group has revealed that the cytochrome P450 (CYP) gene family encodes key enzyme proteins involved in the biosynthesis of indole alkaloids such as indole glycosides. EVM0003075 The gene belongs to the CYP gene family, and previous work showed that it was significantly overexpressed in tissues with high isatis glycoside content. However, regarding isatis glycosides... EVM0003075 No reports have yet been published regarding genes and their application in regulating the synthesis of compounds such as indigo glycosides.

[0024] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains.

[0025] The term "conversion" refers to the process of transforming woad into a more complex and efficient form. EVM0003075 Gene transfer involves the genetic transformation of polynucleotides or polypeptides into plants by introducing genes into plant cells. Methods for introducing these polynucleotides or polypeptides into plants are well known in the art, including but not limited to stable transformation, transient transformation, and virus-mediated transformation. "Stable transformation" refers to the integration of the introduced polynucleotide construct into the genome of a plant cell and its inheritance through its progeny; "transient transformation" refers to the introduction of a polynucleotide into a plant but its temporary expression or presence within the plant.

[0026] The term "explant" refers to a fragment of an organ or tissue used as in vitro culture material in plant tissue culture, such as seeds, cotyledons, and hypocotyls. In practice, those skilled in the art select appropriate explants for transformation based on different plants.

[0027] The term "regeneration" refers to the further cultivation of transformed callus or explants on a suitable culture medium, allowing them to grow into an independent and complete plant.

[0028] The term "control group" refers to Isatis indigotica plants that have not received the pRI101-GFP vector plasmid and have been treated and treated under the same conditions as the experimental group.

[0029] Unless otherwise specified, all reagents and materials used in the following examples were purchased from the market.

[0030] Example 1 Overexpression EVM0003075 The gene increases the indigo glycoside content of Isatis indigotica.

[0031] Step 1: Isatis indigotica EVM0003075 Construction of gene overexpression vectors First, use the sequence shown in SEQ ID No. 1 ( EVM0003075 The gene sequence was used as a template for artificial chemical synthesis (Sangon Biotech Co., Ltd.), and the plant expression vector pRI101-GFP was constructed using the pRI101-GFP vector. EVM0003075 .

[0032] Used to construct expression of Isatis indigotica EVM0003075 The Agrobacterium-mediated transformation vector for the gene was pRI101-GFP, donated by the laboratory of Yang Yongping, Kunming Institute of Botany, Chinese Academy of Sciences. The nucleotide sequence of the pRI101-GFP vector has been published in Zheng Y, Gao Z, Luo LD, Wang YG, Chen Q, Yang Y, Kong XX, Yang YP. Divergence of the genetic contribution of FRIGIDA homologues in regulating the flowering time in Brassica rapa ssp. rapa[J]. Gene, 2021, 796-797(4):145790.DOI:10.1016 / j.gene.2021.145790.

[0033] Step 2: Agrobacterium-mediated transformation The expression vector pRI101-GFP- was expressed using a freeze-thaw method. EVM0003075 Transplanted into Agrobacterium GV3101 competent cells (available from Beijing Qingke Biotechnology Co., Ltd.). The specific method is as follows: 1) Thaw Agrobacterium GV3101 competent cells stored at -80℃ on ice.

[0034] 2) Use a pipette to draw 10 μL of the solution from step 1 above (Isatis indigotica). EVM0003075 pRI101-GFP- (construction of gene overexpression vector) EVM0003075 The plasmid was added to the thawed Agrobacterium GV3101 competent cells from step 1), and the mixture was gently incubated on ice for 5 min, in liquid nitrogen for 5 min, in a 37°C water bath for 5 min, and in an ice bath for 5 min.

[0035] 3) Add 700 μL of antibiotic-free LB medium to 2), mix well, and incubate at 28°C and 200 rpm for 2 h. Take 200 μL of the incubated bacterial solution and spread it onto a solid culture medium containing 50 μg / mL kanamycin and 20 μg / mL rifampin using a spreader. Invert the plate and incubate at 28°C for 2-3 days.

[0036] Step 3: Genetic transformation Genetic transformation was performed using Agrobacterium-mediated transformation with Isatis tinctoria as the recipient material. The tissue culture process is described in [link to relevant documentation]. Figure 2 .

[0037] The specific method is as follows: 1) Seed germination Take an appropriate amount of mature Isatis indigotica seeds, remove the husks, and disinfect them with 10% sodium hypochlorite for 5-7 minutes, shaking continuously during this time. Then rinse them 7-8 times with sterile distilled water, blot dry the seed surface with sterile filter paper, and then inoculate them. Inoculate the disinfected seeds into MS medium and incubate them in the dark at 25°C for 7-8 days.

[0038] 2) Agrobacterium infection The specific steps are as follows: a) Use a sterile pipette tip to transfer the bacterial plaques grown on the antibiotic medium in step 2 (Agrobacterium transformation) into a liquid antibiotic medium containing 50 μg / mL kanamycin and 20 μg / mL rifampin, and incubate at 28°C and 200 rpm for 2-3 days.

[0039] b) Collect bacterial cells by centrifugation at 6000g and 4℃ for 10 min, discarding the supernatant; add 5 mL of staining buffer (4.74 g / L MS powder, 20 g / L sucrose, pH=5.6, sterilized at 121℃ for 20 min) containing 200 μM acetylsylgenone (AS), suspend the bacterial cells, and gently aspirate with a pipette to ensure complete resuscitation. The final OD of the bacterial solution is... 600 =0.8 and keep it in reserve.

[0040] c) Cut the hypocotyls obtained after seed germination in step 1) into 0.5 mm pieces, transfer them to a petri dish covered with sterile filter paper, and air dry them on a clean bench for 10-20 minutes.

[0041] d) Immerse the dried hypocotyl in a 50 mL centrifuge tube containing the above bacterial solution for 15 min, shaking it once every 5 min during the process; then discard the bacterial solution, remove the infected hypocotyl and place it on sterile filter paper to air dry for 10-20 min.

[0042] 3) Co-culture and transgenic bud regeneration A) Transfer the dried hypocotyls from step 2) after infection to a co-culture medium lined with sterile filter paper and co-culture at 28°C in the dark for 2-3 days. The co-culture medium is MS medium containing 0.1 mg / L thiafenuron (TDZ) and 100 μM AS.

[0043] B) After dark culture, the hypocotyls were transferred to selection medium for further selection 2-3 times, each time for two weeks, under 25°C light (16 hours light / 8 hours dark). The selection medium was MS medium containing 0.1 mg / L TDZ, 100 μM AS, 200 mg / L termetidine (TMT), and 50 mg / L kanamycin, pH=5.8. 4) Elongation of regenerated shoots: After the regenerated shoots grow to 2 cm, they are transferred to shoot subculture medium (MS medium containing 0.1 mg / L TDZ, 100 μM AS, 100 mg / L TMT and 25 mg / L kanamycin, pH=5.8) and cultured at 25℃ under light (16 hours light / 8 hours dark) for about two weeks.

[0044] 5) Rooting: When the shoots grow to 4-5 cm and have 4-5 leaves, the elongated shoots can be transferred to rooting medium (1 / 2 MS medium containing 0.1 mg / L NAA, 100 mg / L TMT and 100 mg / L activated carbon, pH=5.8) and cultured at 25℃ under light (16 hours light / 8 hours darkness) for about four weeks. Rooted seedlings are then directly transplanted into soil after hardening off.

[0045] Step 4: Preliminary identification of transgenic plants, observation of agronomic traits, and determination of indigo glycoside content. A) Preliminary identification of transgenic plants: After hardening off the seedlings, the plants were transferred to soil and planted. Genomic DNA was extracted from the T0 generation transformed seedlings using the EasyPurePlant Genomic DNA Kit. This DNA was then amplified using the corresponding specific PCR primers for preliminary identification of the transgenic plants. Plants exhibiting amplified bands were identified as transgenic plants. The amplification primers were those specified in SEQ ID No. 1 and SEQ ID No. 2 (SEQ ID No. 1, ...). EVM0003075 The nucleotide sequence (1404 bp) was derived from Isatis indigotica and was obtained through artificial synthesis (Sangon Biotech Co., Ltd.). PCR amplification primers were designed based on this sequence, as shown in Table 1. pRI101-GFP- EVM0003075 -F and pRI101-GFP- EVM0003075 -R is EVM0003075 The gene amplification primers, pRI101-GFP-F and pRI101-GFP-R, are the control group amplification primers. For example... Figure 2As shown, plants numbered 1-1 to 1-7 are transgenic positive plants, and plants numbered 0-1 to 0-5 are control group plants.

[0046] B) Transgenic plants EVM0003075 Gene expression level detection: When the transgenic plants transplanted to soil culture reached 120 days of growth, gene expression levels were detected. EVM0003075 Gene expression level detection. First, amplification primers were designed using SEQ ID No. 1 as the template (see Table 1, QtpRI101-GFP-). EVM0003075 -F and QtpRI101-GFP- EVM0003075 For each sample, 100 mg was used. Total RNA was extracted using the RNA Prep Pure Plant Kit (TIANGEN), and genomic DNA was digested. After quantification with Nanodrop, 2 μg of total RNA was used as a template. Then, the RNA was reverse transcribed into cDNA using the TransScript All-in-One kit. Finally, qRT-PCR analysis was performed using the SuperReal PreMix Plus (SYBR Green) kit. The real-time PCR program was: pre-denaturation at 95℃ for 10 min, denaturation at 95℃ for 15 s, annealing at 60℃ for 15 s, extension at 72℃ for 1.5 min, 30 cycles, followed by a final extension of 5 min. All samples were performed in triplicate. Actin 7 was used as an internal control gene (primers are shown in Table 1, ACTIN7-F and ACTIN7-R) for qRT-PCR analysis. Figure 3 As shown, overexpression EVM0003075 Gene expression levels were 5-9 times higher than those in control plants.

[0047] C) Detection of indigo glycoside content in transgenic plants: When the transgenic plants transplanted to soil cultivation reached 120 days of growth, the indigo glycoside content of the transgenic plants was detected by HPLC. The detection method is referenced in: Zhang Libin. Ethnobotanical Investigation and Indigo Production Mechanism Study of Isatis indigotica [D]. Kunming, Kunming Institute of Botany, Chinese Academy of Sciences, 2021. Specifically: Fresh transgenic plant leaves were wrapped in tin foil and immediately placed in liquid nitrogen for 15 min. Approximately 200-500 mg was then quickly weighed and transferred to a 2 mL centrifuge tube. 1 mL of N,N-dimethylformamide (DMF) and two small steel balls (d=4 mm) were added, and the tube was then ground in an automated sample grinder (Tissuelyser-24, Shanghai Jingxin Industrial Development Co., Ltd.). After grinding, 7 mL of DMF was added, and ultrasonic extraction was performed for 30 minutes. After cooling to room temperature, the volume was adjusted to 10 mL, and the tube was centrifuged for 5 minutes (3000 r / min). The supernatant was filtered through a 0.45 μm microporous membrane for later use. The HPLC instrument was an Agilent 1260 high-performance liquid chromatograph, and the chromatographic column was an Agilent Technologies C18 column, model: Agilent ZORBAX Rx-C18, 4.6. 250 mm, 5 μm. Mobile phase A is water, and phase B is methanol, using a gradient elution method: 0 min: 10% methanol / 90% water; 30 min-50 min: 80% methanol / 20% water. Flow rate: 1.0 ml / min, column temperature: 30℃, detection wavelength: 280 nm, injection volume: 10 μL. Figure 4 As shown, testing for indoside on day 120 revealed that indoside was still undetectable in the control group, while it was detectable in the transgenic plants, at levels 10-66 times higher than in the control group. Furthermore, as... Figure 5 As shown, the transgenic plants grew well 120 days after being transplanted into the soil.

[0048] Table 1 Primer information used in this invention In summary, the method of the present invention can not only increase the content of indoside in Isatis indigotica, but also shorten the accumulation period of indoside.

[0049] The various embodiments of this application have been described above. These descriptions are exemplary and not exhaustive, nor are they limited to the disclosed embodiments. Many modifications and variations will be apparent to those skilled in the art without departing from the scope and spirit of the described embodiments. The terminology used herein is chosen to best explain the principles, practical application, or improvement of the technology in the market, or to enable others skilled in the art to understand the embodiments disclosed herein.

[0050] The sequence listing is: SEQ ID No. 1> EVM0003075

Claims

1. An overexpression EVM0003075 A method for cultivating Isatis indigotica plants with genetically modified genes, characterized in that... The cultivation method includes the following steps: (1) Constructing an expression of Isatis indigotica EVM0003075 Agrobacterium-mediated transformation vector for genes; (2) The Agrobacterium transformation vector constructed in step (1) was transformed into Agrobacterium GV3101 competent cells by freezing and thawing to obtain the transformed Agrobacterium strain; (3) Using Isatis indigotica as the recipient material, the Agrobacterium strain obtained in step (2) was genetically transformed to obtain a stable overexpression strain. EVM0003075 Isatis seedlings with genetically modified citronella; (4) The Isatis indigotica seedlings obtained in step (3) are screened and then cultivated to obtain overexpression EVM0003075 Isatis indigotica plants with genetic defects.

2. The cultivation method according to claim 1, characterized in that, In step (1), the expression of Isatis indigotica is constructed using the pRI101-GFP vector. EVM0003075 Agrobacterium-mediated transformation vector for genes.

3. The cultivation method according to claim 1, characterized in that, The specific operation of the freeze-thaw method in step (2) is as follows: 1) Thaw Agrobacterium GV3101 competent cells stored at -80℃ on ice; 2) Add 10 μL of pRI101-GFP- from step 1) to Agrobacterium GV3101 competent cells. EVM0003075 The plasmid was gently mixed and then placed on ice for 5 min, in liquid nitrogen for 5 min, in a 37°C water bath for 5 min, and in an ice bath for 5 min in sequence. 3) Add 700 μL of antibiotic-free LB medium to step 2), mix well, and revive at 28°C and 200 rpm for 2 h; take 200 μL of the revived bacterial solution and spread it on a solid culture medium containing 50 μg / mL kanamycin and 20 μg / mL rifampin using a spreader. Invert the plate and place it in a 28°C incubator for 2-3 days.

4. The cultivation method according to claim 1, characterized in that, The genetic transformation in step (3) is carried out using Agrobacterium-mediated genetic transformation with Isatis tinctoria as the recipient material.

5. The cultivation method according to claim 1, characterized in that, The cultivation in step (4) involves directly transplanting the rooted Isatis indigotica seedlings into the soil after hardening them off.

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