Method for increasing content of ginsenoside Rg1 in panax notoginseng cells
By targeting and knocking out the competitive enzyme CAS in Panax notoginseng cells using CRISPR/Cas9 gene editing technology, the content of ginsenoside Rg1 was increased, solving the problem of low Rg1 content in traditional agricultural production and realizing efficient and standardized production of Panax notoginseng cells.
Patent Information
- Application Number
- CN202511423040.1
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-09-30
- Publication Date
- 2026-01-16
AI Technical Summary
In existing technologies, the content of ginsenoside Rg1 in Panax notoginseng cell culture systems is low and cannot meet market demand. Furthermore, traditional agricultural production models are limited by long cycles, continuous cropping obstacles, and environmental influences, resulting in unstable yield and quality. There is a lack of effective genetic regulation methods to increase Rg1 content.
CRISPR/Cas9 gene editing technology was used to target and knock out CAS, a key enzyme that competitively inhibits the synthesis of ginsenoside Rg1 in Panax notoginseng cells. The knockout was then introduced into Panax notoginseng cells via Agrobacterium-mediated genetic transformation, and cell lines with high Rg1 content were screened out.
It significantly increased the content of ginsenoside Rg1 in Panax notoginseng cells, overcame the limitations of traditional agriculture, realized year-round and standardized production, provided an efficient genetic regulation method, increased the Rg1 content of cell lines, and met market demand.
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Abstract
Description
Technical Field
[0001] This invention relates to the field of biotechnology, and in particular to a method for increasing the content of ginsenoside Rg1 in Panax notoginseng cells. Background Technology
[0002] Sanqi ( Panax notoginseng Panax notoginseng (Panax notoginseng) is a perennial herb belonging to the genus Panax in the family Araliaceae. It is a precious traditional Chinese medicine unique to my country, often referred to as "priceless" or "the divine herb of the South." Its medicinal part is primarily the root, which possesses remarkable effects in dispersing blood stasis, stopping bleeding, reducing swelling, and relieving pain, demonstrating significant application potential in the treatment of modern cardiovascular and cerebrovascular diseases. Studies have shown that saponins are the main active components of Panax notoginseng, with ginsenoside Rg1 (Rg1) being one of the most important monomeric components. Rg1 has been proven to possess various significant pharmacological activities, including neuroprotection, promoting angiogenesis, anti-inflammation, anti-oxidation, and improving learning and memory functions. It is a core indicator component for evaluating the quality of Panax notoginseng and its products.
[0003] However, the Rg1 content of Panax notoginseng produced under traditional agricultural production methods is severely constrained by various factors, making it unable to meet the ever-increasing market demand. First, Panax notoginseng has an extremely long growth cycle, typically requiring 3 to 7 years of cultivation before harvest, resulting in high production costs and a long capital recovery period. Second, Panax notoginseng cultivation suffers from severe continuous cropping obstacles, meaning that land previously used for Panax notoginseng is unlikely to be successfully cultivated again for decades. This not only leads to a sharp reduction in suitable arable land but also exacerbates the conflict between humans and land, driving up raw material prices. Third, Panax notoginseng grown in natural environments is susceptible to pests and diseases, as well as climate change (such as drought and frost), resulting in unstable yield and quality. Furthermore, the Rg1 content varies significantly between different origins and batches of Panax notoginseng; this uncontrollable quality presents a significant challenge to the standardized production and clinical application of the medicine.
[0004] To overcome resource bottlenecks, plant cell culture technology, as a modern production technology that is independent of land and climate and can be controlled on a large scale, is widely used in the production of rare medicinal active ingredients. This technology induces callus formation in plant explants under sterile conditions, thereby establishing suspension cell lines, which are then cultured on a large scale in bioreactors to obtain target metabolites. Theoretically, this technology can achieve year-round, standardized, and industrialized production of Panax notoginseng saponins. However, despite significant progress in Panax notoginseng cell culture research, its practical application still faces a key bottleneck: in unoptimized cell culture systems, the content of ginsenoside Rg1 is generally significantly lower than that of Panax notoginseng roots cultivated in the field. The root cause is that during the rapid proliferation of cells in vitro, their metabolic flow is mainly directed towards primary metabolism (such as growth and division) rather than secondary metabolic pathways such as saponin synthesis. The biosynthetic pathway of Rg1 is complex, involving multiple enzymatic reaction steps and their fine regulation. Under conventional cell culture conditions, the expression levels of these key enzyme genes are often suppressed, leading to low synthesis efficiency and insufficient accumulation of the final product Rg1.
[0005] In recent years, studies have successfully increased the yield of specific secondary metabolites in plants using CRISPR technology. However, there are no reports, either domestically or internationally, of applying CRISPR / Cas9 technology to Panax notoginseng cells to precisely edit key negative control factors in its saponin synthesis regulatory network, thereby specifically and significantly increasing the content of ginsenoside Rg1, a single high-value component. Current technology lacks an effective method to genetically reconstruct the metabolic network of Panax notoginseng cells and efficiently and directionally increase Rg1 content.
[0006] Therefore, this paper proposes a method to increase the content of ginsenoside Rg1 in Panax notoginseng cells. This new method combines advanced gene editing technology with cell culture techniques, overcomes the shortcomings of existing technologies, and achieves a significant increase in the content of ginsenoside Rg1 in Panax notoginseng cells. This has significant scientific and application value for ensuring the sustainable supply of Panax notoginseng resources and promoting the modernization and internationalization of traditional Chinese medicine. Summary of the Invention
[0007] To overcome the problems existing in the background technology, the present invention provides a method for increasing the content of ginsenoside Rg1 in Panax notoginseng cells. The method utilizes CRISPR / Cas9 gene editing technology to target and knock out CAS, a key enzyme that competitively inhibits the synthesis of ginsenoside Rg1 in Panax notoginseng cells, and introduces it into Panax notoginseng cells through Agrobacterium-mediated genetic transformation. This allows for the screening of Panax notoginseng cell lines with high ginsenoside Rg1 content, thereby achieving a significant increase in the content of ginsenoside Rg1 in Panax notoginseng cells.
[0008] To achieve the above objectives, the present invention is implemented through the following technical solution: This invention provides a method for increasing the content of ginsenoside Rg1 in Panax notoginseng cells, comprising the following steps: (a) Construct a CRISPR / Cas9 expression vector designed to target and knock out CAS, a key enzyme that competitively inhibits the synthesis of ginsenosides in Panax notoginseng cells, thereby relieving its negative regulation on the synthesis of ginsenoside Rg1. (b) Using Agrobacterium-mediated genetic transformation, the CRISPR / Cas9 expression vector constructed in step (a) was introduced into pre-cultured Panax notoginseng cells for infection and co-culture to achieve effective gene transfer. (c) The co-cultured Panax notoginseng cells were sterilized and selectively cultured to screen out Panax notoginseng positive cells that had been successfully introduced into the CRISPR / Cas9 expression vector and had specific resistance; (d) Molecular identification of the selected Panax notoginseng positive cells was performed to confirm that the CRISPR / Cas9 system had been transferred into the plant; (e) The identified Panax notoginseng positive cells were cultured and their ginsenoside Rg1 content was determined.
[0009] In the above technical solution, the Panax notoginseng cells are Panax notoginseng embryonic cell lines.
[0010] In the above technical solution, the specific steps for constructing the CRISPR / Cas9 expression vector in step (a) include: (a1) Targeting the stated gene CAS Design target sites for knockout; (a2) The target fragment was amplified using the cloning vector plasmid pUC57-DT1T2 as a template; (a3) The gel recovery product of the target fragment is ligated with the enzyme-digested pHSE401 linear vector to construct a recombinant plasmid; (a4) Transform the recombinant plasmid into Escherichia coli TOP10 to obtain a positive clone and extract the plasmid; (a5) Transform the extracted recombinant plasmid into Agrobacterium EHA105.
[0011] In the above technical solution, the Agrobacterium-mediated genetic transformation method described in step (b) specifically includes the following steps: (b1) Preparation of Agrobacterium bacterial suspension: Agrobacterium containing the CRISPR / Cas9 expression vector was activated on LB solid medium containing 50 mg·L⁻¹ kanamycin and 20 mg·L⁻¹ rifampicin selective pressure, and cultured in an incubator at 28℃ for 60 h; Agrobacterium was scraped and cultured in LB liquid medium containing 50 mg·L⁻¹ kanamycin and 20 mg·L⁻¹ rifampicin at 30℃ with shaking at 200 rpm until the OD value was 0.6-0.8; centrifuged at 5000 rpm for 10 min, the supernatant was discarded, the invading dye was added, and the suspension was resuspended until the OD value was 0.4-0.5 to obtain the resuspended bacterial suspension; (b2) Infection: The relatively dispersed Panax notoginseng cells with good growth status that have been pre-cultured for 3-5 days are completely immersed in the resuspended bacterial solution and cultured at 25℃ and 100 rpm for 20 min with shaking. (b3) Co-culture: After the infection was completed, the Panax notoginseng cells were filtered and recovered. Excess bacterial solution on the surface of the stem cells was absorbed with sterile filter paper. After blowing the bacterial solution on the surface of the stem cells in a clean bench, the cells were inoculated into a co-culture medium and cultured in the dark at 25°C for 3 days. (b4) Sterilization and screening: After co-culture, the Panax notoginseng cells were transferred to WPM liquid medium containing 400 mg·L⁻¹ cephalosporin and shaken at 25°C and 100 rpm for 30 min. Then, the Panax notoginseng cells were washed 5-7 times with WPM liquid medium until the washing solution was clear. The cells were transferred to sterile filter paper to absorb the water, and after being dried with sterile air on a clean bench for 1 h, they were inoculated onto selective medium containing cephalosporin and hygromycin for continuous selective culture.
[0012] In the above technical solution, the specific steps for molecular identification of the screened Panax notoginseng positive cells in step (d) include: extracting their DNA using the CTAB method, and using the extracted DNA as a template to verify whether the material contains the Cas9 gene.
[0013] In the above technical solution, step (e) of determining the content of ginsenoside Rg1 includes: (e1) Grind the Panax notoginseng positive cell material with liquid nitrogen, accurately weigh 0.2g of powder, put it into a centrifuge tube, and add 2mL of methanol at a material-to-liquid ratio of 1:10 (g / mL); (e2) Extract once by sonication in an ultrasonic instrument for 30 minutes; (e3) After being placed in a refrigerator at 4°C overnight, the sample was sonicated again for 30 min, and then filtered through a 0.22 μm microporous membrane to obtain the saponin sample extract. (e4) The content of ginsenoside Rg1 in the saponin sample extract was determined by high performance liquid chromatography using Rg1 standard. Compared with the prior art, the beneficial effects of the present invention are as follows: 1. The cultivation process of this invention is completely detached from soil, avoiding risks such as pesticide and heavy metal pollution and the influence of external climate and environment. Furthermore, the short cycle overcomes the problems associated with large-scale field cultivation. Simultaneously, by utilizing gene editing techniques, a new direction is provided for increasing the content of ginsenoside Rg1 in Panax notoginseng cells. The obtained high-saponin-content positive cell lines can also provide new materials for subsequent large-scale production.
[0014] 2. This invention starts with the synthetic pathway of Panax notoginseng saponins. Based on Panax notoginseng embryonic cell lines, it utilizes CRISPR / Cas9 technology to knock out the key enzyme CAS, which competitively inhibits the synthesis of Panax notoginseng saponins, ultimately increasing the content of ginsenoside Rg1 and obtaining a Panax notoginseng cell line with high Rg1 content. Ginsenoside Rg1 is a triterpenoid saponin compound, a representative active ingredient of saponins, and one of the components with relatively high content. It possesses multiple biological functions, including antioxidant, immunomodulatory, anti-inflammatory, apoptosis-inhibiting, and angiogenesis-promoting effects, and has significant potential and value in the pharmaceutical field. Attached Figure Description
[0015] To more clearly illustrate the technical solutions in the embodiments of this application or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on the structures shown in these drawings without creative effort.
[0016] Figure 1 This is a diagram of the CRISPR expression vector construction of the present invention; A: PCR amplification of the target fragment; M: DNA marker; B: E. coli PCR detection; 1-8: represent 8 different single clones; C: Agrobacterium PCR detection; 1-7: represent 7 different single clones.
[0017] Figure 2 It is the genomic DNA of the Panax notoginseng cell line of this invention; Figure 3 This invention relates to the PCR detection of the Cas9 gene in positive Panax notoginseng cell lines. Figure 4 This is a comparison of the content of ginsenoside Rg1 in different cell lines of Panax notoginseng. Detailed Implementation
[0018] To make the objectives, technical solutions, and advantages of the present invention clearer, the embodiments of this application will be described in further detail below with reference to the accompanying drawings.
[0019] Where the following description relates to the accompanying drawings, unless otherwise indicated, the same numbers in different drawings denote the same or similar elements. The embodiments described in the following exemplary embodiments do not represent all embodiments consistent with the present invention. Rather, they are merely examples of apparatuses and methods consistent with some aspects of the invention as detailed in the appended claims.
[0020] In the description of this invention, it should be understood that the terms "first," "second," etc., are used for descriptive purposes only and should not be construed as indicating or implying relative importance. Those skilled in the art can understand the specific meaning of these terms in this invention based on the specific circumstances. Furthermore, in the description of this invention, unless otherwise stated, "multiple" refers to two or more. "And / or" describes the relationship between related objects, indicating that three relationships can exist. For example, A and / or B can represent: A existing alone, A and B existing simultaneously, and B existing alone. The character " / " generally indicates that the preceding and following related objects have an "or" relationship.
[0021] 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. The terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention. The term "and / or" as used herein includes any and all combinations of one or more of the associated listed items.
[0022] Examples 1-4 This application provides a method for increasing the content of ginsenoside Rg1 in Panax notoginseng cells, including the following steps: (1) Construction of CRISPR expression vector First, a target site was designed to knock out the target gene. Then, the target fragment was amplified using the cloning vector plasmid pUC57-DT1T2 as a template. Next, the gel recovery product of the target fragment was ligated with the enzyme-digested pHSE401 linear vector. Subsequently, it was transformed into E. coli TOP10 to obtain positive clones and extract plasmids. Finally, the recombinant plasmid was transformed into Agrobacterium EHA105.
[0023] (2) Agrobacterium-mediated genetic transformation of Panax notoginseng cells Preparation of Agrobacterium bacterial suspension: The Agrobacterium bacterial suspension containing the target expression vector was activated on LB solid medium containing 50 mg·L⁻¹ kanamycin and 20 mg·L⁻¹ rifampicin under selective pressure, and cultured at 28 °C for 60 h. Agrobacterium was scraped and cultured in LB liquid medium containing kanamycin and rifampicin at 30 °C with shaking at 200 rpm until the OD value reached 0.6-0.8. The medium was then centrifuged at 5000 rpm for 10 min, the supernatant was discarded, and the invading dye was added. The suspension was resuspended until the OD value reached 0.4-0.5 to obtain the resuspended bacterial suspension.
[0024] Infection: Select Panax notoginseng cells that have been pre-cultured for 3-5 days and are relatively dispersed and in good growth condition as the infection material for genetic transformation. During infection, the Panax notoginseng cells are completely immersed in the above Agrobacterium bacterial solution and cultured at 25°C and 100 rpm for 20 min with shaking.
[0025] Co-culture: After infection, cells were collected by filtration, excess bacterial solution on the surface of stem cells was absorbed with sterile filter paper, and the bacterial solution on the surface of stem cells was blown off in a clean bench before being inoculated into co-culture medium and cultured in the dark at 25°C for 3 days.
[0026] Sterilization and screening: After co-culture, cells were transferred to a medium containing 400 mg / L of [unspecified solution]. -1 In WPM liquid medium containing cephalosporin, incubate at 25°C and shake at 100 rpm for 30 min. Then wash the cells 5-7 times with WPM liquid medium until the wash buffer is clear. Transfer the cells to sterile filter paper to blot dry, and air dry them under sterile air in a laminar flow hood for 1 h before inoculating them onto selective medium containing cephalosporin and hygromycin for continuous selective culture.
[0027] (3) Identification of positive cells After successfully obtaining Panax notoginseng cells resistant to hygromycin, their DNA was extracted using the CTAB method. The extracted DNA was used as a template to verify whether the material contained the Cas9 gene, ensuring that the expression vector CRISPR / Cas9 system had been transferred into the plant.
[0028] (4) Determination of saponin content by HPLC Four positive cell lines, namely Panax notoginseng cell lines “1-7, 1-8, 1-9, and 1-18”, were selected and ground with liquid nitrogen. 0.2 g of powder was weighed and placed in a centrifuge tube. 2 mL of methanol was added at a material-to-liquid ratio of 1:10 (g / mL), and the mixture was sonicated for 30 min in an HS10260D ultrasonic instrument for extraction once. After incubation overnight at 4℃, the mixture was sonicated again for 30 min and filtered through a 0.22 μm microporous membrane to obtain the saponin sample extract. Each sample was extracted three times. The ginsenoside Rg1 content of the four positive cell lines was determined using a standard Rg1, as shown in Table 1 below.
[0029] The specific HPLC conditions were as follows: the instrument was an Agilent 1260, the column was an Eicoee WTC18 column (4.6 × 250 mm, 5 μm), the mobile phase was acetonitrile (A)-water (B), gradient elution (0–20 min, 80–78% A, 20–22% B; 20–45 min, 78–46% A, 22–54% B; 45–60 min, 46–10% A, 54–90% B), the flow rate was 0.8 mL / min, the detection wavelength was 203 nm, the column temperature was 25 ℃, and the injection volume was 10 μL.
[0030] Comparative Example 1 A single strain of Panax notoginseng embryonic cells cultured concurrently without Agrobacterium infection was selected, ground with liquid nitrogen, and 0.2 g of powder was weighed and placed in a centrifuge tube. 2 mL of methanol was added at a material-to-liquid ratio of 1:10 (g / mL), and the mixture was sonicated for 30 min in an HS10260D ultrasonic instrument for extraction once. After incubation overnight at 4℃, the mixture was sonicated again for 30 min and filtered through a 0.22 μm microporous membrane to obtain the saponin sample extract. Each sample was extracted three times. The content of ginsenoside Rg1 was determined using the Rg1 standard, as shown in Table 1 below. HPLC conditions were the same as in Examples 1-4.
[0031] Table 1. Comparison of Ginsenoside Rg1 Content between Examples 1-4 and Comparative Examples As shown in the table above, the four cell lines "1-7, 1-8, 1-9, and 1-18" are all positive cell lines obtained through genetic transformation and screening. The Rg1 saponin content of these four cell lines is increased compared with the control wild-type cell lines. The four cell lines were treated with the same method, but the differences in the editing sites and efficiency after gene editing may lead to different degrees of change in their target metabolite saponins.
[0032] In summary, after CRISPR / Cas9 treatment of Panax notoginseng cells, PnCAS The content of ginsenoside Rg1 in positive Panax notoginseng cell lines was significantly increased compared with that in wild-type Panax notoginseng cells. The "1-8" transgenic Panax notoginseng cell line showed the largest increase, approximately 13 times that of wild-type cells. The "1-7" and "1-9" transgenic Panax notoginseng cell lines also showed an increase of approximately 12 times, indicating that ginsenoside Rg1 was knocked out in Panax notoginseng cells through gene editing. PnCAS This can significantly improve the synthesis of ginsenoside Rg1. Finally, it should be noted that the above preferred embodiments are only used to illustrate the technical solution of the present invention and not to limit it. Although the present invention has been described in detail through the above preferred embodiments, those skilled in the art should understand that various changes in form and detail can be made without departing from the scope defined by the claims of the present invention.
Claims
1. A method for increasing the content of ginsenoside Rg1 in Panax notoginseng cells, characterized in that: The method specifically comprises the following steps: (a) constructing a CRISPR / Cas9 expression vector, which is designed to target knockout CAS, a key enzyme competitively inhibiting ginsenoside synthesis in Panax notoginseng cells, to relieve its negative regulation on ginsenoside Rg1 synthesis; (b) introducing the CRISPR / Cas9 expression vector constructed in step (a) into pre-cultured Panax notoginseng cells by using an agrobacterium-mediated genetic transformation method, and performing infection and co-culture to realize effective gene transfer; (c) performing sterilization and selection culture on the Panax notoginseng cells after co-culture, so as to screen out Panax notoginseng positive cells successfully introducing the CRISPR / Cas9 expression vector and having specific resistance; (d) performing molecular identification on the screened Panax notoginseng positive cells to confirm that the CRISPR / Cas9 system has been transferred into the plant; (e) culturing the identified Panax notoginseng positive cells and determining the content of ginsenoside Rg1.
2. The method for increasing the content of ginsenoside Rg1 in Panax notoginseng cells according to claim 1, characterized in that: The Panax notoginseng cells are Panax notoginseng embryonic cell lines.
3. The method for increasing the content of ginsenoside Rg1 in Panax notoginseng cells according to claim 1, characterized in that: The specific steps of constructing the CRISPR / Cas9 expression vector in step (a) comprise: (a1) for the purpose gene CAS Designing target sites for knock-out; (a2) amplifying the target fragment by using the cloning vector plasmid pUC57-DT1T2 as a template; (a3) connecting the gel recovery product of the target fragment with the enzyme-digested pHSE401 linear vector to construct a recombinant plasmid; (a4) transforming the recombinant plasmid into Escherichia coli TOP10 to obtain positive clones and extract plasmids; (a5) transforming the extracted recombinant plasmid into agrobacterium EHA105.
4. The method for increasing the content of ginsenoside Rg1 in Panax notoginseng cells according to claim 1, characterized in that: The specific steps of the agrobacterium-mediated genetic transformation method in step (b) comprise the following steps: (b1) preparing an agrobacterium liquid: activating the agrobacterium containing the CRISPR / Cas9 expression vector on an LB solid culture medium containing 50 mg·L⁻¹ kanamycin and 20 mg·L⁻¹ rifampicin selection pressure, and culturing in a 28°C incubator for 60 h; scraping the agrobacterium in an LB liquid culture medium containing 50 mg·L⁻¹ kanamycin and 20 mg·L⁻¹ rifampicin, and culturing at 30°C and 200 rpm for 10 min; centrifuging at 5000 rpm for 10 min, discarding the supernatant, adding an infection liquid, resuspending to an OD value of 0.4-0.5, and obtaining a resuspended bacterial liquid; (b2) infection: completely immersing the pre-cultured 3-5 d Panax notoginseng cells with good growth state in the resuspended bacterial liquid, and culturing at 25°C and 100 rpm for 20 min; (b3) co-culture: after the infection is completed, the Panax notoginseng cells are recovered by a sieve, the excess bacterial liquid on the surface of the cells is absorbed by sterile filter paper, the bacterial liquid on the surface of the cells is blown dry in a clean bench, and then the cells are inoculated in a co-culture medium and cultured at 25°C in the dark for 3 d; (b4) Decontamination and screening: after the co-culture, the Panax notoginseng cells are transferred to WPM liquid medium containing 400 mg / L cefotaxime, and shaken at 25 °C and 100 rpm for 30 min; then the cells are washed with WPM liquid medium for 5-7 times until the washing liquid is clear; the cells are transferred to sterile filter paper to dry the water, and then inoculated on the selection medium containing cefotaxime and hygromycin after blowing with sterile air for 1 h on the clean bench.
5. The method for increasing the content of ginsenoside Rg1 in Panax notoginseng cells according to claim 1, characterized in that: The specific steps of the molecular identification of the screened Panax notoginseng positive cells in step (d) include: extracting the DNA of the cells by CTAB method, and using the extracted DNA as a template to verify whether the cas9 gene exists in the material.
6. The method for increasing the content of ginsenoside Rg1 in Panax notoginseng cells according to claim 1, characterized in that: The steps of the determination of the content of ginsenoside Rg1 in step (e) include: (e1) grinding the Panax notoginseng positive cell material with liquid nitrogen, accurately weighing 0.2 g of the powder, and placing it in a centrifuge tube, and then adding 2 mL of methanol according to the solid-liquid ratio of 1:10 (g / mL); (e2) extracting once in an ultrasonic instrument for 30 min; (e3) after overnight storage in a 4 °C refrigerator, ultrasonic extraction is performed again for 30 min, and then the ginsenoside sample extract is obtained by filtering through a 0.22 μm microporous filter membrane; (e4) the content of ginsenoside Rg1 in the ginsenoside sample extract is determined by high performance liquid chromatography using a Rg1 standard.