CN114752621A - Method for establishing genetic transformation system of hairy roots of morinda officinalis - Google Patents
Method for establishing genetic transformation system of hairy roots of morinda officinalis Download PDFInfo
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Abstract
A method for establishing a hairy root genetic transformation system of morinda officinalis comprises the steps of taking young stem sections of morinda officinalis aseptic tissue culture seedlings as explants, transferring the young stem sections into an infection solution after preculture, sucking bacterial liquid on the surfaces of the explants after infection is finished, then carrying out co-culture, cleaning the explants forming dental plaques in the co-culture process with sterile water containing cefotaxime sodium and timentin, and transferring the cleaned explants to 1/2 MS solid culture medium containing cefotaxime sodium, timentin and acetosyringone to induce hairy roots; when the hairy roots grow to about 2-3 cm, the hairy roots are cut off and transferred to 1/2 MS solid culture medium containing screening antibiotics, and hairy root systems which have kanamycin resistance, high growth speed and more branches are screened out. The method has higher transformation efficiency, and provides good technical support for subsequent gene function research and utilization of genetic engineering means to improve the content of active ingredients of the medicinal effect of the morinda officinalis.
Description
Technical Field
The invention belongs to the field of plant genetic engineering, and particularly relates to a method for establishing a genetic transformation system of morinda officinalis hairy roots.
Background
Morinda officinalis (Morinda officinalis)Morinda officinalis How) is perennial vine of morinda of rubiaceae, and has the functions of tonifying kidney yang, resisting osteoporosis, resisting depression, resisting inflammation, relieving pain, dispelling wind and removing dampness by using root as medicine. Clinically, radix Morindae officinalis is often combined with other medicines to treat kidney deficiency and osteoporosisFor arthralgia due to wind-dampness, such as jin gang Wan, Ba Ji Wan, Erxian Tang, Xian Teng Tang and san Teng Tang, etc.
Agrobacterium rhizogenes (Agrobacterium rhizogenes) is a gram-negative soil bacterium of the genus Agrobacterium of the family Rhizobiaceae, capable of infecting most dicots, a few monocotyledons and individual gymnosperms. The key role of Agrobacterium rhizogenes is its carried Ri plasmid, containing genes responsible for autonomous growth of hairy roots and opine synthesis. Ri plasmid is similar to Ti plasmid in structure, and mainly has toxic region (Vir), T-DNA region and opine synthesis function region. Ri plasmids are divided into 4 types, depending on the synthesis of opines: 1) a mannopine form; 2) cucumber alkali type; 3) alkali type of agricultural stalk; 4) miqi spear type. Agrobacterium rhizogenes strain ATCC15834 was derived from American Type Culture Collection (ATCC) and was the original strain of Agrobacterium rhizogenes. ATCC15834 contains Ri plasmid of pRi15834 agropine type, which contains rolC gene for inducing hairy root, and causes a large amount of hairy root after infecting plants. The agrobacterium rhizogenes ATCC15834 has wide host range (gramineae, leguminosae, tobacco and the like), has wide application prospect in plant genetic transformation, and is particularly suitable for inducing hairy roots of medicinal plants such as paclitaxel, sweet wormwood and the like.
The pBI121 plasmid is a high-efficiency plant binary expression vector and is derived from pB221 and Bin19 vectors. The pBI121 vector contains ori replication elements derived from ColE1, pROK1 elements derived from CaMV, Neomycin phosphotransferase II Gene (Neomycin phosphotransferase II Gene, NptII), Neomycin resistance Gene (NeoR), and the like. The vector carries a kanamycin resistance gene Kan selection marker, and the kanamycin resistance gene (Kan) is often used as a selection marker gene in the research of cultivating transgenic plants, namely, only a transformed plant containing the kanamycin resistance gene can grow on a kanamycin culture medium.
Hairy roots are also called hairy roots, are a pathological state generated by induced cells after agrobacterium rhizogenes infects plants, have the characteristics of multiple branches, loss of geotropism and the like, and also have the advantages of hormone autonomy, short growth cycle, strong accumulation capacity of stable heredity and secondary metabolites and the like. Therefore, the hairy root genetic transformation system is an important technical means for researching gene functions and constructing efficient oriented biosynthesis drug effect monomers by utilizing synthetic biology, and is particularly suitable for Chinese medicinal materials taking roots as medicines. The hairy root genetic transformation technology is well researched in medicinal plants such as ginseng, salvia miltiorrhiza, panax notoginseng and the like, and is widely applied to the research of functional genomics and the development and utilization of natural medicinal components. However, only the current reports of Zheng's transmission and the like are that the condition of inducing the hairy root of the south medicinal morinda officinalis is researched by taking the plumule as an explant, the research on the hairy root genetic transformation system of the morinda officinalis is not reported at present, and the research and the application of the functional genomics of the morinda officinalis are severely restricted.
Disclosure of Invention
Aiming at the defects of the prior art, the invention provides a method for establishing a genetic transformation system of morinda officinalis hairy roots, which optimizes agrobacterium rhizogenes, explants, a treatment method and the like with higher transformation efficiency, and provides better technical support for subsequent gene function research and utilization of genetic engineering means to improve the content of active ingredients of morinda officinalis drug effect.
In order to achieve the purpose, the invention adopts the following technical scheme:
a method for establishing a genetic transformation system of morinda officinalis hairy roots comprises the following steps:
(1) pre-culture of explants
Placing young stem sections of the morinda officinalis aseptic tissue culture seedlings as explants in an MS solid culture medium containing 80-120 mu M acetosyringone, and performing dark culture at 22-28 ℃ for 2-3 days;
(2) preparing an infection liquid:
transforming a plant expression vector containing a target gene into agrobacterium rhizogenes ATCC15834, and preparing an infection solution;
(3) infection with Agrobacterium rhizogenes
Transferring the stem sections after the pre-culture to the infection solution obtained in the step (2), and oscillating for 10-30 min at the temperature of 25-30 ℃ and at the speed of 50-100 r/min to ensure that the explants after the pre-culture are fully contacted with the agrobacterium rhizogenes;
(4) co-cultivation
After infection is finished, sucking the bacterial liquid on the surface of the explant by using sterile filter paper, transferring the bacterial liquid to an MS solid culture medium containing 80-120 mu M acetosyringone, and co-culturing for 2-3 d under the dark culture condition at 22-28 ℃;
(5) washing bacteria
Washing the explant forming bacterial plaque in the co-culture process with sterile water containing 250-350 mg/L cefotaxime sodium and 150-250 mg/L timentin for 2-3 times, and then sucking water on the surface of the explant with sterile filter paper;
(6) induction of hairy roots
Transferring the washed explants to 1/2 MS solid culture medium containing 250-350 mg/L cefotaxime sodium, 150-250 mg/L timentin and 80-120 mu M acetosyringone, and placing the culture medium in a constant temperature incubator at 28 ℃; subculturing once every other week, and gradually reducing the concentration of the degerming antibiotics until bacterial plaque around the explant completely disappears;
(7) screening of Positive hairy root
When the induced hairy roots grow to about 2-3 cm, shearing down the hairy roots, transferring the sheared hairy roots to 1/2 MS solid culture medium containing screening antibiotics, carrying out dark culture at 25-30 ℃, and screening hairy root hair roots which have kanamycin resistance, high growth speed and more branches.
Preferably, the explants in the step (1) are from sterile tissue culture seedlings of morinda officinalis, young stem sections are stem sections with more than 2-3 leaves on the upper portion of the tissue culture seedling plant, the surface layers of the stem sections are slightly scratched along the oblique downward 30-degree angle of the young stem sections, and the stem sections do not need to be scratched.
Preferably, the preparation method of the infection solution in the step (2) is as follows:
a. transforming the plant expression vector containing the target gene into agrobacterium rhizogenes ATCC15834 by a freeze-thawing method;
b. in a sterile operation platform, selecting the transformed agrobacterium rhizogenes ATCC15834 by using an inoculating loop, streaking to a YEP solid culture medium containing 40-60 mg/L kanamycin, and carrying out inverted culture at 25-30 ℃ for 48-72 h;
c. selecting a single colony, inoculating the single colony into 1.5 mL YEB liquid culture medium containing 40-60 mg/L kanamycin, and carrying out overnight culture at the temperature of 25-30 ℃ for 16-18 h at the speed of 200-250 r/min;
d. inoculating the mixture into 50 mL of YEB liquid culture medium containing 40-60 mg/L kanamycin according to the inoculation amount of 0.5-1%, and performing shake culture at 25-30 ℃ at 200-250 r/min;
e. when the concentration of the bacterial liquid reaches OD600When the concentration is about 0.6-0.8, centrifuging, pouring out supernate and collecting thalli;
f. adding an equal volume of fresh MS liquid culture medium to resuspend the agrobacterium, centrifuging, pouring out supernatant, and collecting thalli;
g. adding an equal volume of fresh MS liquid culture medium to resuspend the agrobacterium, adding 80-120 mu M acetosyringone, and standing at normal temperature for 1-2 h to obtain the infection solution.
Preferably, the plant expression vector containing the target gene is pBI121-bHLH 25.
Preferably, the explant in the step (1) is a tender stem section of a sterile morinda officinalis tissue culture seedling;
preferably, the agrobacterium rhizogenes strain in the step (2) is ATCC 15834; this preferred condition is critical for transformation, and the transformation efficiency of different explants of different strains varies greatly.
Preferably, the subculture in step (6) is performed under the following conditions: the culture temperature is 25-30 ℃, and the illumination condition is 14 h illumination and 10 h darkness every day.
Preferably, the screening antibiotic in step (7) is kanamycin.
Preferably, the concentration of the screening antibiotic kanamycin is 28-32 mg/L.
Preferably, the method for culturing the aseptic tissue culture seedlings of morinda officinalis in step (1) is as follows:
selecting strong branches of morinda officinalis plants, removing leaves, sterilizing with 70% alcohol for 20-30 s, soaking in 3% sodium hypochlorite solution for 20-30 min, and washing with sterile water for 5-6 times; cutting into stem segments of about 1 cm according to the position of the axillary buds, cutting into an MS solid culture medium containing 0.5 mg/L6-BA, culturing in an illumination incubator at 28 ℃, illuminating for 16 h/8 h in darkness, and cutting and infecting explants when the axillary buds grow to 4-5 cm.
Cefotaxime sodium is Cefotaxime sodium, Cef, Timentin, Timm. Kanamycin is Kanamycin, Kan.
Compared with the prior art, the invention has the following beneficial effects:
1. the method has the advantages of easily-obtained materials, simplicity, practicability, short period and easy obtainment of sufficient hairy roots, and the obtained hairy roots can be applied to the gene function research of morinda officinalis and the efficient and directional production of pharmacodynamic active monomers through synthetic biology; solves the technical problem that the traditional morinda officinalis stable plant transformation takes a long time from genetic transformation to T1 generation plants which can be used for gene function identification. In comparison, the genetic transformation system of the hairy roots of the morinda officinalis shortens the research period obviously.
2. The morinda officinalis hairy root genetic transformation system established by the invention defines the explant type, agrobacterium strain, infection time, hairy root culture conditions and the like suitable for morinda officinalis hairy root genetic transformation, and accelerates the research process of morinda officinalis molecular biology.
Drawings
FIG. 1 is a drawing of a sterile Morinda citrifolia tissue culture;
FIG. 2 is a graph showing the results of an experiment in which leaves and young stem sections were precultured as explants;
FIG. 3 (a) is a diagram showing the stem and leaf as explants, after Agrobacterium infection, the rooting lines induced by 10 days were cultured; (b) adopting stem segments and leaves as explants, and culturing 20 d of induced hairy root system after agrobacterium infection;
FIG. 4 is a graph showing the results of experiments in which the propagation of Agrobacterium around stem segments after infection resulted in the death of explants, as indicated by arrows;
in FIG. 5, (a) is a graph of the experimental results of infecting tender stem sections of Morinda citrifolia with Agrobacterium rhizogenes ATCC15834 and culturing for 10 days after infection; (b) is an experimental result chart of infecting tender stem segments of morinda officinalis by agrobacterium rhizogenes ATCC15834 and culturing for 20 days after infection;
FIG. 6 is a morphological feature map of transgenic hairy root system;
in FIG. 7, (a) is the verification of hairy root by detecting rolC (768 bp) gene of Ri plasmid, 1: root system of tissue culture seedling; 2-8: transgenic hairy root hair root systems; 9: ddH 2O; marker DL 2000; (b) for detecting a target gene bHLH25 (461 bp) in the transgenic hairy root, 1, tissue culture seedling root systems are added; 2-8: transgenic hairy root hair root system; 9: ddH 2O; and (5) Marker DL 2000.
Detailed Description
The technical solution of the present invention is further illustrated by the following examples.
Example 1 obtaining sterile tissue culture seedlings of Morinda officinalis
Selecting strong branches of morinda officinalis plants, removing leaves, sterilizing with 70% alcohol for 20-30 s, soaking in 3% sodium hypochlorite solution for 20-30 min, and washing with sterile water for 5-6 times; cutting into stem segments of about 1 cm according to the position of the axillary buds, cutting into an MS solid culture medium containing 0.5 mg/L6-BA, culturing in an illumination incubator (28 ℃, 16 h illumination/8 h darkness), and when the axillary buds grow to 4-5 cm, cutting the explants to infect (figure 1).
Example 2 selection of optimal explants and hairy root strains
(1) Selection of explants
Taking leaves and young stem sections of the morinda officinalis aseptic tissue culture seedling as explants, wherein the young stem sections are stem sections of parts above 2-3 leaves on the upper part of a tissue culture seedling plant.
(2) Agrobacterium rhizogenes
MSU440 (streptomycin resistance), ar. qual (streptomycin and chloramphenicol resistance), C58C1 (streptomycin and rifampin resistance), K599 (streptomycin resistance) and ATCC15834 were selected as the infecting strain.
(3) Preparing an infection liquid:
a. in an aseptic operation table, selecting the agrobacterium rhizogenes by using an inoculating loop, streaking to a YEP solid culture medium containing corresponding antibiotics, and carrying out inverted culture at 28 ℃ for 48-72 h;
b. selecting a single colony, inoculating the single colony into 1.5 mL YEB liquid culture medium (containing corresponding antibiotics), and culturing at 220 r/min and 28 ℃ for 16-18 h;
c. inoculating into 50 mL YEB liquid culture medium (containing corresponding antibiotics) according to the inoculation amount of 0.5-1%, and performing inoculation at 28 ℃ for 220 r/min;
d. when the concentration of the bacterial liquid reaches OD600 of about 0.6-0.8, centrifuging at 5000 rpm for 5min, and pouring out the supernatant;
e. adding an equal volume of fresh MS liquid culture medium, resuspending the agrobacterium, centrifuging at 5000 rpm for 5min, and collecting thalli;
f. Adding an equal volume of fresh MS liquid culture medium to resuspend the agrobacterium, adding 100 mu M acetosyringone, and standing at normal temperature for 1-2 h (for inducing the activation of the Vir region gene of the agrobacterium and improving the infectivity), thereby obtaining the infection solution.
(4) Hairy root induction
a. Pre-culture of explants
Taking leaves and young stem sections of the morinda aseptic tissue culture seedling as explants, slightly scratching the surface layer of the stem sections along the oblique 30-degree angle of the young stem sections by using a scalpel, slightly scratching the leaves along the direction vertical to veins (figure 2), placing the scratched leaves in an MS solid culture medium containing 100 mu M acetosyringone, and carrying out dark culture at 25 ℃ for 2 days;
b. infection with Agrobacterium rhizogenes
Transferring the pre-cultured explant to the prepared agrobacterium rhizogenes staining solution, placing the explant in a shaking table, shaking for 10-30 min at the temperature of 28 ℃, and enabling the pre-cultured explant to be in full contact with the agrobacterium rhizogenes; each treatment group was infected with 30 explants respectively.
c. Co-cultivation
After infection is finished, the bacterial liquid on the surface of the explant is sucked to be dry by using sterile filter paper, then the explant is transferred to an MS solid culture medium containing 100 mu M acetosyringone, and co-culture is carried out for 2-3 d under the dark culture condition of 25 ℃.
c. Washing bacteria
Washing the explant forming bacterial plaque in the co-culture process with sterile water (containing 300 mg/L cefotaxime sodium and 200 mg/L timentin) for 2-3 times, and then sucking water on the surface of the explant by using sterile filter paper;
d. Induction of hairy roots
The explants were transferred to 1/2 MS solid medium (containing 300 mg/L cefotaxime sodium, 200 mg/L timentin and 100. mu.M acetosyringone), cultured under the conditions of 28 ℃ and long-day (14 h light/10 h dark) and subcultured once every other week, and the antibiotic concentration was gradually reduced until the bacterial plaque around the explants completely disappeared.
(5) Counting the number of hair roots
And (4) after infection, culturing for 20 d, counting the number of hairy roots induced by different hairy-root agrobacterium infected leaves and stem segments, and screening suitable hairy-root agrobacterium and explants for genetic transformation of morinda officinalis according to the induction rate of hairy roots. After the agrobacterium infection, the cultivation lasts for 10 d, tender morinda officinalis stems and tender morinda officinalis stems grow into hairy roots, and leaves do not exist (figure 3 a); after 20 d of culture, the stem-induced hairy root line rapidly elongated, grew along the surface of the medium, and exhibited negative geotropism (typical characteristics of hairy roots), while no leaf was produced from the hairy root line (FIG. 3 b).
As can be seen from table 1, after 20 days of culture, the probability of inducing hairy roots by using morinda officinalis leaves as explants is very low, and only one hairy root system (3.33%) is obtained by ar.qual and K599; the tender stem section is used as an explant, 5 agrobacterium rhizogenes strains can induce a starting root system, the induction rate is 13.33% -26.67%, and the root induction rate of the strain ATCC15834 is the highest (26.67%). Therefore, ATCC15834 was subsequently selected as the inducing strain and young stem segments were used as explants for the genetic transformation of hairy roots of morinda officinalis.
TABLE 1 statistics of hairy root number of different Agrobacterium rhizogenes infected leaves and stem segments
Example 3 genetic transformation of hairy roots:
(1) selection and Pre-culture of explants
Taking a tender stem section of the morinda officinalis aseptic tissue culture seedling as an explant, wherein the tender stem section is a stem section of a part with more than 2-3 leaves on the upper part of a tissue culture seedling plant. Taking a tender stem section of the morinda officinalis aseptic tissue culture seedling as an explant, slightly scratching the surface layer of the stem section by using a scalpel along the inclined downward angle of 30 degrees of the tender stem section, placing the scratched stem section in an MS solid culture medium containing 100 mu M acetosyringone, and carrying out dark culture at 25 ℃ for 2 days. The process of pre-culture can make the explant adapt to the condition of in vitro culture, promote cell division and easily integrate exogenous DNA, and improve the transformation efficiency.
(2) Preparing an infection liquid:
a. selecting ATCC15834 as an infecting strain, and transforming the constructed target gene vector pBI121-bHLH25 to Agrobacterium rhizogenes ATCC15834 by a freeze-thawing method;
b. in a sterile operation platform, selecting the transformed agrobacterium rhizogenes ATCC15834 by using an inoculating loop, streaking to a YEP solid culture medium containing 50 mg/L kanamycin, and carrying out inverted culture at 28 ℃ for 48-72 h;
c. selecting a single colony, inoculating the single colony into 1.5 mL YEB liquid culture medium (containing 50 mg/L kanamycin), and culturing at 220 r/min and 28 ℃ for 16-18 h;
d. Inoculating into 50 mL YEB liquid culture medium (containing 50 mg/L kanamycin) according to the inoculation amount of 0.5-1%, and performing inoculation at 28 ℃ for 220 r/min;
e. when the concentration of the bacterial liquid reaches OD600When the speed is about 0.6-0.8, centrifuging at 5000 rpm for 5 min, and pouring out the supernatant;
f. adding an equal volume of fresh MS liquid culture medium, resuspending the agrobacterium tumefaciens, and centrifuging at 5000 rpm for 5 min to collect thalli;
g. after resuspending the bacteria by using an equal volume of fresh MS liquid culture medium, adding 100 mu M acetosyringone, and standing at room temperature for 1-2 h to obtain an infection solution;
(3) infection with Agrobacterium rhizogenes
a. Transferring the stem segments after the pre-culture into the prepared staining solution, placing the stem segments in a shaking table, shaking at 90 r/min and 28 ℃ for 10-30 min to ensure that the stem segments after the pre-culture can be fully contacted with the agrobacterium rhizogenes;
(4) co-cultivation
After infection is finished, the bacteria liquid on the surface of the stem section is sucked up by sterile filter paper, and then the stem section is transferred to an MS solid culture medium containing 100 mu M acetosyringone for dark culture at 25 ℃ for 2-3 days. Acetosyringone is added into the co-culture medium to continuously induce the activation of Vir region genes of the agrobacterium rhizogenes, promote the integration of exogenous genes and improve the transformation efficiency.
(5) Washing bacteria
The explants forming bacterial plaques in the co-culture process are washed with sterile water (containing 300 mg/L cefotaxime sodium and 200 mg/L timentin) for 2-3 times, and then the water on the surface of the stem segments is sucked up by sterile filter paper.
(6) Induction of hairy roots
The explants were transferred to 1/2 MS solid medium (containing 300 mg/L cefotaxime sodium, 200 mg/L timentin and 100. mu.M acetosyringone), cultured under long-day (14 h light/10 h dark) conditions at 28 ℃ and subcultured every other week, and the concentration of the sterilization antibiotics was gradually reduced until the bacterial plaque around the stem segments was completely disappeared to prevent the death of the explants due to the propagation of Agrobacterium (FIG. 4). Infecting morinda officinalis stem sections by agrobacterium rhizogenes ATCC15834, culturing for 10 d after infection, wherein hairy roots grow out at wounds of the stem sections, and the stem sections have typical characteristics of being fluffy, negative in geotropism and the like (figure 5 a); after 20 d of culture, the induced hairy root line showed rapid elongation, thickening and typical negative geotropism in the culture medium (FIG. 5 b).
(7) Screening of Positive hairy root
When the induced hairy roots grow to about 2-3 cm, cutting off the hairy roots, transferring the cut hairy roots to 1/2 MS solid culture medium containing screening antibiotics (30 mg/L kanamycin), carrying out dark culture at 28 ℃, and screening to obtain transgenic hairy root hairy roots with kanamycin resistance, high growth speed and more branches in the culture process. As shown in FIG. 6, the hairy root system obtained by screening has the typical characteristics of multiple branches, rapid growth, negative geotropism (the root system creeps on the surface of the culture medium) and the like.
(8) Detection and transformation efficiency of Positive hairy roots
Randomly selecting 8 transgenic hairy root systems with kanamycin resistance, high growth speed and more branches obtained by screening in the step (7) of the embodiment as detection objects, taking the roots of the tissue culture seedlings as negative control, and taking ddH (dichloro-diphenyl-trichloroethane) as negative control2O as blank control. Identifying hairy roots according to rolC genes on pRi15834 plasmid in Agrobacterium rhizogenes ATCC15834, designing primers between 35S promoter and target gene bHLH25, detecting target gene bHLH25, and detecting the primersAs shown in table 2. The PCR reaction system is as follows: 2 XTaq Master Mix 25 uL, upstream and downstream primers 2 uL each, template DNA 1 uL, ddH2O20. mu.L. The reaction procedure is as follows: 3 min at 95 ℃; 30 s at 95 ℃, 30 s at 60 ℃, 60 s at 72 ℃ and 33 cycles; 10 min at 72 ℃.
The PCR detection result shows that: 5 of 8 induced hairy root systems can detect rolC gene, and the efficiency is 62.5%; and the roots and ddH of the tissue culture seedlings2No band was detected in any of O, and the ability to detect rolC gene in hairy root line indicates that hairy root is induced by Agrobacterium rhizogenes ATCC15834 (FIG. 7 a). The target gene detection result shows that the target gene bHLH25 can be detected in 4 hairy root strains, and the result shows that the exogenous target gene bHLH25 is successfully integrated into morinda officinalis hairy roots through agrobacterium rhizogenes ATCC15834 mediation, and the transformation efficiency is about 50% (fig. 7 b).
TABLE 2 PCR detection primers for positive hairy roots
Example 4
A method for establishing a genetic transformation system of morinda officinalis hairy roots comprises the following steps:
(1) pre-culture of explants
Placing young stem of Morinda citrifolia aseptic tissue culture seedling as explant in MS solid culture medium containing 100 μ M acetosyringone, and culturing at 25 deg.C in dark for 2 d;
(2) preparing an infection liquid:
transforming a plant expression vector pBI121-bHLH25 containing a target gene into Agrobacterium rhizogenes ATCC15834, and preparing an invasion solution;
(3) infection with Agrobacterium rhizogenes
Transferring the stem section after the pre-culture into the infection solution obtained in the step (2), and oscillating for 10-30 min at the temperature of 28 ℃ and at the speed of 90 r/min to ensure that the explant after the pre-culture is fully contacted with the agrobacterium rhizogenes;
(4) co-cultivation
After infection is finished, sucking the bacterial liquid on the surface of the explant by using sterile filter paper, transferring the bacterial liquid into an MS solid culture medium containing 100 mu M acetosyringone, and co-culturing for 2-3 d under the dark culture condition at 25 ℃;
(5) washing bacteria
Washing the explant forming bacterial plaque in the co-culture process with sterile water containing 300 mg/L cefotaxime sodium and 200 mg/L timentin for 2-3 times, and then sucking water on the surface of the explant by using sterile filter paper;
(6) Induction of hairy roots
Transferring the washed explants to 1/2 MS solid culture medium containing 300 mg/L cefotaxime sodium, 200 mg/L timentin and 100 mu M acetosyringone, and placing the culture medium in a constant temperature incubator at 28 ℃; subculturing once every other week, and gradually reducing the concentration of the degerming antibiotics until bacterial plaque around the explant completely disappears; the conditions of the subculture were as follows: the culture temperature is 28 deg.C, and the light condition is 14 h light and 10 h dark per day.
(7) Screening of Positive hairy root
When the induced hairy roots grow to about 2-3 cm, the hairy roots are cut off and transferred to 1/2 MS solid culture medium containing screening antibiotic 30 mg/L kanamycin, dark culture is carried out at 28 ℃, and hairy root systems with kanamycin resistance, high growth speed and more branches are screened out.
The explant in the step (1) is from a sterile tissue culture seedling of morinda officinalis, a young stem section is a stem section with more than 2-3 leaves on the upper part of a tissue culture seedling plant, the surface layer of the stem section is slightly scratched at an angle of 30 degrees downwards along the young stem section, and the stem section does not need to be scratched.
The preparation method of the infection liquid in the step (2) comprises the following steps:
a. transforming a plant expression vector pBI121-bHLH25 containing a target gene into agrobacterium rhizogenes ATCC15834 by a freeze-thawing method;
b. In an aseptic operation platform, picking the transformed agrobacterium rhizogenes ATCC15834 by using an inoculating loop, streaking to a YEP solid culture medium containing 50 mg/L kanamycin, and carrying out inverted culture at 28 ℃ for 48-72 h;
c. selecting a single colony, inoculating the single colony into 1.5 mL YEB liquid culture medium containing 50 mg/L kanamycin, and carrying out overnight culture at 220 r/min and 28 ℃ for 16-18 h;
d. inoculating the mixture into 50 mL YEB liquid culture medium containing 50 mg/L kanamycin according to the inoculation amount of 0.5-1%, and carrying out shake culture at 28 ℃ at 220 r/min;
e. when the concentration of the bacterial liquid reaches OD600When the speed is about 0.6-0.8, centrifuging for 5min at 5000 rpm, pouring out supernate and collecting thalli;
f. adding an equal volume of fresh MS liquid culture medium to resuspend the agrobacterium, centrifuging at 5000 rpm for 5min, pouring out supernatant and collecting thalli;
g. adding an equal volume of fresh MS liquid culture medium to resuspend the agrobacterium, adding 100 mu M acetosyringone, and standing at normal temperature for 1-2 h to obtain the infection solution.
Example 5
A method for establishing genetic transformation system of hairy root of Morinda officinalis comprises the following steps:
(1) pre-culture of explants
Placing young stem of Morinda citrifolia aseptic tissue culture seedling as explant in MS solid culture medium containing 120 μ M acetosyringone, and culturing at 28 deg.C in dark for 3 d;
(2) Preparing an infection liquid:
transforming a plant expression vector pBI121-bHLH25 containing a target gene into agrobacterium rhizogenes ATCC15834, and preparing an infection solution;
(3) infection with Agrobacterium rhizogenes
Transferring the stem sections after the pre-culture into the infection solution obtained in the step (2), and oscillating for 30 min at 30 ℃ at 100 r/min to ensure that the explants after the pre-culture are fully contacted with the agrobacterium rhizogenes;
(4) co-cultivation
After infection is finished, sucking the bacterial liquid on the surface of the explant by using sterile filter paper, transferring the bacterial liquid into an MS solid culture medium containing 120 mu M acetosyringone, and co-culturing for 3 d under the dark culture condition of 28 ℃;
(5) washing bacteria
Washing explants forming dental plaques in the co-culture process with sterile water containing 350 mg/L cefotaxime sodium and 250 mg/L timentin for 3 times, and then sucking water on the surfaces of the explants by using sterile filter paper;
(6) induction of hairy roots
Transferring the washed explants to 1/2 MS solid culture medium containing 350 mg/L cefotaxime sodium, 250 mg/L timentin and 120 mu M acetosyringone, and placing the solid culture medium in a constant temperature incubator at 28 ℃; subculturing once every other week, and gradually reducing the concentration of the degerming antibiotic until bacterial plaques around the explants completely disappear;
(7) Screening of Positive hairy root
When the induced hairy roots grow to about 3 cm, the hairy roots are cut off and transferred to a kanamycin 1/2 MS solid culture medium containing screening antibiotic 32 mg/L, dark culture is carried out at 30 ℃, and hairy root systems with kanamycin resistance, high growth speed and more branches are screened out.
The explant in the step (1) is a sterile tissue culture seedling from morinda officinalis, a young stem section is a stem section with more than 3 leaves on the upper part of a tissue culture seedling plant, the surface layer of the stem section is slightly scratched at an angle of 30 degrees obliquely downwards along the young stem section, and the stem section does not need to be scratched.
The preparation method of the infection liquid in the step (2) comprises the following steps:
a. transforming a plant expression vector pBI121-bHLH25 containing a target gene into agrobacterium rhizogenes ATCC15834 by a freeze-thawing method;
b. in an aseptic operation platform, picking the transformed agrobacterium rhizogenes ATCC15834 by using an inoculating loop, streaking to a YEP solid culture medium containing 60 mg/L kanamycin, and carrying out inverted culture at 30 ℃ for 48-72 h;
c. picking a single colony, inoculating the single colony into 1.5 mL YEB liquid culture medium containing 60 mg/L kanamycin, and culturing at 250 r/min and 30 ℃ overnight for 18 h;
d. inoculating the strain into 50 mL YEB liquid culture medium containing 60 mg/L kanamycin according to the inoculation amount of 0.5-1%, and performing shake culture at 30 ℃ at 250 r/min;
e. When the concentration of the bacterial liquid reaches OD600When the concentration is about 0.6-0.8, centrifuging, pouring out supernate and collecting thalli;
f. adding an equal volume of fresh MS liquid culture medium to resuspend the agrobacterium, centrifuging, pouring out supernatant, and collecting thalli;
g. adding an equal volume of fresh MS liquid culture medium to resuspend the agrobacterium, adding 120 mu M acetosyringone, and standing at normal temperature for 2 h to obtain the infection solution.
The subculture conditions in step (6) are as follows: the culture temperature is 30 deg.C, and the light condition is 14 h light and 10 h dark per day.
Example 6
A method for establishing a genetic transformation system of morinda officinalis hairy roots comprises the following steps:
(1) pre-culture of explants
Placing young stem of Morinda citrifolia aseptic tissue culture seedling as explant in MS solid culture medium containing 80 μ M acetosyringone, and dark culturing at 30 deg.C for 2 d;
(2) preparing an infection liquid:
transforming a plant expression vector pBI121-bHLH25 containing a target gene into Agrobacterium rhizogenes ATCC15834, and preparing an invasion solution;
(3) infection with Agrobacterium rhizogenes
Transferring the stem section after the pre-culture into the infection solution obtained in the step (2), and oscillating for 10 min at 25 ℃ and 50 r/min to ensure that the explant after the pre-culture is fully contacted with the agrobacterium rhizogenes;
(4) Co-cultivation
After infection is finished, sucking the bacterial liquid on the surface of the explant by using sterile filter paper, transferring the bacterial liquid into an MS solid culture medium containing 80 mu M acetosyringone, and co-culturing for 2 d under the dark culture condition of 22 ℃;
(5) washing bacteria
Washing explants forming bacterial plaques in the co-culture process with sterile water containing 250 mg/L cefotaxime sodium and 150 mg/L timentin for 2 times, and then sucking water on the surfaces of the explants by sterile filter paper;
(6) induction of hairy roots
Transferring the washed explants to 1/2 MS solid culture medium containing 250 mg/L cefotaxime sodium, 150 mg/L timentin and 80 mu M acetosyringone, and placing the culture medium in a constant temperature incubator at 28 ℃; subculturing once every other week, and gradually reducing the concentration of the degerming antibiotics until bacterial plaque around the explant completely disappears;
(7) screening of Positive hairy root
When the induced hairy roots grow to about 2 cm, the hairy roots are cut off and transferred to a kanamycin 1/2 MS solid culture medium containing screening antibiotic 28 mg/L, dark culture is carried out at 25 ℃, and hairy root systems with kanamycin resistance, high growth speed and more branches are screened out.
The explant in the step (1) comes from a sterile tissue culture seedling of morinda officinalis, a young stem section is a stem section with more than 2-3 leaves on the upper part of a tissue culture seedling plant, the surface layer of the stem section is slightly scratched at an angle of 30 degrees obliquely downwards along the young stem section, and the stem section does not need to be scratched.
The preparation method of the infection liquid in the step (2) comprises the following steps:
a. transforming a plant expression vector pBI121-bHLH25 containing a target gene into agrobacterium rhizogenes ATCC15834 by a freeze-thawing method;
b. in a sterile operation platform, selecting the transformed agrobacterium rhizogenes ATCC15834 by using an inoculating loop, streaking to a YEP solid culture medium containing 40 mg/L kanamycin, and carrying out inverted culture at 25 ℃ for 48-72 h;
c. selecting a single colony, inoculating the single colony into 1.5 mL YEB liquid culture medium containing 40 mg/L kanamycin, and carrying out overnight culture at the temperature of 25-30 ℃ at 200 r/min for 16-18 h;
d. inoculating the mixture into 50 mL YEB liquid culture medium containing 40 mg/L kanamycin according to the inoculation amount of 0.5-1%, and performing shake culture at 25 ℃ at 200 r/min;
e. when the concentration of the bacterial liquid reaches OD600When the concentration is about 0.6-0.8, centrifuging, pouring out supernate and collecting thalli;
f. adding an equal volume of fresh MS liquid culture medium to resuspend the agrobacterium, centrifuging, pouring out supernatant and collecting thalli;
g. adding an equal volume of fresh MS liquid culture medium to resuspend the agrobacterium, adding 80 mu M acetosyringone, and standing at normal temperature for 1-2 h to obtain the infection solution.
The subculture in the step (6) is carried out under the following conditions: the culture temperature is 25 deg.C, and the illumination condition is 14 h illumination and 10 h dark each day.
Claims (9)
1. A method for establishing a genetic transformation system of morinda officinalis hairy roots is characterized by comprising the following steps:
(1) pre-culturing explants:
placing young stem sections of the morinda officinalis aseptic tissue culture seedlings as explants in an MS solid culture medium containing 80-120 mu M acetosyringone, and performing dark culture at 22-28 ℃ for 2-3 days;
(2) preparing an infection liquid:
transforming a plant expression vector containing a target gene into agrobacterium rhizogenes and preparing into an infection solution;
(3) infection with Agrobacterium rhizogenes:
transferring the stem sections after the pre-culture to the infection solution obtained in the step (2), and oscillating for 10-30 min at the temperature of 25-30 ℃ and at the speed of 50-100 r/min to ensure that the explants after the pre-culture are fully contacted with the agrobacterium rhizogenes;
(4) co-culturing:
after infection is finished, sucking a bacterial liquid on the surface of the explant by using sterile filter paper, transferring the bacterial liquid into an MS solid culture medium containing 80-120 mu M acetosyringone, and co-culturing for 2-3 d under a dark culture condition at 22-28 ℃;
(5) washing bacteria:
washing the explant forming bacterial plaque in the co-culture process with sterile water containing 250-350 mg/L cefotaxime sodium and 150-250 mg/L timentin for 2-3 times, and then sucking water on the surface of the explant by using sterile filter paper;
(6) Induction of hairy roots:
transferring the washed explants to 1/2 MS solid culture medium containing 250-350 mg/L cefotaxime sodium, 150-250 mg/L timentin and 80-120 mu M acetosyringone, and placing the culture medium in a constant temperature incubator at 28 ℃; subculturing once every other week, and gradually reducing the concentration of the degerming antibiotics until bacterial plaque around the explant completely disappears;
(7) screening positive hairy roots:
when the induced hairy roots grow to about 2-3 cm, the hairy roots are cut off and transferred to 1/2 MS solid culture medium containing screening antibiotics, dark culture is carried out at 25-30 ℃, and hairy root systems with kanamycin resistance, high growth speed and more branches are screened out.
2. The method for establishing the genetic transformation system for the hairy root of morinda officinalis how in claim 1, wherein the explant in step (1) is from a sterile tissue culture seedling of morinda officinalis how, and the young stem is a stem with more than 2-3 leaves on the upper part of the tissue culture seedling plant, and the surface layer of the stem is slightly scratched along the oblique 30-degree angle of the young stem without cutting the stem.
3. The method for establishing the genetic transformation system for the morinda officinalis hairy root according to claim 1, wherein the infection solution prepared in the step (2) is prepared by the following steps:
a. Transforming the plant expression vector containing the target gene into agrobacterium rhizogenes ATCC15834 by a freeze-thawing method;
b. in a sterile operation platform, selecting the transformed agrobacterium rhizogenes ATCC15834 by using an inoculating loop, streaking to a YEP solid culture medium containing 40-60 mg/L kanamycin, and carrying out inverted culture at 25-30 ℃ for 48-72 h;
c. selecting a single colony, inoculating the single colony into 1.5 mL YEB liquid culture medium containing 40-60 mg/L kanamycin, and carrying out overnight culture at the temperature of 25-30 ℃ for 16-18 h at the speed of 200-250 r/min;
d. inoculating the mixture into 50 mL of YEB liquid culture medium containing 40-60 mg/L kanamycin according to the inoculation amount of 0.5-1%, and performing shake culture at 25-30 ℃ at 200-250 r/min;
e. when the concentration of the bacterial liquid reaches OD600When the concentration is 0.6-0.8%, centrifuging, pouring out supernate and collecting thalli;
f. adding an equal volume of fresh MS liquid culture medium to resuspend the agrobacterium, centrifuging, pouring out supernatant and collecting thalli;
g. adding an equal volume of fresh MS liquid culture medium to resuspend the agrobacterium, adding 80-120 mu M acetosyringone, and standing at normal temperature for 1-2 h to obtain the infection solution.
4. The method for creating a genetic transformation system for Morinda citrifolia hairy roots according to claim 1 or 3, wherein said plant expression vector containing said target gene is pBI121-bHLH 25.
5. The method for establishing the genetic transformation system for morinda citrifolia hairy root according to claim 1, wherein the conditions of said subculture in step (6) are as follows: the culture temperature is 25-30 ℃, and the illumination condition is 14 h illumination and 10 h darkness every day.
6. The method for establishing the genetic transformation system for the morinda citrifolia hairy root according to claim 1, wherein the selection antibiotic in the step (7) is kanamycin.
7. The method for establishing the genetic transformation system for the morinda officinalis hairy root according to claim 6, wherein the concentration of the screened antibiotic kanamycin is 28-32 mg/L.
8. The method for establishing the genetic transformation system for morinda citrifolia hairy root as claimed in claim 1, wherein said morinda citrifolia tissue culture seedling of step (1) is cultured by the following steps:
selecting strong branches of morinda officinalis plants, removing leaves, disinfecting with 70% alcohol for 20-30 s, soaking in 3% sodium hypochlorite solution for 20-30 min, and washing with sterile water for 5-6 times; cutting into stem segments of about 1 cm according to the position of the axillary buds, cutting into an MS solid culture medium containing 0.5 mg/L6-BA, culturing in an illumination incubator at 28 ℃, illuminating for 16 h/8 h in darkness, and cutting and infecting explants when the axillary buds grow to 4-5 cm.
9. The method for establishing the genetic transformation system for morinda citrifolia hairy roots according to claim 1, wherein the agrobacterium rhizogenes in step (2) is agrobacterium rhizogenes ATCC 15834.
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Cited By (2)
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| CN115725644A (en) * | 2022-12-13 | 2023-03-03 | 安徽农业大学 | Genetic transformation method and application of polygonatum cyrtonema |
| CN116411016A (en) * | 2023-04-21 | 2023-07-11 | 安徽农业大学 | Method for rapid screening of wild-root genetic transformation and gene editing gRNA targets of kiwi fruits without screening markers |
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| CN105748655A (en) * | 2014-12-31 | 2016-07-13 | 陈深源 | Method for enhanced production of morinda metabolites |
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN115725644A (en) * | 2022-12-13 | 2023-03-03 | 安徽农业大学 | Genetic transformation method and application of polygonatum cyrtonema |
| CN116411016A (en) * | 2023-04-21 | 2023-07-11 | 安徽农业大学 | Method for rapid screening of wild-root genetic transformation and gene editing gRNA targets of kiwi fruits without screening markers |
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