CN114561419A - Agrobacterium tumefaciens-mediated phellinus igniarius genetic transformation method - Google Patents
Agrobacterium tumefaciens-mediated phellinus igniarius genetic transformation method Download PDFInfo
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Abstract
The invention provides a genetic transformation method of agrobacterium tumefaciens-mediated phellinus igniarius, which comprises the following steps: 1) carrying out enzymolysis and purification on phellinus igniarius hyphae by a muramidase enzyme solution to obtain phellinus igniarius protoplasts, 2) carrying out genetic transformation treatment on the phellinus igniarius protoplasts obtained in the step 1) by using recombinant agrobacterium tumefaciens containing exogenous genes, and 3) carrying out regeneration culture and resistance screening culture on the treated phellinus igniarius protoplasts, thereby obtaining phellinus igniarius transformant strains integrated with the exogenous genes. The method can effectively mediate the integration of the exogenous gene into the genome of the phellinus igniarius, and the phellinus igniarius is stably copied and expressed, thereby establishing a transformation system of the phellinus igniarius. The phellinus igniarius conversion system established by the method opens up a wide application field for phellinus igniarius, and thus the phellinus igniarius, a valuable resource, is utilized more fully.
Description
Technical Field
The invention belongs to the field of microbiology in the field of biotechnology, and particularly relates to a genetic transformation method of agrobacterium tumefaciens-mediated phellinus igniarius.
Background
Phellinus igniarius is a basic source strain of medicinal and edible fungi Phellinus igniarius, Phellinus igniarius sporocarp can grow in wild environment to generate a plurality of unique bioactive morin components, and the Phellinus igniarius sporocarp has medicinal functions of resisting cancer, resisting lipid peroxidation, regulating immunity, protecting liver, resisting cirrhosis, resisting mutagenesis and the like, has attracted the attention of the international medical field, and has widely developed researches on aspects of biochemical pharmacology, clinical application and the like.
However, due to the physiological and ecological characteristics and complexity of Phellinus linteus, and the change of harsh wild environmental conditions, the number of wild fruiting bodies formed in the natural world is very small, and it takes at least three years to form medicinal fruiting bodies. Under the conditions that the wild fruiting body resources are seriously deficient and the yield is extremely low when the wild fruiting body is cultured under the laboratory conditions, the improvement of the existing strains by a biotechnology means to obtain high-yield strains becomes a hot point of another research.
Disclosure of Invention
The invention aims to provide an agrobacterium tumefaciens-mediated phellinus igniarius genetic transformation method.
The invention provides an agrobacterium tumefaciens-mediated phellinus igniarius genetic transformation method, which comprises the following steps:
1) carrying out enzymolysis and purification on the phellinus igniarius hypha by a muramidase solution to obtain phellinus igniarius protoplast,
2) carrying out genetic transformation treatment on the phellinus igniarius protoplast obtained in the step 1) by using recombinant agrobacterium tumefaciens containing exogenous genes,
3) and performing regeneration culture and resistance screening on the treated phellinus igniarius protoplast to obtain the phellinus igniarius transformant integrated with the exogenous gene.
In the step 1), the adding amount of the lywallzyme solution is 2-10 mL per gram of wet hypha; the enzymolysis condition is enzymolysis for 1.5 to 3 hours at the temperature of 30 to 35 ℃; and the purification method in the step 1 comprises the steps of filtering the enzymolysis liquid by using a G3 sand core funnel, centrifuging the filtrate to remove supernatant, and washing the filtrate for 1-3 times by using 0.5-0.8 mol/L osmotic pressure stabilizer to obtain the purified phellinus igniarius protoplast.
The recombinant agrobacterium tumefaciens containing the exogenous gene in the step 2) is a recombinant vector obtained by introducing a recombinant vector pCH-hph into agrobacterium AGL-1 to obtain the hygromycin screening marker-containing agrobacterium AGL-1-pCH-hph expressed in fungi, wherein the sequence of EcoRI and SacI sites of the recombinant vector pCH-hph is a pDHt2 vector, the sequence is replaced by a nucleotide sequence shown in the sequence 1, and other sequences are kept unchanged. The pDHt2 vector was based on the pCAMBIA1300 vector, and the hygromycin resistance gene on the pCAMBIA1300(CAMBIA, Canberra, Australia) plasmid and the 35S promoter sequence of cauliflower mosaic virus were removed by Xho I digestion, and the digested plasmids were self-ligated to produce a heavy-duty vector without inserts.
Wherein, the construction method of the agrobacterium AGL-1-pCH-hph in the step 2) comprises the following steps:
s1) inoculating the bacterial liquid of the agrobacterium AGL-1 into an LB liquid culture medium containing rifampicin for overnight culture;
s2) inoculating the culture solution obtained in the step S1) into LB liquid culture medium of rifampicin, and culturing to OD600Reaching 0.5, centrifuging, and adding CaCl2Suspending the thalli in the solution to obtain agrobacterium-mediated cells;
s3) taking the competent cells obtained in the step S2), adding a recombinant vector pCH-hph, uniformly mixing, cooling, performing heat shock, adding an LB liquid culture medium without antibiotics for culture, coating the LB liquid culture medium on a solid LB culture medium flat plate containing kanamycin, and screening resistant colonies to obtain an AGL-1 monoclonal containing pCH plasmids;
s4) inoculating single AGL-1 clone containing pCH plasmid into minimal medium containing rifampicin and kanamycin at 1% inoculation amount, culturing overnight, and culturing withDilution of liquid Induction Medium to OD600Reaching 0.15, and obtaining the agrobacterium AGL-1-pCH-hph.
The method specifically comprises the following steps:
s1) inoculating the bacterial liquid of the agrobacteria AGL-1 into LB liquid culture medium with the concentration of 50mg/L rifampicin, and culturing overnight at 28 ℃;
s2) taking 2mL of the culture solution obtained in the step S1), inoculating the culture solution into 50mL of LB liquid culture containing 50mg/L rifampicin, and culturing at 28 ℃ to OD600Up to 0.5. Standing on ice for 10min, centrifuging at 4 deg.C and 3000g for 10min, pre-cooling with 1mL ice of 20mM CaCl2Suspending the thallus in the solution to obtain agrobacterium-infected cells;
s3) taking 100 mu L of competent cells obtained in the step S2), adding a proper amount of recombinant vector pCH-hph, uniformly mixing, freezing for 30min, liquid nitrogen for 5min, performing heat shock at 37 ℃ for 25min, adding 1mL of LB liquid culture medium without antibiotics, shaking bacteria at 28 ℃ and 150 r/min for 3h, coating the solution on a solid LB culture medium plate containing kanamycin with the concentration of 50mg/L, culturing for 1-2d at 28 ℃, and screening resistant colonies to obtain AGL-1 monoclonal containing pCH plasmids;
s4) inoculating single AGL-1 clone containing pCH plasmid at 1% inoculum size in minimal medium containing 50mg/L rifampicin and 50mg/L kanamycin, culturing at 28 deg.C overnight, diluting with liquid induction medium to OD600Culturing at the temperature of 0.15 ℃ and 200 rpm for 3 hours at the temperature of 28 ℃ to obtain the agrobacterium AGL-1-pCH-hph.
Wherein the LB liquid medium containing rifampicin at a concentration of 50mg/L is obtained by adding rifampicin to LB liquid medium to make the concentration of rifampicin in the mixture 50 mg/L; the preparation method of the LB liquid culture medium comprises the steps of adding 10g of tryptone, 5g of yeast extract, 10g of sodium chloride and 1mL of 1M NaOH into 950mL of deionized water, fixing the volume to 1L, and adjusting the pH value to 7.2 to obtain the LB liquid culture medium.
Wherein the conversion treatment in the step 2) comprises the following steps: uniformly mixing agrobacterium AGL-1-pCH-hph and purified phellinus igniarius protoplast, and coating the mixture on a solid induction culture medium; culturing for 2-3 days at 20-30 ℃, transferring to a resistance screening culture medium containing hygromycin, cefamycin, streptomycin and tetracycline, and continuously culturing for 8-12 days to obtain the phellinus igniarius transformant.
The resistance screening culture medium containing hygromycin, cefamycin, streptomycin and tetracycline is a mixed solution obtained by adding hygromycin, cefamycin, streptomycin and tetracycline into the resistance screening culture medium, wherein the concentration of the hygromycin in the mixed solution is 30 mg/L; the concentration of the cefuroxime is 200 mu M; the concentration of streptomycin is 100 mg/L; the concentration of the tetracycline is 50 mg/L; the resistance screening culture medium is a PDA culture medium, and the preparation method of the PDA culture medium comprises the steps of adding water into 200g of peeled potatoes, 20g of glucose and 15g of agar to reach a constant volume of 1L.
Wherein, in the step 3), the modified martin agar culture medium containing hygromycin is used for resistance screening culture, and the modified martin agar culture medium containing hygromycin is prepared by adding a hygromycin solution into the modified martin agar culture medium so that the concentration of the hygromycin in the culture medium is 30 mg/L; the improved martin agar culture medium is 50g of peptone, 20g of yeast extract, 10g of dipotassium hydrogen phosphate, 5g of magnesium sulfate, 20g of glucose and 15g of agar, and water is added to the mixture to be constant volume to 1L.
The present invention also provides a biomaterial related to the genetic transformation of phellinus igniarius, which is the recombinant vector pCH-hph or the Agrobacterium AGL-1-pCH-hph according to claim 3.
The application of the biological material related to the genetic transformation of the phellinus igniarius in the genetic transformation of the phellinus igniarius is also within the protection scope of the invention.
The method is suitable for phellinus igniarius. The Phellinus igniarius protoplast treated by the agrobacterium transformation system by the method grows a plurality of strains on a PDA (personal digital assistant) plate containing HmB (hygromycin), and the strains can still keep good HmB resistance after 4 generations of passage under the condition of no selective pressure; whereas, Phellinus igniarius protoplasts that were not treated by the method of the present invention were all unable to grow on the modified Martin agar plates containing HmB (as shown in FIG. 5): the method of the invention is proved to effectively integrate the hph gene into the genome of the phellinus igniarius and stably express the hph gene in the phellinus igniarius, so that the phellinus igniarius transformant expresses the HmB resistance of the foreign gene, and the transformant still has strong HmB resistance after multi-generation passage under the condition of no selective pressure, thereby proving that the expression of the foreign gene in the transformant has strong stability.
The results show that the method can effectively mediate the integration of the exogenous gene into the genome of the phellinus igniarius, and the phellinus igniarius can be stably copied and expressed, so that a transformation system of the phellinus igniarius is established. The phellinus igniarius conversion system established by the method opens up a wide application field for phellinus igniarius, and thus the phellinus igniarius, a valuable resource, is utilized more fully. Specifically, the method mainly comprises the following steps:
1. phellinus igniarius has high protein secretion capacity, can correctly perform post-translational processing of an expression product, including skin chain shearing, glycosylation and the like, has a glycosylation mode similar to that of higher eukaryotes, is a confirmed safe strain, is beneficial and harmless to a human body, and has mature fermentation and post-treatment processes, so that exogenous genes from higher organisms with important economic values can be introduced into the Phellinus igniarius to for expression by utilizing a Phellinus igniarius transformation technology. Thus, the fermentation method of Phellinus igniarius can be used for producing active exogenous protein rapidly, in large scale and at low cost.
2. Certain genes related to excellent characters are introduced into the phellinus igniarius through a phellinus igniarius transformation technology, the phellinus igniarius strain can be rapidly and directionally improved in a short time, and a new high-quality and high-yield phellinus igniarius variety is cultured.
3. In the aspect of basic research, the transformation of phellinus igniarius is used as a gene cloning and expression system of fungi, can be used for the research on aspects of gene separation, genome structure, gene expression, regulation and control of the gene expression and the like, and can provide theoretical and experimental basis for establishing transformation systems of other large-scale fungi.
Drawings
FIG. 1: is a schematic diagram of pCH plasmid;
FIG. 2: is pCH vector single enzyme digestion (EcoRI), wherein Lane 1-4 is pCH vector single enzyme digestion with EcoRI, Lane 5 is pCH original vector which is not enzyme digested;
FIG. 3: is a pCH vector single digested enzyme (XbaI), wherein lane 1 is pCH-5 'original vector not digested, and lanes 2-4 are pCH-5' digested with XbaI;
FIG. 4: phellinus igniarius transformation subgraph growing on PDA culture medium containing hygromycin 30 mug/mL, cefamycin 200 mug/mL, streptomycin 100 mug/mL and tetracycline 50 mug/mL;
FIG. 5: phellinus igniarius transformed subgraph grown on modified Martin medium containing hygromycin 30. mu.g/mL.
Detailed Description
The present invention is described in further detail below with reference to specific embodiments, which are given for the purpose of illustration only and are not intended to limit the scope of the invention. The examples provided below serve as a guide for further modifications by a person skilled in the art and do not constitute a limitation of the invention in any way.
The experimental procedures in the following examples are conventional unless otherwise specified. Materials, reagents and the like used in the following examples are commercially available unless otherwise specified.
Description of materials:
plasmid pCH in the following examples
Agrobacterium AGL-1 of the following examples is disclosed in the document Lei Zhou, Jun Zhoo, Wangzhen Guo, Tianzhen Zhang, Functional Analysis of Autophagy Genes via Agrobacterium-Mediated Transformation in the Vascular wild Fungus Verticillium dahliae, Journal of Genetics and Genetics 40(2013)421e431 publicly available from the Ningxia medical university.
Phellinus igniarius hyphae in the examples described below are disclosed in the documents Xiuli Wu, Sheng Lin, Chenggen Zhu, Homo-and Heptanor-sterols and Tremulane Sesquiterpens from Cultures of Phellinus igniarius, J.Nat.Prod.2010,73, 1294-.
Example 1:
sensitivity detection of Phellinus igniarius to hygromycin: different strains have different sensitivity degrees to hygromycin, and in order to screen transformants, the hygromycin concentration needs to be screened, and the screening method comprises the following steps: taking the strong Phellinus igniarius hyphae growing on the improved martin agar slant, inoculating the hyphae into an improved martin culture medium, and culturing at 28 ℃ and 180 r/min. After 10 days, the cultured hyphae are washed clean by sterile water, the surface water is absorbed by sterile filter paper, 1mL of enzyme solution is added into every 300mg of wet hyphae, and the zymohydrolysis is carried out for 3 hours in a water bath with the rotating speed of 100 r/min at the temperature of 30 ℃. Filtering the enzymolysis solution with sterilized G3 sand core funnel, centrifuging at 3400 rpm for 10min to remove supernatant, and washing with osmotic pressure stabilizer for 2 times to obtain purified white protoplast. Diluting protoplasm with osmotic pressure stabilizer to obtain suspension, counting with blood counting plate, and adjusting concentration to 1 × 105one/mL. 10 μ L of protoplast suspension was pipetted and inoculated into a suspension containing hygromycin at the concentrations: 0, 15, 30, 50. mu.g/mL PDA medium, 28 ℃,3 replicates of each treatment. The germination and growth of protoplasts were observed every 1 day until day 10, and the minimum concentration capable of inhibiting the germination of the protoplast suspension was the final concentration of hygromycin.
Under the condition of reaching 30 mu g/mL of hygromycin, the protoplast does not germinate at all, so the invention takes 30 mu g/mL as the lowest screening concentration of the hygromycin for inhibiting the protoplast, namely the proper concentration for screening positive transformants.
Example 2:
1. preparation of Phellinus igniarius protoplast
Taking the strong phellinus igniarius hyphae growing on the improved martin agar slant, inoculating the phellinus igniarius hyphae into an improved martin liquid culture medium, culturing for 10 days at 28 ℃, then centrifuging for 10 minutes at 5000 r/min, removing supernatant, and washing for 3 times by using 0.5mol/L glycerol solution to obtain fresh phellinus igniarius wet hyphae;
taking 1g of Phellinus igniarius wet hyphae, adding 3mL of muramidase solution, and carrying out enzymolysis for 2 hours at 35 ℃. Filtering the enzymolysis solution with sterilized G3 sand core funnel, centrifuging at 3000 rpm for 10min, removing supernatant, washing with 0.5mol/L mannitol for 2 times to obtain purified Phellinus igniarius protoplast.
2. Construction of plasmid pCH-hph
On the basis of the pCAMBIA1300 vector, the hygromycin resistance gene on the pCAMBIA1300(CAMBIA, Canberra, Australia) plasmid and the 35S promoter sequence of cauliflower mosaic virus were removed by Xho I digestion, the digested plasmids were self-ligated to produce pDHt2 without an insert, the sequences of EcoRI and SacI sites of the pDHt2 vector were replaced with the nucleotide sequences shown in sequence 1, and the other sequences were kept unchanged to obtain a recombinant vector as shown in FIG. 1, and the recombinant vector was named pCH-hph.
3. Construction of Agrobacterium AGL-1-pCH-hph:
agrobacterium, AGL-1, is a gram-negative Agrobacterium, which contains a helper plasmid conferring rifampicin resistance, which contains a Ti plasmid conferring rifampicin resistance to induce infected plant or fungal cells; the Ti plasmid has a section of transfer DNA, and when the agrobacterium infects the host, the section of DNA can be inserted into the host genome, so that the carried gene can be expressed in the host. The recombinant vector pCH-hph was introduced into AGL-1 as follows:
(a) inoculating AGL-1 in an amount of 1% into 50mL LB liquid culture containing rifampicin at a concentration of 50mg/L, and culturing overnight at 28 ℃;
(b) inoculating 2mL of the culture solution obtained in step (a) into 50mL of LB liquid culture medium containing rifampicin at a concentration of 50mg/L, and culturing at 25 ℃ to OD600Up to 0.5. The mixture was placed on ice for 10 minutes, centrifuged at 3000g for 10 minutes at 4 ℃ and precooled with 1mL ice of 20mM CaCl2Suspending the thalli to obtain agrobacterium-induced cells;
(c) taking 100 mu L of agrobacterium tumefaciens competent cells in the step (b), adding a proper amount of recombinant vector pCH-hph, uniformly mixing, freezing for 30min, using liquid nitrogen for 5min, thermally shocking for 25min at 37 ℃, adding 1mL of LB liquid culture medium without antibiotics, shaking the bacteria for 3h at 28 ℃ and 150 rpm, coating the bacteria on a solid LB culture medium plate containing kanamycin with the concentration of 50mg/L, culturing at 28 ℃, and screening transformants;
(d) the AGL-1 introduced with the recombinant vector pCH-hph as described above was inoculated in an amount of 1% to a basal medium containing rifampicin at a concentration of 25 mg/L and kanamycin at a concentration of 50mg/L, cultured overnight at 28 ℃ and then induced with a liquid induction mediumDiluting to OD600Culturing at 28 deg.C for 3 hr to obtain Agrobacterium AGL-1-pCH-hph;
4. transformation of
And (3) uniformly mixing 100 mu L of purified phellinus igniarius protoplast with 100 mu L of the prepared agrobacterium AGL-1-pCH-hph bacterial liquid. Coating on a solid induction culture medium, culturing at 28 ℃ for 2 days, transferring to a resistance screening culture medium plate (containing four antibiotics simultaneously in the resistance screening culture medium) containing hygromycin with the concentration of 30mg/L, cefamycin with the concentration of 200 mu M, streptomycin with the concentration of 100mg/L and tetracycline with the concentration of 50mg/L, and culturing at the same temperature for 10 days to obtain phellinus igniarius transformants with the transformation efficiency of about 100 transformants/1X 107And (4) protoplasts.
The LB liquid medium in example 2 was: adding 10g of tryptone, 5g of yeast extract, 10g of sodium chloride and 1mL of 1M NaOH into 950mL of deionized water, fixing the volume to 1L, and adjusting the pH to 7.2; the LB solid medium is: 15g of agar was added to the above LB liquid medium.
The basic medium in this example 2 was: 950mL of deionized water with 2g of glucose, 1.45g K2HPO4， 2.05g KH2PO4，0.5g(NH4)NO3，0.01g CaCl2，0.6g MgSO4·7H2O, 0.3g NaCl, 5mL Z-Salts (l 0mL Z-Salts contained: 0.001g MgSO4·7H2O，0.001g CuSO4·5H2O,0.001g H3BO4， 0.5g(NH4)2SO4，0.001g MnSO4·H2O，0.001g NaMoO4·H2O), adding the solution to a constant volume of 1L, and adjusting the pH value to 6.7-7.0 by using HCl.
The liquid induction medium in this example 2 was: 950mL of deionized water was added with 8.7g of MM salt, 98% MES, 2g of glucose, 5g of glycerol, and 200. mu.M acetosyringone AS, and the volume was adjusted to 1L, and the pH was adjusted to 5.4 with HC 1.
The muramidase solution in the embodiment 2 comprises the following components in parts by weight: lywallzyme 1.5% (w/v), dissolved in 100mL of mannitol (osmotic pressure stabilizer) solution at a concentration of 0.6mo 1/L.
The solid induction medium in this example 2 was: 950mL of deionized water was added with 8.7g of MM salt, 98% MES, 2g of glucose, 5g of glycerol, 200. mu.M acetosyringone AS, and 15g of agar, and the volume was adjusted to 1L, and the pH was adjusted to 5.4 with HC 1.
The resistance screening culture medium in the embodiment 2 is a PDA culture medium containing hygromycin, cefamycin, streptomycin and tetracycline, wherein the PDA culture medium is prepared by boiling potato 200g for 30 minutes, filtering with four layers of gauze, adding glucose 20g and agar 15g, and adding water to a constant volume of 1L.
The modified Martin agar medium in this example 2 was: 5g of peptone, 1g of dipotassium phosphate, 2g of yeast extract powder, 0.5g of magnesium sulfate, 20g of glucose and 15g of agar, and adding water to a constant volume of 1000 mL.
The modified Martin liquid medium in example 2 is: 5g of peptone, 1g of dipotassium phosphate, 2g of yeast extract powder, 0.5g of magnesium sulfate and 20g of glucose, and adding water to a constant volume of 1000 mL.
Example 3:
taking the strong phellinus igniarius hyphae growing on the improved martin agar slant, inoculating the phellinus igniarius hyphae into an improved martin liquid culture medium, culturing for 12 days at 27 ℃, then centrifuging for 10 minutes at 4000 rpm, removing supernatant, and washing for 3 times by using 0.5mol/L glycerol solution to obtain fresh phellinus igniarius wet hyphae;
taking 2g of Phellinus igniarius wet hyphae, adding 8mL of lywallzyme solution, and carrying out enzymolysis for 2.5 hours at 30 ℃. Filtering the enzymolysis solution with sterilized G3 sand core funnel, centrifuging at 4000 rpm for 10min, removing supernatant, and washing with 0.5mol/L mannitol for 2 times to obtain purified Phellinus igniarius protoplast.
Collecting purified 100 μ L Phellinus igniarius protoplast (1 × 10)8piece/mL) was mixed with 100. mu.L of AGL-1 prepared above. Coating on a solid induction culture medium, culturing at 30 ℃ for 2 days, transferring to a resistance screening culture medium plate containing hygromycin with the concentration of 30mg/L, cefamycin with the concentration of 200 mu M, streptomycin with the concentration of 100mg/L and tetracycline with the concentration of 50mg/L, and culturing at the same temperature for 10 days to obtain phellinus igniarius transformants with the transformation efficiency of about 120 transformants/1X 107And (3) protoplasts.
The LB liquid medium and LB solid medium in this example 3 are the same as in example 2. The MM liquid medium in this example 3 is the same as that in example 2. The IM liquid medium in this example 3 was the same as that in example 2.
The components and the mixture ratio of the lywallzyme liquid in the embodiment 3 are as follows: lywallzyme 2.5% (w/v), dissolved in 100mL of a 0.8mo1/L mannitol (osmotic pressure stabilizer) solution.
The solid induction medium in this example 3 was the same as in example 2.
The regeneration medium in this example 3 was PDA medium, which was the same as that in example 2.
Example 4:
taking the strong phellinus igniarius hyphae growing on the improved martin agar slant, inoculating the phellinus igniarius hyphae into an improved martin liquid culture medium, culturing for 10 days at 26 ℃, then centrifuging for 10 minutes at 3000 r/min, removing supernatant, and washing for 3 times by using 0.8mol/L glycerol solution to obtain fresh phellinus igniarius wet hyphae;
taking 4 g of Phellinus igniarius wet hyphae, adding 10mL of lywallzyme solution, and carrying out enzymolysis for 3 hours at 30 ℃. Filtering the enzymolysis solution with sterilized G3 sand core funnel, centrifuging at 3500 rpm for 10min, removing supernatant, washing with 0.8mol/L mannitol for 2 times to obtain purified Phellinus igniarius protoplast.
Collecting purified 100 μ L Phellinus igniarius protoplast (1 × 10)8one/mL) was mixed with 100. mu.L of AGL-1 prepared above. Coating on a solid induction culture medium, culturing at 24 ℃ for 2 days, transferring to a resistance screening culture medium plate containing hygromycin with the concentration of 30mg/L, cefamycin with the concentration of 200 mu M, streptomycin with the concentration of 100mg/L and tetracycline with the concentration of 50mg/L, and culturing at the same temperature for 10 days to obtain phellinus igniarius transformants with the transformation efficiency of about 150 transformants/1X 107And (4) protoplasts.
The LB liquid medium and LB solid medium in this example 4 are the same as in example 2. The MM liquid medium in this example 4 is the same as that in example 2. The IM liquid medium in this example 4 was the same as that in example 2.
The muramidase solution in the embodiment 4 comprises the following components in parts by weight: lywallzyme 2% (w/v), dissolved in 100mL of a 0.8mo1/L mannitol (osmotic pressure stabilizer) solution.
The solid induction medium in this example 4 was the same as in example 2.
The regeneration medium in this example 4 was PDA medium, which was the same as that in example 2.
The hygromycin concentration in this example 4 is unpredictable experimental data, and if it is too high, the transformation efficiency will be low or even fail, and the phellinus igniarius protoplasts will not germinate at all up to 30. mu.g/mL hygromycin.
Example 5 validation test
The genetic transformant of the phellinus igniarius transformed with the exogenous target gene is inoculated on a PDA (PDA) solid culture medium without HmB (hygromycin) and is continuously subcultured for 4 times, and the strains can still grow on an improved Martin agar plate containing HmB after 4 generations under the condition of no selective pressure and keep good HmB resistance (as shown in figure 5); whereas, Phellinus igniarius protoplasts not treated by the method of the present invention were all unable to grow on the modified Martin agar plates containing HmB (as shown in FIG. 5): the method of the invention is proved to effectively integrate the hph gene into the phellinus igniarius genome and stably express the hph gene in the phellinus igniarius, so that the phellinus igniarius transformant expresses the HmB resistance of the foreign gene, and the transformant still has strong HmB resistance after multi-generation passage under the condition of no selective pressure, thereby proving that the expression of the foreign gene in the transformant has strong stability.
The present invention has been described in detail above. It will be apparent to those skilled in the art that the invention can be practiced in a wide range of equivalent parameters, concentrations, and conditions without departing from the spirit and scope of the invention and without undue experimentation. While the invention has been described with reference to specific embodiments, it will be appreciated that the invention can be further modified. In general, this application is intended to cover any variations, uses, or adaptations of the invention following, in general, the principles of the invention and including such departures from the present disclosure as come within known or customary practice within the art to which the invention pertains. The use of some of the essential features is possible within the scope of the claims attached below.
Sequence listing
<110> Ningxia medical university
<120> Agrobacterium tumefaciens-mediated phellinus igniarius genetic transformation method
<160> 1
<170> SIPOSequenceListing 1.0
<210> 1
<211> 1406
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 1
tgatattgaa ggagcatttt ttgggcttgg ctggagctag tggaggtcaa caatgaatgc 60
ctattttggt ttagtcgtcc aggcggtgag cacaaaattt gtgtcgtttg acaagatggt 120
tcatttaggc aactggtcag atcagcccca cttgtagcag tagcggcggc gctcgaagtg 180
tgactcttat tagcagacag gaacgaggac attattatca tctgctgctt ggtgcacgat 240
aacttggtgc gtttgtcaag caaggtaagt ggacgacccg gtcatacctt cttaagttcg 300
cccttcctcc ctttatttca gattcaatct gacttaccta ttctacccaa gcatccaaat 360
gaaaaagcct gaactcaccg cgacgtctgt cgagaagttt ctgatcgaaa agttcgacag 420
cgtctccgac ctgatgcagc tctcggaggg cgaagaatct cgtgctttca gcttcgatgt 480
aggagggcgt ggatatgtcc tgcgggtaaa tagctgcgcc gatggtttct acaaagatcg 540
ttatgtttat cggcactttg catcggccgc gctcccgatt ccggaagtgc ttgacattgg 600
ggagttcagc gagagcctga cctattgcat ctcccgccgt gcacagggtg tcacgttgca 660
agacctgcct gaaaccgaac tgcccgctgt tctccagccg gtcgcggagg ccatggatgc 720
gatcgctgcg gccgatctta gccagacgag cgggttcggc ccattcggac cgcaaggaat 780
cggtcaatac actacatggc gtgatttcat atgcgcgatt gctgatcccc atgtgtatca 840
ctggcaaact gtgatggacg acaccgtcag tgcgtccgtc gcgcaggctc tcgatgagct 900
gatgctttgg gccgaggact gccccgaagt ccggcacctc gtgcatgcgg atttcggctc 960
caacaatgtc ctgacggaca atggccgcat aacagcggtc attgactgga gcgaggcgat 1020
gttcggggat tcccaatacg aggtcgccaa catcctcttc tggaggccgt ggttggcttg 1080
tatggagcag cagacgcgct acttcgagcg gaggcatccg gagcttgcag gatcgccgcg 1140
cctccgggcg tatatgctcc gcattggtct tgaccaactc tatcagagct tggttgacgg 1200
caatttcgat gatgcagctt gggcgcaggg tcgatgcgac gcaatcgtcc gatccggagc 1260
cgggactgtc gggcgtacac aaatcgcccg cagaagcgcg gccgtctgga ccgatggctg 1320
tgtagaagta ctcgccgata gtggaaaccg acgccccagc actcgtccga gggcaaagga 1380
atagagtaga tgccgaccgg gaacca 1406
Claims (9)
1. An agrobacterium tumefaciens-mediated phellinus igniarius genetic transformation method is characterized by comprising the following steps of:
1) carrying out enzymolysis and purification on the phellinus igniarius hypha by a muramidase solution to obtain phellinus igniarius protoplast,
2) carrying out genetic transformation treatment on the phellinus igniarius protoplast obtained in the step 1) by using recombinant agrobacterium tumefaciens containing exogenous genes,
3) and performing regeneration culture and resistance screening culture on the treated phellinus igniarius protoplast to obtain the phellinus igniarius transformant integrated with the exogenous gene.
2. The agrobacterium tumefaciens-mediated phellinus igniarius genetic transformation method according to claim 1, wherein in the step 1), the addition amount of the lywallzyme solution is 2-10 mL per gram of wet hypha; the enzymolysis condition is enzymolysis for 1.5 to 3 hours at the temperature of 30 to 35 ℃; and the purification method in the step 1 comprises the steps of filtering the enzymolysis liquid by using a G3 sand core funnel, centrifuging the filtrate to remove supernatant, and washing for 1-3 times by using 0.5-0.8 mol/L osmotic pressure stabilizer to obtain the purified phellinus igniarius protoplast.
3. The method for genetic transformation of Phellinus igniarius mediated by Agrobacterium tumefaciens according to claim 1, wherein the recombinant Agrobacterium tumefaciens containing the foreign gene in step 2) is an Agrobacterium AGL-1-pCH-hph obtained by introducing a recombinant vector pCH-hph into Agrobacterium AGL-1 to obtain a hygromycin screening marker expressed in fungi, wherein the recombinant vector pCH-hph is a recombinant vector obtained by replacing the sequences at the EcoRI and SacI sites of the vector pDHt2 with the nucleotide sequences shown in sequence 1 and leaving the other sequences unchanged.
4. The method for Agrobacterium tumefaciens-mediated genetic transformation of Phellinus igniarius according to claim 3, wherein the method for constructing Agrobacterium AGL-1-pCH-hph in step 2) comprises:
s1) inoculating the bacterial liquid of the agrobacterium AGL-1 into an LB liquid culture medium containing rifampicin for overnight culture;
s2) inoculating the culture solution obtained in the step S1) into LB liquid culture medium of rifampicin, and culturing to OD600Reaching 0.5, centrifuging, adding CaCl2Suspending the thalli in the solution to obtain agrobacterium-mediated cells;
s3) taking the competent cells obtained in the step S2), adding a recombinant vector pCH-hph, uniformly mixing, cooling, then thermally shocking, adding an LB liquid culture medium without antibiotics for culture, coating the mixture on a solid LB culture medium plate containing kanamycin, and screening resistant colonies to obtain an AGL-1 monoclonal containing pCH plasmids;
s4) inoculating single AGL-1 clone containing pCH plasmid at 1% inoculation amount in minimal medium containing rifampicin and kanamycin, culturing overnight, and diluting to OD with liquid induction medium600Reaching 0.15 to obtain the agrobacterium AGL-1-pCH-hph.
5. The Agrobacterium tumefaciens-mediated genetic transformation method of Phellinus igniarius according to claim 4, wherein said LB liquid medium containing rifampicin is added to LB liquid medium so that the concentration of rifampicin in the mixture is 50 mg/L; the preparation method of the LB liquid culture medium comprises the steps of adding 10g of tryptone, 5g of yeast extract, 10g of sodium chloride and 1mL of 1M NaOH into 950mL of deionized water, fixing the volume to 1L, and adjusting the pH value to 7.2 to obtain the LB liquid culture medium.
6. The method for Agrobacterium tumefaciens-mediated genetic transformation of Phellinus igniarius according to claim 4, wherein the transformation treatment in step 2) is performed by: uniformly mixing agrobacterium AGL-1-pCH-hph and purified phellinus igniarius protoplast, and coating the mixture on a solid induction culture medium; culturing for 2-3 days at 20-30 ℃, transferring to a resistance screening culture medium containing hygromycin, cefamycin, streptomycin and tetracycline, and continuously culturing for 8-12 days to obtain the phellinus igniarius transformant.
7. The agrobacterium tumefaciens-mediated phellinus igniarius genetic transformation method according to claim 6, wherein the selective culture medium containing hygromycin, cefamycin, streptomycin and tetracycline is a mixed solution obtained by adding hygromycin, cefamycin, streptomycin and tetracycline to the selective culture medium, and the concentration of hygromycin in the mixed solution is 30 mg/L; the concentration of the cefuroxime is 200 mu M; the concentration of streptomycin is 100 mg/L; the concentration of tetracycline is 50 mg/L; the resistance screening culture medium is a PDA culture medium, and the preparation method of the PDA culture medium comprises the steps of adding water into 200g of peeled potatoes, 20g of glucose and 15g of agar to a constant volume of 1L; .
8. A biomaterial associated with genetic transformation of Phellinus igniarius, which is the recombinant vector pCH-hph of claim 3, or the Agrobacterium AGL-1-pCH-hph of claim 3.
9. Use of the biological material related to the genetic transformation of phellinus igniarius according to claim 8 for the genetic transformation of phellinus igniarius.
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| CN1342752A (en) * | 2001-10-11 | 2002-04-03 | 李宝健 | Process for transforming ganoderma by agrobacterium to mediate foreign gene |
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| CN111909953A (en) * | 2020-06-05 | 2020-11-10 | 东北林业大学 | Recombinant vector for genetic expression of phellinus igniarius, construction method and genetic transformation method |
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| CN102492715A (en) * | 2011-12-12 | 2012-06-13 | 河南省农业科学院 | Genetic transformation method for agrobacterium tumefacien-mediated fusarium oxysporum sesame specialized strain |
| CN111909953A (en) * | 2020-06-05 | 2020-11-10 | 东北林业大学 | Recombinant vector for genetic expression of phellinus igniarius, construction method and genetic transformation method |
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