CN105296534A - Method of establishing Lycium Ruthenicum genetic transformation system and application of method - Google Patents
Method of establishing Lycium Ruthenicum genetic transformation system and application of method Download PDFInfo
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Abstract
The invention discloses a method of establishing a Lycium Ruthenicum genetic transformation system and application of the method. The method comprises following steps: (1), introducing plasmids containing target DNA into Agrobacterium to obtain recombinant Agrobacterium; (2), culturing the recombinant Agrobacterium for the transformation of impregnating liquid; (3), soaking Lycium Ruthenicum explants by using the impregnating liquid; (4), co-culturing the explants in a co-culture medium; (5), transferring the co-cultured explants to a callus induction and screening medium for culturing, and removing the Agrobacterium; (6), inducing resistant calli by using kanamycin as screening pressure; (7), transferring the resistant calli into a differentiation medium for culturing to obtain cluster buds; (8), inducing rooting to obtain resistant regenerated seedlings of Lycium Ruthenicum, and determining the resistant seedlings as positive transgenic seedlings by PCR (polymerase chain reaction) identification. The genetic transformation method of Lycium Ruthenicum is simple to operate and feasible and gives high survival rate of calli.
Description
Technical field
The present invention relates to the genetic transforming method of biological field, the method for particularly black fruit lyceum genetic transformation foundation and application thereof.
Background technology
Black fruit lyceum (LyciumruthenicumMurr.) is the barbed wild bush of one of Solanaceae Lycium, plant height is 20 ~ 50cm about, mainly be distributed in the Desert Area such as North of Shanxi, Ningxia, Gansu, Qinghai, Xinjiang, Tibet, to grow thickly in the form of sheets distribution, to salinization soil, there is very strong adaptive faculty.Research at present for black fruit lyceum mainly concentrates on nutrient constituents of fruit, medicinal ingredients, pigment and polysaccharide extracting process and pharmacology analysis aspect, yet there are no the report about black fruit lyceum Establishment of Agrobacterium-Mediated Transformation System.
Summary of the invention
The object of this invention is to provide the method for black fruit lyceum Establishment of Agrobacterium-Mediated Transformation System.
The method that black fruit lyceum genetic transformation provided by the present invention is set up, comprises the steps:
(1) plasmid containing target DNA is imported Agrobacterium and obtain recombinational agrobacterium; By described recombinational agrobacterium in the LB liquid nutrient medium containing kantlex (Kanamycin, Kan) in 28 DEG C of shaking culture 24-36 hour;
(2) get the bacterium liquid of described step (1) in the LB liquid nutrient medium containing kantlex, 28 DEG C of shaking culture are to OD
600value is 0.5-0.7, collected by centrifugation thalline;
(3) with the thalline that the resuspended described step (2) of LB liquid nutrient medium is collected, 28 DEG C are continued shaking culture to bacterium liquid OD
600value is respectively 0.5,0.6 and 0.7, as infecting liquid;
(4) steep black fruit lyceum explant 10-30 minute by the immersion that infects of described step (3), obtain the black fruit lyceum explant after infecting;
(5) described step (4) is obtained infect after black fruit lyceum explant to be positioned on Dual culture substratum Dual culture 2-4 days in dark, obtain coculture;
(6) coculture described step (5) obtained is with containing 500mg/L cephamycin (Cefotaxime, Cef) be transferred to after sterile water wash in callus induction containing 20mg/L kantlex and 300mg/L cephamycin and screening culture medium and cultivate 10-14 days, obtain the callus after removing Agrobacterium;
(7) callus after the removal Agrobacterium described step (6) obtained cultivates 2-3 week on the callus induction and screening culture medium of cephamycin descending concentrations, obtains resistant calli;
(8) resistant calli that described step (7) obtains is proceeded on selection and division culture medium and cultivate 6-8 week, obtain Multiple Buds;
(9) the Multiple Buds root induction on root induction substratum described step (8) obtained, namely obtains the resistance regrowth of black fruit lyceum.
In described step (1) containing the concentration of each composition in the LB liquid nutrient medium of kantlex be: kantlex 50mg/L, Tryptones 10g/L, yeast extract 5g/L and sodium-chlor 10g/L.
In described step (2) containing the concentration of each composition in the LB liquid nutrient medium of kantlex be: kantlex 50mg/L, Tryptones 10g/L, yeast extract 5g/L and sodium-chlor 10g/L.
The OD of bacterium liquid described in described step (3)
600value is 0.6.
In described step (4), described black fruit lyceum explant is black fruit lyceum blade; Described soak time is 20 minutes.
Described in described step (5), Dual culture substratum is: MS minimum medium, additional 30g/L sucrose, 6.0g/L agar powder and 0.2mg/L2,4-D.
Described in described step (6), callus induction and screening culture medium are: MS minimum medium, additional 30g/L sucrose, 6.0g/L agar powder, 0.2mg/L2,4-D, 20mg/L kantlex and 300mg/L cephamycin.
Described in described step (8), selection and division culture medium are: MS minimum medium, additional 30g/L sucrose, 6.0g/L agar powder, 2.0mg/L6-BA, 20mg/L kantlex and 150mg/L cephamycin.
Described in described step (9), root induction substratum is: MS minimum medium, additional 30g/L sucrose, 6.0g/L agar powder and 0.2mg/L α-naphthaleneacetic acid.
The described application of method in acquisition black fruit lyceum transgenic line of setting up black fruit lyceum genetic conversion system also belongs to protection scope of the present invention.
The genetic transforming method of black fruit lyceum provided by the present invention, operation is simple, and the callus survival rate obtained after conversion is high, and the genetically engineered research and the molecular breeding that can be black fruit lyceum provide material.
Accompanying drawing explanation
Fig. 1 is plasmid extraction detected result; Swimming lane 1-4: plasmid pET21b; Swimming lane M:100bpplusDNAladder.
Fig. 2 is NahG gene High fidelity PCR result; Swimming lane 1 and 2:NahG band; Swimming lane M:100bpDNAladder.
Gene and carrier double digestion for the purpose of Fig. 3; Swimming lane 1:NahG gene double digestion; Swimming lane 2: carrier pBI121 double digestion; Swimming lane M:100bpDNAladder.
Fig. 4 is the result utilizing M13F/R primer to carry out Trans1-T1 bacterium liquid PCR; Swimming lane 1 and 2:NahG band; Swimming lane M:DL1000DNAladder.
Fig. 5 is the GV3101 bacterium liquid PCR detected result carrying NahG; Swimming lane 1 and 2:NahG band; Swimming lane M:100bpplusDNAladder.
Fig. 6 is construction of eukaryotic expression vector schematic diagram.
Fig. 7 is black fruit lyceum blade Kan resistance test; Wherein, a:A in Fig. 7: contrast, B:50mg/LKan, C:100mg/LKan, D:200mg/LKan; B:A:10mg/LKan, B:20mg/LKan, C in Fig. 7: contrast, D:30mg/LKan, E:50mg/LKan.
Fig. 8 is the process that black fruit lyceum genetic transformation obtains resistance regrowth; A: explant infects the callus of rear generation, B: callus breaks up in screening culture medium, C: the resistance Multiple Buds of differentiation, D: the resistance regeneration plant of taking root.
Embodiment
Embodiment 1, set up black fruit lyceum genetic conversion system
One, the structure of carrier for expression of eukaryon
1, the acquisition of goal gene NahG
(1) plasmid extraction
E.coli bacterial strain DH5 α containing carrier PET21b-NahG is so kind as to give by Logistics College of Chinese People's Armed Police Force (Tianjin occupation and environmental hazard Fang Zhi key lab) teacher Zhao Huabing.NahG gene (AY20891ND09189257-90561) is from naphthalene degradation bacteria strains Pseudomonassp.ND6 (Zhaoetal.2005).DH5 α (pET21b-NahG) is seeded to LB liquid nutrient medium, 37 DEG C, 200rpm/min shaking culture is spent the night, and use the little extraction reagent kit of sky root plasmid (TIANprepMiniPlasmidKit) to extract plasmid, method is as follows:
1. add the balance liquid BL of 500 μ L to (adsorption column puts into collection tube) in adsorption column CP3, the centrifugal 1min of 12,000rpm, outwells the waste liquid in collection tube, is placed back in by adsorption column in collection tube, equilibrium adsorption post;
2. get the bacterium liquid of 1-5mL incubated overnight, add in centrifuge tube, use conventional desktop whizzer, the centrifugal 1min of 12,000rpm, absorbs supernatant as far as possible;
3. in the centrifuge tube leaving bacterial sediment, add 250 μ L solution P1 (adding RNaseA before use), the thorough suspended bacterial precipitation of turbula shaker;
4. in centrifuge tube, add 250 μ L solution P2, leniently spin upside down and make the abundant cracking of thalline for 6-8 time;
5. in centrifuge tube, add 350 μ L solution P3, leniently spin upside down 6-8 time immediately, fully mix, now will occur white flock precipitate, the centrifugal 10min of 12,000rpm;
6. the supernatant liquor pipettor that previous step is collected is transferred to (adsorption column puts into collection tube) in adsorption column CP3, the centrifugal 30-60s of 12,000rpm, outwells the waste liquid in collection tube, and adsorption column CP3 is put into collection tube;
7. in adsorption column CP3, add 600 μ L rinsing liquids PW (adding dehydrated alcohol before use), the centrifugal 30-60s of 12,000rpm, outwells the waste liquid in collection tube, and adsorption column CP3 is put into collection tube;
8. repetitive operation step 7;
9. adsorption column CP3 is put into collection tube, the centrifugal 2min of 12,000rpm, rinsing liquid remaining in adsorption column is removed.Adsorption column CP3 is uncapped, is placed in room temperature and places several minutes, thoroughly to dry rinsing liquid remaining in sorbing material;
10. adsorption column CP3 is placed in a clean centrifuge tube, the middle part to adsorption film drips 50-100 μ L elution buffer EB, and room temperature places the centrifugal 2min of 2min, 12,000rpm, is collected in centrifuge tube by plasmid solution.
The plasmid of extraction is carried out agarose gel electrophoresis detected result as Fig. 1.
(2) design of primers, synthesis and PCR method amplifying target genes NahG
With PrimerPremier5 software design primer, and add the restriction enzyme digestion sites corresponding to expression vector, synthesized by raw work (SangonBiotec), final primer sequence is as follows:
NG-F5’-CGC
GGATCCATGAAAAACAATAAACC-3’
NG-R5’-CCAATCTCGAGCCCTTGACGTAGCAC-3’
Wherein underscore part is BamHI restriction enzyme site, and italicized item is XhoI restriction enzyme site.With the plasmid vector pET21b extracted for template, carry out PCR with high-fidelity DNA polymerase Pyrobest, obtain goal gene NahG, 50 μ L reaction systems are as follows:
Pcr amplification program:
PCR primer agarose gel electrophoresis detected result is shown in Fig. 2.
Reclaim test kit with TransgenDNA glue and reclaim goal gene fragment, method is as follows:
1. cut the sepharose containing DNA fragmentation under ultraviolet lamp, put into 1.5mL centrifuge tube;
2. add the sol solutions GSB of 3 times of gel volumes, 55 DEG C of water-baths are until gel dissolves completely;
3. colloidal sol is transferred in DNA adsorption tube centrifugal column after being cooled to room temperature, leaves standstill 1min;
4.12,000rpm, centrifugal 30 – 60s; Abandoned stream fluid;
5. add 650 μ L rinsing liquid WB, 12,000rpm, centrifugal 30 – 60s, abandoned stream fluid;
6.12,000rpm, empty centrifugal 2min, remove residual WB;
7. adsorption column is uncapped in Bechtop 2min, WB is volatilized completely;
8. add 30-50 μ L elutriant TE, room temperature leaves standstill 1min; Adsorption column puts into new 1.5mL centrifuge tube;
9.12,000rpm, centrifugal 2min, be placed in-20 DEG C and save backup.
(3) PCR primer sequencing
Determine 3 positive monoclonals, its sequence is completely the same, has the difference of 2 bases with the NahG sequence delivered, i.e. 43 and 609 (identity=99.58%), can think that the gene obtained that increases is NahG.
(4) goal gene is cut with expression vector enzyme, reclaim and is connected
PBI121 plasmid and NahG (being connected to pEASY-T3 carrier) are carried out double digestion with restriction enzyme BamHI and XhoI respectively, and 20 μ L enzymes cut system:
Double digestion the results are shown in Figure 3.
Agarose gel electrophoresis is separated and cuts required DNA fragmentation, and reclaim test kit with DNA glue and reclaim product, method is the same.By recovery fragment T
4dNA ligase connects, 10 μ L linked systems:
3, product amplification and sequencing is connected
Utilize heat shock method will connect product conversion to 50 μ L competent escherichia coli cell Trans1-T1, coating kalamycin resistance Selective agar medium flat board (LB solid medium+50mg/LKan).The positive single bacterium colony of picking, is seeded to LB liquid nutrient medium, 37 DEG C, 200rpm/min shaking culture 14h, extracts plasmid and check order.
Bacterium liquid PCR method qualification positive colony, 15 μ L systems are as follows:
Pcr amplification program:
PCR primer is carried out agarose gel electrophoresis, and result is as Fig. 4.
4, heat shock method transformation Agrobacterium GV3101 competent cell
Adopt heat shock method that the connection product of goal gene and expression vector is proceeded to Agrobacterium GV3101 bacterial strain (purchased from ocean bio tech ltd of Beijing CHMC, catalog number WaryongGT707), method is as follows:
1) connection product is added 50 μ L competent cells, softly mix, ice bath 30min;
2) quick-frozen 1min in liquid nitrogen, 37 DEG C of water-bath 3min, add 500 μ L LB liquid medium, 28 DEG C of shaking culture 4h;
3) get 500 μ L bacterium liquid to coat on kalamycin resistance screening culture medium flat board, dry up, be inverted for 28 DEG C and cultivate 2-3 days;
4) PCR method is utilized to identify positive colony.
Bacterium liquid PCR method is the same, and agarose gel electrophoresis the results are shown in Figure 5.To positive colony order-checking, institute check order arrange consistent with former sequence.So far, the structure completing carrier for expression of eukaryon and the Agrobacterium of carrying goal gene (engineering bacteria) build, and Fig. 6 is shown in by carrier for expression of eukaryon schematic diagram.
Two, the genetic transformation of black fruit lyceum
1, black fruit lyceum is to the resistance test of kantlex
Black fruit lyceum seedling leaves is seeded on the MS substratum containing different concns kantlex, observes blade to the tolerance of kantlex.The concentration arranging kantlex is respectively: 0,50,100,150,200,250 and 300mg/L, and result: when kantlex concentration is greater than 200mg/L, namely explant occurs albinism in inoculation after 3 days, after 5 days, almost all albefaction is dead; When kantlex concentration is 50mg/L, explant does not have significant difference with contrasting; When kantlex concentration be 100 and 150mg/L time, explant obviously turns yellow after inoculation two weeks, half explant albefaction death after three weeks.The results are shown in Figure 7a.
Afterwards, implement black fruit lyceum explant further at callus inducing medium (MS+30g/L sucrose+6.0g/L agar powder+0.2mg/L2, kantlex resistance test 4-D), explant is seeded in kantlex concentration and is followed successively by 0,10, on the callus inducing medium of 20,30,50mg/L.Result shows, kantlex concentration be 0 control group explant inoculate 10 days after start to form callus; Kantlex concentration be 10 and the explant of 20mg/L start to occur callus when inoculation 15 days, and the former Callus formation rate is higher than the latter; Kantlex concentration is higher than after 30mg/L, and explant becomes dry, and Callus formation reduces, of poor quality, and formation time seriously postpones.Therefore this experimental selection 20mg/L kantlex is as screening pressure, the results are shown in Figure 7b.
2, test is infected
1) mono-clonal being the positive by PCR detection and sequence verification is inoculated in LB liquid nutrient medium (Tryptones 10g/L, yeast extract 5g/L, sodium-chlor 10g/L containing 50mg/L kantlex; PH7.0) in, 28 DEG C of shaking culture 24-36h;
2) the above-mentioned bacterium liquid of 1mL contains 50mg/L kantlex LB liquid nutrient medium (Tryptones 10g/L, yeast extract 5g/L, sodium-chlor 10g/L in 50mL is got; PH7.0), in, 28 DEG C of shaking culture are to OD
600value is 0.5-0.7, then 4,000rpm, 4 DEG C of centrifugal 10min, collects thalline;
3) with LB liquid nutrient medium (Tryptones 10g/L, yeast extract 5g/L, sodium-chlor 10g/L; PH7.0) resuspended thalline, 28 DEG C are continued shaking culture to OD
600value is respectively 0.5,0.6 and 0.7, as infecting liquid;
4) get the black fruit lyceum blade of seedling age 20-25 days and stem section as explant, infect immersion bubble explant different time with above-mentioned: 10,20 and 30min; Carry out same infecting experiment with the black fruit lyceum callus that early stage cultivates simultaneously;
5) with aseptic thieving paper, unnecessary bacterium liquid on the blade after infecting is blotted, then blade is positioned over Dual culture substratum MS
1on (MS+30g/L sucrose+6.0g/L agar powder+0.2mg/L2,4-D) in dark Dual culture appropriate time; Infect test and carry out orthogonal design with different explants type, bacterial concentration, time of infection and Dual culture time 4 factors, infect scheme (see table 1) to obtain the best.
Table 1. black fruit lyceum infects test orthogonal design
Note: G0 represents conversion empty carrier pBI121; GV represents conversion recombinant vectors pBI121-NahG
6) Dual culture terminates after rear use contains the sterile water wash of 500mg/L cephamycin, blade to be transferred to callus induction and screening culture medium MS
2(MS+30g/L sucrose+6.0g/L agar powder+0.2mg/L2,4-D+20mg/L kantlex+300mg/L cephamycin), cultivates 10-14 days, obtains the callus after removing Agrobacterium;
7) replaced medium after two weeks, is down to 200mg/L by cephamycin concentration in substratum, and within later every 10 days, change a subculture, cephamycin concentration is successively decreased successively;
8) after one month, the resistant calli induced is proceeded to selection and division culture medium MS
3(MS+30g/L sucrose+6.0g/L agar powder+2.0mg/L6-BA+20mg/L kantlex+150mg/L cephamycin) continues cultivate, within every 20 days, change a subculture;
9) after two months Multiple Buds at root induction substratum MS
4(MS+30g/L sucrose+6.0g/L agar powder+0.2mg/LNAA) upper root induction, namely obtains the resistance regrowth of black fruit lyceum.
10) resistance seedling PCR is accredited as positive transgenic seedling
With black fruit lyceum resistance regrowth genomic dna for template, carry out PCR with Kan resistant gene (npt II) primer of pBI121 carrier, whether be integrated into plant genome to detect pBI121 expression vector; Carry out PCR with the 35S promoter upstream primer on carrier/NahG downstream of gene primer (35SF/NGR), whether testing goal gene NahG is integrated into plant genome simultaneously.
Kan resistant gene (npt II) primer sequence:
Kan-F:5’-TATGACTGGGCACAACAGACAA
Kan-R:5’-AGCGGCGATACCGTAAAGCAC
Goal gene detects primer sequence:
35SF:5’-ACCCACAGATGGTTAGAGAGG
NGR:5’-CCAATCTCGAGCCCTTGACGTAGCAC
PCR system is identical with program with the system being connected product amplification and sequencing above with program.
Above two PCR reaction results show, NahG gene integration enters black fruit lyceum genome, and the resistance regrowth obtained is transgenic positive seedling.
Genetic transformation obtains the process of resistance regrowth as shown in Figure 8.
3. the determination of black fruit lyceum genetic conversion system method
From the result infecting experiment (table 1), for the genetic transformation of black fruit lyceum, carry out infecting its effect with blade as explant and be better than stem section, and it is the poorest directly to infect effect with callus, trace it to its cause may be after infecting callus absorption thalline too much, and not easily remove, when making Dual culture, callus is lethal by fouled by microzyme.Compared with bacterial concentration 0.5, time of infection 30min, the Dual culture experiment combination of 4 days are combined with bacterial concentration 0.6, time of infection 20min, the Dual culture experiment of 2 days, the two transformation efficiency does not have significant difference.Therefore, the operability of Considering experimental and time factor, we establish the preferred method of black fruit lyceum genetic conversion system, that is: take blade as explant, infect bacterium liquid OD
600be 0.6, bacterium liquid soak time is 20min, and thalline and explant Dual culture time are 2 days; Each period used medium type and composition be respectively:
Dual culture substratum MS
1: MS minimum medium, additional 30g/L sucrose, 6.0g/L agar powder and 0.2mg/L2,4-D.
Callus induction and Selective agar medium MS
2: MS minimum medium, additional 30g/L sucrose, 6.0g/L agar powder, 0.2mg/L2,4-D, 20mg/L kantlex and 300mg/L cephamycin.
Select and division culture medium MS
3: MS minimum medium, additional 30g/L sucrose, 6.0g/L agar powder, 2.0mg/L6-BA, 20mg/L kantlex and 150mg/L cephamycin.
Root induction substratum MS
4: MS minimum medium, additional 30g/L sucrose, 6.0g/L agar powder and 0.2mg/L α-naphthaleneacetic acid.
Claims (10)
1. set up a method for black fruit lyceum genetic conversion system, comprise the steps:
(1) plasmid containing target DNA is imported Agrobacterium and obtain recombinational agrobacterium; By described recombinational agrobacterium in the LB liquid nutrient medium containing kantlex in 28 DEG C of shaking culture 24-36 hour;
(2) get the bacterium liquid of described step (1) in the LB liquid nutrient medium containing kantlex, 28 DEG C of shaking culture to OD600 values are 0.5-0.7, collected by centrifugation thalline;
(3) with the thalline that the resuspended described step (2) of LB liquid nutrient medium is collected, 28 DEG C are continued shaking culture and are respectively 0.5,0.6 and 0.7, as infecting liquid to bacterium liquid OD600 value;
(4) steep black fruit lyceum explant 10-30 minute by the immersion that infects of described step (3), obtain the black fruit lyceum explant after infecting;
(5) described step (4) is obtained infect after black fruit lyceum explant to be positioned on Dual culture substratum Dual culture 2-4 days in dark, obtain coculture;
(6) coculture that described step (5) obtained cultivates 10-14 days with containing in the callus induction be transferred to after the sterile water wash of 500mg/L cephamycin containing 20mg/L kantlex and 300mg/L cephamycin and screening culture medium, obtains the callus after removing Agrobacterium;
(7) callus after the removal Agrobacterium described step (6) obtained cultivates 2-3 week on the callus induction and screening culture medium of cephamycin descending concentrations, obtains resistant calli;
(8) resistant calli that described step (7) obtains is proceeded on selection and division culture medium and cultivate 6-8 week, obtain Multiple Buds;
(9) the Multiple Buds root induction on root induction substratum described step (8) obtained, namely obtains the resistance regrowth of black fruit lyceum.
2. method according to claim 1, is characterized in that: in described step (1) containing the concentration of each composition in the LB liquid nutrient medium of kantlex be: kantlex 50mg/L, Tryptones 10g/L, yeast extract 5g/L and sodium-chlor 10g/L.
3. method according to claim 1 and 2, is characterized in that: in described step (2) containing the concentration of each composition in the LB liquid nutrient medium of kantlex be: kantlex 50mg/L, Tryptones 10g/L, yeast extract 5g/L and sodium-chlor 10g/L.
4., according to described method arbitrary in claim 1-3, it is characterized in that: described in described step (3), the OD600 value of bacterium liquid is 0.6.
5., according to described method arbitrary in claim 1-4, it is characterized in that: in described step (4), described black fruit lyceum explant is black fruit lyceum blade; Described soak time is 20 minutes.
6. according to the method described in claim 1-5, it is characterized in that: described in described step (5), Dual culture substratum is: MS minimum medium, additional 30g/L sucrose, 6.0g/L agar powder and 0.2mg/L2,4-D.
7. according to described method arbitrary in claim 1-6, it is characterized in that: described in described step (6), callus induction and screening culture medium are: MS minimum medium, additional 30g/L sucrose, 6.0g/L agar powder, 0.2mg/L2,4-D, 20mg/L kantlex and 300mg/L cephamycin.
8. according to described method arbitrary in claim 1-7, it is characterized in that: described in described step (8), selection and division culture medium are: MS minimum medium, additional 30g/L sucrose, 6.0g/L agar powder, 2.0mg/L6-BA, 20mg/L kantlex and 150mg/L cephamycin.
9. according to described method arbitrary in claim 1-8, it is characterized in that: described in described step (9), root induction substratum is: MS minimum medium, additional 30g/L sucrose, 6.0g/L agar powder and 0.2mg/L α-naphthaleneacetic acid.
10. in claim 1-9, the method for arbitrary described black fruit lyceum Establishment of Agrobacterium-Mediated Transformation System is obtaining the application in black fruit lyceum transgenic line.
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| CN108728483A (en) * | 2018-06-08 | 2018-11-02 | 石河子大学 | A kind of agriculture bacillus mediated efficient glycyrrhiza glabra genetic transforming method |
| CN112592925A (en) * | 2021-02-24 | 2021-04-02 | 沈阳农业大学 | Lycium ruthenicum stable genetic transformation system and application thereof |
| CN113265421A (en) * | 2021-05-11 | 2021-08-17 | 浙江农林大学 | Method for establishing agrobacterium-mediated shortstem ephedra stem callus transgenic system |
| CN113265421B (en) * | 2021-05-11 | 2022-06-21 | 浙江农林大学 | Method for establishing agrobacterium-mediated shortstem ephedra stem callus transgenic system |
| CN116606880A (en) * | 2023-05-25 | 2023-08-18 | 中国科学院华南植物园 | Method for producing anthocyanin by using lycium ruthenicum callus |
| CN116606880B (en) * | 2023-05-25 | 2023-12-08 | 中国科学院华南植物园 | Method for producing anthocyanin by using lycium ruthenicum callus |
| CN116904506A (en) * | 2023-08-31 | 2023-10-20 | 中国科学院华南植物园 | Lycium ruthenicum LrANT1 gene and application of coded protein thereof |
| CN116904506B (en) * | 2023-08-31 | 2023-12-12 | 中国科学院华南植物园 | Lycium ruthenicum LrANT1 gene and application of coded protein thereof |
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