CN109496865A - Improve the induced medium and method of macleaya cordata sanguinarine and chelerythrine alkali content - Google Patents
Improve the induced medium and method of macleaya cordata sanguinarine and chelerythrine alkali content Download PDFInfo
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- CN109496865A CN109496865A CN201811523946.0A CN201811523946A CN109496865A CN 109496865 A CN109496865 A CN 109496865A CN 201811523946 A CN201811523946 A CN 201811523946A CN 109496865 A CN109496865 A CN 109496865A
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01H—NEW PLANTS OR NON-TRANSGENIC PROCESSES FOR OBTAINING THEM; PLANT REPRODUCTION BY TISSUE CULTURE TECHNIQUES
- A01H4/00—Plant reproduction by tissue culture techniques ; Tissue culture techniques therefor
- A01H4/001—Culture apparatus for tissue culture
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01H—NEW PLANTS OR NON-TRANSGENIC PROCESSES FOR OBTAINING THEM; PLANT REPRODUCTION BY TISSUE CULTURE TECHNIQUES
- A01H4/00—Plant reproduction by tissue culture techniques ; Tissue culture techniques therefor
- A01H4/005—Methods for micropropagation; Vegetative plant propagation using cell or tissue culture techniques
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Abstract
The present invention discloses a kind of induced medium and method for improving macleaya cordata sanguinarine and chelerythrine alkali content, the induced medium is mainly made of MS culture medium and elicitor, the elicitor is one or both of methyl jasmonate, salicylic acid, and the concentration of the elicitor is 50~150 μm of ol/L.The method is by 16 hours optical cultures and 8 hours dark cultures, and intensity of illumination is 4500~9000Lx, and the sterile tissue-cultured seedling of culture 60 days is placed in above-mentioned induced medium, at 25 DEG C under 16 hour photoperiod 24~120h of induction culturing；Then quantitative analysis metabolite content and gene expression amount.The present invention by adding elicitor methyl jasmonate or salicylic acid in the medium, it can promote the expression of P6H gene and DBOX gene in macleaya cordata, and then promote the synthesis of sanguinarine and Chelerythrine, to improve the content of sanguinarine and Chelerythrine in macleaya cordata tissue-cultured seedling.
Description
Technical field
The present invention relates to a kind of induced mediums and method for improving macleaya cordata sanguinarine and chelerythrine alkali content.
Technical background
Macleaya cordata [Macleaya cordata (Willd)R.B] it is Papaveraceae Macleaya perennial plant, is a kind of heavy
The traditional medicinal plant wanted, active constituent are mainly isoquinoline alkaloid.Benzophenanthridine alkaloid (abbreviation BIAs) is a kind of
The alkaloid of wide variety, these compounds have extensive bioactivity, including sanguinarine, Chelerythrine, Biflorine,
Allocryptopine etc..Wherein sanguinarine has an extensive bioactivity, including powerful antitumor, antibacterial and anti-inflammatory etc..In addition, sanguinarine
With Chelerythrine as spontaneous growth promotor, the antibiotic growth promoter being widely used in substitution animal husbandry.2006
Year, antibiotic growth promoter is added in raising as European Union completely forbids, therefore the spontaneous growth promotor quilt of plant source
It is widely used as the substitute of antibiotic in husbandry sector.Its market increases year by year.Currently, the product containing sanguinarine is in more than 70 a states
Family and area sale extensively.In addition to this, sanguinarine or a kind of active drug of anti-schistosome.Currently, sanguinarine and it is white bend
The red alkali of dish is mainly extracted from macleaya cordata.However, macleaya cordata is a kind of wild resource and also not by domestication, can not also at present
Extensive artificial growth.Therefore, the mode for collecting resource now brings the increasing pressure to macleaya cordata resource, causes to provide
Source atrophy year by year.In addition, the content of sanguinarine influenced by genetic constitution and environment it is very big.Therefore, a kind of new stabilization is established
It is had a very important significance with the development of resources mode of sustainable development.
Plant Tissue Breeding has become the sustainable and effective alternative strategy of commercial scale secondary metabolite.It is medicinal
Plant Tissue Breeding has the advantage that (1) not to be influenced by environment or geographical conditions.It (2) can strict control production process and production
Quality.(3) shorten the growth cycle of plant.(4) it avoids occupying large amount of land resources.There are many Plant Secondary Materials generations at present
It thanks to product to produce by this kind of mode in the past few decades, such as alkannin, ginsenoside and taxol.In Plant Tissue Breeding
On the basis of, then pass through some elicitors and induce the secondary metabolites that can be further enhanced in plant.Such as, methyl jasmonate treatment
Enhance the content of camptothecine in Ophiorrhiza japonica.And salicylic acid can then increase the accumulation of secondary metabolites in Radix Salviae Miltiorrhizae cell.Example again
Such as: CN107360971A discloses a kind of method for promoting Siraitia grosvenorii SS gene expression, and Luohanguo With Plantlets of Tissue Culture is seeded in jasmonic
Methyl acetate concentrations are that 28~30d is cultivated in the induced medium of 260~345 μm of ol/L.It can induce the SS base in Luohanguo With Plantlets of Tissue Culture
Because of expression, and then promote the synthesis of momordica glycoside V.But there is presently no about elicitor in macleaya cordata tissue cultures
The report that BIAs has an impact.
Summary of the invention
The technical problem to be solved in the present invention is to provide a kind of raising macleaya cordata sanguinarine and chelerythrine alkali content to lure
Culture medium and method are led, the culture medium and method can promote (protopine -6- hydroxylase) gene of P6H in macleaya cordata and DBOX
The expression of (dihydrobenzo phenanthridines oxidizing ferment) gene, and then promote the synthesis of sanguinarine and Chelerythrine, and then improve rich fall
Return the content of sanguinarine and Chelerythrine and other benzophenanthridine alkaloids (abbreviation BIAs) in tissue-cultured seedling.
In order to solve the above-mentioned technical problem, the invention adopts the following technical scheme:
A kind of induced medium improving macleaya cordata sanguinarine and chelerythrine alkali content, the induced medium master are provided
It to be made of MS culture medium and elicitor, the elicitor is methyl jasmonate (Methyl Jasmonate, abbreviation MJ), bigcatkin willow
One or both of sour (Salicylic acid, abbreviation SA), the concentration of the elicitor are 50~150 μm of ol/L.
Further,
The concentration of the methyl jasmonate is preferably 100 μm of ol/L.
Further,
The salicylic concentration is preferably 100 μm of ol/L.
Macleaya cordata sanguinarine and chelerythrine alkali content are improved using above-mentioned induced medium the present invention also provides a kind of
Method specifically comprises the following steps:
(1) it cultivates sterile tissue-cultured seedling: choosing according to 16 hours optical cultures and 8 hours dark cultures, intensity of illumination is 4500~
The sterile tissue-cultured seedling of 9000Lx, culture 60 days are spare；
(2) the sterile tissue-cultured seedling that step (1) is cultivated is placed in induced medium, was lured under 16 hour photoperiod at 25 DEG C
Lead 24~120h of cultivation；
The induced medium is mainly made of MS culture medium and elicitor, and the elicitor is methyl jasmonate, bigcatkin willow
One or both of acid；Elicitor filtration sterilization, and it is dilute using dimethyl sulfoxide (Methyl sulfoxide, abbreviation DMSO)
Elicitor is released, the concentration of elicitor is 50~150 μm of ol/L in induced medium；
(3) metabolite in sterile tissue-cultured seedling of the extraction step (2) after induction culturing and quantitative analysis is carried out.
Further,
The concentration of methyl jasmonate described in step (2) is preferably 100 μm of ol/L.
Further,
Salicylic concentration described in step (2) is preferably 100 μm of ol/L.
Further,
Sterile tissue-cultured seedling of the step (2) after induction culturing should be placed in liquid nitrogen before extracting metabolite at once
In stored frozen at -70 DEG C, for use.
Further,
The method also includes the RNA of sterile tissue-cultured seedling of step (4) extraction step (2) after induction culturing, then
Synthesis cDNA simultaneously carries out PCR quantitative analysis.
Further,
Step (1) specifically: first use 75% alcohol to impregnate the macleaya cordata explant of picking, then carried out with 0.1% mercuric chloride
Sterilization processing is completed in disinfection treatment；In aseptic working platform, rowed dry on explant 2~3 wounds with sterile scalpel
Mouthful, being finally inoculated into MS solid medium, (MS directly bought in the market adds water to be adjusted to 4.42g/L, adds 30g/L sucrose, adjusts
PH=5.8 is saved, 0.3% plant gel is added and is tuned into solid medium) on, it is placed in 25 DEG C of dark culture casees and cultivates, wait grow more
Wound, then is placed in 25 DEG C, 16 hours optical cultures and grows sterile tissue-cultured seedling after dark culture in 8 hours.
Further,
Step (1) further includes causing callus by squamous subculture or plant regeneration or explant to the sterile tissue-cultured seedling of acquisition
Means expand it is numerous.
Further,
The macleaya cordata explant includes blade, stem section, in aseptic working platform, with sterile scissors by the blade after disinfection
It cuts to 5mm × 5mm size, stem section is mitered into the segment of 0.4~0.6cm of length, then do scuffing processing.
Beneficial effects of the present invention:
Induced medium provided by the invention, by adding elicitor methyl jasmonate (MJ) or salicylic acid in the medium
(SA), (protopine -6- hydroxylase) gene of P6H in macleaya cordata and DBOX (dihydrobenzo phenanthridines oxidizing ferment) gene can be promoted
Expression, and then promote sanguinarine and Chelerythrine synthesis, and then improve macleaya cordata tissue-cultured seedling in sanguinarine and greater celandine
The content of red alkali.
Through detecting, the expression quantity of P6H gene and DBOX gene is relatively untreated in the macleaya cordata tissue-cultured seedling after Fiber differentiation
9 times and 80 times can be respectively increased in group control；The content of sanguinarine and Chelerythrine can increase separately 10 times and 14
Times.
Detailed description of the invention
In order to more clearly explain the embodiment of the invention or the technical proposal in the existing technology, to embodiment or will show below
There is attached drawing needed in technical description to be briefly described, it should be apparent that, the accompanying drawings in the following description is only this
Some embodiments of invention for those of ordinary skill in the art without creative efforts, can be with
It obtains other drawings based on these drawings.
Fig. 1 is the route of synthesis of sanguinarine and Chelerythrine in macleaya cordata；
Fig. 2 is that induction culturing difference is cultivated the time, allocryptopine in the macleaya cordata tissue-cultured seedling of different elicitors and control group
Content balance figure；
Fig. 3 is that induction culturing difference cultivates time, the macleaya cordata tissue-cultured seedling Protopine of different elicitors and control group
Content balance figure；
Fig. 4 is that induction culturing difference is cultivated the time, and dihydro is white in the macleaya cordata tissue-cultured seedling of different elicitors and control group bends
The content balance figure of the red alkali of dish；
Fig. 5 is that induction culturing difference is cultivated the time, dihydro red root in the macleaya cordata tissue-cultured seedling of different elicitors and control group
The content balance figure of alkali；
Fig. 6 is that induction culturing difference is cultivated the time, chelerythrine in the macleaya cordata tissue-cultured seedling of different elicitors and control group
The content balance figure of alkali；
Fig. 7 is that induction culturing difference is cultivated the time, sanguinarine in the macleaya cordata tissue-cultured seedling of different elicitors and control group
Content balance figure；
Fig. 8 is that induction culturing difference is cultivated the time, P6H gene table in the macleaya cordata tissue-cultured seedling of different elicitors and control group
Up to amount comparison diagram；
Fig. 9 is that induction culturing difference is cultivated the time, DBOX gene in the macleaya cordata tissue-cultured seedling of different elicitors and control group
Expression quantity comparison diagram.
Specific embodiment
In order to preferably illustrate the content of the invention, below by specific embodiment to further verifying of the invention.It is special
Illustrate herein, embodiment is only that more directly description is of the invention, they are a part of the invention, cannot be to structure of the present invention
At any restrictions.
1, sterile tissue-cultured seedling is cultivated:
Based on the research discovery before applicant: experimental implementation process is operated using spray is more more convenient than blade, and spray
Group is easier, faster generates callus, also can faster grow and obtain whole plant.The present invention is using macleaya cordata stem section as nothing
The explant that bacterium tissue-cultured seedling is cultivated.
It first uses 75% alcohol to impregnate the macleaya cordata stem section of picking, then is carried out disinfection processing with 0.1% mercuric chloride, completion sterilizes
Sterilization treatment；In aseptic working platform, stem section is mitered into the segment of 0.4~0.6cm of length first, then uses sterile scalpel
Row dry 2~3 wounds on stem segment, and being finally inoculated into MS solid medium, (MS directly bought in the market adds water to be adjusted to
4.42g/L adds 30g/L sucrose, adjusts pH=5.8, and 0.3% plant gel is added and is tuned into solid medium) on, it is placed in 25 DEG C secretly
It is cultivated in incubator, callus to be grown, then is placed in 25 DEG C, intensity of illumination is 4500~9000Lx, 16 hours optical cultures and 8 hours
Sterile tissue-cultured seedling is grown after dark culture；Sterile tissue-cultured seedling causes the means of callus by squamous subculture or plant regeneration or explant
Expand numerous；The sterile tissue-cultured seedling cultivated 60 days is chosen, for use.
2, MS culture medium and induced medium are prepared
The MS culture medium powder that will be bought on the market, matches according to standard, and 4.42 grams of MS culture medium powders add 1L water to be tuned into
Then plus sucrose basal medium: 30g/L+ plant gel: 3g/L, adjusting pH value is 5.8 to get MS culture medium；
By elicitor methyl jasmonate (MJ), salicylic acid (SA) respectively using 0.22 μM of membrane filter (Millipore,
USA) filtration sterilization, and using dimethyl sulfoxide dilute induction, and be separately added into MS culture medium, it prepares contain 100 μ respectively
The MS culture medium of mol/L MJ and MS culture medium containing 100 μm of ol/L SA.And using no added MS culture medium as control
Group.
Elicitor MJ and SA are purchased from Sigma, USA.
3, induction culturing
Sterile tissue-cultured seedling is respectively placed in the MS culture medium containing 100 μm of ol/L MJ, the MS training containing 100 μm of ol/L SA
Support on base and no added MS culture medium, at 25 DEG C under 16 hour photoperiod induction culturing, and collect 0h respectively, for 24 hours,
The tissue-cultured seedling sample of 72h and 120h.
Sterile tissue-cultured seedling after induction culturing should be placed in liquid nitrogen at once at -70 DEG C before extracting metabolite
Stored frozen, for use.
4, respectively by the different culture medium of collection is cultivated and different time points (0h, for 24 hours, 72h and 120h) are collected freezing
Dry each tissue-cultured seedling sample (0.5mg) mixes with methanol (25mL).Then, by ultrasonic extraction sample 30 minutes, and with
14000rpm is centrifuged 15 minutes, and passes through 0.22mm membrane filter.Finally, solution passes through ultra- using BEH C18 column
1290 instrument of HPLC Agilent carries out chromatographic isolation.Autosampler temperature is set as 6 DEG C.UHPLC and QQQ mass spectrograph
(6460A, Agilent) coupling.Calibration curve, and the absolute quantitation for assessing target compound are generated using 5 points.
5, by the different culture medium of collection is cultivated and different time points (0h, for 24 hours, 72h and 120h) are collected each tissue-cultured seedling
Tissue (100mg) is inoculated with to extract for RNA.By ground in liquid nitrogen in a organized way, and using RNA extracts kit (TaKaRa,
MiniBEST Plant Genomic DNA, China) extract total serum IgE.Meanwhile using PrimeScriptTMRT Master
Mix (TaKaRa, China) synthesizes cDNA.It is listed in the table below shown in 1 by qPCR primer used in gene expression.Using ABI
7300 and SYBR Premix (Roche, Switzerland) carries out quantitative PCR (qPCR).In all applications, a pair of of 18S is used
Make house-keeping gene.
1 primer nucleic acid sequence of table
6, it statisticallys analyze
All experiments in triplicate and in different time points (0h, for 24 hours, 72h and 120h) record data；Use GraphPad
Prism software carries out one-way analysis of variance, then carries out average ratio using the significant sex differernce (HSD) of post-hoc tests Tukey
Compared with.
7, conclusion and analysis
(1) BAIs assay result and analysis in tissue-cultured seedling
The content (mg/g) of allocryptopine in the tissue-cultured seedling of 2 different culture medium different time points of table
| Time | 0h | 24h | 72h | 120h |
| MS culture medium | 0.3±0.06 | / | / | / |
| Containing 100 μm of ol/L MJ | / | 1.1±0.2 | 0.92±0.12 | 0.67±0.2 |
| Containing 100 μm of ol/L SA | / | 1.62±0.24 | 1.47±0.32 | 0.66±0.13 |
The content (mg/g) of the tissue-cultured seedling Protopine of 3 different culture medium different time points of table
| Time | 0h | 24h | 72h | 120h |
| MS culture medium | 1.57±0.2 | / | / | / |
| Containing 100 μm of ol/L MJ | / | 2.17±0.11 | 1.32±0.17 | 1.08±0.23 |
| Containing 100 μm of ol/L SA | / | 2.28±0.35 | 2.46±0.23 | 1.18±0.04 |
The content (mg/g) of dihydrochelerythrine in the tissue-cultured seedling of 4 different culture medium different time points of table
The content (mg/g) of dihydrosanguinarine in the tissue-cultured seedling of 5 different culture medium different time points of table
| Time | 0h | 24h | 72h | 120h |
| MS culture medium | 0.027±0.004 | / | / | / |
| Containing 100 μm of ol/L MJ | / | 0.03±0.006 | 0.025±0.003 | 0.027±0.001 |
| Containing 100 μm of ol/L SA | / | 0.016±0.003 | 0.039±0.006 | 0.027±0.004 |
The content (mg/g) of Chelerythrine in the tissue-cultured seedling of 6 different culture medium different time points of table
The content (mg/g) of sanguinarine in the tissue-cultured seedling of 7 different culture medium different time points of table
| Time | 0h | 24h | 72h | 120h |
| MS culture medium | 0.26±0.016 | / | / | / |
| Containing 100 μm of ol/L MJ | / | 0.57±0.091 | 1.71±0.57 | 2.54±0.51 |
| Containing 100 μm of ol/L SA | / | 0.41±0.18 | 0.44±0.1 | 0.19±0.04 |
Above-mentioned table 2~7 and Fig. 2~7 are as the result is shown: compared with untreated fish group, by MJ treated macleaya cordata tissue culture
The content of sanguinarine and Chelerythrine in seedling increases separately 10 times and 14 times, and its content reaches most for 120 hours in processing
High level, sanguinarine and Chelerythrine yield respectively reach 2.54 ± 0.51mg/g and 0.827 ± 0.104.The result shows that MJ pairs
The influence of red root alkali content is to increase over time in experimental period (within 120h)；Allocryptopine under MJ induction
Reached maximum value at 24 hours with Biflorine；MJ significant can increase (P < 0.05) dihydro chelerythrine at 72-hours post-treatment
The content of alkali and dihydrosanguinarine.
(2) gene expression results and analysis in tissue-cultured seedling
It was found from the route of synthesis of sanguinarine and Chelerythrine in macleaya cordata shown in FIG. 1: from Biflorine (PRO) to
Dihydrosanguinarine (DHSAN) arrives sanguinarine (SAN) again and is respectively necessary for by 6 hydroxylase of Biflorine (P6H) and dihydrobenzo
Phenanthridines oxidizing ferment (DBOX) catalysis；Dihydrochelerythrine (DHCHE) is generated again to generation chelerythrine from allocryptopine (ALL)
Alkali (CHE), it is also desirable to 6 hydroxylases (P6H) and dihydrobenzo phenanthridines oxidizing ferment (DBOX) catalysis.Therefore it can pass through detection
The expression of both genes further analyzes influence of the induction pattern to content.By analyzing P6H and DBOX gene
Gene expression dose, can further analyze effect of the elicitor MJ and SA to gene expression.Testing result such as the following table 8~9
Shown, Fig. 8~9 are respectively the expression quantity pair of P6H gene, DBOX gene in different time and the macleaya cordata tissue-cultured seedling of different disposal
Than figure.Compared with 0h group, (Tukey's is examined No. * expression significant changes, and * indicates that P < 0.05, * * indicate that P < 0.01, * * * indicate P
< 0.005, * * * * indicate P < 0.001).
The expression quantity of P6H gene in the tissue-cultured seedling of 8 different culture medium different time points of table
| Time | 0h | 24h | 72h | 120h |
| MS culture medium | 13.14±0.69 | / | / | / |
| Containing 100 μm of ol/L MJ | / | 2.94±0.113 | 4.53±0.472 | 24.97±4.64 |
| Containing 100 μm of ol/L SA | / | 1.01±0.024 | 35.98±3.64 | 127.29±5.19 |
The expression quantity of DBOX gene in the tissue-cultured seedling of 9 different culture medium different time points of table
| Time | 0h | 24h | 72h | 120h |
| MS culture medium | 1.32±0.1 | / | / | / |
| Containing 100 μm of ol/L MJ | / | 3.23±0.42 | 14.68±0.72 | 107.3±2.85 |
| Containing 100 μm of ol/L SA | / | 1.36±0.06 | 33±5.56 | 60.6±7.09 |
From known to Fig. 8~9, Ji Shangbiao 8~9: compared with untreated fish group, after elicitor SA processing, P6H base in tissue-cultured seedling
Because expression reached peak value at 24 hours and reduced at any time, and DBOX gene expression dose highest in 120h.Elicitor
After MJ processing, in tissue-cultured seedling P6H gene expression dose for 24 hours, 72h even there are also inhibiting effect, be just more than not locate by 120 hours
The level of reason group；And DBOX gene expression dose highest in 120h, it is 80 times of untreated fish group, is nearly 2 times of SA group.
To sum up, different elicitors has different influences to different enzyme genes, but final, passes through elicitor SA, MJ
Processing can improve the content of sanguinarine and Chelerythrine in macleaya cordata tissue-cultured seedling, can also improve other BAIs in various degree
Content.
The above is a specific embodiment of the invention, but any restrictions cannot be constituted to the present invention, therefore need special
It points out, it is all based on the present invention, it is made any modification and is all fallen within the scope of the present invention with improvement.
No.1~6 SEQ ID are respectively primer McP6H-QP-F, McP6H-QP-R, McDBOX-QP-F, McDBOX-QP-
R, the sequence of 18S-QP-F, 18S-QR-R.
SEQUENCE LISTING
<110>Hunan beauty is up to living resources limited liability company
<120>induced medium and method of macleaya cordata sanguinarine and chelerythrine alkali content are improved
<130> 20181210
<160> 6
<170> PatentIn version 3.5
<210> 1
<211> 20
<212> DNA
<213>McP6H-QP-F sequence
<400> 1
catcaaggac gttcgagcct 20
<210> 2
<211> 20
<212> DNA
<213>McP6H-QP-R sequence
<400> 2
ctcctcacca cgcacaatct 20
<210> 3
<211> 20
<212> DNA
<213>McDBOX-QP-F sequence
<400> 3
actgttgcca cggtcgatag 20
<210> 4
<211> 20
<212> DNA
<213>McDBOX-QP-R sequence
<400> 4
tggaggagct tgtcaacacc 20
<210> 5
<211> 20
<212> DNA
<213>18S-QP-F sequence
<400> 5
cttcgggatc ggagtaatga 20
<210> 6
<211> 20
<212> DNA
<213>18S-QR-R sequence
<400> 6
gcggagtcct agaagcaaca 20
Claims (9)
1. a kind of induced medium for improving macleaya cordata sanguinarine and chelerythrine alkali content, which is characterized in that the induction training
Feeding base is mainly made of MS culture medium and elicitor, and the elicitor is one or both of methyl jasmonate, salicylic acid, institute
The concentration for stating elicitor is 50~150 μm of ol/L.
2. the induced medium according to claim 1 for improving macleaya cordata sanguinarine and chelerythrine alkali content, feature
It is, the concentration of the methyl jasmonate is 100 μm of ol/L.
3. the induced medium according to claim 1 for improving macleaya cordata sanguinarine and chelerythrine alkali content, feature
It is, the salicylic concentration is 100 μm of ol/L.
4. a kind of method for improving macleaya cordata sanguinarine and chelerythrine alkali content, which is characterized in that specifically comprise the following steps:
(1) it cultivates sterile tissue-cultured seedling: choosing 16 hours optical cultures and 8 hours dark cultures, intensity of illumination is 4500~9000Lx, training
It is spare to support 60 days sterile tissue-cultured seedling；
(2) the sterile tissue-cultured seedling that step (1) is cultivated is placed in induced medium, induced training at 25 DEG C under 16 hour photoperiod
Educate 24~120h；
The induced medium is mainly made of MS culture medium and elicitor, and the elicitor is methyl jasmonate, in salicylic acid
One or two；Elicitor filtration sterilization, and it is sub using dimethyl sulfoxide dilute induction, and elicitor is dense in induced medium
Degree is 50~150 μm of ol/L；
(3) metabolite in sterile tissue-cultured seedling of the extraction step (2) after induction culturing and quantitative analysis is carried out.
5. the method according to claim 4 for improving macleaya cordata sanguinarine and chelerythrine alkali content, which is characterized in that
The concentration of methyl jasmonate described in step (2) is 100 μm of ol/L.
6. the method according to claim 4 for improving macleaya cordata sanguinarine and chelerythrine alkali content, which is characterized in that
Salicylic concentration described in step (2) is 100 μm of ol/L.
7. the method for raising macleaya cordata sanguinarine and chelerythrine alkali content, feature according to claim 4~6 exist
In,
Sterile tissue-cultured seedling of the step (2) after induction culturing should be placed at once -70 in liquid nitrogen before extracting metabolite
Stored frozen at DEG C, for use.
8. the method for raising macleaya cordata sanguinarine and chelerythrine alkali content, feature according to claim 4~6 exist
In,
The method also includes steps: then the RNA of sterile tissue-cultured seedling of (4) extraction step (2) after induction culturing is synthesized
CDNA simultaneously carries out PCR quantitative analysis.
9. the method for raising macleaya cordata sanguinarine and chelerythrine alkali content, feature according to claim 4~6 exist
In,
Step (1) specifically: first use 75% alcohol to impregnate the macleaya cordata explant of picking, then carried out disinfection with 0.1% mercuric chloride
Sterilization processing is completed in processing；In aseptic working platform, rowed dry on explant 2~3 wounds with sterile scalpel, most
After be inoculated on MS solid medium, be placed in 25 DEG C of dark culture casees and cultivate, callus to be grown, then be placed in 25 DEG C, 16 small time
It cultivates and grows sterile tissue-cultured seedling after dark culture in 8 hours.
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201811523946.0A CN109496865B (en) | 2018-12-13 | 2018-12-13 | Induction medium and method for improving content of macleaya cordata sanguinarine and chelerythrine |
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| CN201811523946.0A CN109496865B (en) | 2018-12-13 | 2018-12-13 | Induction medium and method for improving content of macleaya cordata sanguinarine and chelerythrine |
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| Publication Number | Publication Date |
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