CN113025498A - Method for co-culturing endophyte and dendrobium nobile protocorm with stable and high dendrobine yield - Google Patents
Method for co-culturing endophyte and dendrobium nobile protocorm with stable and high dendrobine yield Download PDFInfo
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Abstract
The invention discloses a method for co-culturing endophyte with dendrobium nobile lindl protocorm with high stable yield, wherein a plant bioreactor system of the dendrobium nobile lindl constructs a product with high dendrobium nobile lindl yield to replace the traditional agricultural production mode of dendrobium nobile lindl, and at present, no literature report on secondary metabolic compounds with important medicinal values such as dendrobium nobile lindl protocorm and endophyte thereof co-culturing the dendrobium nobile lindl protocorm and the endophyte thereof through a plant bioreactor has original innovation, the method and the system which are suitable for stably producing the alkaloids with high efficiency are screened through a new method such as a response surface method and the like by combining the endophyte, the nutrition condition and the growth condition which influence the dendrobium nobile lindl, the existing culture medium formula and the culture condition are optimized, and the yield of the dendrobium noblindl is improved.
Description
Technical Field
The invention relates to the technical field of co-culture of endophytes and dendrobium nobile protocorm, in particular to a co-culture method of endophytes and dendrobium nobile protocorm with stable and high dendrobine yield.
Background
The plant tissue culture comprises different methods such as single cell culture, embryo culture, root tip culture, stem tip culture, callus culture, cell suspension culture, anther culture, protoplast culture and the like, in the dendrobium tissue culture, most of seeds, stems and leaves of dendrobium plants induce tissue culture materials such as callus, protocorm, seedling and the like, wherein the protocorm has wide sources, can be induced from the seeds, stems, leaves, pseudocorm and other tissues, simultaneously has excellent dedifferentiation capacity, is only lower than the seeds in dedifferentiation capacity, is higher than the callus, the pseudocorm, the stems and the like, and is easy to change differentiation ways under the action of hormones.
At present, dendrobine is a secondary metabolite which can be independently synthesized by endophytes and plants of dendrobium nobile, and therefore, the metabolic pathway of dendrobine is supposed to be a junction point of two material metabolic pathways. However, how the synthetic routes of dendrobine in two materials are, how the coordination of the pathways is uniform, and how the mechanism is, the key to solve the problems lies in establishing a dendrobium nobile protocorm and endophyte co-culture system for high yield of dendrobine and analyzing the transcriptome of the system, so that a dendrobium nobile protocorm co-culture method for stably and high yield of dendrobine is provided.
Disclosure of Invention
The invention aims to overcome the defects of the prior art and provides a method for co-culturing endophytic fungi and protocorms to produce dendrobine with high yield.
In order to achieve the purpose, the invention provides the following technical scheme: the method for co-culturing endophyte and dendrobium nobile protocorm comprises the following steps:
s1: preparing dendrobium nobile protocorm, selecting dendrobium nobile capsules, establishing a seed induction protocorm and value-added culture system, drawing a protocorm growth curve on the basis, and identifying the content of dendrobine in the protocorm by using a GC/MS method;
s2: the preparation method of the dendrobium nobile protocorm with higher propagation coefficient has the advantages that the components of the culture medium, the types and the use amount of hormones are one of the main factors influencing the plant tissue culture, and the growth state is described and a growth curve is drawn;
s3: measuring the content of dendrobine in the dendrobium stem protocorm, taking the standard dendrobine as a reference, and making a standard curve;
s4: separating the dendrobium nobile endophytes and identifying the secondary metabolites thereof, streaking, sampling and smearing the purified strains, observing the forms under a microscope, and recording;
s5: identifying secondary metabolites of endophytes in the dendrobium nobile, transferring strains into a fungus liquid culture medium, culturing for 7-15d at 28 ℃, respectively extracting products from a strain sample and a culture solution, performing GC/MS analysis, performing comparative analysis on the products, the secondary metabolites of stem segments of the dendrobium nobile and a dendrobine standard product, and selecting strains containing dendrobine in the secondary metabolites as co-cultured candidate strains;
s6: establishing a co-culture system of the dendrobium nobile protocorms and endophytes, selecting protocorms which are in a rising period in a growth curve, transferring the protocorms into a plant bioreactor, inoculating the endophytes of the dendrobium nobile, adding a liquid culture medium, carrying out co-culture by adopting an intermittent immersion method at the temperature of 25 ℃, detecting the dry-wet weight change of the dendrobium nobile protocorms before and after the co-culture, and detecting the content of dendrobine in the protocorms;
s7: screening excellent strains, transferring the candidate strain obtained in the step S6 into a culture bottle added with dendrobium nobile protocorm, respectively recording the change of the dry weight and the wet weight of the protocorm after 7-15d of inoculation and the change of the content of dendrobine, and taking the strain with the highest dendrobine yield as a tested strain of a co-culture system;
s8: optimizing a co-culture system, and performing orthogonal experimental design and response surface method optimization on the co-culture system by taking the growth period, the relative inoculation amount, the liquid culture medium composition, the culture temperature and the like of protocorms as parameters and combining the yield of the dendrobine obtained by GC/MS detection;
s9: network drawing of endophyte and dendrobium nobile protocorm high-yield dendrobine, and performing transcriptome detection;
s10: transcriptomics analysis, namely comparing all assembled Unigenes with an Nr (non-pi redundant protein database), an Nt (non-redundant nucleic acid database), a Swissprot protein database, a COG database and a KEGG protein database of NCBI (national center of affairs) respectively, performing functional annotation and GO enrichment analysis on Unigenes, and simultaneously further supplementing information such as transcription shearing, monogenic variation and the like in data obtained by a third-generation transcriptome to perfect transcriptome data information;
s11: drawing a functional gene metabolism network for accumulation of dendrobine, relying on databases such as KEGG and the like, combining gene expression difference information disclosed by RNA-Seq, carrying out metabolic pathway analysis, adopting DAVIDDAVID biological function cluster analysis, integrating the functional analysis and signal transmission pathway analysis results, drawing a signal transmission network for high-yield dendrobine under the action of dendrobium nobile and endophyte, and carrying out derivation and verification on secondary metabolic pathway end products by combining identification protein and identification metabolite related information;
s12: and (3) key gene screening and preliminary verification, wherein genes related to dendrobine in a secondary metabolic network annotated by a transcriptome KEGG function are used as candidates, and Q-PCR verification is performed on genes which have high correlation with dendrobine content change in time nodes in metabolome detection and are used as target genes.
Preferably, the inducing and subculture material in the step S1 is 1/2MS +0.2mg/L NAA +30g/L sucrose +6g/L agar +30g/L potato, pH 5.8; 1/2MS +0.3mg/L NAA +1.5 mg/L6-BA 10% potato +30g/L sucrose +6g/L agar, pH 5.8.
Preferably, in the step S2, an orthogonal experiment and a response surface method are adopted to optimize the organic species and concentration, the carbon source species and concentration, and the hormone species and concentration in the cultivation of the dendrobium nobile protocorm.
Preferably, in step S3, 0.5g of dendrobium nobile lindl protocorm dry powder is taken, precisely weighed, placed in a 250mL round bottom flask, added with 20mL of HCl solution with pH 4, shaken for 30min, then added with ammonia water to adjust pH to 10, added with 50mL of chloroform, weighed, extracted under reflux for 2h, weighed, added with chloroform to complement weight loss, precisely taken with 10.0mL of filtrate, decompressed to recover solvent to dryness, added with methanol to dissolve, quantitatively transferred into a 2mL measuring flask, added with methanol to scale, shaken well, filtered through a 0.22 μm microfiltration membrane, and sealed for later use.
Preferably, the dendrobium nobile lindl is collected in the step S4, and roots, stems and leaves are taken to be 0.1% HgCl2Sterilizing for 5-10min, aseptically cutting into segments, transferring into PDA culture medium, culturing at 28 deg.C, transferring grown colony into PDA culture medium for 2-3 days, and further purifying and separating.
Preferably, in the step S8, different time points 0h,1h,2h,4h,8h,16h,24h and 48h of inoculation and points of obvious increase of dendrobine content curve obtained in excellent strain screening are respectively selected, and total RNA is extracted to analyze bioinformation related to dendrobine production and mine bioinformation resources.
Compared with the prior art, the invention has the beneficial effects that:
1. the dendrobium nobile lindl protocorm is characterized in that dendrobium nobile lindl protocorm cultivated by a subject group is combined with specific endophyte to produce dendrobine, and the advantage of a plant bioreactor on the proliferation of dendrobium nobile lindl protocorm is utilized, so that the dendrobium nobile lindl protocorm has positive significance for disclosing a high-efficiency accumulation mechanism of the dendrobine in the dendrobium nobile lindl, and simultaneously is a synthetic accumulation theory and experimental basis for constructing and directionally inducing secondary metabolites by using a dendrobium nobile lindl cell bioreactor;
2. the dendrobium nobile lindl protocorm plant bioreactor system constructs a product with high dendrobium nobile lindl yield so as to replace the traditional dendrobium nobile lindl agricultural production, and at present, no literature report on secondary metabolic compounds with important medicinal values such as dendrobium nobile lindl and the like which are efficiently produced by the dendrobium nobile lindl protocorm and endophyte thereof through the plant bioreactor co-culture exists, and the dendrobium nobile lindl protocorm plant bioreactor system has original innovation;
3. the invention combines the endophytes, the nutritional conditions and the growth conditions which influence the dendrobium nobile lindl, screens a method and a system which are suitable for producing the alkaloid efficiently and stably by a new method such as a response surface method, optimizes the formula and the culture conditions of the existing culture medium, adopts a liquid intermittent immersion culture reactor, and improves the cell proliferation efficiency of the dendrobium nobile lindl protocorm cultured by the plant bioreactor.
Drawings
FIG. 1 is a flow chart of the operation of the present invention;
FIG. 2 is a graph showing the effect of NaCl and KCl added in different amounts on the growth of tissue culture seedlings.
Detailed Description
The technical solutions in the embodiments of the present invention will be described clearly and completely with reference to the accompanying drawings in the embodiments of the present invention, and it is obvious that the described embodiments are only some embodiments of the present invention, not all embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
Example one
Referring to fig. 1-2, the present invention provides a technical solution: a co-culture method of endophytes with high dendrobine yield and dendrobium nobile protocorm comprises the following steps:
s1: preparing dendrobium nobile protocorm, selecting dendrobium nobile capsules, establishing a seed induction protocorm and value-added culture system, drawing a protocorm growth curve on the basis, and identifying the content of dendrobine in the protocorm by using a GC/MS method;
s2: the preparation method of the dendrobium nobile protocorm with higher propagation coefficient has the advantages that the components of the culture medium, the types and the use amount of hormones are one of the main factors influencing the plant tissue culture, and the growth state is described and a growth curve is drawn;
s3: measuring the content of dendrobine in the dendrobium stem protocorm, taking the standard dendrobine as a reference, and making a standard curve;
s4: separating the dendrobium nobile endophytes and identifying the secondary metabolites thereof, streaking, sampling and smearing the purified strains, observing the forms under a microscope, and recording;
s5: identifying secondary metabolites of endophytes in the dendrobium nobile, transferring strains into a fungus liquid culture medium, culturing for 7-15d at 28 ℃, respectively extracting products from a strain sample and a culture solution, performing GC/MS analysis, performing comparative analysis on the products, the secondary metabolites of stem segments of the dendrobium nobile and a dendrobine standard product, and selecting strains containing dendrobine in the secondary metabolites as co-cultured candidate strains;
s6: establishing a co-culture system of the dendrobium nobile protocorms and endophytes, selecting protocorms which are in a rising period in a growth curve, transferring the protocorms into a plant bioreactor, inoculating the endophytes of the dendrobium nobile, adding a liquid culture medium, carrying out co-culture by adopting an intermittent immersion method at the temperature of 25 ℃, detecting the dry-wet weight change of the dendrobium nobile protocorms before and after the co-culture, and detecting the content of dendrobine in the protocorms;
s7: screening excellent strains, transferring the candidate strain obtained in the step S6 into a culture bottle added with dendrobium nobile protocorm, respectively recording the change of the dry weight and the wet weight of the protocorm after 7-15d of inoculation and the change of the content of dendrobine, and taking the strain with the highest dendrobine yield as a tested strain of a co-culture system;
s8: optimizing a co-culture system, and performing orthogonal experimental design and response surface method optimization on the co-culture system by taking the growth period, the relative inoculation amount, the liquid culture medium composition, the culture temperature and the like of protocorms as parameters and combining the yield of the dendrobine obtained by GC/MS detection;
s9: network drawing of endophyte and dendrobium nobile protocorm high-yield dendrobine, and performing transcriptome detection;
s10: transcriptomics analysis, namely comparing all assembled Unigenes with an Nr (non-pi redundant protein database), an Nt (non-redundant nucleic acid database), a Swissprot protein database, a COG database and a KEGG protein database of NCBI (national center of affairs) respectively, performing functional annotation and GO enrichment analysis on Unigenes, and simultaneously further supplementing information such as transcription shearing, monogenic variation and the like in data obtained by a third-generation transcriptome to perfect transcriptome data information;
s11: drawing a functional gene metabolism network for accumulation of dendrobine, relying on databases such as KEGG and the like, combining gene expression difference information disclosed by RNA-Seq, carrying out metabolic pathway analysis, adopting DAVIDDAVID biological function cluster analysis, integrating the functional analysis and signal transmission pathway analysis results, drawing a signal transmission network for high-yield dendrobine under the action of dendrobium nobile and endophyte, and carrying out derivation and verification on secondary metabolic pathway end products by combining identification protein and identification metabolite related information;
s12: and (3) key gene screening and preliminary verification, wherein genes related to dendrobine in a secondary metabolic network annotated by a transcriptome KEGG function are used as candidates, and Q-PCR verification is performed on genes which have high correlation with dendrobine content change in time nodes in metabolome detection and are used as target genes.
In the step S1, the inducing and subculture materials are 1/2MS, 0.2mg/L NAA, 30g/L cane sugar, 6g/L agar and 30g/L potato, and the pH value is 5.8; 1/2MS +0.3mg/L NAA +1.5 mg/L6-BA 10% potato +30g/L sucrose +6g/L agar, pH5.8, in step S2, the organic species and concentration, the carbon source species and concentration, the hormone species and concentration in the cultivation of the dendrobium nobile original bulb are optimized by adopting an orthogonal experiment and response surface method, in step S3, 0.5g of dendrobium nobile original bulb dry powder is taken, precisely weighed, placed in a 250mL round bottom flask, 20mL of HCl solution with pH 4 is added, shaken for 30min, then ammonia water is added to adjust the pH to 10, 50mL of chloroform is added, weighed, reflux extraction is carried out for 2h, weighed, chloroform is used for complementing weight loss, 10.0mL of continuous filtrate is precisely weighed, solvent is recovered to be dry under reduced pressure, methanol is added for dissolution, quantitatively transferred into a 2mL measuring flask, methanol is added to scale, shaken uniformly, a 0.22 mu m microporous filter membrane is filtered, sealing, collecting Dendrobium nobile in step S4, collecting the root, stem and leaf in 0.1% HgCl2Sterilizing for 5-10min, aseptically cutting, transferring into PDA culture medium, purifying and separating at 28 deg.C for 2-3d, inoculating at different time points of 0h,1h,2h,4h,8h,16h,24h,48h and the point of obvious increase of dendrobine content curve obtained in excellent strain screening, and extracting total RNA to analyze the biological information related to dendrobine production and to mine biological information resource.
In the invention, the dendrobium nobile lindl is characterized in that dendrobium nobile lindl which is a rare traditional Chinese medicine with geographical advantages is adopted, specific endophytes of the dendrobium nobile lindl are combined to produce the dendrobine, and the yield of the dendrobium nobile lindl is updated by utilizing the plant bioreactor, so that the dendrobium nobile lindl has positive significance for disclosing the high-efficiency accumulation mechanism of the dendrobine in the dendrobium nobile lindl, and is also a synthetic accumulation theory and experimental basis for constructing and directionally inducing secondary metabolites by using a dendrobium nobile cell bioreactor.
In the invention, the agricultural production of dendrobium nobile lindl is replaced by the product for constructing the dendrobium nobile lindl protocorm plant bioreactor system, and at present, no literature report on the secondary metabolism compounds with important medicinal values such as dendrobium nobile lindl and the like which are efficiently produced by the dendrobium nobile lindl protocorm and endophyte thereof through the plant bioreactor co-culture exists, and the original innovation is realized.
In the invention, the three methods which influence the endophytes, the nutritional conditions and the growth conditions of the dendrobium nobile are combined, and a method and a system which are suitable for efficiently and stably producing the alkaloid are screened by a new method such as a response surface method and the like, so that the formula and the culture conditions of the existing culture medium are optimized, and the cell proliferation efficiency of the dendrobium nobile protocorm cultured by a solid culture and a plant bioreactor is improved.
Example two
Separation of dendrobium nobile endophytes and preliminary identification of fungus products:
sterilizing roots, stems and buds of dendrobium nobile lindl in red water of Guizhou, transferring the dendrobium nobile lindl into a PDA culture medium, culturing the dendrobium nobile lindl at 28 ℃ for 3-5 days, counting strain separation conditions, separating to obtain 98 strains of bacteria and 19 strains of fungi in total, purifying and culturing the grown bacteria, performing microscopic examination, recording, further performing expanded culture on the purified strains, collecting the strains after 7-15 days, extracting, filtering, concentrating and detecting by GC/MS (gas chromatography/mass spectrometry), and comparing the strains with a dendrobine standard product (T ═ 5.91min) to find that peaks which are the same as the peak emergence time of the dendrobine appear in 5 strains; the content of endophytic fungi isolate for producing dendrobine is 26.3%.
EXAMPLE III
Establishment and optimization of a dendrobium nobile protoplast culture system:
placing young leaves of dendrobium nobile lindl in a dark environment for low-temperature plasmolysis pretreatment, adding prepared enzyme liquid, preparing dendrobium nobile lindl protoplasts by an enzyme hydrolysis method, dropwise adding prepared cleaning liquid, cleaning and purifying, counting and detecting the activity of the protoplasts, and knowing that the three-dimensional response surface maps of S1 and S2, S1 and S3, S2 and S3 have the highest points according to contour lines and three-dimensional response surface maps which are drawn by Design-expert8.0.6 software and reflect the interaction relation among variables, which shows that the response values have the highest values. And obtaining the optimal combination condition for culturing the dendrobium nobile protoplast according to the analysis result of the mathematical model of the secondary regression: 2,4-D concentration 1.42mg/L, 6-BA concentration 1.02mg/L, NAA concentration 3.18mg/L, incubation time 19.44h, this time maximum viability model prediction value is 84.62%, this is because in a certain amount of culture medium, the incubation time is very big influence on protoplast viability, in addition, through the results of contour analysis, further discover, 2,4-D concentration and 6-BA concentration interact, 2,4-D concentration and NAA concentration interact, 6-BA concentration and NAA concentration interact has significant influence on the cultivation of Dendrobium nobile protoplast.
Example four
Establishment of the culture system of the protocorm of the golden hairpin: according to the early-stage experimental basis, the mature capsules of the dendrobium nobile are disinfected and sterilized, seeds in the mature capsules are used as experimental materials, 67-V, MS and 1/2MS are used as basic culture media, the proportion of hormones such as 6-BA and NAA is adjusted, substances such as activated carbon, potatoes, coconut juice and bananas are added timely to optimize the formula of the culture media, TDZ substances are added to maintain the genetic stability of seedlings, the optimal formula of the culture media suitable for researching three growth stages of seed germination, protocorm differentiation and rooting and seedling strengthening is screened out at present, a tissue culture rapid propagation technical system of excellent dendrobium nobile strains is constructed, meanwhile, high-quality protocorm materials are inoculated in a plant reactor, and a preliminary propagation system of the dendrobium nobile in the plant reactor is established.
EXAMPLE five
Creation of a hairpin ploidy material:
in the invention, endophytes of dendrobium nobile are separated, secondary metabolites of the endophytes are identified and verified, the optimization results of various factors of the response surface design in the protocorm proliferation research are verified, and a technical scheme for culturing the dendrobium nobile protocorms by using a novel plant bioreactor is established. Analyzing the characterization detection and the detection of total alkaloids of the dendrobium nobile after the co-culture of different dendrobium nobile endophytes and dendrobium nobile protocorm, screening endophyte strains promoting the accumulation of the dendrobium nobile, carrying out GC/MS detection on secondary metabolites after the co-culture of different endophytes and protocorm, and comparing and analyzing the types and the variation trends of the metabolites.
In the invention, a system for producing the dendrobine with a plant reactor is established by using different components of the preferable strain capable of promoting the dendrobium alkaloid in the dendrobium nobile protocorm to continuously and efficiently accumulate, the proliferation rate of the dendrobium nobile protocorm is influenced to be combined with a strain inducer, nutrient components of a culture medium, stress factors, hormones and the like accumulated by the dendrobine, and the production conditions of the plant bioreactor for efficiently accumulating the dendrobine in the dendrobium nobile protocorm are screened.
In the invention, under the treatment of endophytes, a system for efficiently accumulating the dendrobine is subjected to transcriptome detection and analysis, expression profiles and metabolic pathways of functional genes in the system are analyzed, a mechanism is correspondingly and preliminarily revealed with GC/MS results of metabolites, the production conditions of a plant bioreactor for efficiently accumulating the dendrobine are verified, the transcriptome results and key genes analyzed in the metabolic pathways are screened, and the change of the transcriptome results and the key steps of signal identification, signal transduction, functional gene expression and the like in the co-culture induction process are combined, so that data are provided for a mechanism for revealing the target medicinal components identified and accumulated by the endophytes and medicinal plants.
Although embodiments of the present invention have been shown and described, it will be appreciated by those skilled in the art that changes, modifications, substitutions and alterations can be made in these embodiments without departing from the principles and spirit of the invention, the scope of which is defined in the appended claims and their equivalents.
Claims (6)
1. A co-culture method of endophytes with high dendrobine yield and dendrobium nobile protocorm is characterized by comprising the following steps:
s1: preparing dendrobium nobile protocorm, selecting dendrobium nobile capsules, establishing a seed induction protocorm and value-added culture system, drawing a protocorm growth curve on the basis, and identifying the content of dendrobine in the protocorm by using a GC/MS method;
s2: the preparation method of the dendrobium nobile protocorm with higher propagation coefficient has the advantages that the components of the culture medium, the types and the use amount of hormones are one of the main factors influencing the plant tissue culture, and the growth state is described and a growth curve is drawn;
s3: measuring the content of dendrobine in the dendrobium stem protocorm, taking the standard dendrobine as a reference, and making a standard curve;
s4: separating the dendrobium nobile endophytes and identifying the secondary metabolites thereof, streaking, sampling and smearing the purified strains, observing the forms under a microscope, and recording;
s5: identifying secondary metabolites of endophytes in the dendrobium nobile, transferring strains into a fungus liquid culture medium, culturing for 7-15d at 28 ℃, respectively extracting products from a fungus sample and a culture solution, performing GC/MS analysis, performing comparative analysis on the products, the secondary metabolites of stem segments of the dendrobium nobile and a dendrobine standard product, and selecting strains containing dendrobine in the secondary metabolites as co-cultured candidate strains;
s6: establishing a co-culture system of the dendrobium nobile protocorms and endophytes, selecting protocorms which are in a rising period in a growth curve, transferring the protocorms into a plant bioreactor, inoculating the endophytes of the dendrobium nobile, adding a liquid culture medium, carrying out co-culture by adopting an intermittent immersion method at the temperature of 25 ℃, detecting the dry-wet weight change of the dendrobium nobile protocorms before and after the co-culture, and detecting the content of dendrobine in the protocorms;
s7: screening excellent strains, transferring the candidate strain obtained in the step S6 into a culture bottle added with dendrobium nobile protocorm, respectively recording the change of the dry weight and the wet weight of the protocorm after 7-15d of inoculation and the change of the content of dendrobine, and taking the strain with the highest dendrobine yield as a tested strain of a co-culture system;
s8: optimizing a co-culture system, and performing orthogonal experimental design and response surface method optimization on the co-culture system by taking the growth period, the relative inoculation amount, the liquid culture medium composition, the culture temperature and the like of protocorms as parameters and combining GC/MS (gas chromatography/mass spectrometry) detection to obtain the dendrobine yield;
s9: the endophyte and the dendrobium nobile protocorm high-yield dendrobine are drawn through a network and subjected to transcriptomics detection;
s10: transcriptomics analysis, namely comparing all assembled Unigenes with an Nr (non-pi redundant protein database), an Nt (non-redundant nucleic acid database), a Swissprot protein database, a COG database and a KEGG protein database of NCBI (national center of affairs) respectively, performing functional annotation and GO enrichment analysis on Unigenes, and simultaneously further supplementing information such as transcription shearing, monogenic variation and the like in the data obtained by the third-generation transcriptome to perfect transcriptome data information;
s11: drawing a functional gene metabolic network for accumulation of dendrobine, carrying out metabolic pathway analysis by using a database such as KEGG (Kegg-based genetic algorithm) and the like as a support and combining gene expression difference information disclosed by RNA-Seq, integrating functional analysis and signal transmission pathway analysis results by adopting DAVIDDAVID biological function cluster analysis, drawing a high-yield dendrobine signal transmission network for action of dendrobium nobile and endophyte, and deducing and verifying a secondary metabolic pathway final product by combining identification protein and identification metabolite related information;
s12: and (3) key gene screening and preliminary verification, namely performing Q-PCR verification on genes related to dendrobine in a secondary metabolic network annotated by the function of the transcriptome KEGG as a candidate, wherein the genes have high correlation with dendrobine content change in a time node in metabolome detection as target genes.
2. The co-culture method of the endophyte with the dendrobium nobile protocorm for stably and highly producing the dendrobine according to claim 1, which is characterized in that: the inducing and subculture material in the step S1 is 1/2MS +0.2mg/L NAA +30g/L sucrose +6g/L agar +30g/L potato, and the pH value is 5.8; 1/2MS +0.3mg/L NAA +1.5 mg/L6-BA 10% potato +30g/L sucrose +6g/L agar, pH 5.8.
3. The co-culture method of the endophyte with the dendrobium nobile protocorm for stably and highly producing the dendrobine according to claim 1, which is characterized in that: in the step S2, organic species and concentration, carbon source species and concentration, and hormone species and concentration in the cultivation of the dendrobium nobile protocorm are optimized by adopting an orthogonal experiment and a response surface method.
4. The co-culture method of the endophyte with the dendrobium nobile protocorm for stably and highly producing the dendrobine according to claim 1, which is characterized in that: taking 0.5g of dendrobium nobile protocorm dry powder in the step S3, precisely weighing, placing in a 250mL round-bottom flask, adding 20mL of HCl solution with pH being 4, shaking for 30min, then adding ammonia water to adjust pH to 10, adding 50mL of chloroform, weighing, carrying out reflux extraction for 2h, weighing, complementing weight loss with chloroform, precisely taking 10.0mL of subsequent filtrate, recovering solvent under reduced pressure to dryness, adding methanol to dissolve, quantitatively transferring into a 2mL measuring flask, adding methanol to scale, shaking uniformly, filtering with a 0.22 mu m microporous membrane, and sealing for later use.
5. The co-culture method of the endophyte with the dendrobium nobile protocorm for stably and highly producing the dendrobine according to claim 1, which is characterized in that: collecting Dendrobium nobile in step S4, and collecting root, stem and leaf in 0.1% HgCl2Sterilizing for 5-10min, aseptically cutting into segments, transferring into PDA culture medium, culturing at 25 deg.C, transferring grown colony into PDA culture medium for 2-3 days, and further purifying and separating.
6. The co-culture method of the endophyte with the dendrobium nobile protocorm for stably and highly producing the dendrobine according to claim 1, which is characterized in that: in the step S8, inoculating different time points of 0h,1h,2h,4h,8h,16h,24h and 48h and points with obviously increased dendrobine content curves obtained in excellent strain screening are respectively selected, and total RNA is extracted to analyze biological information related to dendrobine production and mine biological information resources.
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