CN116606880A - Method for producing anthocyanin by using lycium ruthenicum callus - Google Patents
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- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
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Abstract
The invention discloses a method for producing anthocyanin by using lycium ruthenicum callus, which is characterized by comprising the following steps: and (3) performing suspension culture on the lycium ruthenicum callus which is over-expressed with the LrAN2 gene for 20-22 days, collecting the lycium ruthenicum callus, and extracting anthocyanin. According to the invention, through over-expressing the LrAN2 gene in the tissue culture of lycium ruthenicum, a callus line OE-LrAN2 capable of synthesizing and accumulating a large amount of anthocyanin is screened out, so that the anthocyanin can be directly synthesized and accumulated on the cellular level; and further, by optimizing the culture mode and culture conditions of the calluses, the bidirectional multiplication of the biomass and anthocyanin yield of the lycium ruthenicum calluses is realized, more possibilities are created for the industrialized production of anthocyanin, the partial requirements of anthocyanin in the market can be met, and the ecological environment of the wild lycium ruthenicum medicinal plant resources and the production place is protected.
Description
Technical Field
The invention belongs to the technical field of biology, and particularly relates to a method for producing anthocyanin by using lycium ruthenicum callus.
Background
Lycium ruthenicum (Lycium ruthenicum Murr) fruits are rich in anthocyanin, mainly petuniin, with content as high as 2.5-18.8mg/g (DW) accounting for 53.4-66.2% of fruit anthocyanin, and small amount of delphinidin and malvidin (Tang et al Journal of Functional Foods,2017, 30:97-107). Anthocyanin is an active secondary metabolite, is safe and nontoxic, has various effects of improving eyesight and benefiting liver (Yao et al, chemistry & Biodiversity,2011, 8:976-1010), reducing blood sugar (Celli et al, encyclopedia of Food Chemistry,2019,3:218-223; jang et al, bioorganic Chemistry,2019, 87:803-809), resisting oxidation (Fan, food Research International,2018, 109:232-249), resisting tumors (Munagala et al, cancer Letters,2017, 393:94-102) and the like in human bodies, and is an important natural water-soluble plant pigment in the food, health-care product and cosmetic industries. Anthocyanin supply in the consumer market is not required, but due to long growth period of lycium ruthenicum, low fruit yield and high extraction cost, development and utilization of lycium ruthenicum anthocyanin are limited.
The anthocyanin is extracted from a plurality of raw materials with wide sources, but the plant growth period is longer, the anthocyanin is greatly influenced by environment and plant diseases and insect pests depending on natural conditions, and the extracted raw materials have complex components, high pigment purification difficulty and high cost. The secondary metabolite produced by plant cell culture has the advantages of short period, strong controllability, little influence by natural environment, easy extraction and purification of target substances, and the like, and becomes an important mode for anthocyanin production.
At present, the genetic transformation method of lycium ruthenicum has not been mature, the genetic transformation time is long, the structural genes or the regulatory factors for regulating and controlling the synthesis of anthocyanin by the calli are fewer, and no report on the use of the calli of lycium ruthenicum for producing anthocyanin exists.
Disclosure of Invention
Based on this, the present invention aims to provide a method for producing anthocyanin by using lycium ruthenicum callus.
The technical scheme for realizing the aim of the invention comprises the following steps.
In a first aspect of the present invention, there is provided a method for producing anthocyanin using lycium ruthenicum callus, comprising the steps of: and (3) performing suspension culture on the lycium ruthenicum callus which is over-expressed with the LrAN2 gene for 20-22 days, collecting the lycium ruthenicum callus, and extracting anthocyanin.
According to the invention, through over-expression of the LrAN2 gene in the tissue culture of lycium ruthenicum, a callus line OE-LrAN2 capable of synthesizing and accumulating a large amount of anthocyanin is obtained, and the direct synthesis and accumulation of anthocyanin from the cellular level are realized; and further, by optimizing the culture mode (comprising hormone level and elicitor) and culture conditions (inoculum size, liquid loading amount, shaking table speed and illumination time) of the callus, the bidirectional multiplication of the biomass of the callus of the lycium ruthenicum and the anthocyanin yield is realized, more possibilities are created for industrialized production of anthocyanin, part of the requirements of anthocyanin on the market can be met, and the wild lycium ruthenicum medicinal plant resource and the ecological environment of the production place are protected.
Drawings
FIG. 1 is a morphology of wild-type Lycium ruthenicum and OE-LrAN2 calli from example 1 according to the invention.
FIG. 2 is a comparison of biomass of OE-LrAN2 calli cultured with different hormone combinations according to example 2 of the present invention.
FIG. 3 shows comparison of anthocyanin content in OE-LrAN2 calli cultured with different inducers according to example 3 of the present invention.
FIG. 4 shows the effect of different inoculum sizes on the growth of OE-LrAN2 calli in example 3 according to the invention.
FIG. 5 shows the effect of different loading amounts on the growth of OE-LrAN2 calli in example 3 according to the invention.
FIG. 6 shows the effect of different shaking table rotational speeds on OE-LrAN2 callus growth in example 3 of the invention.
FIG. 7 shows the effect of different illumination conditions on the growth of OE-LrAN2 calli in example 3 according to the invention.
Detailed Description
The present invention will be described more fully hereinafter in order to facilitate an understanding of the present invention. This invention may be embodied in many different forms and is not limited to the embodiments described herein. Rather, these embodiments are provided so that this disclosure will be thorough and complete.
Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. The terminology used in the description of the invention herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention. The term "and/or" as used herein includes any and all combinations of one or more of the associated listed items.
The experimental procedures, which do not address the specific conditions in the examples below, are generally followed by conventional conditions, such as those described in Green and Sambrook et al, molecular cloning, an experimental guideline (Molecular Cloning: A Laboratory Manual, 2013), or by the manufacturer's recommendations. The various chemicals commonly used in the examples are commercially available.
In the invention, firstly, transcriptomic data of lycium ruthenicum fruits are analyzed, and key regulatory genes LrAN2 synthesized by anthocyanin of lycium ruthenicum are screened out from R2R3-MYB transcription factor families; GFP expression system is used to construct an over-expression plant binary expression vector, genetic transformation (agrobacterium-mediated method) is carried out on lycium ruthenicum explant (plant cells, plant tissues, plant organs and cluster buds obtained under aseptic culture) to obtain lycium ruthenicum callus which over-expresses LrAN2, the lycium ruthenicum callus is subjected to synthesis and accumulation of a large amount of anthocyanin from almost no anthocyanin, and a UV spectrophotometry screening method is used to produce lycium ruthenicum callus OE-LrAN2 with high anthocyanin content (the content of which can reach 249.19 +/-11.76 mg/g DW Petunidin-3-O-glucoside chloride); then optimizing hormone level (0.45-0.55 mg/L6-BA and 0.8-1.2 mg +.L2,4-D to obtain more efficient multiplication of OE-LrAN2 callus), adding elicitors (200 mM NaCl and/or 10mM NaHCO) 3 ) And optimizing culture conditions (culture mode, inoculation amount, liquid loading amount, rotation speed of a shaking table and illumination time, so that OE-LrAN2 callus grows stably and rapidly to adapt to wider culture conditions, and improving the anti-browning and anthocyanin synthesis capacities of OE-LrAN2 callus) and the like to improve the biomass and anthocyanin content of OE-LrAN2 callus. The specific experimental results are as follows:
1. adding 200mM NaCl or/and 10mM NaHCO into solid MS culture medium 3 The anthocyanin content of the OE-LrAN2 callus can be improved to 26.70 +/-0.81 mg/g FW to the maximum, and the anthocyanin content of the inducer is 1.55 times that of the inducer without addition.
2. The liquid MS culture medium is used for suspension culture, the culture density is high, the OE-LrAN2 suspension cells are stable in cell growth and free of damage in a culture period, can be subjected to infinite subculture (subculture once every half month and stable still after being continuously used for more than one year), and are suitable for large-scale reactor culture of plant cells to realize industrialization.
3. 0.5mg/L6-BA and 0.8mg/L2,4-D hormone, 10% of inoculation amount, 20mL/100mL of liquid loading amount, and continuous full illumination at the rotating speed of a shaking table of 90-100 rpm, and is most beneficial to improving the biomass and anthocyanin content of OE-LrAN2 callus. And culturing the OE-LrAN2 callus under optimal culture conditions, wherein the biomass of the OE-LrAN2 callus under suspension culture in a culture period is 1.45+/-0.02 g/21dDW after 21 days of culture, the biomass is 1.14 times that of the OE-LrAN2 callus before optimization, and the anthocyanin content is 256.66 +/-11.75 mg/g DW.
4. Compared with the black fruit medlar with long growth period and high fruit acquisition cost, the black fruit medlar OE-LrAN2 suspension cell is a customizable and sustainable substitute of the traditional anthocyanin production platform, widens the anthocyanin raw material source, has great potential in the anthocyanin industry and medical application, and realizes the production of high-yield anthocyanin in plant cell factories by suspension culture of OE-LrAN2 callus, and finally obtains a large amount of anthocyanin.
In some embodiments of the present invention, a method for producing anthocyanin using lycium ruthenicum callus is disclosed, comprising the following steps: and (3) performing suspension culture on the lycium ruthenicum callus which is over-expressed with the LrAN2 gene for 20-22 days, collecting the lycium ruthenicum callus, and extracting anthocyanin.
In some of these embodiments, the suspension culture medium is an MS medium containing 0.45mg/L to 0.55 mg/L6-BA and 0.8mg/L to 1.2 mg/L2, 4-D.
In some of these embodiments, the suspension culture medium is an MS medium containing 0.45mg/L to 0.55 mg/L6-BA and 0.75mg/L to 0.85 mg/L2, 4-D.
In some embodiments, the suspension medium further comprises 180 mM-220 mM NaCl and/or 8 mM-12 mM NaHCO 3 。
In some embodiments, the suspension culture is one in which the amount of Lycium ruthenicum callus that overexpresses the LrAN2 gene is 5% -20%.
In some embodiments, the suspension culture may be performed with an inoculum size of 10% to 15% for Lycium ruthenicum callus that overexpresses the LrAN2 gene.
In some embodiments, the suspension culture is performed with an inoculum size of 9% to 11% for Lycium ruthenicum callus that overexpresses the LrAN2 gene.
In some embodiments, the liquid loading amount in the suspension culture is 18 mL-22 mL/100mL.
In some embodiments, the shaker speed for the suspension culture is 85rpm to 105rpm.
In some embodiments, the shaker speed of the suspension culture is 90rpm to 100rpm.
In some of these embodiments, the light conditions of the suspension culture are 16h light/8 h darkness or full light.
In some embodiments, the light conditions of the suspension culture are full light.
In some embodiments, the method for preparing the lycium ruthenicum callus overexpressing the LrAN2 gene comprises the following steps:
(1) Taking lycium ruthenicum fruit cDNA as a template, taking SEQ ID NO.2 and SEQ ID NO.3 as primers, and amplifying to obtain a target gene LrAN2;
(2) The PCR product of the target gene LrAN2 is connected with a pSuper 1300:GFP vector to obtain a recombinant plasmid pSuper 1300:LrAN 2-GFP;
(3) The recombinant plasmid pSuper1300 is obtained by introducing LrAN2-GFP into aseptic explant of lycium ruthenicum, and culturing by co-culture medium, screening culture medium and callus induction culture medium to induce callus.
In the following examples, the materials used include the following:
and (3) dyeing liquid: 1M MgCl 2 (0.5mL)+0.5M MES(1.0mL，pH 5.6)+0.2M AS(50.0μL)+dH 2 O(48.5mL)。
Solid MS medium: 4.43g/L MS (Murashige & Skoog 15Basal Medium with Vitamins), 30.0g/L sucrose, 0.5g/L MES (morpholinoethanesulfonic acid) and 8.0g/L agar powder, and adjusting the pH to 5.8-6.0.
Liquid MS medium: 4.43g/LMS, 30.0g/L of cross and 0.5g/LMES, and adjusting the pH to 5.8-6.0.
Co-culture medium: solid MS medium+0.5 mg/L6-BA+0.4 mg/LNAA.
Screening the culture medium: solid MS medium +0.5 mg/L6-BA +0.4mg/LNAA +200mg/Lcef +5mg/L HygB.
Callus induction medium: solid MS medium +0.5 mg/L6-BA +1.0 mg/L2,4-D +200mg/L cef +5mg/L HygB.
Callus growth medium: solid MS medium+0.5 mg/L6-BA+1.0 mg/L2, 4-D.
1mg/mL of 6-BA mother liquor: weighing 1.0mg of 6-BA solid powder, dissolving a proper amount of concentrated hydrochloric acid, adding deionized water to a volume of 1.0ml, filtering by a 0.22 mu m filter, and sterilizing.
1mg/mLNAA mother liquor: weighing 1.0mg NAA solid powder, dissolving with appropriate amount of 95% alcohol, adding deionized water to volume to 1.0ml, filtering with 0.22 μm filter, and sterilizing.
200mg/mL Cef mother liquor: 200.0mg of Cef solid powder is weighed, deionized water is fixed to 1.0ml, and the solid powder is filtered and sterilized by a 0.22 mu m filter, thus obtaining the Cef solid powder.
5mg/mL HygB mother liquor: weighing 5.0mg of HygB solid powder, fixing the volume to 1.0ml with deionized water, and filtering and sterilizing by a 0.22 μm filter.
1mg/mL of 2,4-D mother liquor: weighing 1.0mg of 2,4-D solid powder, dissolving a proper amount of NaOH, adding deionized water to a volume of 1.0ml, filtering by a 0.22 mu m filter, and sterilizing.
1M NaCl mother liquor preparation: weighing 2.922g of NaCl solid powder, adding 50mL of deionized water for dissolution, and filtering and sterilizing by a 0.22 mu m filter.
1M NaHCO 3 Preparing mother solution: weighing NaHCO 3 4.200g of solid powder is added with 50mL of deionized water for dissolution, filtered and sterilized by a 0.22 mu m filter, and the product is obtained.
Anthocyanin extract: 3% concentrated hydrochloric acid methanol solution.
In the following examples, the total anthocyanin content of the calli was determined by ultraviolet spectrophotometry, as follows:
1. extraction of total anthocyanin from callus
(1) And (3) freeze-drying: and grinding the sample stored in the refrigerator at the temperature of minus 80 ℃ into powder by using an ultra-low temperature grinding instrument in liquid nitrogen, and freeze-drying the powder, wherein the powder is used immediately after freeze-drying or is stored in a constant-temperature drying oven at the temperature of 26 ℃ for standby.
(2) Weighing: weighing a proper amount of freeze-dried sample by a ten-thousandth balance, loading into a 2mL centrifuge tube, wrapping the centrifuge tube by tinfoil paper, and avoiding light.
(3) Extracting: adding 2mL anthocyanin extract, mixing, performing ultrasonic treatment at 4deg.C for 30min, performing ultrasonic treatment, centrifuging at 4deg.C for 5min, removing callus residues, and transferring supernatant to a new tin foil wrapped centrifuge tube.
2. Drawing anthocyanin standard curve
(1) Standard stock solution: 1mg of anthocyanin standard substance chlorinated petuniin-3-O-glucoside (petuniin-3-O-glucoside chloride, wuhan Zhongzhi Techno Co., ltd., 6988-81-4) was weighed, and 10mL of anthocyanin extract was added to prepare a standard stock solution of 100.00 g/mL.
(2) Standard stock solutions of 0.01, 0.05, 0.10, 0.20, 0.40, 0.60 and 0.80mL are respectively taken, 3% concentrated hydrochloric acid methanol solution is fixed to 1mL to prepare serial anthocyanin standard solutions with the concentrations of 1.00, 5.00, 10.00, 20.00, 40.00, 60.00 and 80.00g/mL respectively, the absorbance A of the maximum absorption wavelength max corresponding to each mass concentration is taken as an ordinate, the mass concentration of the corresponding standard substance is taken as an abscissa, a chlorinated petuniin-3-O-glucoside standard curve is drawn, and a linear regression equation (Liu Shuiying and the like, food science, 2014, 35:84-91) of the anthocyanin standard curve is obtained by fitting by a linear least square method.
3. Calculating determination and yield of lycium ruthenicum anthocyanin
Under the same condition as anthocyanin standard substance chlorinated petuniin-3-O-glucoside, determining light absorption value A of callus anthocyanin of sample to be detected 0 Substituting the anthocyanin concentration into a regression equation of a standard curve to obtain the mass concentration rho of the anthocyanin in the sample to be detected.
The yield (ω) of lycium ruthenicum anthocyanin was calculated using the following formula (Liu Shuiying et al, food science, 2014, 35:84-91):
wherein: omega is the yield (mug/g) of lycium ruthenicum anthocyanin; ρ is the mass concentration of lycium ruthenicum anthocyanin (μg/mL); n is dilution multiple; v is the volume (mL) of the anthocyanin diluent of the lycium ruthenicum; m is the mass (g) of lycium ruthenicum.
The invention is described in detail below with reference to the drawings and the specific embodiments.
Example 1 candidate Gene screening, construction of overexpression vector and screening of OE-LrAN2 callus capable of producing high-yield anthocyanin
1. Candidate Gene screening
The transcriptome data analysis and the evolutionary tree homology analysis show that the relative expression quantity of the lycium ruthenicum gene LrAN2 is positively correlated with the synthesis and accumulation of anthocyanin in the development process of lycium ruthenicum fruits, so that the LrAN2 (SEQ ID NO. 1) is selected as a candidate gene for genetic transformation.
LrAN2 gene (SEQ ID NO. 1):
ATGACTTTTAATAAAGTTGAGGAAGGAACAAGCCATACCCCCAACAAGCAGGCCAACACACTAAGTGTGACCCGTATCATGATGAATACTAGTGTTACTATTACTAAATCATCTGGAGTGAGGAAAGGTGCATGGACTGAAGAAGAAGATCATCTTTTGAGAAAATGCATTCAAAAGTACGGTGAAGGAAAATGGCATCAAGTTCCCATTAGAGCTGGTCTAAATAGATGCAGGAAGAGTTGTAGACTGAGGTGGCTGAATTATCTAAGGCCACATATAAAGAGAGGTGACTTCTCTTCTGAGGAAGTTGACCTTATCTTGAGGCTTCATAAGCTCTTAGGCAACAGATGGTCACTCATTGCGGGTAGACTTCCGGGAAGAACAGCAAACGATGTCAAAAACTACTGGAACACACACCTACAGAGGAAGTTAACTGCTCCTCATCAACAAGAGAGAAAGTACAATAATGCCCTCAAGATCACAGAAAACACCATACTAAGACCTCGACCTCGAACCTTCACATCAAGTAGTGCAAAGAATGTTTCTTTTTGCAGCAACAAAAGTATCACAAACACTGTAGATAAAAACGCACACAACAATGAAATACTAAATATTTGTGAGAAGCCAACAGGTGAAACGACGTCGGTAGACGAGGGAGTTCAATGGTGGACAAGTTTACTGGAAAATTGCAATGAAACTGAGGAAGAAGCAGAAGCATTTGGGAGCTTTGATGAAGAAAATATGTTACAAAGTTTGTTGCATGAGGAAATTTCACCACCCATGCAACAAGGACAAAGTGGTAATTGGGATGACTTTTCCGCTGATATTGACCTATGGAATCTACTGAATTAG
2. construction of overexpression vector
Designing an upstream primer LrAN2-F of a target gene LrAN2 by taking cDNA of lycium ruthenicum fruits as a template: 5'-TCCCCCGGGATGATGAATACTAGTGTTACTATTACTAAATC-3' (SEQ ID NO. 2) and LrAN2-R:5'-CGGGGTACCATTCAGTAGATTCCATAGGTCAATATCAG-3' (SEQ ID NO. 3), amplifying the target gene to obtain a PCR product containing SmaI and KpnI cleavage sites, double cleavage of the purified PCR amplification product with SmaI and KpnI, and ligation with a pSuper1300 (GFP vector (teaching and giving away from Chinese university of agriculture Yang Shuhua, which can be constructed by conventional methods in the art)) digested with the same enzyme under the action of a DNA ligation kit (NEB) to obtain a recombinant plasmid pSuper1300 (LrAN 2-GFP (SEQ ID NO. 4).
Recombinant plasmid pSuper 1300:LrAN 2-GFP (SEQ ID NO. 4):
GGATCCCTGAAAGCGACGTTGGATGTTAACATCTACAAATTGCCTTTTCTTATCGACCATGTACGTAAGCGCTTACGTTTTTGGTGGACCCTTGAGGAAACTGGTAGCTGTTGTGGGCCTGTGGTCTCAAGATGGATCATTAATTTCCACCTTCACCTACGATGGGGGGCATCGCACCGGTGAGTAATATTGTACGGCTAAGAGCGAATTTGGCCTGTAGGATCCCTGAAAGCGACGTTGTGTTAACATCTACAAATTGCCTTTTCTTATCGACCATGTACGTAAGCGCTTACGTTTTTGGTGGACCCTTGAGGAAACTGGTAGCTGTTGTGGGCCTGTGGTCTCAAGATGGATCATTAATTTCCACCTACGATGGGGGGCATCGCACCGGTGAGTAATATTGTACGGCTAAGAGCGAATTTGGCCTGTAGGATCCCTGAAAGCGACGTTGGATGTTAACATCTACAAATTGCCTTTTCTTATCGACCATGTACGTAAGCGCTTACGTTTTTGGTGGACCCTTGAGGAAACTGGTAGCTGTTGTGGGCCTGTGGTCTCAAGATGGATCATTAATTTCCACCTTCACCTACGATGGGGGGCATCGCACCGGTGAGTAATATTGTACGGCTAAGAGCGAATTTGGCCTGTAGGATCCGCGAGCTGCTCAATCCCATTGCTTTTGAAGCAGCTCAACATTGATCTCTTTCTCGATCGAGGGAGATTTTTCAAATCAGTGCGCAAGACGTGACGTAAGTATCCGAGTCAGTTTTTATTTTTCTACTAATTTGGTCGTTTATTTCGGCGTGTAGGACATGGCAACCGGGCCTGAATTTCGCGGGTATTCTGTTTCTATTCCAACTTTTTCTTGATCCGCAGCCATTAACGACTTTTGAATAGATACGCTGACACGCCAAGCCTCGCTAGTCAAAAGTGTACCAAACAACGCTTTACAGCAAGAACGGAATGCGCGTGACGCTCGCGGTGACGCCATTTCGCCTTTTCAGAAATGGATAAATAGCCTTGCTTCCTATTATATCTTCCCCCAAATTACCAATACATTACACTAGCATCTGAATTTCATAACCAATCTCGATACACCAAATCGACTCTAGAAAGCTTCTGCAGGGGCCCGGGATGATGAATACTAGTGTTACTATTACTAAATCATCTGGAGTGAGGAAAGGTGCATGGACTGAAGAAGAAGATCATCTTTTGAGAAAATGCATTCAAAAGTACGGTGAAGGAAAATGGCATCAAGTTCCCATTAGAGCTGGTCTAAATAGATGCAGGAAGAGTTGTAGACTGAGGTGGCTGAATTATCTAAGGCCACATATAAAGAGAGGTGACTTCTCTTCTGAGGAAGTTGACCTTATCTTGAGGCTTCATAAGCTCTTAGGCAACAGATGGTCACTCATTGCGGGTAGACTTCCGGGAAGAACAGCAAACGATGTCAAAAACTACTGGAACACACACCTACAGAGGAAGTTAACTGCTCCTCATCAACAAGAGAGAAAGTACAATAATGCCCTCAAGATCACAGAAAACACCATACTAAGACCTCGACCTCGAACCTTCACATCAAGTAGTGCAAAGAATGTTTCTTTTTGCAGCAACAAAAGTATCACAAACACTGTAGATAAAAACGCACACAACAATGAAATACTAAATATTTGTGAGAAGCCAACAGGTGAAACGACGTCGGTAGACGAGGGAGTTCAATGGTGGACAAGTTTACTGGAAAATTGCAATGAAACTGAGGAAGAAGCAGAAGCATTTGGGAGCTTTGATGAAGAAAATATGTTACAAAGTTTGTTGCATGAGGAAATTTCACCACCCATGCAACAAGGACAAAGTGGTAATTGGGATGACTTTTCCGCTGATATTGACCTATGGAATCTACTGAATGGTACCATGAGCAAGGGCGAGGAGCTGTTCACCGGGGTGGTGCCCATCCTGGTCGAGCTGGACGGCGACGTAAACGGCCACAAGTTCAGCGTGTCCGGCGAGGGCGAGGGCGATGCCACCTACGGCAAGCTGACCCTGAAGTTCATCTGCACCACCGGCAAGCTGCCCGTGCCCTGGCCCACCCTCGTGACCACCCTGACCTACGGCGTGCAGTGCTTCAGCCGCTACCCCGACCACATGAAGCAGCACGACTTCTTCAAGTCCGCCATGCCCGAAGGCTACGTCCAGGAGCGCACCATCTTCTTCAAGGACGACGGCAACTACAAGACCCGCGCCGAGGTGAAGTTCGAGGGCGACACCCTGGTGAACCGCATCGAGCTGAAGGGCATCGACTTCAAGGAGGACGGCAACATCCTGGGGCACAAGCTGGAGTACAACTACAACAGCCACAACGTCTATATCATGGCCGACAAGCAGAAGAACGGCATCAAGGTGAACTTCAAGATCCGCCACAACATCGAGGACGGCAGCGTGCAGCTCGCCGACCACTACCAGCAGAACACCCCCATCGGCGACGGCCCCGTGCTGCTGCCCGACAACCACTACCTGAGCACCCAGTCCGCCCTGAGCAAAGACCCCAACGAGAAGCGCGATCACATGGTCCTGCTGGAGTTCGTGACCGCCGCCGGGATCACTCTCGGCATGGACGAGCTGTACAAGTAAGAGCTCGAATTTCCCCGATCGTTCAAACATTTGGCAATAAAGTTTCTTAAGATTGAATCCTGTTGCCGGTCTTGCGATGATTATCATATAATTTCTGTTGAATTACGTTAAGCATGTAATAATTAACATGTAATGCATGACGTTATTTATGAGATGGGTTTTTATGATTAGAGTCCCGCAATTATACATtTAATACGCGATAGAAAACAA
3. genetic transformation of Lycium ruthenicum Murr, and screening of OE-LrAN2 callus capable of producing high-yield anthocyanin
The recombinant plasmid is introduced into a black matrimony vine aseptic plant body through an agrobacterium mediating method, and the black matrimony vine OE-LrAN2 callus which is over-expressed by LrAN2 is obtained through co-culture medium, screening culture medium and callus induction culture medium, wherein the OE-LrAN2 callus is black purple, has metallic luster and hard texture, cells are mostly spherical (figure 1), and the black matrimony vine OE-LrAN2 callus cultured on a solid MS culture medium is increased from almost no anthocyanin synthesis to synthesis and accumulation of a large amount of anthocyanin, and the content of the anthocyanin is increased from 7.51+/-0.08 mg/g DW to 249.19 +/-11.76 mg/g DW. The calli were subcultured for later study.
EXAMPLE 2 optimization of conditions for culture of OE-LrAN2 calli under solid culture
1. Hormone proportioning
Preparing a sterilized solid MS culture medium, preparing 0.3mg/L, 0.5mg/L and 0.7mg/L of 6-BA and 0.8mg/L, 1.0mg/L and 1.2mg/L of 2,4-D, respectively adding the two to the solid MS culture medium after combination, weighing 1.0g of OE-LrAN2 callus fresh sample, inoculating to the culture medium containing different hormone combinations for culture, repeating three biological steps, collecting the sample after 21 days of culture and weighing.
As a result, as shown in FIG. 2, it was found that the combination of 0.5 mg/L6-BA+0.8 mg/L2,4-D hormone was most suitable for the proliferation and growth of OE-LrAN2 callus, and the final biomass in the culture period was 7.79.+ -. 0.38g/21dFW. Therefore, the callus growth medium was adjusted to solid MS medium+0.5 mg/L6-BA+0.8 mg/L2,4-D (hereinafter referred to as optimization medium) for use in further optimization experiments.
2. Addition of an inducer
The lycium ruthenicum is mainly grown in a saline-alkali soil environment, and it is presumed that salt treatment, alkali treatment and saline-alkali treatment are beneficial to synthesis and accumulation of anthocyanin by lycium ruthenicum callus.
Using the Lycium ruthenicum OE-LrAN obtained in example 1 2 Callus was added with (1) 200mM NaCl based on the optimized medium; (2) 10mM NaHCO 3 And (3) 200mM NaCl+10mM NaHCO 3 As an inducer.
Weighing OE-LrAN 2 1.0g of fresh callus is inoculated into the three culture media, other culture conditions are the same as those in the step 1, the fresh OE-LrAN2 callus is collected after 14 days of culture, and the change condition of anthocyanin content of the callus after the culture of a control group and the three culture media is measured by an ultraviolet spectrophotometry, and the result is shown in a figure 3.
As can be seen from FIG. 3, the control OE-LrAN2 callus anthocyanin content was 17.25.+ -. 0.29mg/g FW, and (1) 200mM NaCl was added; (2) 10mM NaHCO 3 And (3) 200mM NaCl+10mM NaHCO 3 After the elicitor, the content of the OE-LrAN2 callus is 25.95+/-0.40 mg/g, 23.56+/-0.71 mg/g and 26.70 +/-0.81 mg/g FW respectively in the culture period, and the anthocyanin content is extremely higher than that of the OE-LrAN2 callus of the control group.
Therefore, after optimizing the culture conditions of OE-LrAN2 callus, the combination of 0.5mg/L6-BA and 0.8mg/L2,4-D hormone was found to be most suitable for OE-LrAN2 callus growth; 200mM NaCl and/or 10mM NaHCO were added 3 The inducer can obviously improve the anthocyanin content of OE-LrAN2 calli, which is 1.50 times, 1.37 times and 1.55 times of that of a control group.
Example 3 establishment and optimization of high anthocyanin production culture System for suspension cells
1. Inoculum size
Using the Lycium ruthenicum OE-LrAN2 calli obtained in example 1, fresh samples of OE-LrAN2 calli of different inoculum sizes were inoculated in jars containing equal amounts of suspension medium and cultured at 5%, 10%, 15% and 20% (inoculum size W/V, fresh weight (g)/culture fluid volume (mL)), each for 21 days, and proliferation factors were calculated after removal.
As shown in FIG. 4, the results showed that 10% of the inoculum size showed the best effect on proliferation of OE-LrAN2 calli compared with the other inoculum sizes.
2. Liquid loading amount
1.0g of the fresh OE-LrAN2 callus obtained in example 1 was weighed and cultured in wide-mouth flasks of different liquid loadings of 10, 20, 30 and 35mL/100mL, each for 21 days, and the dry weight was weighed after taking out.
As a result, as shown in FIG. 5, the cell biomass was extremely significantly different from the other liquid loading levels at 20mL/100mL, and the biomass reached 1.15.+ -. 0.07g/21 dDW. Therefore, a loading of 20mL/100mL was most suitable for OE-LrAN2 callus proliferation.
3. Rotation speed of shaking table
1.0g of fresh OE-LrAN2 callus obtained in example 1 was weighed and cultured on shaking tables of different rotational speeds, at 60, 90, 100 and 120rpm, for 21 days each, and dry weight was weighed after taking out.
As a result, FIG. 6 shows that the culture medium was optimized for the proliferation of OE-LrAN2 callus at 90rpm to 100rpm, and the biomass was 1.07.+ -. 0.09g/21d DW at 90rpm and 1.15.+ -. 0.03g/21d DW at 100rpm.
4. Illumination conditions
1.0g of the fresh OE-LrAN2 callus obtained in example 1 was weighed and inoculated onto a liquid MS medium +0.5 mg/L6-BA +0.8mg/L2,4-D (suspension medium) for dark culture, 16h light/8 h dark culture and full light culture, respectively.
As a result, as shown in FIG. 7, the proliferation amount of OE-LrAN2 calli in the whole-light culture was extremely remarkably different from that in the dark culture and the 16h light/8 h dark culture, and the biomass reached 1.45.+ -. 0.02g/21 dDW after 21 days of culture. Thus, full-light culture is most suitable for OE-LrAN2 callus proliferation.
5. Optimized culture mode
The Lycium ruthenicum Murr OE-LrAN2 callus obtained in example 1 is adopted, 2.0g of fresh OE-LrAN2 callus is respectively weighed and inoculated into a solid MS culture medium and a liquid MS culture medium, 0.5mg/L6-BA and 0.8mg/L2,4-D are respectively added, the rotation speed of a shaking table for liquid culture is 100rpm, the liquid loading amount is 20mL/100mL, continuous full light culture is carried out for 21 days, dry weight is weighed after taking out, anthocyanin content is measured, and each treatment is repeated three times.
TABLE 1
| Culture mode | Biomass (g/21 dDW) | Anthocyanin content (mg/gDW) |
| Liquid culture | 1.45±0.02 A | 256.66±11.75 A |
| Solid culture | 1.26±0.02 B | 249.19±11.76 A |
Note that: the difference between groups of different capital letters indicates that the difference is very significant (P < 0.01)
As a result, as shown in Table 1, the biomass of OE-LrAN2 calli in liquid culture was significantly improved compared with that in solid culture, and anthocyanin content was increased, but there was no significant difference, and the biomass of OE-LrAN2 calli in suspension culture in the culture period was 1.45.+ -. 0.02g/21 dDW, and anthocyanin content was 256.66.+ -. 11.75mg/g DW.
In combination, the liquid MS culture medium, 10% of inoculation amount, 20mL/100mL of liquid loading amount, 100rpm of shaking table rotating speed, 0.5mg/L of 6-BA and 0.8mg/L of 2,4-D hormone are combined, continuous full illumination is realized, and the improvement of OE-LrAN2 callus biomass and anthocyanin content is most facilitated. And culturing OE-LrAN2 callus under optimal culture conditions with 10% of inoculum size, wherein the biomass of the OE-LrAN2 callus under suspension culture in a culture period is 1.45+/-0.02 g/21dDW and the anthocyanin content is 256.66 +/-11.75 mg/g DW after 21 days of culture.
The technical features of the above-described embodiments may be arbitrarily combined, and all possible combinations of the technical features in the above-described embodiments are not described for brevity of description, however, as long as there is no contradiction between the combinations of the technical features, they should be considered as the scope of the description.
The above examples illustrate only a few embodiments of the invention, which are described in detail and are not to be construed as limiting the scope of the invention. It should be noted that it will be apparent to those skilled in the art that several variations and modifications can be made without departing from the spirit of the invention, which are all within the scope of the invention. Accordingly, the scope of protection of the present invention is to be determined by the appended claims.
Claims (10)
1. A method for producing anthocyanin by using lycium ruthenicum callus, which is characterized by comprising the following steps: and (3) performing suspension culture on the lycium ruthenicum callus which is over-expressed with the LrAN2 gene for 20-22 days, collecting the lycium ruthenicum callus, and extracting anthocyanin.
2. The method for producing anthocyanin by using lycium ruthenicum callus as claimed in claim 1, wherein the culture medium of suspension culture is MS culture medium containing 0.45mg/L to 0.55mg/L of 6-BA and 0.8mg/L to 1.2mg/L of 2,4-D.
3. The method for producing anthocyanin by using lycium ruthenicum callus as claimed in claim 2, wherein the culture medium of suspension culture is MS culture medium containing 0.45mg/L to 0.55mg/L of 6-BA and 0.75mg/L to 0.85mg/L of 2,4-D.
4. A method for producing anthocyanin by using lycium ruthenicum callus according to any one of claims 1 to 3, wherein the culture medium of the suspension culture further comprises 180mM NaCl-220 mM NaCl and/or 8 mM-12 mM NaHCO 3 。
5. The method according to any one of claims 1 to 3, wherein the amount of the seed of lycium ruthenicum callus over-expressing the LrAN2 gene in the suspension culture is 5% to 20%.
6. The method for producing anthocyanin by using lycium ruthenicum callus as claimed in claim 5, wherein the inoculation amount of the lycium ruthenicum callus which overexpresses the LrAN2 gene in the suspension culture is 10% -15%; more preferably, the inoculation amount is 9% -11%.
7. The method according to any one of claims 1 to 3, wherein the amount of the liquid to be filled in the suspension culture is 18 to 22mL/100mL.
8. A method for producing anthocyanin by using lycium ruthenicum callus according to any one of claims 1 to 3, wherein the shaking speed of the suspension culture is 85rpm to 105rpm, more preferably 90rpm to 100rpm.
9. A method according to any one of claims 1 to 3, wherein the suspension culture is under light conditions of 16h light/8 h darkness or full light, more preferably full light.
10. A method for producing anthocyanin by using lycium ruthenicum callus according to any one of claims 1 to 3, wherein the preparation method of lycium ruthenicum callus over-expressing LrAN2 gene comprises the following steps:
(1) Taking lycium ruthenicum fruit cDNA as a template, taking SEQ ID NO.2 and SEQ ID NO.3 as primers, and carrying out PCR amplification to obtain a target gene LrAN2;
(2) The target gene LrAN2 is connected with a GFP vector to obtain a recombinant plasmid pSuper1300 which contains LrAN2-GFP;
(3) The recombinant plasmid pSuper1300 is obtained by introducing LrAN2-GFP into aseptic explant of lycium ruthenicum and inducing callus.
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