CN109055424A - A kind of method improving polysaccharide content of dendrobium candidum and the dendrobium candidum obtained using this method - Google Patents
A kind of method improving polysaccharide content of dendrobium candidum and the dendrobium candidum obtained using this method Download PDFInfo
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- CN109055424A CN109055424A CN201810934404.6A CN201810934404A CN109055424A CN 109055424 A CN109055424 A CN 109055424A CN 201810934404 A CN201810934404 A CN 201810934404A CN 109055424 A CN109055424 A CN 109055424A
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
- C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
- C12N15/8241—Phenotypically and genetically modified plants via recombinant DNA technology
- C12N15/8242—Phenotypically and genetically modified plants via recombinant DNA technology with non-agronomic quality (output) traits, e.g. for industrial processing; Value added, non-agronomic traits
- C12N15/8243—Phenotypically and genetically modified plants via recombinant DNA technology with non-agronomic quality (output) traits, e.g. for industrial processing; Value added, non-agronomic traits involving biosynthetic or metabolic pathways, i.e. metabolic engineering, e.g. nicotine, caffeine
- C12N15/8245—Phenotypically and genetically modified plants via recombinant DNA technology with non-agronomic quality (output) traits, e.g. for industrial processing; Value added, non-agronomic traits involving biosynthetic or metabolic pathways, i.e. metabolic engineering, e.g. nicotine, caffeine involving modified carbohydrate or sugar alcohol metabolism, e.g. starch biosynthesis
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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
- A01H4/008—Methods for regeneration to complete plants
Abstract
The invention discloses a kind of methods for improving polysaccharide content of dendrobium candidum, belong to genetic engineering field, and step includes being overexpressed DcUGP vector construction, actication of culture, infecting, co-culture, resistance screening and obtaining resistant plant；The method that the application utilizes mediated by agriculture bacillus, DcUGP is transferred in dendrobium candidum, the dendrobium candidum that glucose, sucrose and polyoses content are above wild type is obtained；This method is at low cost, easy to operate, to the dendrobium candidum breeding time limit is shortened, improves its quality and is of great significance.
Description
Technical field
The present invention relates to gene engineering technology fields, more particularly to a kind of pass through to be overexpressed gene raising Dendrobium officinale polysaccharide
The method of content and the dendrobium nobile obtained using this method.
Background technique
Dendrobium candidum (Dendrobium catenatum Lindl), also known as ribbed hedyotis herb are that orchid family Dendrobium is perennial often
Green herbaceous plant.Its is many kinds of, and whole world Dendrobium Sw just has more than 1000 kinds, and wherein China has more than 70 kinds, most of point
Cloth is in south China and southwest.Dendrobium nobile is the civil medicinal material for having high value, scapus in the country in China and Southeast Asia
As " maple bucket ", according to " only giving birth to person's victory on stone also has person on raw oak, and name wood dry measure used in former times is unbearably used " in " Bencao Tujing ", and know
Dendrobium candidum is top grade in Dendrobium, and good quality, medical value is high, and medicinal ingredient is good.Dendrobium candidum contains human body must
The multiple beneficial ingredient needed, cold nature sweet in flavor belong to the yin tonics in tonic；Not only there is voice-clearing and throat-moistening, nourishing Yin and clearing heat, promote the production of body fluid
Beneficial stomach and other effects can also enhance immunity of organisms, prevention angiocarpy and ophthalmology disease.
The effective component of dendrobium nobile includes polysaccharide, alkaloid, Flavonoid substances, luxuriant and rich with fragrance class compound, amino acid and microelement
Deng wherein polysaccharide is main active, and the power of dendrobium nobile physiological activity is closely related with its polyoses content, therefore is often contained with polysaccharide
The height of amount judges the quality of Shihu " medicinal materials quality.
At present it is known that raising dendrobium candidum in the method for polyoses content include:
1. by matrix --- the tree species type for changing plantation dendrobium candidum, to improve polyoses content.
Disadvantage: needing domesticated seedlings, and proliferation rate is low, and polyoses content improves simultaneously few, only improves than dendrobium officinale
5.94% or so；
2. improving polyoses content by changing fertilizing method.
Disadvantage: needing domesticated seedlings, and fertilising need to be in harvesting the previous year, and method is cumbersome, and the period is long；
3. controlling the conditions such as light temperature increases polyoses content.
Disadvantage: at high cost, the control period is long.
In addition, UGP (uridine diphosphate glucose pyrophosphorylase gene) expression product
For UDPglucose pyrophosphorylase (UDP-glucose pyrophosphorylase, UGPase), UGPase catalysis
Cori's eater Cori and uridine triphosphate synthesis UDP-glucose and pyrophosphoric acid, and UDP-glucose is that polysaccharide synthesizes main sugar
Base donor.UGP gene has been cloned in numerous researchs from the plants such as Radix Astragali, sugarcane and potato.Minority is to dendrobium nobile UGP gene
Research also only rest on cDNA clone and expression analysis stage, the research of Lv Nan et al. has also only related to the gene in iron sheet stone
Dry measure used in former times different year, the expression quantity in different tissues.But not yet the appearance overexpression gene can cause dendrobium candidum itself at present
The research of which kind of influence.
Summary of the invention
An object of the present invention, in that a kind of method for improving polysaccharide content of dendrobium candidum is provided, it is above-mentioned to solve
Problem.
To achieve the goals above, the technical solution adopted by the present invention is that such: a kind of raising Dendrobium officinale polysaccharide contains
The method of amount, comprising the following steps:
(1) it is overexpressed DcUGP vector construction:
A. according to the DcUGP cDNA sequence of known dendrobium candidum, design primer；
B. it extracts dendrobium candidum RNA reverse transcription and obtains total cDNA, PCR is carried out according to the primer that design obtains in step (a)
Amplification obtains DcUGP target fragment；
C. DcUGP target fragment carrier pFGC1008 and step (b) obtained with SpeI and AscI limitation restriction endonuclease into
Row digestion；
D. by the DcUGP target fragment and carrier pFGC1008 progress electrophoresis detection after step (c) digestion, to required segment
It is recycled；Recovery product addition ligase is attached reaction, obtains connection product pFGC1008-DcUGP；
E. by after the resulting connection product pFGC1008-DcUGP conversion Escherichia coli of step (d), plasmid is extracted
PFGC1008-DcUGP is simultaneously verified；
(2) pFGC1008-DcUGP converts Agrobacterium GV3101
The resulting plasmid pFGC1008-DcUGP of step (1) is converted into Agrobacterium GV3101, then carries out clone PCR, into
The verifying of row Agrobacterium；
(3) actication of culture:
The preservation Agrobacterium GV3011 containing pFGC1008-DcUGP is activated, infected liquid is made；
(4) it infects:
It chooses greener robust growth, color and finer and close dendrobium candidum wild type protocorm and cuts, step is then added
Suddenly infected liquid obtained by (3), is infected；
(5) it co-cultures:
Protocorm after step (4) is infected is transferred to co-culture and co-culture on base；
(6) resistance screening:
Protocorm after step (5) are co-cultured is transferred to screening and culturing medium and carries out resistance screening, until differentiating new original
Bulb, i.e. completion resistance screening；
(7) plant regeneration:
The resulting new protocorm of step (6) is transferred to the culture medium without hygromycin, after its differentiation budding, is transferred to
It takes root in root media, obtains intact plant.
As a preferred technical solution, in step (3), the activation method of Agrobacterium GV3011 are as follows: by Agrobacterium GV3011
It is cultivated twice with YEP culture solution, thallus is collected after culture with MS fluid nutrient medium, infected liquid is made.
As further preferred technical solution, in step (3), the composition of the YEP culture solution are as follows: 50 μ g/mL+ of Gen
Rif 10μg/mL+Cm 25μg/mL。
As a preferred technical solution, in step (4), before infecting, concentration tune is carried out to the resulting infected liquid of step (3)
It is whole.
As a preferred technical solution, in step (4), the condition infected is that 28 DEG C of shaking table 50rpm infect 30min.
As a preferred technical solution, in step (5), the condition that co-cultures is dark culturing 4d at 20 DEG C.
As a preferred technical solution, in step (6), the screening and culturing based formulas are as follows: MS+6-BA1.0mg/L+NAA
Add cephalo 500mg/L+ hygromycin 20mg/L after 0.2mg/L+ sucrose 30g/L+ agar 4.8g/L+ potato 20g/L+ sterilizing.
As a preferred technical solution, in step (6), the condition of screening and culturing are as follows: 22-25 DEG C, intensity of illumination 1800-
2000Lx, periodicity of illumination are 14h illumination/d, the culture of the constant temperature incubation room 10h dark/d.
As a preferred technical solution, in step (7), the formula of the root media are as follows: MS+ sucrose 30g/L+ agar
Add cephalo 500mg/L after 4.8g/L+ potato 20g/L+ sterilizing.
The second object of the present invention is to provide dendrobium candidums obtained by the above method.
Compared with the prior art, the advantages of the present invention are as follows:
1. DcUGP gene is transferred in dendrobium candidum by the method using mediated by agriculture bacillus, method is easy, easy to operate and cost
It is low；
2. obtaining the increased dendrobium candidum of polyoses content by the DcUGP gene for being overexpressed dendrobium candidum itself, overcome
Extraneous some uncertain factors, such as temperature, illumination, the influence of PH；
3. to solve to provide a kind of effective ways the problems such as the production cycle present in traditional breeding method is long, poor quality；
4. the tissue-cultured seedling obtained it is clean nontoxic and can massive amplification, can be directly used for Polyose extraction, can also transplant to nature
Environment plantation, lays the foundation to be widely popularized；
5. combining the method for some external conditions that polyoses content may be made to improve more after transplanting；
6. can be obtained the increased dendrobium candidum of polyoses content in the tissue-cultured seedling stage, it is not required to tame, it is more from infecting to obtaining
The increased dendrobium candidum plant of sugared content only needs 1 year or so.
That is, the method that the application utilizes mediated by agriculture bacillus, DcUGP is transferred in dendrobium candidum, glucose, sucrose are obtained
And polyoses content is above the dendrobium candidum of wild type；This method is at low cost, easy to operate, to shorten the dendrobium candidum breeding time limit,
Its quality is improved to be of great significance.
Detailed description of the invention
Fig. 1 is pFGC1008-DcUGP vector construction electrophoresis result figure；
Fig. 2 is pFGC1008 carrier figure；
Fig. 3 is that Agrobacterium colonies PCR verifies electrophoresis result figure；
Fig. 4 is conversion and the selection result schematic diagram；
Fig. 5 is dendrobium candidum seedling quantitative fluorescence analysis, and in Fig. 5, WT is wild type；T1, T2 and T3 are transgenic；
Fig. 6 is dendrobium candidum protocorm glucose and cane sugar content comparative result schematic diagram；
Fig. 7 is dendrobium candidum plant polyoses content comparative result schematic diagram.
Specific embodiment
The present invention will be further described with reference to the accompanying drawings.
Embodiment 1:
A method of improving polysaccharide content of dendrobium candidum, comprising the following steps:
(1) it is overexpressed DcUGP vector construction:
A. according to the DcUGP cDNA sequence of known dendrobium candidum, design primer；
Using the CDS sequence and cDNA sequence of NCBI query site dendrobium candidum DcUGP, nucleotide sequence is respectively such as
Shown in SEQ ID NO:1 and SEQ ID NO:2, according to above-mentioned nucleic acid sequence, primer-design software primer 3.0 with
Design primer in DNAman, resulting primer sequence is as shown in table 1,
1 primer sequence of table
B. dendrobium candidum RNA reverse transcription is extracted using the prior art and obtains total cDNA, obtained according to design in step (a)
Primer carries out PCR amplification, first obtains the DcUGP gene target fragment containing 5 ' UTR and the 3 ' parts UTR, as shown in Figure 1A；Again with this
Segment is template, and the specific primer containing restriction enzyme site is added and carries out PCR amplification, obtains the DcUGP mesh containing restriction enzyme site
Segment；Wherein, the positive sequence and reverse sequence such as SEQ ID of the primer when amplification contains the DcUGP of 5 ' UTR and 3 ' UTR
Shown in NO:5 and SEQ ID NO:6, the positive sequence and backward sequence of specific primer used when DcUGP containing restriction enzyme site are expanded
Column are as shown in SEQ ID NO:3 and SEQ ID NO:4；
C. DcUGP mesh carrier pFGC1008 and step (b) obtained with two kinds of AscI limitation restriction endonucleases with SpeI simultaneously
Segment be that DcUGP containing restriction enzyme site carries out digestion；
Wherein, SpeI and AscI is purchased from Beijing NEW ENGLAND Biolabs；Carrier pFGC1008 comes fromhttps:// Www.arabidopsis.org/servlet/TairObject? id=500300074&type=vector,
Its structure as shown in Fig. 2, restriction enzyme site as shown in the box in Fig. 2,
D. the DcUGP and pFGC1008 after above-mentioned digestion is subjected to 1% agarose gel electrophoresis detection, electrophoresis result is as schemed
Shown in 1-B, it was demonstrated that digestion is correct, recycles to required segment glue, and the connection for next step is reacted；Recovery product is attached,
T4DNA ligase (being purchased from Beijing NEW ENGLAND Biolabs) is added, target fragment is determined according to the concentration that glue recycles segment
The adding proportion of DcUGP and carrier pFGC1008 react 20 μ L of total volume, and then 5h is reacted in 16 DEG C of connections, obtain connection product
pFGC1008-DcUGP；
E. by after the resulting connection product pFGC1008-DcUGP conversion Escherichia coli of step (d), plasmid is extracted
PFGC1008-DcUGP is simultaneously verified, specific steps are as follows:
Competent cell (Escherichia coli) are taken out in ultra low temperature freezer) it is put into ice, plasmid (100 μ are added after it melts
The resulting connection product pFGC1008-DcUGP of 20mL step d is added in L Escherichia coli), after ice bath 30min, 42 DEG C of thermal shock 1min
Ice bath 2min in ice is put back to rapidly；YEP culture solution 1mL is added, 37 DEG C on shaking table, 200rpm rocks the coated plate (Cm (present invention after 1h
In, unless stated otherwise, write a Chinese character in simplified form " Cm " and refer both to chloramphenicol) 25 μ g/mL) it is statically placed in 37 DEG C of overnight incubations, 8-12h；
Each plate picking single colonie carries out clone PCR reaction, detection recombinant DNA whether successful conversion.By successful conversion
Escherichia coli be transferred in YEP fluid nutrient medium of the 5mL containing 25 μ g/mL chloramphenicol, 200rpm shakes bacterium at 37 DEG C overnight；Later
It filters out bacterium containing recombinant plasmid and extracts Plasmid DNA, and with SpeI/AscI digestion verification, as a result shown in following Fig. 1-C, out
The band of existing 10496bp and 1433bp, meets expected results, illustrates that connection converts successfully；Correct plasmid will be finally verified to send
It is sequenced toward company；Whether checking carrier constructs successfully the positive sequence and reverse sequence such as SEQ ID NO:7 and SEQ of the primer
Shown in IDNO:8；
(2) pFGC1008-DcUGP converts Agrobacterium GV3101
By the resulting plasmid pFGC1008-DcUGP conversion Agrobacterium GV3101 of step (1) (purchased from Beijing Bo Maide biology
Technology Co., Ltd.), then carry out clone PCR, PCR product electrophoresis result as shown in figure 3, there is the band of 769bp size,
Meet with expected results, shows connection product conversion Agrobacterium success, can be used for dendrobium candidum and infect；
(3) actication of culture:
Activate Agrobacterium GV3011；
The Agrobacterium GV3011 of preservation is first taken to the 5mL liquid YEP culture solution (composition are as follows: Gen containing corresponding antibiotic
(present invention in, unless stated otherwise, write a Chinese character in simplified form " Gen " and refer both to gentamicin) 50 μ g/mL+Rif are (in the present invention, except non-specifically saying
Bright, write a Chinese character in simplified form " Rif " and refer both to rifampin) 10 μ g/mL+Cm, 25 μ g/mL) in, sealing is placed in 28 DEG C, and 200rpm shaking table is trained overnight
It supports；It transfers to big in 250mL liquid YEP culture solution (composition are as follows: 50 μ g/mL+Rif of Gen, 10 μ g/mL+Cm, 25 μ g/mL)
Thallus is collected after amount culture, infected liquid is made with MS fluid nutrient medium；
(4) it infects:
The light green and finer and close dendrobium candidum wild type protocorm of robust growth, color is chosen as transformation receptor and to cut
It cuts, infected liquid obtained by step (3) is then added, is infected, detailed process are as follows:
A. spectrophotometer is opened, 600nm is adjusted to, measures agrobacterium liquid concentration；
B. the Agrobacterium YEP solution of aforementioned amplification cultivation is sub-packed in 2 centrifuge tubes, 4000rpm is centrifuged 10min；
C. supernatant is abandoned after being centrifuged, 10mL MS culture solution is added, and is suspended on ice complete molten；
It is 0.6 or so that d.MS culture solution, which dilutes bacterium solution to OD value,；
C. AS (acetosyringone), which is added, makes the final concentration of 100 μm of ol/L of solution A S, shakes up；
E. robust growth, the greener and finer and close dendrobium candidum wild type protocorm of color are chosen (as shown in Fig. 4-A)
It is cut to diameter 3-5mm or so, is put into sterile conical flask, the infected liquid that abovementioned steps are made is added；
F.28 a DEG C shaking table 50rpm infects 30min；
(5) it co-cultures:
By protocorm, filter paper blots extraction raffinate in sterile petri dish after step (4) is infected, and is transferred to and co-cultures on base,
Milky Agrobacterium is enclosed with one around protocorm after dark culturing 4d at 20 DEG C, as shown in Fig. 4-B；
(6) resistance screening:
Specific steps are as follows:
After 4d, the protocorm that picking co-cultures rocks number with aseptic water washing 4-5 times in the sterile triangular flask of 100mL every time
It is secondary, until water is no longer muddy, as clear as crystal state is presented, is finally shaken again with the 50mL water containing 100 μ L cephalos (500mg/L)
Cleaning about 1min is shaken, liquid is outwelled, protocorm is placed in sterile petri dish on filter paper, then suck dry moisture is transferred quickly to
Screening and culturing medium (screening and culturing based formulas are as follows: MS+6-BA (6-benzyl aminopurine) 1.0mg/L+NAA (methyl α-naphthyl acetate) 0.2mg/L+
Add cephalo 500mg/L+ hygromycin 20mg/L after sucrose 30g/L+ agar 4.8g/L+ potato 20g/L+ sterilizing)；It is placed on 22-
25 DEG C, intensity of illumination 1800-2000Lx, periodicity of illumination is 14h illumination/d, the culture of the constant temperature incubation room 10h dark/d；Screening
Period, the survival condition of periodic statistical protocorm, and be transferred in the screening and culturing medium newly prepared, until differentiating new protocorm
Stem completes resistance screening；
Situation is screened after about 50d as shown in Fig. 4-C, brown or transparent to convert failed protocorm, because it does not have
Resistant gene and cannot survive in screening and culturing medium, it is gradually dead；The protocorm color of arrow meaning is still in Fig. 4-C
Green, can survive in screening and culturing medium, it was demonstrated that successfully be transferred to DcUGP gene, transformation efficiency 16.9%；
(7) plant regeneration:
Screening is transferred into the culture medium without hygromycin after obtaining new protocorm, is transferred to after its differentiation budding
In root media (formula are as follows: add cephalo 500mg/L after MS+ sucrose 30g/L+ agar 4.8g/L+ potato 20g/L+ sterilizing)
It takes root and obtains intact plant；
After protocorm after screening is stable continues squamous subculture, protocorm color is dark green, fine and close full, and part protocorm has
Break up (as shown in Fig. 4-D)；After squamous subculture 2 months, protocorm differentiation goes out dense and light green color seedling (such as Fig. 4-E
It is shown)；The seedling of robust growth is moved in root media, after 3 months, seedling development is increased at plant, stem
Slightly, and successfully it takes root (as shown in Fig. 4-F).
The sucrose, agar of the present embodiment are purchased from the extensive Youtech Co., Ltd. in Chengdu,
NAA, 6-BA, cephalo, hygromycin, rifampin, gentamicin, chloramphenicol are purchased from Beijing Solarbio.
Embodiment 2qRT-PCR detection
The resulting intact plant of embodiment 1 is subjected to qRT-PCR detection, wherein DcUGP primer when qRT-PCT is detected
Positive sequence and reverse sequence as shown in SEQ ID NO:9 and SEQ ID NO:10, the positive sequence of internal control primer and anti-
To sequence as shown in SEQ ID NO:11 and SEQ ID NO:12, as a result as shown in figure 5, the DcUGP base of transgenosis dendrobium candidum
Because of expression quantity significantly increasing than wild type dendrobium candidum.
The detection of 3 glucose of embodiment and sucrose
Precision weighs DEXTROSE ANHYDROUS reference substance and is configured to the reference substance deposit mother liquor that mass concentration is 10mg/L, draws Portugal
Grape Standard for Sugars liquid is configured to be packed into sample injection bottle after concentration gradient solution crosses 0.45 μm of filter membrane.It takes and (weighs open country using Hot water extraction
Each 5g of fresh goods protocorm of raw type and transgenic, remaining step are identical with the extraction of following plant polysaccharide) transgenosis of extraction
Protocorm polysaccharide solution 5mL crosses 0.45 μm of filter membrane, is packed into sample injection bottle.It is bent that standard is drawn after being detected according to HPAEC-PAD method
Line and the content for calculating dextrose and saccharose in sample solution, as a result as shown in Figure 6, the results showed that: DcUGP is overexpressed iron sheet
Increase 107.8% (Fig. 6-A) of the dendrobium nobile protocorm glucose content than wild type, cane sugar content DcUGP overexpression dendrobium candidum
Protocorm increases 325.9% (Fig. 6-A) than wild type.
The detection of 4 polyoses content of embodiment
It takes DEXTROSE ANHYDROUS reference substance appropriate, adds water that the glucose solution that concentration is 0.1mg/mL is made.
The preparation of standard curve: precision measure reference substance solution 0.2mL, 0.4mL, 0.6mL, 0.8mL, 1.0mL, 1.2mL,
1.4mL, 1.6mL, 1.8mL, 2.0mL are set respectively in 10mL tool plug test tube, and respectively plus water is mended to 2.0mL, add anthracene containing 1mg/mL
80% sulfuric acid 6mL of ketone, shakes up, sets and heat 20min in boiling water bath, takes out, and sets cooling 5min in ice bath, is sky with corresponding reagent
It is white, absorbance is measured at the wavelength of 620nm, using absorbance as ordinate, concentration is abscissa, draws standard curve.
The preparation of sample solution: wild type and transformant dendrobium candidum plant about 3g are taken respectively, adds water 20mL.80 DEG C of water-baths
2h takes supernatant to deposit in another clean centrifuge tube after standing.Precipitating continues according to the above ratio plus water, so repetition are extracted more
Sugar 2 times.It takes gained supernatant solution 5mL that dehydrated alcohol to concentration of alcohol is added to be 80%, is placed in precipitate polysaccharides in 4 DEG C of refrigerators and is no less than
For 24 hours, 7000rpm is centrifuged 5min, abandons supernatant, and hot water dissolving's precipitating is added, sample solution can be obtained.
Measuring method: measuring test solution 1mL, set in 10mL tool plug test tube, the method under sighting target directrix curve preparation, from
" the 80% sulfuric acid 6mL that the anthrone containing 1mg/mL is added in precision " rises, and measures absorbance in accordance with the law, and it is molten that test sample is read from standard curve
The amount of DEXTROSE ANHYDROUS in liquid calculates to get as a result as shown in fig. 7, showing that transgenic plant polyoses content is higher than wild type
21.6%.
The foregoing is merely illustrative of the preferred embodiments of the present invention, is not intended to limit the invention, all in essence of the invention
Made any modifications, equivalent replacements, and improvements etc., should all be included in the protection scope of the present invention within mind and principle.
Claims (10)
1. a kind of method for improving polysaccharide content of dendrobium candidum, which comprises the following steps:
(1) it is overexpressed DcUGP vector construction:
A. according to the DcUGP cDNA sequence of known dendrobium candidum, design primer；
B. it extracts dendrobium candidum RNA reverse transcription and obtains total cDNA, PCR amplification is carried out according to the primer that design obtains in step (a)
Obtain DcUGP target fragment；
C. enzyme is carried out to the obtained DcUGP target fragment of carrier pFGC1008 and step (b) with SpeI and AscI limitation restriction endonuclease
It cuts；
D. by the DcUGP target fragment and carrier pFGC1008 progress electrophoresis detection after step (c) digestion, required segment is carried out
Recycling；Recovery product addition ligase is attached reaction, obtains connection product pFGC1008-DcUGP；
E. by after the resulting connection product pFGC1008-DcUGP conversion Escherichia coli of step (d), plasmid pFGC1008- is extracted
DcUGP is simultaneously verified；
(2) pFGC1008-DcUGP converts Agrobacterium GV3101
The resulting plasmid pFGC1008-DcUGP of step (1) is converted into Agrobacterium GV3101, clone PCR is then carried out, carries out agriculture
Bacillus verifying；
(3) actication of culture:
The preservation Agrobacterium GV3011 containing pFGC1008-DcUGP is activated, infected liquid is made；
(4) it infects:
It chooses greener robust growth, color and finer and close dendrobium candidum wild type protocorm and cuts, step (3) then are added
Gained infected liquid, is infected；
(5) it co-cultures:
Protocorm after step (4) is infected is transferred to co-culture and co-culture on base；
(6) resistance screening:
Protocorm after step (5) are co-cultured is transferred to screening and culturing medium and carries out resistance screening, until new protocorm is differentiated,
Complete resistance screening；
(7) plant regeneration:
The resulting new protocorm of step (6) is transferred to the culture medium without hygromycin, after its differentiation budding, is transferred to and takes root
It takes root in culture medium, obtains intact plant.
2. the method according to claim 1 for improving polysaccharide content of dendrobium candidum, which is characterized in that in step (3), agriculture bar
The activation method of bacterium GV3011 are as follows: Agrobacterium GV3011 is cultivated twice with YEP culture solution, thallus MS liquid is collected after culture
Infected liquid is made in culture medium.
3. the method according to claim 2 for improving polysaccharide content of dendrobium candidum, which is characterized in that described in step (3)
The composition of YEP culture solution are as follows: 50 μ g/mL+Rif of Gen, 10 μ g/mL+Cm, 25 μ g/mL.
4. the method according to claim 1 for improving polysaccharide content of dendrobium candidum, which is characterized in that in step (4), infect
Before, concentration adjustment is carried out to the resulting infected liquid of step (3).
5. the method according to claim 1 for improving polysaccharide content of dendrobium candidum, which is characterized in that described in step (4)
The condition infected is that 28 DEG C of shaking table 50rpm infect 30min.
6. the method according to claim 1 for improving polysaccharide content of dendrobium candidum, which is characterized in that in step (5), train altogether
The condition of supporting is dark culturing 4d at 20 DEG C.
7. the method according to claim 1 for improving polysaccharide content of dendrobium candidum, which is characterized in that described in step (6)
Screening and culturing based formulas are as follows: MS+6-BA 1.0mg/L+NAA 0.2mg/L+ sucrose 30g/L+ agar 4.8g/L+ potato 20g/L
Add cephalo 500mg/L+ hygromycin 20mg/L after+sterilizing.
8. the method according to claim 1 for improving polysaccharide content of dendrobium candidum, which is characterized in that in step (6), screening
The condition of culture are as follows: 22-25 DEG C, intensity of illumination 1800-2000Lx, periodicity of illumination is 14h illumination/d, 10h dark/d perseverance
Warm culturing room's culture.
9. the method according to claim 1 for improving polysaccharide content of dendrobium candidum, which is characterized in that described in step (7)
The formula of root media are as follows: add cephalo 500mg/L after MS+ sucrose 30g/L+ agar 4.8g/L+ potato 20g/L+ sterilizing.
10. the dendrobium candidum that method described in any one of claim 1-9 obtains.
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| CN113943744A (en) * | 2021-11-02 | 2022-01-18 | 云南农业大学 | Application of RCA gene of cymbidium floribundum and vector construction method thereof |
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| CN115873891A (en) * | 2022-08-09 | 2023-03-31 | 四川农业大学 | Application of dendrobium officinale DoObgC and variable spliceosome thereof in promotion of elongation of embryonic axis |
| WO2024031835A1 (en) * | 2022-08-09 | 2024-02-15 | 四川农业大学 | Use of dendrobium officinale doobgc and variable spliceosome thereof in promoting embryonic axis elongation |
| CN115873891B (en) * | 2022-08-09 | 2024-05-07 | 四川农业大学 | Dendrobium candidum DoObgC and application of alternative spliceosome thereof in promotion of hypocotyl elongation |
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