CN107630032A - The plant expression vector of the 3g genes of tobacco 14 3 and its application - Google Patents
The plant expression vector of the 3g genes of tobacco 14 3 and its application Download PDFInfo
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Abstract
The invention discloses one to grow tobacco14‑3‑3gGene plant expression vector and its application, belong to genetic engineering field, and the present invention uses tobacco14‑3‑3gThe cDNA structure plant expression vectors pK of gene2‑35S‑14‑3‑3g, and be transferred to by agriculture bacillus mediated in plant, plant is improved absorption and the tolerance of PARA FORMALDEHYDE PRILLS(91,95) (HCHO), overcome plant to absorb the shortcomings that tolerance HCHO abilities are low in itself；Test result indicates that the overexpression in tobacco14‑3‑3gGene, make tobacco in 4 mmolL‑1Chlorophyll degradation under liquid HCHO stress slows down, MDA (MDA) and hydrogen peroxide (H2O2) accumulation also slow down, therefore enhance tobacco leaf for HCHO resistance while promote absorption of the tobacco leaf to liquid HCHO, illustrate overexpression14‑3‑3gTransgene tobacco is better than wild type to liquid HCHO resistance and absorbability.
Description
Technical field
The invention belongs to plant genetic engineering field, and in particular to tobacco14-3-3gGene plant expression vector and its
Prepare the application in the genetically modified plants for absorbing the enhancing of formaldehyde ability.
Background technology
Formaldehyde (Formaldehyde, HCHO) is the volatile organic matter all toxic to all biologies, and HCHO is to human body
Have many toxic actions and have certain correlation with many diseases of people, it to skin, mucous membrane, liver, reproductive system with
And nervous system etc. all shows significant toxic action.Exogenous HCHO is entered by way of suction or skin and mucosa contact
Human body, there is certain an excitant and sensitization to skin and mucous membrane first, and HCHO concentration is more than 0.6 mg/m in air3Can be to eyes
Stimulation is produced, if concentration is higher, red swelling of the skin and pain can be caused.Plant, which is exposed in HCHO environment, can cause its body
It is interior to produce excessive active oxygen (Reactive oxygen species, ROS), if largely accumulation will be in plant cell by ROS
Film fat, protein, DNA etc. important large biological molecule produces oxidative damage, or even makes the rupture of cell membrane, causes cell cytosol
The outer relative conductivity increase for blending cell, ROS accumulation can also make MDA (Malondialdehyde, MDA) and mistake
Hydrogen oxide (Hydrogen peroxide, H2O2) content rises, and accelerates the decomposition of chloroplaset Determination of Chlorophyll.
14-3-3 albumen is one group of highly conserved acid protein dimer in eukaryotic cells, in plant, 14-
3-3 albumen has been found to be distributed widely on cytoplasm, nucleus, mitochondrial matrix, chloroplast stroma and thylakoid membrane.Each
14-3-3 protein protomers can independently combine 1 target protein, or two subunits while two domain knots with a target protein
Close, in the bind profile of 14-3-3 albumen, most serines for containing phosphorylation with the protein bound target proteins of 14-3-3
(Ser) or threonine (Thr) site, only a small number of recognition sequences containing non-phosphorylating.14-3-3 albumen is in plant cell
Very important modulin, it participates in the regulation and control of a variety of biological processes in plant, including plant carbon/nitrogen metabolism and
Stress response.
The content of the invention
It is an object of the invention to provide one kind to contain tobacco14-3-3gThe plant expression vector pk of gene2-35S-14- 3-3g, while the construction method of the carrier is provided, and prepare the quick transgenosis for absorbing and being resistant to HCHO using the carrier and plant
Thing.
Carrier provided by the present invention contains tobacco14-3-3gGene, CaMV35S strong promoters, it is described14-3-3gBase
The cDNA of cause derives from tobacco（Nicotiana tabacum）, GenBank accession number is AF299256.1.
In above-mentioned carrier, the initial vector for building affiliated plant expression vector (is purchased from Dalian TaKaRa for pMD18-T
Biotech firm), the final carrier for building affiliated plant expression vector is pK2GW7（Purchased from Flanders
Interuniversity Institute for Biotechnology, VIB）.
In order to realize the above-mentioned purpose of the present invention, technical scheme is as follows：
1st, the structure of plant expression vector
（1）Tobacco is searched from GenBank14-3-3gThe cDNA sequence of gene, and design the following primer of a pair of sequences.
Sense primer 5'GTCGACTCTAAACATGGCGGTGGC3'（ContainSal I site）；
Anti-sense primer 5'GATATCTCAATTTTTGGCTTCATCAGG3'（ContainEcoR V sites）；
The end of sense primer 5 ' addition Sal I（GTCGAC）Restriction enzyme site；The end of anti-sense primer 5 ' addition EcoR V（GATATC）Digestion
Site, using tobacco the first chain cDNA as template amplification, obtain14-3-3gThe full-length cDNA of gene；
（2）Reclaim and purify14-3-3gFull-length gene cDNA fragments, and be connected on pMD18-T carriers, extract plasmid
DNA, recombinant plasmid pMD18-T- is obtained by PCR detections and digestion detection14-3-3g；
（3）Structure introduction cloning vector pENTR-2B-14-3-3g, useEcoR V andSalI digestions pMD18-T-14-3-3gWith
PENTR-2B, recovery14-3-3gCDNA fragments, pENTR-2B is subcloned into by ligase, obtains entry clones carrier
pENTR-2B-14-3-3g；
（4）Build plant over-express vector pK2-35S-14-3-3g, handle is reacted by the LR of Gateway technologies14-3-3gYa Ke
It is grand to arrive plant expression vector pK2GW7In, obtain14-3-3gThe plant expression vector pK of gene2-35S-14-3-3g。
The plant expression vector of the present invention is all suitable for the plant that can implement transgeneic procedure, such as tobacco, arabidopsis, short is led
Ox, African Chrysanthemum etc., illustrate the application process of the expression vector by taking tobacco as an example in an embodiment of the present invention.
2nd, the conversion of tobacco
Using electroporated method conversion plasmid pK2-35S-14-3-3gInto Agrobacterium C58C1（pMP90）Competent cell, apply
Whether cloth grows two days later on Spe flat boards, be transformed into bacterium colony PCR detection plasmids in Agrobacterium.By agriculture bacillus mediated
Leaf disc transformation method preparation tobacco (Nicotiana tabacumCv.Xanthi aseptic seedling), is then obtained by tissue cultures
Seedling, screening have kanamycins（Kan）Resistant plant, it is transferred into MS culture mediums（Contain 3 % sucrose）On continue to cultivate,
So as to obtain sterile transgenic tobacco plant.
3、14-3-3gThe detection of gene transcription level
For testing goal gene14-3-3gExpression quantity of the gene in transgene tobacco, resistance seedling RNA is extracted, utilized14-3- 3gGene upstream and downstream primer carries out RT-PCR detections, filters out14-3-3gThe tobacco plant that gene expression amount significantly raises.
4th, overexpression14-3-3gTransgenic tobacco leaf detects to liquid HCHO absorbability
Use 4 mmolL-1HCHO solution (contain 5 mmolL-1 KHCO3, 1% MES), it is new that each treatment fluid adds 2 g
Fresh tobacco leaf, it is placed in 25 DEG C/24 h (100 μm of olm-2·s-1) illumination, the rpm of the shaking speed 100 progress HCHO sides of body
Compel processing, the content of remaining HCHO in treatment fluid is determined respectively at 6,12,24,48 and 72 h, respectively from each after being disposed
The g of tobacco leaf 0.2 is taken in experimental group, is saved backup with being placed in -80 DEG C after liquid nitrogen flash freezer.
5th, overexpression14-3-3gResistance detecting of the transgene tobacco to liquid HCHO
（1）The measure of chlorophyll content：Take and the tobacco leaf after 72 h is handled in above-mentioned experiment, with the sterile ddH of precooling2O is rushed
Wash blade 3 ~ 4 times, paper handkerchief blots the ddH of blade surface residual2O, 95% ethanol extraction chlorophyll, and with ultraviolet spectrophotometry
Determine the absorbance A at 665 and 649 nm649And A665, chlorophyll a and b content are calculated according to below equation.Chlorophyll a
Content (mgg-1) =13.95×A665–6.88×A649；Content of chlorophyll b (mgg-1)=24.96×A649–7.32×
A665。
（2）MDA（MDA）And hydrogen peroxide（H2O2）The measure of content：Take 4 mmolL-1Liquid HCHO stress at
The tobacco leaf for managing 48 h is used for MDA（MDA）And hydrogen peroxide（H2O2）The measure of content, wherein MDA assays use
TBA methods, H2O2Content is determined using xylenol orange method
The recombinant vector pK of the present invention2-35S-14-3-3gApplication in transfer-gen plant can improve its speed of absorption to HCHO
Rate and resistance；Test result indicates that the overexpression in tobacco14-3-3gGene, make tobacco in 4 mmolL-1Liquid HCHO is coerced
Chlorophyll degradation under compeling slows down, MDA (MDA) and hydrogen peroxide (H2O2) accumulation also slows down, therefore enhances tobacco leaf
For HCHO resistance while promote absorption of the tobacco leaf to liquid HCHO, illustrate overexpression14-3-3gTransgenosis
Tobacco is better than wild type to liquid HCHO resistance and absorbability.
Brief description of the drawings
Fig. 1 is tobacco14-3-3gGene TA clones schematic diagram；
Fig. 2 is14-3-3gPlant Overexpression vector pK2-35S-14-3-3gBuild schematic diagram；
Fig. 3 is overexpression14-3-3gTransgene tobacco transcriptional level testing result；Using the cDNA of wild-type tobacco as control mould
Plate（WT）, kg1 ~ kg5 is the transgene tobacco strain gone out through antibiotic-screening.
Fig. 4 is overexpression14-3-3gAbsorption schematic diagram of the tobacco leaf to 4 liquid HCHO；
Fig. 5 is 4 mmolL-1Liquid HCHO coerces the situation of change of WT and transgene tobacco chlorophyll content after 72 h；
Fig. 6 is 4 mmolL-1Liquid HCHO coerces WT and transgenic tobacco leaf MDA after 48 h（A）And H2O2（B）Content
Situation of change.
Embodiment
Reagent and instrument：
Reagent is broadly divided into molecular biology experiment reagent, the culture medium needed for Genetic Transformation in Higher Plants and genetically modified plants identification
With the various reagents needed for detection.Restriction enzymeEcoR V andSalI, Taq archaeal dna polymerases, reverse transcriptase, RNase suppression
Preparation, dNTP, plasmid extraction kit etc. are precious bioengineering Co., Ltd (Dalian) product, and TRIzoL Reagent RNA are carried
Kit, Gateway LR clonase Enzyme Mix kit is taken to be purchased from Invitrogen companies.Remaining reagent is domestic
Analyze pure.
Instrument is molecular biology and genetic engineering laboratories common instrument.
All primer sequences synthesize in Shuo Qing biotech firms.
Method therefor is conventional method unless otherwise instructed in the embodiment of the present invention.
Embodiment 1：14-3-3gThe PCR amplifications of gene cDNA and TA clones
Tobacco is searched from GenBank14-3-3gThe CDS sequences of gene, and design the following primer of a pair of sequences：
Sense primer 5'GTCGACTCTAAACATGGCGGTGGC3'（ContainSal I site）；
Anti-sense primer 5'GATATCTCAATTTTTGGCTTCATCAGG3'（ContainEcoR V sites）
The addition of the end of sense primer 5 'Sal I（GTCGAC）Restriction enzyme site；The addition of the end of anti-sense primer 5 'EcoR V（GATATC）Digestion
Site, using tobacco the first chain cDNA as template amplification, obtain14-3-3gThe full-length cDNA of gene.
With TRIzoL Reagent（Invitrogen）Total serum IgE is extracted from tobacco leaf, takes the g of young leaf of plant about 0.1,
The TRIzoL extract solutions for adding 1mL are ground in mortar, are moved into centrifuge tube after being stored at room temperature 5 min, are added 0.2 mL chloroforms,
Vibration mixes, and centrifuges 15 min（12000 rpm）, transfer supernatants to new pipe, 0.5 mL isopropanols are added, room temperature is mixed and places
10 min, 4 DEG C of centrifugation 10min（12000 rpm）, supernatant is abandoned, precipitation is cleaned with the mL of 75% ethanol 1,4 DEG C of 5 min of centrifugation
（7500 rpm）, ethanol vacuum drying precipitation or naturally dry are abandoned, with 20 μ l pyrocarbonic acid diethyl esters（DEPC）Handle water dissolving
RNA.Use M-MuLV Reverse Transcriptase Kit（TaKaRa）CDNA synthesis is carried out, takes plant total serum IgE about
0.1 ~ 5 μ g, oligo (dT) 50ng, 10 mmolL-1The μ l of dNTP mix 1,10 μ l are complemented to DEPC processing water, are mixed
After even, it is collected in ttom of pipe by of short duration centrifugation, is placed in 65 DEG C of heating 5 min, the min of ice bath 10, is added the μ l of reactant mixture 9（5
× reaction buffer 4 μ l, 25 mmolL-1 MgCl24 μ l, 0.1 molL-1The μ l of DTT 2, RNase suppress
The μ l of agent 1）, said mixture is mixed, it is collected in ttom of pipe by of short duration centrifugation, 25 DEG C of 2 min of insulation, adds 1 μ l M-MuLV
Reverse Transcriptase, said mixture is mixed, and it is collected in ttom of pipe by of short duration centrifugation, and 25 DEG C are incubated 20 min,
Then 42 DEG C of 70 min of insulation, synthesize cDNA.
TA cloning vectors pMD18-T-14-3-3gStructure（Fig. 1）, using wild-type tobacco cDNA as template, first using cigarette
Careless 18s internal references detect cDNA reverse transcriptions effect to determine the concentration of template, then expand14-3-3gGene C DS code areas, use
Above-mentioned primer is extended, and is determined extension of time by 1k bp/1 min standard according to gene size, is determined by grads PCR
Annealing temperature.PCR system（20µl）：(1) 94 DEG C, the min of pre-degeneration 5；(2) 94 DEG C, 30 s；(3) 55 DEG C, 45 s；(4)
72 DEG C, 1 min；(5) 72 DEG C, 10 min；From (2) ~ (4), 31 circulations are set.
PCR primer carries out TA clones, TA clone's systems after glue reclaim and gel electrophoresis（10 µl）：pMD18-T
The μ L of Vector 0.5 (25 ng), Insert DNA 4.5 μ L (about 150 ng), the μ L of Solution I 5, in 16 DEG C of metals
8 h are bathed, TA cloned plasmids are converted by the α of DH 5 using thermostimulation conversion method, after 37 DEG C, 200 rpm/50 min cultures, room temperature
11000 rpm centrifuge 5 min, be coated with the LB culture mediums of resistance containing AMP in, 37 DEG C cultivate 8 h.6-8 single bacterium colony of picking,
It is inoculated into LB liquid medium containing AMP and cultivates 8 h, extraction plasmid warpEcoR V andSalI double digestions and PCR detections are correct,
DNA Song Shuoqing biotech firms sequencing analysis are extracted, after DNAMAN softwares gene order and protein sequence are correctly used for afterwards
Continuous vector construction.
Embodiment 2：14-3-3gEntry clones carrier pENTR-2B-14-3-3gWith plant over-express vector pK2-35S-14-3-3gStructure
TA in embodiment 1 is cloned and is sequenced and compares successful pMD18-T-14-3-3gIt is correct with being examined before laboratory
PENTR-2B is unloaded to be carried outEcoR V andSalThe double enzymes of I and recovery of tapping rubber, and will14-3-3gGene reclaims fragment, passes through connection
Enzyme, which is subcloned on pENTR-2B, obtains entry clones carrier pENTR-14-3-3g, plant expression vector is constructed by
Gateway LR reaction technologies build plant over-express vector pK2-35S-14-3-3g, LR, which reacts plasmid, need to pass through matter
Grain purification kit is purified, and the recombinant vector after converting successfully needs the resistance LB culture mediums by Spe (spectinomycin)
Screened.Construction strategy as shown in Fig. 2 plant expression vector can T-DNA insertion by way of, recombination and integration to tobacco-based
Because in group, and express14-3-3gGene, over-express vector pK2-35S-14-3-3gContaining composing type strong promoter CaMV 35S,
So as to obtain overexpression14-3-3gThe transgenic tobacco plant of gene.
Embodiment 3：During Agrobacterium tobacco and the culture and screening of transgene tobacco
Using leaf disc transformation method, with Agrobacterium tumefaciems C58C1（pMP90）For medium, the min of tobacco 15 ~ 20 is transfected, is contaminated successfully
Tobacco explant afterwards, is positioned over MS1 culture mediums（Containing 3% sucrose）48 h are co-cultured under dark condition, transfer to MS4（Containing 3%
Sucrose, 50 μ gmL-1Kanamycins and 100 μ gmL-1Cephalosporin）Break up callus on germination culture medium and induce
Budding, 25 DEG C/24 h (100 μm of olm-2·s-1) after d, transfecting successful explant can differentiate illumination cultivation about 25
Transgenic plant, clip seedling and subculture and MS（Containing 2% sucrose, 50 μ gmL-1Kanamycins and 100 μ gmL-1Cephalo is mould
Element）Secondary Culture is carried out in agarose media once, and to suppress Agrobacterium growth, passage then can be in the life of non-resistant later
Cultivated in root culture medium MS agarose medias.
Embodiment 4：Overexpression14-3-3gTransgene tobacco transcriptional level detects
After the transgenic plant growth and maturity of Secondary Culture, each 0.1 g tobacco leaf materials of 2 parts of clip, throw in liquid nitrogen respectively
Middle freezing simultaneously preserves in -80 DEG C, detects for RT-PCR and tobacco RNA is extracted using TRI-zol methods, carried out by PCR
Detection.
In 5 plants detected14-3-3gIn overexpression transgene tobacco（Fig. 3）, kg1 tobacco lines14-3-3gBase
Because up-regulated expression is the most notable, final choice kg1 is used for the material as next step experiment.
Embodiment 5：Overexpression14-3-3gMeasure of the transgene tobacco to liquid HCHO absorbabilities
HCHO can be with Nash reagents（Ammonium Acetate：15%, glacial acetic acid：0.3%, acetylacetone,2,4-pentanedione：0.2%）Generation chemical reaction has
The material of color, a length of 410 nm of maximum absorption wave of the material, therefore standard curve can be prepared, marked according to HCHO-Nash
Directrix curve can calculate the content of HCHO in reaction solution, and absorption rate of the plant to HCHO can be determined using this method.
Use 4 mmolL-1HCHO solution (contain 5 mmolL-1 KHCO3, 1% MES), each treatment fluid adds 2 g
Fresh tobacco leaf, it is placed in 25 DEG C/24 h (100 μm of olm-2·s-1) illumination, the rpm of shaking speed 100 progress HCHO
Stress treatment, determine the content of remaining HCHO in treatment fluid respectively at 6,12,24,48 and 72 h, after being disposed respectively from
The g of tobacco leaf 0.2 is taken in each experimental group, is saved backup with being placed in -80 DEG C after liquid nitrogen flash freezer.
As shown in Figure 4, during 0 ~ 12 h, WT is suitable to liquid HCHO absorption efficiency with kg1 tobacco leafs, and 24 ~ 72
The HCHO absorption efficiencies of kg1 tobacco leafs are considerably better than WT during h, and when handling 72 h, kg1 is to 4 mmolL-1Liquid
HCHO absorption efficiencies compare WT and improve 13.1%.
Embodiment 6：Overexpression14-3-3gDetection of the transgene tobacco to liquid HCHO resistances
（1）The measure of chlorophyll content：HCHO solution handles and rinses blade 4 ~ 5 with pre- cold sterile distilled water after tobacco leaf terminates
It is secondary, paper handkerchief blot blade surface residual distilled water, 95% ethanol extraction chlorophyll, the nm of determined by ultraviolet spectrophotometry 665 and
Absorbance value A at 649 nm665And A649, chlorophyll a and b content are calculated according to below equation.Chlorophyll-a Content (mg
g-1) =13.95×A665–6.88×A649；Content of chlorophyll b (mgg-1)=24.96×A649–7.32×A665。
As shown in Figure 5, compareed with what unused liquid HCHO was handled（CK）Compare, 4 mmolL-1It is each after 72 h of HCHO processing
Being remarkably decreased occurs in WT and kg1 experimental group tobacco leaf Determination of Chlorophyll a content of chlorophyll b, but the leaf in kg1 tobacco leafs
Green plain a contents are slightly above WT, and chlorophyll content b is then significantly higher than WT, about the 1.5 of WT tobaccos times.
（2）Take 4 mmolL-1Liquid HCHO handles the g of blade 0.5 after 48 h with being fully ground after liquid nitrogen flash freezer,
This-HCl buffer solutions（1 mol·L-1, pH 7.5）Fully extracting, extract can be used for soluble sugar and every oxidative stress
The measure of relative physiologic index and analysis.MDA（MDA）Content is determined using thiobarbituricacidα- method, is contained in 0.4 mL
0.6% TBA（Thiobarbituricacidα-）20% TCA（Trichloroacetic acid）Solution in add 0.4 mL extracts, the min of boiling water bath 15
It is placed in cooled on ice at once afterwards.(12000 rpm/10 min), read the light absorption value OD of supernatant532And OD450.Substitute into formula：
MDA concentration（µmol·L-1）= 6.45∙OD532-0.56∙OD450Calculate MDA concentration；Hydrogen peroxide（H2O2）Assay uses
Xylenol orange method, uses ddH2O water carrys out reagent preparation A（3.3 mmol·L-1 FeSO4, 3.3 mmolL-1 (NH4)2SO4,
412.5 mmol·L-1 H2SO4）With reagent B（165 µmol·L-1Xylenol orange, 165 mmolL-1Sorbierite）.Before use
By reagent A and reagent B according to 1：10 ratio is mixed into working reagent.This working reagent and H2O2Prepare liquid is according to 2：1 ratio
After example mixing, after 30 DEG C of 30 min of colour developing of water-bath, absorbance value OD at its 560 nm is determined560, reference standard curve y=
0.1734x-0.0055（x：H2O2Concentration（mg/mL）, y：OD560, R2=0.9995）Calculate H2O2Content.
It can be seen from Fig. 6 A in the CK not coerced by HCHO, the MDA contents of tobacco leaf are relatively low, 4 mmol
MDA content has certain rise in WT and kg1 tobacco leafs after 48 h of L-1 liquid HCHO processing, and wherein WT rises are CK's
1.6 times, kg1 MDA contents are about 1.1 times of control group, substantially less than WT tobaccos.
From Fig. 6 B, after liquid HCHO handles 48 h, there are different journeys in H2O2 contents in WT and kg1 tobacco leafs
The rise of degree, WT H2O2 contents rise is 1.4 times of CK, and kg1 is about then about 1.1 times of CK, substantially less than WT.
Sequence table
<110>Kunming University of Science and Technology
<120>Tobacco14-3-3gThe plant expression vector of gene and its application
<160> 2
<170> PatentIn version 3.3
<210> 1
<211> 24
<212> DNA
<213>Artificial sequence（Artificial）
<400> 1
gtcgactcta aacatggcgg tggc 24
<210> 2
<211> 27
<212> DNA
<213>Artificial sequence（Artificial）
<400> 2
gatatctcaa tttttggctt catcagg 27
Claims (2)
1. tobacco14-3-3gThe plant expression vector pK of gene2-35S-14-3-3g, it is characterised in that：The tobacco14-3-3g
The GenBank accession number of gene is AF299256.1, and the carrier contains CaMV35S strong promoters.
2. the tobacco described in claim 114-3-3gThe plant expression vector of gene absorbs turn of formaldehyde ability enhancing preparing
Application in gene plant.
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| CN114524428A (en) * | 2022-01-24 | 2022-05-24 | 江苏师范大学 | Water-soluble biomass-derived carbon dot and preparation method and application thereof |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102363790A (en) * | 2011-10-11 | 2012-02-29 | 昆明理工大学 | Plant expression vector of arabidopsis heat shock factor gene AtHsfA1d, and application thereof |
| CN104745619A (en) * | 2015-03-03 | 2015-07-01 | 昆明理工大学 | Plant expression vector of arabidopsis formate dehydrogenase FDH gene and application of plant expression vector |
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2017
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| CN102363790A (en) * | 2011-10-11 | 2012-02-29 | 昆明理工大学 | Plant expression vector of arabidopsis heat shock factor gene AtHsfA1d, and application thereof |
| CN104745619A (en) * | 2015-03-03 | 2015-07-01 | 昆明理工大学 | Plant expression vector of arabidopsis formate dehydrogenase FDH gene and application of plant expression vector |
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| CN114524428A (en) * | 2022-01-24 | 2022-05-24 | 江苏师范大学 | Water-soluble biomass-derived carbon dot and preparation method and application thereof |
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