CN105198967B - Protein VdAL is improving the application in plant product quality - Google Patents
Protein VdAL is improving the application in plant product quality Download PDFInfo
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- CN105198967B CN105198967B CN201510691295.6A CN201510691295A CN105198967B CN 105198967 B CN105198967 B CN 105198967B CN 201510691295 A CN201510691295 A CN 201510691295A CN 105198967 B CN105198967 B CN 105198967B
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- 241001515965 unidentified phage Species 0.000 description 1
- 239000013603 viral vector Substances 0.000 description 1
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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- 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
- C12N15/8261—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield
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Abstract
The invention discloses protein VdAL to improve the application in plant product quality.In application provided by the present invention, protein VdAL is following A 1) or A2) or A3): A1) amino acid sequence is the protein of sequence 1；A2) in the amino acid sequence of sequence 1 through substitution and/or be deleted and/or added one or several amino acid residues obtain it is with the same function as A1) derived from protein；A3) in A1) or the obtained fused protein of N-terminal A2) or/and C-terminal connection label.It is demonstrated experimentally that plant product quality can be improved in protein VdAL of the invention and its biological agent.
Description
Technical field
The present invention relates to protein VdAL in field of biotechnology to improve the application in plant product quality.
Background technique
China's large area grain and the Sustainable high yield of industrial crops rely primarily on largely using chemical fertilizer, prevention and treatment pest and disease damage at present
It relies primarily on and largely sprays chemical pesticide.Chinese Academy of Agricultural Sciences's agricultural economy and Institute of Development Studies researcher Hu Dinghuan, are being talked
And he points out when agricultural pollution problem, our countries are present still quite serious, and chemical fertilizer and pesticide are excessively used, Yi Jiyang
Serious pollution will all be caused to agricultural production by growing the antibiotic largely used in industry and heavy metal additive.
The current chemical fertilizer year usage amount of the province is counted at 30,000 tons or more according to Liaoning Province, unit area chemical fertilizer application intensity about 340
Kilogram per hectare, national ecotope construction requirements per hectare limit value is then 250 kilograms of per hectare, the light phosphate fertilizer potash fertilizer phenomenon of diazonium fertilizer
Universal problem of fertilization lack of standardization is also more prominent, and Liaoning Province's pesticide annual consumption is more than 1.4 ten thousand tons and is in rise year by year trend.Residual
Pesticide, which is entered by way of atmospheric sedimentation and rain drop erosion in environment and agricultural product, easily causes Environment Pollution Event.Liaoning Province
Environmentally friendly Room rural area department director Wang Jun is indicated recently as the outstanding day by day environment letters and calls case of rural environmental pollution problem in high-incidence
Situation.Wherein case concerning farmers is close for the every annual 4310 of 74, Liaoning Province Jin Liangnian counties and districts' environment letters and calls case concerning farmers according to statistics
2795 account for 65% or so of letters and calls case.
Excessive fertilization, which will cause, enhances antagonism between ion when crops absorb nutrient, if certain nutrient ion
The presence of high concentration is able to suppress the activity of one or more nutrient ions, to influence crops to another nutrient ions
Absorption.The sowing amount of nitrogenous fertilizer is excessive such as in acid soil, and crop absorbs calcium ion with regard to highly difficult.If excessively applying calcium
Fertilizer can induce the microelement deficiencies such as zinc, boron, iron, magnesium, the manganese of crops.Potassium Fertilizer excessively also will affect crops to calcium from
The absorption of son, magnesium ion；And excessive fertilization easily causes crops to be poisoned, and because applying a large amount of fertilizer, increases the soil liquid
Concentration makes crop root system absorb moisture difficulty, causes overground part to wilt, plant is withered.In addition, excessive fertilization has an effect on agricultural production
The quality of product, crop growth intermediary and later stages can make the reduction of agricultural product organ sugar content, intolerant to storage if largely applying chemical fertilizer
Hiding, to influence commodity value, reduces the economic benefit of crops.
Summary of the invention
The technical problem to be solved by the present invention is to how improve plant product quality.The plant product is mankind's cultivation
The product that the necessary for human of plant production is wanted.The evaluation criterion of the quality is different because of the purposes of product difference.As food
The quality of product can withdraw deposit in the nutritional quality and edible quality of product；The quality of product as clothing raw material can withdraw deposit
On the fiber quality of product.Product quality is evaluated, two kinds of indexs are generally used, first is that biochemical composition and harmful substance contain
Amount, such as nutrition content and micronutrient levels；Second is that physical index, such as the shape of product, size, flavour, fragrance, color
Pool, skin depth is thin for kind, uniformity, fibre length, fibre strength etc., such as the ginning outturn of cotton.
In order to solve the above technical problems, present invention firstly provides protein to improve the application in plant product quality.
Protein provided by the present invention improve plant product quality in application in, the protein it is entitled
VdAL, be following A1) A2) or A3):
A1) amino acid sequence is the protein of sequence 1；
A2) residual by replacing and/or one or several amino acid being deleted and/or added in the amino acid sequence of sequence 1
Base obtains with the same function as A1) derived from protein；
A3) in A1) or the obtained fused protein of N-terminal A2) or/and C-terminal connection label.
Wherein, sequence 1 is made of 297 amino acid.
In order to make A1) in protein convenient for purifying, can in sequence table the amino terminal of protein shown in sequence 1 or
Carboxyl terminal connects upper label as shown in Table 1.
The sequence of table 1, label
| Label | Residue | Sequence |
| Poly-Arg | 5-6 (usually 5) | RRRRR |
| Poly-His | 2-10 (usually 6) | HHHHHH |
| FLAG | 8 | DYKDDDDK |
| Strep-tag II | 8 | WSHPQFEK |
| c-myc | 10 | EQKLISEEDL |
Above-mentioned A2) in VdAL can be artificial synthesized, can also first synthesize its encoding gene, then carry out biological expression and obtain.On
State A2) in the encoding gene of VdAL can be by the way that one or several amino will be lacked in DNA sequence dna shown in sequence 2 in sequence table
The codon of sour residue, and/or the missense mutation of one or several base-pairs is carried out, and/or connect at its 5 ' end and/or 3 ' ends
The coded sequence of label shown in table 1 obtains.
For above-mentioned VdAL in improving the application in plant product quality, the raising plant product quality can be for described in improve
The content of microelement and/or the raising plant in the content and/or the raising plant product of plant product nutritional ingredient
The palatability of product.The plant product nutritional ingredient concretely vitamin C, soluble sugar and/or protein.It is described micro
Element concretely Cu, Fe, K, Mn and/or Zn.The palatability may be embodied in dry rate and/or the decline of coarse-fibred content
On.
For above-mentioned VdAL in improving the application in plant product quality, the raising plant product quality can be to pass through promotion
The maturation of the plant product and the quality for improving the plant product.In one embodiment of the invention, the application of VdAL
Promote the maturation of cotton production, flower yield increases before the frost of cotton, and then improves cotton quality.
In improving the application in plant product quality, the plant can be planted above-mentioned VdAL for dicotyledon or unifacial leaf
Object.
Above-mentioned VdAL improve plant product quality in application in, the dicotyledon can for it is following a), b), c) or
D):
A) cucurbitaceous plant；
B) watermelon；
C) malvaceae plant；
D) cotton.
In order to solve the above technical problems, the present invention also provides biomaterials relevant to VdAL to improve plant product product
Application in matter.
Biomaterial relevant to VdAL provided by the present invention is improving the application in plant product quality；
The biomaterial is following B1) any one of to B20):
B1 the nucleic acid molecules of VdAL) are encoded；
B2) contain B1) expression cassettes of the nucleic acid molecules；
B3) contain B1) recombinant vectors of the nucleic acid molecules；
B4) contain B2) recombinant vector of the expression cassette；
B5) contain B1) recombinant microorganisms of the nucleic acid molecules；
B6) contain B2) recombinant microorganism of the expression cassette；
B7) contain B3) recombinant microorganism of the recombinant vector；
B8) contain B4) recombinant microorganism of the recombinant vector；
B9) contain B1) the transgenic plant cells systems of the nucleic acid molecules；
B10) contain B2) the transgenic plant cells system of the expression cassette；
B11) contain B3) the transgenic plant cells system of the recombinant vector；
B12) contain B4) the transgenic plant cells system of the recombinant vector；
B13) contain B1) Transgenic plant tissues of the nucleic acid molecules；
B14) contain B2) Transgenic plant tissue of the expression cassette；
B15) contain B3) Transgenic plant tissue of the recombinant vector；
B16) contain B4) Transgenic plant tissue of the recombinant vector；
B17) contain B1) the genetically modified plants organs of the nucleic acid molecules；
B18) contain B2) the genetically modified plants organ of the expression cassette；
B19) contain B3) the genetically modified plants organ of the recombinant vector；
B20) contain B4) the genetically modified plants organ of the recombinant vector.
Above-mentioned biomaterial relevant to VdAL is in improving the application in plant product quality, B1) nucleic acid molecules
Can be following b1) or b2) or b3) gene:
B1) nucleotide sequence is the cDNA molecule or DNA molecular of sequence 2 in sequence table；
B2 the nucleotide sequence) and b1) limited has 75% or 75% or more identity, and encodes the cDNA molecule of VdAL
Or genomic DNA molecule；
B3) the nucleotide sequence hybridization limited under strict conditions with b1), and encode the cDNA molecule or genome of VdAL
DNA molecular.
Wherein, the nucleic acid molecules can be DNA, such as cDNA, genomic DNA or recombinant DNA；The nucleic acid molecules can also
To be RNA, such as mRNA or hnRNA.
Wherein, sequence 2 is made of 894 nucleotide, protein shown in coded sequence 1.
Those of ordinary skill in the art can easily adopt by known method, such as the side of directed evolution and point mutation
Method is mutated the nucleotide sequence of coding VdAL of the invention.Those have and separate with the present invention by manually modified
The nucleotide sequence 75% of obtained VdAL or the nucleotide of higher identity, as long as encoding VdAL and having the function of VdAL,
It is derived from nucleotide sequence of the invention and to be equal to sequence of the invention.
Term " identity " used herein refers to the sequence similarity with native sequence nucleic acid." identity " includes and this hair
The nucleotide sequence of the protein of amino acid sequence composition shown in bright coding VdAL has 75% or higher or 85% or more
The nucleotide sequence of height or 90% or higher or 95% or higher identity.Identity can with the naked eye or computer software into
Row evaluation.Using computer software, the identity between two or more sequences can be indicated with percentage (%), can be used
To evaluate the identity between correlated series.
Above-mentioned biomaterial relevant to VdAL improve plant product quality in application in, the stringent condition be
In the solution of 2 × SSC, 0.1%SDS, hybridizes at 68 DEG C and wash film 2 times, each 5min, and in 0.5 × SSC, 0.1%SDS
Solution in, hybridize at 68 DEG C and wash film 2 times, each 15min；Or, 0.1 × SSPE (or 0.1 × SSC), 0.1%SDS
In solution, hybridizes under the conditions of 65 DEG C and wash film.
Above-mentioned 75% or 75% or more identity can be 80%, 85%, 90% or 95% or more identity.
Above-mentioned biomaterial relevant to VdAL improve plant product quality in application in, B2) described in containing compile
The expression cassette (VdAL expression casette) of the nucleic acid molecules of code VdAL is the DNA for referring to express VdAL in host cell, should
DNA not only may include the promoter for starting VdAL genetic transcription, may also include the terminator for terminating VdAL genetic transcription.Into one
Step, the expression cassette may also include enhancer sequence.Promoter for use in the present invention includes but is not limited to: composing type starting
Son is organized, the promoter and inducible promoter that organ and development are special.The example of promoter includes but is not limited to: cauliflower
The constitutive promoter 35S: the wound-inducible promoter from tomato of mosaic virus, leucine aminopeptidase (" LAP ",
Chao et al. (1999) Plant Physiol 120:979-992)；Chemical inducible promoter from tobacco, pathogenesis
1 (PR1) of correlation (is induced) by salicylic acid and BTH (diazosulfide -7- carbothioic acid S-methyl ester)；Tomato protease inhibitors
II promoter (PIN2) or LAP promoter (available methyl jasmonate induction)；Heat-shock promoters (United States Patent (USP) 5,187,
267)；Tetracycline inducible promoter (United States Patent (USP) 5,057,422)；Seed specific promoters, such as Millet Seed specificity
Promoter pF128 (CN101063139B (Chinese patent 200710099169.7)), the special promoter of seed storage protein matter
(for example, promoter (Beachy et al. (1985) of phaseolin, napin, oleosin and soybean beta conglycin
EMBO is J.4:3047-3053)).They can be used alone or are used in combination with other plant promoters.Institute cited herein
There is bibliography to be cited in full text.Suitable transcription terminator includes but is not limited to: Agrobacterium nopaline syntase terminator
(NOS terminator), cauliflower mosaic virus CaMV 35S terminator, tml terminator, pea rbcS E9 terminator and kermes ammonia
Sour and octopine synthase terminator (see, e.g.: Odell et al. (I985)Nature 313:810；Rosenberg et al.
(1987)Gene,56:125；Guerineau et al. (1991) Mol.Gen.Genet, 262:141；Proudfoot(1991)
Cell,64:671；Sanfacon et al. Genes Dev., 5:141；Mogen et al. (1990) Plant Cell, 2:1261；
Munroe et al. (1990) Gene, 91:151；Ballad et al. (1989) Nucleic Acids Res.17:7891；Joshi etc.
People (1987) Nucleic Acid Res., 15:9627).
The recombinant vector of the VdAL expression casette can be contained with existing expression vector establishment.The plant expression carries
Body includes double base agrobacterium vector and the carrier etc. that can be used for plant micropellet bombardment.As pAHC25, pBin438,
PCAMBIA1302, pCAMBIA2301, pCAMBIA1301, pCAMBIA1300, pBI121, pCAMBIA1391-Xa or
PCAMBIA1391-Xb (CAMBIA company) etc..The plant expression vector also may include 3 ' end non-translational regions of foreign gene
Domain, i.e., comprising polyadenylation signals and any other DNA fragmentation for participating in mRNA processing or gene expression.The polyadenylic acid letter
Number bootable polyadenylic acid is added to 3 ' ends of mRNA precursor, as Agrobacterium crown gall nodule induces (Ti) plasmid gene (such as nopaline
Synthase gene Nos), plant gene (such as soybean storage protein genes) 3 ' end transcription non-translational region all have similar functions.
When using gene constructed plant expression vector of the invention, enhancer, including translational enhancer or transcriptional enhancer also can be used,
These enhancer regions can be ATG initiation codon or neighboring region initiation codon etc., but must read with coded sequence
Frame is identical, to guarantee the correct translation of entire sequence.The source of the translation control signal and initiation codon be it is extensive,
Can be it is natural, be also possible to synthesis.Translation initiation region can come from transcription initiation region or structural gene.In order to just
In transgenic plant cells or plant are identified and screened, plant expression vector used can be processed, it can as being added
The coding expressed in plant can produce the enzyme of color change or gene (gus gene, luciferase genes of luminophor
Deng), the marker gene of antibiotic (if assigned the nptII gene to kanamycins and associated antibiotic resistance, assigns to herbicide
The bar gene of phosphinothricin resistance assigns the hph gene to antibiotic hygromycin resistance, and assigns to methotrexate resistance
Dhfr gene is assigned to the EPSPS gene of glyphosate) or (such as anti-herbicide base such as anti-chemical reagent marker gene
Cause), provide metabolism mannose ability mannose-6-phosphate isomerase gene.It, can not from the security consideration of genetically modified plants
Add any selected marker, transformed plant is directly screened with adverse circumstance.
For above-mentioned biomaterial relevant to VdAL in improving the application in plant product quality, the carrier can be matter
Grain, sticking grain, bacteriophage or viral vectors.The plasmid can be carrier pET42a (+).
For above-mentioned biomaterial relevant to VdAL in improving the application in plant product quality, the microorganism can be ferment
Mother, bacterium, algae or fungi, such as Escherichia coli.The Escherichia coli can be e. coli jm109.
For above-mentioned biomaterial relevant to VdAL in improving the application in plant product quality, the genetically modified plants are thin
Born of the same parents system, Transgenic plant tissue and genetically modified plants organ do not include propagation material.
In an embodiment of the invention, the expression cassette that the encoding gene of VdAL passes through the encoding gene containing VdAL
Recombinant vector import e. coli jm109 in obtain recombinant microorganism.The recombinant vector is by carrier pET42a's (+)
Sequence between NdeI and KpnI recognition site replaces with DNA molecular shown in 1-894 nucleotide of sequence 2 in sequence table
(i.e. VdAL gene), obtained recombinant vector.VdAL shown in the recombinant vector expressed sequence 1.The recombinant microorganism table
Up to VdAL shown in sequence 1.
Above-mentioned biomaterial relevant to VdAL is in improving the application in plant product quality, the raising plant product
Quality can be the content of microelement in the content and/or the raising plant product of the raising plant product nutritional ingredient
And/or improve the palatability of the plant product.The plant product nutritional ingredient concretely vitamin C, soluble sugar and/
Or protein.The microelement concretely Cu, Fe, K, Mn and/or Zn.The palatability may be embodied in dry rate and/or
In coarse-fibred content decline.
Above-mentioned biomaterial relevant to VdAL is in improving the application in plant product quality, the raising plant product
Quality can be for by promoting the maturation of the plant product to improve the quality of the plant product.In an implementation of the invention
In example, the application of VdAL promotes the maturation of cotton production, and flower yield increases before the frost of cotton, and then improves cotton quality.
For above-mentioned biomaterial relevant to VdAL in improving the application in plant product quality, the plant can be Shuangzi
Leaf plant or monocotyledon.
For above-mentioned biomaterial relevant to VdAL in improving the application in plant product quality, the dicotyledon can
For it is following a), b), c) or d):
A) cucurbitaceous plant；
B) watermelon；
C) malvaceae plant；
D) cotton.
In order to solve the above technical problems, the present invention also provides a kind of methods for improving plant product quality.
The method provided by the present invention for improving plant product quality, including VdAL, the biology are applied to purpose plant
Material or biological agent improve the purpose plant product quality；The active constituent of the biological agent is VdAL or the life
Object material.
In the method for above-mentioned raising plant product quality, the biological agent can be according to method 1) preparation, the method 1)
Are as follows: the recombinant microorganism is cultivated, the encoding gene of VdAL is expressed, obtains the biological agent.
In the method for above-mentioned raising plant product quality, the method 1) concretely it is following 11) and 12):
11) recombinant microorganism is cultivated, the encoding gene is expressed, obtains the micro- life of recombination for expressing the protein
Object culture；
12) thallus being crushed in the culture of microorganism obtains the biological agent.
In the method for above-mentioned raising plant product quality, the method 1) it may also include and the biological agent is done
It is dry.
In the method for above-mentioned raising plant product quality, the encoding gene can be B1) nucleic acid molecules.
In an embodiment of the invention, the recombinant microorganism is that will contain the expression cassette of the encoding gene of VdAL
Recombinant vector import e. coli jm109 obtained in recombinant microorganism.The recombinant vector is by carrier pET42a's (+)
Sequence between NdeI and KpnI recognition site replaces with DNA molecular shown in 1-894 nucleotide of sequence 2 in sequence table
(i.e. VdAL gene), obtained recombinant vector.VdAL shown in the recombinant vector expressed sequence 1.The recombinant microorganism table
Up to VdAL shown in sequence 1.
It is described to purpose plant application VdAL, the biomaterial or life in the method for above-mentioned raising plant product quality
Object preparation is concretely to purpose plant leaf blade application VdAL, the biomaterial or the biological agent, such as to purpose
Plant-spraying VdAL, the biomaterial or the biological agent, it is also possible to VdAL, the biomaterial or the biological agent
The seed of the purpose plant is handled, such as with the liquid preparation of VdAL, the biomaterial or the biological agent to the mesh
The seed of plant soak seed.The liquid preparation of VdAL, the biomaterial or the biological agent can be directly to use clear water
The liquid that dissolution or suspension VdAL, the biomaterial or the biological agent obtain.VdAL, the life are applied to purpose plant
The amount of object material or biological agent can be determined according to the type and/or growth period of the purpose plant.
In the method for above-mentioned raising plant product quality, the raising plant product quality can be the raising plant product
The content of microelement and/or fitting for the raising plant product in the content and/or the raising plant product of nutritional ingredient
Mouth property.The plant product nutritional ingredient concretely vitamin C, soluble sugar and/or protein.The microelement is specific
It can be Cu, Fe, K, Mn and/or Zn.The palatability may be embodied in dry rate and/or the decline of coarse-fibred content.
In the method for above-mentioned raising plant product quality, the raising plant product quality can be for by promoting the plant
The maturation of product and the quality for improving the plant product.In one embodiment of the invention, the application of VdAL promotes cotton
The maturation of product, flower yield increases before the frost of cotton, and then improves cotton quality.
In the method for above-mentioned raising plant product quality, the plant can be dicotyledon or monocotyledon.
In the method for above-mentioned raising plant product quality, the dicotyledon can for it is following a), b), c) or d):
A) cucurbitaceous plant；
B) watermelon；
C) malvaceae plant；
D) cotton.
In order to solve the above technical problems, the present invention also provides the biological agents to improve answering in plant product quality
With.
For above-mentioned biological agent in improving the application in plant product quality, the raising plant product quality can be raising
In the content and/or the raising plant product of the plant product nutritional ingredient described in the content and/or raising of microelement
The palatability of plant product.The plant product nutritional ingredient concretely vitamin C, soluble sugar and/or protein.It is described
Microelement concretely Cu, Fe, K, Mn and/or Zn.The palatability may be embodied under dry rate and/or coarse-fibred content
In drop.
For above-mentioned biological agent in improving the application in plant product quality, the raising plant product quality can be to pass through
Promote the maturation of the plant product and improves the quality of the plant product.In one embodiment of the invention, VdAL
Application promotes the maturation of cotton production, and flower yield increases before the frost of cotton, and then improves cotton quality.
For above-mentioned biological agent in improving the application in plant product quality, the plant can be dicotyledon or list
Leaf plant.
Above-mentioned biological agent improve plant product quality in application in, the dicotyledon can for it is following a), b),
C) or d):
A) cucurbitaceous plant；
B) watermelon；
C) malvaceae plant；
D) cotton.
In the present invention, VdAL or the biological agent can be because of VdAL or the biological agent concentration to the effect of different plants
Difference and there is different results.When applying VdAL or the biological agent, the concentration of VdAL or the biological agent can
(to be sought in purpose plant product as described in improving according to the purpose of crop or its kind and application VdAL or the biological agent
Support the content of substance) adjust the concentration of VdAL or the biological agent.
In an embodiment of the present invention, the biological agent, which dilutes 2000 times of obtained liquid, can be improved nutrition in watermelon
The content of substance and microelement.The biological agent, which dilutes 3000-5000 times of obtained liquid, can be improved the ginning outturn of cotton.
It is demonstrated experimentally that pacifying biological agent (VDLA) as the Lynn Vidali of active constituent using VdAL in the present invention can be improved plant
Product quality can also promote fruit maturation:
1, after the processing of Lynn Vidali peace, main nutrient composition and micronutrient levels increase in watermelon, and palatability improves.Victor
After sharp peace processing, Vc content is significantly improved, and improves 50.41%；Soluble sugar content improves 8.88%, albumen in dry matter
Matter content improves 14.2%, and protein content improves 0.62% in fresh watermelon, and dry rate decline 12.4% is coarse-fibred
Content has a small amount of decline.After the processing of Lynn Vidali peace, the quality of Ca, Cu, Fe, K, Mg, Mn, Na, P and Zn of every liter of fresh watermelon are equal
Have an increase, incrementss are respectively 12.75%, 16.54%, 19.21%, 16.46%, 1.05%, 20.45%, 2.13%,
10.02%, 19.73%；The mass content of Cu, Fe, K, Mn and Zn have increase in fresh watermelon, and incrementss are respectively
2.09%, 19.09%, 1.88%, 5.72% and 5.07%.
2, after the processing of Lynn Vidali peace, flower yield and ginning outturn increase before cotton frost.Shandong is handled with 3000 times of dilution of VDLA
No. 30, output increased 19.48% is spent before white；When being handled with 5000 times of VDLA of dilution, flower output increased before the frost of Shandong 30
21.25%, flower output increased 19.06% before 616 frosts, flower output increased 16.37% before the frost of Shandong 25.With 3000 times of dilution
When VDLA processing, ginning outturn increment rate is 0.24%；When being handled with 5000 times of dilution of VDLA, ginning outturn increment rate is 0.49%.
It is demonstrated experimentally that plant product quality can be improved in protein VdAL of the invention and its biological agent.
Detailed description of the invention
Fig. 1 is the western-blot testing result of VdAL in the sight of Lynn Vidali Anwai and Lynn Vidali peace.Wherein, A is Victor
Li Anwaiguan；B is the western-blot testing result of VdAL in Lynn Vidali peace, and blower expression enters in sample drying process
The relatively fine powder at air outlet position, king-tower indicate the normal powder in sample drying process.
Fig. 2 is the nutrient growth that Lynn Vidali peace can promote watermelon.Wherein, CK indicates that untreated watermelon, VdAL indicate
The watermelon of Lynn Vidali peace processing.
Fig. 3 is that Lynn Vidali peace can promote cucumber fruit setting and expand.Wherein, CK indicates untreated cucumber, and 300 indicate dilute
The cucumber of 300 times of VdAL processing is released, 600 indicate to dilute the cucumber of 600 times of VdAL processing, and 900 indicate to dilute 900 times
The cucumber of VdAL processing.
Fig. 4 is the growing way of the cucumber of different disposal.Wherein, CK indicates untreated cucumber, and 300 indicate to dilute 300 times
The cucumber of VdAL processing.
Fig. 5 is the aging situation of the cucumber of different disposal.Wherein, CK indicates untreated cucumber, 2000 × expression dilution
The cucumber of 2000 times of VdAL processing.
Fig. 6 is the fruit setting situation of the cherry tomato of different disposal.Wherein, CK indicates untreated cherry tomato, 2000 × expression
The cherry tomato of the VdAL processing of 2000 times of dilution.
Fig. 7 is the strawberry of different disposal.Wherein, CK indicates untreated strawberry, and 2000 × expression dilutes 2000 times
The strawberry of VdAL processing.
Fig. 8 is the muskmelon of different disposal.Wherein, CK indicates untreated muskmelon, and 2000 × expression dilutes 2000 times
The muskmelon of VdAL processing.
Fig. 9 is the pimento of different disposal.Wherein, CK indicates untreated pimento, and 1000 × expression dilutes 1000 times
The pimento of VdAL processing.
Figure 10 is the germination of the soya seeds of different disposal.Wherein, CK indicates untreated soya seeds, 1000 tables
Show dilution 1000 times VdAL processing soya seeds, 3000 indicate dilute 3000 times VdAL processing soya seeds, 5000
Indicate the soya seeds that the VdAL for diluting 5000 times is handled, 10000 indicate to dilute the soya seeds of 10000 times of VdAL processing,
20000 indicate to dilute the soya seeds of 20000 times of VdAL processing.
Figure 11 is the Soybean Seedlings Growth situation of different disposal.Wherein, CK indicates untreated soybean seedling, 5000 × table
Show the soybean seedling of the VdAL processing of 5000 times of dilution.
Specific embodiment
The present invention is further described in detail With reference to embodiment, and the embodiment provided is only for explaining
The bright present invention, the range being not intended to be limiting of the invention.
Experimental method in following embodiments is unless otherwise specified conventional method.
The materials, reagents and the like used in the following examples is commercially available unless otherwise specified.
E. coli jm109 in following embodiments is Beijing Suo Laibao Science and Technology Ltd product, and catalog number is
C1300。
Carrier pET42a (+) in following embodiments is Beijing Bi Chenglan Biotechnology Co., Ltd product, catalogue
Number be S18-16.
Early No. 58 of new land in following embodiments is Xinjiang brocade cotton seed industry Science and Technology Co., Ltd. product, Shandong 30,
616, Shandong 25 and Shandong grind cotton No. 24 as Shandong Nong Xingzhong industry Co., Ltd product.
Variety of watermelon gold Lido in following embodiments is that Shouguang is accumulated virtue commerce and trade Co., Ltd product.
Middle wheat 816 in following embodiments is Beijing Long Shengyuan development in science and technology Co., Ltd product.
Tomato variety Fuchs in following embodiments is Zhong Ye Co., Ltd of Jinpeng of Shouguang City product.
Magnificent green stem vegetable F1 in following embodiments is dragon species industry Co., Ltd of Hongqiao in Shanghai day product.
Cucumber variety saliva miscellaneous No. 1 in following embodiments is Shandong Nong Xingzhong industry Co., Ltd product.
Red No. 2 of cherry tomato kind capital in following embodiments is Beijing North Nong Lvheng development in science and technology Co., Ltd product.
Strawberry cultivars Feng Xiang in following embodiments is Beijing North Nong Lvheng development in science and technology Co., Ltd product.
Super emerald green precious fragrant melon variety in following embodiments is Jinan Rui Mao agricultural science and technology development corporation, Ltd. product.
Sweet Pepper Varieties farsighted excellent 816 in following embodiments are Jinan Rui Mao agricultural science and technology development corporation, Ltd. product.
Soybean varieties China soybean king in following embodiments is Jinan Rui Mao agricultural science and technology development corporation, Ltd. product.
The preparation of embodiment 1, Lynn Vidali peace
Lynn Vidali peace is developed by China Agricultural University, by Shandong Huimin Hu Hao Biotechnology Co., Ltd development and production.
1, the building of recombinant vector and recombinant bacterium
DNA molecular shown in 1-894 nucleotide of sequence 2, i.e. VdAL gene in artificial synthesized sequence table.By carrier
Sequence between NdeI the and KpnI recognition site of pET42a (+) replaces with shown in 1-894 nucleotide of sequence 2 in sequence table
DNA molecular (i.e. VdAL gene), keep pET42a (+) other sequences it is constant, obtain recombinant vector, by obtained recombination carry
Body is named as pET42a-VdAL.Protein VdAL shown in sequence 1 in recombinant vector pET42a-VdAL expressed sequence table.
Wherein, sequence 2 is made of 894 nucleotide, amino acid sequence shown in coded sequence 1.
PET42a-VdAL is imported in Agrobacterium tumefaciems e. coli jm109, obtains recombinant bacterium, the recombinant bacterium that will be obtained
It is named as JM109-pET42a-VdAL, protein shown in JM109-pET42a-VdAL expressed sequence 1.
2, the preparation of Lynn Vidali peace
JM109-pET42a-VdAL is fermented under the conditions of 37 DEG C and obtains pre-fermentation broth to OD value for 0.6, to pre- hair
IPTG is added in zymotic fluid, makes the concentration 1mM of IPTG, obtains induction liquid, induction liquid is fermented 6 hours under the conditions of 25 DEG C and is obtained
Fermentation liquid.Fermentation liquid is centrifuged, supernatant is abandoned, obtained bacterial sediment is crushed, is then spray-dried at 150 DEG C
Dry powder formulations (A in Fig. 1) is obtained, Lynn Vidali peace is named as.
The VdAL in Lynn Vidali peace is detected using the method for western-blot, primary antibody is VdAL antibody (VdAL
Antibody is that protein shown in sequence 2 is the polyclonal antibody that immunogene is prepared), the results showed that, contain in Lynn Vidali peace
VdAL (B in Fig. 1), Lynn Vidali peace in VdAL content be 7.28mg/g.
Lynn Vidali is pacified with clear water and is dissolved, respectively obtaining Lynn Vidali peace concentration is respectively 10g/3kg, 10g/6kg, 10g/
The liquid of 9kg, 1g/kg, 1g/2kg, 1g/2.5kg, 1g/3kg, 1g/4kg, 1g/5kg, 1g/7.5kg, 1g/10kg and 1g/20kg
These liquid are respectively designated as the VdAL of 300 times of dilution, the VdAL for diluting 600 times of VdAL, diluting 900 times, dilution by body
1000 times of VdAL, the VdAL for diluting 2000 times, the VdAL for diluting 2500 times, the VdAL for diluting 3000 times, 4000 times are diluted
VdAL, the VdAL for diluting 5000 times, the VdAL for diluting 7500 times, the VdAL for diluting 10000 times and the VdAL for diluting 20000 times.
Embodiment 2, Lynn Vidali peace can promote the growth of cotton
1, Lynn Vidali peace can promote the germination of cotton seeds
Respectively with 5000 times of dilution of VdAL of embodiment 1, dilute 7500 times of VdAL and dilute 10000 times VdAL and
Clear water impregnates new land early No. 58 seed 24 hours, and germination test is carried out in big flowerpot, and temperature is 25 DEG C, in sowing the 8th day, system
Count Cotton Seed rate.100 seeds of every kind of processing, experiment is in triplicate.
The results show that at the VdAL and clear water of 10000 times of the VdAL of 5000 times of dilution, the VdAL of 7500 times of dilution and dilution
The average germination percentage of cotton is respectively 74.00%, 75.50%, 71.00% and 70.23% after reason cotton seeds, dilutes 5000 times
VdAL, dilute 7500 times VdAL and dilute 10000 times VdAL processing cotton seeds after cotton average germination percentage difference
1.05 times, 1.08 times, 1.01 times that the average germination percentage of cotton after cotton seeds is handled for clear water, show that Lynn Vidali peace can be with
The germination percentage of cotton seeds is improved, the ability that Lynn Vidali peace improves Cotton Seed rate becomes with the variation of Lynn Vidali peace concentration
Change.
2, the yield of cotton can be improved in Lynn Vidali peace
Experiment one:
Field experimental design is as follows: experiment uses RANDOMIZED BLOCK DESIGN, 3 duplicate blocks is arranged, each duplicate block is set at random
Set nine treatment regions, the respectively VdAL of Shandong 30 handles 1st area, the VdAL of Shandong 30 handles 2nd area, the control treatment area of Shandong 30,
616VdAL handles 1st area, 616VdAL handles 2nd area, 616 control treatment areas, the VdAL of Shandong 25 handles 1st area, the VdAL of Shandong 25 handles 2
Area and the control treatment area of Shandong 25, the area of each treatment region are 0.2 mu.
Shandong 30, VdAL handles the seedling stage of the Shandong 30 in 1st area and Hua Ling is handled early period in the following manner respectively:
3000 times of dilution of VdAL for spraying embodiment 1 to cotton leaf by the way of top spray (machinery sprays), sprays 45- per acre
50kg obtains the Shandong 30 of the VdAL processing of 3000 times of dilution.Seedling stage and Hua Ling are respectively sprayed 1 time early period；Spraying time is in the morning
Or at dusk, preferably avoid the high temperature period at high noon；Forbid to mix with drug and fertilizer when spraying；If raining in 2 hours after spraying needs
Filling spray, it is primary to apply.
The VdAL for diluting 3000 times is replaced with 5000 times of dilution by the processing method that 1st area is handled according to the VdAL of Shandong 30
VdAL handles 2nd area to the VdAL of Shandong 30 and handles, other steps are constant, obtains the Shandong 30 of the VdAL processing of 5000 times of dilution
Number.
The VdAL for diluting 3000 times is replaced with clear water to Shandong 30 by the processing method that 1st area is handled according to No. 30 VdAL in Shandong
Control treatment area is handled, other steps are constant, obtains untreated Shandong 30.
The processing method in 1st area is handled according to the VdAL of Shandong 30, and 1st area and the VdAL processing 1 of Shandong 25 are handled to 616VdAL respectively
Area is handled, other steps are constant, respectively obtain dilution 3000 times VdAL processing 616, Shandong 25.
The VdAL for diluting 3000 times is replaced with 5000 times of dilution by the processing method that 1st area is handled according to the VdAL of Shandong 30
VdAL handles 2nd area to 616VdAL respectively and the VdAL of Shandong 25 handles 2nd area and handles, other steps are constant, respectively obtain
Dilution 5000 times VdAL processing 616, Shandong 25.
The VdAL for diluting 3000 times is replaced with clear water by the processing method that 1st area is handled according to the VdAL of Shandong 30, right respectively
616 control treatment areas and the control treatment area of Shandong 25 are handled, other steps are constant, respectively obtain untreated 616, Shandong
No. 25.
Statistics dilutes the Shandong 30, not of the Shandong 30 of 3000 times of VdAL processing, the VdAL processing for diluting 5000 times respectively
The Shandong 30 of processing, dilute 3000 times VdAL processing 616, dilute 5000 times VdAL processing 616, it is untreated 616,
Shandong 25, the Shandong 25 of the VdAL processing of 5000 times of dilution and the frost of untreated Shandong 25 of the VdAL processing of 3000 times of dilution
Preceding colored yield is with flower yield and total output after, frost, and the results are shown in Table 1.
Table 1, different disposal cotton average product
Note: processing 1 indicates that the processing that the VdAL for diluting 3000 times is carried out, processing 2 indicate that the VdAL for diluting 5000 times is carried out
Processing, CK indicate untreated (processing carried out with clear water).
The results show that the yield of cotton can be improved in the Lynn Vidali peace of debita spissitudo: with the VdAL processing for diluting 3000 times
Shandong 30, spends output increased 19.48% before white, total output improves 17.04%；When being handled with 5000 times of VdAL of dilution,
Flower output increased 21.25% before the frost of Shandong 30, total output improve 20.00%, flower output increased 19.06%, total output before 616 frosts
19.69% is improved, flower output increased 16.37%, total output improve 17.60% before the frost of Shandong 25.
Experiment two:
Field experimental design is as follows: experiment uses RANDOMIZED BLOCK DESIGN, 3 duplicate blocks is arranged, each duplicate block is set at random
Four treatment regions are set, respectively VdAL handles 1st area, VdAL handles 2nd area, VdAL handles 3rd area and control treatment area, each treatment region
Area be 10 mu.
Cotton No. 24 seedling stages are ground in the Shandong that VdAL handles 1st area and Hua Ling is handled early period in the following manner respectively: being adopted
3000 times of dilution of VdAL for spraying embodiment 1 to cotton leaf with the mode of top spray (machinery sprays), sprays 45- per acre
50kg, for locomotive shunting speed control at 6 kilometers of speed per hour or so, the Shandong for obtaining the VdAL processing of 3000 times of dilution grinds cotton No. 24.Seedling
Phase and Hua Ling are respectively sprayed 1 time early period；Spraying time in the morning or at dusk, preferably avoid the high temperature period at high noon；Forbid when spraying with
Drug and fertilizer mixing；It is applied once if raining to need to fill spray in 2 hours after spraying.
The VdAL for diluting 3000 times is replaced with VdAL pairs of 5000 times of dilution by the processing method that 1st area is handled according to VdAL
VdAL handles 2nd area and is handled, other steps are constant, and the Shandong for obtaining the VdAL processing of 5000 times of dilution grinds cotton No. 24.
The VdAL for diluting 3000 times is replaced with VdAL pairs of 10000 times of dilution by the processing method that 1st area is handled according to VdAL
VdAL handles 3rd area and is handled, other steps are constant, and the Shandong for obtaining the VdAL processing of 10000 times of dilution grinds cotton No. 24.
The processing method that 1st area is handled according to VdAL, will dilute 3000 times of VdAL replace with clear water to control treatment area into
Row processing, other steps are constant, obtain untreated Shandong and grind cotton No. 24.
Count each processing Shandong grind cotton No. 24 numbers of plant per acre, single-strain bell-forming number, the total bell number of mu, Single boll weight, ginning outturn and
Per mu yield, the average value of projects and increment compared with the control are as shown in table 2 and table 3.
Table 2, the Shandong respectively handled grind the average value and increment of No. 24 projects of cotton
Note: processing 1 indicates that the processing that the VdAL for diluting 3000 times is carried out, processing 2 indicate that the VdAL for diluting 5000 times is carried out
Processing, processing 3 indicate dilute 10000 times VdAL carry out processing, CK indicate untreated (processing carried out with clear water).
Table 3, the Shandong respectively handled grind the average value and increment of No. 24 projects of cotton
Note: processing 1 indicates that the processing that the VdAL for diluting 3000 times is carried out, processing 2 indicate that the VdAL for diluting 5000 times is carried out
Processing, processing 3 indicate dilute 10000 times VdAL carry out processing, CK indicate untreated (processing carried out with clear water).
The results show that the number of plant per acre, single-strain bell-forming number, the total bell of mu of cotton can be improved in the Lynn Vidali peace of debita spissitudo
Number, Single boll weight, ginning outturn and per mu yield: when with diluting 3000 times of VdAL processing, the total bell number of single-strain bell-forming number, mu, ginning outturn and mu
Yield has increase, and increment rate is respectively 4.5%, 1.98%, 0.24% and 1.76%；With 5000 times of dilution of VdAL processing
When, the total bell number of plant number, single-strain bell-forming number, mu, Single boll weight, ginning outturn and per mu yield have increase per acre, and increment rate is respectively
1.4%, 3.41%, 4.41%, 1.9%, 0.49% and 5.22%；When being handled with 10000 times of dilution of VdAL, the increasing of projects
Incrementss of the dosage not with projects when the VdAL processing of 5000 times of VdAL processing and dilution diluting 3000 times are high.Show
The quality and yield of cotton can be improved in the Lynn Vidali peace of application debita spissitudo.
The quality and yield of watermelon can be improved in embodiment 3, Lynn Vidali peace
Field experimental design is as follows: experiment uses RANDOMIZED BLOCK DESIGN, 3 duplicate blocks is arranged, each duplicate block is set at random
2 treatment regions, respectively VdLA treatment region and control treatment area are set, the area of each treatment region is 0.2 mu.
It is handled in the following manner in the variety of watermelon gold Lido of VdLA treatment region: using top spray (machinery sprays)
Mode sprays 2000 times of dilution of VdLA of embodiment 1 to water melon leaf, sprays 30kg per acre, obtains the VdAL of 2000 times of dilution
The watermelon of processing.The Seedling Stage from watermelon is sprayed, and is sprayed again after spraying 15 days for the first time 1 time, is sprayed 2 times altogether；Spraying time
In the morning or at dusk, the high temperature period at high noon is preferably avoided；It is not mixed with drug and fertilizer preferably when spraying；If 2 hours after spraying
Interior rainy needs, which fill spray, to be applied once.
According to the processing method of VdAL treatment region, the VdAL for diluting 2000 times is replaced with into clear water, control treatment area is carried out
Processing, other steps are constant, obtain untreated watermelon.
As a result, it has been found that Lynn Vidali peace has facilitation (Fig. 2) to the nutrient growth of watermelon and fruit setting.Count different disposal
The yield of watermelon in area, as a result, it has been found that, per plant number is 1.43 ± 0.5 after the VdAL processing watermelon of 2000 times of dilution, yield
Be 5200.2 ± 500kg/ mus, and the per plant number of untreated watermelon be 1.21 ± 0.5, yield be 3898.2 ±
500kg/ mus.Compared with untreated watermelon, the per plant number of the watermelon of Lynn Vidali peace processing improves 18.2%, and yield mentions
It is high by 33.4%.
The watermelon maturity period measure respectively the water content of different disposal watermelon, dry rate, Vc content, soluble sugar content,
Titratable acid content, soluble solid content, melon skin thickness, protein content, crude fiber content (table 4) and microelement
The content (table 5) of Ca, Cu, Fe, K, Mg, Mn, Na, P and Zn.Wherein, (it is fresh to account for watermelon to water content for dry matter in watermelon with dry rate
The percentage of weight) it will be measured after watermelon drying, Vc content is measured according to 2,6- dichloroindophenol titration, soluble
Sugared content is measured according to anthrone colorimetry, and titratable acid content is measured according to colorimetric method, soluble solid content,
Protein content is measured according to spectrophotometric metering method, and crude fiber content is carried out according to Wen Defa detection crude fibre method
Measurement, trace elements of Ca, the content of Cu, Fe, K, Mg, Mn, Na, P and Zn are measured using microelement analyzer.
The average content of table 4, each index of different disposal watermelon
| Index | It is untreated | VdLA processing |
| Water content (%) | 90.22 | 91.43 |
| Dry rate (%) | 9.78 | 8.57 |
| Vc(mg/100g) | 12.1 | 18.2 |
| Soluble sugar (%) | 6.83 | 7.44 |
| Titratable acid (%) | 0.09 | 0.09 |
| Soluble solid (%) | 9.1 | 9.1 |
| Melon skin thickness (cm) | 0.97 | 1.23 |
| Protein (%DW) | 5.54 | 6.33 |
| Protein (%FW) | 0.54 | 0.54 |
| Crude fibre (%DW) | 2.06 | 2.02 |
| Crude fibre (%FW) | 0.2 | 0.17 |
The average content of table 5, each microelement of different disposal watermelon
| Number | Index | It is untreated | VdAL processing |
| 1 | Moisture content (%) | 90.22 | 91.43 |
| 2 | Ca(mg/L) | 20.13 | 22.70 |
| 3 | Ca content (mg/100gFw) | 9.83 | 9.71 |
| 4 | Cu(mg/L) | 0.13 | 0.15 |
| 5 | Cu content (mg/100gFw) | 0.06 | 0.06 |
| 6 | Fe(mg/L) | 0.50 | 0.60 |
| 7 | Fe content (mg/100gFw) | 2.50 | 2.98 |
| 8 | K(mg/L) | 158.00 | 184.00 |
| 9 | K content (mg/100gFw) | 77.19 | 78.64 |
| 10 | Mg(mg/L) | 22.27 | 22.50 |
| 11 | Mg content (mg/100gFw) | 10.88 | 9.63 |
| 12 | Mn(mg/L) | 0.10 | 0.12 |
| 13 | Mn content (mg/100gFw) | 0.05 | 0.05 |
| 14 | Na(mg/L) | 7.20 | 7.35 |
| 15 | Na content (mg/100gFw) | 3.52 | 3.14 |
| 16 | P(mg/L) | 18.30 | 20.13 |
| 17 | P content (mg/100gFw) | 8.93 | 8.65 |
| 18 | Zn(mg/L) | 0.25 | 0.30 |
| 19 | Zn content (mg/100gFw) | 0.12 | 0.13 |
Note: 3, in 5,7,9,11,13,15,17 and 19 rows the content of each microelement refer both to it is each micro- in every liter of fresh watermelon
The quality of secondary element, the content of each microelement refers both to each micro in fresh watermelon in 2,4,6,8,10,12,14,16 and 18 rows
The content of element.
The results show that the water content of watermelon is increased slightly, and increases 1.34% after the processing of Lynn Vidali peace；Vc content is significant
It improves, improves 50.41%；Soluble sugar content improves 8.88%, and protein content improves 14.2% in dry matter, newly
Protein content improves 0.62% in fresh watermelon, and the content of titratable acid and soluble solid does not change, and dry
Rate decline 12.4%, coarse-fibred content has a small amount of decline.After showing the processing of Lynn Vidali peace, the main nutrient composition in watermelon
Content increases；And acidity does not obviously increase, dry rate and coarse-fibred content have decline, improve the palatability of watermelon.Dimension
After the processing of Dary peace, the quality of Ca, Cu, Fe, K, Mg, Mn, Na, P and Zn of every liter of fresh watermelon have increase, incrementss difference
It is 12.75%, 16.54%, 19.21%, 16.46%, 1.05%, 20.45%, 2.13%, 10.02%, 19.73%；It is fresh
The mass content of Cu, Fe, K, Mn and Zn have an increase in watermelon, incrementss are respectively 2.09%, 19.09%, 1.88%,
5.72% and 5.07%.Show Lynn Vidali peace can significantly improve in watermelon to human body healthy trace elements with household Cu, Fe, K, Mn and
The content of Zn.Above it is demonstrated experimentally that the quality of watermelon can be improved in Lynn Vidali peace.
Embodiment 4, Lynn Vidali peace can promote the growth of wheat, improve the yield of wheat
1, Lynn Vidali peace can promote the germination and nutrient growth of wheat
Experiment one:
By middle 816 seed of wheat respectively 1000 times of the dilution of embodiment 1 VdAL, dilute 2000 times VdAL, dilution
It soaks seed 12 hours in 5000 times of VdAL, the VdAL and clear water of 10000 times of dilution, then (is covered with two in germination box in germination box
Layer sterile water-soaked aseptic filter paper) at 25 DEG C vernalization 96 hours, statistics wheat seed germinating rate (wheat seed germinating
Standard are as follows: seed bud is a length of or half more than seed length, and root long is or is more than seed length).Every kind handles 100
Seed, experiment is in triplicate.
The results show that control and 1000 times of dilution of VdAL, diluting 2000 times of VdAL, the VdAL, dilute of 5000 times of dilution
The germination percentage for releasing 10000 times of VdAL and the wheat seed of clear water processing is respectively 95.31 ± 1%, 83.26 ± 1%, 92.51
± 1%, 97.67 ± 1% and 99.50 ± 1%, the VdAL of 1000 times of dilution, dilutes 2000 times of VdAL, dilutes 5000 times
VdAL, dilute 10000 times VdAL processing wheat seed after, germination percentage has been respectively increased -12.64%, -0.00%, 2.48%
With 4.40%.Show that the Lynn Vidali peace of suitable concentration can promote the germination of wheat seed.
Experiment two:
By middle 816 seed of wheat respectively in 5000 times of the dilution of embodiment 1 of VdAL, dilute 10000 times of VdAL and clear water
It is middle seed soaking 24 hours, be then seeded in soil, will sowing the same day be denoted as sowing the 1st day, sowing the 3rd day by wheat seedling from
It is taken out in soil, weighs wheat seedling fresh weight.
The results show that fresh with the single plant of the wheat of 5000 times of dilution of VdAL, the VdAL of 10000 times of dilution and clear water processing
Again it is respectively 0.128 ± 0.022g, 0.141 ± 0.022g and 0.111 ± 0.022g, dilutes 5000 times of VdAL and dilution
After 10000 times of VdAL seed soaking, 15.32% and 27.03%, the VdAL of 10000 times of dilution has been respectively increased in wheat single-strain fresh weight
Single-strain fresh weight after seed soaking is significantly higher than the wheat single-strain fresh weight after clear water seed soaking.Show that Lynn Vidali peace can promote wheat
Nutrient growth.
2, the yield of wheat can be improved in Lynn Vidali peace
Field experimental design is as follows: experiment uses RANDOMIZED BLOCK DESIGN, 3 duplicate blocks is arranged, each duplicate block is set at random
Five treatment regions are set, respectively VdAL handles 1st area, VdAL handles 2nd area, VdAL handles 3rd area, VdAL handles 4th area and control treatment
Area, the area of each treatment region are 0.5 mu.
It carries out handling wheat 1 time in the following manner in the blooming stage that VdAL handles the middle wheat 816 in 1st area: using top spray
The mode of (artificial spraying) sprays 2500 times of dilution of VdAL of embodiment 1 to wheat leaf blade, sprays 45-50kg per acre, obtains
The wheat of the VdAL processing of 2500 times of dilution.Spraying time in the morning or at dusk, preferably avoid the high temperature period at high noon；It is tight when spraying
Taboo is mixed with drug and fertilizer；It is applied once if raining to need to fill spray in 2 hours after spraying.
The VdAL for diluting 2500 times is replaced with VdAL pairs of 5000 times of dilution by the processing method that 1st area is handled according to VdAL
VdAL handles 2nd area and is handled, other steps are constant, obtains the wheat of the VdAL processing of 5000 times of dilution.
The VdAL for diluting 2500 times is replaced with VdAL pairs of 10000 times of dilution by the processing method that 1st area is handled according to VdAL
VdAL handles 3rd area and is handled, other steps are constant, obtains the wheat of the VdAL processing of 10000 times of dilution.
The VdAL for diluting 2500 times is replaced with VdAL pairs of 20000 times of dilution by the processing method that 1st area is handled according to VdAL
VdAL handles 4th area and is handled, other steps are constant, obtains the wheat of the VdAL processing of 20000 times of dilution.
The processing method that 1st area is handled according to VdAL, will dilute 2500 times of VdAL replace with clear water to control treatment area into
Row processing, other steps are constant, obtain untreated wheat.
The wheat yield of each processing is counted, the results show that the yield of wheat does not have after the VdAL processing wheat of 2500 times of dilution
Be improved, yield 8679kg/ha, dilution 5000 times VdAL, dilute 10000 times VdAL, dilute 20000 times VdAL and
The average product of the wheat of clear water processing is respectively 8942kg/ha, 9485kg/ha, 9231kg/ha and 8772kg/ha, dilution
5000 times of VdAL, the VdAL for diluting 10000 times, after diluting 20000 times of VdAL processing wheat, yield is respectively increased
1.94%, 8.13%, 5.23%.
Mass of 1000 kernel, number of productive ear, grain number per spike and the spike length of the wheat of different disposal are counted, the results are shown in Table 6.As a result it shows
Show, after the VdAL processing wheat of 5000 times of dilution, number of productive ear increases 1.5%, and grain number per spike increases 0.5%；Dilution 10000
VdAL again, number of productive ear increase 7%, and grain number per spike increases 3.5%；The VdAL of 20000 times of dilution, number of productive ear increase
2.3%, grain number per spike increases 3.7%.
Table 6, the mass of 1000 kernel of the wheat of different disposal, number of productive ear, grain number per spike and spike length average value
Note: processing 1 indicate dilute 20000 times VdAL carry out processing, processing 2 indicate dilute 10000 times VdAL into
Capable processing, processing 3 indicate that the processing that the VdAL for diluting 5000 times is carried out, processing 4 indicate what the VdAL for diluting 2500 times was carried out
Processing, CK indicate untreated (processing carried out with clear water).
Embodiment 5, Lynn Vidali peace can increase tomato per plant number
Field experimental design is as follows: experiment uses RANDOMIZED BLOCK DESIGN, 3 duplicate blocks is arranged, each duplicate block is set at random
Three treatment regions are set, respectively VdAL handles 1st area, VdAL handles 2nd area and control treatment area, the area of each treatment region are
0.1 mu.
After VdAL handles the coming into bloom and handle for the first time of the tomato variety Fuchs in 1st area the 15th day respectively under
The mode of stating sprays VdAL: spraying 1000 times of dilution of embodiment 1 to tomato leaf by the way of top spray (artificial spraying)
VdAL sprays the tomato that 45-50kg obtains the VdAL processing of 1000 times of dilution per acre, is denoted as after spraying on the day of the 1st time is sprayed
0th day.Spraying time in the morning or at dusk, preferably avoid the high temperature period at high noon；Forbid to mix with drug and fertilizer when spraying；Spray
It is applied once if raining to need to fill spray in 2 hours after applying.
The VdAL for diluting 1000 times is replaced with VdAL pairs of 3000 times of dilution by the processing method that 1st area is handled according to VdAL
VdAL handles 2nd area and is handled, other steps are constant, obtains the tomato of the VdAL processing of 3000 times of dilution.
The processing method that 1st area is handled according to VdAL, will dilute 1000 times of VdAL replace with clear water to control treatment area into
Row processing, other steps are constant, obtain untreated tomato.
The per plant number of the 20th day statistics tomato after spraying, the results show that the VdAL of 1000 times of dilution, dilution 3000
Times VdAL and clear water processing tomato after the average per plant number of tomato be respectively 34.9/plant, 40.4/plant and 33.3
After a/strain, the VdAL of 1000 times of dilution and the VdAL processing tomato of 3000 times of dilution, the per plant number of tomato is respectively increased
4.8% and 21.3%.Show that Lynn Vidali peace can promote tomato result.
Embodiment 6, Lynn Vidali peace can promote the growth of green stem vegetable
Field experimental design is as follows: experiment uses RANDOMIZED BLOCK DESIGN, 3 duplicate blocks is arranged, each duplicate block is set at random
Five treatment regions are set, respectively VdAL handles 1st area, VdAL handles 2nd area, VdAL handles 3rd area, VdAL handles 4th area and control treatment
Area, the area of each treatment region are 0.2 mu.
In one heart stage of two leaves that VdAL handles the magnificent hybridization green stem vegetable in 1st area, VdAL is sprayed 1 time in the following manner: being used
The mode of top spray (artificial spraying) sprays 2000 times of dilution of VdAL of embodiment 1 to tomato leaf, sprays 45-50kg per acre and obtains
To the green stem vegetable of 2000 times of dilution of VdAL processing, processing the 0th day is denoted as on the day of Lynn Vidali peace is sprayed.Spraying time is in the morning
Or at dusk, preferably avoid the high temperature period at high noon；Forbid to mix with drug and fertilizer when spraying；If raining in 2 hours after spraying needs
Filling spray, it is primary to apply.
The VdAL for diluting 2000 times is replaced with VdAL pairs of 2500 times of dilution by the processing method that 1st area is handled according to VdAL
VdAL handles 2nd area and is handled, other steps are constant, obtains the green stem vegetable of the VdAL processing of 2500 times of dilution.
The VdAL for diluting 2000 times is replaced with VdAL pairs of 3000 times of dilution by the processing method that 1st area is handled according to VdAL
VdAL handles 3rd area and is handled, other steps are constant, obtains the green stem vegetable of the VdAL processing of 3000 times of dilution.
The VdAL for diluting 2000 times is replaced with VdAL pairs of 4000 times of dilution by the processing method that 1st area is handled according to VdAL
VdAL handles 4th area and is handled, other steps are constant, obtains the green stem vegetable of the VdAL processing of 4000 times of dilution.
The processing method that 1st area is handled according to VdAL, will dilute 2000 times of VdAL replace with clear water to control treatment area into
Row processing, other steps are constant, obtain untreated green stem vegetable.
In processing the 20th day, the fresh weight of 50 plants of green stem vegetables of different disposal was counted respectively, the results show that 2000 times of dilution
Average 50 plants of blueness after VdAL, the VdAL for diluting 2500 times, the VdAL for diluting 3000 times, the VdAL for diluting 4000 times and clear water processing
Stalk dish fresh weight is respectively 2.960kg, 3.270kg, 3.835kg, 3.895kg and 1.912kg, the VdAL of 2000 times of dilution, dilution
After the VdAL processing green stem vegetable of 4000 times of 2500 times of VdAL, the VdAL for diluting 3000 times and dilution, 50 plants of green stem vegetable fresh weights point
54.8%, 71.0%, 100.6% and 103.7% is not improved.Show that Lynn Vidali peace can promote the growth of green stem vegetable.
The effect of embodiment 7, Lynn Vidali in other crops
1, Lynn Vidali peace can promote cucumber growth, improve yield, delay senescence
Field experimental design is as follows: experiment uses RANDOMIZED BLOCK DESIGN, 3 duplicate blocks is arranged, each duplicate block is set at random
Five treatment regions are set, respectively VdAL handles 1st area, VdAL handles 2nd area, VdAL handles 3rd area, VdAL handles 4th area and control treatment
Area, the area of each treatment region are 0.1 mu.
After VdAL handles cucumber variety saliva miscellaneous No. 1 of 1st area come into bloom and handle for the first time the 15th day respectively according to
Following manner sprays VdAL: by the way of top spray (artificial spraying) cucumber leaves are sprayed with 300 times of dilution of embodiment 1
VdAL sprays 45-50kg per acre, obtains the cucumber of the VdAL processing of 300 times of dilution, and the 1st time is sprayed Lynn Vidali peace and sprayed and is worked as
It, which is denoted as, sprays the 0th day.Spraying time in the morning or at dusk, preferably avoid the high temperature period at high noon；Forbid when spraying with drug and
Fertilizer mixing；It is applied once if raining to need to fill spray in 2 hours after spraying.
The VdAL for diluting 300 times is replaced with VdAL pairs of 600 times of dilution by the processing method that 1st area is handled according to VdAL
VdAL handles 2nd area and is handled, other steps are constant, obtains the cucumber of the VdAL processing of 600 times of dilution.
The VdAL for diluting 300 times is replaced with VdAL pairs of 900 times of dilution by the processing method that 1st area is handled according to VdAL
VdAL handles 3rd area and is handled, other steps are constant, obtains the cucumber of the VdAL processing of 900 times of dilution.
The VdAL for diluting 300 times is replaced with VdAL pairs of 2000 times of dilution by the processing method that 1st area is handled according to VdAL
VdAL handles 4th area and is handled, other steps are constant, obtains the cucumber of the VdAL processing of 2000 times of dilution.
The VdAL for diluting 300 times is replaced with clear water and carried out to control treatment area by the processing method that 1st area is handled according to VdAL
Processing, other steps are constant, obtain untreated cucumber.
It is spraying the 20th day, is counting the result situation of cucumber, as a result, it has been found that, Lynn Vidali pacifies the fruit setting that cucumber can be improved
Rate promotes expanding for cucumber, improves cucumber yield (Fig. 3), shows that Lynn Vidali peace can promote the nutrient growth and reproduction of cucumber
Growth.It is spraying the 20th day, is observing the growing way of cucumber, as a result, it has been found that, the growing way of the cucumber of Lynn Vidali peace processing is better than untreated
Cucumber growing way (Fig. 4), show that Lynn Vidali peace can promote the nutrient growth of cucumber.It is spraying the 45th day, is observing cucumber
Aging situation, as a result, it has been found that, the aging situation of untreated cucumber (figure more serious than the aging situation of the cucumber of Lynn Vidali peace processing
5), show that Lynn Vidali peace can delay the aging of cucumber.
2, Lynn Vidali peace can be improved the percentage of fertile fruit of cherry tomato, improve yield
Field experimental design is as follows: experiment uses RANDOMIZED BLOCK DESIGN, 3 duplicate blocks is arranged, each duplicate block is set at random
Two treatment regions, respectively VdAL treatment region and control treatment area are set, the area of each treatment region is 1 mu.
In the cherry tomato kind capital of VdAL treatment region red No. 2 florescence, VdAL is sprayed 1 time in the following manner: using top
The mode of (artificial spraying) is sprayed to 2000 times of dilution of VdAL of cherry tomato blade spraying embodiment 1, sprays 45-50kg per acre,
The cherry tomato of the VdAL processing of 2000 times of dilution is obtained, is denoted as processing the 0th day on the day of Lynn Vidali peace is sprayed.Spraying time is in morning
Morning or dusk preferably avoid the high temperature period at high noon；Forbid to mix with drug and fertilizer when spraying；If raining in 2 hours after spraying
Needing to fill spray, it is primary to apply.
According to the processing method of VdAL treatment region, the VdAL for diluting 2000 times is replaced with into clear water, control treatment area is carried out
Processing, other steps are constant, obtain untreated cherry tomato.
In processing the 7-10 days, the result situation of cherry tomato is observed, as a result, it has been found that, cherry tomato can be improved in Lynn Vidali peace
Percentage of fertile fruit improves cherry tomato yield (Fig. 6), shows that Lynn Vidali peace can promote the growth of cherry tomato.
3, Lynn Vidali peace can promote the growth of strawberry
Field experimental design is as follows: experiment uses RANDOMIZED BLOCK DESIGN, 3 duplicate blocks is arranged, each duplicate block is set at random
Two treatment regions, respectively VdAL treatment region and control treatment area are set, the area of each treatment region is 1 mu.
In the phase in florescence that the strawberry cultivars of VdAL treatment region are Feng Xiang, VdAL is sprayed 1 time in the following manner: using top
The mode of spray (artificial spraying) sprays 2000 times of dilution of VdAL of embodiment 1 to Strawberry Leaves, sprays 45-50kg per acre, obtains
To the strawberry of 2000 times of dilution of VdAL processing, processing the 0th day is denoted as on the day of Lynn Vidali peace is sprayed.Spraying time in the morning or
At dusk, the high temperature period at high noon is preferably avoided；Forbid to mix with drug and fertilizer when spraying；If needs are rained after spraying in 2 hours
Filling spray, it is primary to apply.
According to the processing method of VdAL treatment region, the VdAL for diluting 2000 times is replaced with into clear water, control treatment area is carried out
Processing, other steps are constant, obtain untreated strawberry.
In processing the 7-10 days, the growing state of strawberry is observed, as a result, it has been found that, Lynn Vidali pacifies the nutrition that can promote strawberry
It grows (Fig. 7).
4, Lynn Vidali peace can promote the growth of muskmelon
Field experimental design is as follows: experiment uses RANDOMIZED BLOCK DESIGN, 3 duplicate blocks is arranged, each duplicate block is set at random
Two treatment regions, respectively VdAL treatment region and control treatment area are set, the area of each treatment region is 1 mu.
In the seedling stage of the precious fragrant melon variety of the super kingfisher of VdAL treatment region, VdAL is sprayed 1 time in the following manner: using top
The mode of spray (artificial spraying) sprays 2000 times of dilution of VdAL of embodiment 1 to muskmelon blade, sprays 45-50kg per acre, obtains
To the muskmelon of 2000 times of dilution of VdAL processing, processing the 0th day is denoted as on the day of Lynn Vidali peace is sprayed.Spraying time in the morning or
At dusk, the high temperature period at high noon is preferably avoided；Forbid to mix with drug and fertilizer when spraying；If needs are rained after spraying in 2 hours
Filling spray, it is primary to apply.
According to the processing method of VdAL treatment region, the VdAL for diluting 2000 times is replaced with into clear water, control treatment area is carried out
Processing, other steps are constant, obtain untreated muskmelon.
In processing the 30th day, the growing state of muskmelon is observed, as a result, it has been found that, Lynn Vidali peace can promote the nutrition of muskmelon raw
Long (Fig. 8).
5, Lynn Vidali peace can promote the growth of pimento
Field experimental design is as follows: experiment uses RANDOMIZED BLOCK DESIGN, 3 duplicate blocks is arranged, each duplicate block is set at random
Two treatment regions, respectively VdAL treatment region and control treatment area are set, the area of each treatment region is 1 mu.
In the Sweet Pepper Varieties of VdAL treatment region farsighted excellent 816 florescence, VdAL sprays 1 time in the following manner: using top spray
The mode of (artificial spraying) sprays 45-50kg per acre, obtains to 1000 times of dilution of VdAL of pimento blade spraying embodiment 1
The pimento of the VdAL processing of 1000 times of dilution, is denoted as processing the 0th day on the day of Lynn Vidali peace is sprayed.Spraying time in the morning or is close to
Evening preferably avoids the high temperature period at high noon；Forbid to mix with drug and fertilizer when spraying；It needs to mend if raining in 2 hours after spraying
It sprays primary.
According to the processing method of VdAL treatment region, the VdAL for diluting 1000 times is replaced with into clear water, control treatment area is carried out
Processing, other steps are constant, obtain untreated pimento.
In processing the 7-10 days, the growing state of pimento is observed, as a result, it has been found that, after spraying Lynn Vidali peace, pimento growing way is grown sturdily
It is strong, blade is dark green (Fig. 9), show that Lynn Vidali peace can promote the nutrient growth of pimento.
6, Lynn Vidali peace can promote the emergence and growth of soybean
By soybean varieties China soybean king the seed VdAL at 1000 times of the dilution of embodiment 1,3000 times of dilution respectively
VdAL, it the VdAL for diluting 5000 times, the VdAL for diluting 10000 times, dilutes and soaks seed 24 hours in 20000 times of VdAL and clear water,
Then in pallet (being covered with two layers of aseptic filter paper with sterile water-soaked in pallet) at 25 DEG C vernalization 72 hours, count soybean
Percentage of seedgermination.100 seeds of every kind of processing, experiment is in triplicate.
After (Figure 10) as the result is shown, the VdAL of 3000 times of dilution and the VdAL soya seeds of 5000 times of dilution, germination percentage is equal
It is improved.Show that Lynn Vidali peace can promote the germination of soya seeds.
Experiment two:
Soybean varieties China soybean king seed is soaked seed 24 in 5000 times of the dilution of embodiment 1 of VdAL and clear water respectively
Hour, be then seeded in soil, will sowing the same day be denoted as sowing the 1st day, sowing the 20th day by soybean seedling from soil
It takes out, weighs soybean seedling fresh weight (Figure 11).
The results show that being respectively 1.59 ± 0.3g with the single-strain fresh weight of 5000 times of dilution of the VdAL and clear water soybean handled
With 1.36 ± 0.3g, after the VdAL seed soaking of 5000 times of dilution, soybean per plant fresh weight improves 16.91%；With 5000 times of dilution
The aerial part single-strain fresh weight of the soybean of VdAL and clear water processing is respectively 1.36 ± 0.3g and 1.06 ± 0.3g, dilutes 5000 times
VdAL seed soaking after, soybean aerial part single-strain fresh weight improves 28.30%, be significantly higher than clear water processing soybean overground part
Divide single-strain fresh weight.Show that Lynn Vidali peace can promote the nutrient growth of soybean.
Claims (11)
1. protein is improving the application in plant product quality；The protein is the protein that amino acid sequence is sequence 1；
The plant product quality that improves is to improve the content and/or the raising plant product of the plant product nutritional ingredient
The content of middle microelement and/or the palatability for improving the plant product；
The plant product nutritional ingredient is vitamin C, soluble sugar and/or protein；
The microelement is Cu, Fe, K, Mn and/or Zn；
The palatability is embodied in dry rate and/or the decline of coarse-fibred content.
2. applying according to claim 1, it is characterised in that: the plant is dicotyledon or monocotyledon.
3. application according to claim 2, it is characterised in that: the dicotyledon be it is following a), b) or c):
A) cucurbitaceous plant；
B) watermelon；
C) malvaceae plant.
4. a kind of method for improving plant product quality, including to protein or life described in purpose plant application claim 1
Object preparation；The active constituent of the biological agent is protein described in claim 1；
The plant product quality that improves is to improve the content and/or the raising plant product of the plant product nutritional ingredient
The content of middle microelement and/or the palatability for improving the plant product；
The plant product nutritional ingredient is vitamin C, soluble sugar and/or protein；
The microelement is Cu, Fe, K, Mn and/or Zn；
The palatability is embodied in dry rate and/or the decline of coarse-fibred content.
5. according to the method described in claim 4, it is characterized by: the biological agent is according to method 1) preparation, the method
1) are as follows: recombinant microorganism of the culture containing protein coding gene described in claim 1 makes the encoding gene of the protein
Expression, obtains the biological agent.
6. according to the method described in claim 5, it is characterized by: the method 1) be it is following 11) and 12):
11) recombinant microorganism containing protein coding gene described in claim 1 is cultivated, the encoding gene is expressed,
Obtain expressing the recombinant microorganism culture of the protein；
12) thallus being crushed in the culture of microorganism obtains the biological agent.
7. according to any the method for claim 4-6, it is characterised in that: the plant is that dicotyledon or unifacial leaf are planted
Object.
8. according to the method described in claim 7, it is characterized by: the dicotyledon be it is following a), b) or c):
A) cucurbitaceous plant；
B) watermelon；
C) malvaceae plant.
9. biological agent described in claim 4 or 5 is improving the application in plant product quality；
The plant product quality that improves is to improve the content and/or the raising plant product of the plant product nutritional ingredient
The content of middle microelement and/or the palatability for improving the plant product；
The plant product nutritional ingredient is vitamin C, soluble sugar and/or protein；
The microelement is Cu, Fe, K, Mn and/or Zn；
The palatability is embodied in dry and/or the decline of coarse-fibred content.
10. applying according to claim 9, it is characterised in that: the plant is dicotyledon or monocotyledon.
11. application according to claim 10, it is characterised in that: the dicotyledon be it is following a), b) or c):
A) cucurbitaceous plant；
B) watermelon；
C) malvaceae plant.
Priority Applications (6)
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| CN201510691295.6A CN105198967B (en) | 2015-10-22 | 2015-10-22 | Protein VdAL is improving the application in plant product quality |
| PCT/CN2016/088074 WO2017067214A1 (en) | 2015-10-22 | 2016-07-01 | Applications of protein vdal in improving output, product quality and drought resistance of plant and in improving fruit coloring of plant |
| EP16856654.5A EP3351555A4 (en) | 2015-10-22 | 2016-07-01 | Applications of protein vdal in improving output, product quality and drought resistance of plant and in improving fruit coloring of plant |
| US15/769,746 US10806145B2 (en) | 2015-10-22 | 2016-07-01 | Applications of protein VdAL in improving output, product quality and drought resistance of plant and in improving fruit coloring of plant |
| CA3002881A CA3002881A1 (en) | 2015-10-22 | 2016-07-01 | Applications of protein vdal in improving output, product quality and drought resistance of plant and in improving fruit coloring of plant |
| CL2018001026A CL2018001026A1 (en) | 2015-10-22 | 2018-04-20 | Applications of the protein vdal in the improvement of the yield, quality of the product and resistance to the drought of plants and in the promotion of the coloration of the fruits of the plant |
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| EP3351555A4 (en) * | 2015-10-22 | 2019-08-21 | Pherobio Technology Co., Ltd | Applications of protein vdal in improving output, product quality and drought resistance of plant and in improving fruit coloring of plant |
| CN115413678A (en) * | 2022-09-20 | 2022-12-02 | 中捷四方生物科技股份有限公司 | Wettable powder containing verticillium dahliae activator protein and application |
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| allergen Asp f2-like protein [Verticillium dahliae]，Accession No: AY524789.1;Wang，J. Y. et al;《Genbank数据库》;20060130;全文 |
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| Purification of protein from culture filtrates of Fusarium oxysporum that induces ethylene and necrosis in leaves of Erythroxylum coca;Bryan A. Bailey;《Phytopatholgogy》;19951231;第85卷(第10期);第1250-1255页 |
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