CN105567709A - Agaricus bisporus PPO gene segment and application thereof in lowering of PPO enzyme activity - Google Patents
Agaricus bisporus PPO gene segment and application thereof in lowering of PPO enzyme activity Download PDFInfo
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Abstract
The invention discloses an agaricus bisporus PPO gene segment which is shown as a sequence table SEQ ID NO:1. Application of the segment in lowering of the PPO enzyme activity is achieved through the four following steps that 1, a cDNA segment sequence for cloning an agaricus bisporus PPO gene is separated; 2, an antisense expression vector of the agaricus bisporus PPO gene is constructed; 3, genetic transformation of agaricus bisporus is performed; 4, the PPO enzyme activity level and browning degree of transgenic agaricus bisporus are tested and determined. Accordingly, the PPO enzyme activity level in the transgenic agaricus bisporus fruiting body obtained through the method is lowered by 41%-62%, postharvest storage browning is inhibited, the purpose of regulating and controlling the PPO enzyme activity level through the antisense PPO gene to inhibit postharvest browning of the agaricus bisporus is achieved, and the wide prospect is achieved when the gene segment is applied to improvement of the quality that the agaricus bisporus is prone to be browned.
Description
Technical field
The present invention relates to the field of transgenic technology of fungi, be specifically related to a kind of Twospore Mushroom (Agaricusbisporus) PPO gene cDNA fragment and reducing the application in PPO enzymic activity.
Background technology
Polyphenoloxidase (polyphenoloxidase), referred to as PPO, is the key enzyme causing Twospore Mushroom to adopt rear brown stain.Brown stain can cause Twospore Mushroom to be adopted, and rear commodity declines, shortening shelf-lives, and the mushroom caused because of brown stain every year adopts rear rate of loss up to 30% ~ 35%.PPO tool cresolase (cresolase in Twospore Mushroom, and catechol-oxydase (catecholase EC1.14.1.18), EC1.10.3.1) active, single phenol and pyrocatechol can be acted on, there is the reaction of non-enzymatic electrophilic polymerization in reaction product quinones, form melanochrome, cause brown stain.Research shows, by reducing the activity of PPO enzyme, fruits and vegetables class can be suppressed to adopt rear brown stain.Such as, utilize the physico-chemical processes such as low temperature, Cu sequestrant, Glabrene to suppress the activity of PPO enzyme, double-spore mushroom browning can be alleviated, prolong storage period.In potato, by suppressing PPO genetic expression, can brown stain in inhibition of potato.Therefore, as the activity by suppressing the expression of PPO gene to reduce PPO enzyme, realize the suppression of Twospore Mushroom being adopted to rear brown stain, can be the improvement that Twospore Mushroom adopts rear brown stain and technical basis is provided, to raising Twospore Mushroom Post-harvest quality, prolong storage period, be useful, feasible, come out through the retrieval document announcement that so far there are no is correlated with and new strains.
Summary of the invention
The technical problem to be solved in the present invention is to provide a kind of Twospore Mushroom PPO gene cDNA fragment and is reducing the application in PPO enzymic activity.
Solve the problems of the technologies described above and adopt following technical scheme:
This PPO gene cDNA fragment nucleotide sequence is as shown in SEQ ID NO:1, and aminoacid sequence is as shown in SEQ ID NO:2.
Twospore Mushroom PPO gene cDNA fragment and the application in reduction PPO enzymic activity are carried out as follows:
(1) separating clone Twospore Mushroom PPO gene cDNA fragment sequence: extract Twospore Mushroom sporophore total serum IgE, reverse transcription synthesis cDNA, to be compared design degenerate primer by the Twospore Mushroom PPO gene order known with GenBank, reverse transcription PCR is utilized to obtain amplified production, amplified production reclaims through sepharose, to reclaim in product cloning to pMD18-T carrier and to check order, biology tool is utilized to analyze sequencing result, with the comparison of Twospore Mushroom PPO gene, be defined as the conserved regions fragment in PPO gene and copper desmoenzyme active centre, and called after antiPPO;
(2) structure of Twospore Mushroom Anti-sense PPO gene antisense expression vector: utilize restriction enzyme to be connected with the expression vector pCamG cut through enzyme by antiPPO gene with ligase enzyme, antiPPO gene is oppositely inserted pCamG, obtain Twospore Mushroom restructuring binary expression vector, called after pCamG-antiPPO;
(3) genetic transformation of Twospore Mushroom: above-mentioned expression vector pCamG-antiPPO is utilized Agrobacterium-mediated transformation Twospore Mushroom, obtain the transgenosis mushroom with hygromycin resistance, extract transgenosis mushroom mycelium DNA, utilize special upper primer and lower primer to carry out pcr amplification qualification, determine to obtain Twospore Mushroom transgenosis mycelium;
(4) mensuration of transgenosis Twospore Mushroom PPO enzyme activity level and BPH resistant rice variety degree: utilize straw-manure compost half sponge process to carry out cultivation fruiting, utilize spectrophotometry transgenosis Twospore Mushroom and PPO enzyme activity level in non-transgenosis mushroom, transgenosis Twospore Mushroom PPO enzyme running water is flat significantly to be reduced; Transgenosis Twospore Mushroom and non-non-transgenic control 15 DEG C carry out the contrast experiment of browning degree when storing, during transgenosis Twospore Mushroom 15 DEG C storage, browning degree is suppressed, illustrate and proceed to Anti-sense PPO gene in Twospore Mushroom, remarkable reduction PPO enzyme activity level, transgenosis mushroom BPH resistant rice variety quality is obviously improved.
The invention has the beneficial effects as follows, separating clone PPO gene cDNA sequence from Twospore Mushroom, the coding region of this gene is inserted into Twospore Mushroom binary expression vector in an inverse manner and proceeds in Twospore Mushroom, compared with non-transgenosis mushroom, transgenosis mushroom PPO enzyme activity level decline 41%-62%, adopts rear storage brown stain and is suppressed, therefore, present invention achieves and utilize Anti-sense PPO gene to regulate and control Twospore Mushroom PPO enzyme activity level, significant to improvement Twospore Mushroom BPH resistant rice variety quality.
Accompanying drawing explanation
Fig. 1 is that the present invention's Twospore Mushroom binary expression vector used pCamG-antiPPOb builds schematic diagram.
Fig. 2 is transgenosis agaricus bisporus mycelia (1) (2) and sporophore (3) (4) aspect graph.
Fig. 3 is that the PCR of transgenosis mushroom detects electrophorogram.
Fig. 4 is transgenosis Twospore Mushroom and contrast PPO enzymic activity comparison diagram in mushroom fruitbody.
Fig. 5 is transgenosis Twospore Mushroom and contrast brown stain value (L value) comparison diagram when mushroom fruitbody 15 DEG C is stored.
Embodiment
The present invention is also described in further detail with reference to accompanying drawing below in conjunction with embodiment.Following experimental technique, if no special instructions, is ordinary method.Reagent used, if no special instructions, is the conventional reagent purchased from biochemical reagents company.Described contrast Twospore Mushroom is all non-transgenosis Twospore Mushroom.
This PPO gene cDNA fragment nucleotide sequence is as shown in SEQ ID NO:1, and aminoacid sequence is as shown in SEQ ID NO:2.
Twospore Mushroom PPO gene cDNA fragment and the application in reduction PPO enzymic activity are carried out as follows:
(1) separating clone Twospore Mushroom Anti-sense PPO gene cDNA fragment sequence
Adopt improved Trizol method to extract Twospore Mushroom sporophore total serum IgE, by 0.1g sporophore liquefied ammonia grind into powder, add the mixing of 1mLTrizol reagent (Invitrogen company) concuss; Leave standstill 20min, 12000rpm on ice, 4 DEG C of centrifugal 15min; Get supernatant, add 1/5 volume of chloroform, vortex mixes, and room temperature leaves standstill 5min, 12000rpm, 4 DEG C of centrifugal 20min; Get supernatant, add equal-volume chloroform, vortex mixes, and room temperature leaves standstill 5min, 12000rpm, 4 DEG C of centrifugal 20min; Get supernatant, add 1/2 volume isopropanol, 1/2 volume high-salt buffer (0.8M Trisodium Citrate, 1.2MNaCl), reversing mixing, place 30min, 12000rpm, 4 DEG C of centrifugal 20min for-20 DEG C; Abandon supernatant, add the ethanol of 1mL75%, 12000rpm, 4 DEG C, 10min; Abandon supernatant, Air drying; Add 30 μ lDEPC water dissolution, carry out electrophoresis and UV spectrophotometer measuring;
Utilize ThermoScript II that the first chain cDNA is synthesized in total serum IgE reverse transcription, in the 1.5mL centrifuge tube of DEPC process, first add Oligo (dT) 18primer of about 1 μ g total serum IgE and 1 μ l0.5 μ g/ μ l, careful mixing, 70 DEG C of insulation 5min, are placed on ice immediately; Add following reagent respectively in order: 4 μ l5 × Frist-strandBuffer, 1 μ ldNTPmix (10mM), 2 μ l100mMDTT, 1 μ lRNase inhibitor (40U/ μ l), 1 μ lSuperScript
tMiIReverseTranscriptase, then adds DEPC water to 20 μ l; Careful mixing, centrifugal 5 seconds of room temperature, at the bottom of all solution collection to pipe, 42 DEG C of insulation 50min; 72 DEG C of reaction 15min, cooled on ice ,-20 DEG C save backup;
To be compared design degenerated primer by the fungi PPO gene order known with GenBank, search at NCBI again and download fungi PPO gene order, Clustalx is utilized to carry out sequence alignment, judge that the copper of fungi PPO gene is in conjunction with CuA and CuB conserved regions according to sequence similarity, according to conserved regions sequences Design degenerated primer, primer sequence is as follows: upstream primer AbPPO-F:5 '-CAACGGCCTCTTCCCC (C/G) (C/G) (A/C) TGGCA (C/T) (A/C) G-3 ' (SEQIDNO:3); Downstream primer AbPPO-R:5 '-GGTCGACGTTGCAGTGGTG (A/C) A (G/T) (A/G) (C/T) A (A/G) AA-3 ' (SEQIDNO:4).With above-mentioned cDNA first chain for template, carry out pcr amplification by this to primer, PCR system is as follows:
10×EXTaqbuffer5μl
AbPPO-F(10mM)1μl
AbPPO-R(10mM)1μl
dNTPs(200μM)4μl
EXTaqpolymerase(5U/μl)0.5μl
Reverse transcription cDNA product 2 μ l
PCRwater36.5μl
Amplification condition is: PCR parameter is: 94 DEG C of 3min1cycle; 94 DEG C of 30sec, 63 DEG C of 1min, 72 DEG C of 1min, 15cycles; 94 DEG C of 30sec, 56 DEG C of 1min, 72 DEG C of 1min, 20cycles; 72 DEG C of 10min1cycle.Pcr amplification product reclaims through sepharose, reclaims product cloning in pMD18-T carrier, obtains pMD18-T-antiPPOa carrier, order-checking, obtains Twospore Mushroom PPO gene conserved regions fragment; Be antiPPO by this unnamed gene.
(2) structure of Twospore Mushroom antiPPO gene binary expression vector pCamG-antiPPO
According to the Twospore Mushroom PPO gene coding region design primer obtained in step (1), upstream primer be antiPPO-BEIIF:5 '-
gCAGGTCACCaACGGCCTCTTCCCCGGATGGCATCG-3 ' (SEQIDNO:5); Downstream primer be antiPPO-BgIIR:5 '-
tCGAGATCTgGTCGACGTTGCAGTGGTGCAGGTAGAA-3 ' (SEQIDNO:6). the sequence of underlined region is appended sequence, for cloning with the connection of expression vector.With the carrier pMD18-T-antiPPOa plasmid DNA of step (1) for template, amplification coding region sequence, PCR system is as follows:
2×PrimeSTARMaxDNAPolymerase25μl
antiPPO-BgIIF(10mM)1μl
antiPPO-BEIIR(10mM)1μl
PMD18-T-antiPPOa plasmid DNA (20ng/ μ L) 1 μ l
PCRwater22μl
Amplification condition is: 94 DEG C of 3min1cycle; 98 DEG C of 10sec, 56 DEG C of 15sec, 72 DEG C of 5sec, 35cycles; 72 DEG C of 10min1cycle.Object fragment is reclaimed according to TaKaRaAgaroseGelDNAExtractionKit after PCR reaction terminates.
The DNA two sections reclaimed adds A, and reaction system is as follows:
10×ExTaqBuffer1μl
dNTPs(2.5mM)0.4μl
Reclaim DNA product 5 μ l
EXTaqpolymerase(5U/μl)0.2μl
ddH
2O3.4μl。
Reaction conditions is: 95 DEG C of reaction 2min, 72 DEG C of reaction 15min.Reclaim product and add A rear clone in pMD18-T carrier, obtain pMD18-T-antiPPOb carrier.
With Bgl II and BstE II double digestion pMD18-T-antiPPOb carrier, it is as follows that enzyme cuts system:
PMD18-T-antiPPOb vector plasmid (200ng/ μ l) 10 μ l
10×HBuffer5μl
BglⅡ1μl
BstEⅡ1μl
ddH
2O33μl
37 DEG C of enzymes are cut 2 hours rear electrophoresis and are cut glue recovery;
With Bg1 II and BstE II double digestion pCamG carrier, it is as follows that enzyme cuts system:
PCamG vector plasmid (200ng/ μ l) 5 μ l
10×HBuffer5μl
Bg1Ⅱ1μl
BstEⅡ1μl
ddH
2O38μl
37 DEG C of enzymes are cut 2 hours rear electrophoresis and are cut glue recovery;
Object fragment be connected with carrier, reaction system is as follows:
10×T4DNAligasebuffer1μl
T4DNALigase(350U/μl)0.5μl
DNA fragmentation (100ng/ μ l) 4 μ l
PCamGvector(BglⅡ/BstEⅡ)(50ng/μl)2μl
ddH
2O2.5μl
16 DEG C connect 30min and are placed on ice, get 8 μ l reaction solution transformed competence colibacillus cell DH5 α.The sequence information of sequence verification recombinant clone Insert Fragment, the correct plasmid called after pCamG-antiPPO (binary expression vector structure is shown in Fig. 1) containing goal gene sequence.
(3) genetic transformation of Twospore Mushroom
The fungi binary expression vector pCamG-antiPPO of above-mentioned structure is converted into agrobacterium strains LBA4404,28 DEG C of cultivations, picking carries the single bacterium colony of Agrobacterium of binary expression vector pCamG-antiPPO plasmid, be seeded in 3-5ml containing in the YEB liquid nutrient medium of 50mg/LRif and 50mg/LKan, 28 DEG C, 220rpm shaking culture is spent the night, until logarithmic growth OD
600for 0.6-0.8; Activate the Agrobacterium spent the night to be seeded in identical 20 ~ 50mlYEB liquid nutrient medium in the ratio of 1:100, continue to be cultured to logarithmic phase; Get Twospore Mushroom lamella tissue, the tissue block being cut into 3mm, in aseptic bottle, adds Agrobacterium bacterium liquid, 28 DEG C of 120rpm vibration 30min; After taking-up explant sterilized water rinses repeatedly, filter paper blots lamella tissue block surface-moisture, and proceed on the CM substratum of band filter paper, 25 DEG C of dark culturing cultivate 1 day; Lamella proceeds on the CM substratum containing Hyg35mg/L and Cef200mg/L, 25 DEG C of dark culturing 3-4 weeks; After resistance mycelium grows, cultivating once containing the CM substratum of Hyg35mg/L and Cef200mg/L continuing tube, obtain hygromycin resistance mycelium (transgenic strain is shown in Fig. 2).
The resistance mycelium obtained further is identified, get 0.1g mycelium, liquid nitrogen grinding powdered, adds DNA extraction liquid (2%SDS, 0.5MNaCl, 50mMTris-HCl, 50mMEDTA, pH8.0,1% beta-mercaptoethanol) 700 μ l, proceed in 1.5ml centrifuge tube, 65 DEG C of insulation 50min; 12000rpm, 4 DEG C of centrifugal 15min; Get supernatant, add equal-volume phenol/chloroform/primary isoamyl alcohol (25:24:1), put upside down mixing, the centrifugal 15min of 12000rpm; Get supernatant, add equal-volume chloroform: primary isoamyl alcohol (24:1) mixes, the centrifugal 15min of 12000rpm; Get the new centrifuge tube of supernatant liquor to, add 1/10 volume NaAc (pH5.2) and 2 times of volume dehydrated alcohols (ice-cold), precipitate DNA, centrifugally remove supernatant, precipitation washed by 70% ethanol, is dissolved in 100 μ lTE after drying.With Totomycin hyg primers, upstream primer is 5 '-CTGCTCCATACAAGCCAACCA-3 ' (SEQIDNO:7), downstream primer is 5 '-GACAGCGTCTCCGACCTGAT-3 ' (SEQIDNO:8), with the above-mentioned DNA extracted for template, carry out pcr amplification, what band (600bp) detected is positive transformants mycelium, i.e. transgenic strain (PCR of transgenic strain detects electrophorogram and sees Fig. 3).Transgenic strain proceeds on wheat substratum and cultivates, and utilizes straw-manure compost half sponge process to carry out cultivation fruiting, obtains transgenosis Twospore Mushroom sporophore.
(4) change of transgenosis Twospore Mushroom PPO enzymic activity and browning degree measure
Get in step (3) and obtain transgenosis Twospore Mushroom sporophore freeze-drying sample 100mg, add 1mLpH6.5 phosphoric acid extraction damping fluid after liquid nitrogen grinding, mixing puts 25min on ice, 12,000rpm, 4 DEG C of centrifugal 10min; Get 0.1mL supernatant liquor, add 0.9mL substrate reactions liquid (15mmol/LL-DOPA, 100mmol/LpH6.5 sodium phosphate buffer), 25 DEG C of reaction 2min, utilize spectrophotometer determined wavelength 478nm light absorption value.A PPO enzyme activity unit (U) is defined as per minute light absorption value under condition determination and changes the enzyme amount needed for 0.01.Transgenosis Twospore Mushroom and non-non-transgenic control PPO activity level figure are shown in Fig. 4.
Transgenosis double-spore mushroom browning level determinations: transgenosis Twospore Mushroom and non-non-transgenic control are 15 DEG C of storages, and by WSC-S type full-automatic colour examining colour-difference-metre periodic measurement mushroom mushroom lid brown stain value L value, transgenosis Twospore Mushroom and non-non-transgenic control L value are shown in Fig. 5.
Below, the accompanying drawing situation relevant with the application is described as follows:
Still it should be noted that Fig. 1 represents that the application binary expression vector pCamG-antiPPO used builds schematic diagram, wherein marked (1) represents Hygromycin resistance marker's gene, (2) gpd promotor is represented, (3) antiPPO gene is represented, (4) NOS terminator is represented, (5) represent that Bgl II enzyme cuts insertion point, (6) represent that BstE II enzyme cuts insertion point.AntiPPO gene is positioned at gpd promotor downstream, NOS terminator upstream.
Fig. 2 is the growth figure of transgenosis Twospore Mushroom different times, wherein marked (1) for Agrobacterium infect after lamella tissue morphology figure, (2) the Twospore Mushroom resistance mycelium morphology figure on Totomycin CM substratum is being contained, (3) the sporophore shape figure that obtains after carrying out mushroom producing culture of transgenosis agaricus bisporus mycelia, the sporophore shape figure of (4) transgenosis Twospore Mushroom maturation.
Fig. 3 is that the PCR of transgenosis agaricus bisporus mycelia detects electrophorogram, from left side, the first swimming lane is negative control (not containing template, only adding amplimer), DL2000Markers molecular weight marker, positive control (plasmid DNA containing antiPPO gene), wild-type Twospore Mushroom, transgenosis mycelium 1,2,3,4,5,6,7.Fig. 3 demonstrates, and positive control and transfer-gen plant all can amplify 600bp band, and negative control and non-transgenosis Twospore Mushroom all do not amplify this band.
Fig. 4 is transgene tobacco and contrast Twospore Mushroom sporophore PPO enzyme activity level comparison diagram, ordinate zou represents PPO enzymic activity (unit OD/g/min), and X-coordinate represents wild-type Twospore Mushroom, transgenosis Twospore Mushroom 1,2,3,4 respectively from left side.Fig. 4 demonstrates PPO enzyme running water in transgenosis Twospore Mushroom sporophore on average lower than wild-type Twospore Mushroom.
Fig. 5 is transgenosis Twospore Mushroom and contrast mushroom lid L value (browning index) comparison diagram when Twospore Mushroom sporophore 15 DEG C is preserved, ordinate zou represents mushroom cap surface L value (browning index), and X-coordinate represents wild-type Twospore Mushroom, transgenosis Twospore Mushroom 1,2,3,4 respectively from left side; Corresponding 3 the 15 DEG C of storage time sections of each X-coordinate, are respectively: 0d, 1d, 2d from left to right.When Fig. 5 demonstrates 15 DEG C of storages, transgenosis Twospore Mushroom mushroom cap surface L value is on average higher than wild-type Twospore Mushroom, and mushroom lid browning degree is lower than wild-type Twospore Mushroom.
Claims (2)
1. a PPO gene cDNA fragment, it is characterized in that the nucleotide sequence of this gene is as shown in SEQ ID NO:1, aminoacid sequence is as shown in SEQ ID NO:2.
2. Twospore Mushroom PPO gene cDNA fragment is reducing the application in PPO enzymic activity, it is characterized in that carrying out as follows:
(1) separating clone Twospore Mushroom PPO gene fragment order: extract Twospore Mushroom sporophore total serum IgE, reverse transcription synthesis cDNA, to be compared design degenerate primer by the Twospore Mushroom PPO gene order known with GenBank, reverse transcription PCR is utilized to obtain amplified production, amplified production reclaims through sepharose, to reclaim in product cloning to pMD18-T carrier and to check order, biology tool is utilized to analyze sequencing result, with the comparison of Twospore Mushroom PPO gene, be defined as the conserved regions fragment in PPO gene and copper desmoenzyme active centre, and called after antiPPO;
(2) structure of Twospore Mushroom PPO gene antisense expression vector: utilize restriction enzyme and ligase enzyme to enter to connect by antiPPO gene and the expression vector pCamG cut through enzyme, antiPPO gene is oppositely inserted pCamG, obtain Twospore Mushroom restructuring binary expression vector, called after pCamG-antiPPO;
(3) genetic transformation of Twospore Mushroom: expression vector pCamG-antiPPO is utilized Agrobacterium-mediated transformation Twospore Mushroom, obtain the transgenosis mushroom with hygromycin resistance, extract transgenosis mushroom mycelium DNA, utilize special upper primer and lower primer to carry out pcr amplification qualification, determine to obtain Twospore Mushroom transgenosis mycelium; Utilize straw-manure compost half sponge process to carry out cultivation fruiting, transgenosis Twospore Mushroom sporophore is identified;
(4) mensuration of transgenosis Twospore Mushroom PPO enzyme activity level and browning degree: transgenosis Twospore Mushroom significantly reduces than non-transgenosis mushroom PPO enzymic activity; During 15 DEG C of storages, transgenosis Twospore Mushroom is lower than non-non-transgenic control browning degree, illustrates and proceed to Anti-sense PPO gene in Twospore Mushroom, significantly reduces PPO enzymic activity, and during 15 DEG C of storages, brown stain is suppressed, and transgenosis mushroom BPH resistant rice variety quality is obviously improved.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107916273A (en) * | 2017-10-23 | 2018-04-17 | 上海市农业科学院 | A kind of method of agaricus bisporus genetic transformation |
| CN112662686A (en) * | 2021-01-26 | 2021-04-16 | 贵州大学 | Cloning method and application of Yunwu tribute tea CsPPO gene |
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107916273A (en) * | 2017-10-23 | 2018-04-17 | 上海市农业科学院 | A kind of method of agaricus bisporus genetic transformation |
| CN112662686A (en) * | 2021-01-26 | 2021-04-16 | 贵州大学 | Cloning method and application of Yunwu tribute tea CsPPO gene |
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