CN1285721C - Gibberella gene engineering bacterium and its preparation and application - Google Patents
Gibberella gene engineering bacterium and its preparation and application Download PDFInfo
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- CN1285721C CN1285721C CN 200410082305 CN200410082305A CN1285721C CN 1285721 C CN1285721 C CN 1285721C CN 200410082305 CN200410082305 CN 200410082305 CN 200410082305 A CN200410082305 A CN 200410082305A CN 1285721 C CN1285721 C CN 1285721C
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Abstract
The present invention provides a gibberellin gene engineering strain. The gibberellin gene engineering strain is obtained by that rate-limiting enzyme genes cps/ks and a strong promoter Pgpd or PtrpC are respectively amplified by using PCR technology, and the rate-limiting enzyme genes cps/ks are synthetized by gibberellin; the rate-limiting enzyme genes cps/ks and the strong promoter Pgpd or PtrpC are cloned to corresponding sites of objective plasmid which can be expressed in filamentous fungi to obtain a cps/ks gene overexpression carrier driven by the strong promoter; the overexpression carrier is converted into gibberella, and thus, the gibberellin gene engineering strain can be obtained. The present invention uses gene engineering technology to improve the gibberella and has a specific breeding objective and high efficiency; the obtained gibberella engineering strain has strong gibberellin synthesis capability which is raised by more than 40 percent in comparison with the gibberellin synthesis capability of a starting gibberella strain of the engineering strain.
Description
(1) technical field
The invention provides a kind of Plant hormones regulators,gibberellins genetic engineering bacterium and preparation and application that produces Plant hormones regulators,gibberellins, belong to the genetically engineered field.
(2) background technology
(gibberellins is a plant growth regulators GAs) to Plant hormones regulators,gibberellins, and important effect is arranged in growth and development of plants.It can promote the internode elongation of plant, removes the dormancy of seed, stem tuber, bud, promotes flowering of plant, can induce parthenocarpy and suppress aging etc.Plant hormones regulators,gibberellins is widely used on crops such as fruit, vegetables, paddy rice.Plant hormones regulators,gibberellins can also be used for beer production industry.In recent years, along with promoting the use of of Plant hormones regulators,gibberellins, market demand constantly increases.
Plant hormones regulators,gibberellins mainly relies on microbial fermentation production, and producing bacterium is fusarium moniliforme gibberella fujikuroi (Gibberella fujikuroi), and Plant hormones regulators,gibberellins is the isoprenoid secondary metabolite of this bacterium.The production of Plant hormones regulators,gibberellins is the history of existing nearly half a century so far, between decades, in order to improve Plant hormones regulators,gibberellins output, mainly adopts traditional methods such as selection by mutation and zymotechnique improvement.But traditional bacterial strain breeding method mainly relies on physics and chemistry mutagenesis at random, and target is indeterminate, and it is not obvious to waste time and energy and produce effects.At present very limited by adopting these methods to improve the ability of the synthetic Plant hormones regulators,gibberellins of gibberella fujikuroi, potentiality are little.
(3) summary of the invention
The present invention promptly is that a kind of breeding objective is clear and definite, efficient is high in order to provide, the strong Plant hormones regulators,gibberellins genetic engineering bacterium of ability of synthetic Plant hormones regulators,gibberellins, and the preparation method of this engineering bacteria and application.
For reaching goal of the invention the technical solution used in the present invention be:
A kind of Plant hormones regulators,gibberellins genetic engineering bacterium, described Plant hormones regulators,gibberellins genetic engineering bacterium obtains by the following method: utilize round pcr increase respectively Plant hormones regulators,gibberellins synthetic rate-limiting enzyme gene cps/ks and strong promoter Pgpd or PtrpC, and they are cloned into the corresponding site of the target plasmid that can express in filamentous fungus, obtain the cps/ks gene overexpression vector that strong promoter drives, overexpression vector is transformed into gibberella again, promptly obtains described Plant hormones regulators,gibberellins genetic engineering bacterium.
Described gibberella is a gibberella fujikuroi.
Described target plasmid is one of following:
①pCB1004②pCB1003③pCSN43④pCSN44⑤pSH75
⑥pBARGEM5-1⑦pBARGEM7-1⑧pBARKS1
⑨pBARGEM7-2⑩pBARMTE1
The method for preparing described Plant hormones regulators,gibberellins genetic engineering bacterium, described method is as follows: utilize round pcr increase respectively Plant hormones regulators,gibberellins synthetic rate-limiting enzyme gene cps/ks and strong promoter Pgpd or PtrpC, and they are cloned into corresponding site in the target plasmid, obtain the cps/ks gene overexpression vector that strong promoter drives, described overexpression vector is transformed into gibberella again, promptly obtains described Plant hormones regulators,gibberellins genetic engineering bacterium.
Concrete, described method is carried out as follows:
(1) behind pcr amplification Pgpd or the PtrpC Pgpd or PtrpC are cloned into target plasmid, obtain recombinant plasmid;
(2) pcr amplification Plant hormones regulators,gibberellins synthase gene cps/ks, the PCR product reclaims the corresponding site of rear clone to target plasmid through gel, promptly after promotor Pgpd or the PtrpC, obtains cps/ks gene overexpression vector;
(3) the gibberella inoculation culture washed spore and is forwarded to liquid nutrient medium 20～25 ℃ of 100～300rpm cultivations 24～48 hours after 5～15 days;
(4) it is centrifugal that spore precipitates that step (3) gained nutrient solution filters the back, and the spore suspension is made with 0.1M Lithium Acetate solution in the washing back;
(5) get step (2) gained overexpression vector, be added in the spore suspension, add spermidine, behind 4 ℃ of incubation 30min, add 40%PEG8000 and 0.1M Lithium Acetate and continue incubation 1h for 4 ℃ to 4mM;
(6) step (5) spore suspension will be inoculated on the PDA flat board after the spore washing with 37 ℃ of water-bath heat shock 5min, cultivates 16～24h for 20～25 ℃;
(7) on step (6) PDA flat board, cover 1% nutrient agar that one deck contains Totomycin 100～200mg/L, continue to cultivate 5～10 days, promptly get described Plant hormones regulators,gibberellins genetic engineering bacterium.
Further, described method is carried out as follows:
(1) behind the pcr amplification Pgpd Pgpd is cloned into target plasmid, obtains recombinant plasmid;
(2) pcr amplification Plant hormones regulators,gibberellins synthase gene cps/ks, the PCR product reclaims rear clone through gel and after the Pgpd site, obtain cps/ks gene overexpression vector to target plasmid;
(3) gibberella is inoculated into the PDA substratum, cultivates after 5～15 days for 20～25 ℃, washes spore with sterilized water, is transferred to the YpSs liquid nutrient medium, 20～25 ℃, 100～300rpm shaking culture 24～48 hours;
(4) step (3) gained nutrient solution filters back 4000rpm and got the spore precipitation in centrifugal 5 minutes, after sterilized water washing 2～4 times, makes the spore suspension with 0.1M Lithium Acetate solution;
(5) get step (2) gained overexpression vector, be added in the spore suspension, add spermidine, behind 4 ℃ of incubation 30min, add 40%PEG8000 and 0.1M Lithium Acetate and continue incubation 1h for 4 ℃ to 4mM;
(6) 37 ℃ of water-bath heat shock 5min with aseptic washing 1～3 time, are inoculated into spore on the PDA flat board, cultivate 16～24h for 20～25 ℃;
(7) on step (6) PDA flat board, cover 1% nutrient agar that one deck contains Totomycin 100～200mg/L, continue to cultivate 5～10 days, promptly get described Plant hormones regulators,gibberellins genetic engineering bacterium.
Especially, described method is carried out as follows:
(1) the strong promoter Pgpd on the pcr amplification plasmid pAN7-1 by the corresponding site that restriction enzyme Not I/BamH I is cloned into plasmid pCB1004, obtains recombinant plasmid pCB1004Pgpd with Pgpd;
(2) the Plant hormones regulators,gibberellins synthase gene cps/ks in the pcr amplification gibberella fujikuroi, the PCR product is cloned into the Pgpd site of pCB1004Pgpd by restriction enzyme BamH I/Xho I after gel reclaims after, obtain cps/ks gene overexpression vector pCB1004:Pgpd+cps/ks, be abbreviated as pCBcps;
(3) gibberella is inoculated into the PDA substratum, cultivates after 10 days for 24 ℃, washes spore with sterilized water, is transferred to the YpSs liquid nutrient medium, 24 ℃, 200rpm shaking culture 36 hours;
(4) step (3) gained nutrient solution filters back 4000rpm and got the spore precipitation in centrifugal 5 minutes, after sterilized water washing 3 times, makes the spore suspension with 0.1M Lithium Acetate solution;
(5) get step (2) gained overexpression vector pCBcps, be added in the spore suspension, add spermidine, behind 4 ℃ of incubation 30min, add 40%PEG8000 and 0.1M Lithium Acetate and continue incubation 1h for 4 ℃ to 4mM;
(6) 37 ℃ of water-bath heat shock 5min with aseptic washing 2 times, are inoculated into spore on the PDA flat board, cultivate 20h for 24 ℃;
(7) on step (6) PDA flat board, cover 1% nutrient agar that one deck contains Totomycin 100mg/L, continue to cultivate 5～10 days, promptly get described Plant hormones regulators,gibberellins genetic engineering bacterium.
Described Plant hormones regulators,gibberellins genetic engineering bacterium is applied to prepare Plant hormones regulators,gibberellins.
Described application is that described Plant hormones regulators,gibberellins genetic engineering bacterium is inoculated into fermention medium with 5～20% inoculum sizes, after 7～9 days, obtains described Plant hormones regulators,gibberellins through separation and purification at 25～35 ℃, 200～300rpm condition bottom fermentation.
Usually, be that described Plant hormones regulators,gibberellins genetic engineering bacterium is inserted seed culture medium after cultivating 24～48 hours under 25～35 ℃ with bacterium dish or spore suspension, change fermention medium over to 5～20% inoculum sizes, after 7～9 days, obtain described Plant hormones regulators,gibberellins at 25～35 ℃, 150～300rpm condition bottom fermentation through separation and purification.
Substratum described in the present invention can be all conventional substratum that can be used for cultivating gibberella.Used substratum is as follows among the present invention:
PDA substratum (g/l): potato, 200; Sucrose, 20; Agar powder, 15 (agar powder is 10 in the 1%PDA substratum); The pH nature.Sterilized 18 minutes down at 121 ℃.
YpSs liquid nutrient medium (g/L): yeast extract, 4; Zulkovsky starch, 15; KH
2PO
4, 1; MgSO
47H
2O, 0.5; The pH nature.Sterilized 18 minutes down at 121 ℃.
Seed culture medium: groundnut meal, 1.5%; Dextrin, 1%; MgSO
47H
2O, 0.1%; Glucose, 1.5%; KH
2PO
4, 0.1%; The PH nature.Sterilized 18 minutes down at 121 ℃.
Fermention medium: Semen Maydis powder, 1.5%; Trisun Oil R 80,6%; (NH
4)
2SO
4, 0.05%; KH
2PO
4, 0.1%; The pH nature.Sterilized 18 minutes down at 121 ℃.
Plant hormones regulators,gibberellins class material is a plant-growth regulator, and it has multiple homologue, has found kind more than 100 at occurring in nature at present, and according to the morning and evening of finding, these homologues are named as A
1(GA
1), GA
2, GA
3, GA
4, GA
5....Realized the mass-produced GA of being at present
3, its structural formula is as follows:
Indication Plant hormones regulators,gibberellins is GA among the present invention
3
Plant hormones regulators,gibberellins genetic engineering bacterium of the present invention and preparation thereof and the beneficial effect of using are mainly reflected in: (1) utilization genetic engineering technique is improved gibberella, and breeding objective is clear and definite, efficient is high; (2) ability of the synthetic Plant hormones regulators,gibberellins of gained engineering bacteria is strong, has improved more than 40% than the gibberella bacterial strain that sets out of engineering bacteria.
(4) description of drawings
Fig. 1 is the overexpression vector pCBcps collection of illustrative plates that embodiment 1 makes up, and hyg is a hygromycin gene; CamR is a chloramphenicol resistance gene;
Fig. 2 is GA
3Standard model HPLC analytical results collection of illustrative plates, GA
3Retention time 16.965min;
Fig. 3 is non-engineering bacteria GA
3HPLC analytical results collection of illustrative plates, GA
3Retention time 16.898min;
Fig. 4 is embodiment 3 gained genetic engineering bacterium GA
3HPLC analytical results collection of illustrative plates, GA
3Retention time 16.665min.
(5) embodiment
The present invention is described further below in conjunction with specific embodiment:
Embodiment 1: the structure of overexpression vector
Step:
(1) strong promoter PgPd is used pcr amplification from plasmid pAN7-1 (Punt et al.1987Gene56:117-124), be cloned into the corresponding site of carrier pCB1004 (Carroll et al.1994Fungal Genet.Newsl.41:22) by restriction enzyme Not I/BamH I, form recombinant plasmid pCB1004Pgpd.Strong promoter Pgpd also can contain any DNA of this sequence or directly synthetic from other.Promotor except that Pgpd, constructive expression's in filamentous fungus such as available PtrpC promotor also.The plasmid that is used to make up cps/ks gene overexpression vector is gone back plasmid such as pCSN43, pCSN44, pCB1004, pCB1003, pSH75, pMYX100, pMSN1 and pAN26 etc. that available energy is expressed in filamentous fungus except that pCB1004.
(2) according to Plant hormones regulators,gibberellins synthase gene cps/ks (the GeneBank accession number: sequences Design primer (forward primer: GG AJ810802) of G.fujikuroi
GGATCCGCCGTACGTATGAAACACACCT, the sequence of underscore is restriction enzyme BamH I point of contact; Reverse primer: CC
CTCGAGTACATATCAAAATTACCAGCT, the sequence of underscore is restriction enzyme Xho I point of contact), utilize round pcr amplification coding region sequence.PCR reaction conditions: 94 ℃ of sex change 5min; Then carry out 32 circulations, parameter is 94 ℃, 1min; 55 ℃, 30sec and 72 ℃, 2min; Extend 5min at 72 ℃ at last.
(3) the PCR product is after gel reclaims, and is cloned into the corresponding site of pCB1004Pgpd with restriction enzyme BamH I/Xho I, and formation cps/ks gene overexpression vector pCB1004:Pgpd+cps/ks is abbreviated as pCBcps, and its collection of illustrative plates is seen Fig. 1.
Embodiment 2: the structure of overexpression vector
Step:
(1) with strong promoter PtrpC from plasmid PZPnat1 pcr amplification, be cloned into the corresponding site of carrier pBARKS1 by restriction enzyme XbaI/BamH I, form recombinant plasmid pBARtrpC.
(2) according to Plant hormones regulators,gibberellins synthase gene cps/ks (the GeneBank accession number: sequences Design primer (forward primer: GG AJ810802) of G.fujikuroi
GGATCCGCCGTACGTATGAAACACACCT, the sequence of underscore is restriction enzyme BamH I point of contact; Reverse primer: CC
CTCGAGTACATATCAAA TTACCAGCT, the sequence of underscore is restriction enzyme Xho I point of contact), utilize round pcr amplification coding region sequence.PCR reaction conditions: 94 ℃ of sex change 5min; Then carry out 32 circulations, parameter is 94 ℃, 1min; 55 ℃, 30sec and 72 ℃, 2min; Extend 5min at 72 ℃ at last.
(3) the PCR product is after gel reclaims, and is cloned into the corresponding site of pBARtrpC with restriction enzyme BamH I/Xho I, and formation cps/ks gene overexpression vector p BARKS1:ptrpC+cps/ks is abbreviated as pBARcps.
Embodiment 3: the acquisition of engineering bacteria
Step:
(1) gibberella fujikuroi is inoculated into the PDA culture medium flat plate, cultivated 10 days for 24 ℃;
(2) wash spore with sterilized water, be transferred to YpSs liquid nutrient medium 100mL, 24 ℃, 200rpm shaking culture 36 hours;
(3) nutrient solution is filtered back 4000rpm and got the spore precipitation in centrifugal 5 minutes, it is inferior to give a baby a bath on the third day after its birth with aseptic deionized water;
(4) with the 0.1M Lithium Acetate solution suspension spore of 200 μ l;
(5) get embodiment 1 gained plasmid pCBcps 5 μ l (concentration 2 μ g/ μ l), join in the spore suspension, adding spermidine again, to make its final concentration be 4mM, 4 ℃ of incubation 30min;
(6) the 0.1M Lithium Acetate solution of adding 150 μ l 40%PEG8000,4 ℃ are continued incubation 1h;
(7) 37 ℃ of water-bath heat shocks 5 minutes wash twice with spore with aseptic deionized water, receive on the PDA flat board, cultivate 20h for 24 ℃;
(8) on the PDA flat board, cover one deck 1% nutrient agar (containing Totomycin 100mg/l) and continue to cultivate 5-10 days, be transferred on the PDA substratum (containing Totomycin 100mg/l) after growing bacterium colony, repeat subculture screening 6 times, preserve bacterial strain.
PDA substratum (g/l): potato, 200; Sucrose, 20; Agar powder, 15; The pH nature.Sterilized 18 minutes down at 121 ℃.
Embodiment 3: Plant hormones regulators,gibberellins synthesis capability contrast experiment
The red mould engineering bacteria of embodiment 2 gained after cultivating 7 days on the PDA substratum, is inserted among the seed culture medium 50mL, cultivated 36 hours at 30 ℃, 240rpm shaking table.Change fermention medium over to 10% inoculum size then, the bottled 30ml fermention medium of 250ml triangle was 30 ℃, 240rpm fermentation 8 days; Fermented liquid transfers to 2.0 with filtrate pH behind the individual layer filter paper filtering, filtrate is through 0.45 μ m filtering with microporous membrane, and the gained sample carries out efficient liquid phase chromatographic analysis.
Adopt above-mentioned same procedure that the bacterium gibberella fujikuroi bacterium that sets out of engineering bacteria is carried out respective handling, the gained sample carries out efficient liquid phase chromatographic analysis.
Analyze Plant hormones regulators,gibberellins GA with rp-hplc
3Output.High performance liquid chromatograph is Tianjin, island SPD-10A VP (UV-VIS) type, analyzes with Zhejiang University's chromatographic working station.Moving phase consists of: 30% methyl alcohol, 0.01M phosphoric acid is regulated pH to 3 with potassium hydroxide.Detect wavelength 206nm, flow velocity 1ml/min.Plant hormones regulators,gibberellins GA with Sigma company
3Make standard model, standard model is dissolved in moving phase, and concentration is (mg/ml): 0.1,0.5,0.75,1.Retention time according to the standard substance chromatographic peak is qualitative.Make the typical curve of peak area-Plant hormones regulators,gibberellins concentration, Plant hormones regulators,gibberellins in the sample is carried out quantitatively according to typical curve.Sample size 20 μ l.
PDA substratum (g/l): potato, 200; Sucrose, 20; Agar powder, 15 (agar powder is 10 in the 1%PDA substratum); The pH nature.Sterilized 18 minutes down at 121 ℃.
Seed culture medium: groundnut meal, 1.5%; Dextrin, 1%; MgSO
47H
2O, 0.1%; Glucose, 1.5%; KH
2PO
4, 0.1%; The PH nature.Sterilized 18 minutes down at 121 ℃.
Fermention medium: Semen Maydis powder, 1.5%; Trisun Oil R 80,6%; (NH
4)
2SO
4, 0.05%; KH
2PO
4, 0.1%; The pH nature.Sterilized 18 minutes down at 121 ℃.
GA
3Standard model HPLC analytical results collection of illustrative plates is seen Fig. 2; Non-genomic engineering gibberella fujikuroi bacterium GA
3HPLC analytical results collection of illustrative plates see Fig. 3; Embodiment 2 gained genetic engineering bacterium GA
3HPLC analytical results collection of illustrative plates see Fig. 4;
Compound sample high-efficient liquid phase chromatogram analytical results sees Table 1:
Table 1
| Non-engineering bacteria | Engineering bacteria | Engineering bacteria GA 3Content raising rate (%) | |
| Peak area | 1042240.438 | 1550715.625 | 52.6 |
| GA 3Content (mg/ml) | 420 | 641 |
Embodiment 4: the acquisition of engineering bacteria
Step:
(1) gibberella fujikuroi is inoculated into the PDA culture medium flat plate, cultivated 15 days for 22 ℃;
(9) wash spore with sterilized water, be transferred to YpSs liquid nutrient medium 100mL, 20 ℃, 100rpm shaking culture 48 hours;
(10) nutrient solution is filtered back 4000rpm and got the spore precipitation in centrifugal 5 minutes, it is inferior to give a baby a bath on the third day after its birth with aseptic deionized water;
(11) the 0.1M Lithium Acetate solution suspension spore of usefulness 0.3mL;
(12) get embodiment 2 gained plasmid pBARcps 5 μ l (concentration 2 μ g/ μ l), join in the spore suspension, add spermidine again to 4mM, 4 ℃ of incubation 30min;
(13) add the 0.1M Lithium Acetate solution that 200 μ L contain 40%PEG8000,4 ℃ are continued incubation 1h;
(14) 37 ℃ of water-bath heat shocks 5 minutes with aseptic washing twice, are received spore on the PDA flat board, cultivate 24h for 20 ℃;
(15) on the PDA flat board, cover one deck 1% nutrient agar (containing Totomycin 150mg/l) and continue to cultivate 7 days, be transferred on the PDA substratum (containing Totomycin 150mg/l) after growing bacterium colony, repeat subculture screening 6 times, preserve bacterial strain.
PDA substratum (g/l): potato, 200; Sucrose, 20; Agar powder, 15; The pH nature.Sterilized 18 minutes down at 121 ℃.
Embodiment 6: Plant hormones regulators,gibberellins is synthetic
Analyze Plant hormones regulators,gibberellins GA with rp-hplc
3Output.High performance liquid chromatograph is Tianjin, island SPD-10A VP (UV-VIS) type, analyzes with Zhejiang University's chromatographic working station.Moving phase consists of: 30% methyl alcohol, 0.01M phosphoric acid is regulated pH to 3 with potassium hydroxide.Detect wavelength 206nm, flow velocity 1ml/min.The result shows GA in the engineering bacteria
3The content ratio bacterium that sets out has improved 49.8%.PDA substratum (g/l): potato, 200; Sucrose, 20; Agar powder, 15; The pH nature.Sterilized 18 minutes down at 121 ℃.
Seed culture medium: groundnut meal, 1.5%; Dextrin, 1%; MgSO
47H
2O, 0.1%; Glucose, 1.5%; KH
2PO
4, 0.1%; The PH nature.Sterilized 18 minutes down at 121 ℃.
Fermention medium: Semen Maydis powder, 1.5%; Trisun Oil R 80,6%; (NH
4)
2SO
4, 0.05%; KH
2PO
4, 0.1%; The pH nature.Sterilized 18 minutes down at 121 ℃.
Claims (10)
1. Plant hormones regulators,gibberellins genetic engineering bacterium, it is characterized in that described Plant hormones regulators,gibberellins genetic engineering bacterium obtains by the following method: utilize pcr amplification or restriction enzyme clone or chemical synthesising technology clone Plant hormones regulators,gibberellins synthetic rate-limiting enzyme gene cps/ks and strong promoter Pgpd or PtrpC, and they are cloned into corresponding site in the target plasmid that can express in filamentous fungus, obtain the cps/ks gene overexpression vector that strong promoter drives, overexpression vector changes gibberella again over to, promptly obtains described Plant hormones regulators,gibberellins genetic engineering bacterium.
2. Plant hormones regulators,gibberellins genetic engineering bacterium as claimed in claim 1 is characterized in that described gibberella is a gibberella fujikuroi.
3. Plant hormones regulators,gibberellins genetic engineering bacterium as claimed in claim 2 is characterized in that described target plasmid is one of following:
①pCB1004 ②pCB1003 ③pCSN43 ④pCSN44 ⑤pSH75
⑥pBARGEM5-1 ⑦pBARGEM7-1 ⑧pBARKS1
⑨pBARGEM7-2 ⑩pBARMTE1。
4. preparation is as the method for the described Plant hormones regulators,gibberellins genetic engineering bacterium of one of claim 1～3, it is characterized in that described method is as follows: utilize pcr amplification or restriction enzyme clone or chemical synthesising technology clone Plant hormones regulators,gibberellins synthetic rate-limiting enzyme gene cps/ks and strong promoter Pgpd or PtrpC, and they are cloned into corresponding site in the target plasmid, obtain the cps/ks gene overexpression vector that strong promoter drives, described overexpression vector is transformed into gibberella again, promptly obtains described Plant hormones regulators,gibberellins genetic engineering bacterium.
5. the preparation method of Plant hormones regulators,gibberellins genetic engineering bacterium as claimed in claim 4 is characterized in that described method carries out as follows:
(1) behind pcr amplification Pgpd or the PtrpC Pgpd or PtrpC are cloned into target plasmid, obtain recombinant plasmid;
(2) pcr amplification Plant hormones regulators,gibberellins synthase gene cps/ks, the PCR product reclaims the corresponding site of rear clone to target plasmid through gel, obtains cps/ks gene overexpression vector;
(3) the gibberella inoculation culture washed spore and is forwarded to 20～25 ℃ of 100～300rpm cultivations of liquid nutrient medium 24～48 hours after 5～15 days;
(4) step (3) gained nutrient solution filter the back centrifugal the spore precipitation, the washing back with 0.1M Lithium Acetate solution dissolve the spore suspension;
(5) get step (2) gained overexpression vector, be added in the spore suspension, add spermidine again to 4mM, 4 ℃ of incubations added 40%PEG8000 and 0.1M Lithium Acetate and continued incubation 1 hour for 4 ℃ after 30 minutes;
(6) step (5) spore suspension will be inoculated on the PDA flat board after the spore washing with 37 ℃ of water-bath heat shocks 5 minutes, cultivates 16～24 hours for 20～25 ℃;
(7) on step (6) PDA flat board, cover 1% nutrient agar that one deck contains Totomycin 100～200mg/L, continue to cultivate 5～10 days, promptly get described Plant hormones regulators,gibberellins genetic engineering bacterium.
6. the preparation method of Plant hormones regulators,gibberellins genetic engineering bacterium as claimed in claim 5 is characterized in that described method carries out as follows:
(1) behind the pcr amplification Pgpd Pgpd is cloned into target plasmid, obtains recombinant plasmid;
(2) pcr amplification Plant hormones regulators,gibberellins synthase gene cps/ks, the PCR product reclaims rear clone through gel and after the Pgpd site, obtain cps/ks gene overexpression vector to target plasmid;
(3) gibberella is inoculated into the PDA substratum, cultivates after 5～15 days for 20～25 ℃, washes spore with sterilized water, is transferred to the YpSs liquid nutrient medium, 20～25 ℃, 100～300rpm shaking culture 24～48 hours;
(4) step (3) gained nutrient solution filters back 4000rpm and got the spore precipitation in centrifugal 5 minutes, after sterilized water washing 2～4 times, makes the spore suspension with 0.1M Lithium Acetate solution;
(5) get step (2) gained overexpression vector, be added in the spore suspension, add spermidine again to 4mM, 4 ℃ of incubations added 40%PEG8000 and 0.1M Lithium Acetate and continued incubation 1 hour for 4 ℃ after 30 minutes;
(6) 37 ℃ of water-bath heat shocks 5 minutes with aseptic washing 1～3 time, are inoculated into spore on the PDA flat board, cultivate 16～24 hours for 20～25 ℃;
(7) on step (6) PDA flat board, cover 1% nutrient agar that one deck contains Totomycin 100～200mg/L, continue to cultivate 5～10 days, promptly get described Plant hormones regulators,gibberellins genetic engineering bacterium.
7. the preparation method of Plant hormones regulators,gibberellins genetic engineering bacterium as claimed in claim 5 is characterized in that described method carries out as follows:
(1) the strong promoter Pgpd on the pcr amplification plasmid pAN7-1 by the corresponding site that restriction enzyme Not I/BamH I is cloned into plasmid pCB1004, obtains recombinant plasmid pCB 1004Pgpd with Pgpd;
(2) the Plant hormones regulators,gibberellins synthase gene cps/ks in the pcr amplification gibberella fujikuroi, the PCR product is cloned into the corresponding site of pCB 1004Pgpd by restriction enzyme BamH I/Xho I after gel reclaims, obtain cps/ks gene overexpression vector pCB1004:Pgpd+cps/ks, be abbreviated as pCBcps;
(3) gibberella is inoculated into the PDA substratum, cultivates after 10 days for 24 ℃, washes spore with sterilized water, is transferred to the YpSs liquid nutrient medium, 24 ℃, 200rpm shaking culture 36 hours;
(4) step (3) gained nutrient solution filter centrifugal 5 minutes of back 4000rpm the spore precipitation, after sterilized water washing 3 times, with 0.1M Lithium Acetate solution dissolve the spore suspension;
(5) get step (2) gained overexpression vector pCBcps, be added in the spore suspension, add spermidine again to 4mM, 4 ℃ of incubations added 40%PEG8000 and 0.1M Lithium Acetate and continued incubation 1 hour for 4 ℃ after 30 minutes;
(6) 37 ℃ of water-bath heat shocks 5 minutes with aseptic washing 2 times, are inoculated into spore on the PDA flat board, cultivate 20 hours for 24 ℃;
(7) on step (6) PDA flat board, cover 1% nutrient agar that one deck contains Totomycin 100mg/L, continue to cultivate 5～10 days, promptly get described Plant hormones regulators,gibberellins genetic engineering bacterium.
8. as the application of the described Plant hormones regulators,gibberellins genetic engineering bacterium of one of claim 1～3 in preparation Plant hormones regulators,gibberellins.
9. the application of Plant hormones regulators,gibberellins genetic engineering bacterium as claimed in claim 8 in preparation Plant hormones regulators,gibberellins, it is characterized in that described application is that described Plant hormones regulators,gibberellins genetic engineering bacterium is inoculated into fermention medium with 5～20% inoculum sizes, after 7～9 days, obtain described Plant hormones regulators,gibberellins at 25～35 ℃, 200～300rpm condition bottom fermentation through separation and purification.
10. the application of red mould basic procatarxis engineering bacteria in preparation Plant hormones regulators,gibberellins of stating as claim 8, it is characterized in that described application is that described Plant hormones regulators,gibberellins genetic engineering bacterium is inserted seed culture medium after cultivating 24～48 hours under 25～35 ℃ with bacterium dish or spore suspension, change fermention medium over to 5～20% inoculum sizes, after 7～9 days, obtain described Plant hormones regulators,gibberellins at 25～35 ℃, 150～300rpm condition bottom fermentation through separation and purification.
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|---|---|---|---|
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| CN105441340A (en) * | 2016-01-07 | 2016-03-30 | 南京工业大学 | Gibberellin GA4+7High-yield strain and application thereof |
| CN110527630B (en) * | 2019-05-24 | 2021-04-20 | 浙江工业大学 | Aleurites lutescens mutant strain bred by ARTP mutagenesis technology and application thereof |
| CN114517161B (en) * | 2022-03-10 | 2024-04-19 | 浙江工业大学 | High yield gibberellin GA3Genetically engineered bacterium of (2), construction method and application |
| CN115786140A (en) * | 2022-09-15 | 2023-03-14 | 浙江工业大学 | High-yield gibberellin GA 3 The gene engineering bacterium, the construction method and the application |
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