CN104017824A - Culture method of transgenic soybean with OsPT6 phosphorus being efficiently utilized - Google Patents

Abstract

The invention belongs to the field of the molecular biology and biotechnology, relates to phosphorus translocator gene OsPT6 and an application of the gene in transgenic crops with phosphorus being efficiently utilized, provides a culture method of the transgenic soybean with phosphorus being efficiently utilized by using the gene, an expression carrier and construction containing the gene, as well as host bacteria used in the culture process as well as a measurement method and a kit for measuring relative expression quantity of the transgenic soybean OsPT6 gene. The blank that the phosphorus translocator OsPT6 gene in the soybean breeding in the prior art is solved, a plant expression carrier containing the OsPT6 gene is constructed, a CaMV35S promoter and an NOS (nitric oxide synthase) terminator are introduced into the OsPT6 gene to increase the expression regulation of the OsPT6 gene in foreign genes; the carrier further contains a herbicide resistance gene bar gene, the resistance of the transgenic plant to herbicides can be conveniently detected and is increased, the transgenic soybean produced by using the culture method provided by the invention is capable of efficiently utilizing phosphorus.

Description

OsPT6 phosphorus efficiency utilizes the method for cultivation of genetically engineered soybean

Technical field

The invention belongs to molecular biology and biological technical field, relate to a kind of phosphate transporter gene OsPT6 and this gene and utilize the application in genetically modified crops at phosphorus efficiency, be specifically related to utilize this gene to carry out phosphorus efficiency and utilize the method for cultivation of genetically engineered soybean, in cultivating process, use containing the expression vector of this gene and measuring method and the test kit of structure, Host Strains and genetically engineered soybean OsPT6 gene relative expression quantity.

Technical background

Soybean is rich in albumen and vegetables oil, its nutritive value profundity is liked by human consumer, along with market demand increases year by year, it has become important cash crop (Wang XR, Yan XL, Liao H.Genetic improvement for phosphorus efficiency in soybean:a radical approach.Annals of Botany, 2010,106:215-222.).At present, China is in the world main soybean country of consumption and importer, wherein 5,838 ten thousand tons of imported soybeans in 2012, because the impact soybean in China area and the output that are subject to imported soybean decline year by year, within 2012, soybean ultimate production is 1,301 ten thousand tons, and the soybean of the annual consumption of China approximately has 80% to depend on external import (State Statistics Bureau: http://data.stats.gov.cn/).South China area there is serious soil nutrient problem and restrict Soybean production (Li Jie, Shi Yuanliang, Chen Zhiwen, Red Soils in Southern China phosphorus element research overview. soil circular, 2011.42 (3): 763-768).Due to phosphoric in soil easily by Fe ³⁺, Al ³⁺ion is fixed, or be combined with organism, make available phosphate concentration become one of limiting factor of Soybean production (Raghothama K G and Karthikeyan A S.Phosphate acquisition.Plant and Soil, 2005,274:37-49.) lower than 10 μ M.Lack phosphorus problem for solving soil, peasant habit is in blindness increasing the p application rate, but most of phosphate fertilizer is by soil fixing, utilization ratio is low, can not solve cost-effectively the scarce phosphorus problem of soil and cause again the wasting of resources and environmental pollution (Shen JB, Yuan LX, Zhang JL, Li HG, Bai ZH, Chen XP, Zhang WF, Zhang FS.Phosphorus Dynamics:From Soil to Plant.Plant physiology, 2011,156:997-1005.).And phosphorus ore is Nonrenewable resources, by current phosphorate rock mining speed, phosphate rock resource will be at the approach exhaustion end of this century (Gilbert N.Environment:The disappearing nutrient.Nature, 2009,461:716-718.).

Improve phosphorus element efficiency from plant itself, cultivate phosphorus efficiency and utilize plant new germ plasm, in conjunction with the nutrient administrative skill of science, for reducing using of phosphate fertilizer, alleviate environmental pollution, increase economic efficiency, promote agriculture Sustainable development, have great importance.In prior art, have been found that difference overexpression OsPT1, OsPT2 and OsPT8 in paddy rice, under low-phosphorous condition, can increase the transport of phosphorus from underground part to overground part, thereby improve the resistance to low-phosphorous ability of plant (Jia HF, Ren HY, Gu M, Zhao JN, Sun SB, Zhang X, Chen JY, Wu P, Xu GH.The phosphate transporter gene OsPht1; 8is involved in phosphate homeostasis in rice.Plant Physiol.2011,156:1164-1175; Sun SB, Gu M, Cao Y, Huang XP, Zhang X, Ai PH, Zhao JN, Fan XR, Xu GH.A constitutive expressed phosphate transporter, OsPht1; 1, modulates phosphate uptake and translocation in phosphate-replete rice.Plant Physiology.2012,159:1571-1581; Wu P, Shou HX, Xu GH, Lian XM.Improvement of phosphorus efficiency in rice on the basis of understanding phosphate signaling and homeostasis.Current Opinion in Plant Biology, 2013,16:1-8.).Tobacco (Nicotiana tabacum L.) NtPT1 gene overexpression in paddy rice, under low-phosphorous and high phosphorus condition, all can significantly increase phosphorus content (the Park MR of transfer-gen plant, Baek SH, Reyes BG, Yun SJ.Overexpression of a high-affinity phosphate transporter gene from tobacco (NtPT1) enhances phosphate uptake and accumulation in transgenic rice plants.Plant Soil, 2007,292:259-269.).Overexpression Pht1 in Arabidopis thaliana (Arabidopsis); 5 genes can change remobilization (Nagarajan, V.K., A.Jain, M.D.Poling, A.J.Lewis, K.G.Raghothama, the and A.P.Smith.Arabidopsis Pht1 of phosphorus in plant body; 5mobilizes phosphate between source and sink organs and influences the interaction between phosphate homeostasis and ethylene signaling.Plant Physiology, 2011.156 (3): 1149-1163.).More than studies have shown that, in plant, overexpression phosphate transporter gene can increase the absorption of phosphorus and improve resistance to low-phosphorous ability under low-phosphorous condition.Therefore, utilizing transgenic technology can improve in the Gene Into Soybean of phosphate use efficiency, is a kind of effective way of cultivating phosphorus efficiency new soybean varieties.

Rice Os PT6 gene is the phosphate transporter gene of a strong expression under low-phosphorous induction, proves that by yeast function complementation experiment result OsPT6 has mediated the phosphate cotransporter of yeast cell film, for high-affinity phosphorus transporter albumen, has phosphorus efficiency receptivity.In paddy rice, suppressing OsPT6 by RNAi expresses, under low-phosphorus stress condition, in transgenic line, the total content of phosphorus is compared wild-type and is sharply reduced, prove that this phosphate cotransporter albumen is that paddy rice obtains phosphorus from low-phosphorus stress environment requisite, in absorption to phosphorus and body, transport (the Ai PH that plays an important role, Sun SB, Zhao JN, Fan XR, Xin WJ, Guo Q, Yu L, Shen QR, Wu P, Miller AJ, Xu GH.Two rice phosphate transporters, OsPht1; 2and OsPht1; 6, have different functions and kinetic properties in uptake and translocation.The Plant Journal, 2009,57:798-809.).Utilize biotechnology means by this gene transferred crop, can improve crop phosphorus and absorb and utilising efficiency, effectively reduce the amount of application of phosphate fertilizer.But up to the present, not yet find that there is the report that this gene is expressed in soybean, utilize biotechnology to improve the utilization of soybean phosphorus efficiency and also rarely have report.

Summary of the invention

The present invention seeks to solve the blank of phosphate transporter gene OsPT6 in genetically engineered soybean breeding in prior art, realize the transhipment of soybean phosphorus efficiency, reduce the excessive use of phosphate fertilizer, thereby reduce Soil degradation speed.

For achieving the above object, the invention provides following technology:

The invention provides a kind of plant expression vector pCAMBIA3301-OsPT6, contain phosphorus transporter albumen OsPT6 gene and herbicide resistance gene bar gene, rice phosphorus translocator OsPT6 gene 5' end and 3' end are introduced respectively CaMV35S promotor and NOS terminator.

The present invention also provides a kind of OsPT6 phosphorus efficiency to utilize the method for cultivation of genetically engineered soybean, the method builds up to rice phosphorus transporter gene OsPT6 in plant expression vector pCAMBIA3301, obtain recombinant plant expression vector pCAMBIA3301-OsPT6, import soybean by agriculture bacillus mediated method.

The present invention also provides the construction process of plant expression vector pCAMBIA3301-OsPT6, comprises the steps:

1) design primer, extension increasing sequence is the OsPT6 gene fragment shown in SEQ ID NO.1; Wherein, upstream primer P6s: as shown in SEQ ID NO.2; Downstream primer P6a: as shown in SEQ ID NO.3; By amplification, OsPT6 gene fragment is introduced SacI and XbaI enzyme cutting site;

2) OsPT6 gene is connected to pMD19-T plasmid vector, builds cloning vector pMD19-T-OsPT6, transformed competence colibacillus cell, extracts positive recombinant plasmid;

3) the synthetic T800 fragment that contains CaMV35S promotor, multiple clone site, NOS terminator of complete sequence, sequence, as shown in SEQ ID NO.4, is introduced respectively EcoRI and HindIII restriction enzyme site at the 3' of the 5' of composition sequence CaMV35S promotor end and NOS terminator end;

4) method of cutting connection by enzyme is inserted into T800 fragment between the EcoRI and HindIII restriction enzyme site of carrier is carrier pCAMBIA3301, obtains intermediate carrier pCAMBIA3301-T800;

5) with SacI and XbaI enzyme cutting clone carrier pMD19-T-OsPT6 and intermediate carrier pCAMBIA3301-T800 plasmid respectively, connect the OsPT6 gene fragment and the pCAMBIA3301-T800 that reclaim with ligase enzyme, OsPT6 gene is inserted between intermediate carrier CaMV35S promotor and NOS terminator, enzyme is cut checking, builds plant expression vector pCAMBIA3301-OsPT6.

Above-mentioned OsPT6 phosphorus efficiency utilizes the method for cultivation of genetically engineered soybean, specifically comprises the steps:

1) acquisition of OsPT6 genetically engineered soybean transformant: taking soybean cotyledon node as explant, by agrobacterium-mediated transformation, plant expression vector pCAMBIA3301-OsPT6 is imported in soybean gene group, obtain T by herbicide screening ₀for transformant;

2) OsPT6 genetically engineered soybean T ₂acquisition for positive plant: T ₀obtain T for plant by self-pollination ₁for seed, T ₁for identifying positive plant by PCR and GUS dyeing after seed germination, positive plant self-pollination obtains T ₂for seed; T ₂for identifying positive plant by same method after seed germination, obtain phosphorus efficiency and utilize genetically engineered soybean.

The present invention also provides phosphorus transporter albumen OsPT6 gene to utilize the application in transgenic plant at phosphorus efficiency, preferably, utilizes the application in genetically engineered soybean at phosphorus efficiency.

The plant expression vector pCAMBIA3301-OsPT6 that the present invention is above-mentioned, utilizes the application in transgenic plant cultivation at phosphorus efficiency, preferably, utilizes the application in genetically engineered soybean cultivation at phosphorus efficiency.

The present invention also provides a kind of Host Strains, contains above-mentioned plant expression vector pCAMBIA3301-OsPT6.

The present invention also provides above-mentioned Host Strains to utilize the application in transgenic plant cultivation at phosphorus efficiency, preferably, utilizes the application in genetically engineered soybean cultivation at phosphorus efficiency.

The measuring method that the present invention also provides a kind of phosphorus efficiency to utilize genetically engineered soybean OsPT6 gene relative expression quantity, the method, using soybean GmSKIP16 as reference gene, is measured OsPT6 gene by qRT-PCR and is utilized the expression amount in genetically engineered soybean at phosphorus efficiency;

Wherein, detect OsPT6 upstream region of gene primers F PT6: as shown in SEQ ID No.15, downstream primer RPT6: as shown in SEQ ID No.16;

Detect the upstream primer FSKIP16 of GmSKIP16 gene: as shown in SEQ ID No.13; Downstream primer RSKIP16: as shown in SEQ ID No.14.

The present invention also provides a kind of phosphorus efficiency to utilize the test kit of genetically engineered soybean OsPT6 gene relative expression flow measurement, and this test kit comprises following sequence:

OsPT6 gene test primer, upstream primer FPT6: as shown in SEQ ID No.15, downstream primer RPT6: as shown in SEQ ID No.16;

The detection primer of GmSKIP16 gene, upstream primer FSKIP16: as shown in SEQ ID No.13; Downstream primer RSKIP16: as shown in SEQ ID No.14.

Beneficial effect of the present invention:

1. the recombinant plant expression vector pCAMBIA3301-OsPT6 of the rice phosphorus transporter gene OsPT6 that the present invention builds, OsPT6 gene 5' end and 3' end are introduced respectively CaMV35S promotor and NOS terminator, increase the expression regulation of OsPT6 gene in foreign gene, improved the expression level of OsPT6 gene; This carrier can be applied to multiple phosphorus efficiency and utilize cultivation, the especially phosphorus efficiency of transfer-gen plant to utilize the cultivation of genetically engineered soybean, has filled up blank of the prior art.

2. the genetically engineered soybean that contains OsPT6 gene that adopts phosphorus efficiency provided by the invention to utilize the method for cultivation of genetically engineered soybean to produce, have the advantages that phosphorus efficiency utilizes, can under low-phosphorous environment, survive, pass through test for identification, the genetically engineered soybean of producing by method of cultivation provided by the invention, even under 75 days low-phosphorous growing environments, upgrowth situation is still good, output is also significantly higher than wild-type plant.

3. the recombinant plant expression vector pCAMBIA3301-OsPT6 of the rice phosphorus transporter gene OsPT6 that the present invention builds, contain herbicide resistance gene bar gene, make the plant expression vector that carries phosphate transporter gene OsPT6 in host cell, express more easily detection, the extra herbicide resistance gene bar gene increasing, has given transfer-gen plant Tolerance To Herbicides.

4. the present invention is by suitable reference gene GmSKIP16 gene, using this as benchmark, use fluorescent quantitative PCR technique, analyze OsPT6 gene relative expression quantity, can quantitative assay OsPT6 gene expression dose by the method, thereby select the genetically engineered soybean of suitable expression, for transgenic breeding is provided convenience.

Brief description of the drawings

The qualification of Fig. 1 plant expression vector pCAMBIA3301-OsPT6

A figure: OsPT6 fragment agarose gel electrophoresis is analyzed:

M:DNA Marker electrophoretic band, size is respectively 2Kb, 1Kb, 0.75Kb, 0.5Kb, 0.25Kb, 0.1Kb;

1:OsPT6 fragment electrophoretic band;

B figure: plant expression vector pCAMBIA3301-OsPT6 plasmid SacI, XbaI double digestion agarose gel electrophoresis are analyzed:

M1:DNA Marker electrophoretic band, size is respectively 15Kb, 10Kb, 7.5Kb, 5Kb, 2.5Kb, 1Kb, 0.25Kb;

1:pCAMBIA3301-OsPT6 electrophoretic band

2:pCAMBIA3301-T800 is through SacI, XbaI double digestion electrophoretic band

3:pCAMBIA3301-OsPT6 is through SacI, XbaI double digestion electrophoretic band;

M2:DNA Marker electrophoretic band, size is respectively 2Kb, 1Kb, 0.75Kb, 0.5Kb, 0.25Kb, 0.1Kb;

C figure: plant expression vector pCAMBIA3301-OsPT6 plasmid EcoRI, HindIII double digestion agarose gel electrophoresis are analyzed:

M1:DNA Marker electrophoretic band, size is 15Kb, 10Kb, 7.5Kb, 5Kb, 2.5Kb, 1Kb, 0.25Kb respectively,

1:pCAMBIA3301-OsPT6 electrophoretic band,

2:pCAMBIA3301-T800 is through EcoRI, HindIII double digestion electrophoretic band,

3:pCAMBIA3301-OsPT6 is through EcoRI, HindIII double digestion electrophoretic band,

M2:DNA Marker, size is respectively 2Kb, 1Kb, 0.75Kb, 0.5Kb, 0.25Kb, 0.1Kb.

Fig. 2 plant expression vector pCAMBIA3301-OsPT6 collection of illustrative plates

Fig. 3 Agrobacterium-mediated Transformation Systems of Soybean Cotyledonary Node process

A figure: 5 days seedling age aseptic seedling; B figure: cultivate altogether explant; C figure: differentiation; D figure: bud extends; E figure: bud is stretched to 3-4cm; F figure: half leaf method GUS dyeing; G figure: GUS is positive, and bud root grows to 1-2cm; H figure: T ₀transfer-gen plant

Fig. 4 genetically engineered soybean T ₀for plant PCR and Southern hybridization qualification

A figure: PCR qualification:

A figure: the pcr amplification rear electrophoresis figure of gus gene; B figure: the pcr amplification rear electrophoresis figure of bar gene; C figure: the pcr amplification rear electrophoresis figure of OsPT6 gene;

M:DNA Marker electrophoretic band: size is respectively 2Kb, 1Kb, 0.75Kb, 0.5Kb, 0.25Kb, 0.1Kb; P: plasmid DNA positive control; WT: wild-type plant negative control; 1 and 2: be respectively T ₀for transfer-gen plant 12PT6-1 and 12PT6-2;

B figure: Southern hybridization analysis:

P: plasmid DNA positive control; WT: wild-type plant negative control; 1 and 2: be respectively T ₀for transfer-gen plant 12PT6-1 and 12PT6-2.

Fig. 5 genetically engineered soybean GUS qualification of dyeing

A figure: stem cross section (the positive bud of GUS); B figure: T ₀for plant leaf; C figure: T ₀for plant; D figure: T ₀for plant flower pesticide; E figure: T ₀for plant beanpod; F figure: sprout the T of 24 hours ₁for seed; G figure: sprout the T of 72 hours ₂for seedling; H figure: T ₂for plant lateral root.

Fig. 6 turns OsPT6 gene soybean leaves Basta Resistance Identification

A figure: genetically engineered soybean blade has Basta resistance; B figure: wild-type soybean leaves does not have Basta resistance.

OsPT6 is at T for Fig. 7 fluorescence quantitative PCR detection ₂for the relative expression quantity in Transgenic soybean plants

WT: wild-type plant negative control; 1,2,3: be respectively T ₂for genetically engineered soybean strain 12PT6-1-1,12PT6-1-14,12PT6-2-8

Under the low-phosphorous water planting condition of Fig. 8, genetically engineered soybean growing state

A-C figure: represent low-phosphorous processing plant growth condition after 30 days; D-F figure: represent low-phosphorous processing plant growth condition after 60 days; G figure: represent low-phosphorous processing plant growth condition after 75 days.

OsPT6T ₂: represent T ₂transfer-gen plant; WT: represent wild-type plant negative control.Wherein, each vertical line length represents that ratio scale Bar:A, D, G short-term length represent actual Bar=15cm; B, C, the long line length of E and F represents actual Bar=1cm

Nutrient fluid cultivation plant Different Organs phosphorus accumulation under the low-phosphorous and normal phosphorus condition of Fig. 9

A figure: nutrient fluid cultivation is plant root, stem, leaf available phosphorus content after 60 days; B figure: nutrient fluid cultivation is plant root, stem, leaf total phosphorous after 60 days;

12PT6-1-1,12PT6-1-14 and 12PT6-2-8: represent respectively OsPT6 genetically engineered soybean T ₂for different strains; WT: represent wild-type plant negative control; Each strain selects 8 plant for analyzing altogether, different letter representation Duncan ' s multiple comparisons significant difference in P<0.05 level.

Embodiment

If method therefor is the usual technique means in this area without specified otherwise in following embodiment, reagent, material used, all can obtain by commercial sources if no special instructions.

One, the structure of plant expression vector pCAMBIA3301-OsPT6

The constructed plant expression vector of the present invention is taking pCAMBIA3301 carrier as carrier is carrier, and embodiment is as follows: 1. the clone of rice phosphorus transporter gene OsPT6 gene:

Selecting the rice leaf of scarce phosphorus processing is material, with reference to TaKaRa company total RNA extraction reagent (catalog number (Cat.No.): D9108A) specification sheets method, extracts the total RNA of blade reverse transcription and becomes cDNA, residual to remove RNA with RNase digested cdna product.With reference to rice phosphorus transporter gene OsPT6 (hereinafter to be referred as OsPT6) sequence information (NCBI accession number: AF536966.1), the OsPT6 gene fragment of design primer amplification sequence as shown in SEQ ID NO.1;

Upstream primer P6s: as shown in SEQ ID NO.2;

Downstream primer P6a: as shown in SEQ ID NO.3.

Taking above-mentioned rice cDNA as template, carry out PCR reaction, 50 μ L reaction systems comprise: 10 × PCR Buffer5.0 μ L, 2.5mmolL ^-1dNTP mix4.0 μ L, 25mmolL ^-1mgCl ₂3 μ L, 20 μ molL ^-1the each 1.0 μ L of P6s, P6a primer, Taq DNA Polymerase0.2 μ L, cDNA template 1.0 μ L, ddH ₂o mends to 50 μ L; Response procedures: 94 DEG C of denaturation 4min, 94 DEG C of sex change 1min, 61.4 DEG C of renaturation 1min, 72 DEG C are extended 2min, after 30 circulations, 72 DEG C of total elongation 10min; PCR product reclaims test kit (AXYGEN with gel, catalog number (Cat.No.): AP-GX-50) recovery purifying, be connected to pMD19-T carrier (TaKaRa, catalog number (Cat.No.): D102A), build cloning vector pMD19-T-OsPT6, transform intestinal bacteria TOP10 competent cell, carry out sequencing;

2. the transformation of intermediate carrier pCAMBIA3301-T800:

The synthetic T800 fragment that contains CaMV35S promotor, multiple clone site, NOS terminator of complete sequence, its sequence, as shown in SEQ ID NO.4, is introduced respectively EcoRI and HindIII restriction enzyme site (prosperous bio tech ltd of Beijing ancient cooking vessel state) at two ends; Get plasmid vector pCAMBIA3301 (CAMBIA, and the each 15 μ L of T800 fragment Australia), use respectively EcoRI (Promega) and HindIII (Promega) double digestion, 50 μ L enzymes are cut system: 10 × Buffer E5 μ L, BSA0.5 μ L, plasmid pCAMBIA3301 or T80015 μ L, EcoRI1.25 μ L, HindIII1.25 μ L, ddH ₂o27 μ L; 37 DEG C of endonuclease reaction 3h, enzyme is cut product and is carried out agarose gel electrophoresis analysis, reclaims test kit (AXYGEN) reclaim plasmid pCAMBIA3301 large fragment with gel, and size is 11256bp and T800 small segment, and size is 849bp, according to the ratio T of 1:4 ₄dNA ligase (TaKaRa, catalog number (Cat.No.): D2011A) connects, and 25 μ L ligation systems contain: 10 × T ₄ligase Buffer2.5 μ L, pCAMBIA3301 large fragment 2 μ L, T800 small segment 8 μ L, T ₄dNA ligase 1 μ L, ddH ₂o11.5 μ L; 16 DEG C of reaction overnight, get 10 μ L and connect product conversion TOP10 competent cell, 37 DEG C of incubated overnight of coated plate; After picking mono-clonal enlarged culturing, extract plasmid, pCAMBIA3301-T800 intermediate carrier has been transformed;

3. the structure of plant expression vector pCAMBIA3301-OsPT6

Get the intermediate carrier pCAMBIA3301-T800 of reincarnate and the plasmid of the cloning vector pMD19-T-OsPT6 that the 1st step obtains is used respectively SacI (Promega) and XbaI (Promega) double digestion.

50 μ L pCAMBIA3301-T800 double digestion systems contain: 10 × Buffer E5 μ L, BSA0.5 μ L, plasmid pCAMBIA3301-T800 or pMD19-T-OsPT615 μ L, SacI1.25 μ L, XbaI1.25 μ L, ddH ₂o27 μ L; 37 DEG C of reaction 3h;

50 μ L pMD19-T-OsPT6 double digestion systems contain: 10 × Buffer E5 μ L, BSA0.5 μ L, plasmid pMD19-T-OsPT615 μ L, SacI1.25 μ L, XbaI1.25 μ L, ddH ₂o27 μ L; 37 DEG C of reaction 3h;

Respectively above-mentioned double digestion product is carried out to agarose gel electrophoresis analysis, reclaim test kit (AXYGEN) with gel and reclaim plasmid pCAMBIA3301-T800 large fragment and OsPT6 small segment; The plasmid pCAMBIA3301-T800 large fragment and the OsPT6 small segment that reclaim, according to the ratio of 1:4, use T ₄dNA ligase (TaKaRa) connects, and the ligation system of 25 μ L contains: 10 × T ₄ligase Buffer2.5 μ L, pCAMBIA3301-T800 large fragment 2 μ L, OsPT6 small segment 8 μ L, T ₄dNA ligase 1 μ L, ddH ₂o11.5 μ L; 16 DEG C of ligations of spending the night, get 10 μ L and connect product conversion intestinal bacteria TOP10 competent cell.37 DEG C of incubated overnight, picking mono-clonal enlarged culturing, extracts plasmid, carries out PCR agarose gel electrophoresis analysis (seeing Figure 1A) and enzyme and cuts checking (seeing Figure 1B and 1C).Plant expression vector pCAMBIA3301-OsPT6 (seeing Fig. 2) successfully constructs.Two, plant expression vector pCAMBIA3301-OsPT6 transfection Agrobacterium EHA105

1. get the plasmid of the plant expression vector pCAMBIA3301-OsPT6 of 2 μ g (10 μ L) purifying, add in 200 μ L Agrobacterium EHA105 competence, mix;

2. ice bath 5min, proceeds to liquid nitrogen freezing 1min;

3. add 800 μ L YEB liquid nutrient mediums, 250rpm, 28 DEG C, 4-5h;

4. with pipettor, bacterium liquid being moved to YEB solid selects substratum (containing 50mgL ^-1kantlex) evenly coating;

Cultivate 1-2d for 5.28 DEG C, picking list bacterium colony, upgrading grain, PCR detects and obtains the Agrobacterium engineering bacteria transforming.

Three, agriculture bacillus mediated OsPT6 expression vector soybean transformation

1. the preparation of soybean cotyledon node explant and infecting

Get soybean ' NY-1001 ' seed of mature and plump, after surface sterilization, be inoculated in germination medium [B5+3% (w/v) sucrose+0.8% (w/v) agar, pH5.8], under 25 DEG C of conditions, illumination (16h illumination/8h dark, intensity 90 μ mol m ^-2s ^-1) 5 days acquisition aseptic seedling (as shown in Fig. 3-A) of cultivation.The hypocotyl that retains cotyledon below 2-3mm, vertically separates cotyledon along lower shaft embryo, and the former base of 5-7 axillalry bud of horizontal bar, obtains cotyledonary node explant.

Agrobacterium EHA105 containing plant expression vector pCAMBIA3301-OsPT6 is lined to flat board, cultivate 24h for 28 DEG C, picking mono-clonal liquid culture 24h, then gets 2mL bacterium liquid and adds in 200mL YEP liquid nutrient medium, and enlarged culturing is to OD ₆₅₀for 0.6-0.8, after bacterium liquid is centrifugal, with liquid culture medium [1/10B5 substratum+1.67mgL altogether ^-16-benzylaminopurine+0.25mgL ^-1plant hormones regulators,gibberellins+200 μ molL ^-1syringylethanone+3% (w/v) sucrose+0.7% (w/v) agar, pH5.4] resuspended, regulate OD ₆₅₀value is 1, obtains resuspended bacterium liquid.

Explant is put in the resuspended bacterium liquid of 50mL, infects 30min.Infect after end, blot unnecessary bacterium liquid, explant adaxial and its surface is positioned over downwards on the common substratum that is covered with one deck filter paper and (adds 3mmolL ^-1cys, 1mmolL ^-1sulfothiorine and dithiothreitol (DTT)), 4 days (as shown in Fig. 3-B) of 25 DEG C of dark cultivations.

2. the regeneration of Transgenic soybean plants

Cultivate altogether the explant after finishing, with liquid bud inducing culture [B5 medium+1.67mgL ^-16-benzylaminopurine+500mgL ^-1pyocianil+3% (w/v) sucrose+3mmolL ^-1mES, pH5.6] shake to wash and remove surperficial unnecessary thalline for 4-5 time, explant adaxial and its surface is upwards inoculated in to solid bud inducing culture (liquid bud inducing culture+0.8% (w/v) agar).25 DEG C of illumination (16h illumination/8h dark, 90 μ mol m ^-2s ^-1) cultivate 10 days.After 10 days, explant is inoculated in bud induction screening culture medium (bud inducing culture+4mgL ^-1two the third ammonia phosphorus), screening and culturing 4 weeks, every 2 weeks subcultures are once.

After screening and culturing 4 weeks, by there being the explant (as shown in Fig. 3-C) of differentiation, transfer to bud elongation medium [MS substratum+1mgL ^-1zeatin+0.5mgL ^-1plant hormones regulators,gibberellins+0.1mgL ^-1indolylacetic acid+100mgL ^-1pyrrolidonecarboxylic acid+50mgL ^-1altheine+300mgL ^-1pyocianil+2mgL ^-1two third ammonia phosphorus+3% (w/v) sucrose+3mmolL ^-1mES+0.8% (w/v) agar, pH5.6], bud extends (as shown in Fig. 3-D) to be cultivated, and every two weeks subcultures 1 time, bud extends 6-8 week.

In the time that bud is stretched to 3-4cm (as shown in Fig. 3-E), carry out half leaf method GUS dyeing qualification (as shown in Fig. 3-F), the positive bud of GUS of elongation cuts from base portion with scalpel, proceeds to root media [1/2B5 substratum+0.5mgL ^-1iBA+3% (w/v) sucrose+0.8% (w/v) agar, pH5.6], when the positive bud root of GUS grows to 1-2cm length (as shown in Fig. 3-G), open culturing bottle bottle cap domestication 2-3d, then carefully seedling is taken out with tweezers, clean the substratum of root, transplant as in the plastic tub that contains vermiculite: the peat composed of rotten mosses=1:1, in illumination box, tame for 25 DEG C, after 1 week, move into greenhouse, Routine Management, until ripe results obtain T ₀transfer-gen plant (as shown in Fig. 3-H).Obtain T by weedicide (Basta) screening ₀for transformant;

3. turn OsPT6 gene soybean T ₂for the acquisition of positive plant

T ₀obtain T for plant by self-pollination ₁for seed, T ₁for identifying positive plant by PCR and GUS dyeing after seed germination, positive plant self-pollination obtains T ₂for seed; T ₂for identifying positive plant by same method after seed germination.

Four, the qualification of genetically engineered soybean

1. Transgenic soybean plants PCR qualification

Get T ₀for genetically engineered soybean young leaflet tablet, SDS method is extracted genomic dna, and taking genomic dna as template, the primer that designs respectively gus, bar and OsPT6 gene carries out PCR, and agarose gel electrophoresis detects (as shown in Fig. 4-A).Detect the transgenosis characteristic of tentative confirmation plant by the PCR to foreign gene in transformed plant.

Upstream primer GUS1: as shown in SEQ ID No.5;

Downstream primer GUS2: as shown in SEQ ID No.6;

The pcr amplification program of gus gene: 94 DEG C of denaturation 5min, 94 DEG C of sex change 45sec, 55 DEG C of renaturation 1min, 72 DEG C are extended 1min, after 30 circulations, 72 DEG C of total elongation 10min;

Upstream primer bar1: as shown in SEQ ID No.7;

Downstream primer bar2: as shown in SEQ ID No.8;

The pcr amplification program of bar gene: 94 DEG C of denaturation 5min, 95 DEG C of sex change 1min, 55 DEG C of renaturation 1min, 72 DEG C are extended 1min, after 30 circulations, 72 DEG C of total elongation 10min;

Upstream primer PT61: as shown in SEQ ID No.9;

Downstream primer PT62: as shown in SEQ ID No.10.

The pcr amplification program of OsPT6 gene: 95 DEG C of denaturation 5min, 95 DEG C of sex change 1min, 63 DEG C of renaturation 30sec, 72 DEG C are extended 1min, after 30 circulations, 72 DEG C of total elongation 10min.

2. Transgenic soybean plants Southern blot qualification

Adopt SDS method, extract in a large number T ₀for genetically engineered soybean leaves genomic DNA.30 μ g genomic dnas are analyzed for Southern blot after being cut digestion by EcoRI enzyme; The mark of 421-bp OsPT6 probe and hybridizing method carry out (Roach, production number: 11745832910) according to Roach DIG-High Prime DNA Labeling and Detection Starter Kit I specification sheets.As shown in Fig. 4-B, OsPT6 gene is successfully incorporated in soybean gene group with the form of single copy.

421-bp OsPT6 fragment PCR obtains:

Upstream primer F: as shown in SEQ ID No.11;

Downstream primer R: as shown in SEQ ID No.12;

PCR response procedures: 95 DEG C of 5min; 68 DEG C of 2min; 72 DEG C of 30s; 30 circulations; 72 DEG C of 10min.3. the GUS of the each organ of genetically engineered soybean dyeing qualification

Get T ₀for organ samples and the T of genetically engineered soybean and wild-type soybean ₁, T ₂the seed that generation sprouts, is placed in respectively GUS staining fluid [50mmolL ^-1sodium phosphate buffer (pH7.0), 1% (v/v) Triton X100,20% (v/v) methyl alcohol, 50mmolL ^-1six cyanogen close ferrous acid potassium, 50mmolL ^-1hexacyanoferrate potassium, 1mM X-gluc (GOLDBIO, article No.: G1281C)] in, 37 DEG C of dyeing 12h.After 70% ethanol decolorization, the observation (as shown in Figure 5) of taking pictures.Compared with control group, each organ of transformed plant has gus genetic expression, and foreign gene can entail offspring.

In above substratum or staining fluid, the concentration of composition, quality volume percent are all for corresponding substratum or staining fluid volume.

4. genetically engineered soybean Basta smears qualification

Choose the T of healthy growth ₀for genetically engineered soybean and wild-type soybean leaves, dip 0.05%Basta by swab stick and be applied in half blade, after five days, observe blade performance, Taking Pictures recording (as shown in Figure 6).Shown in Fig. 6, half blade table that non-transgenic plant is smeared Basta reveals chlorosis, and the blade of transfer-gen plant is normal, shows that transfer-gen plant has Herbicid resistant, further proves the transgenosis characteristic of plant.

5. genetically engineered soybean T ₂for the mensuration of OsPT6 gene relative expression quantity in plant

Quantitative fluorescent PCR reaction, GmSKIP16 is as reference gene.Design respectively the qRT-PCR primer of GmSKIP16 and OsPT6 gene:

The qRT-PCR primer of GmSKIP16:

Upstream primer FSKIP16: as shown in SEQ ID No.13,

Downstream primer RSKIP16: as shown in SEQ ID No.14;

The qRT-PCR primer of OsPT6 gene:

Upstream primer FPT6: as shown in SEQ ID No.15,

Downstream primer RPT6: as shown in SEQ ID No.16.

For detecting soybean T ₂for the expression of OsPT6 gene in plant, get T ₂for the blade of transfer-gen plant and wild-type plant, extract total RNA (with reference to TaKaRa Trizol Reagent specification sheets), according to reverse transcription test kit (TaKaRa, article No.: DRR047A) method be cDNA by RNA reverse transcription, carry out qRT-PCR reaction, measure Ct value.Reaction system is with reference to TaKaRa premix Ex Taq ^tMiI (TaKaRa, article No.: RR820A) specification sheets 25 μ L reaction systems comprise: premix Ex Taq ^tMiI12.5 μ L, each 1.0 μ L (the 20 μ molL of upstream primer and downstream primer (GmSKIP16 or OsPT6 gene) ^-1), cDNA template 2 μ L, ddH ₂o8.5 μ L.Response procedures: after 95 DEG C of denaturation 30sec, 95 DEG C of sex change 5sec, 60 DEG C of annealing 20sec, 40 circulations.OsPT6 gene △ CT=C _{t, OsPT6}-C _{t, GmSKIP16}, wherein C _{t, OsPT6}for the Ct value of OsPT6 gene, C _{t, gmSKIP16}for the Ct value of reference gene GmSKIP16 gene.

OsPT6 gene relative expression quantity (as shown in Figure 7) in genetically engineered soybean, in figure, 1,2 and 3 represent respectively T ₂for transgenic line strain 12PT6-1-1,12PT6-1-14 and 12PT6-2-8, show that OsPT6 gene is at T ₂for all there being high expression level in rotaring gene plant blade.

6. the resistance to low-phosphorous qualification of genetically engineered soybean

Turn OsPT6 gene soybean T with above-mentioned 3 overexpressions ₂for strain (strain 12PT6-1-1,12PT6-1-14 and 12PT6-2-8) for resistance to phosphorus deprivation test.Above-mentioned 3 overexpressions of getting respectively 12 days seedling ages turn OsPT6 gene soybean T ₂for each 1 strain of seedling and a strain wild-type soybean seedling, WT in contrast, is fixed on porose cystose with sponge, is positioned on the plastic tank of Hoagland nutritive medium that improvement is housed.Two phosphorus concentrations, 10 μ M and 1000 μ M are set, in low-phosphorous nutritive medium, use NH ₄cl Substitute For Partial NH ₄h ₂pO ₄making final phosphorus concentration in solution is 10 μ M; Other ionic concns are constant; EC=2.61dSm ^-1.Nutritive medium is carried out to ventilation oxygen-supplying, within every three days, change one time of nutrition liquid (regulating pH value to 6.5).Each processing repeats 8 times.Greenhouse adopts Artificial Control, diurnal temperature: 28 DEG C/20 DEG C, and the photoperiod: (12h illumination/12h dark, HPS lamp, PPFD:600 μ mol.m ^-2.s ^-1), relative humidity: 70 – 80%, CO ₂concentration: 400 μ M.

During low-phosphorous processing, observe each plant strain growth performance (as shown in Figure 8).The low-phosphorous processing of nutrient fluid cultivation plant is after 30 days, and wild-type contrasts leaf chlorosis (as Fig. 8 A, shown in 8C); Low-phosphorous processing is after 60 days, and withered spot (as Fig. 8 D, shown in 8F) appears in wild-type adjoining tree blade; And rotaring gene plant blade normal (as Fig. 8 B, shown in 8E).Low-phosphorous processing is after 75 days, wild-type adjoining tree blade all come off (as shown in Fig. 8 G), and still on plant (as shown in Fig. 8 G) of rotaring gene plant blade major part shows that OsPT6 transfer-gen plant growth conditions under low-phosphorous condition is significantly better than wild-type contrast.

It is long that water planting is measured plant height and the root of plant for 50 days afterwards, and water planting is measured plant root biomass (as shown in table 1) after 90 days.As shown in Table 1, under low-phosphorus stress, the plant height of transfer-gen plant, root length and root weight average are significantly higher than wild-type contrast.Under normal phosphorus supply condition, transfer-gen plant and wild-type plant growth indexes are without significant difference.

In the plant reproductive growth later stage, effective pod number is measured; After seed results, measure single-strain seed number and seed weight (as shown in table 2).As shown in Table 2, under low-phosphorous condition, transfer-gen plant single-strain legumen number, seed grain number and seed weight are all significantly higher than wild-type contrast, show that OsPT6 transfer-gen plant can significantly increase the output of plant under low-phosphorous condition.Under normal phosphorus supply condition, transfer-gen plant and wild-type plant growth indexes are without significant difference.

Water planting was processed after 60 days, got transgenosis and wild-type plant leaf, stem and root sample determination available phosphorus and total phosphorous.

Available phosphorus content is measured: get 0.2 gram of fresh sample liquid nitrogen grinding powdered, to sample freeze thawing, add perchloric acid (PCA) the grinding homogenate of 1mL10% (w/v) in 4 DEG C of placements (on ice or refrigerator); Homogenate dilutes 10 times with the perchloric acid (PCA) of 5% (w/v), in placing 30 minutes on ice; 4 DEG C, centrifugal 10 minutes of 10000g, supernatant liquor is measured available phosphorus content with molybdenum blue method, calculates available phosphorus content.

Total phosphorous is measured: 105 DEG C of Plant samples, the 30min that completes, toasts two days in 70 DEG C of baking ovens, grinds, and mixes, and carries out sample total phosphorus concentration mensuration.Take about 0.1g plant dry sample, use dense H ₂sO ₄-H ₂o ₂mixing disappears boils.After cooling constant volume, by molybdenum antimony resistance colorimetric method mensuration, and calculate total phosphorous.

Be can be observed by Fig. 9, under low-phosphorous and normal phosphorus condition, transfer-gen plant available phosphorus and total phosphorous are all significantly higher than wild-type plant.

Plant vegetative growth index under table 1 water planting condition

Note: plant height and root length are measured in water planting for 50 days, root fresh weight and dry weight are measured in water planting for 90 days.

In table, data are mean value ± SD (n=8), adopt Duncan ' s multiple comparisons to carry out significance of difference analysis, and after same column data, the not identical person of lowercase represents significant difference (P ﹤ 0.05).

Plant reproductive growth index under table 2 water planting condition

Note: measure for 90 days in water planting.In table, data are mean value ± SD (n=8), adopt Duncan ' s multiple comparisons to carry out significance of difference analysis, and after same column data, the not identical person of lowercase represents significant difference (P ﹤ 0.05).

Claims (10)

1. a plant expression vector pCAMBIA3301-OsPT6, it is characterized in that containing phosphorus transporter albumen OsPT6 gene and herbicide resistance gene bar gene, rice phosphorus translocator OsPT6 gene 5' end and 3' end are introduced respectively CaMV35S promotor and NOS terminator.

2. an OsPT6 phosphorus efficiency utilizes the method for cultivation of genetically engineered soybean, it is characterized in that rice phosphorus transporter gene OsPT6 to build up in plant expression vector pCAMBIA3301, obtain recombinant plant expression vector pCAMBIA3301-OsPT6, import soybean by agriculture bacillus mediated method.

3. the construction process of plant expression vector pCAMBIA3301-OsPT6, is characterized in that, comprises the steps:

1) design primer, extension increasing sequence is the OsPT6 gene fragment shown in SEQ ID NO.1; Wherein, upstream primer P6s: as shown in SEQ ID NO.2; Downstream primer P6a: as shown in SEQ ID NO.3; By amplification, OsPT6 gene fragment is introduced SacI and XbaI enzyme cutting site;

2) OsPT6 gene is connected to pMD19-T plasmid vector, builds cloning vector pMD19-T-OsPT6, transformed competence colibacillus cell, extracts positive recombinant plasmid;

3) the synthetic T800 fragment that contains CaMV35S promotor, multiple clone site, NOS terminator of complete sequence, sequence, as shown in SEQ ID NO.4, is introduced respectively EcoRI and HindIII restriction enzyme site at the 3' of the 5' of composition sequence CaMV35S promotor end and NOS terminator end;

4) method of cutting connection by enzyme is inserted into T800 fragment between the EcoRI and HindIII restriction enzyme site of carrier is carrier pCAMBIA3301, obtains intermediate carrier pCAMBIA3301-T800;

5) with SacI and XbaI enzyme cutting clone carrier pMD19-T-OsPT6 and intermediate carrier pCAMBIA3301-T800 plasmid respectively, connect the OsPT6 gene fragment and the pCAMBIA3301-T800 that reclaim with ligase enzyme, OsPT6 gene is inserted between intermediate carrier CaMV35S promotor and NOS terminator, enzyme is cut checking, builds plant expression vector pCAMBIA3301-OsPT6.

4. OsPT6 phosphorus efficiency claimed in claim 2 utilizes the method for cultivation of genetically engineered soybean, specifically comprises the steps:

1) acquisition of OsPT6 genetically engineered soybean transformant: taking soybean cotyledon node as explant, by agrobacterium-mediated transformation, plant expression vector pCAMBIA3301-OsPT6 is imported in soybean gene group, obtain T by herbicide screening ₀for transformant;

2) OsPT6 genetically engineered soybean T ₂acquisition for positive plant: T ₀obtain T for plant by self-pollination ₁for seed, T ₁for identifying positive plant by PCR and GUS dyeing after seed germination, positive plant self-pollination obtains T ₂for seed; T ₂for identifying positive plant by same method after seed germination, obtain phosphorus efficiency and utilize genetically engineered soybean.

5. phosphorus transporter albumen OsPT6 gene utilizes the application in transgenic plant at phosphorus efficiency, preferably, utilizes the application in genetically engineered soybean at phosphorus efficiency.

6. plant expression vector pCAMBIA3301-OsPT6 claimed in claim 1, utilizes the application in transgenic plant cultivation at phosphorus efficiency, preferably, utilizes the application in genetically engineered soybean cultivation at phosphorus efficiency.

7. a Host Strains, is characterized in that containing plant expression vector pCAMBIA3301-OsPT6 claimed in claim 1.

8. Host Strains claimed in claim 7 utilizes the application in transgenic plant cultivation at phosphorus efficiency, preferably, utilizes the application in genetically engineered soybean cultivation at phosphorus efficiency.

9. phosphorus efficiency utilizes a measuring method for genetically engineered soybean OsPT6 gene relative expression quantity, it is characterized in that: using soybean GmSKIP16 as reference gene, measure OsPT6 gene utilize the expression amount in genetically engineered soybean at phosphorus efficiency by qRT-PCR;

Wherein, detect OsPT6 upstream region of gene primers F PT6: as shown in SEQ ID No.15, downstream primer RPT6: as shown in SEQ ID No.16;

Detect the upstream primer FSKIP16 of GmSKIP16 gene: as shown in SEQ ID No.13; Downstream primer RSKIP16: as shown in SEQ ID No.14.

10. phosphorus efficiency utilizes a test kit for genetically engineered soybean OsPT6 gene relative expression flow measurement, it is characterized in that: this test kit comprises following sequence:

OsPT6 gene test primer, upstream primer FPT6: as shown in SEQ ID No.15, downstream primer RPT6: as shown in SEQ ID No.16;

The detection primer of GmSKIP16 gene, upstream primer FSKIP16: as shown in SEQ ID No.13; Downstream primer RSKIP16: as shown in SEQ ID No.14.