CN104004755A - Method for breeding transgenosis soybean capable of efficiently using phosphorus by using OsPT2 - Google Patents

Abstract

The invention belongs to the molecular biology and the technical field of biotechnologies, relates to a phosphorus transfer protein gene OsPT2 and application of the gene for breeding transgenic crops and provides an expression cassette comprising the gene, and building of a recombinant plant expression vector and host bacteria comprising the vector, a method for breeding transgenosis soybean capable of efficiently using phosphorus, a method and a kit for detecting relative expression quantity of OsPT2 gene. The method for breeding transgenosis soybean by using OsPT2 based on efficient utilization of phosphorus fills in the blank of the application of phosphorus transfer protein gene OsPT2 in soybean breeding; by selecting an appropriate enzyme cutting site and introducing a 35s promoter and a terminator into the OsPT2 gene, expression regulation of the OsPT2 gene in an exogenous gene is improved; the built recombinant plant expression vector comprises a herbicide resistance gene which is a bar gene, thus facilitating the detection and improving the resistance of the transgenosis plant to the herbicide; the soybean produced by the breeding method is capable of efficiently using the phosphorus.

Description

OsPT2 phosphorus efficiency utilizes genetically engineered soybean method of cultivation

Technical field

The invention belongs to molecular biology and biological technical field, relate to a kind of phosphate transporter gene OsPT2 and the application of this gene in genetically modified crops breeding, be specifically related to the structure of a kind of phosphate transporter gene OsPT2 expression cassette, this expression cassette, and utilize that this expression cassette carries out that phosphorus efficiency utilizes the method for cultivation of genetically engineered soybean, the expression vector that uses in cultivating process and detection kit and the quantitative detecting method of Host Strains and application and this kind of genetically engineered soybean.

Technical background

Phosphorus (P) is one of required macroelement of growth and development of plants, almost participates in lived important Metabolic activity, in the processes such as transmission ofenergy, signal conduction, photosynthesis and respiration in plant materials, plays a key effect; In addition the important composition composition of phosphorus or nucleus and phosphatide (Raghothama K G, Phosphate transport and signaling.Current Opinion in Plant Biology, 2000.3 (3): 182-187).Plant obtains phosphorus and only obtains with phosphoric acid salt (Pi) form from soil.Although total phosphorous is higher in soil, but because phosphate concn lower (0.1 – 10 μ M) forms as one of limiting plant growth limiting factor (Raghothama K G and Karthikeyan A S, Phosphate acquisition.Plant and Soil, 2005.274:37-49).Approximately there is the 2/3 serious phosphorus that lacks in China farmland, wherein red soil is as the main anthropogenic soil of south China, the physico-chemical property that it is unique, phosphorus element wherein is more easily fixed, thereby show serious phosphorus phenomenon (Li Jie, Shi Yuanliang, the Chen Zhiwen of lacking, Red Soils in Southern China phosphorus element research overview. soil circular, 2011.42 (3): 763-768).In order to obtain high yield, the annual approximately 3,000 ten thousand tons of phosphate fertilizers in the whole world are in arable soil, wherein be wasted (L ó pez-Bucio J up to 80%, de la Vega OM, Guevara-Garc í a A and Herrera-Estrella L.Enhanced phosphorus uptake in transgenic tobacco plants that overproduce citrate.Nature Biotechnology, 2000.18:450-453).Soil degradation and environmental pollution are accelerated in using of a large amount of phosphate fertilizer, and cause phosphor resource to waste, it is estimated that phosphate rock resource will be at approach exhaustion (the Hata S end of this century, Kobae Y and Banba M, Interactions between plants and arbuscular mycorrhizal fungi.International Review of Cell and Molecular Biology, 2010.281:1-48).

Soybean is the crop that needs phosphorus amount more, one of crop that generally plant in the Ye Shi world, the topmost vegetables oil of the mankind and dietary protein origin (Herridge D F, Peoples M B and Boddey R M, Global inputs of biological nitrogen fixation in agricultural systems.Plant and Soil, 2008.311:1-18).Low-phosphorous output (the Vance C P that is limiting soybean of soil, Symbiotic nitrogen fixation and phosphorus acquisition.Plant nutrition in a world of declining renewable resources.Plant Physiology, 2001.127:390-397).And the countries in the world including China increase rapidly the consumption of soybean in recent years, make the imbalance between supply and demand of Soybean production more and more outstanding, particularly in China, be directly connected to national economic development and grain security (Cheng Fengxian, Tu Panfeng, Yan little Long, Wang Xiurong, Liao Hong, the phosphorus nutrition characteristic of phosphorus efficiency soybean new germ plasm in characteristic of acid red soil. plant nutrition and fertilizer journal, 2010.16 (1): 71-81).

In recent years, affine height phosphate transporter gene is transferred in suitable economy and farm crop, the transgenic line that obtains phosphorus efficiency is a kind of effective way of cultivating phosphorus efficiency New Crop Varieties, as overexpression GmPT5 gene in soybean, the phosphorus content of transfer-gen plant all significantly increases (Qin L than the phosphorus content of contrast under low-phosphorous and high phosphorus condition, Zhao J, Tian J, Chen L Y, Sun Z A, Guo Y X, Lu X, Gu M, Xu G H, Liao H.The high-affinity phosphate transporter GmPT5regulates phosphate transport to nodules and nodulation in soybean.Plant Physiology, 2012, 159:1634-1643).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 H F, Ren H Y, Gu M, Zhao J N, Sun S B, Zhang X, Chen J Y, Wu P and Xu G H, The phosphate transporter gene OsPht1; 8is involved in phosphate homeostasis in rice.Plant Physiology, 2011.156:1164-1175; Wu P, Shou H X, Xu G H and Lian X M, 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 overexpression in paddy rice, under low-phosphorous and high phosphorus condition, all can significantly increase phosphorus content (the Park M R of transfer-gen plant, Baek S H, Reyes B G and Yun S J, Overexpression of a high-affinity phosphate transporter gene from tobacco (NtPT1) enhances phosphate uptake and accumulation in transgenic rice plants.Plant and Soil, 2007.292:259-269).As can be seen here, in plant, overexpression phosphate transporter gene can increase the absorption of phosphorus and improve resistance to low-phosphorous ability under low-phosphorous condition.

The phosphate transporter gene OsPT2 of one of paddy rice (Oryza sativa L.) Pht1 family gene is mainly at root center pillar, leaf phloem, in xylem, express, it is the response gene of phosphate starvation signal, be responsible for absorption and phosphorus transport process (the Wu P in vivo of plant to phosphorus, Shou H X, Xu G H and Lian X M, Improvement of phosphorus efficiency in rice on the basis of understanding phosphate signaling and homeostasis.Current Opinion in Plant Biology, 2013.16:1-8).Utilize RNA silent technology to knock out that OsPT2 significantly increases the absorption of phosphorus and from root to over-ground part long-distance transportation, and overexpression OsPT2 can cause the accumulation of the excessive phosphorus of paddy rice overground part, thereby cause phosphorism.Due to its special expression pattern, OsPT2 is considered to play important effect (Ai P H, Sun S B in plant phosphorus transport process, Zhao J N, Fan X R, Xin W J, Guo Q, Yu L, Shen Q R, Wu P, Miller A J and Xu G H, Two rice phosphate transporters, OsPht1; 2and OsPht1; 6, have different functions and kinetic properties in uptake and translocation.The Plant Journal, 2009.57 (5): 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 OsPT2 gene in soybean breeder 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 phosphorus transporter albumen OsPT2 expression casette, its sequence is SEQ ID NO.2, has EcoRI and HindIII double enzyme site.

The present invention also provides the construction process of above-mentioned phosphorus transporter albumen OsPT2 expression casette, comprises the steps:

1) design primer, extension increasing sequence is the OsPT2 gene fragment shown in SEQ ID NO.1; Wherein, upstream primer P1: as shown in SEQ ID NO.3; Downstream primer P2: as shown in SEQ ID NO.4;

2) OsPT2 gene is connected to pMD19-T plasmid vector, transforms TOP10 competent cell, extract positive recombinant plasmid, carry out SacI and XbaI double digestion, reclaim OsPT2 gene fragment;

3) carry out plant expression vector pBI121SacI and XbaI double digestion, reclaim pBI121 large fragment;

4) connect OsPT2 gene fragment and the pBI121 large fragment of above-mentioned recovery with ligase enzyme, form and connect product, transform TOP10 competent cell to connect product, 37 DEG C of incubated overnight, picking mono-clonal enlarged culturing, filters out transformant;

5) extraction step 4) in the positive recombinant plasmid of transformant carry out EcoRI and HindIII double digestion, reclaim small segment, obtain OsPT2 expression casette;

Wherein, step 2), 3) and step 5) in double digestion system be: 10 × Buffer15 μ L in 50 μ L systems, BSA0.5 μ L, DNA15 μ L, SacI1.0 μ L, XbaI1.0 μ L, ddH ₂o mends to 50 μ L; 37 DEG C of reaction 2h; Double digestion product carries out agarose gel electrophoresis analysis, reclaims object fragment;

Step 4) in ligation system be: 10 × T ₄ligase Buffer2.5 μ L, pBI121 large fragment 2 μ L, OsPT2 gene fragment 8 μ L, T ₄dNA ligase 1 μ L, ddH ₂o mends to 25 μ L.

The invention provides OsPT2 gene and utilize the application in transgenic plant at phosphorus efficiency, preferably, utilize the application in genetically engineered soybean at phosphorus efficiency.

The method of cultivation that the present invention also provides a kind of phosphorus efficiency to utilize genetically engineered soybean, builds up to rice phosphorus transporter gene OsPT2 in plant expression vector pCAMBIA3301-OsPT2, imports soybean by agriculture bacillus mediated method; Specifically comprise the steps:

1) structure of recombinant plant expression vector pCAMBIA3301-OsPT2: rice phosphorus translocator OsPT2 expression casette is built up in plant expression vector pCAMBIA3301, form recombinant plant expression vector pCAMBIA3301-OsPT2;

2) acquisition of OsPT2 genetically engineered soybean transformant: taking soybean cotyledon node as explant, by agrobacterium-mediated transformation, plant expression vector pCAMBIA3301-OsPT2 is imported in soybean gene group, obtain T by weedicide Bialaphos screening ₀for transformant;

3) turn OsPT2 gene 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 a kind of recombinant plant expression vector pCAMBIA3301-OsPT2, contain above-mentioned phosphorus transporter albumen OsPT2 expression casette and herbicide resistance gene bar gene, rice phosphorus translocator OsPT2 expression casette is between the EcoRI and HindIII restriction enzyme site of plant expression vector pCAMBIA3301.

Above-mentioned plant expression vector pCAMBIA3301-OsPT2, 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-OsPT2.

Host Strains provided by the invention 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 phosphorus efficiency to utilize the measuring method of genetically engineered soybean OsPT2 gene relative expression quantity, extend factor EF-laX56856 gene TefS1 as reference gene using soybean, measure OsPT2 gene by sxemiquantitative RT-PCR and utilize the expression amount in genetically engineered soybean at phosphorus efficiency;

Wherein, use the primer of preparing of 444-bp OsPT2 gene probe, upstream primer F: as shown in SEQ ID No.9, downstream primer R: as shown in SEQ ID No.10;

Detect the upstream primer FT of TefS1 gene: as shown in SEQ ID No.11; Downstream primer RT: as shown in SEQ ID No.12.

The present invention also provides a kind of phosphorus efficiency to utilize the test kit of genetically engineered soybean OsPT2 gene relative expression flow measurement, and this test kit comprises following sequence:

444-bp OsPT2 gene probe prepare primer, upstream primer F: as shown in SEQ ID No.9, downstream primer R: as shown in SEQ ID No.10;

The detection primer of TefS1 gene, upstream primer FT: as shown in SEQ ID No.11; Downstream primer RT: as shown in SEQ ID No.12.

Beneficial effect of the present invention:

1. the present invention, by selecting suitable restriction enzyme site, introduces 35s promotor and terminator by the OsPT2 gene through artificial pcr amplification, has increased the expression regulation of OsPT2 gene in foreign gene, has improved the expression level of OsPT2 gene; Simultaneously, by introducing EcoRI and HindIII double enzyme site, the phosphorus transporter albumen OsPT2 expression casette forming 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 soybean that contains OsPT2 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, there is not significance and reduce in output.

3. the recombinant plant expression vector pCAMBIA3301-OsPT2 of the rice phosphorus transporter gene OsPT2 that the present invention builds, contain herbicide resistance gene bar gene, make the plant expression vector that carries phosphate transporter gene OsPT2 in host cell, express more easily detection, the extra herbicide resistance gene bar gene increasing has improved the resistance of transfer-gen plant to weedicide, has increased quality.

4. the present invention is that soybean extends factor EF-laX56856 gene (TefS1) as reference gene by selecting suitable house-keeping gene, using this as benchmark, use integrated gel imaging system, gel image analysis OsPT2 gene is with respect to the expression amount of TefS1 gene, can quantitative assay OsPT2 gene transformation rate by the method, thereby select the genetically engineered soybean of suitable expression, for transgenic breeding is provided convenience.

Brief description of the drawings

Fig. 1 OsPT2 expression casette

The T-DNA district of Fig. 2 plant expression vector pCAMBIA3301-OsPT2

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

A: 5 days seedling ages of aseptic seedling; B: cotyledonary node is cultivated altogether; The C:Bialaphos screening explant of 2 months; D: resistant buds is stretched to 3-4cm; The positive blastogenesis root of E:GUS; F: transgenic positive plant.

Fig. 4 Transgenic soybean plants PCR and Southern hybridization qualification

A:PCR identifies T ₀for plant: a-gus gene; B-bar gene; C-OsPT2 gene; M-DNA Marker: size is respectively 2Kb, 1Kb, 0.75Kb, 0.5Kb, 0.25Kb, 0.1Kb; P-plasmid DNA positive control; WT-wild-type plant negative control; 1～10-be respectively T ₀for transfer-gen plant 12PT2-1～12PT2-10;

B:Southern hybridization analysis: M-DNA Marker: size is respectively 15Kb, 7.5Kb, 0.75Kb, 0.5Kb, 0.25Kb, 0.1Kb; P-plasmid DNA positive control; WT-wild-type plant negative control; 1～10-be respectively T ₀for transfer-gen plant 12PT2-1～12PT2-10.

Fig. 5 genetically engineered soybean GUS qualification of dyeing

A:T ₀for plant leaf; B:T ₀for plant; C: sprout the T of 3 days ₁for seed; D: sprout the T of 24 hours ₂for seed: 1-3-be respectively 12PT2-1,12PT2-2,12PT2-4, WT-wild-type plant negative control.

Fig. 6 turns OsPT2 gene plant Basta Analysis of Resistance

A: black arrow represents that transfer-gen plant has Basta resistance; B: white arrow represents that negative plant does not have Basta resistance.

Fig. 7 sxemiquantitative RT-PCR detects OsPT2 at T ₂for the expression in plant

A: sxemiquantitative RT-PCR detects OsPT2 genetic expression electrophoresis result.B: photodensitometry OsPT2 gene relative expression quantity.WT-wild-type plant negative control; 1,2,3-be respectively transfer-gen plant 12PT2-1,12PT2-2,12PT2-4; TefS1-house-keeping gene

Nutrient fluid cultivation plant strain growth situation under the low-phosphorous condition of Fig. 8

A-C: low-phosphorous processing is after 10 days; D-F: low-phosphorous processing is after 45 days; G: low-phosphorous processing is after 75 days; OsPT2T ₂-T ₂transfer-gen plant; WT-wild-type plant negative control, wherein, each vertical line length represents 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: nutrient fluid cultivation is plant root, stem, leaf available phosphorus content after 30 days; B: nutrient fluid cultivation is plant root, stem, leaf total phosphorous after 30 days; C: total phosphorous in fallen leaves; D: total phosphorous in seed; Each strain selects 5 plant for analyzing altogether, and different letter representation LSD are significant difference in P<0.05 level relatively.

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-OsPT2

1. the clone of rice phosphorus transporter gene OsPT2 gene:

Taking rice leaf as material, with reference to TaKaRa company total RNA extraction reagent box (catalog number (Cat.No.): D9108A) specification sheets method, extract the total RNA of blade reverse transcription and become cDNA, with RNase digested cdna product, with reference to rice phosphorus transporter gene gene order information (NCBI accession number: AF536962), design primer carries out PCR reaction, amplifies rice phosphorus transporter gene OsPT2; Wherein, the sequence of gene OsPT 2 is as SEQ ID NO.1; Primer sequence: upstream primer P1 sequence is as shown in SEQ ID NO.3, and downstream primer P2 sequence is as shown in SEQ ID NO.4.

The cDNA becoming taking above-mentioned reverse transcription, as template, carries out PCR reaction;

PCR reaction system: 10 × PCR Buffer5.0 μ L, dNTP mix4.0 μ L (2.5mmolL ^-1), each 1.0 μ L (the 20 μ molL of primer P1 and P2 ^-1), PrimeSTAR ^tMhS DNA Polymerase0.4 μ L, cDNA template 1 μ L, ddH ₂o mends to 50 μ L;

Two-step approach PCR response procedures: 94 DEG C, 5min, 65 DEG C, 2min, 72 DEG C, 45s, after 30 circulations, 72 DEG C of total elongation 10min;

PCR product reclaims test kit (AXYGEN, catalog number (Cat.No.): AP-GX-50) with gel and reclaims purifying, uses T ₄dNA ligase (TaKaRa, catalog number (Cat.No.): D2011A) is connected to pMD19-T carrier (TaKaRa, catalog number (Cat.No.): D102A), transforms TOP10 competent cell, obtains positive recombinant plasmid, carries out sequencing;

2. the structure of phosphorus transporter albumen OsPT2 expression casette

Extract above-mentioned positive recombinant plasmid and carry out SacI and XbaI double digestion, reclaim OsPT2 gene fragment;

Plant expression vector pBI121 is carried out to SacI and XbaI double digestion simultaneously, reclaim pBI121 large fragment;

Double digestion system (50 μ L): 10 × Buffer15 μ L, BSA0.5 μ L, DNA15 μ L, SacI1.0 μ L, XbaI1.0 μ L, ddH ₂o mends to 50 μ L; 37 DEG C of reaction 2h; Double digestion product carries out agarose gel electrophoresis analysis, reclaims object fragment;

The pBI121 large fragment of above-mentioned recovery and OsPT2 gene fragment, according to the ratio of 1:4, use T ₄dNA ligase connects, ligation system: 10 × T ₄ligase Buffer2.5 μ L, pBI121 large fragment 2 μ L, OsPT2 gene fragment 8 μ L, T ₄dNA ligase 1 μ L, ddH ₂o mends to 25 μ L; 16 DEG C of reactions are spent the night, and get 10 μ L and connect product conversion TOP10 competent cell.37 DEG C of incubated overnight, picking mono-clonal enlarged culturing, screening transformant;

Extract the positive recombinant plasmid of transformant, carry out EcoRI and HindIII double digestion according to the above-mentioned enzyme system of cutting, reclaim small segment, obtain OsPT2 expression casette, sequence is as shown in SEQ ID NO.2;

3. the structure of recombinant plant expression vector pCAMBIA3301-OsPT2

Plant expression vector pCAMBIA3301 is carried out to EcoRI and HindIII double digestion according to the above-mentioned enzyme system of cutting, reclaim large fragment.

The pCAMBIA3301 large fragment reclaiming after double digestion and OsPT2 expression casette, according to the ratio of 1:4, use T ₄dNA ligase connects, ligation system: 10 × T ₄ligase Buffer2.5 μ L, pCAMBIA3301 large fragment 2 μ L, OsPT2 expression casette 8 μ L, T ₄dNA ligase 1 μ L, ddH ₂o mends to 25 μ L; 16 DEG C of reactions are spent the night, and get 10 μ L and connect product conversion TOP10 competent cell.37 DEG C of incubated overnight, picking mono-clonal enlarged culturing, extracts plasmid, carries out double digestion checking, and recombinant plant expression vector pCAMBIA3301-OsPT2 successfully constructs.

Two, recombinant plant expression vector pCAMBIA3301-OsPT2 transfection Agrobacterium EHA105

1. extract 10 μ L, i.e. the recombinant plant expression vector pCAMBIA3301-OsPT2 of 2 μ g purifying, adds 200 μ L Agrobacterium EHA105 competence, mixes;

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 is moved to YEB solid and select substratum, wherein containing 50mgL ^-1kantlex, evenly coating;

Cultivate 1-2d for 5.28 DEG C, picking list bacterium colony, extracts plasmid, and enzyme is cut qualification and obtained the Agrobacterium engineering bacteria transforming.

Three, agriculture bacillus mediated OsPT2 expression vector soybean transformation

1. the preparation of soybean cotyledon node explant and infecting

Get soybean ' new the Liao Dynasty the is fresh ' seed of mature and plump, after surface sterilization, be inoculated in germination medium [B5 medium+3% (w/v)+0.8% (w/v) agar, pH5.8], under 25 DEG C of conditions, illumination (time 16h daytime/8h night, intensity 90 μ mol m ^-2s ^-1) 5-6 days acquisition aseptic seedling (as shown in Fig. 3-A) of cultivation.The hypocotyl that retains cotyledon below 2-3cm, vertically separates cotyledon along lower shaft embryo, and the former base of 5-7 axillalry bud of horizontal bar, obtains cotyledonary node explant.

To line flat board containing recombinant plant expression vector pCAMBIA3301-OsPT2 Agrobacterium EHA105, and 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 substratum [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.

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 inoculated in to common substratum (the interpolation 3mmolL that is covered with one deck filter paper downwards ^-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 transfer-gen plant

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).(16h illumination/8h dark, intensity of illumination is 90 μ mol m in 25 DEG C of illumination ^-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 once.

Cultivate after 4 weeks, by there being the explant (as shown in Fig. 3-C) of differentiation, be inoculated into 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] on cultivate, every two weeks subcultures 1 time, cultivate about 6-8 week.

In the time that bud is stretched to 3-4cm (as shown in Fig. 3-D), cut from base portion with scalpel, proceed to root media [1/2B5 substratum+0.5mgL ^-1iBA+3% (w/v) sucrose+0.8% (w/v) agar, pH5.6], when root grows to 1-2cm length (as shown in Fig. 3-E), domestication moves into greenhouse, and Routine Management, until ripe results.Obtain T by weedicide (Bialaphos) screening ₀for transformant;

3. turn OsPT2 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 blade, SDS method is extracted genomic dna, taking genomic dna as template, respectively gus, bar and OsPT2 gene is carried out to PCR detection, and agarose gel electrophoresis detects (as shown in Fig. 4-A).Detect by the PCR to foreign gene gus, bar and OsPT2 in transformed plant, tentative confirmation OsPT2 gene success transformed plant also obtains restructuring.

OsPT2 gene PCR detects primer sequence, upstream primer P1: as shown in SEQ ID NO.3; Downstream primer P2: as shown in SEQ ID NO.4;

Gus gene PCR detects primer sequence, upstream primer GUS1: as shown in SEQ ID No.5; Downstream primer GUS2: as shown in SEQ ID No.6;

Bar gene PCR detects primer sequence, 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 gus gene: 94 DEG C, 5min, 94 DEG C, 45sec, 55 DEG C, 1min, 72 DEG C, 1min, after 30 circulations, 72 DEG C of total elongation 10min; The pcr amplification program of bar gene: 94 DEG C, 5min, 94 DEG C, 45sec, 58 DEG C, 1min, 72 DEG C, 1min, after 30 circulations, 72 DEG C of total elongation 10min; 94 DEG C of the pcr amplification programs of OsPT2 gene, 5min, 65 DEG C, 2min, 72 DEG C, 45s, 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 EcoRI enzyme is cut digestion; The mark of 444-bp OsPT2 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.Shown in Fig. 4-B, transfer-gen plant contains 1-3 copy.

444-bp OsPT2 probe obtains by pcr amplification reaction:

PCR reaction upstream primer F: as shown in SEQ ID No.9, downstream primer R: as shown in SEQ ID No.10.PCR response procedures: 95 DEG C, 4min; 95 DEG C, 2min, 55 DEG C, 30s; 72 DEG C, 30s; 72 DEG C, 10min.

3. the GUS dyeing qualification of transfer-gen plant flower and seed

Get T ₀for genetically engineered soybean and wild-type soybean leaves, flower and seed, be 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, production number: G1281C)], 37 DEG C of dyeing 12h.After 70% ethanol decolorization, the observation (as shown in Figure 5) of taking pictures.Shown in Fig. 5, T ₀gus genetic expression, T are all detected for transfer-gen plant flower with seed ₁and T ₂after sprouting for transgenic seed, also detect the expression of gus gene, illustrate that 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 5 days, observe blade Taking Pictures recording (as shown in Figure 6).Shown in Fig. 6, the 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 OsPT2 gene relative expression quantity in plant

Sxemiquantitative RT-PCR (reverse transcription PCR) measures OsPT2 gene at the above-mentioned OsPT2 gene plant T that turns ₂for the expression amount in plant, using soybean house-keeping gene: soybean extends factor EF-laX56856 gene (TefS1) as reference gene.The gel images that uses integrated gel imaging system (GenoSens1880, Shanghai Qin Xiang Science and Technology Ltd., China) to carry out after ethidium bromide staining obtains and analyzes.The optical density(OD) of setting house-keeping gene TefS1 gene is that the optical density(OD) of 1, OsPT2 gene and the Reinhoit Zahl of house-keeping gene TefS1 gene are the relative expression quantity of OsPT2 gene.

Wherein, in reverse transcription PCR reaction, the primer that detects OsPT2 gene adopts preparation 444-bp OsPT2 probe primer, upstream primer F: as shown in SEQ ID No.9; Downstream primer R: as shown in SEQ ID No.10;

Detect house-keeping gene TefS1 gene, upstream primer FT: as shown in SEQ ID No.11; Downstream primer RT: as shown in SEQ ID No.12.

For detecting soybean T ₂for the expression of OsPT2 gene in plant, get 3 single copy transfer-gen plant (12PT2-1,12PT2-2 and 12PT2-4) and blade and the root of wild-type adjoining tree, extract respectively total RNA (with reference to TaKaRaTrizol Reagent specification sheets), according to reverse transcription test kit (TaKaRa, article No.: DRR047A) method be cDNA by RNA reverse transcription, carry out sxemiquantitative RT-PCR reaction.20 μ L reaction systems comprise: 10 × PCR Buffer2.0 μ L, dNTP mix1.6 μ L (2.5mmolL- ¹), each 1.0 μ L (the 20 μ molL of the upstream primer FT of TefS1 upstream primer F, downstream primer R and OsPT2 gene and downstream primer RT ^-1), PrimeSTAR ^tMhS DNA Polymerase0.2 μ L, cDNA template 1 μ L, ddH ₂o mends to 20 μ L.Response procedures: 95 DEG C, 4min; 95 DEG C, 2min, 55 DEG C, 30s; 72 DEG C, 30s; 30 rear 72 DEG C of extension 10min of circulation.Blade and root are carried out to the mensuration (as shown in Figure 7) of TefS1 gene expression amount and OsPT2 gene relative expression quantity.Be can be observed by Fig. 7, OsPT2 gene is at T ₂for being overexpression in rotaring gene plant blade and root.

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

Turn OsPT2 gene soybean T with above-mentioned 3 overexpressions ₂be used for resistance to phosphorus deprivation test for strain.Three overexpressions turn OsPT2 gene soybean T ₂for the positive plant of 10 days seedling ages of strain (12PT2-1,12PT2-2 and 12PT2-4) with contrast (WT) 10 days seedling age plant and be colonizated in the Hoagland solution that contains improvement and (use NH ₄cl Substitute For Partial NH ₄h ₂pO ₄making final P concentration in solution is 20 μ M; Other ionic concns are constant; EC=2.61dSm ^-1) 18-L bucket in (above enclosing diameter is 30cm), every barrel of T that contains three different strains ₂for plant and an adjoining tree (four plant are square pitch arrangement, and diagonal distance is 26cm), nutritive medium is led to oxygen continuously, and replacing in every three days (by HCl or NaOH adjusting pH value to 6.5).Each T ₂repeat 10 times for strain.Greenhouse adopts Artificial Control, diurnal temperature: 28 DEG C of daytime/20 DEG C nights, photoperiod: 12h daytime/12h night (HPS lamp, PPFD:600 μ mol.m ^-2.s ^-1), relative humidity: 70 – 80%, CO ₂concentration: 400 μ M.Taking standard Hoagland nutritive medium as contrast.

During low-phosphorous processing, observe each Symptoms (as shown in Figure 8).The low-phosphorous processing of nutrient fluid cultivation plant is after 10 days, and wild-type contrast blade edge starts to occur withered spot (as Fig. 8 A, shown in 8C); Low-phosphorous processing is after 30 days, and the withered spot of wild-type adjoining tree blade edge increases, and withered spot (as Fig. 8 D, shown in 8F) also appears in vein; And there is not all the time withered spot (as Fig. 8 B, shown in 8E) in rotaring gene plant blade.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 to turn OsPT2 gene plant growth conditions under low-phosphorous condition and is significantly better than wild-type contrast.

Plant height, root length, biomass and the phosphorus content (as table 1 and Fig. 9 A, shown in 9B) of plant measured in low-phosphorous processing for 30 days afterwards.As shown in Table 1, under low-phosphorus stress, the plant height of transfer-gen plant, root length and biomass are all significantly higher than wild-type contrast.Be can be observed by Fig. 9, under low-phosphorus stress, in transfer-gen plant root, stem, leaf, available phosphorus and content of tatal phosphorus are all significantly higher than wild-type contrast.After plant maturation, measure phosphorus content (as Fig. 9 C, shown in 9D) in fallen leaves and seed.By Fig. 9 C, shown in 9D, in transfer-gen plant fallen leaves and seed, phosphorus content is significantly higher than wild-type contrast, shows that transfer-gen plant has higher phosphate use efficiency.

Under low-phosphorous processing, plant enters reproductive stage, to spending number, pod number to measure; 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 spends number, pod number and output to be all significantly higher than wild contrast; Under normal condition, OsPT2 transfer-gen plant spends number, pod number and output and wild-type plant and transfer-gen plant without significant difference, shows the better tolerance of OsPT2 transfer-gen plant in low-phosphorous condition, under low-phosphorous condition, can significantly increase output.

The comparison of plant vegetative growth index under table 1. nutrient fluid cultivation condition

Each parameter is cultivated and is measured afterwards for 30 days plant; Compare and do test of significance in P<0.05 level with LSD, the different significant differences that represent of letter.

The comparison of plant reproductive growth index under table 2. nutrient fluid cultivation condition

Compare and do test of significance in P<0.05 level with LSD, the different significant differences that represent of letter.

Claims (10)

1. a phosphorus transporter albumen OsPT2 expression casette, is characterized in that: this expression cassette sequence is as shown in SEQ ID NO.2.

2. the construction process of phosphorus transporter albumen OsPT2 expression casette claimed in claim 1, is characterized in that, comprises the steps:

1) design primer, extension increasing sequence is the OsPT2 gene fragment shown in SEQ ID NO.1; Wherein, upstream primer P1: as shown in SEQ ID NO.3; Downstream primer P2: as shown in SEQ ID NO.4;

2) OsPT2 gene is connected to pMD19-T plasmid vector, transforms TOP10 competent cell, extract positive recombinant plasmid, carry out SacI and XbaI double digestion, reclaim OsPT2 gene fragment;

3) carry out plant expression vector pBI121SacI and XbaI double digestion, reclaim pBI121 large fragment;

4) connect OsPT2 gene fragment and the pBI121 large fragment of above-mentioned recovery with ligase enzyme, form and connect product, transform TOP10 competent cell to connect product, 37 DEG C of incubated overnight, picking mono-clonal enlarged culturing, filters out transformant;

5) extraction step 4) in the positive recombinant plasmid of transformant carry out EcoRI and HindIII double digestion, reclaim small segment, obtain OsPT2 expression casette;

Wherein, step 2), 3) and step 5) in double digestion system be: 10 × Buffer15 μ L in 50 μ L systems, BSA0.5 μ L, DNA15 μ L, SacI1.0 μ L, XbaI1.0 μ L, ddH ₂o mends to 50 μ L; 37 DEG C of reaction 2h; Double digestion product carries out agarose gel electrophoresis analysis, reclaims object fragment;

Step 4) in ligation system be: 10 × T ₄ligase Buffer2.5 μ L, pBI121 large fragment 2 μ L, OsPT2 gene fragment 8 μ L, T ₄dNA ligase 1 μ L, ddH ₂o mends to 25 μ L.

3.OsPT2 gene utilizes the application in transgenic plant at phosphorus efficiency, preferably, utilizes the application in genetically engineered soybean at phosphorus efficiency.

4. a phosphorus efficiency utilizes the method for cultivation of genetically engineered soybean, it is characterized in that rice phosphorus transporter gene OsPT2 to build up in plant expression vector pCAMBIA3301, obtain in recombinant plant expression vector pCAMBIA3301-OsPT2, import soybean by agriculture bacillus mediated method; Specifically comprise the steps:

1) structure of recombinant plant expression vector pCAMBIA3301-OsPT2: rice phosphorus translocator OsPT2 expression casette is built up in plant expression vector pCAMBIA3301, form recombinant plant expression vector pCAMBIA3301-OsPT2;

2) acquisition of OsPT2 genetically engineered soybean transformant: taking soybean cotyledon node as explant, by agrobacterium-mediated transformation, plant expression vector pCAMBIA3301-OsPT2 is imported in soybean gene group, obtain T by weedicide Bialaphos screening ₀for transformant;

3) OsPT2 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. a recombinant plant expression vector pCAMBIA3301-OsPT2, it is characterized in that containing phosphorus transporter albumen OsPT2 expression casette claimed in claim 1 and herbicide resistance gene bar gene, rice phosphorus translocator OsPT2 expression casette arrives between the EcoRI and HindIII restriction enzyme site of plant expression vector pCAMBIA3301.

6. plant expression vector pCAMBIA3301-OsPT2 claimed in claim 5, 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 recombinant plant expression vector pCAMBIA3301-OsPT2 claimed in claim 4.

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. a phosphorus efficiency utilizes the measuring method of genetically engineered soybean OsPT2 gene relative expression quantity, it is characterized in that: extend factor EF-laX56856 gene TefS1 as reference gene using soybean, measure OsPT2 gene by sxemiquantitative RT-PCR and utilize the expression amount in genetically engineered soybean at phosphorus efficiency;

Wherein, use the primer of preparing of 444-bp OsPT2 gene probe, upstream primer F: as shown in SEQ ID No.9, downstream primer R: as shown in SEQ ID No.10;

Detect the upstream primer FT of TefS1 gene: as shown in SEQ ID No.11; Downstream primer RT: as shown in SEQ ID No.12.

10. phosphorus efficiency utilizes a test kit for genetically engineered soybean OsPT2 gene relative expression flow measurement, it is characterized in that: this test kit comprises following sequence:

444-bp OsPT2 gene probe prepare primer, upstream primer F: as shown in SEQ ID No.9, downstream primer R: as shown in SEQ ID No.10;

The detection primer of TefS1 gene, upstream primer FT: as shown in SEQ ID No.11; Downstream primer RT: as shown in SEQ ID No.12.