CN101381730A - Use of gene OsPT2 in controlling phosphate absorption and transport - Google Patents

Abstract

The invention discloses application of a gene OsPT2 to the control of absorption and transport of paddy phosphate, wherein an OsPT2 gene is utilized to establish an excessive expression vector; the vector is transferred into a paddy variety to obtain a transgenic paddy variety of the excessive expression OsPT2; a full-length coding region of the OsPT2 gene is amplified by the PCR method and is connected to an excessive expression vector pCAMBIA1301s; and the established vector undergoes genetic transformation and is excessively expressed in the paddy variety. Under the condition of culture by a normal nutrient solution, the maximum absorption rate of transgenic positive plants on phosphor is 4.05 times of that of negative plants; the effective phosphor concentration on the overground part is 5.5 times of that of the negative plants, and the effective phosphor concentration on the underground part is 2 times of that of the negative plants; and the total phosphorous concentration on the overground part of the transgenic positive plants is 4 times of that of the negative plants, and the total phosphorous concentration on the underground part is 1.5 times of that of the negative plants.

Description

The purposes of gene OsPT 2 in the control phosphate absorption and transport

Technical field

The present invention relates to the plant gene engineering technology field.Be specifically related to a kind of application of controlling the gene OsPT 2 of phosphate absorption.

Background technology

Paddy rice (Oryza sativa L.) is one of China's staple food crop.Phosphorus is one of essential macronutrient of plant-growth institute, and phosphoric is significant to the paddy rice good quality and high output.By transgenic method, the OsPT2 gene of overexpression coding phosphor hydrochlorate translocator in rice varieties Hejiang 19 finds that paddy rice available phosphorus and full phosphorus concentration all significantly improve.Therefore, overexpression OsPT2 has great importance for the absorption that improves the paddy rice phosphoric in paddy rice.

Phosphorus is one of 17 kinds of macronutrients of necessary for plant growth, and the intravital phosphorus of plant accounts for 0.05% to 0.5% of dry weight.Phosphorus is not only the important composition composition of cellular elements such as ATP, nucleic acid, phosphatide, and in many metabolic processes, play critical regulating effect, these processes comprise: energy transfer, protein activation and carbon and amino acid metabolism (Marschner, 1995).

Phosphoric is significant to the paddy rice good quality and high output, however the common issue with that the shortage of soil available phosphorus is a worldwide to be faced.China's soil content of tatal phosphorus major part changes (Xiong Yi, 1987) between 200～1100 μ g/g.The soil content of tatal phosphorus only shows that the phosphorus element of soil stocks, and it is relevant little to the phosphorus supply ability of crop with soil.In soil, the phosphorus procatarxis for absorption, deposit and change reason such as organophosphorus into and cause the phosphorus more than 80% can not directly be utilized (Daniel et al., 1998) by plant materials.In the world, about 43% soil lacks phosphorus (Abelson, 1999) in 13.19 hundred million hectares of arable lands, and China is even more serious, and about 2/3 arable land lacks phosphorus.Plant mainly by root to the absorption of phosphorus in the soil needed phosphorus that obtains to grow, yet the concentration of available phosphorus is very low in most of soil, (Raghothama about about 2 μ M, 1999), be no more than 10 μ M (Bieleski, 1973), therefore have only by constantly use phosphate fertilizer in soil, crop could obtain or keep high yield.Yet phosphorus is Nonrenewable resources, and according to statistics such as Raghothama (1999), world's phosphate rock resource can only be kept 60～90 years, thereby the human constantly exhausted situation of earth phosphor resource that is being faced with.Using in a large number of phosphate fertilizer can be caused the eutrophication of water body, destroys ecotope.Therefore, plant is excavated the potentiality of self, and fully activate and utilize potential phosphorus, be the important mechanisms of self carrying out the phosphate starvation rescue.If utilizing the approach of genetic breeding introduces a fine variety or the genotype (promptly can obtain the crop gene type of high yield on low-phosphorous soil) of seed selection phosphorus efficiency, can not only effectively improve crop yield, can also overcome in the traditional fertilization method because the problem of environmental pollution (Yan Xiaolong etc., 1992) that excessive use chemical fertilizer causes.So, research plant tolerant to low-phosphorus stress characteristic and that it is carried out genetic improvement is significant.

The present invention in rice varieties Hejiang 19, overexpression the OsPT2 gene of coding phosphor hydrochlorate translocator, improved available phosphorus and the full phosphorus concentration of paddy rice, for the quality breeding of paddy rice provides new thinking.

Summary of the invention

The objective of the invention is to be to provide the purposes of a kind of gene OsPT 2 in the control phosphate absorption and transport, find and proved that one absorbs phosphoric acid salt to the control paddy rice and has the function of vital role gene OsPT 2.In paddy rice behind the OsPT2 gene of overexpression coding phosphor hydrochlorate translocator, find under the normal nutritive medium cultivation condition, positive plant is 94nmol/g/min to phosphatic absorption maximum absorption speed, and the utmost point is significantly higher than the 23.2nmol/g/min of cloudy negative control, is 4.05 times of negative plant.Available phosphorus level in positive plant overground part and the underground part and full phosphorus level all are significantly higher than negative plant: the available phosphorus concentration of overground part is 8.3mg Pi/gFW in the transgenic positive plant, is 5.5 times of negative plant concentration of its correspondence; The available phosphorus concentration of underground part is 1.8mg Pi/g FW in the transgenic positive plant, is 2 times of negative plant concentration of its correspondence.The full phosphorus concentration of finding overground part in the transgenic positive plant is 38.5mg P/g DW, is 4 times of negative plant concentration of its correspondence; The full phosphorus concentration of underground part is 16.5mg P/g DW in the transgenic positive plant, is 1.5 times of negative plant concentration of its correspondence.

The present invention is achieved in that

The present invention utilizes the genomic fragment of OsPT2 as applying gene, carries out the checking of transgenosis overexpression, changes this gene over to rice varieties Hejiang 19, and transfer-gen plant shows the phenomenon that significantly improves at the available phosphorus level and the full phosphorus level utmost point.

Phosphate cotransporter albumen is responsible for phosphatic absorption and transhipment in the plant materials, and is significant to the metabolism of plant phosphoric.The applicant goes up input " OsPT2 " and " rice " at NCBI website (www.ncbi.nlm.nih.gov), obtain sequence number and be the dna sequence dna of gene of one section coding phosphor hydrochlorate translocator of AF536962, on the softberry website, predict total length ORF, thereby obtain the full-length gene group sequence of OsPT2 gene.OsPT2 full-length gene group sequence is 1587bp, and full-length cDNA is 1587bp, 528 amino acid of encoding.This gene comprises 1 exon and intronless.The present invention is by PCR method, amplifies the full length coding region of OsPT2 gene, is connected on the overexpression carrier pCAMBIA1301s.Carry out genetic transformation again, in rice varieties Hejiang 19, this gene of overexpression obtains the overexpression plant.At transgenosis T ₂For finding in the plant that available phosphorus in the positive plant and full phosphorus level all are significantly higher than negative plant.

The invention has the advantages that:

(1) the present invention has proved a kind of concrete function of controlling the gene OsPT 2 of rice phosphate transhipment.The applicant behind the OsPT2 gene of overexpression coding phosphor hydrochlorate translocator, finds that under the normal nutritive medium cultivation condition, positive plant has improved 4.05 times to phosphatic absorption maximum absorption speed with respect to negative plant in rice varieties Hejiang 19.Available phosphorus level in positive plant overground part and the underground part and full phosphorus level all are significantly higher than negative plant: the available phosphorus concentration of overground part is 8.3mg Pi/g FW in the transgenic positive plant, is 5.5 times of negative plant concentration of its correspondence; The available phosphorus concentration of underground part is 1.8mg Pi/g FW in the transgenic positive plant, is 2 times of negative plant concentration of its correspondence.The full phosphorus concentration of finding overground part in the transgenic positive plant is 38.5mg P/g DW, is 4 times of negative plant concentration of its correspondence; The full phosphorus concentration of underground part is 16.5mgP/g DW in the transgenic positive plant, is 1.5 times of negative plant concentration of its correspondence.

(2) the present invention's gene OsPT 2 that influences paddy rice phosphorus absorption and transport of overexpression in paddy rice first.For the genotype of rice genetic breeding or seed selection phosphorus efficiency provides new thinking.

(3) gene of using among the present invention can provide support for the phosphoric absorption and transport research of cereal crop such as paddy rice and other crop.

Description of drawings

Fig. 1 is a kind of utilisation technology route synoptic diagram of controlling the gene OsPT 2 of rice grain quality.

Fig. 2 is a carrier pCAMBIA1301s structural representation.Fig. 2 a is a carrier pCAMBIA1301 structural representation; Fig. 2 b is in multiple clone site, inserts segmental structural representation; Fig. 2 c is the structural representation of improved carrier pCAMBIA1301s.

Fig. 3 is the structural representation of overexpression conversion carrier.As seen the OsPT2 gene is by the 35S promoter overexpression.

Embodiment

Below in conjunction with accompanying drawing the present invention is further described in detail:

Phosphate cotransporter albumen is responsible for phosphatic absorption and transhipment in the plant materials, and is significant to the metabolism of plant phosphoric.The applicant goes up input " OsPT2 " and " rice " at NCBI website (www.ncbi.nlm.nih.gov), obtain sequence number and be the dna sequence dna of gene of one section coding phosphor hydrochlorate translocator of AF536962, on the softberry website, predict total length ORF, thereby obtain the full-length gene group sequence of OsPT2 gene.OsPT2 full-length gene group sequence is 1587bp, and full-length cDNA is 1587bp, 528 amino acid of encoding.This gene comprises 1 exon and intronless.The present invention passes through PCR method, total DNA with rice varieties Nipponbre is a template, amplification has obtained comprising the sequence of total length OsPT2 gene coding region 1587bp, this fragment is connected to overexpression vector plasmid pCAMBIA1301s, and (overexpression vector pCAMBIA1301s is that this experiment is at the plasmid of carrier pCAMBIA1301[from open report of Australia and use, referring to http://www.cambia.org] the basis on transform and to obtain: at first carrier pCAMBIA1301 is carried out enzyme with two restriction endonuclease HindIII of multiple clone site outermost end and EcoRI and cuts, remove multiple clone site, reconnect one section and comprise the CaMV35S promotor, the sequence of multiple clone site and polyA terminator, thereby obtained to apply to the novel vector pCAMBIA1301s of gene overexpression, this carrier comprises the reporter gene of GUS and the screening-gene of Totomycin) on, (EHA105 is provided by Australian CAMBIA laboratory with Agrobacterium, referring to: New Agrobacterium helperplasmids for gene transfer to plants, 1993, Transgenic Res 2:208-218) Jie Dao method, overexpression OsPT2 gene in rice varieties Hejiang 19.Observe transfer-gen plant offspring (T ₂Generation), find that transfer-gen plant shows the proterties variation of expectation, promptly available phosphorus and full phosphorus level significantly rise in the plant.

Following examples further define the present invention, and have described separation OsPT2 gene, genetic transformation, and the measuring method of amino acid levels and protein content and the expression pattern of this gene in paddy rice.Implement example according to following description and these, those skilled in the art can determine essential characteristic of the present invention, and under the situation that does not depart from spirit and scope of the invention, can make various changes and modification, so that its suitable various uses and condition to the present invention.

Embodiment 1:OsPT2 gene is determined the acquisition with sequence

Go up input " OsPT2 " and " rice " at NCBI website (www.ncbi.nlm.nih.gov), obtain sequence number and be the dna sequence dna of gene of one section coding phosphor hydrochlorate translocator of AF536962, on the softberry website, predict total length ORF, thereby obtain the full-length gene group sequence of OsPT2 gene.

Embodiment 2: the structure of overexpression conversion carrier

According to the known full length sequence design primer (seeing Table 1) of gene OsPT 2, be template with total DNA of rice varieties Nipponbre, amplification obtains comprising the 1825bp fragment of OsPT2 full length gene coding region.When the applicant increases the OsPT2 gene, on primer, added the enzyme cut-grafting head of SacI and PstI, therefore the fragment that obtains of amplification can be carried out enzyme with restriction endonuclease SacI and PstI and cut, be connected to overexpression vector pCAMBIA1301s then and go up (the carrier of this experimental reconstruction, the overexpression that can be used for gene, carrier figure is referring to Fig. 2), and then with the primer shown in the table 1 clone who obtains is checked order, determine that gene is connected on the pCAMBIA1301s carrier by correct direction.

To be this experiment transform on the basis of carrier pCAMBIA1301 (from open report of Australia and the plasmid that uses) overexpression vector pCAMBIA1301s obtains.At first carrier pCAMBIA1301, carrying out enzyme with two restriction endonuclease HindIII of multiple clone site outermost end and EcoRI cuts, remove multiple clone site, reconnect one section sequence that comprises CaMV35S promotor, multiple clone site and polyA terminator, thereby obtained to apply to the novel vector pCAMBIA1301s of gene overexpression.This carrier comprises the reporter gene of GUS and the screening-gene of Totomycin.

Table 1 is used for the primer of design voluntarily of the present invention

Embodiment 3: the transgenic experiments of overexpression OsPT2

After the fragment that comprises the OsPT2 gene of coding phosphor hydrochlorate translocator is connected to carrier pCAMBIA1301s and goes up, adopt agriculture bacillus mediated transgenic method, obtain genetically modified rice plant, transgenosis concrete steps of the present invention are as follows:

The correct clone's that obtains plasmid is situated between by Agrobacterium, and (EHA105 is provided by Australian CAMBIA laboratory, referring to: New Agrobacterium helper plasmids for gene transfer to plants, 1993, Transgenic Res 2:208-218) water guide rice genetic conversion system imports in the rice varieties Hejiang 19, through the callus of cultivating in advance, infecting, cultivating altogether, screening having hygromycin resistance, break up, take root, acclimatization and transplants, obtain transfer-gen plant.Paddy rice (japonica rice subspecies) genetic conversion system of Agrobacterium (EHA105) mediation is mainly used people's reported method such as Hiei (referring to Efficient transformation of rice, Oryza sativa L., mediated by Agrobacterium and sequence analysis of the boundaries of theT-DNA, 1994, Plant Journal 6:271-282) is optimized on the basis.

The method of the key step of genetic transformation of the present invention, substratum and preparation thereof is as described below:

(1) reagent and solution abbreviation

The abbreviation of the used plant hormone of substratum is expressed as follows among the present invention: 6-BA (6-BenzylaminoPurine, 6-benzyladenine); CN (Carbenicillin, Pyocianil); KT (Kinetin, kinetin); NAA (Napthalene acetic acid, naphthylacetic acid); IAA (Indole-3-acetic acid, indolylacetic acid); 2,4-D (2,4-Dichlorophenoxyaceticacid, 2,4 dichlorophenoxyacetic acid); AS (Acetosringone, Syringylethanone); CH (CaseinEnzymatic Hydrolysate, caseinhydrolysate); HN (Hygromycin B, Totomycin); DMSO (Dimethyl Sulfoxide, dimethyl sulfoxide (DMSO)); N6max (N6 macroelement composition solution); N6mix (N6 trace element composition solution); MSmax (MS macroelement composition solution); MSmix (MS trace element composition solution)

(2) solution formula

1) N6 substratum macroelement mother liquor (according to 10 times of concentrated solutions (10X) preparation):

Saltpetre (KNO ₃) 28.3 grams

Potassium primary phosphate (KH ₂PO ₄) 4.0 grams

Ammonium sulfate ((NH ₄) ₂SO ₄) 4.63 grams

Sal epsom (MgSO ₄7H ₂O) 1.85 grams

Calcium chloride (CaCl ₂2H ₂O) 1.66 grams

Mentioned reagent is dissolved one by one, be settled to 1000 milliliters with distilled water then.

2) N6 substratum trace element mother liquor (is prepared according to 100 times of concentrated solutions (100X)

Potassiumiodide (KI) 0.08 gram

Boric acid (H ₃BO ₃) 0.16 gram

Manganous sulfate (MnSO ₄4H ₂O) 0.44 gram

Zinc sulfate (ZnSO ₄7H ₂O) 0.15 gram

Mentioned reagent is settled to 1000 milliliters 20-25 degree centigrade of following dissolving and with distilled water.

3) molysite (Fe ₂EDTA) stock solution (according to the preparation of 100X concentrated solution)

With 3.73 gram b diammonium disodium edta (Na ₂EDTA2H ₂O) and 2.78 the gram FeSO ₄7H ₂O dissolves respectively, mixes and is settled to 1000 milliliters with distilled water, bathes 2 hours to 70 ℃ of temperature, and 4 ℃ of preservations are standby.

4) VITAMIN stock solution (according to the preparation of 100X concentrated solution)

Nicotinic acid (Nicotinic acid) 0.1 gram

VITMAIN B1 (Thiamine HCl) 0.1 gram

Vitamin B6 (Pyridoxine HCl) 0.1 gram

Glycine (Glycine) 0.2 gram

Inositol (Inositol) 10 grams

Adding distil water is settled to 1000 milliliters, and 4 ℃ of preservations are standby.

5) MS substratum macroelement mother liquor (MSmax mother liquor) (according to the preparation of 10X concentrated solution)

Ammonium nitrate (NH ₄NO ₃) 16.5 grams

Saltpetre 19.0 grams

Potassium primary phosphate 1.7 grams

Sal epsom 3.7 grams

Calcium chloride 4.4 grams

Mentioned reagent is dissolved under 20-25 ℃ of temperature, and be settled to 1000 milliliters with distilled water.

6) MS substratum trace element mother liquor (MSmin mother liquor) (according to the preparation of 100X concentrated solution)

Manganous sulfate (MnSO ₄4H ₂O) 2.23 grams

Zinc sulfate (ZnSO ₄7H ₂O) 0.86 gram

Boric acid (H ₃BO ₃) 0.62 gram

Potassiumiodide (KI) 0.083 gram

Sodium orthomolybdate (Na ₂MoO ₄2H ₂O) 0.025 gram

Copper sulfate (CuSO ₄5H ₂O) 0.0025 gram

Cobalt chloride (CoCl ₂6H ₂O) 0.0025 gram

Mentioned reagent is dissolved under 20-25 ℃ of temperature, and be settled to 1000 milliliters with distilled water.

7) 2, the preparation of 4-D stock solution (1 mg/ml):

Weigh 2,100 milligrams of 4-D dissolved 5 minutes with 1 milliliter of 1N potassium hydroxide, added then to be settled to 100 milliliters after 10 ml distilled waters dissolve fully, preserved under 20-25 ℃ of temperature.

8) preparation of 6-BA stock solution (1 mg/ml):

Weigh 100 milligrams of 6-BA, with 1 milliliter of 1N potassium hydroxide dissolving 5 minutes, be settled to 100 milliliters after adding the dissolving fully of 10 ml distilled waters then, 20-25 ℃ of temperature preserved.

9) preparation of naphthylacetic acid (NAA) stock solution (1 mg/ml):

Weigh 100 milligrams of NAA, with 1 milliliter of 1N potassium hydroxide dissolving 5 minutes, be settled to 100 milliliters after adding the dissolving fully of 10 ml distilled waters then, 4 ℃ of preservations are standby.

10) preparation of indolylacetic acid (IAA) stock solution (1 mg/ml):

Weigh 100 milligrams of IAA, with 1 milliliter of 1N potassium hydroxide dissolving 5 minutes, be settled to 100 milliliters after adding the dissolving fully of 10 ml distilled waters then, 4 ℃ of preservations are standby.

11) preparation of glucose stock solution (0.5 grams per milliliter):

Weigh glucose 125 grams, be settled to 250 milliliters with dissolved in distilled water then, the back 4 ℃ of preservations of sterilizing are standby.

12) preparation of AS stock solution:

Weigh the AS0.392 gram, add the dissolving of DMSO10 milliliter, divide to be filled in 1.5 milliliters of centrifuge tubes, 4 ℃ of preservations are standby.

13) 1N potassium hydroxide stock solution

Weigh potassium hydroxide 5.6 grams, be settled to 100 milliliters with dissolved in distilled water, 20-25 ℃ of temperature preserved standby.

(3) be used for the culture medium prescription that rice genetic transforms

1) inducing culture

100 milliliters in N6max mother liquor (getting the 10X concentrated solution that has prepared, down together)

10 milliliters in N6mix mother liquor (getting the 100X concentrated solution that has prepared, down together)

Fe ²⁺10 milliliters of EDTA stock solutions (getting the 100X concentrated solution that has prepared, down together)

10 milliliters of VITAMIN stock solutions (getting the 100X concentrated solution that has prepared, down together)

2,2.5 milliliters of 4-D stock solutions (get above-mentioned prepare)

Proline(Pro) (Proline) 0.3 gram

CH 0.6 gram

Sucrose 30 grams

Phytagel 3 grams

Adding distil water to 900 milliliter, 1N potassium hydroxide is regulated pH value to 5.9, boil and be settled to 1000 milliliters, divide and install to 50 milliliters of triangular flasks (25 milliliters/bottle), sterilization according to a conventional method after sealing (sterilized 25 minutes down for 121 ℃, following medium sterilization method is identical with the sterilising method of basal culture medium).

2) subculture medium

100 milliliters in N6max mother liquor (10X)

10 milliliters in N6mix mother liquor (100X)

Fe ²⁺10 milliliters of EDTA stock solutions (100X)

10 milliliters of VITAMIN stock solutions (100X)

2,2.0 milliliters of 4-D stock solutions

Proline(Pro) 0.5 gram

CH 0.6 gram

Sucrose 30 grams

Phytagel 3 grams

Adding distil water to 900 milliliter, 1N potassium hydroxide is regulated pH value to 5.9, boils and is settled to 1000 milliliters, divides to install to 50 milliliters of triangular flasks (25 milliliters/bottle), seals, as stated above sterilization.

3) pre-culture medium

12.5 milliliters in N6max mother liquor (10X)

1.25 milliliters in N6mix mother liquor (100X)

Fe ²⁺2.5 milliliters of EDTA stock solutions (100X)

2.5 milliliters of VITAMIN stock solutions (100X)

2,0.75 milliliter of 4-D stock solution

CH 0.15 gram

Sucrose 5 grams

Agar powder 1.75 grams

Adding distil water to 250 milliliter, 1N potassium hydroxide is regulated pH value to 5.6, seals, as stated above sterilization.

Use preceding heating for dissolving substratum and add 5 milliliters of glucose stock solutions and 250 microlitre AS stock solutions, (25 milliliters/ware) in the culture dish are poured in packing into.

4) be total to substratum

12.5 milliliters in N6max mother liquor (10X)

1.25 milliliters in N6mix mother liquor (100X)

Fe ²⁺2.5 milliliters of EDTA stock solutions (100X)

2.5 milliliters of VITAMIN stock solutions (100X)

2,0.75 milliliter of 4-D stock solution

CH 0.2 gram

Sucrose 5 grams

Agar powder 1.75 grams

Adding distil water to 250 milliliter, 1N potassium hydroxide is regulated pH value to 5.6, seals, as stated above sterilization.

Use preceding heating for dissolving substratum and add 5 milliliters of glucose stock solutions and 250 microlitre AS stock solutions, (25 milliliters/every ware) in the culture dish are poured in packing into.

5) suspension culture base

5 milliliters in N6max mother liquor (10X)

0.5 milliliter in N6mix mother liquor (100X)

Fe ²⁺0.5 milliliter of EDTA stock solution (100X)

1 milliliter of VITAMIN stock solution (100X)

2,0.2 milliliter of 4-D stock solution

CH 0.08 gram

Sucrose 2 grams

Adding distil water to 100 milliliter is regulated pH value to 5.4, divides in the triangular flask that installs to two 100 milliliters, seals, as stated above sterilization.

Add 1 milliliter of aseptic glucose stock solution and 100 microlitre AS stock solutions before using.

6) select substratum

25 milliliters in N6max mother liquor (10X)

2.5 milliliters in N6mix mother liquor (100X)

Fe ²⁺2.5 milliliters of EDTA stock solutions (100X)

2.5 milliliters of VITAMIN stock solutions (100X)

2,0.625 milliliter of 4-D stock solution

CH 0.15 gram

Sucrose 7.5 grams

Agar powder 1.75 grams

Adding distil water to 250 milliliter is regulated pH value to 6.0, seals, as stated above sterilization.

Dissolving substratum before using adds 250 microlitre HN (50 mg/ml) and (25 milliliters/ware) in the culture dish are poured in 400 microlitre CN (250 mg/ml) packing into.(annotate: selecting substratum Pyocianil concentration for the first time is 400 mg/litre, and selecting substratum Pyocianil concentration for the second time and later on is 250 mg/litre).

7) break up substratum in advance

25 milliliters in N6max mother liquor (10X)

2.5 milliliters in N6mix mother liquor (100X)

Fe ²⁺2.5 milliliters of EDTA stock solutions (100X)

2.5 milliliters of VITAMIN stock solutions (100X)

0.5 milliliter of 6-BA stock solution

0.5 milliliter of KT stock solution

NAA stock solution 50 microlitres

IAA stock solution 50 microlitres

CH 0.15 gram

Sucrose 7.5 grams

Agar powder 1.75 grams

Adding distil water to 250 milliliter, 1N potassium hydroxide is regulated pH value to 5.9, seals, as stated above sterilization.

Dissolving substratum before using, 250 microlitre HN (50 mg/ml), 250 microlitre CN (250 mg/ml), (25 milliliters/ware) in the culture dish are poured in packing into.

8) division culture medium

100 milliliters in N6max mother liquor (10X)

10 milliliters in N6mix mother liquor (100X)

Fe ²⁺10 milliliters of EDTA stock solutions (100X)

10 milliliters of VITAMIN stock solutions (100X)

2 milliliters of 6-BA stock solutions

2 milliliters of KT stock solutions

0.2 milliliter of NAA stock solution

0.2 milliliter of IAA stock solution

CH 1 gram

Sucrose 30 grams

Phytagel 3 grams

Adding distil water to 900 milliliter, 1N potassium hydroxide is regulated pH value to 6.0.

Boil and be settled to 1000 milliliters, divide to install to 50 milliliters of triangular flasks (50 milliliters/bottle), seal, as stated above sterilization with distilled water.

9) root media

50 milliliters in MSmax mother liquor (10X)

5 milliliters in MSmix mother liquor (100X)

Fe ²⁺5 milliliters of EDTA stock solutions (100X)

5 milliliters of VITAMIN stock solutions (100X)

Sucrose 20 grams

Phytagel 3 grams

Adding distil water to 900 milliliter is regulated pH value to 5.8 with 1N potassium hydroxide.

Boil and be settled to 1000 milliliters, divide to install to (25 milliliters/pipe) in the pipe of taking root, seal, as stated above sterilization with distilled water.

(4) agriculture bacillus mediated genetic transformation step (EHA105 is provided by Australian CAMBIA laboratory)

3.1 callus of induce

1) sophisticated Hejiang 19 rice paddy seeds is shelled, used 70% Ethanol Treatment then successively 1 minute, 0.15% mercury chloride (HgCl ₂) seed-coat sterilization 15 minutes;

2) wash seed 4-5 time with sterilization;

3) seed is placed on the inducing culture;

4) postvaccinal substratum is placed dark place cultivate 4 weeks, 25 ± 1 ℃ of temperature.

3.2 callus subculture

Select the embryo callus subculture of glassy yellow, consolidation and relatively dry, be put in dark 2 weeks, 25 ± 1 ℃ of the temperature of cultivating down on the subculture medium.

3.3 pre-the cultivation

Select the embryo callus subculture of consolidation and relatively dry, be put in dark 2 weeks, 25 ± 1 ℃ of the temperature of cultivating down on the pre-culture medium.

3.4 Agrobacterium is cultivated

1) (the LA culture medium preparation is with reference to J. Sa nurse Brooker etc. having the LA substratum that corresponding resistance selects, the molecular cloning experiment guide, the third edition, Jin Dongyan etc. (translating), Science Press, 2002, Beijing) went up the pre-Agrobacterium EHA105 of cultivation (this bacterial strain is from the agrobacterium strains of CAMBIA company public use) two days, 28 ℃ of temperature;

2) Agrobacterium is transferred in the suspension culture base, cultivated 2-3 hour on 28 ℃ of shaking tables.

3.5 Agrobacterium is infected

1) pre-incubated callus is transferred in the bottle of the bacterium of having gone out;

2) regulate the suspension of Agrobacterium to OD ₆₀₀0.8-1.0;

3) callus was soaked in agrobacterium suspension 30 minutes;

4) shifting callus blots to the good filter paper of sterilization; Be placed on then on the common substratum and cultivated temperature 19-20 ℃ 3 days.

3.6 callus washing and selection are cultivated

1) aqua sterilisa washing callus is to cannot see Agrobacterium;

2) be immersed in the aqua sterilisa that contains 400 milligrams/L Pyocianil (CN) 30 minutes;

3) shifting callus blots to the good filter paper of sterilization;

4) shift callus to selecting to select on the substratum cultivation 2-3 time, each 2 weeks.

3.7 differentiation

1) kanamycin-resistant callus tissue is transferred on the pre-differentiation substratum in dark place cultivation 5-7 days;

2) callus that shifts pre-differentiation cultivation is to division culture medium, and illumination is cultivated down, 26 ℃ of temperature.

3.8 take root

1) cuts the root that differentiation phase produces;

Then it is transferred to and cultivates 2-3 week, 26 ℃ of temperature in the root media under the illumination.

3.9 transplant

Wash the residual substratum on the root off, the seedling that will have good root system changes the greenhouse over to, divides moistening at initial several Tian Bao water holding simultaneously.

Transform japonica rice variety Hejiang 19, obtain transgenosis individual plant T ₀For plant.Detect copy number with southern, detect gene at the intravital expression amount of plant with northern.Obtain the plant of single copy, overexpression, and breed, obtain T ₁And T ₂For transfer-gen plant.

Embodiment 4: the Function Identification of the transfer-gen plant of overexpression OsPT2

Measure T ₁For available phosphorus concentration in the transfer-gen plant and full phosphorus concentration, find that T1 compares with the wild-type plant for transfer-gen plant, the phenotype that shows expectation changes, and promptly available phosphorus concentration and full phosphorus concentration are significantly higher than contrast in the transfer-gen plant.Measured T simultaneously ₂For available phosphorus concentration in the transfer-gen plant and full phosphorus concentration, find T ₂Negative plant for transgenic positive plant and its correspondence is compared, and the phenotype that also shows expectation changes.This has proved that also this gene can improve phosphatic absorption greatly by genetic transformation simultaneously.In addition, by the absorption dynamics test determination, show that positive plant has improved 4.05 times to phosphatic absorption maximum absorption speed with respect to negative plant.

1. phosphorus absorption test measuring method

Prepare the supporting lid of little alms bowl (light tight) of about one liter of some volume. four seedlings of every alms bowl (germinateed back about 40 days, according to the experiment material of oneself, reach certain root amount. by regulating the root biomass, phosphorus concentration during beginning, these factors of liquor capacity make plant the phosphorus in the little alms bowl solution to be exhausted at 4-5 hour)

Young plant begins and can cultivate in big bread box earlier, and per week changes one time of nutrition liquid. and forward in the little alms bowl the last week of experiment beginning, changes one time of nutrition liquid every day. and change without phosphorus nutrition the day before yesterday of experiment into and cultivated one day.

Be ready to a collection of little alms bowl (volume is 1 liter) during experiment, each little alms bowl is measured isopyknic nutritive medium with graduated cylinder, simultaneously the plant of needs test is moved on to (identical earthen bowl in these little alms bowls then, so only need lid is moved on in another earthen bowl together with seedling), get 1.0 milliliters of nutritive mediums every half an hour, test phosphorus concentration wherein. replenish isopyknic water after getting nutritive medium at every turn.

The testing method of phosphorus concentration is with the anti-method of molybdenum antimony (method see for details back available phosphorus measure) in the nutritive medium.

Table 2 the present invention clone's OsPT2 transgenic line phosphorus absorption test

Sample	Time (hour)	The concentration (mg/L) of phosphoric residue (v=0.9L) in the later solution of different time points	Phosphorus absorbs total amount (mg)	Root dry weight (g)	Every gram root dry weight absorbs the amount (mg Pi/g) of phosphorus
						The initial phosphorus concentration of solution (mg/L)	0	2.766
Negative control	7	2.02	0.828	0.177	4.682±0.0003
						OsPT2 overexpression transgenic positive plant	7	0.966	2	0.136	14.705±0.0008 **

Note ^*Expression T ₂The probable value of positive and negative tree characteristics differences t test of generation is in 1% level utmost point significant difference.

Table 3 the present invention clone's OsPT2 transgenic line absorption dynamics is measured

The absorption dynamics index	OsPT2 overexpression transgenic positive plant	Negative control
			Imax(nmol/g/min)	94±15.7**	23.2±1
Km(uM)	63.7±10**	5.9±3
			Cmin(uM)	0.7±0	0.6±0

Note ^*Expression T ₂The probable value of positive and negative tree characteristics differences t test of generation is in 1% level utmost point significant difference.

This experimental result shows 14.705 milligrams in the root absorption phosphorus of OsPT2 overexpression transgenic positive plant every gram dry weight in 7 hours time period, and the utmost point is significantly higher than 4.682 milligrams of cloudy negative control.The kinetic determination result shows that OsPT2 overexpression transgenic positive plant is 94nmol/g/min to the uptake rate of phosphorus, and the utmost point is significantly higher than the 23.2nmol/g/min of cloudy negative control.

2, the measuring method of available phosphorus concentration

The sample treatment step is as follows:

1. fresh plant is washed with tap water earlier,, dry with thieving paper then using distilled water flushing.

2. get 0.5 gram bright sample liquid nitrogen grinding powdered, to the sample freeze thawing, perchloric acid (PCA) grinding that adds 1ml10% (w/v) is even in 4 ℃ of placement (on ice or refrigerator).

3. homogenate dilutes 10 times with the perchloric acid (PCA) of 5% (w/v), in placing 30 minutes on ice.

4. in 4 ℃, centrifugal 10 minutes of 10000g, supernatant liquor are used for the mensuration (the anti-method of molybdenum antimony sees full phosphorus determination method for details) of available phosphorus content.

5. get the 2ml working solution and mix, in 40 ℃ of incubations 20 minutes with 1ml sample supernatant liquor.

6. reaction solution is measured absorption value down in the 820nm visible wavelength after cooled on ice.Too high as sample concentration, should suitably dilute, its OD value is dropped in the linearity range of graticule.

The making of phosphorus typical curve:

1. phosphorus standardized solution preparation (60ppm P): dissolving 0.230g primary ammonium phosphate (NH4H2PO4) is in 100ml distilled water, promptly get the phosphorus standardized solution of 600ppmP, phosphorus standardized solution with 600ppmP dilutes 10 times with extraction agent again, gets the phosphorus standardized solution of 60ppmP.

2. typical curve is drawn: the standard phosphorus solution of 60ppmP is diluted with extraction agent, make 0.6,1.2,2.4,3.6,4.8 and the standard serial solution of 6ppmP respectively.Extraction agent is with the perchloric acid of the perchloric acid and 5% (w/v) of 10% (w/v) 1:9 mixed preparing by volume.Do blank with the reaction solution of extraction agent and working solution.

3. gained typical curve (2.5ml quartz colorimetric utensil, light path 1cm, BACKMAN DU460 spectrophotometer) as shown in the table:

Table 4 plant available phosphorus bioassay standard curve

Preparation mark liquid is long-pending	Required mother liquor volume	PPM	Final concentration PPM (diluting 3 times)	OD820
					1ml	10 1	0.6	0.2	0.1625
1ml	20 1	1.2	0.4	0.3256
					1ml	40 1	2.4	0.8	0.6808
1ml	60 1	3.6	1.2	1.0128
					1ml	80 1	4.8	1.6	1.3637
1ml	100 1	6.0	2.0	1.7171

Y＝0.8634X-0.0151R2＝0.9999(Y＝OD820，X＝PPM Pi)

Plant available phosphorus (Pi) content (mgPi/g FW)=OD value * (V/m) * (V2/V1) * C

The mass concentration (PPM:mg/L) of phosphorus in OD value-conversion back liquid to be measured

The ml number of V-specimen preparation solution, promptly sample with the volume (this is 0.01L) of extraction agent

M-sample fresh weight (g) (according to reality claim Mass Calculation)

V1-absorption reaction used volume (1ml)

The overall product of V2-reaction solution (3ml)

C-dilution of sample multiple (, need be diluted to reaction again behind the 1ml) if sample concentration is too high

3, the measuring method of full phosphorus concentration

Boil 1.H2SO4-H2O2 disappear: take by weighing plant sample 0.3～0.5g (accurate to 0.0002g) and put into 100ml and disappear and boil pipe, it is moistening to add 1ml distilled water, and 4ml is dense in adding, respectively adds 2ml at twice, shake up after each the adding, after question response finished, placing disappears boiled on the stove heating and disappears and boil, and treated the H2SO4 cigarette that turns white, when solution becomes brown, stop heating, (general 180 ℃, 30min, 270 ℃, 30min, 360 ℃, 30min), be cooled to a bottle wall non-scald on hand, add H2O2 2ml, the continuation heating disappears and boils about 5～10min, cooling, adding H2O22ml again disappears and boils, (it is about 8 ~ 10ml) generally to add the H2O2 total amount, continues heating 5～10min again, to eliminate remaining H2O2 to be colourless or limpid back to solution so repeatedly.Cooling, constant volume.Sample is done blank test.

2. drawing 0.50～1.00ml disappears and boils liquid in 10 centrifuge tubes, add the less water dilution, add 1 dinitrophenol indicator, dropping 6mol/L NaOH solution is neutralized to and just is yellow, adding the 0.5mol/L dilution heat of sulfuric acid again is adjusted to yellow and just takes off, add the anti-developer 1.00ml of molybdenum antimony then, add the water constant volume, add a cover and shake up.Place 30mim under the room temperature, the 700nm colorimetric.(a large amount of samples can be measured with microplate reader) does blank, is that reference is regulated instrument zero with the blank solution.

3. typical curve: accurately draw 5mg/L P standard operation solution 0,0.5,1,2,3,4,5,6,8ml, put into the 50ml volumetric flask respectively, add water to 30ml, the same step colour developing and constant volume, promptly get 0,0.05,0.1,0.2,0.3,0.4,0.5,0.6,0.8mg/L P standard serial solution, measure simultaneously with liquid to be measured, read absorbancy.Drawing standard curve and linear regression equation.

Full P%=c (P) * V1/m * (V3/V2) * 10-4

C in the formula (P)---phosphorus concentration from the colour developing liquid that regression equation is tried to achieve, mg/L

V3---colour developing liquid is long-pending, ml

It is long-pending that V2---disappearing of draw measuring boiled liquid, ml

V1---disappear and boil liquid constant volume, ml

M---sample weighting amount, g

The performance of table 5 the present invention clone's OsPT2 gene transgenic T2 individual plant

	Transgenic positive plant overground part	The negative plant overground part of corresponding its transgenosis	The multiple that rises	Transgenic positive plant underground part	The negative plant underground part of corresponding its transgenosis	The multiple that rises
							Available phosphorus concentration (mg Pi/g FW)	8.31± 0.81 **	1.51± 0.07	5.5	1.80± 0.25 **	0.84± 0.02	1.8
Full phosphorus concentration (mg Pi/g DW)	38.53± 1.81 **	9.49± 0.69	4	16.47± 0.93 **	10.74± 0.74	1.5

Note ^*Expression T ₂The probable value of positive and negative tree characteristics differences t test of generation is in 1% level utmost point significant difference.

Claims (1)

1, the application of a kind of gene OsPT 2 in control rice phosphate absorption and transport.

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