CN102925480B

CN102925480B - Plant expression vector for suppressing rice 2-PGK gene expression and method for culturing low phytic acid rice

Info

Publication number: CN102925480B
Application number: CN201210430894.9A
Authority: CN
Inventors: 李文旭; 舒庆尧; 赵海军; 谭媛媛; 卢海平
Original assignee: Zhejiang University ZJU
Current assignee: Zhejiang University ZJU
Priority date: 2012-11-02
Filing date: 2012-11-02
Publication date: 2014-05-07
Anticipated expiration: 2032-11-02
Also published as: CN102925480A

Abstract

The invention discloses a plant expression vector and a method for culturing low phytic acid rice. The plant expression vector comprises an amiRNA structure unit inserted into the original vector and a specific promoter, wherein the specific promoter is the promoter Oleosin 18, and the nucleotide sequence is shown by SEQ ID No.1. The method comprises the following steps of: (1) establishing the plant expression vector; (2) performing agrobacterium tumefaciens-mediated transformation on the plant expression vector into rice callus; and (3) transferring the rice callus to a selective medium for further culture; when the rice callus is differentiated into seedlings, transplanting the seedlings to a field; and screening to obtain low phytic acid rice plants. According to the invention, the gene expression is controlled by use of the seed specific promoter Oleosin 18, thus the gene silencing only occurs in the seed embryo and the aleurone layer, and the influence on the phytic acid synthesis at other tissue parts of the rice can be effectively avoided.

Description

A kind of plant expression vector of paddy rice 2-PGK genetic expression and method of cultivating low phytic acid paddy rice of suppressing

Technical field

The invention belongs to genetically engineered field, relate in particular to a kind of plant expression vector of paddy rice 2-PGK genetic expression and method of cultivating low phytic acid paddy rice of suppressing.

Background technology

Phytic acid (PA, phytic acid), have another name called phytinic acid (myo-inositol-1,2,3,4,5,6-hexakisphosphate, write a Chinese character in simplified form InsP6 or IP6), be the major storage form of phosphorus in the crop seeds such as Cereal, beans, oil plant, conventionally account for 1.0% of seed dry weight, account for 75 ± 10% of the full phosphorus amount of seed.Using the seeds of the crops such as Cereal, beans as the mankind and the feeding animal of staple food, and owing to lacking the enzyme of digestion phytic acid, in movement, the content of phosphorus is very high, very easily causes soil and environment water eutrophication; While phytic acid and Zn ²⁺, Ca ²⁺, Fe ³⁺deng the phytate forming, can not be absorbed by the mankind, the asian population of edible Cereal is easily suffered from trace element deficiency for a long time.Although, in aquaculture, can meet the demand of animal to phosphorus by add phytase or phosphorus in feed, this has increased cost undoubtedly, will cause potential environment phosphorus to pollute simultaneously.How solving phytic acid exists the environmental problem and the mankind's trace element deficiency that cause to become the difficult problem of pendulum in face of scientific worker.

By genetic breeding or genetic engineering technique, obtain the crop that phytic acid content suitably declines, other economical characters do not have noticeable change, not only can on source, solve the problem of environmental pollution that phytic acid causes, for reducing livestock industry, produce input, the social reality meaning of increasing farmers' income important simultaneously.As far back as 1996, the employings such as V.Raboy were processed corn with regard to chemical mutagen and have been obtained two class low phytic acid mutant lpa1 and lpa2, and phytic acid content declines respectively 66% and 30%.Subsequently, breeder applies gamma-rays and chemical mutagen processing both at home and abroad, has obtained a series of low phytic acid mutants and kind in barley, paddy rice, wheat, Soybean and Other Crops.Yet, further research finds to be accompanied by the decline of seed phytic acid content, other economical characters of crop also can change, as: crop yield decline, seed vitality reduce, plant forms morphs, even also can have influence on the resistance of plant to biology or abiotic stress, serious also can be lethal.Therefore, obtain the top priority that crop seed phytic acid content declines, significant variation does not occur other economical characters low phytic acid kind becomes breeder simultaneously.

Up to the present, to after deliberation more clearly of plant phytic acid metabolism route of synthesis, a plurality of functional genes that participate in phytic acid metabolism are cloned: MIPS(myo-inositol-3-phosphate synthase, inositol-3-phosphate synthase) gene is the first enzyme of phytic acid route of synthesis, its effect is that photosynthetic crop product G-6-P is changed into 1D-inositol-3-phosphoric acid, and inositol ring is provided for phytic acid is synthetic; MIK(myo-inositol kinase, inositol kinase) gene and IMP(inositol monophosphatase, inositol monophosphatase) gene is responsible for transforming between inositol and 3-phosphoinositide, to the regulation and control of two genes, may play regulatory role the route of synthesis of phytic acid; The IPK(inositol phosphatekinase of plant, inositol monophosphate kinases) be mainly responsible for the phosphorylation of inositol, by comparing the IPK activity of various crop, find that it has the ability of the multiple inositol substrate of phosphorylation, but do not there is the ability of phosphorylation inositol ring 2 ' position; IPK1 (inositol-pentakisphosphate 2-kinase, inositol polyphosphate-2 kinases) finds at present unique enzyme that can phosphorylation IP5 inositol ring 2 ' position; MRP(multidrug resistance protein, multi-medicine resistance albumen) be a kind of translocator, it is being born and is transporting phytic acid to the function of storage vesicle, in paddy rice, corn, soybean, clones and obtains; Except said gene, also having responsible is the phytase gene of inositol and inorganic phosphorus by hydrolysis of phytic acid, in various crop, finds at present.Up to the present, studying the most detailed phytic acid synthetic gene is MIPS, has obtained corresponding low phytic acid mutant by gene silent technology in various crop.Ruth Keller etc. (1998) application constitutive promoter and Antisense RNA Technique, obtained the transgenosis potato that MIPS gene expression dose declines, and analyzes and find that the content of transgenosis potato blade mysoinositol declines; AlineC.S.Nunes etc. (2005) make the phytic acid content in soybean decline 94.5% by the reticent MIPS gene of RNAi technology; Mio Kuwano etc. (2008) under the driving of seed specific promoters, have obtained the transgenic paddy rice that phytic acid content decline, other agronomic traitss do not have significant variation by Antisense RNA Technique.In addition, Shi etc. (2007) application RNAi technology, under corn seed specificity promoter Ole16 and Glb driving, obtains by reticent MRP4 gene corn and the soybean that seed phytic acid content declines.Also not by the reticent phytic acid anabolism of amiRNA technology genes involved, cultivate the report of low phytic acid paddy rice or other crops at present.

Summary of the invention

The invention provides a kind of plant expression vector, utilize this plant expression vector can suppress specifically the expression of corresponding phytic acid anabolism genes involved in rice paddy seed, thus the stable low phytic acid rice plant of acquired character.

AmiRNA suppresses a plant expression vector for paddy rice 2-PGK genetic expression, comprises the amiRNA structural unit and the specificity promoter that insert initial carrier, and described specificity promoter is promotor Oleosin 18, and its nucleotide sequence is as shown in SEQ ID No.1.The Gramene position sequence of described paddy rice 2-PGK gene number is LOC_Os02g57400.1.

Plant expression vector of the present invention is used seed specific promoters Oleosin18, can make gene silencing event occur over just seed embryo and aleurone layer, can effectively avoid other tissue site phytic acid to synthesize and be affected.

Described initial carrier can be that pCAMBIA1301-35SN or other can be used for the binary vector of Agrobacterium-mediated Transformation.

The stem of the ring in the middle of described amiRNA structural unit comprises and formation hairpin structure, described stem suppresses fragment by amiRNA and its incomplete complementary fragment forms.It is the partial sequence of 2-PGK gene cDNA sequence that described amiRNA suppresses fragment, and its base sequence can be as shown in SEQ ID No.2.

The present invention also provides a kind of method of cultivating low phytic acid paddy rice, comprising:

(1) build described plant expression vector;

(2) described plant expression vector is passed through to agrobacterium mediation converted Rice Callus;

(3) Rice Callus is transferred on selective medium and continued to cultivate, after seedling differentiation, transplant to land for growing field crops, screening obtains low phytic acid rice plant.

Wherein, the construction process of described plant expression vector is:

(1) amiRNA is suppressed to fragment and its incomplete complementary fragment and replace the stem in paddy rice endogenous miRNA precursor osaMIR528, obtain amiRNA structural unit;

(2) amiRNA structural unit is inserted to the downstream of the promotor of initial carrier;

(3) promotor of promotor Oleosin 18 being replaced to initial carrier.

Described Agrobacterium can be chosen as EHA105 agrobacterium tumefaciens.

For the low phytic acid plant of the stable transgenosis of acquired character, can be to the many generation plantations of the low phytic acid rice plant obtaining, the stable individual plant of screening proterties.

Wherein, the concrete steps that described amiRNA structural unit builds are:

(1) utilize WMD3 platform to obtain the amiRNA primer (I, II, III, IV) that builds reticent 2-PGK gene;

(2) take pNW55(containing paddy rice osaMIR528) be template, utilize respectively primer I I+ universal primer G-4368, primer I+primer I V, primer I II+ universal primer G-4369 to carry out pcr amplification, the ring structure, the amiRNA that obtain amiRNA structural unit suppress fragment and incomplete complementary fragment thereof;

(3) take above-mentioned PCR product is template, utilizes universal primer G-4368+G-4369 to carry out overlapping PCR, obtains described amiRNA structural unit.

Wherein, the base sequence of primer I, II, III, IV is:

I：5’-agTAAGTATTAATCAAGACCCTGcaggagattcagtttga-3’；

II：5’-tgCAGGGTCTTGATTAATACTTActgctgctgctacagcc-3’；

III：5’-ctCAGGGACTTCATTAATACTTAttcctgctgctaggctg-3’；

IV：5’-aaTAAGTATTAATGAAGTCCCTGagagaggcaaaagtgaa-3’；

The base sequence of universal primer G-4368, G-4369 is:

G-4368：5’-CTGCAAGGCGATTAAGTTGGGTAAC-3’；

G-4369：5’-GCGGATAACAATTTCACACAGGAAACAG-3’。

The preparation method of described promotor Oleosin 18 is:

(1) extract paddy rice mature leaf genomic dna;

(2) take described total DNA is template, utilizes primer Ole18-F and Ole18-R to carry out pcr amplification.

Described primer Ole18-F and Ole18-R are as follows:

Ole?18-F：5’-AAGCTTATGTCTGCCAGCATTGTGAAG-3’；

Ole18-R：5’-GGTACCTGCTAAGCTAGCTAGCAAGATGA-3’。

Compared with prior art, beneficial effect of the present invention is:

(1) method provided by the invention utilizes seed specific promoters Oleosin 18 and amiRNA technology to control the expression of phytic acid synthesis related gene in rice paddy seed, make gene silencing event occur over just seed embryo and aleurone layer, can effectively avoid affecting other tissue site phytic acid of paddy rice synthetic.

(2) utilize method provided by the invention can obtain the low phytic acid rice strain of stable transgenosis, and tie up to other agronomy aspects such as plant height, spike length, thousand seed weight without significant difference with other non-transformed feminine genders, guaranteed the overall quality of the low phytic acid paddy rice of transgenosis.

Accompanying drawing explanation

Figure 1A is the colony PCR amplification the result figure of Oleosin 18-T carrier.

Figure 1B is the bacterium colony PCR the result figure of mi-2-PGK-T carrier.

Fig. 2 is pmi-2-PGK carrier structure schematic diagram.

Fig. 3 is the GUS colour developing figure of rice transformation callus.Wherein, "+" represents transgenic positive, and "-" represents that non-transgenic is negative.

Fig. 4 is transgenic paddy rice blade GUS colour developing figure.Wherein, "+" represents transgenic positive, and "-" represents that non-transgenic is negative.

Fig. 5 is seed inorganic phosphorus color reaction schematic diagram.Wherein, "+" represents transgenic positive, and "-" represents that non-transgenic is negative.

Embodiment obtains low phytic acid paddy rice by building the amiRNA of 2-PGK gene

1, the acquisition of mi-2-PGK structural unit

(1) design of mi-2-PGK primer

On Gramene website, search rice varieties Japan fine 2-PGK gene order number (LOC_Os02g57400.1), Os02g57400.1 is input to Web MicroRNA Designer platform (WMD3) platform and obtains the amiRNA primer that builds reticent 2-PGK gene, the base sequence of primer is respectively:

I：5’-agTAAGTATTAATCAAGACCCTGcaggagattcagtttga-3’；

II：5’-tgCAGGGTCTTGATTAATACTTActgctgctgctacagcc-3’；

III：5’-ctCAGGGACTTCATTAATACTTAttcctgctgctaggctg-3’；

IV：5’-aaTAAGTATTAATGAAGTCCCTGagagaggcaaaagtgaa-3’；

Universal primer:

G-4368：5’-CTGCAAGGCGATTAAGTTGGGTAAC-3’；

G-4369：5’-GCGGATAACAATTTCACACAGGAAACAG-3’；

Primer is responsible for synthetic by giving birth to work biotechnology (Shanghai) limited-liability company.

(2) amplification of mi-2-PGK structural unit

Take pNW55 as template, according to following table, by pcr amplification, obtain ring structure, ami-2-PGK inhibition fragment and the incomplete complementary fragment thereof that builds mi-2-PGK structural unit; And then take that ring structure, mi-2-PGK suppress fragment and incomplete complementary fragment is template 1:1:1 mixed in molar ratio, and by overlapping PCR, obtaining ami-2-PGK structural unit, construction procedures is in Table 1.The base sequence of ami-2-PGK fragment is as shown in SEQ ID No.2.

The construction procedures of table 1mi-2-PGK structural unit

PCR reaction system is:

2, the amplification of Oleosin18 promotor

(1) oryza sativa genomic dna extracts

Adopt Plant Genome to extract in a small amount test kit (rich day) and extract the fine mature leaf genomic dna of paddy rice Japan.Concrete steps are:

1. in liquid nitrogen or ice bath, plant tissue is ground.

2. get the tissue of no more than 100mg through grinding, put into 1.5 or 2.0mL Eppendorf tube.

3. add 450 μ L LP Buffer, and mix.(optional: the 100mg/mLRNase A that adds 4 μ L).

4. in 65 ℃ of environment temperature bathe in 15min(temperature bath process can between or centrifuge tube 2-3 time of vibrating), then shift out.

5. add 150 μ L DA Buffer, after mixing, in ice bath, place 5min.

6. mixture is all transferred to Shredder spin column, in the centrifugal 3min of 14,000g (also can be prior to the centrifugal 3min of 14,000g, then supernatant liquor is transferred to Shredder spincolumn, the centrifugal 1min of 12,000g).

7. filtrate is transferred to a new 1.5mL centrifuge tube.

8. add the P Binding Buffer of 1.5 times of 750 μ L or filtrate volumes, and mix.

9. mixing liquid is transferred to spin column, in the centrifugal 1min of 6,000g, and discarded the liquid in adapter.Because mixing liquid volume is greater than 750 μ L, minute 2 centrifugal posts of crossing.

10. to the G Binding Buffer that adds 500 μ L in spin column, in the centrifugal 30s of 10,000g, and discard liquid in adapter.

wash Buffer to adding 600 μ L in spin column, in the centrifugal 30s of 10,000g, and discards liquid in adapter.

repeating step

once.

again by spin column in the centrifugal 1min of 10,000g, and spin column is transferred on a new 1.5mL centrifuge tube.

to the Elution Buffer that adds 100 μ L-200 μ L in spin column, and in room temperature incubation 1min.

in the centrifugal 1min of 12,000g, and discard spin column.In the liquid connecing in 1.5mL centrifuge tube, contain DNA.

the DNA extracting can be directly used in various downstream tests, if do not used immediately, is stored in-20 ℃.

(2) amplification of Oleosin18 promotor

According to Gramene website relevant rice varieties Japan fine grease albumen (Oleosin18kDa) gene upstream sequence, design primer:

Ole?18-F：5’-AAGCTTATGTCTGCCAGCATTGTGAAG-3’；

Ole?18-R：5’-GGTACCTGCTAAGCTAGCTAGCAAGATGA-3’。

The fine genomic dna of paddy rice Japan of take is template, and with high-fidelity enzyme KOD-Plus, amplification obtains Oleosin18 promoter fragment, and its base sequence is as shown in SEQ ID No.1.Concrete operations are as follows:

PCR reaction system is:

PCR response procedures is:

3, goal gene fragment is cloned into T-vector and sequence verification

(1) connect

Goal gene fragment (Oleosin18 promoter fragment and mi-2-PGK structural unit) is connected to respectively pMD-18T carrier after purifying end adds A.

Linked system is:

16 ℃ of connections are spent the night.

(2) conversion of competent escherichia coli cell

1. the centrifuge tube that DH5 α competent cell (precious biotechnology company limited) is housed is placed in to 10min left and right on ice, after it all melts, adds the above-mentioned connection product of 10 μ L, with rifle head, mix gently.

2. after ice bath 30min, the centrifuge tube that competent cell is housed is proceeded in 42 ℃ of water-baths, thermal shock 1min, will be equipped with the centrifuge tube ice bath 5min of competent cell rapidly.

3. every pipe adds 800 μ LLB liquid nutrient mediums, 37 ℃, in 180rpm shaking table, cultivates 45min.

4. by the intestinal bacteria that transformed in the centrifugal 5min of 5000rpm, supernatant discarded, retains the LB substratum of 100-200 μ L, rifle head is applied to the dull and stereotyped upper 37 ℃ of incubated overnight of screening that are added with corresponding resistant after repeatedly inhaling and beating re-suspended cell.

(3) positive colony is identified

On the LB solid medium that contains penbritin, screen after 12-14 hour, picking colony PCR verifies that positive clone delivers to Nanjing Genscript Biotechnology Co., Ltd.'s order-checking, and the result as shown in FIG. 1A and 1B.Sequencing result shows Oleosin18 promoter fragment, mi-2-PGK structural unit to be successfully cloned into respectively to T carrier, and the sequence that amplification obtains is consistent with online announcement sequence.

(4) structure of p35S-mi-2-PGK carrier

The pCAMBIA1301-35SN carrier that is connected into same processing after the mi-2-PGK structural unit of sequence verification is cut by SalI+KpnI enzyme is formed on the plant expression vector p35S-mi-2-PGK under 35S promoter regulation and control with amiRNA structural unit.

It is as follows that SalI+KpnI enzyme is cut system:

Enzyme Qie Wendu is 37 ℃, and the enzyme time of cutting is no less than 4hr, adds afterwards 10 * loading Buffer termination reaction, after recovery purifying, connects.

(5) structure of pmi-2-PGK carrier

By HindIII+KpnI enzyme, cut Oleosin18 promotor is connected into p35S-mi-2-PGK carrier, displace its original 35S promoter and obtain pmi-2-PGK carrier, whole mi-2-PGK structure is placed under the regulation and control of promotor Oleosin18, obtains pmi-2-PGK carrier (Fig. 2).

It is as follows that HindIII+KpnI enzyme is cut system:

Enzyme Qie Wendu is 37 ℃, and the enzyme time of cutting is no less than 4hr, adds afterwards 10 * loading Buffer termination reaction, after recovery purifying, connects and transforms intestinal bacteria.

(6) transform Agrobacterium

1. get 10 μ Lpmi-2-PGK plasmids and evenly mix with EHA105 agrobacterium tumefaciens competent cell, place 30min on ice.

2. the centrifuge tube that competent cell is housed is immersed to liquid nitrogen 5min, 37 ℃ of water-bath 5min, repeat once.

3. in centrifuge tube, add 800 μ LYEB substratum, 28 ℃ of 180r/min cultivate after 1h, are applied on the YEP substratum that contains kantlex and Rifampin and cultivate 2 days, and bacterium colony PCR identifies.

(7) the Mature Embryos of Rice genetic transformation method of agrobacterium tumefaciens mediation

1) transform the bacterium enrichment culture of agriculture bar

1. the Agrobacterium bacterium liquid that picking contains conversion carrier, is scoring to YEB substratum (containing 50mg/L kantlex and 30mg/L Rifampin) upper, cultivates 2 days for 28 ℃.

2. the Agrobacterium bacterium liquid 100 μ L of picking mono-clonal or the preservation of absorption institute are in 4mL YEP (containing 50mg/L kantlex and 30mg/L Rifampin) nutrient solution, and 28 ℃, 250rpm shaken overnight is cultivated.

3. next day, from the YEP nutrient solution that contains Agrobacterium, draw 1-2mL, proceed in 25-50mLAB (containing 30 μ mol/L kantlex+100, μ mol/L Rifampin+50 μ mol/L Syringylethanones) liquid nutrient medium and cultivate, 28 ℃, 250rpm, cultivate about approximately 4 hours, until OD600 reaches 0.5 left and right.

2) infect-Rice Callus and Agrobacterium are cultivated altogether

1. with the bacterium liquid containing obtaining in the resuspended previous step of equal-volume AAM of 100 μ mol/L Syringylethanones.

2. picking circle, beige, quality densification at the fine callus of the NBD substratum succeeding transfer culture paddy rice of 3-7 days Japan to the aseptic Erlenmeyer flask of 100mL.

3. the activation bacterium liquid in 1. walking is added to ready callus, 28 ℃, 150rpm infects after 30min, forwards callus on filter paper dry 30min.

4. the callus of drying being transferred to the NBD substratum (containing 100 μ mol/L Syringylethanones, pH 5.2) of Surface mulch one deck filter paper cultivates 3 days altogether.

3) screening and culturing

1. the Rice Callus of cultivating 3 days is transferred to the aseptic Erlenmeyer flask of 100mL, add about 50mL sterilized water, oscillation cleaning, each 3-5min, washes 5-6 time, until scavenging solution becomes as clear as crystal.

2. the sterile water wash callus that use contains Pyocianil (500mg/mL) again three times, each 3-5min.

3. callus is forwarded on filter paper and be dried after 30min, callus is transferred to upper 28 ℃ of dark the cultivations 2 weeks of NBD substratum (containing 25mg/L Totomycin and 500mg/L Pyocianil), and succeeding transfer culture is to 28 ℃ of dark cultivations 2-3 week of new NBD substratum (containing 50mg/L Totomycin and 500mg/L Pyocianil) afterwards.

4) seedling differentiation

1. picking diameter is that 0.5-1.0mm Rice Resistance callus subculture is to division culture medium (NB substratum, containing 1mg/L naphthylacetic acid and 2mg/L 6-benzyladenine), 28 ℃ of illumination (5000lx) cultivation 2-3 week.

2. the indefinite bud until callus grows up to after the seedling of high about 2-4cm, is transferred in the 1/2MS root media that contains 0.5mg/L naphthylacetic acid, and 28 ℃ of illumination (5000lx) are cultivated.After little seedling rooting, its root substratum is cleaned and is transplanted to land for growing field crops.

(8) detection of transgenic paddy rice and screening

First stage, the group training stage: Rice Callus is before proceeding to division culture medium, and the part callus of each conversion system of picking carries out GUS color reaction (Fig. 3), and the callus subculture of test positive is to division culture medium.

Subordinate phase, the hardening stage: the rice leaf that takes a morsel, carry out GUS color reaction (Fig. 4) detailed record, the little seed of paddy rice of test positive is in field.

Phase III, after results: individual plant results rice paddy seed, each strain is got 8-12 grain seed, adopts half granule seed inorganic phosphorus color reaction (Fig. 5), analyzes content of inorganic phosphorus in seed, filters out transgenic positive strain.To filtered out positive strain, many generation plantations, carry out half granule seed inorganic phosphorus color reaction to each seed from generation to generation, filter out the low phytic acid paddy rice of stable transgenosis.

According to GUS developing technology, screen 15 transgenic positive systems, wherein 12 tie up to the demonstration positive in half granule seed inorganic phosphorus detection reaction, and part strain is stable after cultivating for 3 generations.

(9) investigation of the low phytic acid paddy rice of transgenosis agronomic shape and phytic acid content are measured

To cultivating the T obtaining ₀seed for the low phytic acid paddy rice of transgenosis carries out inorganic phosphorus color developing detection, the strain of high inorganic phosphorus phenotype is carried out to many cultures, through 3 generation cultured continuously obtained 2 parts of low phytic acid paddy rice of stable transgenosis, and they have been carried out to economical character investigation and seed phytic acid content mensuration.

Table 2 transgenic positive system and non-transgenic feminine gender are Agronomic Traits in Rice investigation

(note: "+" represents transgenic positive system, "-" represents the negative system of non-transgenic.）

From table 2, negative system compares with non-transgenic, transgenic positive ties up to plant height, spike number, thousand grain weigth aspect without significant variation, there is obvious decline (approximately 15%) in seed phytic acid content, this shows can, not changing under the prerequisite of Other Main Agronomic Characters, effectively reduce the content of rice paddy seed phytic acid by the present invention.

Claims

1. cultivate a method for low phytic acid paddy rice, it is characterized in that, comprising:

(1) build the plant expression vector that amiRNA suppresses paddy rice 2-PGK genetic expression;

(3) Rice Callus is transferred on selective medium and continued to cultivate, after seedling differentiation, transplant to land for growing field crops, screening obtains low phytic acid rice plant;

Described plant expression vector comprises amiRNA structural unit and the specificity promoter that inserts initial carrier, and described specificity promoter is promotor Oleosin18, and its nucleotide sequence is as shown in SEQ ID No.1;

The preparation method of described promotor Oleosin18 is:

(a) extract paddy rice mature leaf genomic dna;

(b) take described genomic dna as template, utilize primer Ole18-F and Ole18-R to carry out pcr amplification;

Described primer Ole18-F and Ole18-R are as follows:

Ole18-F：5’-AAGCTTATGTCTGCCAGCATTGTGAAG-3’；

Ole18-R：5’-GGTACCTGCTAAGCTAGCTAGCAAGATGA-3’；

Described initial carrier is pCAMBIA1301-35SN;

The stem of the ring in the middle of described amiRNA structural unit comprises and formation hairpin structure, described stem suppresses fragment by amiRNA and its incomplete complementary fragment forms, and described amiRNA suppresses the base sequence of fragment as shown in SEQ ID No.2;

The concrete steps that described amiRNA structural unit builds are:

(i) utilize WMD3 platform to obtain the amiRNA primer that builds reticent 2-PGK gene, described amiRNA primer is comprised of primer I, II, III, IV;

(ii) the pNW55 of take containing paddy rice osaMIR528 is template, utilize respectively primer I I+ universal primer G-4368, primer I+primer I V, primer I II+ universal primer G-4369 to carry out pcr amplification, the ring structure, the amiRNA that obtain amiRNA structural unit suppress fragment and incomplete complementary fragment thereof;

(iii) the above-mentioned PCR product of take is template, utilizes universal primer G-4368+G-4369 to carry out overlapping PCR, obtains described amiRNA structural unit;

Wherein, the base sequence of primer I, II, III, IV is:

I：5’-agTAAGTATTAATCAAGACCCTGcaggagattcagtttga-3’；

II：5’-tgCAGGGTCTTGATTAATACTTActgctgctgctacagcc-3’；

III：5’-ctCAGGGACTTCATTAATACTTAttcctgctgctaggctg-3’；

IV：5’-aaTAAGTATTAATGAAGTCCCTGagagaggcaaaagtgaa-3’；

The base sequence of universal primer G-4368, G-4369 is:

G-4368：5’-CTGCAAGGCGATTAAGTTGGGTAAC-3’；

G-4369：5’-GCGGATAACAATTTCACACAGGAAACAG-3’。

2. the method for claim 1, is characterized in that, described Agrobacterium is agrobacterium tumefaciens EHA105.

3. the method for claim 1, is characterized in that, by the many generation plantations of low phytic acid rice plant, acquired character is stablized plant.