CN102925480A - Plant expression vector for suppressing rice 2-PGK gene expression and method for culturing low phytic acid rice - Google Patents
Plant expression vector for suppressing rice 2-PGK gene expression and method for culturing low phytic acid rice Download PDFInfo
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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
Technical field
The invention belongs to the 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, usually account for 1.0% of seed dry weight, account for 75 ± 10% of the full phosphorus amount of seed.With the seeds of the crops such as Cereal, the beans human and feeding animal as staple food, owing to lacking the enzyme of digestion phytic acid, the content of phosphorus is very high in the movement, very easily causes soil and environment water eutrophication; While phytic acid and Zn
2+, Ca
2+, Fe
3+Can not be absorbed by the mankind Deng the phytate that forms, the asian population of edible Cereal is easily suffered from trace element deficiency for a long time.Although, in aquaculture, can satisfy animal to the demand of phosphorus by interpolation phytase or phosphorus in feed, this has increased cost undoubtedly, will cause simultaneously potential environment phosphorus to pollute.How solving phytic acid exists the environmental problem and the human trace element deficiency that cause to become the difficult problem of pendulum in face of the scientific worker.
Obtain the crop that phytic acid content suitably descends, other economical characters do not have noticeable change by genetic breeding or genetic engineering technique, not only can solve in the source problem of environmental pollution that phytic acid causes, produce input, the social reality meaning of increasing farmers' income important for reducing livestock industry 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 descends respectively 66% and 30%.Subsequently, the breeder uses 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 plant to the resistance of biology or abiotic stress, serious also can cause death.Therefore, obtain simultaneously the top priority that the crop seed phytic acid content descends, significant variation does not occur other economical characters low phytic acid kind becomes the breeder.
Up to the present, to after deliberation more clearly of plant phytic acid metabolism route of synthesis, the a plurality of functional genes that participate in the phytic acid metabolism are cloned: MIS(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 the 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 the 3-phosphoinositide, may play regulating effect to the route of synthesis of phytic acid to the regulation and control of two genes; The IPK(inositol phosphatekinase of plant, the inositol monophosphate kinases) mainly is responsible for the phosphorylation of inositol, find that by the IPK that compares various crop is active it has the ability of the multiple inositol substrate of phosphorylation, but do not have the ability of phosphorylation inositol ring 2 ' position; IPKl (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, has cloned to obtain in paddy rice, corn, soybean; Except said gene, also having responsible is the phytase gene of inositol and inorganic phosphorus with hydrolysis of phytic acid, finds in various crop 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.(1998) such as Ruth Keller use constitutive promoter and Antisense RNA Technique, have obtained the transgenosis potato that the MIPS gene expression dose descends, and analyze the content decrease of finding transgenosis potato blade mysoinositol; AlineC.S.Nunes etc. (2005) make the phytic acid content in the soybean descend 94.5% by the reticent MIPS gene of RNAi technology; (2008) such as Mio Kuwano 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) use the RNAi technology, under corn seed specificity promoter Ole16 and Glb driving, obtain corn and the soybean that the seed phytic acid content descends by reticent MRP4 gene.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 the rice paddy seed, thus the stable low phytic acid rice plant of acquired character.
A kind of amiRNA suppresses the plant expression vector of 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 Oleosin18, and its nucleotide sequence is 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 uses seed specific promoters Oleosin18, can make the 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 consists of.It is the partial sequence of 2-PGK gene cDNA sequence that described amiRNA suppresses fragment, and its base sequence can be shown in SEQ ID No.2.
The present invention also provides a kind of method of cultivating low phytic acid paddy rice, comprising:
(1) makes up described plant expression vector;
(2) described plant expression vector is passed through the agrobacterium mediation converted Rice Callus;
(3) Rice Callus is transferred to continuation cultivation on the selective medium, behind seedling differentiation, transplanted to the land for growing field crops, screening obtains to hang down the phytic acid rice plant.
Wherein, the construction process of described plant expression vector is:
(1) amiRNA is suppressed the incomplete complementary fragment of fragment and its and replace stem among paddy rice endogenous miRNA precursor osaMIR528, obtain the amiRNA structural unit;
(2) the amiRNA structural unit is inserted the downstream of the promotor of initial carrier;
(3) promotor Oleosin18 is replaced the promotor of initial carrier.
Described Agrobacterium can be chosen as the EHA105 agrobacterium tumefaciens.
For the low phytic acid plant of the stable transgenosis of acquired character, can be many for plantation to the low phytic acid rice plant that obtains, the stable individual plant of screening proterties.
Wherein, the concrete steps of described amiRNA structural unit structure are:
(1) utilize the WMD3 platform to obtain to make up the amiRNA primer (I, II, III, IV) of reticent 2-PGK gene;
(2) contain paddy rice osaMIR528 take pNW55() 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 the amiRNA structural unit suppress fragment and incomplete complementary fragment thereof;
(3) take above-mentioned PCR product as template, utilize universal primer G-4368+G-4369 to carry out overlapping PCR, obtain 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 Oleosin18 is:
(1) extracts paddy rice mature leaf genomic dna;
(2) take described total 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’。
Compared with prior art, beneficial effect of the present invention is:
(1) method provided by the invention is utilized the expression of phytic acid synthesis related gene in seed specific promoters Oleosin18 and the amiRNA technology control rice paddy seed, make the 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.
Description of drawings
Figure 1A is the colony PCR amplification the result figure of Oleosin18-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, "+" expression transgenic positive, "-" expression non-transgenic is negative.
Fig. 4 is transgenic paddy rice blade GUS colour developing figure.Wherein, "+" expression transgenic positive, "-" expression non-transgenic is negative.
Fig. 5 is seed inorganic phosphorus color reaction schematic diagram.Wherein, "+" expression transgenic positive, "-" expression non-transgenic is negative.
Embodiment obtains low phytic acid paddy rice by the amiRNA that makes up the 2-PGK gene
1, the acquisition of mi-2-PGK structural unit
(1) design of mi-2-PGK primer
Search rice varieties Japan fine 2-PGK gene order number (LOC_Os02g57400.1) in the Gramene website, Os02g57400.1 is input to the amiRNA primer that Web MicroRNA Designer platform (WMD3) platform obtains to make up reticent 2-PGK gene, and 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 worker's biotechnology (Shanghai) limited-liability company.
(2) amplification of mi-2-PGK structural unit
Take pNW55 as template, suppress fragment and incomplete complementary fragment thereof according to following table by ring structure, the ami-2-PGK that pcr amplification obtains to make up the mi-2-PGK structural unit; And then take ring structure, mi-2-PGK inhibition fragment and incomplete complementary fragment thereof as template 1:1:1 mixed in molar ratio, obtaining the ami-2-PGK structural unit by overlapping PCR, construction procedures sees Table 1.The base sequence of ami-2-PGK fragment is shown in SEQ ID No.2.
The construction procedures of table 1 mi-2-PGK structural unit
The 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 the 2.0mL Eppendorf tube.
3. add 450 μ L LP Buffer, and mix.(optional: the 100mg/mLRNaseA that adds 4 μ L).
4. in 65 ℃ of environment temperature bathe in the 15min(temperature bath process can between or centrifuge tube 2-3 time of vibrating), then shift out.
5. add 150 μ LDA Buffer, in ice bath, place 5min after mixing.
6. mixture is all transferred to Shredder spin column, in the centrifugal 3min of 14,000g (also can prior to the centrifugal 3min of 14,000g, again supernatant liquor be 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 and connect
Liquid in the liquid pipe.Because the mixing liquid volume greater than 750 μ L, divides the centrifugal posts of crossing 2 times.
10. the G Binding Buffer that adds 500 μ L in the spin column, in 10,000g from
Heart 30s, and discard liquid in the adapter.
The DNA that extracts can be directly used in various downstream tests, if do not use immediately, is stored in-20 ℃.(2) amplification of Oleosin18 promotor
Design primer according to the relevant rice varieties in Gramene website Japan fine grease albumen (Oleosin18kDa) gene upstream sequence:
Ole?18-F：5’-AAGCTTATGTCTGCCAGCATTGTGAAG-3’；
Ole?18-R：5’-GGTACCTGCTAAGCTAGCTAGCAAGATGA-3’。
Take the fine genomic dna of paddy rice Japan as template, with high-fidelity enzyme KOD-Plus, amplification obtains the Oleosin18 promoter fragment, and its base sequence is shown in SEQ ID No.1.Concrete operations are as follows:
The PCR reaction system is:
The PCR response procedures is:
3, the goal gene fragment is cloned into T-vector and sequence verification
(1) connects
Goal gene fragment (Oleosin18 promoter fragment and mi-2-PGK structural unit) is connected to the pMD-18T carrier after adding A through the purifying end respectively.
Linked system is:
16 ℃ of connections are spent the night.
(2) conversion of competent escherichia coli cell
The centrifuge tube that DH5 α competent cell (precious biotechnology company limited) 1. will be housed places on ice about 10min, adds the above-mentioned connection products of 10 μ L after it all melts, with rifle head mixing gently.
2. behind the ice bath 30min, the centrifuge tube that competent cell is housed is changed in 42 ℃ of water-baths, thermal shock 1min will be equipped with rapidly the centrifuge tube ice bath 5min of competent cell.
3. every pipe adds 800 μ L LB liquid nutrient mediums, 37 ℃, cultivates 45min in the 180rpm shaking table.
4. will transform good intestinal bacteria in the centrifugal 5min of 5000rpm, supernatant discarded, the LB substratum of reservation 100-200 μ L, rifle head are applied to the dull and stereotyped upper 37 ℃ of incubated overnight of the screening that is added with corresponding resistant after repeatedly inhaling and beating re-suspended cell.
(3) positive colony is identified
After 12-14 hour, the positive clone of picking colony PCR checking delivers to Nanjing Genscript Biotechnology Co., Ltd.'s order-checking in the LB solid medium screening that contains penbritin, and the result is shown in Figure 1A and Figure 1B.Sequencing result shows Oleosin18 promoter fragment, mi-2-PGK structural unit successfully is cloned into respectively the T carrier, and to announce sequence consistent with online for the sequence that amplification obtains.
(4) p35S-mi-2-PGK Vector construction
The pCAMBIA1301-35SN carrier that is connected into same processing after the mi-2-PGK structural unit of sequence verification cut by the SalI+KpnI enzyme is formed on the lower plant expression vector p35S-mi-2-PGK with amiRNA structural unit of 35S promoter regulation and control.
It is as follows that the 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, connects behind the recovery purifying.
(5) pmi-2-PGK Vector construction
Cut by the HindIII+KpnI enzyme Oleosin18 promotor is connected into the p35S-mi-2-PGK carrier, displace its original 35S promoter and obtain the pmi-2-PGK carrier, whole mi-2-PGK structure is placed under the regulation and control of promotor Oleosin18, obtain pmi-2-PGK carrier (Fig. 2).
It is as follows that the 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, connects behind the recovery purifying to transform 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.
The centrifuge tube that 2. competent cell will be housed immerses liquid nitrogen 5min, and 37 ℃ of water-bath 5min repeat once.
3. add 800 μ LYEB substratum in centrifuge tube, after 28 ℃ of 180r/min cultivate 1h, be applied on the YEP substratum that contains kantlex and Rifampin and cultivated 2 days, bacterium colony PCR identifies.
(7) the Mature Embryos of Rice genetic transformation method of agrobacterium tumefaciens mediation
1) the bacterium enrichment culture of the agricultural bar of conversion
1. picking contains the Agrobacterium bacterium liquid of conversion carrier, is scoring on the YEB substratum (containing 50mg/L kantlex and 30mg/L Rifampin), 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 4mLYEP (containing 50mg/L kantlex and 30mg/L Rifampin) nutrient solution, and 28 ℃, the 250rpm shaken overnight is cultivated.
3. next day, from the YEP nutrient solution that contains Agrobacterium, draw 1-2mL, change in 25-50mL AB (the containing 30 μ mol/L Rifampins+50 μ mol/L kantlex+100 μ mol/L Syringylethanones) liquid nutrient medium and cultivate, 28 ℃, 250rpm, cultivate approximately about 4 hours, until OD600 reaches about 0.5.
2) infect-Rice Callus and Agrobacterium cultivate altogether
1. with containing the bacterium liquid that obtains in the resuspended previous step of equal-volume AAM of 100 μ mol/L Syringylethanones.
2. the picking circle, beige, quality densification at the fine callus of 3-7 days paddy rice of NBD substratum succeeding transfer culture Japan to the aseptic Erlenmeyer flask of 100mL.
3. the activation bacterium liquid in will 1. going on foot adds ready callus, 28 ℃, after 150rpm infects 30min, forwards callus on the filter paper dry 30min.
4. will be the callus of the drying NBD substratum (contain 100 μ mol/L Syringylethanones, pH 5.2) of transferring to Surface mulch one deck filter paper cultivated altogether 3 days.
3) screening and culturing
1. the Rice Callus that will cultivate 3 days is transferred to the aseptic Erlenmeyer flask of 100mL, adds approximately 50mL sterilized water, oscillation cleaning, and each 3-5min washes 5-6 time, until scavenging solution becomes as clear as crystal.
2. again with the sterile water wash callus three times that contains Pyocianil (500mg/mL), each 3-5min.
3. callus is forwarded on the filter paper behind the dry 30min, callus is transferred to upper 28 ℃ of dark the cultivations for 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. the picking diameter be 0.5 – 1.0mm Rice Resistance callus subculture to division culture medium (the NB substratum contains 1mg/L naphthylacetic acid and 2mg/L 6-benzyladenine), 2-3 week is cultivated in 28 ℃ of illumination (5000lx).
2. after the indefinite bud of callus grows up to the seedling of high approximately 2-4cm, it is transferred in the 1/2MS root media that contains the 0.5mg/L naphthylacetic acid, 28 ℃ of illumination (5000lx) are cultivated.Behind the little seedling rooting, with the clean land for growing field crops that is transplanted to of its root substratum.
(8) detection of transgenic paddy rice and screening
Fs, the group training stage: Rice Callus is before changing division culture medium over to, 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) and detail record, the little seed of the paddy rice of test positive is in the field.
Phase III, after the 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 the seed, filters out the transgenic positive strain.To the positive strain that filters out, many generation plantations are carried 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.
Screen 15 transgenic positive systems according to the GUS developing technology, wherein 12 tie up to the demonstration positive in the half granule seed inorganic phosphorus detection reaction, and the part strain is stable after cultivating for 3 generations.
(9) the low phytic acid paddy rice agronomic shape investigation of transgenosis and phytic acid content are measured
To cultivating the T that obtains
0Seed for the low phytic acid paddy rice of transgenosis carries out the inorganic phosphorus color developing detection, the strain of high inorganic phosphorus phenotype is carried out many cultures, through 3 generation cultured continuously obtained the low phytic acid paddy rice of 2 parts of stable transgenosiss, and they have been carried out economical character investigation and seed phytic acid content mensuration.
Table 2 transgenic positive system and non-transgenic feminine gender are the Agronomic Traits in Rice investigation
(annotate: "+" expression transgenic positive system, the negative system of "-" expression non-transgenic.）
By as seen 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, obvious decline (approximately 15%) has then occured in the seed phytic acid content, this shows by the present invention can under the prerequisite that does not change Other Main Agronomic Characters, reduce the content of rice paddy seed phytic acid effectively.
Claims (7)
1. an amiRNA suppresses the plant expression vector of paddy rice 2-PGK genetic expression, comprise the amiRNA structural unit and the specificity promoter that insert initial carrier, it is characterized in that, described specificity promoter is promotor Oleosin 18, and its nucleotide sequence is shown in SEQ ID No.1.
2. plant expression vector as claimed in claim 1 is characterized in that, described initial carrier is pCAMBIA1301-35SN.
3. plant expression vector as claimed in claim 1, it is characterized in that, 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 consists of, and described amiRNA suppresses the base sequence of fragment shown in SEQ IDNo.2.
4. a method of cultivating low phytic acid paddy rice is characterized in that, comprising:
(1) makes up such as the arbitrary described plant expression vector of claim 1～3;
(2) described plant expression vector is passed through the agrobacterium mediation converted Rice Callus;
(3) Rice Callus is transferred to continuation cultivation on the selective medium, behind seedling differentiation, transplanted to the land for growing field crops, screening obtains to hang down the phytic acid rice plant.
5. method as claimed in claim 4 is characterized in that, the construction process of described plant expression vector is:
(1) amiRNA is suppressed the incomplete complementary fragment of fragment and its and replace stem among paddy rice endogenous miRNA precursor osaMIR528, obtain the amiRNA structural unit;
(2) the amiRNA structural unit is inserted the downstream of the promotor of initial carrier;
The promotor of (3) promotor Oleosin 18 being replaced initial carrier.
6. method as claimed in claim 4 is characterized in that, described Agrobacterium is agrobacterium tumefaciens EHA105.
7. method as claimed in claim 4 is characterized in that, will hang down the many generation plantations of phytic acid rice plant, and acquired character is stablized plant.
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105063047A (en) * | 2015-08-12 | 2015-11-18 | 安徽省农业科学院水稻研究所 | Plant seed specific expression promotor OsSee1 |
| CN105063047B (en) * | 2015-08-12 | 2018-08-28 | 安徽省农业科学院水稻研究所 | Vegetable seeds specific expression promoter OsSee1 |
| CN105087588A (en) * | 2015-09-21 | 2015-11-25 | 安徽省农业科学院水稻研究所 | Plant embryo and aleurone layer specific promoter OsEmb3 and corresponding acquiring method |
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Inventor after: Li Wenxu Inventor after: Shu Qingyao Inventor after: Zhao Haijun Inventor after: Tan Huanhuan Inventor after: Lu Haiping Inventor before: Li Wenxu Inventor before: Shu Qingyao Inventor before: Zhao Haijun Inventor before: Tan Yuanyuan Inventor before: Lu Haiping |