CN103710346A - RNA (ribonucleic acid) interference vector and application thereof in culture of high nodulation nitrogen-fixing plants - Google Patents
RNA (ribonucleic acid) interference vector and application thereof in culture of high nodulation nitrogen-fixing plants Download PDFInfo
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Abstract
The invention discloses an RNA (ribonucleic acid) interference vector for regulating and controlling nodulation nitrogen-fixing capability of plants. The RNA interference vector comprises a backbone vector and a hairpin structure, wherein the hairpin structure is constituted by utilizing a gene fragment as shown in SEQ (sequence) ID (identity) NO: 2, and the gene fragment is inserted between enzyme digestion recognition sites of restriction endonucleases Kpn I/Spe I and BamH I/Sac I of a pTCK 303 vector to obtain the recombinant RNA interference vector. The obtained RNA interference vector can transform receiver plants, silence corresponding genes in the receiver plants and enable root tissues of the receiver plants to have the enhanced nodulation nitrogen-fixing capability. By utilizing the RNA interference vector constructed according to the invention to transform receiver soybean plants to obtain transgenic plants, the nodulation nitrogen-fixing capability of soybeans can be significantly improved, and the RNA interference vector further has great significance for improving the yield of the soybeans.
Description
Technical field
The present invention relates to gene engineering technology field, particularly relate to a kind of rna interference vector for Papilionaceae and uses thereof.
Background technology
Soybean is crop very important, that have special economic value.Soybean root raw root nodule can be converted into the ammonia-state nitrogen that plant can absorb airborne nitrogen, thereby provides essential nitrogen nutriment for soybean growth.The symbiotic nitrogen fixation being mediated by root nodule not only affects normal growth and the output of soybean, also helps save energy, reduces environmental chemistry and pollutes.At present, improve nitrogen-fixing efficiency and become one of important channel of improving soybean yields and assurance agricultural sustainable development.
Soybean root system nodulation and nitrogen fixation is a very complicated biological procedures that is subject to a plurality of gene regulatings, and at present increasing regulatory gene is in the news.The people's such as Madsen in 2003 result of study shows that NFR1 and NFR5 participate in leguminous plants to the response of nod factor (Madsen et al., 2003) as nod factor acceptor.Afterwards, research in soybean report points out, the sudden change of NFR gene causes root system non-nodulating, and cross, expresses the nodule number (Indrasumunar et al., 2010,2011) that NFR1 α gene can increase soybean.In soybean, cross and express the dross (Kalsi et al., 2000) that phytohemagglutinin phosphatase nucleic acid lytic enzyme LNP can significantly increase soybean root system.
ARF8 is the important growth hormone response factors in growth hormone signal path.ARF8 is regulating and controlling thaliana flower growth course (Nagpal et al., 2005, Wu et al., 2006).Mutant arf8 shows hypocotylar elongation (Tian et al., 2004) under illumination condition.ARF8 lowers to express and causes four-wheel floral organ to occur extremely (Ru et al., 2006).ARF8 participates in the lateral root growth (Gifford et al., 2008) of regulation and control Arabidopis thaliana root system when corresponding nitrogen nutrition lacks.Have not yet to see the report of growing about ARF8 regulation and control Legume nodule.In soybean, ARF8 Functional identification of genes provides foundation by the molecular mechanism of illustrating soybean nodulation growth for us, thereby looks for another way for improve the nitrogen-fixing efficiency of soybean by biological nitrogen fixation.
Summary of the invention
The application that the technical problem to be solved in the present invention is to provide a kind of rna interference vector and cultivates high nodulation and nitrogen fixation plant, especially provides a kind of cultivation to have the method for the new soybean varieties of high nitrogen fixing capacity.
For solving the problems of the technologies described above, the technical solution used in the present invention is as follows.
For building a gene fragment for rna interference vector, this gene fragment is for building the hairpin structure of rna interference vector, and the nucleotide sequence of described gene fragment is as shown in the SEQ ID NO:2 in sequence table, and this fragment is as positive-sense strand.
The complementary nucleotide sequence of said gene fragment, with the antisense strand of positive-sense strand complementation.
For a rna interference vector for regulating plant nodulation and nitrogen fixation ability, comprise skeleton carrier and hairpin structure, described hairpin structure utilizes the gene fragment described in claim 1 or 2 to build.
As a preferred technical solution of the present invention, described skeleton carrier is pTCK303 carrier.
As a preferred technical solution of the present invention, described hairpin structure be spaced apart the paddy rice intron on pTCK303 carrier.
As a preferred technical solution of the present invention, positive-sense strand is arranged in the upstream of pTCK303 carrier paddy rice intron, and antisense strand is positioned at the downstream of paddy rice intron, and integral body has formed take the double-stranded RNA interference carrier of the formed hairpin structure that paddy rice intron is interval.The restriction enzyme Kpn I/Spe I and the BamH I/Sac I enzyme that gene fragment are inserted into pTCK303 are cut between recognition site, obtain recombinant expression vector.
The purposes of above-mentioned rna interference vector, for cultivating the plant with high nodulation and nitrogen fixation ability.
The purposes of above-mentioned rna interference vector, makes rna interference vector transformation receptor plant, and the corresponding gene in reticent recipient plant makes the root tissue of recipient plant have the nodulation and nitrogen fixation ability of enhancing.
The purposes of above-mentioned rna interference vector, the method for described transformation receptor plant is to utilize agriculture bacillus mediated hairly root conversion method; The described gene being silenced is the gene shown in SEQ ID NO:1.
The purposes of above-mentioned rna interference vector, described recipient plant is Papilionaceae plant.
The beneficial effect that adopts technique scheme to produce is: referring to following embodiment 3, utilize the rna interference vector transformation receptor soybean plant strain that the present invention builds to obtain transgenic plant, can significantly improve the nodulation and nitrogen fixation ability of soybean, to improving soybean yields, be significant.
Accompanying drawing explanation
Fig. 1 is the expression pattern analysis that GmARF8L1 gene response short-term root nodule bacterium are infected, root nodule bacterium, infect in early stage Soybean Root as seen, the expression of GmARF8L1 gene is significantly inhibited, and this presentation of results GmARF8L1 gene has participated in the soybean root system dross response process to root nodule bacterium in early days.
Fig. 2 is that GmARF8L1 responds the expression pattern analysis that long-term root nodule bacterium are infected, the visible long period of infecting root nodule bacterium, be in the process of root nodule genesis and development, also there is obvious downward in the expression level of GmARF8L1 gene, result shows, the genesis and development process of GmARF8L1 participation regulating and controlling soybean root system after root nodule bacterium are infected.
Fig. 3 is the phenotype analytical to soybean nodulation after downward expression GmARF8L1, under identical experimental situation, lowering the transgenosis root of expression GmARF8L1 compares with the transgenosis root system (EV) of empty carrier conversion, root nodule numbers significantly increases (Fig. 3 A and 3B), Fig. 3 C is the expression level analysis to GmARF8L1 in transgenosis root system, and the expression that result is presented at GmARF8L1 in transgenosis root system is significantly to lower.
Embodiment
Following embodiment is convenient to understand better the present invention, but does not limit the present invention.Experimental technique in following embodiment, if no special instructions, is ordinary method.Test materials used in following embodiment, if no special instructions, is and purchases available from routine biochemistry reagent company.% in following embodiment, if no special instructions, is quality percentage composition.Quantitative test in following examples, all arranges and repeats experiment, results averaged for three times.
The rna interference vector that the present invention builds can be special reticent soybean in GmARF8L1 gene.This gene belongs to ARF8 gene family, and it is positioned on No. 2 karyomit(e)s of soybean, and real-time fluorescence quantitative PCR detected result shows, the expression of GmARF8L1 gene in Soybean Root is subject to the inhibition that root nodule bacterium are infected; The dross analysis that transforms chimeric seedling by hairly root shows, the increase that can significantly promote soybean root system dross number is expressed in the downward of GmARF8L1, shows that GmARF8L1 gene has important effect in soybean nodulation is grown.
1) material obtains: experiment material therefor is that WILLIAMS-DARLING Ton 82(is called for short W82); Material carries out according to following flow process: 70% the smart sterilizing 30S that spills for soybean seeds, be seeded in vermiculite perlite (3:1) mixed-matrix of low nitrogen nutrition immersion bubble, and in culturing room, cultivate, 16h light/8h is dark, light intensity 7000LUX, 26 ℃ of temperature, relative humidity is 70%.After planting 7 days, the slow raw type root nodule bacterium USDA110 bacterium liquid (OD of every strain inoculation
600: 0.08) 30ml gets respectively the root of soybean for 0,1,3,6,12 and 24 hour after connecing bacterium;
2) separation of mRNA: adopt Trizol method to extract the total RNA of soybean, 1. first tissue is put into grinding liquid nitrogen grinding 3 times, by ground, to organize 50-100mg to add in 1.5mL centrifuge tube then to add 1ml RNAVzol, split product should be the transparent thick liquid of clarification. room temperature is placed 5 minutes; 2. add 200 μ l chloroforms, vibration mixes, the standing 5min of room temperature, 12,000r/min, 4 ℃ of centrifugal 10min; 3. get 500 μ l supernatants in another centrifuge tube, add equal-volume Virahol, room temperature is placed 10min, 12,000r/min, 4 ℃ of centrifugal 10min; 4. add 1ml75% ethanol to wash precipitation, 8,000r/min, 4 ℃ of centrifugal 5min, repeat twice; The air-dry 10min of room temperature left and right, adds RNase Free water (DEPC water) dissolution precipitation that 20 μ l left and right DEPC processed;
3) reverse transcription is cDNA: by the mRNA of extraction, with the ThermoScript II AMV reverse transcription of Promega company, be cDNA;
4) real-time fluorescence quantitative PCR analysis: the SYBR Premix Ex TagTM that uses TaKaRa company to produce carries out quantitative PCR analysis; In 20 μ l systems, add and walk gained cDNA template 2 μ l, forward and reverse primer each 0.4 μ l, SYBR Primix Ex taqTM (2 *) 10 μ l, ROX Reference Dye II (50 *) 0.4 μ l, ddH2O 6.8 μ l; Amplification program is: 95 ℃ of 30s; 95 ℃ of 5s, 60 ℃ of 34s, 45cycles; 95 ℃ of 15s, 60 ℃ of 1min, 95 ℃ of 15s; Wherein,
Forward primer is: GTATTCGTCGACAGGGAGAAC(SEQ ID NO:3);
Reverse primer is: TCAGTATTCAAGGGAGCCAATT(SEQ ID NO:4);
5) result: as shown in Figure 1, infect in early stage Soybean Root root nodule bacterium, the expression of GmARF8L1 gene is significantly inhibited, this presentation of results GmARF8L1 gene has participated in the soybean root system dross response process to root nodule bacterium in early days.
1) material obtains: experiment material therefor is that WILLIAMS-DARLING Ton 82(is called for short W82); Material carries out according to following flow process: 70% the smart sterilizing 30S that spills for soybean seeds, be seeded in vermiculite perlite (3:1) mixed-matrix of low nitrogen nutrition immersion bubble, and in culturing room, cultivate, 16h light/8h is dark, light intensity 7000LUX, 26 ℃ of temperature, relative humidity is 70%.After planting 7 days, the slow raw type root nodule bacterium USDA110 bacterium liquid (OD of every strain inoculation
600: 0.08) 30ml, gets respectively Soybean Root and leaf texture for 0,1,3,5,10 day after connecing bacterium;
2) separation of mRNA: adopt Trizol method to extract the total RNA of soybean, 1. first tissue is put into grinding liquid nitrogen grinding 3 times, by ground, to organize 50-100mg to add in 1.5mL centrifuge tube then to add 1ml RNAVzol, split product should be the transparent thick liquid of clarification. room temperature is placed 5 minutes; 2. add 200 μ l chloroforms, vibration mixes, the standing 5min of room temperature, 12,000r/min, the centrifugal 10min of 4C; 3. get 500 μ l supernatants in another centrifuge tube, add equal-volume Virahol, room temperature is placed 10min, 12,000r/min, 4 ℃ of centrifugal 10min; 4. add 1ml 75% ethanol to wash precipitation, 8,000r/min, 4 ℃ of centrifugal 5min, repeat twice; The air-dry 10min of room temperature left and right, adds RNase Free water (DEPC water) dissolution precipitation that 20 μ l left and right DEPC processed;
3) reverse transcription is cDNA: by the mRNA of extraction, with the ThermoScript II AMV reverse transcription of Promega company, be cDNA;
4) real-time fluorescence quantitative PCR analysis: the SYBR Premix Ex TagTM that uses TaKaRa company to produce carries out quantitative PCR analysis; In 20 μ l systems, add and walk gained cDNA template 2 μ l, forward and reverse primer each 0.4 μ l, SYBR Primix Ex taqTM (2 *) 10 μ l, ROX Reference Dye II (50 *) 0.4 μ l, ddH2O 6.8 μ l; Amplification program is: 95 ℃ of 30s; 95 ℃ of 5s, 60 ℃ of 34s, 45cycles; 95 ℃ of 15s, 60 ℃ of 1min, 95 ℃ of 15s; Wherein,
Forward primer is: GTATTCGTCGACAGGGAGAAC(SEQ ID NO:3);
Reverse primer is: TCAGTATTCAAGGGAGCCAATT(SEQ ID NO:4);
5) result: as shown in Figure 2, the long period of infecting root nodule bacterium,, in the process of root nodule genesis and development, also there is obvious downward in the expression level of GmARF8L1 gene, result shows, the genesis and development process of GmARF8L1 participation regulating and controlling soybean root system after root nodule bacterium are infected.
1) extraction of total RNA in Wilimas82 soybean function leaf texture
Mortar pollutes to eliminate RNA enzyme through 180 ℃ of pyroprocessing 8 hours or through burn processing; Chloroform, Virahol, the reagent such as ethanol are used new Kaifeng unpolluted; Other equipment is if rifle head, centrifuge tube and reagent are as ultrapure water, NaAc, equal 121 ° of high-temperature heat sterilizations 30 minutes after 1 ‰ DEPC water treatments are spent the night, rifle head, 65 ° of dry for standby of centrifuge tube; Adopt Trizol method to extract the total RNA of soybean:
(1) get 50mg material (blade) and use liquid nitrogen grinding material, add 1ml redzol reagent, fully, after homogenate, homogenate is sucked to 1.5ml centrifuge tube, room temperature is placed 5min;
(2) add 200 μ l chloroforms, concussion mixes, standing 2-3min, 12000g4 ℃ of centrifugal 10min;
(3) get supernatant in another centrifuge tube, add equal-volume Virahol, place 30min, 12000g4 ℃ of centrifugal 10min for-20 °;
(4) 1ml 75% ethanol is washed precipitation, the centrifugal 5min of 7500g, twice repetition; The air-dry 10min of room temperature left and right, adds the sterilized water dissolution precipitation that 20 μ l left and right DEPC processed.
2) reverse transcription PCR
(1) with DEPC, processed 200 μ l PCR pipes in order add 5 μ L RNA and 3 μ L oligo (dt) 18; 4 μ L dNTPs, are placed in rapidly cooled on ice after 65 ℃ of incubation 5min;
(3) add in the following order following solution: 5XM-MLV buffer(invitrogen company produces) 4 μ l, RNase inhibitor 1 μ l, M-MLV 1 μ l, 0.1MDTT 2 μ l;
(4) above-mentioned reaction solution is mixed, 37 ℃ are reacted 1.5 hours;
(5), after reaction finishes, process 10min deactivation reverse transcriptase activity for 70 ℃; The cDNA article one chain that reacts synthetic can be used as PCR reaction template.
3) build recombinant expression vector
(1) clone of GmARF8L1 gene fragment
Vector construction according to the encoding sequence of GmARF8L1 (SEQ ID NO:1) design primer pair for gene silencing (RNAi), according to the multiple clone site on carrier pTCK303, primer end introduces respectively Kpn I/Spe I and BamH I/Sac I enzyme is cut recognition site; The cDNA of the soybean of take order-checking kind W82 carries out PCR as template, and amplification GmARF8L1 length is the gene fragment of 435bp (SEQ ID NO:2); Primer sequence is:
Forward primer: GGGGTACCACTAGTTTGTAACTCGGACACCCA(SEQ ID NO:5);
Reverse primer: CGGGATCCGAGCTCGATTCCCACGGATCGTCT(SEQ ID NO:6);
Amplification program is: 95 ℃ 5 minutes; 95 ℃ 30 seconds, 57 ℃ 30 seconds, 72 ℃ 40 seconds, 26 circulations; 72 ℃ 70 seconds;
Pcr amplification product carries out 1% agarose gel electrophoresis, adopts the raw work glue in Shanghai to reclaim the band that test kit reclaims purifying 435bp left and right;
The DNA fragmentation reclaiming is connected with pMD19-T carrier (Takara company), in 10 μ l systems, add T vector 1 μ l, solution I 5 μ l, reclaim fragment 4 μ l, mix mixed solution, 16 ℃ of connections are spent the night, thermal shock method proceeds to E.coli competent escherichia coli cell, incubated overnight, chooses positive colony, delivers the order-checking of invitrogen company.
(2) structure of recombinant expression vector
A. extract the T vector plasmid of the GmARF8L1 435bp gene order that contains correct order-checking, with Spe I and Sac I enzyme, cut and obtain just nucleotide sequence, same PCR product is cut the nucleotide sequence of rear acquisition antisense by BamH I and Kpn I enzyme;
B. just fragment is first cloned into the upstream of paddy rice intron in pTCK303 carrier;
C. then antisense fragment is cloned into the downstream of paddy rice intron in the restructuring pTCK303 carrier that contains just fragment, has finally formed and take the dsRNA interference carrier of the formed hairpin structure that paddy rice intron is interval;
D. by the connection product heat shock transformed competence colibacillus e.colistraindh5α of step 3,37 ℃ of incubated overnight, picking positive colony checks order; Sequencing result shows, has obtained recombinant plasmid GmARF8L1-RNAi.
4) hairly root of Agrobacterium K599 mediation transforms:
(1) conversion of Agrobacterium, adopts liquid nitrogen freeze-thaw method to transform Agrobacterium, and concrete operations are:
A. take out 200 frozen μ l competent cells, after thawing, add 5-10 μ l plasmid DNA, flick tube wall and mix, put 20-30min on ice;
B. after putting into liquid nitrogen 5min, take out, pipe proceeded to after 37 ℃ (5min) thawing, add 800 μ l LB(non-resistants) liquid nutrient medium, 28 ℃ of low-speed oscillations (150rpm) 4-5h;
C.10000rpm, 30sec, removes supernatant, adds 100 μ l LB liquid nutrient mediums, coated plate after suspension thalline (containing 50mg/ml kantlex);
D. put 28 ℃ and be cultured to white transformant and grow, for the conversion of hairly root;
(2) hairly root of Agrobacterium K599 mediation transforms
Take soybean varieties W82 as material, get seed material and use disinfection by chlorine after 10 hours, at B
5substratum (culture medium prescription: 2% sucrose, 0.8 agar powder (sigma), 1 * GAMBORG B-5BASAL(Phyto Technology Laboratories, article No.: G398), pH is adjusted to 5.7 left and right; ) upper sprouting 5 days, when just will opening, cuts cotyledon cotyledon, and in cotyledon lower end right-angled intersection cutting, be immersed in the Agrobacterium K599 of activation and infect 30min(OD
600: 0.6 left and right), explant is gone to 1/2MS substratum (culture medium prescription: 2% sucrose, 0.8 agar powder (sigma), 0.5 * MURA SHIGE & SKOOG BASAL MEDIOM w/VITAMINS (Phyto Technology Laboratories, article No.: G519), pH is adjusted to 5.7 left and right; ) above train altogether growth after 3 days, go to again on 1/2MS substratum and induce hairly root, after 7 days, hairly root grows, treat that soybean true leaf grows, hairly root reaches after 7-8cm, selects the compound seedling of approaching hairly root of etap to move into soilless substrate (vermiculite: perlite=3:1), cultivate Rhizobium Inoculation USDA110(OD after 1 week
600: 0.08) 20ml/ strain, cultivate and collect root nodule after 28 days, statistics root nodule numbers.
5. result is observed
Result as shown in Figure 3, show under identical experimental situation, lowering the transgenosis root of expression GmARF8L1 compares with the transgenosis root system (EV) of empty carrier conversion, root nodule numbers significantly increases (Fig. 3 A and 3B), Fig. 3 C is the expression level analysis to GmARF8L1 in transgenosis root system, and the expression that result is presented at GmARF8L1 in transgenosis root system is significantly to lower.
Foregoing description only proposes as the enforceable technical scheme of the present invention, not as the Single restriction condition to its technical scheme itself.
Claims (10)
1. for building a gene fragment for rna interference vector, this gene fragment, for building the hairpin structure of rna interference vector, is characterized in that: the nucleotide sequence of described gene fragment is as shown in the SEQ ID NO:2 in sequence table.
2. the complementary nucleotide sequence of gene fragment described in claim 1.
3. for a rna interference vector for regulating plant nodulation and nitrogen fixation ability, comprise skeleton carrier and hairpin structure, it is characterized in that: described hairpin structure utilizes the gene fragment described in claim 1 or 2 to build.
4. rna interference vector according to claim 3, is characterized in that: described skeleton carrier is pTCK303 carrier.
5. rna interference vector according to claim 3, is characterized in that: described hairpin structure be spaced apart the paddy rice intron on pTCK303 carrier.
6. rna interference vector according to claim 5, it is characterized in that: positive-sense strand is arranged in the upstream of pTCK303 carrier paddy rice intron, antisense strand is positioned at the downstream of paddy rice intron, and integral body has formed take the double-stranded RNA interference carrier of the formed hairpin structure that paddy rice intron is interval.
7. the purposes of the rna interference vector described in claim 3-6 any one, for cultivating the plant with high nodulation and nitrogen fixation ability.
8. purposes according to claim 7, is characterized in that: make rna interference vector transformation receptor plant, the corresponding gene in reticent recipient plant, makes the root tissue of recipient plant have the nodulation and nitrogen fixation ability of enhancing.
9. purposes according to claim 7, is characterized in that: the method for described transformation receptor plant is to utilize agriculture bacillus mediated hairly root conversion method; The described gene being silenced is the gene shown in SEQ ID NO:1.
10. purposes according to claim 7, is characterized in that: described recipient plant is Papilionaceae plant.
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| CN108586592A (en) * | 2018-04-23 | 2018-09-28 | 中国科学院上海生命科学研究院 | The gene for regulating and controlling the nodule number of root nodule plant and its application in terms of high-efficiency nitrogen-fixing |
| CN109694880A (en) * | 2019-02-26 | 2019-04-30 | 华中农业大学 | A method of based on the high dross genetically modified plants of RNA interference technique to breed |
| CN109912702A (en) * | 2017-12-13 | 2019-06-21 | 中国科学院遗传与发育生物学研究所 | Application of the protein OsARE1 in regulation plant low nitrogen resisting |
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Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN109912702A (en) * | 2017-12-13 | 2019-06-21 | 中国科学院遗传与发育生物学研究所 | Application of the protein OsARE1 in regulation plant low nitrogen resisting |
| CN109912702B (en) * | 2017-12-13 | 2021-03-16 | 中国科学院遗传与发育生物学研究所 | Application of protein OsARE1 in regulation and control of low nitrogen resistance of plants |
| CN108586592A (en) * | 2018-04-23 | 2018-09-28 | 中国科学院上海生命科学研究院 | The gene for regulating and controlling the nodule number of root nodule plant and its application in terms of high-efficiency nitrogen-fixing |
| CN108586592B (en) * | 2018-04-23 | 2022-08-02 | 中国科学院分子植物科学卓越创新中心 | Gene for regulating and controlling root nodule number of root nodule plant and application of gene in aspect of efficient nitrogen fixation |
| CN109694880A (en) * | 2019-02-26 | 2019-04-30 | 华中农业大学 | A method of based on the high dross genetically modified plants of RNA interference technique to breed |
| CN109694880B (en) * | 2019-02-26 | 2022-04-01 | 华中农业大学 | Method for cultivating high-nodulation transgenic plant based on RNA interference technology |
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