CN102220330A - MiRNA-gma-miR56b related to drought resistance of plants and application thereof - Google Patents
MiRNA-gma-miR56b related to drought resistance of plants and application thereof Download PDFInfo
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Abstract
The invention discloses miRNA-gma-miR56b related to the drought resistance of plants, which has a sequence shown as SEQ ID NO:1 in a sequence table; in soybeans, the miRNA shown as the SEQ ID NO:1 is overexpressed or the expression of miRNA shown as the SEQ ID NO:1 is inhibited so as to cultivate transgenic soybeans with high drought resistance; and therefore, the miRNA-gma-miR56b has great significance for increasing the output of soybeans.
Description
Technical field
The present invention relates to a kind of miRNA relevant, especially a kind ofly derive from miRNA soybean, that can regulate soybean drought resisting with plant drought resistance; The invention still further relates to precursor and the purposes of this miRNA.
Background technology
Soybean is a crop very important, that have special economic value.Environment-stress such as arid, saline and alkaline has material impact to growing of soybean, can cause the extensive underproduction of soybean when serious.Resolve the molecular mechanism of soybean opposing adverse circumstance, the using gene engineering breeding has become and has strengthened the crop resistance of reverse, and cultivating the strong soybean varieties of resistance of reverse is one of major objective of present soybean planting industry.Higher plant has started the multiple network of replying when replying environment-stress, microRNA(miRNA) also have important effect (Leung and Sharp, 2007) therein.
MiRNA is the non-encoding histone RNA of endogenous of a kind of length 20～24 nt of Recent study discovery.The miRNA gene is transcribed at first and is produced the pre-miRNAs with neck ring structure.Pre-miRNA is transported out nuclear subsequently, by the effect of Dicer enzyme, is processed into sophisticated miRNA in tenuigenin.Combine with the mRNA of particular target gene by sequence is complementary, thereby cause that mRNA degrades or inhibition mRNA translation is played down regulation (Llave et al., 2002 to expression of gene; Palatnik et al., 2003; Tang et al., 2003).
Summary of the invention
The technical problem to be solved in the present invention provides a kind of miRNA relevant with plant drought resistance and precursor sequence thereof, utilizes this miRNA can improve the drought resistance of soybean, is significant to improving soybean yields.
For solving the problems of the technologies described above, the technical solution used in the present invention is as follows.
A kind of miRNA relevant with plant drought resistance, it has the sequence shown in the SEQ ID NO:1 in the sequence table.
The precursor sequence of the above-mentioned miRNA relevant with plant drought resistance, it has the sequence shown in the SEQ ID NO:2 in the sequence table.
Above-mentioned miRNA the application during cultivating drought resisting transgenic plant relevant with plant drought resistance.
As a kind of optimal technical scheme of above-mentioned application, described drought resisting transgenic plant are soybean.
As a kind of optimal technical scheme of above-mentioned application, in soybean, cross and express the miRNA shown in the SEQ ID NO:1, or suppress the expression of miRNA shown in the SEQ ID NO:1, cultivate genetically engineered soybean with high drought resistance.
Adopt the beneficial effect that technique scheme produced to be: the expression of miRNA of the present invention is subjected to the adjusting of drought stress, illustrate that it plays an important role in soybean adaptation drought stress mechanism, based on this, the genetically engineered soybean that can utilize its cultivation to have high drought resistance is significant to improving soybean yields.
Description of drawings
Fig. 1 is the loop-stem structure figure of gma-miR156b precursor sequence, and as seen its precursor sequence is folded into a kind of stable loop-stem structure, belongs to the typical secondary structure of miRNA precursor, meets the constitutional features of miRNA precursor.
Fig. 2 is the expression characteristic of gma-miR156b under the different stress conditions of real-time quantitative PCR detection.
Fig. 3 utilizes soybean hairly root system to analyze the response of gma-miR156b to drought stress.
Embodiment
Following examples describe the present invention in detail.Various raw material used in the present invention and items of equipment are conventional commercially available prod, all can buy directly by market to obtain.Quantitative test in following examples all is provided with repeated experiments three times, results averaged.
Embodiment 1, real-time fluorescence quantitative PCR are analyzed the expression characterization of gma-miR156b
One, coerces processing
Testing used soybean material kind is that Wilimas82(is called for short W82).The seedling phase coerces material and carries out according to following flow process: soybean seeds is with 70% smart sterilization 30 S that spill, and is seeded in the sterile petri dish after the dH2O flushing 6 times, puts 2 aseptic filter papers at the bottom of the ware, and dH2O soaks, 28 ℃ of dark sproutings 3 days.When bud grows to 2 cm, low N water planting, 10,000 lux, temperature is 26 ℃, relative humidity is 70%, cultivates 15 days, carries out the seedling phase and coerces processing, the root of drawing materials, stem, leaf.
(1) Dui Zhao processing: the soybean material of directly getting the home cultivation respectively is organized in-80 ℃ frozen (0 hour) in contrast;
(2) arid is handled: be taken at each tissue of soybean material of cultivating 0.5,3,12 and 24 hour in the nutrient solution that contains 10% PEG8000, in liquid nitrogen flash freezer ,-80 ℃ of preservations are standby;
(3) dormin is handled: seedling phase or one-tenth strain phase material are handled 0.5,3,12 and 24 hour in the nutritive medium that contains 100 μ M dormins (ABA) after, get its each tissue, in liquid nitrogen flash freezer ,-80 ℃ of preservations are standby;
Two, the separation of miRNA
Get leaf, extract test kit (hundred Imtech) (Cat # RP5301) with microRNA and extract small fragment RNA, extracting method is as follows:
(1) homogenized
Leaf grinds fast with mortar in the liquid nitrogen, and per 50～100 mg tissue adds homogenate behind the 1 ml lysate.
(2) with homogenate sample concuss mixing, under 15～30 ℃ of conditions, hatch 5 min so that ribosome decomposes fully.
Centrifugal 10 min of 12,000 rpm under (3) 4 ℃ the condition carefully get in the centrifuge tube that supernatant changes a new RNase free over to.
When sample rich in proteins, fat, polysaccharide, can centrifugal 10 min, remove undissolved material in the homogenate, include epicyte, polysaccharide and high-molecular-weight DNA in the remaining precipitation, and the super seston on upper strata contains RNA.
(4) per 1 ml lysate adds 0.2 ml chloroform.Cover tight sample hose lid, thermal agitation 15 s are also at room temperature hatched 3min with it.
(5) sample can be divided into three layers in 4 ℃ of 12,000 centrifugal 10 min of rpm: lower floor's organic phase, and the water that middle layer and upper strata are colourless, RNA is present in aqueous phase.The capacity of aqueous phase layer be approximately add 60% of lysate volume, water is transferred in the new pipe, carry out next step operation.
(6) add 0.6 times of volume 70% ethanol, put upside down mixing (precipitation may appear in this moment).The solution that obtains changes among the adsorption column RA together with precipitating.
(7) 10, centrifugal 45 s of 000 rpm collect filtrate (containing microRNA in the following filtrate) down, accurately estimate the volume of filtrate down, add the dehydrated alcohol of 2/3 times of volume, put upside down mixing several times, mixed solution is poured among the adsorption column RB, and 10,000 rpm are centrifugal, and 30 s discard waste liquid.
(8) add 700 μ l rinsing liquid RW, centrifugal 60 s of 12,000 rpm discard waste liquid.
(9) add 500 μ l rinsing liquid RW, centrifugal 60 s of 12,000 rpm discard waste liquid.
(10) adsorption column RB is put back in the sky collection tube, centrifugal 2 min of 12,000 rpm remove rinsing liquid as far as possible, in order to avoid residual ethanol suppresses downstream reaction in the rinsing liquid.
(11) take out adsorption column RB, put into a RNase free centrifuge tube, add 60-80 μ l RNase free water(in the adsorption film middle part according to expection RNA output and in 65-70 ℃ of water-bath, heat in advance), room temperature is placed 2 min, centrifugal 1 min of 12,000 rpm then.Collection obtains pure microRNA and is stored in-80 ℃ of refrigerators.
Three, reverse transcription is cDNA
Is cDNA with the sky root miRNA cDNA first chain synthetic agent box with the miRNA reverse transcription of purifying.
1. the Poly of little RNA (A) tailing
The reaction solution prescription is as follows: little RNA, 6 μ l; E-PAP(5U/ μ l), 0.4 μ l; 10 * PAP Buffer, 2 μ l; 5 * rATP solution, 4 μ l; Nuclease-free Water, 7.6 μ l; Cumulative volume, 20 μ l; The reaction solution of the above-mentioned preparation of mixing gently, of short duration centrifugal after, 37 ℃ of reaction 60 min.The gained reaction solution can directly carry out the downstream experiment, also can place-20 ℃ of of short duration preservations, and suggestion is deposited in-80 ℃ as the need prolonged preservation.
2.Poly (A) miRNA of Xiu Shiing carries out reverse transcription reaction
The reaction solution prescription is as follows: Poly (A) reaction solution, 2 μ l; 10 * RT primer, 2 μ l; 10 * RT Buffer, 2 μ l; Ultrapure dNTP Mixture (2.5 mM), 1 μ l; Rnasin (40U/ μ l), 1 μ l; Quant RTase, 0.5 μ l; RNase-free ddH
2O, 1.5 μ l; Cumulative volume, 20 μ l; The reaction solution of the above-mentioned preparation of mixing gently, of short duration centrifugal after, 37 ℃ of reaction 60 min.Synthetic cDNA reaction solution is placed-20 ℃ of preservations, also can directly carry out the downstream fluorescent quantitation and detect.
Four, real-time fluorescence quantitative PCR
By following reaction system, mixed system, divide in the 96 hole optical sheets of packing into then, and cover upward blooming.The instrument that amplification is used is the quantitative real time PCR Instrument of ABI company.
Amplification program is: 95 ℃ of 30s; 95 ℃ of 5 s, 60 ℃ of 34s, 45 cycles; 95 ℃ of 15 s, 60 ℃ of 1min, 95 ℃ of 15 s;
20 μ l reaction systems are formulated as follows: dna profiling, 2 μ l; SYBR
Primix Ex taq TM (2 *), 10 μ l; PCR Forward Primer(10 μ Μ), 0.4 μ l; PCR Reverse Primer(10 μ Μ), 0.4 μ l; ROX Reference Dye II (50 *), 0.4 μ l; DdH
2O, 6.8 μ l; Cumulative volume, 20 μ l;
Primer sequence is: forward primer: TGACAGAAGAGAGAGAGCACAA(SEQ ID NO:4); 5.8S rRNA-F:ACGCCTGCCTGGGTGTCACAC(SEQ ID NO:5); AMRM:GCGAGCACAGAATTAATACGACT(SEQ ID NO:6); (5.8S rRNA is confidential reference items, and AMRM is a reverse primer);
The result as shown in Figure 2, wherein A and B soybean root that to be respectively gma-miR156b handle at PEG and the expression in the leaf.As can be seen from the figure, gma-miR156b be expressed in root and Ye Zhongdou has been subjected to significantly inducing of PEG mimic drought stress.The result shows that gma-miR156b participates in the response of regulating and controlling soybean to drought stress.Like this, in soybean, cross expression gma-miR156b, or suppress the expression of gma-miR156b,, cultivate genetically engineered soybean, the output that improves soybean is significant with high drought resistance with influencing the adaptive faculty of soybean to drought stress.
Embodiment 2, utilize soybean hairly root conversion system that gma-miR156b is analyzed in the expression under the adverse circumstance
One, pCAMBIA3301-
P MiR156b -GUS vector construction:
Described pCAMBIA3301-
P MiR156b -GUS is preferably the promoter sequence with sequence table 3(for the promoter region with the encoding gene of gma-miR156b precursor is inserted in the recombinant plasmid that the multiple clone site of pCAMBIA3301 obtains) sequence 3' cut the recombinant plasmid that obtains between the recognition site from BamHI and NcoI enzyme that 5' holds the 1st to 1076 dna fragmentation shown in the Nucleotide to insert pcambia3301.
Two, material is prepared
With the W82 kind is material, use disinfection by chlorine after 10 hours material, on B5 medium, sprout and carried out the hairly root conversion in 4 days, after explant goes to and grew 2 days on the common training substratum, go to again on the MS/2 substratum and cultivate, after 15 days, hairly root grows, and selects the about 1-2 of length centimetre approaching hairly root of etap to go to respectively and coerces processing.
Three, coerce processing
(1) contrast: the hairly root of choosing is gone on the filter paper that has soaked the MS/2 liquid nutrient medium, handle 0,0.5,3,12 and 24 hour respectively after, GUS dyeing is carried out in sampling, dyeing time is defined as 3 hours.
(2) arid is handled: the hairly root of choosing is gone to has soaked MS/2 and add on the filter paper of 10% PEG8000 liquid nutrient medium, handle 0,0.5,3,12 and 24 hour respectively after, GUS dyeing is carried out in sampling, dyeing time is defined as 3 hours.
(3) dormin is handled: the hairly root of choosing is gone to has soaked on the filter paper of dormin (ABA) liquid nutrient medium that MS/2 adds 100 μ M, handle 0,0.5,3,12 and 24 hour respectively after, sampling carries out GUS dyeing, dyeing time is defined as 3 hours.
Four, GUS tissue chemical analysis
(1) preparation of GUS dye liquor
At first prepare the GUS reaction buffer: Tris Buffer, comprising 100 mM Tris and 50 mM NaCl
Transfer pH to 7.4 with concentrated hydrochloric acid.Prepare 0.5 mM K with Tris buffer then
3[Fe (CN)
6] solution.Concentration by 1 mg/ml takes by weighing x-glu(5-bromo-4-chloro-3-indoles glucuronide), the ratio dissolving x-glu according to every mg adds 10 μ l DMSO (dimethyl sulfoxide (DMSO)) adds the 0.5 mM K that Tris buffer prepares
3[Fe (CN)
6] the solution constant volume, can add several TritonX-100 to strengthen Color.Keep in Dark Place in-20 ℃ after the packing rapidly.
(2) GUS staining procedure
Hairly root to be determined is got plant sample put into the GUS reaction buffer, submergence, 37 ℃ of insulations in the dark.Under anatomical lens, observe color developing effect every half an hour.Dye after 3 hours, sucking-off GUS reaction buffer adds 70% ethanol termination reaction and 37 ℃ of decolourings, changes once every a few hours.
The result as shown in Figure 3, wherein A transforms hairly root that the gma-miR156b promotor the carries gus reporter gene GUS accumulation phenotype in PEG and ABA handle, B is the statistic data to the hairly root number that the GUS accumulation occurs.As can be seen from the figure, the expression of gma-miR156b has been subjected to significantly inducing of PEG8000 mimic drought stress.Drought stress can be induced the generation of dormin ABA, and the result of Fig. 3 A and 3B shows that also the expression of gma-miR156b also obviously has been subjected to inducing of ABA.This result shows that drought stress is significantly induced the expression of gma-MIR156b, thereby explanation gma-MIR156b participates in regulating the reaction of soybean to drought stress.Like this, in soybean, cross expression gma-miR156b, or suppress the expression of gma-miR156b,, cultivate genetically engineered soybean, the output that improves soybean is significant with high drought resistance with influencing the adaptive faculty of soybean to drought stress.
Foregoing description only proposes as the enforceable technical scheme of the present invention, not as the single restricted condition to its technical scheme itself.
Sequence table
<160> 6
<210> 1
<211> 21
<212> RNA
＜213〉Glycine soybean (Glycine max)
<400> 1
UGACAGAAGA?GAGAGAGCAC?A?21
<210> 2
<211> 120
<212> RNA
＜213〉Glycine soybean (Glycine max)
<400> 2
UGAUGUGAGA?UAUCUCAUGU?UGACAGAAGA?GAGAGAGCAC?AACCCGGGAA?UGGCUAAAGG?60
AGUCUUUGCC?UUUGUUGGGA?GUGUGCCCUC?UCUUCCUCUG?UCAUCAUCAC?AUUCACAUGC?120
<210> 3
<211> 1076
<212> DNA
＜213〉Glycine soybean (Glycine max)
<400> 3
ACTTTCTTTC?CTCTGTTGGC?CCTTTATATA?TTCTTACGGT?CCCTCAATTA?GATCTTCTAT?60
ATTATTTGTT?CTTTAATTCC?TATATACATC?AAGGATCATG?CATAGGTGTA?CAGGAGGAGA?120
GAAGCTTTAA?TTGGTGAGGC?TGATTTGGGA?AGAAGAAGGA?AAAGGATCAG?CATATATGGA?180
GGTGAATTAT?TCATGCTTAA?TTGTTAACTC?CCGTTGATGA?GATATACAGC?ATCATGTACT?240
AATAATCTTT?GTGCACATCA?CCCCCCACAC?CCCATCTCAT?TTCTAAAGAA?TATAGTATAT?300
ATACAAGTAC?ACTAGCTCAT?TCAATAATAT?ATTTCTAATC?AGCATATTTC?TTACATTAAG?360
AGAAGCCTAG?CTACATGTCC?CCCCTACTAA?TATATCCTTT?TGCAAATATA?AGACCTAAGA?420
TCTAAAATCG?ATTGATATTT?TGAAGGAATT?AATATATAGA?TGACAGCATA?TATAAAAGTA?480
AAACATGAAT?TAAATTTTAG?GGATGAAGTA?ATTTTCTGAA?AAACTCACTG?TCACTTGATT?540
GTGATCTACA?TATTCTTGAA?GTTTTAGTAT?TATTTTCTCA?GTTTTAGGGC?TATTATTTCC?600
CAGGATTAGG?GCTAGGGTCA?ATTTTCTCGA?TCAGTTTTAG?TATTATTTCA?AACTTGACTA?660
ATTATGTGTT?AAAAGGAATT?AGTTTCACAT?CTTAACTAAA?AGTTGTAGAG?ATATATAAAA?720
GAAGACGAAG?ATTTAAGTAA?AATTAATTTT?GTTTGGATAG?AAAAACTCCA?TAATAATATA?780
TGATTGATGA?AAAAAACATG?GCTTTTTAGT?ATGTACAGAT?GAATTCAATA?TTTATATCTA?840
TATATATGTA?CATATATAAA?AAAGTCTATT?AAAAGAAGTT?AATATCTTAA?ATAAAATTTA?900
ATATTTTAAA?ATATTAGTCT?TTGTTAGAGG?ATACTCAAGA?TTTGTAATAA?AACTTAGTTA?960
TGTAATTTAA?TTATTTGTAA?ATATAATATA?CATATTTAGA?CCAAGAATAG?AGTAGGGAAA?1020
AAGCAAATTT?AAAGGAATAG?AAATATGCTT?GTTTTGATAC?ACGAAAGGTC?CACTTG 1076
<210> 4
<211> 22
<212> DNA
＜213〉artificial sequence
<400> 4
TGACAGAAGA?GAGAGAGCAC?AA?22
<210> 5
<211> 21
<212> DNA
＜213〉artificial sequence
<400> 5
ACGCCTGCCT?GGGTGTCACA?C?21
<210> 6
<211> 23
<212> DNA
＜213〉artificial sequence
<400> 6
GCGAGCACAG?AATTAATACG?ACT?23
Claims (5)
1. miRNA relevant with plant drought resistance, it is characterized in that: it has the sequence shown in the SEQ ID NO:1 in the sequence table.
2. the precursor sequence of the described miRNA relevant with plant drought resistance of claim 1, it is characterized in that: it has the sequence shown in the SEQ ID NO:2 in the sequence table.
3. described miRNA the application during cultivating drought resisting transgenic plant relevant of claim 1 with plant drought resistance.
4. the purposes of the miRNA relevant with plant drought resistance according to claim 3, it is characterized in that: described drought resisting transgenic plant are soybean.
5. the purposes of the miRNA relevant according to claim 4 with plant drought resistance, it is characterized in that: in soybean, cross and express the miRNA shown in the SEQ ID NO:1, or the expression of miRNA shown in the inhibition SEQ ID NO:1, cultivate genetically engineered soybean with high drought resistance.
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104232682A (en) * | 2014-09-18 | 2014-12-24 | 中国科学院遗传与发育生物学研究所 | Method for cultivating high-yield plant by overexpression of gma-miR156b |
| CN106047881A (en) * | 2016-08-22 | 2016-10-26 | 石家庄市农林科学研究院 | miRNA ghr-MiR17 associated with drought stress in plants, and applications thereof |
| CN106434654A (en) * | 2016-08-22 | 2017-02-22 | 石家庄市农林科学研究院 | MiRNA, namely ghr-miR01, related to plant's response to drought stress and application of miRNA |
| CN109929873A (en) * | 2019-03-20 | 2019-06-25 | 华中农业大学 | A method of cultivating drought resisting genetically modified plants |
| CN116254288A (en) * | 2022-12-28 | 2023-06-13 | 南京林业大学 | Application of cymbidium MIR156b gene in regulating and controlling flowering time of plants |
-
2011
- 2011-06-10 CN CN2011101549116A patent/CN102220330A/en active Pending
Non-Patent Citations (3)
| Title |
|---|
| SUNKAR ET AL: "Identification of novel and candidate miRNAs in rice by high throughput sequencing", 《BMC PLANT BIOLOGY》 * |
| WONG ET AL: "MicroRNAs in the shoot apical meristem of soybean", 《JOURNAL OF EXPERIMENTAL BOTANY》 * |
| 陈锐: "耐旱野生大豆MicroRNA的鉴定与表达分析", 《中国博士学位论文全文数据库 农业科技辑》 * |
Cited By (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104232682A (en) * | 2014-09-18 | 2014-12-24 | 中国科学院遗传与发育生物学研究所 | Method for cultivating high-yield plant by overexpression of gma-miR156b |
| CN106047881A (en) * | 2016-08-22 | 2016-10-26 | 石家庄市农林科学研究院 | miRNA ghr-MiR17 associated with drought stress in plants, and applications thereof |
| CN106434654A (en) * | 2016-08-22 | 2017-02-22 | 石家庄市农林科学研究院 | MiRNA, namely ghr-miR01, related to plant's response to drought stress and application of miRNA |
| CN106047881B (en) * | 2016-08-22 | 2019-03-12 | 石家庄市农林科学研究院 | A kind of miRNA relevant to plant reply drought stress --- ghr-miR17 and its application |
| CN109929873A (en) * | 2019-03-20 | 2019-06-25 | 华中农业大学 | A method of cultivating drought resisting genetically modified plants |
| CN116254288A (en) * | 2022-12-28 | 2023-06-13 | 南京林业大学 | Application of cymbidium MIR156b gene in regulating and controlling flowering time of plants |
| CN116254288B (en) * | 2022-12-28 | 2023-09-22 | 南京林业大学 | Application of cymbidium MIR156b gene in regulating and controlling flowering time of plants |
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Application publication date: 20111019 |