CN108220290A - Applications of the rice microRNA osa-miR171b in water resistant rice stripe - Google Patents
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Abstract
The invention discloses applications of the rice microRNA osa miR171b in water resistant rice stripe, present invention high-flux sequence from rice Nipponbare obtains microRNA osa miR171b, is building up in precursor-gene pre miR528 sequences with the microRNA.MicroRNA precursors overexpression os miR171b in rice, after the small brown rice planthopper virus inoculation for carrying rice stripe virus, transgenic line is compared morbidity and mortality with wild type control and is reduced, and disease symptom is alleviated.Therefore, overexpression os miR171b are of great significance to the tolerance of stripe disease for improving rice in rice, this is resistant to stripe disease molecular breeding for rice and provides new resource and research method.
Description
Technical field：
The present invention relates to plant genetic engineering fields, and in particular to a kind of rice microRNA osa-miR171b is in water
Application in rice tolerance rice stripe virus (Rice stripe virus, RSV).
Background technology
Rice (Oryza sativa L.) is a kind of important cereal crops.Stripe disease is to pass through small brown rice planthopper
The important Rice Virus disease that (Laodelphax striatellus) is propagated, gives agricultural components serious threat.
MicroRNA (microRNA) is prevalent in animal and plant body, and the weight of post-transcriptional control is played to its target gene
It acts on, so as to regulate and control many characters of organism.MicroRNA regulates and controls many target genes of plant, some of target gene ginsengs
With plant to the resistance of virus.
Invention content
Group of the present invention finds, the disease resistance of rice can be improved by overexpressing microRNA, so as to disease-resistant to cultivate
Rice provides a new approach.
On the one hand, the present invention obtains length as 21nt rice maturation microRNA by high-flux sequence, is named as osa-
MiR171b sequences, sequence are：5’UGAUUGAGCCGUGCCAAUAUC 3’,(SEQ ID NO：1).With the microRNA,
Osa-miR528 sequences in osa-MIR528 precursor sequences known to the replacement of osa-miR171b sequences, obtain artificial miR171b
Sequence osa-miR171b (SEQ ID NO：2).After the chemical synthesis sequence DNA, which is connected to double base expression
Carrier pCAMBIA1300UR (Fig. 1).The carrier is converted to EHA105 Agrobacteriums, using Agrobacterium tumefaciens-mediated Transformation in water
Osa-miR171b is overexpressed in rice, after the small brown rice planthopper virus inoculation for carrying rice stripe virus, transgenic line and wild
Type control is reduced compared to morbidity and mortality, and disease symptom is alleviated.Therefore, osa- is overexpressed in rice
MiR171b can improve tolerance of the rice to stripe disease.
On the other hand, the present invention provides one section of rice microRNA osa-miR171b sequence, the nucleosides of the microRNA
Acid sequence is SEQ ID NO:Shown in 1, feature exists, which can prepare tolerance rice by transgenosis water method
The rice that strip virus (Rice stripe virus, RSV) infects.
On the other hand, the present invention provides a kind of tolerance rice stripe virus (Rice stripe virus, RSV) for preparing and invades
The method of the rice of dye, wherein, microRNA nucleotides sequences is allowed to be classified as SEQ ID NO:Gene order shown in 1 is transferred to rice
In.
On the other hand, the present invention provides one section of rice microRNA os-miR171b sequence and is preparing tolerance rice stripe
The purposes on rice that viral (Rice stripe virus, RSV) infects, wherein, the RNA os-miR171b are SEQ
ID NO:Sequence shown in 1.
On the other hand, the present invention provides the artificial microRNA osa-miR171b sequences of rice, the nucleosides of the microRNA
Acid sequence is SEQ ID NO:Shown in 2, which is characterized in that the microRNA can prepare tolerance rice by transgenic method
The rice that strip virus (Rice stripe virus, RSV) infects.
On the other hand, the present invention provides a kind of tolerance rice stripe virus (Rice stripe virus, RSV) for preparing and invades
The method of the rice of dye, wherein, nucleotides sequence is allowed to be classified as SEQ ID NO:Gene order shown in 2 is transferred in rice.
On the other hand, the present invention provides the artificial microRNA osa-miR171b gene orders of rice and is preparing tolerance water
Purposes in the rice that cercosporiosis of rice poison (Rice stripe virus, RSV) infects, wherein, the gene order is SEQ
ID NO:Sequence shown in 2.
Those of ordinary skill in the art are it is believed that any transgene method is all suitable for disclosed base
Because of the transfer of sequence, any rice varieties can be applicable in the gene of the present invention.Group of the present invention finds, different rice varieties or
The different transgenic method of person, although there may be the phenotype of other various traits, for being successfully transferred to announcement of the present invention
Target gene, the phenotype of the water resistant cercosporiosis of rice can be shown, and reach the level of notable resistance.
Advantageous effect
Transgenosis is carried out using sequence provided by the invention and obtains rice positive plant, significantly improves Rice Resistance rice item
The antibody of line virus (Rice stripe virus, RSV).
Description of the drawings
Fig. 1 is the pCAMBIA1300UR-osa-miR171b expression vector structure diagrams of the present invention.
Fig. 2 detects osa-miR171b for Northern blot and overexpresses transgenic paddy rice positive plant osa-miR171b
Expression figure.Fig. 3 detects osa-miR171b for Real-Time PCR and overexpresses transgenic paddy rice positive plant osa-
MiR171b relative expression levels.
(Mock expressions connect phenotypic map before and after Fig. 4 overexpresses transgenic paddy rice Inoculated Rice strip virus for osa-miR171b
Phenotype before kind, RSV represent the phenotype after inoculation).
Morbidity and mortality count after Fig. 5 overexpresses transgenic paddy rice Inoculated Rice strip virus for osa-miR171b
Figure.
Plant height counts before and after Fig. 6 overexpresses transgenic paddy rice positive plant Inoculated Rice strip virus for osa-miR171b
Figure.
Specific embodiment
It needs to specialize, specific embodiment of the invention is used for the purpose of how realizing progress in order to illustrate the present invention
For example, such explanation can not form the present invention any limitation.The scope of the present invention obtains body in claim
It is existing.In order to achieve the object of the present invention, it is carried out using way of example as described below.
According to the sequence for the osa-miR171b that high-flux sequence obtains, particular sequence information is as shown:
5’UGAUUGAGCCGUGCCAAUAUC 3’,(SEQ ID NO：1), with the microRNA, osa-miR171b sequences
Osa-miR528 sequences in osa-MIR528 precursor sequences known to replacement obtain artificial miR171b sequences osa-miR171b
(SEQ ID NO：2), wherein, dashed part replaces the corresponding sequence to be formed for sequence 1 in sequence 2, and replacement here is not
It is simply to replace, but 1 corresponding DNA sequence dna of RNA sequenceTGAT TGAGCCGTGC CAATATCReplace original osa-
Osa-miR528 sequences in MIR528 precursor sequences, italicized item are the complementary series of 1 corresponding DNA sequence dna of sequence:G ATATTGGGGC GGTTCAATCThen A synthesizes new sequence 2 by the way of artificial synthesized：
The sequence is through the artificial microRNA osa-miR171b precursor DNA sequences of chemical synthesis.The DNA is through Sac I, BamH I
PCAMBIA1300UR carriers are connected to after digestion, are named as pCAMBIA1300UR-osa-miR171b (Fig. 1).
Convert Agrobacterium
The Agrobacterium EHA105 competence of -70 DEG C of preservations is taken to put on ice to melt, draws 1 μ l pCAMBIA1300UR-osa-
In miR171b plasmids to 100 μ l competence, mixing adds to precooling 1mm electric shock cups；Voltage 2.3kv, 25 μ F of capacitance, resistance are set
200 Ω are electroporated；900 μ l LB fluid nutrient mediums, 28 DEG C of 200rpm shaking table culture 2h are added in, bacterium solution is applied to mould containing that is blocked
On plain 50mg/L, rifampin 50mg/L LB tablets, 28 DEG C of cultures to formation single bacterium colony.
Be conducted into rice by agriculture bacillus mediated rice transformation, rice paddy seed callus induction, squamous subculture,
It co-cultures, screening and differentiation obtain transgenic paddy rice seedling.
It is as follows：
1. the induction of Rice Callus
Take ripe Nipponbare rice paddy seed, the seed of full bright and clean no bacterial plaque is selected in artificial decladding, seed be put into 25ml without
In tube, add in 75% alcohol and impregnate 1min, it is sterile to wash 3 times；30% liquor natrii hypochloritis is added in, impregnates 30min；It outwells secondary
Sodium chlorate solution, with sterile water wash seed 5 times, sterile water impregnates 30min；Seed is put and is blotted on aseptic filter paper, is transferred to induction
On culture medium, per 6-7, ware；Medical adhesive tape seals culture dish, 27 DEG C of illumination box temperature, and humidity 50% is cultivated about 27 days；
It in the callus that super-clean bench is induced with tweezers picking, is transferred on subculture medium, 27 DEG C of illumination box, humidity 50% is cultivated
7 days.
2. callus and the co-cultivation of Agrobacterium
Picking Agrobacterium single bacterium is dropped down onto in 5ml LB fluid nutrient mediums (50mg/L containing Kan, Rif 50mg/L), 28 DEG C,
180rpm shaking table cultures are to orange-yellow；It draws in 2ml bacterium solutions to 2ml EP pipes, 5,000rpm, centrifuges 5min, abandon supernatant；With suitable
It measures AAM culture mediums to suspend, be transferred in the sterile triangular flasks of 100ml, by 50ml AAM fluid nutrient mediums.It is preferable to select state, color
The callus of the squamous subculture of the micro- Huang in pool is transferred in sterile triangular flask, is shaken up, is placed at room temperature for 30min.Culture medium is outwelled, it will more
Injured tissue, which is gone on aseptic filter paper, sucks extra bacterium solution, is transferred to and co-cultures on base, 27 DEG C of dark culturings 2.5 days.
3. the screening and differentiation of Rice Callus
Callus after co-cultivation is transferred on Selective agar medium, 27 DEG C, 50% illumination cultivation of humidity.It was with 10 days
A cycle co-cultures 3 periods.The callus of picking color cadmium yellow is transferred to the plastic bottle equipped with about 60ml differential mediums
In, every bottle puts 5.27 DEG C in incubator, humidity 50%, illumination cultivation is until differentiate seedling.
4. hardening of taking root and transplanting
When the seedling that callus differentiates is grown to about 10cm, seedling is extracted, removes culture medium, root is cut off, be inserted into
In root media, cultivate 5 days.It treats that root long goes out, washes away culture medium, 27 DEG C of illumination Aquaponics 5 days are transplanted to paddy field.
5. rice transformation used medium：
Inducing culture：N6 is a large amount of, and MS-Fe salt, B5 is micro, and B5 is organic, 2,4-D 2.5mg/L, proline 2800mg/L,
L-Glutamine 500mg/L, caseinhydrolysate 300mg/L, inositol 2g/L, sucrose 30g/L, plant gel 3.0g/L, pH=5.8
7. subculture medium：N6 is a large amount of, and MS-Fe salt, B5 is micro, and B5 is organic, 2,4-D 2.0mg/L, proline 2800mg/
L, L-Glutamine 500mg/L, caseinhydrolysate 300mg/L, inositol 2g/L, sucrose 30g/L, plant gel 3.0g/L, pH=
5.8
8.AAM culture mediums：AA is a large amount of, and MS-Fe salt, B5 is micro, and B5 is organic, 2-morpholine ethane sulfonic acid 3.9g/L, casein ammonia
Base acid 500mg/L, inositol 2g/L, barley-sugar 30g/L PH=5.5,200 μM of acetosyringone
9. co-culture culture medium：N6 is a large amount of, and MS-Fe salt, B5 is micro, and B5 is organic, caseinhydrolysate 500mg/L, inositol 2g/
L, sucrose 30g/L, plant gel 3.0g/L, 2-morpholine ethane sulfonic acid 3.9g/L pH=5.5,100 μM of acetosyringone
10. Selective agar medium：N6 is a large amount of, and MS-Fe salt, B5 is micro, and B5 is organic, 2,4-D 2.0mg/L, proline 500mg/
L, glutamine 500mg/L, caseinhydrolysate 300mg/L, inositol 100mg/L, sucrose 30g/L, plant gel 3.0g/L pH=
5.8, carbenicillin 250mg/L, hygromycin 50mg/L
11. differential medium：N6 is a large amount of, and MS-Fe salt, B5 is micro, and B5 is organic, methyl α-naphthyl acetate 0.5mg/L, proline 500mg/
L, glutamine 500mg/L, caseinhydrolysate 300mg/L, 6-benzyl aminopurine 3mg/L, inositol 100mg/L, sucrose 30g/L,
D-sorbite 20g/L, plant gel 3.0g/L pH=5.8, carbenicillin 250mg/L, hygromycin 50mg/L
12. root media：1/2N6 is a large amount of, and MS-Fe salt, B5 is micro, sucrose 30g/L, inositol 100mg/L, agar
0.8%pH=5.8
The specific proportioning of Transgenic Rice used medium：
N6 a great number of elements (20X)
MS molysite (200X)
Na2.EDTA 7460mg
FeSO4·7H2O 5560mg
Water is added to be settled to 1000mL
B5 trace elements (100X)
B5 organic (100X)
AA a great number of elements (10X)
The molecular Biological Detection of transgenic paddy rice
1) Northern blot are analyzed
To confirm that the artificial microRNA being transferred to is overexpressed, Trizol Reagent reagents extract 3 and turn base respectively
Because of positive strain (OE-#1, OE-#2, OE-#3) total serum IgE, probe (5 ' GATATTGGCACGGCTCAATCA 3 ' of sequence is used
SEQ ID NO：3) Northern blot analyses are carried out.Concrete operations flow is with reference to the DIG High Prime of Roche companies
DNA Lebeling and Detection Starter Kit II specifications.Testing result shows that the overexpression of detection turns base
Because os-miR171b expression raises (Fig. 2).
2) Real-Time PCR are analyzed
To confirm the expression of artificial microRNA being transferred to, the transgenic paddy rice seedling transplanted to paddy field was bred into for two generations
Afterwards, Trizol Reagent reagents extract 3 transgenic positive strain (OE-#1, OE-#2, OE-#3) total serum IgEs, reverse transcription respectively
5 ' GTTGGCTCTGGTGCAGGGTCCGAGGTATTCGCACCAGAGCCAAC of primerGATATT3’(SEQ ID NO：4) it reverses
Record obtains cDNA.With 5 ' GCATCGG of sense primerTGATTGAGCCGTGCC3’(SEQ ID NO：And downstream primer 5 ' 5)
GTGCAGGGTCCGAGGT 3’(SEQ ID NO：6) real-time PCR are carried out, os-miR171b is in transgenic paddy rice for analysis
Internal expression.The result shows that expressions of the os-miR171b in transgenic paddy rice body respectively higher than compares wild type
30 times, 19 times and 39 times of rice plant (Fig. 3)
Transgenic paddy rice is to the Analysis of Resistance of stripe disease
Osa-miR171b overexpression transgenic paddy rice seedlings after two generations of breeding are subjected to RSV inoculated identifications.Concrete operations walk
It is rapid as follows：
The full Nipponbare of grain (control) and osa-miR171b overexpression 27 DEG C of vernalization 4 of transgenic paddy rice seed are chosen respectively
After it, each 30 plants of consistent young shoots of growth of selection are transplanted into seed plate.
Rice seedlings grow to 1.5-2 leaf heart stages, are transferred in virus inoculation cage.With carrying RSV small brown rice planthopper Inoculated Rices
Seedling kills small brown rice planthopper after 3-5 heads/plant, 72 hours of virus inoculation worm amount.Period catches up with worm 2 times daily, it is ensured that the rice seedlings of inoculation are equal
It is even to obtain poison.
Malicious transplantation of seedlings will be connect to crop field, carry out disease survey analysis.Statistical analysis, the results showed that osa-miR171b surpasses
The express transgenic rice death rate and incidence are significantly lower than wild type Nipponbare rice (Fig. 4,5).Non- virus inoculation turns base
Because rice seedlings are slightly short in wild type Nipponbare rice plant, and it is higher than wild type Nipponbare plant (Fig. 4,6) after virus inoculation.It says
Bright osa-miR171b overexpressions alleviate the symptom of rice infection RSV, and therefore, osa-miR171b overexpressions can improve rice pair
The tolerance of RSV.
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<110> OrganizationName :Zhejiang Academy of Agricultural Science
Application Project
-------------------
<120> Title :Applications of the rice microRNA osa-miR171b in water resistant rice stripe
<130> AppFileReference :
<140> CurrentAppNumber :
<141> CurrentFilingDate : _ - -
Sequence
--------
<213> OrganismName :Rice（Oryza sativa）
<400> PreSequenceString :
ugauugagcc gugccaauau c 21
<212> Type : RNA
<211> Length : 21
SequenceName :Rice microRNA os-miR171b sequences
SequenceDescription :
Sequence
--------
<213> OrganismName :It is artificial synthesized
<400> PreSequenceString :
gagctctttg gctgtagcag cagcagtgat tgagccgtgc caatatccag gagattcagt 60
ttgaagctgg acttcacttt tgcctctctg atattggggc ggttcaatca ttcctgctgc 120
taggctgttc ggatcc 136
<212> Type : DNA
<211> Length : 136
SequenceName :The precursor sequence of the artificial microRNA osa-miR171b of rice
SequenceDescription :
Sequence
--------
<213> OrganismName :It is artificial synthesized
<400> PreSequenceString :
gatattggca cggctcaatc a 21
<212> Type : DNA
<211> Length : 21
SequenceName :Probe
SequenceDescription :
Sequence
--------
<213> OrganismName :It is artificial synthesized
<400> PreSequenceString :
gttggctctg gtgcagggtc cgaggtattc gcaccagagc caacgatatt 50
<212> Type : DNA
<211> Length : 50
SequenceName :Reverse transcriptase primer
SequenceDescription :
Sequence
--------
<213> OrganismName :It is artificial synthesized
<400> PreSequenceString :
gcatcggtga ttgagccgtg cc 22
<212> Type : DNA
<211> Length : 22
SequenceName :Sense primer
SequenceDescription :
Sequence
--------
<213> OrganismName :It is artificial synthesized
<400> PreSequenceString :
gtgcagggtc cgaggt 16
<212> Type : DNA
<211> Length : 16
SequenceName :Downstream primer
SequenceDescription :
Claims (6)
1. one section of rice microRNA os-miR171b sequence, the RNA os-miR171b nucleotides sequences are classified as SEQ ID NO:
Shown in 1, feature exists, which can prepare tolerance rice stripe virus (Rice by transgenosis water method
Stripe virus, RSV) rice that infects.
2. a kind of method of rice for preparing tolerance rice stripe virus (Rice stripe virus, RSV) and infecting, wherein, it allows
MicroRNA nucleotides sequences are classified as SEQ ID NO:Gene order shown in 1 is transferred in rice.
3. one section of rice microRNA os-miR171b sequence is preparing tolerance rice stripe virus (Rice stripe
Virus, RSV) purposes on the rice that infects, wherein, the RNA os-miR171b are SEQ ID NO:Sequence shown in 1
Row.
4. the artificial microRNA osa-miR171b sequences of rice, the nucleotides sequence of the microRNA is classified as SEQ ID NO:2 institutes
Show, which is characterized in that the microRNA can prepare tolerance rice stripe virus (Rice stripe by transgenic method
Virus, RSV) rice that infects.
5. a kind of method of rice for preparing tolerance rice stripe virus (Rice stripe virus, RSV) and infecting, wherein, it allows
Nucleotides sequence is classified as SEQ ID NO:Gene order shown in 2 is transferred in rice.
6. the artificial microRNA osa-miR171b gene orders of rice are preparing tolerance rice stripe virus (Rice stripe
Virus, RSV) purposes in the rice that infects, wherein, the gene order is SEQ ID NO:Sequence shown in 2.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111321165A (en) * | 2020-03-13 | 2020-06-23 | 浙江大学 | Method for promoting growth and development of tomatoes |
| CN116837024A (en) * | 2023-08-09 | 2023-10-03 | 中国农业科学院烟草研究所(中国烟草总公司青州烟草研究所) | Method for carrying out gene expression by utilizing agrobacterium to transiently transform wild rice seeds |
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| CN116837024B (en) * | 2023-08-09 | 2024-02-02 | 中国农业科学院烟草研究所(中国烟草总公司青州烟草研究所) | Method for carrying out gene expression by utilizing agrobacterium to transiently transform wild rice seeds |
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