CN105112423A - Clone of miRNA for enhancing disease resistance of mulberry and application thereof - Google Patents
Clone of miRNA for enhancing disease resistance of mulberry and application thereof Download PDFInfo
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- CN105112423A CN105112423A CN201510589342.6A CN201510589342A CN105112423A CN 105112423 A CN105112423 A CN 105112423A CN 201510589342 A CN201510589342 A CN 201510589342A CN 105112423 A CN105112423 A CN 105112423A
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Abstract
The invention relates to the technical field of genetic engineering, and provides a clone of miRNA for enhancing the disease resistance of a mulberry and an application thereof. The miRNA is named mul-miRn2 and is provided with an RNA sequence represented in SEQ. ID. NO. 1 in a sequence table. The precursor RNA and the encoding gene sequence of the miRNA are provided with nucleotide sequences shown in SEQ. ID. NO. 2 and SEQ. ID. NO. 3 respectively. The miRNA shown in the SEQ. ID. NO. 1 is over-expressed in the mulberry, and a transgene mulberry which has high resistance to a mulberry yellow dwarf disease and mulberry epidemic diseases can be cultivated. The mul-miRn2 provides a novel gene resource for cultivating a novel disease-resistant mulberry variety, and the gene can be used for disease-resistant seed breeding study of the mulberry and other plants.
Description
Technical field
The present invention relates to gene engineering technology field, provide a kind of clone and the application thereof that strengthen the miRNA of mulberry tree resistance against diseases.
Background technology
Mulberry tree has thousands of years cultivation histories in China, and mulberry tree, as the feed seeds of silkworm, is the important substance basis of silk industry.Mulberry tree is except can breeding silkworms for picking leaves, and mulberry leaf, mulberry fruit, ramulus mori, mulberry skin, Sang Gen also have very high edible, medicinal and feeding value.In addition, mulberry tree as ecology tree kind defending and controlling sand, Rocky Desertification Control, soil conservation, Saline-alkali Field Control, also played vital role in conceding the land to forestry etc., its economy and the ecological value more and more come into one's own.Need to cut mulberry tree to breed silkworms in sericultural production to cut down picking leaves, lasting cutting is cut down picking leaves and is easily caused tree vigo(u)r to decay, and increases the risk being subject to disease and pest and infecting.The mulberry tree disease that China has found has more than 100 to plant, and can cause harm about 40 kinds that cause disaster.Wherein mulberry tree yellow dwarf disease and mulberry epidemic disease are the important diseases of harm mulberry tree always, generation is had in Ge Can district of China, often cause the reduction of mulberry yield and quality, the development of sericulture there is had a negative impact, also limit the realization of medicinal, the feeding and ecological value of mulberry tree simultaneously.Because silkworm is responsive to agricultural chemicals, conventional agricultural chemicals is difficult to use in mulberry field, and therefore, cultivating broad-spectrum disease resistance new variety is one of fundamental ways addressed this problem.
In order to reduce the injury caused by environment-stress, plant has developed complicated response mechanism during evolution, can the expression of regulatory gene in level after transcribing, transcribing and after translation, strengthens self resistance.MiRNAs in plant materials as the important regulating and controlling factor of genetic expression, can transcribe with post-transcriptional level on regulate genetic expression, its function almost relate to grow, all respects of the life process such as signal transduction and environmental response.Pathogenic bacteria can induce host to produce a large amount of microRNA in the process infecting plant, wherein a part is proved to be miRNA, plant also can resist the invasion of germ by the miRNA key gene regulated and controled in some disease-resistant approach, in the defence of plant basis and biotic response, have important effect.MiR393 is first and is reported in the effective miRNA of tool, this miRNA in pathogenic associated molecular pattern triggering immune process and can improves by Developing restraint element signal path the resistivity that the external derived bacterium of plant infects.Although at present both at home and abroad from various plants, clone obtains multiple miR-96 gene and is applied to molecular breeding and has multinomial achievement in research application patent, but " blade specific expresses the method that artificial microRNA improves Rice Resistant To Bacterial Blight " (the Chinese patent CN101565701A) of the invention such as the rarely seen Chen Hao of having of patent of the miRNA relevant to disease resistance of plant, disclose the microRNA sequence of the engineer of a 21nt, and show that this miRNA expression specifically in rice leaf can strengthen the resistance of transgenic paddy rice to bacterial leaf-blight." cultivating the method for disease resisting rice and dedicated carrier thereof and promotor " (the Chinese patent CN103194484A) of the invention such as Fang Rongxiang, disclose a kind of cultivate disease resisting rice method and dedicated carrier and promotor, the encoding gene of miR393 and the inducible promoter that drives it to express are imported object plant, obtains, to bacterial blight of rice, there are more resistance transgenic plant." suppressing the artificial mi RNA of plant virus and structure thereof and purposes " (the Chinese patent CN102559666A) of Sun Chuanbao and He Yuke invention, disclose and have devised the oligonucleotide of silencing efficiency excellence according to the nucleotide sequence of RNA silencing suppressor in the virus infecting plant, these oligonucleotide import to after in plant materials, miRNA can be formed in plant materials when virus infection plant, be combined with the mRNA of virogene silencing suppressors, affect transcribing of mRNA, thus reach the effect suppressing virus." utilizing artificial microRNA to improve method and the special-purpose double-chain RNA thereof of Rice Resistance black streak dwarf " (the Chinese patent CN102676510A) of the invention such as Wang Xingjun, discloses a kind of method and the special-purpose double-chain RNA thereof that utilize artificial mi RNA raising Rice Resistance black streak dwarf.But about the effect of miRNA in mulberry tree breeding for disease resistance, yet there are no report.
Summary of the invention
The present inventor is for the situation of above-mentioned prior art, provide a kind of clone and the application thereof that strengthen the miRNA of mulberry tree resistance against diseases, this miRNA called after mul-miRn2, it has the RNA sequence in sequence table shown in SEQ.ID.NO.1, and its precursor RNA and coding gene sequence have the nucleotide sequence in sequence table shown in SEQ.ID.NO.2 and SEQ.ID.NO.3 respectively.The miRNA shown in process LAN SEQ.ID.NO.1 in mulberry tree, can cultivate and have more resistance transgenosis mulberry tree to mulberry yellow dwarf disease and mulberry epidemic disease; Mul-miRn2 of the present invention cultivates disease-resistant New Mulberry Variety to provide new genetic resources, and this gene can be used for mulberry tree or the breeding research of other plant disease resistance.
Contriver provide firstly a kind of miRNA strengthening mulberry tree resistance against diseases, this miRNA called after mul-miRn2, and it has the RNA sequence in sequence table shown in SEQ.ID.NO.1;
Its precursor RNA has the nucleotide sequence in sequence table shown in SEQ.ID.NO.2;
Its coding gene sequence is the nucleotide sequence shown in SEQ.ID.NO.3;
Except the nucleotide sequence such as shown in SEQ.ID.NO.3, also comprise and can encode the miRNA precursor RNA sequence shown in SEQ.ID.NO.2 and with sequence shown in SEQ.ID.NO.3, there is the nucleotide sequence of more than 90% similarity in plant materials; Can be transcribed into miRNA precursor after this sequence is imported into vegetable cell, described miRNA precursor can be sheared and be processed into ripe miRNA, therefore also at the row of protection scope of the present invention in vegetable cell.
The present invention constructs the overexpression vector pBI121-mul-miRn2 of mulberry tree mul-miRn2 gene, this expression vector is proceeded to Agrobacterium GV3101 competent cell, screening transformant, adopts leaf disc transformation method to transform mulberry tree.Carry out Disease-resistance Analysis to the transgenosis mulberry tree plant obtained to show, transgenosis mulberry tree plant can significantly improve the resistance to yellow dwarf disease and mulberry epidemic disease, has a good application prospect.
The present invention relates to a kind of plant expression vector, include the nucleotide sequence as shown in SEQ.ID.NO.1 or SEQ.ID.NO.2 or SEQ.ID.NO.3, for improving the resistance of mulberry tree to yellow dwarf disease and mulberry epidemic disease.
The invention provides the method that mulberry tree mul-miRn2 gene is applied in plant, the resistance against diseases of plant can be improved.Step is:
1) mul-miRn2 gene order is placed under CaMV35S strong promoter, builds plant expression vector pBI121-mul-miRn2;
2) the expression vector pBI121-mul-miRn2 transformation Agrobacterium competent cell will built, filters out transformant;
3) 2 are utilized) transformant that obtains transforms mulberry tree, obtains turning mul-miRn2 gene mulberry tree.
Based on above-mentioned discovery, contriver, further by genetic engineering technique overexpression mul-miRn2 gene in mulberry tree, can cultivate and have more resistance transgenosis mulberry tree to mulberry yellow dwarf disease and mulberry epidemic disease; Mul-miRn2 of the present invention cultivates disease-resistant New Mulberry Variety to provide new genetic resources, and this gene can be used for mulberry tree or the breeding research of other plant disease resistance.
Accompanying drawing explanation
Fig. 1 is the secondary structure figure of mul-miRn2 precursor sequence,
MiRNA provided by the invention derives from mulberry tree, called after mul-miRn2, and its precursor sequence can be 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 design of graphics of mulberry tree mul-miRn2 gene plant overexpression vector pBI121mul-miRn2;
Fig. 3 is the electrophoretogram of overexpression vector pBI121mul-miRn2 through BamHI and Sac Ι double digestion,
M:MarkerDL2000 in figure; P: digestion products,
Mulberry tree mul-miRn2 gene nucleotide series is inserted in plant expression vector pBI121 by we as seen from the figure, successfully constructs mul-miRn2 gene plant overexpression vector;
Fig. 4 is the PCR qualification result schematic diagram of transfer-gen plant,
WT in figure: wild-type mulberry tree contrasts; L1-L5: the positive strain of transgenosis mulberry tree; M:MarkerDL2000;
With the DNA of the positive plant filtered out for template, the pcr amplification of corresponding transgenic line is carried out according to 35S promoter sequences Design a pair special primer, result all can increase and arrive about 530bp band, illustrate that mul-miRn2 gene successfully proceeds in mulberry tree body by we, and obtain transfer-gen plant;
Fig. 5 is the detection of expression result histogram of the ripe body of mul-miRn2 in transfer-gen plant,
WT in figure: wild-type mulberry tree contrasts; L1-L5: the positive strain of transgenosis mulberry tree,
In transgenosis mulberry tree plant, the gene expression abundance of the ripe body of mul-miRn2, apparently higher than WT lines, illustrates that the ripe body of mul-miRn2 is able to high expression in transgenosis mulberry tree as seen from the figure.
Embodiment
The present invention is defined further in following examples, according to above description and these embodiments, those skilled in the art can determine essential characteristic of the present invention, and when not departing from spirit and scope of the invention, various change and amendment can be made, to make its applicable various uses and condition to the present invention.Except special indicating, be of the present inventionly state of the art;
The cloning process of embodiment 1. mulberry tree mul-miRn2 gene
1, conventional CTAB method is adopted to extract mulberry leaf DNA.
2, according to the nucleotide sequence of transcript profile order-checking gained mul-miRn2 gene, design a pair special primer, miRn2-5 ' (GTCTTTCAACAAAAACTGCCTTG) its nucleotide sequence as shown in SEQ.ID.NO.4 and miRn2-3 ' (TAGGATTAGGATTTCACTCTCT) its nucleotide sequence as shown in SEQ.ID.NO.5, with mulberry leaf DNA for template carries out pcr amplification, system is as follows:
Response procedures is as follows:
The agarose gel electrophoresis that reaction terminates rear use 1% detects PCR primer, reclaim object fragment and carry out Clone and sequence analysis, sequencing result shows its nucleotide sequence as shown in SEQ.ID.NO.3, this sequence can be folded into the typical loop-stem structure of miRNA precursor (as shown in Figure 1), meet the constitutional features of miRNA precursor, show we successful clone obtain mul-miRn2 gene.
The structure of embodiment 2. mulberry tree mul-miRn2 plant expression vector pBI121mul-miRn2
1, according to the nucleotide sequence of isolated mulberry tree mul-miRn2 gene, design primer miRn2-F (GGATCCGTCTTTCAACAAAAACTG) its nucleotide sequence as shown in SEQ.ID.NO.6 and miRn2-R (GAGCTCTAGGATTAGGATTTCACTCTCT) its nucleotide sequence as shown in SEQ.ID.NO.7, the DNA extracted with mulberry leaf, for template, carries out pcr amplification.
2, get PCR primer to be connected with pMDl8-TSimple cloning vector, connect product conversion bacillus coli DH 5 alpha competent cell, penbritin (50mg/L) resistance screening positive colony, bacterium liquid PCR extracts recombinant plasmid dna after identifying, enzyme cuts the laggard row sequencing of qualification.
3, the recombinant plasmid BamHI and Sac Ι enzyme that contain the correct fragment of mulberry tree mul-miRn2 gene through sequencing are cut, reclaim mul-miRn2 gene fragment and connect (as shown in Figure 2) with the pBI121 expression vector of identical digestion with restriction enzyme.Connect product conversion bacillus coli DH 5 alpha competent cell, kantlex (50mg/L) resistance screening positive colony, the enzyme positive colony chosen being carried out to bacterium liquid PCR qualification and plasmid DNA cuts qualification (as shown in Figure 3).
4, the mulberry tree mul-miRn2 gene plant expression vector transformation Agrobacterium GV3101 competent cell adopting freeze-thaw method to build, kantlex (50mg/L) resistance screening positive colony.
The acquisition of embodiment 3. transgenosis mulberry tree plant
1, picking Agrobacterium (the single bacterium colony of the Agrobacterium of carrying recombinant plasmid obtained in embodiment 2) is inoculated in the LB liquid nutrient medium containing kantlex 50mg/L, 28 DEG C, 250rpm, and shaking culture is about 48h to the logarithmic growth later stage; Bacterium liquid MS nutrient solution is diluted 10 times, for subsequent use;
2, get mulberry tree tests for sterility, be cut into small pieces (0.5 × 0.5cm), as explant, is placed in the pre-division culture medium of MS (MS+TDZ1.0mg/L+NAA0.2mg/L), 28 DEG C, light application time 16h/d, intensity of illumination 2000Lux, preculture 2d;
3, the explant after preculture is immersed bacterium liquid 2-l0min, then blot unnecessary bacterium liquid with the filter paper of sterilizing, access MS minimum medium; Under the low light level, 28 DEG C of Dual culture 2d;
4, the explant after Dual culture first washs 3 times with the sterilized water containing cephalo penicillin 250mg/L, 1 time is washed again with the MS nutrient solution containing cephalo penicillin 250mg/L, then blot with aseptic filter paper, proceed to containing kantlex 100mg/L, on the MS Selective agar medium (MS+6-BA3.0mg/L+NAA0.2mg/L+ kantlex 50mg/L+ cephalo penicillin 250mg/L) of cephalo penicillin 250mg/L, 28 DEG C, light application time 16h/d, intensity of illumination 2000Lux cultivate; Every 15d switching once.
5, when Bud Differentiation grows to about lcm, cut Bud Differentiation and proceed in MS root media (MS+ kantlex 50mg/L+ cephalo penicillin 250mg/L), short its is taken root.
6, when differentiation seedling grows to 5-6 sheet leaf, after root system development is good, moves into and fill in the flowerpot of sterile soil, room temperature Routine Management.
The PCR qualification of embodiment 4. transgenosis mulberry tree plant
1, CTAB method is adopted to extract the DNA of resistant plant and WT lines.
2, according to 35S promoter sequences Design a pair its nucleotide sequence of special primer 35S-5 (GGCCATGGAGTCAAAGATTC) as shown in SEQ.ID.NO.8 and 35S-3 (CCGTGTTCTCCAAATG) its nucleotide sequence as shown in SEQ.ID.NO.9, respectively with the resistant plant extracted and the DNA of WT lines for template, carry out PCR, amplification 35S promoter, reaction system is as follows:
Response procedures is as follows:
The agarose gel electrophoresis that reaction terminates rear use 1% detects PCR primer.By agarose gel electrophoresis detected result, as shown in Figure 4, all can amplify the band of an about 530bp in resistant plant genomic dna, and WT lines genomic dna fails to amplify this band, show us successfully by mul-miRn2 channel genes in mulberry tree genome.
The detection of expression of the ripe body of embodiment 5.mul-miRn2 in transgenosis mulberry tree
1, the expression amount of method to the ripe body of mul-miRn2 in transgenosis mulberry tree of quantitative fluorescent PCR is adopted to detect.
2, extract the total serum IgE of wild-type and transgenosis mulberry tree by Trizol method, use DNaseI to remove genomic dna, with the OneStep of precious biotechnology company limited
miRNAcDNASynthesisKit carries out the reverse transcription of miRNA.
3, with mulberry tree U6 for reference gene, with the reverse transcription product of wild-type and transgenosis mulberry tree miRNAs for template, reaction system and reactions steps reference
premixExTaq
tMiI specification sheets reacts, and maps according to reaction result Excel, analyzes the ripe body of mulberry tree mul-miRn2 success in transgenosis mulberry tree and expresses and gene expression abundance.The ripe body of mul-miRn2 is all able to high expression in transgenosis mulberry tree as seen from Figure 5, show the mulberry tree mul-miRn2 gene that proceeds to can in mulberry tree overexpression, and be successfully processed to form ripe body.
Embodiment 6. transgenosis mulberry tree is to the Analysis of Resistance of yellow dwarf disease
1, with the annotinous branch of transgenosis mulberry tree and wild-type mulberry tree for scion, be stock grafting respectively with the seedling of wild-type Mulberry Seeds breeding, obtain Graft.
2, between the 7-8 month, choose the sick mulberry branch of yellow dwarf disease, first from base portion, twist skin zone, then at the upper and lower girdling cortex of fringe bud, obtain pipe of buddering, then sick fringe germ tube is socketed in the Graft of above-mentioned acquisition.Grafting " Invest, Then Investigate " in January sickness rate.
3, sickness rate finds that transfer-gen plant sickness rate is 12% by inquiry, and WT lines sickness rate is 85%, and it is heavier to fall ill.Therefore, transgenosis mulberry tree has obvious anti-mulberry tree resisting etiolation type wilt disease ability relative to wild-type mulberry tree, has good application prospect.
Embodiment 7 transgenosis mulberry tree anti-mulberry epidemic disease capability analysis
1, picking pseudomonas syringae bacterial strain [Pseudomonassyringaepv.mori (BoyeretLambert) Young, DyeetWikie, 1978] be inoculated in LB liquid nutrient medium, 30 DEG C, 180rpm, shaking culture is about 24h to the logarithmic growth later stage; Bacterium liquid LB nutrient solution is diluted to 3 × 10
8cFU/mL, for subsequent use.
2, inoculate processing selecting to carry out in the mulberry tree vigorous period dusk in summer, mulberry branch apical growth vigorous and the spire launched not yet completely and the tender tip is broken with acupuncture, eachly repeat thorn 6 tender tips, with miniaturised nebuliser spraying bacterium after thorn, squirt as degree to push up the positive back side of tip 2-3 sheet leaf, use plastics bag moisturizing after spray, m seq removes plastics bag.3 repetitions are all established in each process.Investigate incidence after 7 days.
3, sickness rate finds by inquiry, and transfer-gen plant sickness rate is 8%, and morbidity is comparatively light, and WT lines sickness rate is 94%, and it is heavier to fall ill.Therefore, transgenosis mulberry tree has stronger anti-mulberry epidemic disease ability relative to wild-type mulberry tree, has good application prospect.
Claims (4)
1. strengthen a miRNA for mulberry tree resistance against diseases, it is characterized in that: this miRNA called after mul-miRn2, it has the RNA sequence in sequence table shown in SEQ.ID.NO.1.
2. miRNA according to claim 1, is characterized in that: its precursor RNA sequence is as shown in SEQ.ID.NO.2.
3. miRNA according to claim 1, is characterized in that: its coding gene sequence is the nucleotide sequence shown in SEQ.ID.NO.3.
4. miRNA according to claim 1 strengthens the application of mulberry tree to the resistance of mulberry tree yellow dwarf disease and mulberry epidemic disease in mulberry tree.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN111849989A (en) * | 2020-07-30 | 2020-10-30 | 山东农业大学 | Method for enhancing salt tolerance of mulberry by utilizing long-chain non-coding RNA transgenic rootstock |
| CN113136393A (en) * | 2021-06-04 | 2021-07-20 | 中国水稻研究所 | OsmiR398b interference fragment of rice cadmium-tolerant protein OsCSD2 and application thereof |
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| CN103320467A (en) * | 2013-06-06 | 2013-09-25 | 江苏省农业科学院 | Application of GrVe gene providing plant with verticillium wilt resistance |
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111849989A (en) * | 2020-07-30 | 2020-10-30 | 山东农业大学 | Method for enhancing salt tolerance of mulberry by utilizing long-chain non-coding RNA transgenic rootstock |
| CN111849989B (en) * | 2020-07-30 | 2021-11-23 | 山东农业大学 | Method for enhancing salt tolerance of mulberry by utilizing long-chain non-coding RNA transgenic rootstock |
| CN113136393A (en) * | 2021-06-04 | 2021-07-20 | 中国水稻研究所 | OsmiR398b interference fragment of rice cadmium-tolerant protein OsCSD2 and application thereof |
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