CN104059910B - A kind of regulate and control the miRNA of the anti-Rust genetic expression of Hybrid poplar - Google Patents
A kind of regulate and control the miRNA of the anti-Rust genetic expression of Hybrid poplar Download PDFInfo
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- CN104059910B CN104059910B CN201410208679.3A CN201410208679A CN104059910B CN 104059910 B CN104059910 B CN 104059910B CN 201410208679 A CN201410208679 A CN 201410208679A CN 104059910 B CN104059910 B CN 104059910B
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Abstract
Regulate and control a miRNA for the anti-Rust genetic expression of Hybrid poplar, relate to a kind of regulate and control the miRNA of anti-Rust genetic expression.The invention provides a kind of regulate and control the miRNA of the anti-Rust genetic expression of Hybrid poplar, by the research to this miRNA and target gene thereof, there is in willow breeding for disease resistance important biological significance and potential using value.Is the miRNA nucleotide sequence of the anti-Rust genetic expression of regulation and control Hybrid poplar as SEQ? ID? shown in NO:1.Is its precursor sequence as SEQ? ID? shown in NO:2.The miRNA that the present invention regulates and controls the anti-Rust genetic expression of Hybrid poplar can improve the resistance of Hybrid poplar to rust, for breeding for disease resistance.Can be the poplar adjusted and controlled Resistant Gene To Rust infection mechanism of further investigation by the research of genetic engineering means to this miRNA and improve the resistance of Hybrid poplar to rust and lay the foundation.
Description
Technical field
The present invention relates to a kind of regulate and control the miRNA of anti-Rust genetic expression.
Background technology
MicroRNAs (miRNAs) is the endogenous non-coding tiny RNA (smallRNAs) with regulate gene expression effect, they are mainly by 3 ' non-translational region (the 3 '-untranslatedregion in conjunction with target gene mRNA or target gene mRNA, 3 '-UTR) this gene mRNA or hinder it to translate of degrading, thus express at post-transcriptional level controlling gene.The miRNA found the earliest is the RNA molecule of the long 18-22nt of 3 '-UTR complementation that is that observe in nematode and target gene transcript, comprises lin-4 and let-7 gene, and the growth of nematode regulated and controled by these rna genes.Subsequently miRNA be found in comprise from nematode to fruit bat and people Various Tissues in exist, 2002 years are started to the report of Mirnas of plant.Along with modern biotechnology and molecular biological development, the discovery quantity of Mirnas of plant exponentially increases.At present, registered more than 1160 plant miRNA sequence and relevant information in miRNA database miRBase.The discovery of Mirnas of plant and the effect in growth and development of plants and stress response thereof are large focuses of molecular biology of plants research field.Large quantity research finds, Mirnas of plant s mainly relies on complete between target gene or intimate complementary pairing completely cuts target gene or Translational repression realizes its adjusting function, plays a significant role in coordinate plant growth growth, cell proliferation, biology and abiotic stress response.
Willow is important yielding timber plantationsion, has larger economic benefit and ecological benefits.The poplar rust caused by molecular study of Melampsora larici-populina (Melampsoralarici-populina) is one of Major Diseases of willow.In recent years, it is caused harm and is on the rise.Utilizing genetic engineering technique to cultivate Resistant Gene To Rust Poplar Varieties is important diseases prevention means.Utilize genetically engineered to improve willow disease resistance and depend on understanding to willow disease-resistant related gene and controlling element thereof.Plant defense responses is the combined reaction that various physiological processes regulates and controls mutually.At present about the molecular studies of the anti-Rust of willow mainly concentrate in some functional genes participating in the antimycotic infection resistance effect of willow directly and protein, and have ignored the wire-puller causing many gene differential expressions, namely the upstream regulated and control network of these defense response genes involveds is the blind spots in research.Discovered in recent years plant microRNA has important regulating and controlling effect in growth and development of plants and stress response.The Molecular regulator of upstream is sought if reverse, find " the perpetrator "-microRNA be in willow rust-proofing bacterium molecule network, and the microRNA regulation process corresponding with it to willow rust-proofing bacterium gene is furtherd investigate, maybe can disclose the complete procedure of willow gene expression regulation in Rust process and open up new road for the research of willow rust-proofing bacterium is applied to production practice.
Summary of the invention
The invention provides a kind of regulate and control the miRNA of the anti-Rust genetic expression of Hybrid poplar and application thereof.
The present invention regulates and controls the miRNA of the anti-Rust genetic expression of Hybrid poplar, and its nucleotide sequence is as shown in SEQIDNO:1.
The miRNA precursor sequence of the anti-Rust genetic expression of above-mentioned regulation and control Hybrid poplar is as shown in SEQIDNO:2.
The miRNA controllable Hybrid poplar of the anti-Rust genetic expression of above-mentioned regulation and control Hybrid poplar to the resistance of rust, for breeding for disease resistance.
The present invention builds Hybrid poplar (Populussimonii × P.nigra) the tiny RNA library that molecular study of Melampsora larici-populina (Melampsoralarici-populina) infects front and back, adopt the order-checking of Illumina sequencing technologies, obtain a new miRNA, called after Pn-5p-310376_3, this miRNA be up-regulated (log after rest fungus inoculation
2=12.64).By building degraded group, find that its target gene is NB-ARC (nucleotidebindinginAPAF-1, plantdiseaseresistancegeneproductsandCED-4) resistance protein gene (Potri.014G002000.1), shearing site is 752nt.
After Rust, expression amount raises, and shows that this miRNA works in Hybrid poplar rust-proofing bacterium infection processs.The target gene regulated and controled in view of this miRNA is NB-ARC resistance protein gene (NB-ARC resistance protein genes encoding contains the plant disease-resistant albumen of nucleotide binding site), illustrates further this miRNA and plays a key effect in willow resistance mechanism.By the research to this miRNA and target gene thereof, there is in willow breeding for disease resistance important biological significance and potential using value.
Accompanying drawing explanation
Fig. 1 is the loop-stem structure figure of the miRNA precursor sequence of the anti-Rust genetic expression of regulation and control Hybrid poplar; Fig. 2 is the electrophorogram of Hybrid poplar blade total serum IgE in embodiment 1.
Embodiment
Following embodiment is convenient to better understand 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 and equipment, if no special instructions, all bought by market and directly obtain.
The miRNA of the anti-Rust genetic expression of regulation and control Hybrid poplar provided by the invention derives from Hybrid poplar, called after Pn-5p-310376_3, its length is 21nt, its precursor sequence and be folded into a kind of stable loop-stem structure, as shown in Figure 1, the typical secondary structure of miRNA precursor is belonged to.
Embodiment 1: the acquisition of the miRNA of the anti-Rust genetic expression of regulation and control Hybrid poplar, the qualification of miRNA target gene and miRNA functional verification.
(1) Hybrid poplar RNA extracting and quality evalution:
Molecular study of Melampsora larici-populina is inoculated in Hybrid poplar blade, get inoculation molecular study of Melampsora larici-populina 2hpi, 6hpi, 12hpi, 24hpi, 3dpi, 5dpi and 7dpi totally seven groups of Hybrid poplar blades respectively, be divided into fritter, often group is got 100mg plant tissue and is placed in 1.5ml centrifuge tube through DEPC process, and it is freezing for subsequent use to be placed in rapidly liquid nitrogen.Each group of Hybrid poplar blade total serum IgE is extracted with reference to miRNeasyKit (buying from QIAGEN company) operational manual, and the RNA geometric ratio mixing that will often organize.Extract the Hybrid poplar blade total serum IgE not inoculating molecular study of Melampsora larici-populina after the same method.1% agarose gel electrophoresis detects RNA quality, result as shown in Figure 2, in Fig. 2, Rust+ represents the Hybrid poplar blade total serum IgE after inoculating rest fungus, Rust-represents the Hybrid poplar blade total serum IgE not inoculating rest fungus, electrophoretic band is clear, without trailing smear phenomenon, the brightness of 28S band is greater than 18S, and its ratio is close to 2:1.Get 1 μ lRNA sample Eppendorf spectrophotometric determination OD280, OD260 and OD230 value respectively, OD260/280 ratio is respectively at 1.8-2.0, OD260/230 ratio ﹥ 1.8 respectively.Illustrate that extracted RNA quality is better.
Described molecular study of Melampsora larici-populina (Melampsoralarici-populina) in 2003 at " QuantitativeinoculationsofpoplarswithMelampsoralarici-po pulina.EuropeanJournalofPlantPathology " (M.H.Pei, C.Ruiz, J.Harris, T.Hunter., 2003, open 109:269-276.), be so kind as to give by the author M.H.Pei of article.
(2) tiny RNA library construction and high-flux sequence:
IlluminaTruseqSmallRNAPreparationkit test kit is used (to buy from Illumina company, article No. is RS-200-0012) and the tiny RNA library inoculating willow before and after rest fungus is built respectively with reference to its specification sheets, the library IlluminaGAIIX sequenator built carries out the order-checking of the high-throughput degree of depth.
(3) degraded group checks order:
Extract the mRMA (containing polyA end) of willow before and after inoculation rest fungus, connect the RNA joint containing 3 ' MmeI site, the reverse transcription of application ThermoScript II is that cDNA (buy from Promegra company by ThermoScript II, article No. A3801), synthetic double chain cDNA, MmeI enzyme is cut, and (MmeI enzyme is bought from NEB company, article No. R0637S), connect dsDNA joint, gel purified, pcr amplification, thus complete the preparation work of whole library, the library IlluminaGAIIX sequenator built carries out the order-checking of the high-throughput degree of depth.
(4) miRNA bioinformatic analysis
Adopt ACGT101-miRv4.2 analysis software to analyze tiny RNA library sequencing result, application Blast technical Analysis filters out miRNA.
Tiny RNA library sequencing result shows, the library of inoculation rest fungus records 9,736,262 raw sequences altogether, and the library not inoculating rest fungus records 8 altogether, 840,983 raw sequences.After removing redundant sequence, the library of inoculation rest fungus records 4,070 unique sequence altogether, and the library not inoculating rest fungus records 3 altogether, 820 unique sequences.Two libraries before and after infecting obtain 5,135 uniquemiRNA altogether.
Adopt CleaveLand programanalysis degraded group data, in conjunction with the miRNA data that previous step filters out, carry out microRNA target prediction.The short reading frame calibrator of application Oligomap finds out the mRNAs matched with degraded group sequence.The valuable standard sequence of degraded group sequence is compared except de-redundancy in a database with NRPM (every 1,000,000 read).Again apply pairing site upstream 13 sequences and 13, downstream sequence that Oligomap extracts the mRNA of each accurate pairing degraded group sequence, the mRNA of a composition 26nt, Needle program in application EMBOSS bag draws and all sequences that sequence in the tiny RNA storehouse provided matches, then according to Mirnas of plant/target pairing standard, array is marked.
To degraded group carry out analysis and show, altogether obtain 8,409,874raw sequence, wherein 5,077,704uniquereads can find corresponding sequence in willow mRNA database.Analyze further combined with miRNA Data Data, Isosorbide-5-Nitrae 75miRNAs can find the target gene of its correspondence.Respectively BLAST analysis is carried out to these target genes, found that: the target gene of miRNAPn-5p-310376_3 is NB-ARC (nucleotidebindinginAPAF-1, plantdiseaseresistancegeneproductsandCED-4) resistance protein gene (Potri.014G002000.1), shearing site is 752nt.
Tiny RNA library sequencing result relatively before and after inoculation is known, and this miRNAPn-5p-310376_3 (reads=0) expression amount before rest fungus inoculation rear (reads=6) is than inoculation significantly raises (log
2=12.64).
Claims (2)
1. regulate and control a miRNA for the anti-Rust genetic expression of Hybrid poplar, it is characterized in that its nucleotide sequence is as shown in SEQIDNO:1.
2. the precursor sequence of the miRNA of the anti-Rust genetic expression of regulation and control Hybrid poplar as claimed in claim 1, is characterized in that its sequence is as shown in SEQIDNO:2.
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| WO2008115487A1 (en) * | 2007-03-16 | 2008-09-25 | New York University | Methods of affecting plant growth with microrna |
| WO2011067745A3 (en) * | 2009-12-06 | 2011-08-04 | Rosetta Green Ltd. | Compositions and methods for enhancing plants resistance to abiotic stress |
| CN103270158A (en) * | 2010-10-15 | 2013-08-28 | 昆士兰大学 | Small Viral RNA Molecules And Uses Thereof |
| CN103361346A (en) * | 2012-03-26 | 2013-10-23 | 北京林业大学 | Method for cloning and analyzing populus diversifolia micro RNAs (ribonucleic acids) precursor |
| CN103589721A (en) * | 2012-08-15 | 2014-02-19 | 北京命码生科科技有限公司 | Extraction, preparation and application of plant micro ribonucleic acid |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| WO2008115487A1 (en) * | 2007-03-16 | 2008-09-25 | New York University | Methods of affecting plant growth with microrna |
| WO2011067745A3 (en) * | 2009-12-06 | 2011-08-04 | Rosetta Green Ltd. | Compositions and methods for enhancing plants resistance to abiotic stress |
| CN103270158A (en) * | 2010-10-15 | 2013-08-28 | 昆士兰大学 | Small Viral RNA Molecules And Uses Thereof |
| CN103361346A (en) * | 2012-03-26 | 2013-10-23 | 北京林业大学 | Method for cloning and analyzing populus diversifolia micro RNAs (ribonucleic acids) precursor |
| CN103589721A (en) * | 2012-08-15 | 2014-02-19 | 北京命码生科科技有限公司 | Extraction, preparation and application of plant micro ribonucleic acid |
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