CN104830859B - A kind of tea tree miRNA and its application - Google Patents
A kind of tea tree miRNA and its application Download PDFInfo
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- CN104830859B CN104830859B CN201510201694.XA CN201510201694A CN104830859B CN 104830859 B CN104830859 B CN 104830859B CN 201510201694 A CN201510201694 A CN 201510201694A CN 104830859 B CN104830859 B CN 104830859B
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Abstract
The invention discloses a kind of tea tree miRNA and its application, the base sequence of the tea tree miRNA is as shown in SEQ ID NO.1.The present invention passes through Solexa high throughput sequencing technologies, the technologies such as bioinformatic analysis, miR156 and its precursor Cs miR156g is identified from tea tree Zhenong 139 first, and the differential expression of miR156 under the conditions of different nitrogen is demonstrated by different nitrogen processing, draw catechin total amount and the conclusion that miR156 is in notable negative correlativing relation, prove that tea tree miRNA (miR156) of the present invention and its precursor Cs miR156g play an important role in regulation and control tree plant catechin expression, tea tree miRNA (miR156) and its precursor Cs miR156g expression can suppress the synthesis of catechin in tea tree, there is potential using value in the cultivation of high-quality tea kind.
Description
Technical field
The present invention relates to botany technical field, more particularly to a kind of tea tree miRNA and its application.
Background technology
Tealeaves is the necessity in many people's daily lifes, is deeply loved by the public.During Tea Polyphenols is tea leaf quality composition
One of most important chemical composition, accounts for the 18~36% of dry weight of tea leaves, including phenolic acid, flavodic acid, flavan-3-alcohol (catechu
Element), flavones, anthocyanidin and OPC etc., be the important factor in order for improving tea leaf quality.Among these, catechin is again
One of main component of tea leaf quality and health efficacy is determined, the synthesis of catechin is by factors such as illumination, temperature, soil environments
Influence, nitrogen is as one of mostly important nutrient of tea tree, and the catechin synthesis to tea tree also has a major impact.Mesh
Before, cloned from tea tree and obtained the related structural gene of catechin synthesis, such as CHS, DFR, ANR etc.；At the same time, people is studied
Member has found in succession and identifies the transcription factors such as many related controlling gene such as MYB, bHLH, WD40.
The non-coding RNA that microRNA (miRNA) is made up of 18~25 nucleotides, is transcribed by Matrix attachment region first,
Form the primary miRNA (pri-miRNA) with cap-like structure and polya tail.Pri-miRNA through nuclease Drosha at
The premise miRNA (pre-miRNA) of the about 70nt section containing stem ring is formed after reason, maturation is cut into through nuclease again thereafter
miRNA.MiRNA is non-fully matched by base to be combined with the UTR sequences of target gene mRNA 3 ', guiding silencing complex (RNA-
Induced silencing complex, RISC) degraded mRNA or suppress mRNA translation so that the expression of controlling gene.One
Individual miRNA can combine multiple target genes, and a mRNA also can jointly be adjusted by multiple miRNA.This complicated regulating networks
Both the expression of multiple genes can be regulated and controled by a miRNA, can also be by several miRNA combination come finely regulating
The expression of individual gene.
Now, the research for Mirnas of plant and its biological function has become focus.At present, there are 193 things
Kind, miRNA and 25141 maturation miRNA of 21264 precursors is embodied in the (http of miRBase 19.0：//
www.mirbase.org/release 19：, and research object focuses primarily upon woody plant arabidopsis and grass family 2012.8)
Paddy rice, corn, it is less for the report of xylophyta, and be found more in miRBase on the miRNA sequence information of tea tree
It is few.Up to the present, only find to be predicted miRNA analysis and by traditional Direct Cloning side based on tea tree est sequence
Method identifies 6 distinctive miRNA of tea tree (Prashant Mohanpuria Sudesh Kumar
Yadav.Characterization of novel small RNAs from tea.(Camellia sinensis L.)
.Mol Biol Rep.2012,39：3977-3986).
Therefore, it is necessary to further excavate and identify the feature microRNA in tea tree, it can especially regulate and control catechu
The microRNA of cellulose content, so as to regulate and control catechin content on a molecular scale, adapts to different nitrogen conditions, improves tealeaves product
Matter.
The content of the invention
The invention provides a kind of tea tree miRNA and its application, the miRNA can regulate and control the content of catechin in tea tree,
It can be applied to the improvement of tea tree tea leaf quality.
A kind of tea tree miRNA, its base sequence is as shown in SEQ ID NO.1.
The base sequence of above-mentioned tea tree miRNA precursor is as shown in SEQ ID NO.2.Encode before above-mentioned tea tree miRNA
The DNA fragmentation of body, base sequence is as shown in SEQ IDNO.3.
Tea tree miRNA of the present invention can suppress the expression of SPL transcription factor family member genes, and then influence catechin
Expression quantity, the NCBI numbers of logging in of the est sequence of the SPL transcription factors are HP74756, HP73470 and HP74007.
Suppressing the application during tree plant catechin is expressed and described tea present invention also offers described tea tree miRNA
Set application of the miRNA precursor in tree plant catechin expression is suppressed.
The device have the advantages that：
The present invention is by technologies such as Solexa high throughput sequencing technologies, bioinformatic analysis, first from tea tree Zhenong 139
In identify miR156 and its precursor Cs-miR156g, and handled by different nitrogen and demonstrate miR156 under the conditions of different nitrogen
Differential expression, it is in the conclusion of notable negative correlativing relation to draw catechin total amount and miR156, it was demonstrated that tea tree miRNA of the present invention
(miR156) and its precursor Cs-miR156g regulation and control tree plant catechin expression in play an important role, tea tree miRNA
(miR156) and its precursor Cs-miR156g expression can suppress the synthesis of catechin in tea tree, in the cultivation of high-quality tea kind
In have potential using value.
Brief description of the drawings
Fig. 1 is tea tree miR156 and arabidopsis miR156 gene family members affiliation.
Fig. 2 is miR156 precursors Cs-miR156g secondary structure.
Fig. 3 is the expression quantity situation of change of And Development of Tea Shoot Cs-miR156g under the conditions of different nitrogen.
Fig. 4 is target gene (the NCBI numbers of logging in of the est sequence of target gene under the conditions of different nitrogen：HP74756,
HP73470, HP74007) expression quantity situation of change.
Embodiment
With reference to specific embodiment, the invention will be further elaborated, but institute's protection domain of the present invention is not limited
In this.
Experimental method in the following example, is this area conventional method unless otherwise specified.Material used, examination
Agent etc., it is unless otherwise specified, commercially middle to obtain.
The miRNA of embodiment 1 screening and identification
1st, the preparation of vegetable material and sample
The annual cuttage seeding of tea tree (Camellia sinensis) Zhenong 139 kind is taken, artificial climate greenhouse reclaimed water is placed in
Training, plucks after the two leaves and a bud of tea tree seedling, liquid nitrogen flash freezer, is freezed at -70 DEG C standby.
2nd, tea tree miRNA high-flux sequence
Tea tree RNA is extracted using Trizol methods：
1) And Development of Tea Shoot 0.1g or so is taken, rapid liquid feeding chilled nitrogen grinds to form fine-powdered；
2) powder of grinding is filled in the 1.5ml centrifuge tubes handled through DEPC, adds 1000 μ l Trizol reagents, fill
Divide and mix, 5min is placed on ice；
3) 200 μ l chloroforms are added, sample tube cover is covered tightly, firmly shakes and put 15s, it is fully mixed, 5min, 4 DEG C is stood
12000r/min centrifuges 15min；
4) the careful μ l of upper strata aqueous phase 500 that draw are transferred in new centrifuge tube, are added isometric isopropanol, are gently overturned up and down
10 times, room temperature places 10min, and 4 DEG C of 12000r/min centrifuge 10min；
5) supernatant is carefully outwelled, precipitation is left and taken, 1ml is added and now washs RNA once with the vibration of 75% ethanol, 4 DEG C of 7500r/
Min centrifuges 5min；
6) fall ethanol, the centrifuge tube for leaving precipitation is placed in super-clean bench and blows 10min, add 30 μ l DEPC treated
ddH2O dissolves；
7) by micro-spectrophotometer and agarose gel electrophoresis Detection and Extraction RNA mass, RNA OD260/ are extracted
OD280 is 1.8 or so, and electrophoresis showed 28S and 18S band are clear, no degraded, shows that extraction RNA is up-to-standard.Quality is extracted to close
The RNA of lattice is used to build miRNA libraries, and the structure in miRNA libraries is according to Illumina Sample Srepration
Protocol library constructing methods are carried out, and then, using Solexa high-flux sequences, transfer to Lian Chuan Bioisystech Co., Ltd complete
Into 18~30nt of acquisition miRNA sequence.
3rd, miRNA identification
Solexa sequencings are obtained after raw reads data, and biological information is carried out to the initial data obtained by Solexa sequencings
Credit is analysed, first by the way that the original series obtained in step (2) go with joint, low quality is gone, depollutes, removes carrier sequence
Deng processing, the clean sequence available for subsequent analysis is obtained；Then, resulting clean sequence is passed through into associated databases
MiRBase is compared, and is compared and analyzed using species homologies, the sequence of 2 is not more than with other plant material micrRNA mispairing
Row；It is subjected to BLAST with existing tea tree transcript profile database again, the tea tree est sequence compared is subjected to secondary structure
Analysis, the sequence may be considered tea tree if it can form the stable loop-stem structure of similar miRNA precursors (pre-miRNA)
miRNA.With it, identifying the miR156 of tea tree, its mature sequence is：GUGACAGAAGAGAGUGAGCAC (such as SEQ
Shown in IDNO.1).
4th, the prediction of mi156 target genes
Using on-line prediction software program psRNATarget (http：//plantgr.nole.org/psRNATarget/)
The target gene of tea tree is predicted and annotates, the NCBI numbers of logging in for obtaining the corresponding target genes of miR156 are：HP74756, HP73470,
HP74007 three est sequences.
The clone of the tea tree Cs-miR156g precursor sequences of embodiment 2
1st, the preparation of vegetable material and sample
The annual cuttage seeding of tea tree (Camellia sinensis) Zhenong 139 kind is taken, artificial climate greenhouse reclaimed water is placed in
Training, is plucked after the two leaves and a bud of tea tree, liquid nitrogen flash freezer, and being placed in -70 DEG C is used for DNA extraction.
2nd, the extraction of plant genome DNA
Tea tree genomic DNA is extracted using CTAB methods：
1) tea plant material 0.1g or so is taken, mortar liquid nitrogen grinding is put into powder, goes to 1.5ml centrifuge tubes；
2) it is gently agitated for, 65 DEG C of water-bath 1h (every 10min, being gently agitated for)
3) it is cooled to after room temperature, plus isometric chloroform/isoamyl alcohol, jog is mixed, 5min, 10000r/min centrifugations is stood
10min；
4) clear liquid is sucted in new centrifuge tube, plus isometric chloroform/isoamyl alcohol, repeat step 3)；
5) clear liquid is sucted, plus isometric isopropanol is mixed, -20 DEG C of standing 1h；
6) 5000r/min centrifuges 5min, collects precipitation；
7) DNA is washed with 70% ethanol 3 times, centrifuge tube is inverted blotting paper, and DNA is dried, plus 200 μ l ddH2O dissolves；
8) micro-spectrophotometer and agarose gel electrophoresis Detection and Extraction genomic DNA quality.Extracted DNA
OD260/OD280 is 2.0 or so, and electrophoresis has clear band.
3rd, the amplification of miR156 precursor sequences
To extract qualified genomic DNA as template, according to the miR156 precursor sequences of Bioinformatics Prediction, primer is designed
It is as follows：
Sense primer F1：TTTGAAGGGTAAGAGAGGTGAC；
Anti-sense primer R1：AGAGAGAGGAAGAGAGAATGGTT, prepares PCR reaction systems (50 μ l)：
Reaction condition：94 DEG C of pre-degenerations 4min, 94 DEG C of denaturation 30s, 58 DEG C of annealing 30s, 72 DEG C of extension 30s, 30 circulate,
72 DEG C of extension 7min.
Product is detected with 2% agarose gel electrophoresis, gel extraction sequencing is carried out to PCR primer, so as to obtain tea tree
MiR156 precursor sequence, as shown in SEQ ID NO.2.
4th, the analysis of miR156 sequence informations
MiR156 precursor sequence can form stable stem ring secondary structure (Fig. 2), with arabidopsis miR156 family members
MiR156g very high homologies (Fig. 1), therefore, be named as Cs-miR156g.
Applications of the tea tree Cs-miR156g of embodiment 3 in regulation and control tree plant catechin synthesis
1st, tea plant material and treatment conditions
Using the annual cuttage seeding of Zhenong 139, after greenhouse preculture January, nitrogen processing is carried out, sets the N concentration to be respectively
2mM NH4 +, 2mM NO3 -, 2mM NH4 +And NO3 -(NH4 +∶NO3 --1) and zero nitrogen, 4 kinds for the treatment of conditions=1:, collection tea tree is new
Tip two leaves and a bud, is divided into two parts, portion is used to determine catechin content, another is used to detect gene expression amount.
2nd, the method that HPLC determines tree plant catechin content
Carried out using national standard GB/T 8313-2008 methods after the measure of catechin content, fresh leaf precise, 5mL's
70% 70 DEG C of methanol solution water-bath extracts 10min.It is cooled to after room temperature, 3500r/min rotating speeds centrifugation 10min shifts supernatant
To 10mL volumetric flasks.Residue is extracted once with 5mL 70% methanol solution again, repeats to operate above.Merge extract solution to be settled to
10mL, crosses 0.45 μm of film, HPLC detection catechin contents.
HPLC testing conditions are as follows：
Using LC-20A high performance liquid chromatographs (Shimadzu Kyoto, Japan), chromatographic column is Agilent C18 posts,
30 DEG C of column temperature, Detection wavelength 280nm, flow velocity 1.0ml/min；Mobile phase A is that the acetonitrile+96.5% of 0.5% glacial acetic acid+3% is ultrapure
Water, Mobile phase B is the ultra-pure water of+30% acetonitrile of 0.5% glacial acetic acid+69.5%；Gradient：B phases are linear by 20% in preceding 35min
65% is increased to, 5min, the μ l of sample size 10 are then kept with 20%B phases.
3rd, the extraction of total serum IgE
The total serum IgE of tea tree, micro-spectrophotometer and denaturation agar are extracted using Trizol methods (with reference to the method for embodiment 1)
Sugared detected through gel electrophoresis RNA mass.
4th, qRT-PCR detects the expression of miRNA and correspondence target gene
1) miRNA stem rings reverse transcription synthesis cDNA
Using TAKARA the first chain cDNA synthetic agent box, design stem ring primer Stem-loop RT Primer：
GTCGTATCCAGTGCAGGGTCCGAGGTATTCGCACTGGATACGACGTGCTC, prepares following reaction system (20 μ l)：
The reaction solution of mixing is placed in 65 DEG C of heating 5min, 2min in ice is put into afterwards.It is then placed in 16 DEG C of heat of PCR instrument
Swash 30min, afterwards 42 DEG C of 60min, 85 DEG C of 5min make enzyme inactivation stop reverse transcription.
2) reverse transcription of target gene
Using PrimerScriptTMRT reagent Kit with gDNA Eraser(Pefect for Real
Time) kit, prepares following reaction mixture (10 μ l)：
After 42 DEG C of reaction 2min, following inverse transcription reaction liquid (20 μ l) is prepared：
After 37 DEG C of reaction 15min, 85 DEG C of reaction 5min.
3) real-time fluorescence quantitative PCR
Reverse transcription obtains the chains of tea tree cDNA first, using the chains of cDNA first as template, using Takara SYBR
PremixExTaqTM(Tli RnaseH Plus) kit and the CFX-96 quantitative real time PCR Instruments of Bio-Rad companies, primer sequence
Row are with reference to following table：
Configure 20 μ L quantifying systems：
Reaction condition：95 DEG C of pre-degeneration 30s；95 DEG C of denaturation 5s, 58 DEG C of annealing 30s, 40 circulations；Solubility curve expands bar
Part：95 DEG C of 15s, 60 DEG C of 1min, 95 DEG C of 15s.3 repetitions of each sample, respectively using 5.8SrRNA and 26SrRNA as internal reference, number
2 are used according to analysis-ΔΔCTMethod is analyzed.
As shown in Fig. 2 miR156 expresses significantly different under the conditions of different nitrogen.NO3 -Under the conditions of nitrogen stress, Cs-
MiR156g expression is notable to lower.As shown in figure 3, the corresponding target genes of miR156 show as opposite expression trend, NO3 -With lack
Nitrogen condition, target gene is significantly raised.
5th, miR156 and the correlation of tree plant catechin content
Catechin content and tea tree miR156 relative expression quantity correlation analyses are shown that catechin total amount is manifested with miR156
The negative correlativing relation of work, shows miR156 under the conditions of different nitrogen, can regulate and control And Development of Tea Shoot catechin content.
The tea tree Cs-miR156g of table 1. expression and the correlation of catechin content
Note：EGCG：Epigallo-catechin gallate (EGCG)；ECG：L-Epicatechin gallate；EGC：Epi-nutgall
Catechin；EC：Epicatechin；GCG：Nutgall catechin gallic acid ester；CG：Catechin and gallate；C：Catechin.
Claims (2)
1. a kind of applications of tea tree miRNA in tree plant catechin expression is suppressed, it is characterised in that the alkali of the tea tree miRNA
Basic sequence is as shown in SEQ ID NO.1.
2. a kind of application of tea tree miRNA precursor in tree plant catechin expression is suppressed, it is characterised in that the tea tree
The base sequence of miRNA precursor is as shown in SEQ ID NO.2.
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| CN105132417B (en) * | 2015-09-07 | 2017-09-26 | 安徽农业大学 | MiRNA quantitative fluorescent PCRs reference gene and its screening technique and application under tea tree low temperature stress |
| CN105647927A (en) * | 2016-04-06 | 2016-06-08 | 甘肃农业大学 | Potato stu-miR156 member and application thereof |
| CN106191065A (en) * | 2016-08-03 | 2016-12-07 | 吉林大学 | Blue berry miR156 gene and clone products thereof and application |
| CN106399522A (en) * | 2016-10-10 | 2017-02-15 | 天津农学院 | Method for detecting expression quantity of miR156 of malus sieversii |
| CN108441573B (en) * | 2018-03-21 | 2021-08-24 | 湖南科技学院 | Camellia oleifera miRNA and application thereof |
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