CN101993876A - Modified miR169d precursor sequence and application thereof in gene silencing - Google Patents
Modified miR169d precursor sequence and application thereof in gene silencing Download PDFInfo
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- CN101993876A CN101993876A CN2009100913396A CN200910091339A CN101993876A CN 101993876 A CN101993876 A CN 101993876A CN 2009100913396 A CN2009100913396 A CN 2009100913396A CN 200910091339 A CN200910091339 A CN 200910091339A CN 101993876 A CN101993876 A CN 101993876A
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Abstract
The invention relates to the field of gene engineering, in particular to a modified miR169d precursor sequence and an application thereof in gene silencing. The nucleotide sequence of the miR169d precursor sequence amiR169d modified according to the invention is shown in SEQ No.2. In the invention, two specific restriction sites in the neck structure region of the amiR169d precursor sequence are designed, so that the artificially designed target sequence can be conveniently inserted into a carrier of the invention, and plant cells can be introduced to effectively cause the target gene silencing. The transformed plant hosts not only can be dicotyledonous plants such as arabidopsis, tobacco, soybeans and the like but also can be monocotyledonous plants such as corn, rice, wheat and the like.
Description
Technical field
The present invention relates to the genetically engineered field, particularly, the present invention relates to the miR169d precursor sequence and transform and the purposes in gene silencing.
Background technology
It is the instrument of a very useful and effective regulate gene expression that RNA disturbs the gene silencing of (RNAi) mediation, identifies at gene function, the plant virus resistance reaction, crop quality is improved and the aspects such as improvement of economical character have obtained application widely.In the RNAi method, method in common is needing reticent goal gene to be built into inverted repeats, transcribe the back and just form card RNA (hpRNA), being processed into the little RNA of 22nt subsequently by the DCL enzyme, specific reticent homologous target gene at present.Because hpRNA can produce the siRNA of a plurality of～22nt, part siRNA wherein has the potential ability of the non-target gene that acts on other, may influence some other irrelevant expression of gene.
Along with going deep into to miRNA research, it is found that the next specific reticent goal gene of the principle that to utilize miRNA to produce, its primary process is the miRNA precursor (pre-miRNA) that at first obtains wild-type, select the sequence of the special about 21nt of size then according to target gene sequences, be artificial mi RNA (amiRNA, artificial microRNA), at last the miRNA sequence of the wild-type among the pre-miRNA is replaced with aim sequence.Utilize the reticent goal gene that amiRNA people can differential high efficient, all be used for making up artificial little RNA carrier as miR159a precursor, miR164b precursor, miR319a precursor etc., but the precursor of these miRNA is bigger, and structure need carry out repeatedly nested PCR, makes up loaded down with trivial details.
Summary of the invention
Based on this, we analyze existing miRNA and precursor thereof, find that the miR169d characteristics are precursor sequence short (154bp), chain rate in a kind of structure of secondary is less, and can only need to change 5 bases at neck and just can produce 2 special restriction enzyme sites, so we have selected miR169d to carry out the structure of amiRNA carrier.
Therefore, one of purpose of the present invention provides a kind of miR169d precursor sequence amiR169d of transformation.
A further object of the present invention provides the miR169d precursor sequence and transforms the purposes of sequence.
A further object of the present invention provides the gene silencing carrier that contains the miR169d precursor sequence and transform sequence.
A further object of the present invention provides the method for preparing the said gene silent carrier.
The miR169d precursor sequence amiR169d that transforms according to the present invention, its nucleotide sequence are shown in SEQ No.2, and wherein the sequence of being made up of base " n " is any artificial miRNA molecular sequences that designs.Described fragment is that Arabidopis thaliana miR169d precursor sequence (SEQ No.1, wherein the sequence of being made up of base " n " is the miR169 molecular sequences, replaceable arbitrary sequence for artificial design) obtains through transforming.
The invention provides the miR169d precursor sequence and transform the purposes that sequence is used to prepare the gene silencing carrier.
Gene silencing carrier according to the present invention contains the miR169d precursor sequence, or miR169d precursor sequence through transforming, wherein, miR169d molecular sequences in described miR169d precursor sequence or the miR169d precursor sequence through transforming is replaced by specific artificial mi RNA molecule, preferably, the miR169d precursor sequence of described transformation has nucleotide sequence shown in the SEQ No.2.
Can be carrier pAmiR169d according to gene silencing carrier of the present invention, the miR169d precursor sequence through transform of described carrier by nucleotide sequence shown in the above-mentioned SEQ of the having No.2 is cloned into expression vector pCAMBIA1303 and obtains.
The method for preparing the gene silencing carrier according to the present invention comprises the precursor sequence with miR169d, or the miR169d precursor sequence through transforming is cloned into the step of expression vector.
Gene silencing carrier of the present invention be a kind of can be in plant the artificial mi RNA carrier of special reticent target gene.The present inventor successfully utilizes the miR169d precursor molecule first and transforms sequence and prepares the gene silencing carrier, its in the neck structure zone design of amiR169d precursor sequence two special restriction enzyme sites, can be inserted into the aim sequence of artificial design in the carrier of the present invention easily, import vegetable cell, can effectively cause the target gene silence.By the plant transformed host both can be dicotyledonss such as Arabidopis thaliana, tobacco, soybean, also can be monocotyledonss such as corn, paddy rice, wheat.
Description of drawings
Fig. 1 is the secondary structure figure of miRNA precursor, and wherein A is the front body structure of the Arabidopis thaliana miR169d before transforming; B is improved miR169d front body structure; C is the front body structure of artificial amiR-gfp;
Fig. 2 is the artificial mi RNA carrier pAmiR169d of transformation and the design of graphics of pAmiR-gfp; Wherein 35S is cauliflower mosaic virus 35S (CaMV35S) protein gene promoter; Nos is that rouge alkali synthetase (Nos) gene is ended son; Gus reporter gene; LB and RB are border, the T-DNA left and right sides.
Fig. 3 is the reticent effect analysis of pAmiR-gfp to target gene, and wherein, A is the GUS activation analysis, and B is the sxemiquantitative RT-PCR of GUS-GFP transcript, and C is the real-time quantitative PCR of GUS-GFP transcript;
Fig. 4 is the cleavage site analysis of AmiR-gfp to the GUS-GFP transcript, and wherein, A is 5 ' RACE-PCR; B is the shearing site of amiR-gfp.
Embodiment
The acquisition of embodiment 1, pCAMBIA-35SE carrier
Utilize HindIII/EcoRI to scale off the 35S-GUS-Nos fragment of about 3000bp from pBI121 (Clontech), endonuclease bamhi is connected into the HindIII/EcoRI site of pCAMBIA1303 (CAMBIA) behind the sepharose purification kit purifying of Qiagen, form intermediate carrier pCAMBIA-35S.This plasmid is mended flat cohesive end by the T4DNA polysaccharase and is connected certainly after EcoRI digestion, thereby has removed the EcoR I site among the plasmid pCAMBIA-35S, called after pCAMBIA1303-35SE.
Embodiment 2, artificial mi RNA carrier pAmiR169d
Arabidopis thaliana miR169d precursor sequence comprises 154nt, can form a simple neck ring structure (Figure 1A), after its sequence is analyzed, under the prerequisite that does not change its second structure characteristic, modifies transformation.
8 oligonucleotide fragments with overlapping complementary sequence have been synthesized in artificial design, suddenly change respectively in the centre 2 and 3 bases, produce EcoR I and Sal I restriction enzyme site at the two ends of miRNA respectively, but do not changed the secondary structure (Figure 1B) of ath-miR169d precursor sequence.Article 8, the sequence of oligonucleotide fragment is as follows:
oligo?1(5′-gatccGTATCATAGAGTCTTGCATGGA-3′)
oligo2(5′-AAAATTAAAGaattcATTGAGCCAAGGATGACTTGCCGATGTT-3′)
oligo3?5′-ATCAACAAATCTTAACTGATTTTGGTGTCCGGCAAGTTGACCTT-3′)
oligo4?5′-GGCTCTGTCGACTTCTTTTCTTTTCAATGTCAAACTCTAGATATgagct-3′)
oligo5(5′-cATATCTAGAGTTTGACATTGAA-3′)
oligo6?5′-AAGAAAAGAAgtcgacAGAGCCAAGGTCAACTTGCCGGACACCA-3′)
oligo7(5′-AAATCAGTTAAGGATTTGTTGATAACATCGGCAAGTCATCCTTGGC-3′)
oligo8(5′-TCAATCGAATTCTTTAATTTTTCCATGCAAGACTCTATGATACg-3′)
The oligonucleotide fragment of 8 synthetic is formed double-stranded DNA through phosphorylation and annealing, directly be cloned into BamH I and the Sac I site of pCAMBIA1303-35SE, form artificial mi RNA carrier pAmiR169d.
Embodiment 3, utilize the reticent goal gene GUS-GFP of pAmiR169d carrier
Directly synthetic 4 oligonucleotide fragments, annealing forms one section double-stranded DNA (as Fig. 1 C) that comprises the AmiR-gfp sequence, and tip designs is EcoR I and SalI restriction enzyme site, and concrete sequence is as follows:
oligo9(5′-aattcGATtTGTATTCCAaCTTGTGGCCGatgtTAT-3′)
oligo?10(5′-CAAcaAATCttAActGATTTTGGTGtccggccacaagatggaatacatGTcgac-3′)
oligo?11(5′-AAAATCagTTaaGATTtgTTGATAacatCGGCCACAAGtTGGAATACAaATCg-3′)
oligo?12(5′-tcgagtcgACatgtattccatcttgtggccggaCACC-3′)
This annealing product is directly inserted in the plasmid pAmiR169d that EcoR I and Sal I enzyme are cut, form carrier pAmiR-gfp, make up flow process and see Fig. 2.
The reticent effect analysis of embodiment 4, pAmiR-gfp
The carrier pAmiR-gfp and the control vector pCAMBIA1303 that build are transformed Agrobacterium GV3101.Utilize Agrobacterium osmose process transformation of tobacco N.benthamiana blade, method for transformation is as follows: be taken at 24 ℃, grow into the tobacco plant of 6-8 sheet leaf under the 16h photoperiod condition, the Agrobacterium that contains pAmiR-gfp with 0.5ml is soaked into tobacco leaf.Equally, the Agrobacterium that contains pCAMBIA1303 with 0.5ml is soaked into the NC89 blade in contrast.Continue 24 ℃, cultivate 48h under the 16h photoperiod condition.Sampling, liquid nitrogen grinding is extracted total protein, and total protein content is measured by the method for standard Bradford.
Because the GUS-GFP genetic transcription is merged, GFP target sequence is reticent GFP and gus gene simultaneously, so can reflect reticent efficient by detecting the GUS activity.The GUS activity is represented with the ratio nmolMUmg-1 albumen min-1 of MU nmole number and total protein content and time.Analytical instrument is Hoefer DyNA Quant 200 type spectrophotofluorometers, is to measure fluorescent value under 350nm, the emission wavelength 455nm in excitation wavelength.GUS determination of activity result in the tobacco leaf as shown in Figure 3A, the result shows that the GUS activity of the tobacco leaf that soaks into through pAmiR-gfp significantly is lower than contrast, it is about 50% that the GUS activity has reduced, and illustrates that constructed artificial mi RNA carrier pAmiR-GFP can effectively reticent its target gene.
The PCR of the GUS-GFP transcription product of embodiment 5, transformation of tobacco blade measures
Extract tobacco leaf RNA, RNA gents Total RNA Isolation System kit by Promega company carries out the extraction of total RNA, get the total RNA of 2 μ g then and carry out reverse transcription with Invitrogen Corporation's Super Script First-StrandSynthesis System, with the reverse transcription product is template, the transcript of amplification GUS-GFP.Upstream primer forward 5 '-CGATGCGGTCACTCATTAC-3 '; Downstream primer reverse5 '-TTCACACGTGGTGGTGGTGGT-3 '.Reaction conditions is: 94 ℃ 2 minutes, 94 ℃ 20 seconds, 53 ℃ 40 seconds, 72 ℃ 1.5 minutes, 35 circulations; 71 ℃ 10 minutes, the PCR product is 2600bp.With the ACTIN1 of tobacco as confidential reference items, the sequences Design primer NAcfw of the actin1 (AccessionNumber:AB158612) that announces according to GeneBank, 5 '-(forward) and NAcrv of atgagcaagagttggagactg-3 ', 5 '-CAATGGAAGGACCAGATTCAT-3 ' (reverse) carries out PCR, reaction conditions is: 94 ℃ 2 minutes, 94 ℃ 20 seconds, 53 ℃ 40 seconds, 72 ℃ 1 minute, 25 circulations; 71 ℃ 10 minutes, the PCR product is 441bp.The result shows that the comparison of the mRNA level of GUS-GFP is according to significantly reducing (Fig. 3 B) in the tobacco leaf that pAmiR-gfp soaks into.
For the GUS-GFP transcription product is carried out Accurate Analysis, the present invention adopts the method for real-time quantitative PCR that it is measured.Primer: forward GFP FW 5 '-CGACGGGAACTACAAGACAC-3 ' and reverseGFP RV5 '-TTCACACGTGGTGGTGGTGGT-3 '.The PCR reaction conditions is: 95 ℃ of 1min, and 95 ℃ of 15sec, 55 ℃ of 20s, 72 ℃ of 45s (45 circulations), 72 ℃ of 10min, PCR product are 440bp.With above-mentioned tobacco Actin1 as internal control gene.PCR in real time result shows that the mRNA level comparison of GUS-GFP in the tobacco leaf that pAmiR-gfp soaks into is according to low 30 times (Fig. 3 C).
Embodiment 6, artificial mi RNA are to the shearing site analysis of target gene
5 ' RACE is the rapid amplifying of cDNA end, can identify the 5 ' end sequence of RNA, in order to verify the accurate shearing site of amiR-gfp to target gene GUS-GFP, the mRNA of GUS-GFP has been carried out 5 ' RACE analyzed.Specification sheets by 5 ' RACE System2.0 version of GIBCO BRL Life Technologies company carries out the operation of 5 ' RACE.At first get the total RNA of 2 μ g and carry out reverse transcription with the special primer of GFP (GFP RV:5 ' TTCACACGTGGTGGTGGTGGT-3 '), the cDNA that purifying obtains, to remove excessive dNTPs and GFP RV primer, handle (terminal deoxynucleotidyl transferase) at the 3 ' terminal poly C tail that adds with TdT again.CDNA with tailing is a template then, carries out pcr amplification with anchor primer T7-G (5 ' TAATACGACTCACTATAGGGGGGGGGG) and GFP RV, and reaction conditions is: 94 ℃ of 2min, 94 ℃ of 20sec, 65 ℃ of 20sec, 72 ℃ of 45sec (30 circulations), 72 ℃ of 10min.Utilize T7 primer (5 ' TAATACGACTCACTATAGGG) and GFP RV2 (5 ' GTGGTGGTGGTGGCTAGCTTT) to carry out nest-type PRC, reaction conditions is: 94 ℃ of 2min, 94 ℃ of 20sec, 55 ℃ of 20sec, 72 ℃ of 45sec (30 circulations), 72 ℃ of 10min, carry out electrophoresis with 1% agarose then, amplification at the band that a 320bp is arranged, but does not then have amplified production in the sample of empty carrier pCAMBIA1303 infiltration in the sample of carrier pAmiR-gfp infiltration shown in Fig. 4 A.Utilize the dna fragmentation of this 320bp of purification kit purifying of Qiagen, be connected with the pGEM-T carrier, transformed into escherichia coli DH5 α, 5 clones of picking carry out sequential analysis.The sequencing results shows, 5 clones have 5 ' identical terminal nucleotide sequence, the shearing site of this shearing site and expection is in full accord, show the processing that the pAmiR-169d carrier of the transformation among the present invention can be correct in vegetable cell, produce special artificial mi RNA, cause the silence of goal gene.
Sequence table
＜110〉Biological Technology institute, Chinese Academy of Agricultural Sciences
＜120〉the miR169d precursor sequence is transformed and the purposes in gene silencing
<160>14
<210>1
<211>154
<212>DNA
＜213〉Arabidopis thaliana (Arabidopsis thaliana (L.) Heynh)
<400>1
gtatcataga?gtcttgcatg?gaaaaattaa?agaatgagat?nnnnnnnnnn?nnnnnnnnnn 60
natgttatca?acaaatctta?actgattttg?gtgtcnnnnn?nnnnnnnnnn?nnnnnngttt 120
ccttcttttc?ttttcaatgt?caaactctag?atat 154
<210>2
<211>154
<212>DNA
＜213〉Arabidopis thaliana (Arabidopsis thaliana (L.) Heynh)
<400>2
gtatcataga?gtcttgcatg?gaaaaattaa?agaattcgat?nnnnnnnnnn?nnnnnnnnnn 60
natgttatca?acaaatctta?actgattttg?gtgtcnnnnn?nnnnnnnnnn?nnnnnngtcg 120
acttcttttc?ttttcaatgt?caaactctag?atat 154
<210>3
<211>27
<212>DNA
＜213〉artificial sequence
<400>3
gatccgtatc?atagagtctt?gcatgga 27
<210>4
<211>43
<212>DNA
＜213〉artificial sequence
<400>4
aaaattaaag?aattcattga?gccaaggatg?acttgccgat?gtt 43
<210>5
<211>44
<212>DNA
＜213〉artificial sequence
<400>5
atcaacaaat?cttaactgat?tttggtgtcc?ggcaagttga?cctt 44
<210>6
<211>49
<212>DNA
＜213〉artificial sequence
<400>6
ggctctgtcg?acttcttttc?ttttcaatgt?caaactctag?atatgagct 49
<210>7
<211>23
<212>DNA
＜213〉artificial sequence
<400>7
catatctaga?gtttgacatt?gaa 23
<210>8
<211>44
<212>DNA
＜213〉artificial sequence
<400>8
aagaaaagaa?gtcgacagag?ccaaggtcaa?cttgccggac?acca 44
<210>9
<211>46
<212>DNA
＜213〉artificial sequence
<400>9
aaatcagtta?aggatttgtt?gataacatcg?gcaagtcatc?cttggc 46
<210>10
<211>44
<212>DNA
＜213〉artificial sequence
<400>10
tcaatcgaat?tctttaattt?ttccatgcaa?gactctatga?tacg 44
<210>11
<211>36
<212>DNA
＜213〉artificial sequence
<400>11
aattcgattt?gtattccaac?ttgtggccga?tgttat 36
<210>12
<211>54
<212>DNA
＜213〉artificial sequence
<400>12
caacaaatct?taactgattt?tggtgtccgg?ccacaagatg?gaatacatgt?cgac 54
<210>13
<211>53
<212>DNA
＜213〉artificial sequence
<400>13
aaaatcagtt?aagatttgtt?gataacatcg?gccacaagtt?ggaatacaaa?tcg 53
<210>14
<211>37
<212>DNA
＜213〉artificial sequence
<400>14
tcgagtcgac?atgtattcca?tcttgtggcc?ggacacc 37
Claims (7)
1. the miR169d precursor sequence amiR169d of Gai Zaoing is characterized in that, its nucleotide sequence is shown in SEQ No.2, and wherein, the sequence of being made up of base " n " is artificial implementation sequence arbitrarily.
2.miR169d precursor sequence and transformation sequence thereof are used to prepare the purposes of gene silencing carrier.
3. gene silencing carrier, it is characterized in that, described carrier contains the miR169d precursor sequence, or miR169d precursor sequence through transforming, wherein, the miR169d molecular sequences in described miR169d precursor sequence or the miR169d precursor sequence through transforming is replaced by specific artificial implementation sequence.
4. gene silencing carrier according to claim 3 is characterized in that, the miR169d precursor sequence of described transformation is the described sequence of claim 1.
5. a gene silencing carrier pAmiR169d is characterized in that, described carrier is cloned into expression vector pCAMBIA1303 by the miR169d precursor sequence with the described transformation of claim 1 and obtains.
6. a method for preparing the gene silencing carrier is characterized in that, comprises the precursor sequence with miR169d, or the miR169d precursor sequence through transforming is cloned into the step of expression vector.
7. method as claimed in claim 6 is characterized in that, the miR169d precursor sequence of described transformation is the described sequence of claim 1.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103119169A (en) * | 2010-06-09 | 2013-05-22 | 拜尔作物科学公司 | Methods and means to modify a plant genome at a nucleotide sequence commonly used in plant genome engineering |
| CN103194449A (en) * | 2013-04-01 | 2013-07-10 | 吉林农业大学 | Soybean micro RNA (ribonucleic acid) gene gma-miR169d and application thereof in drought control |
-
2009
- 2009-08-18 CN CN2009100913396A patent/CN101993876A/en active Pending
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103119169A (en) * | 2010-06-09 | 2013-05-22 | 拜尔作物科学公司 | Methods and means to modify a plant genome at a nucleotide sequence commonly used in plant genome engineering |
| CN109504700A (en) * | 2010-06-09 | 2019-03-22 | 拜尔作物科学公司 | Plant Genome transformation in commonly on nucleotide sequence modified plant genome Method and kit for |
| CN103194449A (en) * | 2013-04-01 | 2013-07-10 | 吉林农业大学 | Soybean micro RNA (ribonucleic acid) gene gma-miR169d and application thereof in drought control |
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Application publication date: 20110330 |