CN102168086B

CN102168086B - Phalaenopsis miR172 coding sequence and application thereof

Info

Publication number: CN102168086B
Application number: CN201110060055A
Authority: CN
Inventors: 明凤; 严钦骅; 韩颖颖
Original assignee: Fudan University
Current assignee: Fudan University
Priority date: 2011-03-14
Filing date: 2011-03-14
Publication date: 2012-09-05
Anticipated expiration: 2031-03-14
Also published as: CN102168086A

Abstract

The invention belongs to the technical fields of molecular biology and genetic engineering, in particular to a Phalaenopsis miRNA172 sequence and application thereof. A miR172 gene coding sequence cloned from the Phalaenopsis genome, gene expression mode analysis, construction of a transgenic expression vector in Arabidopsis, a primer sequence, a transgenic pure plant and a corresponding phenotype are included. The expression of Phalaenopsis miRNA172 in Arabidopsis can promote apical meristem to be active for presenting premature flowering.

Description

Butterfly orchid miR172 encoding sequence and application thereof

Technical field

The invention belongs to molecular biology, gene engineering technology field, be specifically related to butterfly orchid MiR172And use, comprise butterfly orchid MiR172The butterfly orchid transgene expression vector, primer sequence, transgenic pure lines plant, and the function of this gene of verifying through the pure lines plant is used.

Background technology

Butterfly orchid ( Phalaenopsisamabilis) belong to quilt monocotyledons orchid guiding principle butterfly the orchid family, be a kind of aerial orchid flower, aerial root is arranged, can absorb airborne nutrient and survive.Grow in humid area, the torrid zone, Asia more, adapt to hot and humid airy environment.Single stem is short, and the blade meat is plump, and the butterfly that the flower likeness in form is spread the wings is bright-colored colorful, is a kind of important economic flowers, comes to receive approximately liking of human consumer in recent years more.Has good economic benefits.But butterfly orchid is bloomed the cycle very long, just can bloom later in general 2 years.And its condition that becomes flower to require is very strict, can't bloom in the realization anniversary.The method of at present main flower forcing is through vernalization, and low temperature induction is bloomed.But the current consumption of low temperature flower forcing is big, has increased the cultivation cost, and the luxus consumption of the energy does not meet the STRATEGIES OF SUSTAINABLE DEVELOPMENT requirement.Simultaneously, the low temperature flower forcing needs good closed environment, grows fungal diseases such as botrytis cinerea easily, has reduced economic benefit.Because MiR172Have promoter action to blooming,, can significantly promote flowering of plant once overexpression.Become the flower approach relevant to butterfly orchid MiR172Research help to realize the flower forcing of butterfly orchid anniversary cheaply to have very strong practice significance with further using.

MicroRNA is one type and participates in the important gene that plant-animal grow, time-controllable is broken up in cell fission, is positioned at non-coding region, is processed to form the short nucleic acid molecule of length about 21nt through shearing after transcribing during expression.The MicroRNA gene is coded protein not; Thereby but through coding form microRNA cause the cracking of target gene mRNA or with it specificity combine to suppress expression of gene; This regulatory mechanism depends on the existing complementation (David et al., 2004) in various degree of target gene sequences that the microRNA sequence of microRNA genes encoding is regulated and control with it.The research early start of MicroRNA is in 20th century the nineties (Wightman et al., 1993), but is confined to animal microRNA.People such as Lee had delivered in 2003 MiR172Functional study in the flowering of plant process proves that first there is important effect too in microRNA in the development of plants process, wherein MiR172Be proved to be the important gene (Aukerman et al., 2003) regulating and control to bloom and grow afterwards, belong to the AP2 gene family, and be positioned in the photoperiod path that regulation and control bloom (Schmid et al., 2003).The plant of having cloned at present this gene has Arabidopis thaliana, corn, apple etc. MiR172Effect in different plant species also has hairline; For example in Arabidopis thaliana, mainly show as and promote to bloom and causes arid escape (Schmid et al.; 2003); In corn, participate in flower sex determination and fissional control (Jae-Hoon Jung et al., 2007) more.But in the high ornamental plant of economically valuable, rarely has report.

The present invention obtains butterfly orchid first through the design primer from the full genome of butterfly orchid MiR172Gene; Separation and purification obtains the fragment of the about 250bp of length through the pcr amplification rear electrophoresis; And through in situ hybridization, mRNA separate authentication this expression of gene; With the Agrobacterium plasmid is that carrier will MiR172Transgenic is expressed in Arabidopis thaliana; Go out to have obvious phenotype through the breeding screening and culturing: the transgenic arabidopsis that early blossoming and apical meristem are active has fully proved the function of butterfly orchid miRNA.For MiRNA172The overall understanding of function provides reliable theoretical basis and practical experience in plant.

Summary of the invention

The objective of the invention is to through with the monocotyledons butterfly orchid MiR172Gene changes in the dicotyledons Arabidopis thaliana, the pure lines plant that acquisition has obvious phenotype, and identify PhmiRNA172Expression in transfer-gen plant, the checking butterfly orchid MiRNA172Function.

The present invention provides first, and the clone obtains butterfly orchid MiRNA172Gene order, with and the coding MiRNA172Secondary structure.This gene order is shown in SEQ ID NO.1.

The present invention also is provided for from the butterfly orchid whole genome sequence, according to homology MiRNA172Conservative specific regions two ends non-conservative region designed primer sequence, this primer sequence is shown in SEQ ID NO.2 and SEQ ID NO.3.

The present invention also provides the genetically modified pHB plant expression vector that is used for that contains said gene.This carrier contains just like target gene sequences and the hygromycin resistance marker gene shown in the SEQ ID NO.1, can change Agrobacterium over to and infect Arabidopis thaliana, can genetic stability.

Carrier according to the invention Arabidopis thaliana bloom with apical meristem regulation and control in have an effect, concrete transgeneic procedure step is following:

(1), forms the expression vector that contains SEQ ID NO.1 sequence with the exercisable plant expression vector that is connected in of encoding sequence;

(2) with the expression vector arabidopsis thaliana transformation in the step (1);

(3) through antibiotic-screening, RT-PCR identifies, obtains the transgenic positive individual plant, and selfing obtains the transgenic homozygote, and this homozygote is bloomed in advance, and apical meristem is active.

The present invention also provides a kind of method that is used to detect miR172 expression amount in the butterfly orchid petal, comprises with sequence shown in the SEQ ID NO.1 preparing rna probe and the hybridization of butterfly orchid petal paraffin section sample, and whether detection probes combines.

The present invention also provides a pair of primer sequence of PhmiR172 at the target gene TOE1 of butterfly orchid that be used for increasing.This sequence as:

Ph-TOE1-F:?AAGTTCACAGTATAGAGG （SEQ?ID?NO.4）

Ph-TOE1-R:?GCATGCCTGCAGGTCGAC （SEQ?ID?NO.5）

The present invention also provides a kind of method that is used for identifying butterfly orchid PhmiR172 expression amount; Comprise with SEQ ID NO.2 and SEQ ID NO.3, SEQ ID NO.4 and the said primer of SEQ ID NO.5 and respectively sample is carried out sxemiquantitative PCR reaction, compare miR172 and TOE1 expression in the butterfly orchid Different Organs then.

The present invention also provides a kind of being used for to detect the method whether transgenic arabidopsis exists PhmiR172, comprises the primer with PhmiR172:

PhmiR172-F：5’?GCC?AAG?CTT?GTG?TTT?GCG?GGC?GTG?GCA?TCA?TCA?AGA?TTC?3’ （SEQ?ID?NO.2）

PhmiR172-R：5’GCG?AGC?TCT?TGT?CTG?CGG?ATG?CAG?CAT?CAT?CAA?GAT?3’ （SEQ?ID?NO.3）

Sample is carried out PCR reaction, detect whether amplify the purpose fragment then, if this sample is arranged then from transgenic arabidopsis.

The present invention also comprises having above two kinds of butterfly orchides MiRNA172The Arabidopis thaliana of gene pure lines plant, this transfer-gen plant have obvious phenotypes and are mainly and bloom in advance, and apical meristem shows as a plurality of tops and exists simultaneously at early stage active of vegetative reproduction.

Description of drawings

Fig. 1 is right with software ClustalX At172a, At172b, At172c, At172e, Os172a, Os172b, Os172c, Os172dAnd it is newfound PhmiR172Sequence is compared (B), and makes up evolutionary tree (A).In evolutionary tree, PhmiR172With known Arabidopis thaliana MiR172e, miR172dGet together, two other parallel branch is respectively from paddy rice MiR172Family, AtmiR172C and AtmiR172aWith bFind new PhmiR172Sequence contains above-mentioned known homogenic common conserved sequence, as about length 20bp in the software sectional drawing (B), and region, * labelled notation site.

Fig. 2 software prediction PhmiR172Mature sequence has typical neck ring structure.

Fig. 3 according to PhmiR172The homology target gene PhTOE1The design primer, right PhmiR172And target gene PhTOE1-LIKEThe mRNA level carry out sxemiquantitative PCR.In portion of tissue, PhmiR172Expression and target gene expression complementary effect is arranged.As in root, stem, having PhmiR172Transcript, then do not have PhTOE1-LIKEExpression.In leaf, sepal, do not detect PhmiR172, then detected target gene expression.In addition PhmiR172Higher at bennet and petal expression, it is described has possibly participated in the regulation and control of blooming.

Fig. 4 is with butterfly orchid PhmiR172And target gene PhTOE1Be structured in the pSPT19 carrier, carry out in situ hybridization.As shown in the figure PhmiR172Signal is in early stage floral organs such as outside bract fairly obvious (A, B, C, D). TOE1High expression level is then arranged in pollen granule, expression (E, F, G, H) is also arranged in the foreign steamer organ.Among the figure: po representes pollen granule (pollen); B is bract (bracteole); C is gynostemium (column); S represents sepal (sepal); P is petal (petal); L represents lip (lip); St column cap (stigma); Lpj is lip highlight (lip pre).

Fig. 5 detect T2 for hygromycin gene in the transgenic plant ( Hpt), choosing positive strain is under (A) seed kind, obtains homozygote T3 generation.Extracting PhmiRNA172Transgenic T3 is for plant RNA; Rt becomes cDNA to carry out PCR specific amplified detection its transcript (B) with primer SEQ ID NO.2 and SEQ ID NO.3; With 2 strain wild-type plants is contrast, and 4 transgenic lines detect and all are positive among the figure, and has the transcript of two different sizes.

Fig. 6 is with butterfly orchid MiRNA172Transgenic arabidopsis be experimental group, the empty expression vector and the wild-type that do not contain this gene are control group, relatively in transgenic T3 generation, is at the growing state of contemporaneity apical meristem that grows.The discovery transfer-gen plant is bloomed in advance, and apical meristem is active, a plurality of tops early occur.Among the figure: AWild-type plant 23d does not have the sign of blooming; BTransfer-gen plant 23d is bloomed; CThe enlarged view of bud appears in transfer-gen plant 23d; DThe enlarged view of transfer-gen plant 26d bud; ETransfer-gen plant 26d is bolting; FTransfer-gen plant 29d, bolting is bloomed more vigorous; GThe phenomenon of blooming has just appearred in wild-type 26d.

Embodiment

Below in conjunction with specific embodiment, further illustrate the present invention.Should be understood that these embodiment only to be used to the present invention is described and be not used in the restriction scope of the present invention.The experimental technique of unreceipted actual conditions in the following example; Usually according to normal condition; For example the Sambrook equimolecular is cloned: laboratory manual (New York:Cold Spring Harbor Laboratory Press; 1989) condition of being narrated in, or the condition of advising according to manufacturer.

Embodiment 1 butterfly orchid MiRNA172The clone

1, according to known MiRNA172The conservative region of gene order, the design butterfly orchid MiRNA172Primer such as SEQ ID NO.2 and SEQ ID NO.3.To increasing by organizing the full genome of extractive butterfly orchid to carry out specific PCR;

2, normal temperature 13V gel electrophoresis separated product, detect the size about 250bp band;

3, reclaim the bar zone purification, once more with checking order result such as SEQ ID NO.1 behind the same primer amplification.

Embodiment 2 butterfly orchides MiRNA172Gene order information and homology analysis

1, institute's calling sequence and Arabidopis thaliana among the embodiment 1, paddy rice is known MiR172Gene has carried out the comparison of homology analysis and conservative region, finds that itself and two are from Arabidopis thaliana MiR172Family's family sequence is got together (Fig. 1 A), and has above-mentioned MiR172The homology conservative region sequence (Fig. 1 B) of the total about 20bp of length is tentatively confirmed as MiRNA172Gene;

2, utilize the sequence that obtains to carry out the miRNA secondary structure prediction with RNA-fold software, result such as Fig. 2, this RNA can be formed with typical neck ring structure.

In the embodiment 3 butterfly orchid different tissues PhmiR172And target gene TOE1Expression (mRNA sxemiquantitative PCR)

1, basis MiR172The homology target gene TOE1(sequence such as SEQ ID NO.6) designs primer (SEQ ID NO.4 and SEQ ID NO.5);

2, use above-mentioned primer, and PhmiR172Primer SEQ ID NO.2 and SEQ ID NO.3 increase in each tissue cDNA of butterfly orchid respectively PhmiR172And target gene PhTOE1-LIKE

The result is as shown in Figure 3.In the portion of tissue, PhmiR172Expression and target gene expression complementary effect is arranged.As in root, stem, having PhmiR172Transcript, then do not have TOE1Expression.In leaf, sepal, do not detect PhmiR172, then detected target gene expression.In addition PhmiR172Higher at bennet and petal expression, it is described has possibly participated in the regulation and control of blooming.

In the embodiment 4 butterfly orchid floral organs PhmiR172Gene and target gene PhTOE1Expression pattern analysis and comparison (in situ hybridization)

1, will PhmiR172Gene and target gene PhTOE1Be structured in the pSPT19 carrier respectively, preparation in situ hybridization probe;

2, with butterfly orchid petal dewater embedding sample circuit paraffin section dehydration and embedding, with the fixing 15min (4 oC insulation in advance) of 4%PFA, the residual paraffin of flush away, 50 oC prehybridization 2h (enclose in the blocking solution and add the milt hybridization solution);

3, the probe for preparing is hybridized goal gene.Add antibody after the lowlenthal serum sealing, antibody 37 oC handle 2h, the lucifuge colour developing: 1 normal temperature 1-4h NBT/BCIP+ LEVAMISOLE HCL * L/mL), eliminate the endogenous SEAP;

4, TSM3 5min ' 2 (1MTris pH8.0 10mL+0.5M EDTA 2mL+ddH ₂O 88mL) termination reaction;

5, carry out microscopic examination with blue filter after the mounting, the result is as shown in Figure 4.

Embodiment 5 butterfly orchides MiRNA172Transgenic arabidopsis carries out authentication function

1, contains goal gene PhmiR172The structure of expression vector

1) will PhmiRNA1725 ' the end and the 3 ' end of sequence add respectively HinD III restriction site with SacThe I restriction enzyme site.Be connected into plant expression vector pHB with justice then;

2), change recombinant vectors over to Agrobacterium EH105 with freeze-thaw method, the screening positive bacteria falls enlarged culturing on Rifampin (40mg/L)+Streptomycin sulphate (50mg/L)+kantlex (50mg/L) flat board;

3), the positive single bacterium colony of picking enlarges, and gets lysate with goal gene primer (sequence such as SEQ ID NO.2 and SEQ ID NO.3) PCR after boiling bacterium, the band that contains about 200bp always then is a transgene clone.

, inflorescence dip method arabidopsis thaliana transformation

1) the positive single bacterium colony of picking Agrobacterium is in 3ml LB+ Rifampin (40mg/L)+Streptomycin sulphate (50mg/L)+kantlex (50mg/L) substratum, and 28 ℃, 250rpm shake the bacterium overnight cultures;

2) get 50 μ l bacterium liquid and be inoculated in the LB substratum of the same resistance of 50ml, enlarged culturing is spent the night under the same terms, to 0D600 be 1.2;

3) the centrifugal 5min of 4500rmp under the room temperature removes supernatant, transform the resuspended thalline of damping fluid to 0D600 between 0.8;

Arabidopis thaliana transforms the damping fluid preparation:

1/2MS+6-BA (0.01mg/L)+5% sucrose+0.03% silwet;

4) Agrobacterium spray method arabidopsis thaliana transformation: spending the night before infecting preserves moisture cultivates wild-type Arabidopis thaliana material, and the flower that will open cuts off with the fruit pod.To pour in the little watering can of 50ml with transforming the resuspended bacterium liquid of damping fluid, the still unopened bud of wild-type Arabidopis thaliana (Col.) will be carried out abundant spray painting, transfer normal cultured to after the dark overnight cultures of preserving moisture, and repeat behind the 5d to transform 1 time.

, to the screening of transfer-gen plant

1) pluck mellow fruit folder, 50 ℃ of baking ovens dehydrate a couple of days in batches, take out seed, and it is subsequent use to put back in the moisture eliminator room temperature preservation after cleaning out;

2) with the transgenic seed sterilization, (MS+25mg/Lhyg) sterile culture on the Arabidopis thaliana screening culture medium goes to the phytotron normal cultured behind 4 ℃ of dark cultivation 2d.The resistance seedling that 4 true leaves will be arranged behind the 10d and take root is transferred to basin soil, covers film and preserves moisture, and in the greenhouse, cultivates, and 3d takes off film and transfers normal cultured to, and well-grown plant is transgenic line.

Filter out T1 generation, T2 for transgenic line by above-mentioned steps continuously, checking T2 for hygromycin gene in the plant ( Hpt), if then receiving its seed plantation, the positive obtains T3 for homozygous lines.

, the transfer-gen plant destination gene expression identifies

1) with the SDS method in a small amount extracting T2 Arabidopis thaliana individual plant genomic dna, make blank with the wild-type Arabidopis thaliana, according to hygromycin gene distinguished sequence design primer, carry out PCR and detect, the result sees Fig. 5 A;

2) extract the verticillate leaf RNA that contains hygromycin resistance individual plant filial generation (T3 is for isozygotying), RT-PCR obtains the cDNA sample, in the cDNA sample, detects foreign gene PhmiR172Expression, result such as Fig. 5 B.The foreign gene of three transgenic lines all has obvious expression than the wild-type Arabidopis thaliana.

, the transgenic line phenotypic evaluation

With the wild-type (3 repetitions) of changeing empty carrier as contrast, the flowering time of transgenic homozygous lines (3 repetitions) and wild-type relatively, verticillate leaf number discovery transgenic PhmiR172Arabidopis thaliana the phenotype of blooming has in advance appearred.Verticillate leaf number and wild-type be more or less the same (Fig. 6 A, B).Simultaneously, apical meristem is very active, the situation that has occurred more for a long time growing simultaneously on a plurality of tops (Fig. 6 C, D E), and carry out bolting (Fig. 6 F) in growth in the time of 26 days, and wild-type just begin to bloom this moment (Fig. 6 G).Explanation PhmiR172In transgenic arabidopsis, promote to bloom in advance, and impel apical meristem active.

Reference

[1]David?P.?Bartel?et?al.,?MicroRNAs:?Genomics,?Review?Biogenesis,?Mechanism,?and?Function?[J]. Cell,?2004,116:?281–297.

[2]Jae-Hoon?Jung?et?al.,?The? GIGANTEA-Regulated? MicroRNA172?Mediates?Photoperiodic?Flowering?Independent?of? CONSTANS?in? Arabidopsis?[J].? The?Plant?Cell,?2007,?19:?2736–2748.

[3]Milo?J.?Aukerman?et?al.,?Regulation?of?flowering?time?and?floral?organ?identity?by?a?microRNA?and?itsAPETALA2-Like?target?genes?[J].? The?Plant?Cell,?2004,?15:?2730–2741.

[4] Qian-Hao?Zhu?et?al.,?Over-expression?of? miR172?causes?loss?of?spikelet?determinacy?and?floral?organ?abnormalities?in?rice?( Oryza?sativa)?[J]. BMC?Plant?Biology,?2009,?9:149.

[5] Qian-Hao?Zhu?et?al.,?Regulation?of?flowering?time?and?floral?patterning?by? miR172?[J].? Journal?of?Experimental?Botany,?2011,?62?(2):487-495.

[6]Xuemei?Chen?et?al.,?Novel?Genetic?Mechanisms?in?Development?9,? miR172?modulates?the?output?of?the? AGAMOUS/APETALA2?antagonistic?pair?in?floral?patterning?[J].? Developmental?Biology,?2006,?295:?324?–?326.

[7] Nick?Lauter?et?al.,? MicroRNA172?down-regulates ?glossy15?to?promote?vegetative?phase?change?in?maize?[J]. PNAS,?2005,?102(26):?9412-9417.

Sizolwenkosi?M?lotshwa?et?al.,?Floral?patterning?defects?induced?by? Arabidopsis?APETALA2?and? microRNA172?expression?in? Nicotiana?benthamiana?[J].? Plant?MolBiol,?2006,?61:781–793。

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Claims

1. the gene order that the clone obtains from the butterfly orchid whole genome sequence is characterized in that the gene for butterfly orchid miRNA172, and its dna sequence dna is shown in SEQ ID NO.1, and total length 250bp contains the miRNA172 conservative region.

2. a primer sequence that is used to obtain butterfly orchid miRNA172 gene is characterized in that sequence is shown in SEQ ID NO.2 and SEQ ID NO.3 according to the non-conservative region design in the said miRNA172 gene of claim 1 two ends.

3. one kind contains the plant expression vector of the encoding sequence of gene according to claim 1, contains just like target gene sequences and the hygromycin resistance marker gene shown in the SEQ ID NO.1.

4. the application of carrier as claimed in claim 3 in transgenic plant, the contained gene according to claim 1 of this carrier Arabidopis thaliana bloom with apical meristem regulation and control in have an effect, concrete transgeneic procedure step is following:

5. method that is used to detect miR172 expression amount in the butterfly orchid petal is characterized in that it comprises with sequence shown in the SEQ ID NO.1 and prepares rna probe and butterfly orchid petal paraffin section sample is hybridized whether detection probes combines.

6. a pair of miR172 that is used for increasing is characterized in that shown in SEQ ID NO.4 and SEQ ID NO.5 at the primer sequence of the target gene TOE1 of butterfly orchid.

7. method that is used for identifying butterfly orchid miR172 expression amount; It is characterized in that; It comprises with SEQ ID NO.2 and SEQ ID NO.3, SEQ ID NO.4 and the said primer of SEQ ID NO.5 carries out sxemiquantitative PCR reaction to sample respectively, compares miR172 and TOE1 expression in the butterfly orchid Different Organs then.

8. one kind is used for detecting the method whether transgenic arabidopsis exists butterfly orchid miR172; It is characterized in that it comprises that with SEQ ID NO.2 and the said primer of SEQ ID NO.3 sample being carried out PCR reacts; Detect whether amplify the purpose fragment then, there is butterfly orchid miR172 in this sample if having then.