CN104450770A - Application of larch miR166a in plant development regulation and control - Google Patents
Application of larch miR166a in plant development regulation and control Download PDFInfo
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Abstract
The invention discloses an application of larch miR166a in plant development regulation and control. The application provided by the invention specifically is the application of nucleic acid molecules shown in any of (1)-(4) in plant development regulation and control, namely (1) RNA molecules shown in sequence 1 in a sequence table; (2) RNA molecules shown in sequence 2 in the sequence table; (3) RNA molecules shown in sequence 3 in the sequence table; (4) DNA molecules shown in 27th-539th of the sequence 3 in the sequence table. Experiments prove that the transgenic plants with overexpressed LaMIR166a genes generate lots of lateral roots and grow well when cultured on a rooting culture medium a17 for 4 months; non-transgenic control plants only elongate main roots, do not generate lateral roots and relatively grow slowly. The result indicates that the overexpressed miR166a is capable of promoting the growth speed of larch tissue culture seedling and the generations of the lateral roots, thus increasing the transplanting survival rate.
Description
Technical field
The invention belongs to molecular biology of plants technical field, relate to the application of a kind of tamarack miR166a in regulating development of plants, particularly a kind of tamarack miR166a is in the developmental application of promotion plant lateral roots.
Background technology
Tamarack (Larix spp.) is that the important fast-growing afforestation of northern China uses material coniferous species, can be used for timber and paper making raw material.Globally, tamarack natural distributed is in the Plain of mountain area, temperate zone, cool temperature zone and alpine climate district, and front two bands, roughly between north latitude 48 ° ~ 60 °, have the feature of high latitude High aititude.The tamarack of China's natural distributed has 10 kinds and 5 mutation.They are distributed in northwest, North China, Central China, the mountain region in northeast and southwest and alpine region respectively.The larchen scientific research of Efforts To Develop, the development for northern China Ecological Civilization Construction, promotion national economy is significant.Tamarack as China important needle fast-growing afforestation commerical tree species, it is bred and genetic improvement in a large number, without people's demand growing to wood raw material can be met, also can make tremendous contribution for China's Ecological Civilization Construction.
MicroRNAs(miRNAs) be the endogenic non-coding RNA with adjusting function of a class found in eukaryote, its size is about 20 ~ 25 Nucleotide.Ripe miRNAs is produced by the shearing of longer primary transcript through a series of nuclease, be assembled into the silencing complex of RNA induction subsequently, by the mode identification said target mrna of base pair complementarity, and instruct silencing complex degraded said target mrna according to the difference of complementarity or check the translation of said target mrna.
The target gene of Mirnas of plant is the transcription factor in plant materials mostly, plays regulating and controlling effect widely in all many-sides such as the growing of plant, environment response and disease-resistant responses.By forward genetics screening, Direct Cloning, computer forecast and est sequence analysis, in 67 kind of plant, about 6000 miRNA(miRBase19.0 are identified at present).Along with the development of high throughput sequencing technologies and the decline of order-checking cost, in increasing plant, miRNA in genomic level by Bioinformatics Prediction out.And one of ultimate challenge studied at present is, how to verify the function that the target gene of single miRNA and correspondence thereof plays in vivo.
Summary of the invention
The object of this invention is to provide the novelty teabag of a kind of tamarack miR166a.
Novelty teabag provided by the present invention, be specially arbitrary in following (1)-(4) shown in nucleic acid molecule in the developmental application of Promoting plant growth:
(1) RNA molecule shown in sequence 1 in sequence table;
(2) RNA molecule (tamarack precursor miR166a) in sequence table shown in sequence 2;
(3) DNA molecular (full-length cDNA of tamarack precursor miR166a, called after LaMIR166a gene) in sequence table shown in sequence 3;
(4) DNA molecular shown in 27-539 position of sequence 3 in sequence table.
In above-mentioned application, described Promoting plant growth grows at least one that specifically may be embodied in following (A)-(C):
(A) growth of plant lateral roots is promoted;
(B) plant growth rate is improved;
(C) plant survival rate is improved.
In above-mentioned application, " promoting that plant lateral roots is grown " described in (A) is for promoting the generation of plant lateral roots; (B) " the raising plant growth rate " described in is improve the growth velocity of tissue cultured seedling (as cultivated the tissue cultured seedling of 4 months on root media); (C) " the raising plant survival rate " described in is the surviving rate after improving plant transplantation.
Application in the plant variety of at least one of nucleic acid molecule in seed selection has (I)-(III) as follows shown in arbitrary in (1)-(4) as follows also belongs to protection scope of the present invention:
(1) RNA molecule (the ripe miR166a of tamarack) in sequence table shown in sequence 1;
(2) RNA molecule (tamarack precursor miR166a) in sequence table shown in sequence 2;
(3) DNA molecular (LaMIR166a gene) in sequence table shown in sequence 3;
(4) DNA molecular shown in 27-539 position of sequence 3 in sequence table.
(I) side root well developed root system;
(II) plant growth rate improves;
(III) survival rate of plant improves.
In actual applications, when seed selection has the plant variety of at least one in (I)-(III) as mentioned above, embryogenic cell line that need be higher by the nucleic acid molecule expression amount shown in arbitrary in described (1)-(4), as the genetically modified clone of acquisition object.
Another object of the present invention is to provide a kind of method of cultivating transgenic plant.
The method of cultivation transgenic plant provided by the present invention, specifically can comprise following step a) and b):
A) in object plant, import arbitrary shown nucleic acid molecule in following (1)-(4), obtain the transgenic plant of expressing described nucleic acid molecule;
(1) RNA molecule (the ripe miR166a of tamarack) in sequence table shown in sequence 1;
(2) RNA molecule (tamarack precursor miR166a) in sequence table shown in sequence 2;
(3) DNA molecular (LaMIR166a gene) in sequence table shown in sequence 3;
(4) DNA molecular shown in 27-539 position of sequence 3 in sequence table.
B) obtain from step a) gained transgenic plant compared with described object plant, there are the transgenic plant of at least one in following (I)-(III):
(I) side root well developed root system;
(II) plant growth rate improves;
(III) survival rate of plant improves.
Wherein, sequence 1 is made up of 21 Nucleotide, is the ripe miR166a sequence of tamarack; Sequence 2 is made up of 95 Nucleotide, is tamarack precursor miR166a sequence; Sequence 3 is made up of 884 deoxynucleotides, is the full length cDNA sequence of tamarack precursor miR166a, is LaMIR166a gene.
In the present invention, what adopt when cultivating transgenic plant is imported in described object plant by the DNA molecular shown in the 27-539 position of sequence in sequence table 3.Further, the DNA molecular shown in 27-539 position of sequence 3 in described sequence table is proceeded in described object plant by the form of recombinant expression vector.
Further, the promotor that the DNA molecular shown in 27-539 position described recombinant expression vector starting sequence 3 in described sequence table is transcribed is Super promotor.
More specifically, described recombinant expression vector in multiple clone site (as Hind III and Kpn I) place's insertion sequence table of pSuper1300+ carrier sequence 3 27-539 position shown in DNA molecular after the recombinant plasmid that obtains.
Certainly, described recombinant expression vector also can with existing other plant expression vector establishment, as double base agrobacterium vector and the carrier etc. that can be used for plant micropellet bombardment, more concrete as pGreen0029, pCAMBIA3301, pCAMBIA1300, pBI121, pBin19, pCAMBIA2301, pCAMBIA1301-UbiN or other derivative plant expression vector.Described plant expression vector also can comprise 3 ' end untranslated region of foreign gene, namely comprises the DNA fragmentation of polyadenylation signals and any other participation mRNA processing or genetic expression.The bootable polyadenylic acid of described polyadenylation signals joins 3 ' end of mRNA precursor.When using described gene constructed recombinant expression vector, any one enhancement type, composing type, organizing specific type or inducible promoter can be added before its transcription initiation Nucleotide, such as cauliflower mosaic virus (CAMV) 35S promoter, ubiquitin gene Ubiquitin promotor (pUbi), stress induced promoter rd29A etc., they can be used alone or are combined with other plant promoter; In addition, when using gene constructed recombinant expression vector of the present invention, also enhanser can be used, comprise translational enhancer or transcriptional enhancer, these enhanser regions can be ATG initiator codon or neighboring region initiator codon etc., but must be identical with the reading frame of encoding sequence, to ensure the correct translation of whole sequence.The source of described translation control signal and initiator codon is widely, can be natural, also can be synthesis.Translation initiation region can from transcription initiation region or structure gene.For the ease of identifying transgenic plant cells or plant and screening, can process recombinant expression vector used, the coding can expressed in plant as added can produce enzyme or the gene of luminophor, the antibiotic marker thing with resistance or the chemical resistance reagent marker gene etc. of colour-change.Also any selected marker can not be added, directly with adverse circumstance screening transformed plant.
In the method for above-mentioned cultivation transgenic plant, described recombinant expression vector is imported described object plant, specifically can be: by using Ti-plasmids, Ri plasmid, plant viral vector, directly delivered DNA, microinjection, conductance, conventional biology methods transformed plant cells or the tissue such as agriculture bacillus mediated, and the plant tissue of conversion is cultivated into plant.
In above-mentioned each application or each method, " the side root well developed root system " described in all (I) is side root increased number; " plant growth rate raising " described in all (II) is tissue cultured seedling (as cultivated the tissue cultured seedling of 4 months on root media) growth velocity raising; " survival rate of plant raising " described in all (III) is that the survival rate of plant after transplanting improves.
In above-mentioned each application or each method, described plant can be gymnosperm.
In the present invention, described gymnosperm is larch, specifically can be larch-tree (Larixleptolepis), Changbai larch (Larix olgensis Henry), larch in Xinanlin area (Larix gmelinii) or Larix principis-rupprechtii (Larix principis-rupprechtii Mayr).
Experiment proves, by the DNA molecular process LAN in tamarack shown in the 27-539 position of sequence in sequence table 3, obtains transfer-gen plant.When the transfer-gen plant of result display LaMIR166a gene overexpression cultivates 4 months on root media a17, have a large amount of side roots to occur, and plant strain growth is good; But not non-transgenic control lines, only have main root to extend, do not have side root to occur, and plant strain growth is slow by contrast.This illustrates that process LAN miR166a can promote the growth velocity of tamarack tissue cultured seedling, promote the generation of side root, thus improves its transplanting survival rate.
Accompanying drawing explanation
Fig. 1 is the RNA secondary structure of precursor miR166a.It is the sequence of ripe miR166a in square frame.
Fig. 2 is the larchen PCR detected result of transgenosis.Wherein, the amplification of gene LaMIR166a for the purpose of A; Each swimming lane template DNA is respectively: water, negative control (CK-, not genetically modified tamarack embryogenic cell line), a1 ~ a5, positive control (CK+ imports the transgenosis tamarack embryogenic cell line of recombinant expression vector pSuper::MIR166a); Swimming lane M is DNA molecular amount standard.B is the amplification of hygromycin phosphotransferase gene (HPT); Each swimming lane template DNA is respectively: water, negative control (CK-, not genetically modified tamarack embryogenic cell line), a1 ~ a5, positive control (CK+ imports the transgenosis tamarack embryogenic cell line of recombinant expression vector pSuper::MIR166a); Swimming lane M is DNA molecular amount standard.C is the amplification of Agrobacterium virulent gene VirG; Each swimming lane template DNA is respectively: water, negative control (CK-, not genetically modified tamarack embryogenic cell line), a1 ~ a5, positive control (CK+, Agrobacterium GV3101) swimming lane M are DNA molecular amount standard.
Fig. 3 is the larchen qRT-PCR detected result of transgenosis.
Fig. 4 is the form of transgenosis tamarack tissue cultured seedling when cultivating 4 months on root media a17.Wherein, A is non-transgenosis tamarack tissue cultured seedling; B is the transgenosis tamarack tissue cultured seedling a4 proceeding to recombinant expression vector pSuper::MIR166a.
Embodiment
The experimental technique used in following embodiment if no special instructions, is ordinary method.
Material used in following embodiment, reagent etc., if no special instructions, all can obtain from commercial channels.
PSuper1300+ carrier: be recorded in " Zhu Caihong, Li Shuigen, Qi Liwang, Han Suying. agriculture bacillus mediated larch-tree cells,primordial Study on Genetic Transformation. Chinese biological engineering magazine, 2013,33(5): 75-80 " literary composition.
Agrobacterium GV3101: be recorded in " Zhao Zhan, Li Wensheng. the impact of different Strains on Agrobacterium tumefaciens-mediated Transformation Efficiencyof Trichoderma harzianum. northern gardening, 03 phase in 2006 " literary composition.
The acquisition of embodiment 1, miR166a process LAN transgenic plant and qualification
MiR166a involved in the present embodiment is the miR166a of larch-tree (Larix leptolepis).Wherein, the sequence of ripe miR166a sequence is sequence 1 in sequence table; The sequence of precursor miR166a sequence is sequence 2 in sequence table; The full-length cDNA of precursor miR166a sequence is sequence 3 in sequence table, called after LaMIR166a gene.Wherein, the RNA secondary structure of precursor miR166a as shown in Figure 1.
One, the structure of recombinant expression vector pSuper::MIR166a
1, design of primers
The full-length cDNA (LaMIR166a gene) of the precursor miR166a sequence according to sequence in sequence table 3, each about 200bp place design in the two ends primer of the miR166a loop-stem structure sequence on this cDNA, as follows:
Primer 1:5 '-CCC
aAGCTT(underscore part is the recognition sequence of Hind III to GCAACACCATACTTACGCC-3 ', and sequence is thereafter the 27-45 position of sequence 3;
Primer 2: 5 '-GG
gGTACCcCCTGGAAAACTCACCTATAC-3 ' (underscore part is the recognition sequence of Kpn I, and sequence is thereafter the reverse complementary sequence of the 519-539 position of sequence 3)
2, pcr amplification
Extract the total serum IgE of larch-tree (Larix spp.), after reverse transcription, obtain cDNA sequence.With gained cDNA for template, adopt above-mentioned primer pair, carry out pcr amplification, the nucleotides sequence through order-checking qualification PCR primer is classified as " CCC
aAGCTTthe 27-539 position of the sequence 3 of+sequence table+
gGTACCcC ".
3, vector construction
By restriction enzyme Hind III and Kpn I double digestion step 2 gained PCR primer, be connected with the skeleton large fragment of the pSuper1300+ carrier through same double digestion after glue reclaims, obtain recombinant plasmid.Hind III and the qualification of Kpn I double digestion are carried out to gained recombinant plasmid, qualification correct (obtain size and be about 10kbp and 525bp) sample presentation order-checking afterwards.By the recombinant plasmid obtained after the DNA molecular shown in the 27-539 position showing sequence 3 in insertion sequence table between the multiple clone site Hind III and Kpn I of pSuper1300+ carrier of checking order, called after pSuper::MIR166a.
In recombinant expression vector pSuper::MIR166a, the promotor that in initiating sequence table, the DNA molecular shown in 27-539 position of sequence 3 is transcribed is Super promotor.
Certainly, in the process building recombinant expression vector pSuper::MIR166a, the DNA molecular shown in sequence 3 in sequence table also can be adopted to be that template carries out pcr amplification.
Two, the larchen acquisition of transgenosis
1, the acquisition of transgenosis tamarack embryogenic cell line
(1) preparation of recombinational agrobacterium
YEB liquid nutrient medium (1L): beef extract 5g+ yeast extract paste 5g+ Tryptones 5g+ sucrose 5g+MgSO
47H
2o0.493g, solvent is water; PH7.0.
YEB solid medium (1L): beef extract 5g+ yeast extract paste 5g+ Tryptones 5g+ sucrose 5g+MgSO
47H
2o0.493g+ agar powder 15g, solvent is water; PH7.0.
Recombinant expression vector pSuper::MIR166a step one obtained imports Agrobacterium GV3101.Concrete operations are as follows: joined by 10 μ L recombinant expression vector pSuper::MIR166a in the competent cell of 200 μ L Agrobacterium GV3101, mix gently; Ice bath 30min, liquid nitrogen freezing 3 ~ 5min, 37 DEG C of water-bath 5min; Add 1mL YEB liquid nutrient medium, 28 DEG C, 180rpm shaking culture 4h; The centrifugal 8min of 4000rpm, room temperature, abandons supernatant, adds the resuspended thalline of 200 μ L YEB liquid nutrient medium, coats YEB film solid media (containing Rifampin, gentamicin and each 50mg/L of kantlex); 28 DEG C, dark culturing 2d, picking list bacterium colony PCR detection validation, adopt primer to be primer 1 in step one and primer 2, size will be obtained through pcr amplification be about the recombinational agrobacterium name GV3101/MIR166a of 525bp object band.Setting simultaneously proceeds to the Agrobacterium of pSuper1300+ empty carrier in contrast, by gained corresponding recombinational agrobacterium called after GV3101/pSuper1300+.
(2) preparation of acceptor material and preculture
Tamarack cells,primordial is transferred to new substratum S
0after (being specially the proliferated culture medium recorded in " application number is 200510090282.X, and Authorization Notice No. is the patent of CN100427586C ") upper 15d, the vigorous embryonal connective tissue of picking surface growth is about the acceptor material of 1g as genetic transformation.
(3) agrobacterium liquid preparation.
Substratum Y0(1L): be specially in the proliferated culture medium recorded in " application number is 200510090282.X, and Authorization Notice No. is the patent of CN100427586C " and do not add agar.
Dip-dyeing solution Y1(1L): substratum Y0+AS10-40mg; PH5.75 ~ 5.85.
Take out and be stored in the glycerine Agrobacterium (recombinational agrobacterium GV3101/MIR166a or recombinational agrobacterium GV3101/pSuper1300+) of-80 DEG C, be placed in and make it dissolve on ice, then be placed in 4 DEG C and save backup.Take out the recombinational agrobacterium thawed, at YEB solid medium (adding Rifampin mycin, each 50mg/L of gentamicin, kantlex) upper stroke plate, 25 DEG C of dark culturing.After about 48h, recombinational agrobacterium grows.Picking list bacterium colony from flat board, is inoculated in YEB liquid nutrient medium (adding microbiotic same as described above), 28 DEG C, dark, 180rpm shakes cultivation.After about 18h, recombinational agrobacterium grows to logarithmic phase, and bacterial concentration is about 0.6 ~ 0.8(OD
600nm) time, transfer them in centrifuge tube, transfer to low-temperature and high-speed whizzer after trim, 4000rpm, 4 DEG C of centrifugal 10min.
Carefully remove supernatant, by the bacterium liquid dip-dyeing solution Y collected
1resuspended.Transfer in sterilized triangular flask, and be adjusted to desired concn 0.4(OD
600nm).
(4) infect, Dual culture and resistance screening.
Substratum S
1(1L): substratum S
0+ AS10-40mg; PH5.75 ~ 5.85.
Substratum S
2(1L): substratum S
0+ cephamycin 200-700mg; PH5.75 ~ 5.85.
Substratum S
3(1L): substratum S
0+ cephamycin 200-700mg+ Totomycin 3-20mg; PH5.75 ~ 5.85.
The cells,primordial collected is joined 20-30min in the above-mentioned recombinational agrobacterium liquid prepared.Triangular flask is left standstill, removes supernatant liquid.Cells,primordial is transferred to solid medium S
1on.Postvaccinal material is placed in dark training room, 25 DEG C of Dual culture 3-5d.After growing recombinational agrobacterium around embryonal connective tissue block, with the careful picking of tweezers, be transferred in sterilized triangular flask.Be limpid clear to supernatant liquor after cleaning repeatedly, remove supernatant liquid, with the liquid nutrient medium S containing cephamycin
2cleaning 20min.Collect cells,primordial, be transferred to containing antibacterial antibiotic substratum S
2on, renewal cultivation 3-7d.The callus of renewal cultivation is transferred to screening culture medium S
3on, wait for growing of resistant tissues, resistant tissues is screened again, until resistance, obtain the transgenosis tamarack embryogenic cell line proceeding to recombinant expression vector pSuper::MIR166a, and proceed to the transgenosis tamarack embryogenic cell line of pSuper1300+ empty carrier.Wherein, the transgenosis tamarack embryogenic cell line proceeding to recombinant expression vector pSuper::MIR166a obtains 5 altogether, is designated as pSuper::MIR166a1-5(respectively and is called for short a1-a5).
2, the qualification of transgenosis tamarack embryogenic cell line
(1) PCR detects
5 transgenosis tamarack embryogenic cell line a1-a5 proceeding to recombinant expression vector pSuper::MIR166a obtaining of extraction step 1 respectively, and the genomic dna of transgenosis tamarack embryogenic cell line proceeding to pSuper1300+ empty carrier.Be simultaneously negative control with non-transformed tamarack cells,primordial, with recombinant expression vector pSuper::MIR166a and recombinational agrobacterium GV3101/MIR166a bacterium liquid for positive control.Purpose of design gene (being positioned on carrier, across goal gene LaMIR166a) Auele Specific Primer and hygromycin phosphotransferase gene (HPT) Auele Specific Primer respectively.In addition, in order to get rid of the false positive that positive transformed cells occurs by Agrobacterium pollutes, design VirG gene (being positioned at outside T-DNA) Auele Specific Primer detects Agrobacterium.
The Auele Specific Primer of goal gene LaMIR166a, as follows:
Upstream primer: 5 '-AGATACGCTGACACGCCAA-3 ';
Downstream primer: 5 '-CCGGACACGCTGAACTTG-3 '.
Hygromycin phosphotransferase gene (HPT) Auele Specific Primer, as follows:
Upstream primer: 5 '-ATGGCATCTGCAACATTGTCCA-3 ';
Downstream primer: 5 '-ATAGATACGCTGACACGCCA-3 '.
VirG gene (being positioned at outside T-DNA) Auele Specific Primer, as follows:
Upstream primer: 5 '-GATTTTATTGCCAAGCCTTTT-3 ';
Downstream primer: 5 '-TACTTCTGTCATACACCTCCTCC-3 '.
Adopt Ex-Taq enzyme (Takara) to carry out pcr amplification, reaction system is as follows: Ex-Taq mix10 μ L, each 1 μ L of upstream and downstream primer, template DNA 1 μ L, adds water and mends to 20 μ L.
1.2% agarose gel electrophoresis detects PCR result.As shown in Figure 2, wherein A in goal gene LaMIR166a(Fig. 2) Auele Specific Primer all amplifies the object band that size is about 800bp in 5 transgenosis tamarack embryogenic cell line a1-a5 and positive control proceeding to recombinant expression vector pSuper::MIR166a, and in the transgenosis tamarack embryogenic cell line proceeding to pSuper1300+ empty carrier and non-transgenic tamarack embryogenic cell line, fail to expand object band, consistent with expected results; Hygromycin phosphotransferase gene (HPT) (in Fig. 2 B) Auele Specific Primer proceeds in the transgenosis tamarack embryogenic cell line a1-a5 of recombinant expression vector pSuper::MIR166a, the transgenosis tamarack embryogenic cell line proceeding to pSuper1300+ empty carrier and positive control at 5 the object band all amplifying size and be about 500bp, and fails to expand object band in non-transgenic tamarack embryogenic cell line.Prove that 5 transgenosis tamarack embryogenic cell line a1-a5 proceeding to recombinant expression vector pSuper::MIR166a all carry goal gene and hygromycin phosphotransferase gene fragment thus.C in Agrobacterium virulent gene VirG(Fig. 2) the amplification of Auele Specific Primer show only in Agrobacterium, to amplify object band, therefore 5 transgenosis tamarack embryogenic cell line a1-a5 proceeding to recombinant expression vector pSuper::MIR166a are not all subject to the residual pollution of Agrobacterium, thus eliminate false positive.
(2) qRT-PCR detects
The transgenosis tamarack embryogenic cell line a1-a5 of recombinant expression vector pSuper::MIR166a is proceeded to 5 of step 1 acquisition, proceed to the transgenosis tamarack embryogenic cell line of pSuper1300+ empty carrier, and non-transformed tamarack cells,primordial is experiment material.Extract the total serum IgE of each experiment material.In order to get rid of the interference of genomic dna to follow-up test, in 1 μ g total serum IgE, add 1 μ L DNase(Fermentas, Canada) carry out digesting and remove genomic dna residual in RNA.Add 1 μ L buffer again, add water and mend to 10 μ L; 37 DEG C of reaction 30min; Add 1 μ L EDTA, hatch 10min for 65 DEG C and make DNase enzyme deactivation with termination reaction.
Use Reverse Transcription box (Fermentas), RNA reverse transcription is become cDNA.Following component is added: 1 μ L oligo(dT in the RNA that previous step has digested) primer, 4 μ L Buffer, 1 μ L RNase Inhibitor, 2 μ L dNTPmix, 1 μ L ThermoScript II.Reaction conditions 42 DEG C, 60min; 72 DEG C, 5min; Be stored in after having reacted-80 DEG C for subsequent use.
QRT-PCR adopts the precious biotechnology of test kit SYBR Premix EX Taq Kit(), operate according to test kit specification sheets.By cDNA dilute after, get 2 μ L at 7500Real-time PCR System(Applied Biosystems, USA) on carry out qRT-PCR augmentation detection LaMIR166a gene expression amount.Set up 20 μ L reaction systems as follows: SYBR Premix Ex TaqTM10 μ L, forward primer (concentration 10 μMs) 0.4 μ L, reverse primer (concentration 10 μMs) 0.4 μ L, ROX Reference Dye II0.4 μ L, cDNA2.0 μ L, dH
2o6.8 μ L.
Forward primer: 5 '-CTTACGCCGCCGTCTATTCA-3 ' (the 38-57 position of sequence 3);
Reverse primer: 5 '-GCCAACCATTCCCAAGCTAT-3 ' (reverse complementary sequence of the 193-212 position of sequence 3).
Response procedures: 95 DEG C, 30s; 95 DEG C, 5s, 60 DEG C, 34s, repeats 40 circulations.
Reference gene adopts LaEF1A1(GenBank:JX157845).The amplimer of reference gene is 5 '-GACTGTACCTGTTGGTCGTG-3 ' and 5 '-CCTCCAGCAGAGCTTCAT-3 '.
Use relative quantitation method calculate data and add up, each sample repeats 4 times, averages.By solubility curve analysis (adopting machine default program) and 1.5% agarose gel electrophoresis analysis after PCR, checking PCR result is specific amplification.
The relative expression quantity of LaMIR166a gene (utilizes qRT-PCR technology, adopts 2
-Δ Δ Ctmethod calculate) qRT-PCR detected result as shown in Figure 3,5 expression levels proceeding to LaMIR166a gene in the transgenosis tamarack embryogenic cell line a1-a5 of recombinant expression vector pSuper::MIR166a that step 1 obtains all significantly raise than non-transgenosis tamarack embryogenic cell line in contrast, and expression level in a4 is the highest.For the transgenosis tamarack embryogenic cell line proceeding to pSuper1300+ empty carrier, the expression level of its LaMIR166a gene is basically identical with non-transgenosis tamarack in contrast, without significant difference.
3, the larchen acquisition of transgenosis
By the transgenosis tamarack embryogenic cell line a4 proceeding to recombinant expression vector pSuper::MIR166a the highest for the expression amount through above-mentioned qualification LaMIR166a gene, be transferred to the generation of maturation medium (being specially the maturation medium recorded in " application number is 200510090282.X; Authorization Notice No. is the patent of CN100427586C ") upper somatic embryos with ripe, obtain well-developed mature somatic embryo, be placed on root media a17(1/2MS+IBA1.0mg/L+ sucrose 20g/L+ agar 5.5g/L) until root.By well-grown tissue cultured seedling, be transplanted to greenhouse and continue to cultivate, final acquisition proceeds to the transgenosis tamarack a4 of recombinant expression vector pSuper::MIR166a.
The larchen Function Identification of embodiment 2, transgenosis
What obtain with embodiment 1 proceeds to the transgenosis tamarack a4 of recombinant expression vector pSuper::MIR166a, proceeds to the transgenosis tamarack of pSuper1300+ empty carrier, and not genetically modified tamarack is experiment material.The mature somatic embryo of each experiment material is placed in root media a17(1/2MS+IBA1.0mg/L+ sucrose 20g/L+ agar 5.5g/L) on, in 4 DEG C, process 7d under dark surrounds, then be placed in 25 DEG C, cultivate under illumination (16h/8h) condition, observe and record the plant forms, plant growth rate, root system development situation etc. of each experiment material.After the plant of each experiment material roots, well-grown tissue cultured seedling is transplanted to greenhouse and continues to cultivate.Experiment in triplicate.
Result shows, and when root media a17 cultivates 4 months, not genetically modified larchen tissue cultured seedling only has main root to extend, and do not have side root to occur, plant strain growth is slow; Comparatively speaking, the tissue cultured seedling side root proceeding to the transgenosis tamarack a4 of recombinant expression vector pSuper::MIR166a occurs in a large number, and plant growth rate is significantly faster than not genetically modified adjoining tree.Its form is concrete as shown in Figure 4.And for the transgenosis tamarack proceeding to pSuper1300+ empty carrier, its plant forms, plant growth rate, root system development situation etc. are all basically identical with not genetically modified adjoining tree, without significant difference.These results suggest that, process LAN MIR166a can promote the generation of tamarack tissue cultured seedling side root, thus is beneficial to its transplanting survival rate of raising.
Claims (9)
1. the nucleic acid molecule shown in arbitrary in following (1)-(4) is in the developmental application of Promoting plant growth:
(1) RNA molecule shown in sequence 1 in sequence table;
(2) RNA molecule shown in sequence 2 in sequence table;
(3) DNA molecular shown in sequence 3 in sequence table;
(4) DNA molecular shown in 27-539 position of sequence 3 in sequence table.
2. application according to claim 1, is characterized in that: described Promoting plant growth grows at least one be embodied in following (A)-(C):
(A) growth of plant lateral roots is promoted;
(B) plant growth rate is improved;
(C) plant survival rate is improved.
3. in following (1)-(4), arbitrary shown nucleic acid molecule has the application in the plant variety of at least one in following (a)-(c) in seed selection:
(1) RNA molecule shown in sequence 1 in sequence table;
(2) RNA molecule shown in sequence 2 in sequence table;
(3) DNA molecular shown in sequence 3 in sequence table;
(4) DNA molecular shown in 27-539 position of sequence 3 in sequence table;
(I) side root well developed root system;
(II) plant growth rate improves;
(III) survival rate of plant improves.
4. cultivate the method for transgenic plant, comprise following step a) and b):
A) in object plant, import arbitrary shown nucleic acid molecule in following (1)-(4), obtain the transgenic plant of expressing described nucleic acid molecule;
(1) RNA molecule shown in sequence 1 in sequence table;
(2) RNA molecule shown in sequence 2 in sequence table;
(3) DNA molecular shown in sequence 3 in sequence table;
(4) DNA molecular shown in 27-539 position of sequence 3 in sequence table;
B) obtain from step a) gained transgenic plant compared with described object plant, there are the transgenic plant of at least one in following (I)-(III):
(I) side root well developed root system;
(II) plant growth rate improves;
(III) survival rate of plant improves.
5. method according to claim 4, is characterized in that: the DNA molecular shown in sequence 3 in described sequence table, is to be proceeded in described object plant by the form of recombinant expression vector.
6. method according to claim 5, is characterized in that: described recombinant expression vector starting the promotor that in described sequence table, the DNA molecular shown in sequence 3 is transcribed is Super promotor.
7. application according to claim 6 or method, is characterized in that: described recombinant expression vector in the multiple clone site place insertion sequence table of pSuper1300+ carrier sequence 3 27-539 position shown in DNA molecular after the recombinant plasmid that obtains.
8., according to described application arbitrary in claim 1-7 or method, it is characterized in that: described plant is gymnosperm.
9. application according to claim 8 or method, is characterized in that: described gymnosperm is tamarack.
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| CN114457074B (en) * | 2021-09-27 | 2024-05-24 | 中国林业科学研究院林业研究所 | MiRNA related to ammonium nitrogen response of woody plants and application thereof |
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