CN104450770B - Applications of the larch miR166a in regulating development of plants - Google Patents
Applications of the larch miR166a in regulating development of plants Download PDFInfo
- Publication number
- CN104450770B CN104450770B CN201310452485.3A CN201310452485A CN104450770B CN 104450770 B CN104450770 B CN 104450770B CN 201310452485 A CN201310452485 A CN 201310452485A CN 104450770 B CN104450770 B CN 104450770B
- Authority
- CN
- China
- Prior art keywords
- sequence
- plant
- table shown
- sequence table
- larch
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Expired - Fee Related
Links
Landscapes
- Breeding Of Plants And Reproduction By Means Of Culturing (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
The invention discloses a kind of applications of larch miR166a in regulating development of plants.Application provided by the present invention is specially as follows（1）‑（4）In it is any shown in nucleic acid molecules promote growth and development of plants in application：（1）RNA molecule in sequence table shown in sequence 1；（2）RNA molecule in sequence table shown in sequence 2；（3）DNA molecular in sequence table shown in sequence 3；（4）DNA molecular in sequence table shown in the 27th 539 of sequence 3.It is demonstrated experimentally that when the transfer-gen plant of LaMIR166a gene overexpressions is cultivated 4 months on root media a17, there is substantial amounts of lateral root, and plant strain growth is good；Rather than non-transgenic control lines, only main root elongation, there is no lateral root, and plant strain growth is slow by contrast.This explanation, which is overexpressed miR166a, can promote the growth rate of larch tissue-cultured seedling, promote the generation of lateral root, so as to improve its transplanting survival rate.
Description
Technical field
The invention belongs to molecular biology of plants technical field, it is related to a kind of larch miR166a in regulating development of plants
In application, more particularly to a kind of larch miR166a promote the developmental application of plant lateral roots.
Background technology
Larch（Larix spp.）It is that the important fast-growing afforestation of northern China uses material coniferous species, available for timber and making
Paper material.Globally, larch natural distributed is in temperate zone mountain area, the Plain of cool temperature zone and alpine climate area, preceding two band
Substantially between 48 °~60 ° of north latitude, the characteristics of with high latitude High aititude.The larch of China's natural distributed has 10 kinds and 5
Individual mutation.They are distributed in the mountain region and alpine region in northwest, North China, Central China, northeast and southwest respectively.Efforts To Develop larch
Scientific research, for northern China Ecological Civilization Construction, promote national economy development it is significant.Larch conduct
The important needle fast-growing afforestation commerical tree species of China, are largely bred and genetic improvement to it, without disclosure satisfy that people couple
The growing demand of wood raw material, also can make tremendous contribution for China's Ecological Civilization Construction.
MicroRNAs（miRNAs）It is the endogenic non-coding with adjusting function of the class found in eucaryote
RNA, its size is about 20~25 nucleotides.Ripe miRNAs is to pass through a series of nucleases by longer primary transcript
Shearing and produce, be subsequently assembled into RNA induce silencing complex, target is recognized by way of base pair complementarity
MRNA, and silencing complex degraded said target mrna is instructed according to the difference of complementarity or the translation of said target mrna is checked.
The target gene of Mirnas of plant is the transcription factor in plant mostly, in the growing of plant, environment response
With it is disease-resistant response etc. all many-sides play extensive regulating and controlling effect.Screened by forward genetics, Direct Cloning, computer forecast and
Est sequence is analyzed, and identifies about 6000 miRNA in 67 kinds of plants at present（miRBase19.0）.As high pass is measured
MiRNA is pre- by bioinformatics in genomic level in the development of sequence technology and the decline that cost is sequenced, increasing plant
Measure and.And one of ultimate challenge studied at present is, how to verify single miRNA and its corresponding target gene in biology
The function of playing in vivo.
The content of the invention
It is an object of the invention to provide a kind of larch miR166a new application.
New application provided by the present invention, is specially as follows（1）-（4）In it is any shown in nucleic acid molecules promote plant
Application in growing：
（1）RNA molecule in sequence table shown in sequence 1；
（2）RNA molecule in sequence table shown in sequence 2（Larch precursor miR166a）；
（3）DNA molecular in sequence table shown in sequence 3（Larch precursor miR166a full-length cDNA, is named as
LaMIR166a genes）；
（4）DNA molecular in sequence table shown in 27-539 of sequence 3.
In above-mentioned application, the promotion growth and development of plants specifically may be embodied in as follows（A）-（C）At least one of：
（A）Promote the development of plant lateral roots；
（B）Improve plant growth rate；
（C）Improve plant survival rate.
In above-mentioned application,（A）Described in " promote plant lateral roots development " to promote the generation of plant lateral roots；（B）In
Described " raising plant growth rate " is raising tissue-cultured seedling（The tissue-cultured seedling of culture 4 months such as on root media）Growth
Speed；（C）Described in " raising plant survival rate " for improve plant transplantation after survival rate.
It is as follows（1）-（4）In it is any shown in nucleic acid molecules seed selection have it is as follows（I）-（III）At least one of
Application in plant variety falls within protection scope of the present invention：
（1）RNA molecule in sequence table shown in sequence 1（Larch maturation miR166a）；
（2）RNA molecule in sequence table shown in sequence 2（Larch precursor miR166a）；
（3）DNA molecular in sequence table shown in sequence 3（LaMIR166a genes）；
（4）DNA molecular in sequence table shown in 27-539 of sequence 3.
（I）Lateral root well developed root system；
（II）Plant growth rate is improved；
（III）Survival rate of plant is improved.
In actual applications, seed selection has as described above（I）-（III）At least one of plant variety when, need to be by institute
State（1）-（4）In it is any shown in the higher embryogenic cell line of nucleic acid molecules expression quantity, be used as the cell for obtaining purpose transgenosis
System.
It is also another object of the present invention to provide a kind of method for cultivating genetically modified plants.
The method provided by the present invention for cultivating genetically modified plants, specifically may include following a）And b）The step of：
a）Imported into purpose plant as follows（1）-（4）In it is any shown in nucleic acid molecules, obtain expressing the nucleic acid point
The genetically modified plants of son；
（1）RNA molecule in sequence table shown in sequence 1（Larch maturation miR166a）；
（2）RNA molecule in sequence table shown in sequence 2（Larch precursor miR166a）；
（3）DNA molecular in sequence table shown in sequence 3（LaMIR166a genes）；
（4）DNA molecular in sequence table shown in 27-539 of sequence 3.
b）From step a）Obtained in gained genetically modified plants compared with the purpose plant, with as follows（I）-（III）In
At least one genetically modified plants：
（I）Lateral root well developed root system；
（II）Plant growth rate is improved；
（III）Survival rate of plant is improved.
Wherein, sequence 1 is made up of 21 nucleotides, is larch maturation miR166a sequences；Sequence 2 is by 95 nucleotides
Composition, is larch precursor miR166a sequences；Sequence 3 is made up of 884 deoxynucleotides, is larch precursor miR166a
Full length cDNA sequence, as LaMIR166a genes.
In the present invention, used when cultivating genetically modified plants by shown in 27-539 of sequence in sequence table 3
DNA molecular is imported in the purpose plant.Further, in the sequence table shown in 27-539 of sequence 3 DNA points
Son, is to be transferred to by the form of recombinant expression carrier in the purpose plant.
Further, the DNA shown in 27-539 of sequence 3 in the sequence table is started on the recombinant expression carrier
The promoter of molecule transcription is Super promoters.
More specifically, the recombinant expression carrier is the multiple cloning sites in pSuper1300+ carriers（Such as Hind III
With Kpn I）The recombinant plasmid obtained after DNA molecular in place's insetion sequence table shown in 27-539 of sequence 3.
Certainly, the recombinant expression carrier can also use existing other plant expression vector establishment, and such as double base Agrobacterium carries
Body and carrier available for plant micropellet bombardment etc., it is more specific as pGreen0029, pCAMBIA3301, pCAMBIA1300,
The derivative plant expression vector of pBI121, pBin19, pCAMBIA2301, pCAMBIA1301-UbiN or other.The plant expression
Carrier can also include 3 ' end untranslated regions of foreign gene, i.e., processed comprising polyadenylation signals and any other participation mRNA
Or the DNA fragmentation of gene expression.The bootable polyadenylic acid of polyadenylation signals is added to 3 ' ends of mRNA precursor.Using institute
, can be plus any enhanced, composing type, tissue before its transcription initiation nucleotides when stating gene constructed recombinant expression carrier
Idiotype or inducible promoter, such as cauliflower mosaic virus（CAMV）35S promoter, ubiquitin gene Ubiquitin start
Son（pUbi）, stress induced promoter rd29A etc., they can be used alone or are used in combination with other plant promoters；
In addition, when using the gene constructed recombinant expression carrier of the present invention, enhancer is it is also possible to use, including translational enhancer or transcription increase
Hadron, these enhancer regions can be ATG initiation codon or neighboring region initiation codon etc., but required and coded sequence
Reading frame it is identical, to ensure the correct translation of whole sequence.The source of the translation control signal and initiation codon is wide
General, can be natural or synthesis.Translation initiation region can come from transcription initiation region or structural gene.
For the ease of transgenic plant cells or plant are identified and screened, recombinant expression carrier used can be processed, such as
The coding that adding can express in plant can produce the enzyme of color change or the gene of luminophor, resistant antibiotic
Label or anti-chemical reagent marker gene etc..Also any selected marker can be not added with, is directly screened and converted with adverse circumstance
Plant.
In the method for above-mentioned cultivation genetically modified plants, the recombinant expression carrier is imported into the purpose plant, specifically
Can be：By using Ti-plasmids, Ri plasmids, plant viral vector, directly delivered DNA, microinjection, conductance, agriculture bacillus mediated
Plant cell or tissue are converted Deng conventional biology methods, and the plant tissue of conversion is cultivated into plant.
It is all in above-mentioned each application or each method（I）Described in " lateral root well developed root system " increase for lateral root number；
All（II）Described in " plant growth rate raising " be tissue-cultured seedling（The tissue culture of culture 4 months such as on root media
Seedling）Growth rate is improved；All（III）Described in " survival rate of plant raising " for transplant after survival rate of plant improve.
In above-mentioned each application or each method, the plant can be gymnosperm.
In the present invention, the gymnosperm is larch, concretely larch-tree（Larix
leptolepis）, larix olgensis（Larix olgensis Henry）, larch in Xinanlin area（Larix gmelinii）Or North China
Larch（Larix principis-rupprechtii Mayr）.
It is demonstrated experimentally that the DNA molecular shown in 27-539 of sequence in sequence table 3 is overexpressed in larch, obtain
Obtain transfer-gen plant.As a result show that the transfer-gen plant of LaMIR166a gene overexpressions cultivates 4 on root media a17
During the moon, there is substantial amounts of lateral root, and plant strain growth is good；Rather than non-transgenic control lines, only main root extends, without lateral root
Occur, and plant strain growth is slow by contrast.This explanation be overexpressed miR166a can promote larch tissue-cultured seedling growth rate,
Promote the generation of lateral root, so as to improve its transplanting survival rate.
Brief description of the drawings
Fig. 1 is precursor miR166a RNA secondary structures.It is maturation miR166a sequence in square frame.
Fig. 2 is the larchen PCR testing results of transgenosis.Wherein, A is purpose gene LaMIR166a amplification；Respectively
Swimming lane template DNA is respectively：Water, negative control（CK-, the larch embryogenic cell line of non-transgenosis）, a1~a5, positive control
（CK+, imports recombinant expression carrier pSuper::MIR166a transgenosis larch embryogenic cell line）；Swimming lane M is DNA molecular
Amount standard.B is hygromycin phosphotransferase gene（HPT）Amplification；Each swimming lane template DNA is respectively：Water, negative control
（CK-, the larch embryogenic cell line of non-transgenosis）, a1~a5, positive control（CK+, imports recombinant expression carrier pSuper::
MIR166a transgenosis larch embryogenic cell line）；Swimming lane M is DNA molecular amount standard.C is Agrobacterium virulent gene VirG's
Amplification；Each swimming lane template DNA is respectively：Water, negative control（CK-, the larch embryogenic cell line of non-transgenosis）, a1~
A5, positive control（CK+, Agrobacterium GV3101）Swimming lane M is DNA molecular amount standard.
Fig. 3 is the larchen qRT-PCR testing results of transgenosis.
The form of transgenosis larch tissue-cultured seedling when Fig. 4 is cultivates 4 months on root media a17.Wherein, A is not
Transgenosis larch tissue-cultured seedling；B is to be transferred to recombinant expression carrier pSuper::MIR166a transgenosis larch tissue-cultured seedling a4.
Embodiment
Experimental method used in following embodiments is conventional method unless otherwise specified.
Material, reagent used etc., unless otherwise specified, are commercially obtained in following embodiments.
PSuper1300+ carriers：It is recorded in that " Zhu Caihong, Li Shuigen, Qi Liwang, Han Su English agriculture bacillus mediated Japan falls
Leaf pine cells,primordial Study on Genetic Transformation Chinese biological engineering magazines, 2013,33（5）:The texts of 75-80 " one.
Agrobacterium GV3101：It is recorded in that " the different Agrobacterium tumefaciens strain types of Zhao Zhan, Li Wen life turn to Trichoderma heredity
Change the northern gardening of influence of efficiency, 03 phase in 2006 " text.
Embodiment 1, miR166a are overexpressed the acquisition and identification of genetically modified plants
Involved miR166a is larch-tree in the present embodiment（Larix leptolepis）MiR166a.Its
In, the sequence of ripe miR166a sequences is sequence 1 in sequence table；The sequence of precursor miR166a sequences is sequence 2 in sequence table；
The full-length cDNA of precursor miR166a sequences is sequence 3 in sequence table, is named as LaMIR166a genes.Wherein, precursor miR166a
RNA secondary structures it is as shown in Figure 1.
First, recombinant expression carrier pSuper::MIR166a structure
1st, design of primers
The full-length cDNA of precursor miR166a sequences according to sequence in sequence table 3（LaMIR166a genes）, at this
Primer is designed at each about 200bp in the two ends of miR166a loop-stem structure sequences on cDNA, it is as follows：
Primer 1：5’-CCCAAGCTTGCAACACCATACTTACGCC-3’（Underscore part is Hind III identification sequence
Row, sequence thereafter is 27-45 of sequence 3；
Primer 2：5’-GGGGTACCCCCTGGAAAACTCACCTATAC-3’（Underscore part is Kpn I identification sequence
Row, sequence thereafter is the reverse complementary sequence of 519-539 of sequence 3）
2nd, PCR is expanded
Extract larch-tree（Larix spp.）Total serum IgE, obtain cDNA sequence after reverse transcription.Using gained cDNA as mould
Plate, using above-mentioned primer pair, enters performing PCR amplification, the nucleotides sequence through sequencing identification PCR primer is classified as " CCCAAGCTT+ sequence
27-539 of the sequence 3 of table+GGTACCCC”。
3rd, vector construction
With restriction enzyme Hind III and Kpn I double digestion step 2 gained PCR primers, after glue reclaim with by same
The skeleton large fragment of the pSuper1300+ carriers of sample double digestion is connected, and obtains recombinant plasmid.Hind is carried out to gained recombinant plasmid
III and Kpn I double digestions identify that identification is correct（It is about 10kbp and 525bp to obtain size）Sample presentation is sequenced afterwards.Will be through table be sequenced
It is bright between multiple cloning sites Hind III and the Kpn I of pSuper1300+ carriers in insetion sequence table sequence 3 27-539
The recombinant plasmid obtained after DNA molecular shown in position, is named as pSuper::MIR166a.
In recombinant expression carrier pSuper::In MIR166a, in initiating sequence table shown in 27-539 of sequence 3
The promoter of DNA molecular transcription is Super promoters.
Certainly, recombinant expression carrier pSuper is being built::During MIR166a, it would however also be possible to employ sequence in sequence table
DNA molecular shown in 3 enters performing PCR amplification for template.
2nd, transgenosis is larchen obtains
1st, the acquisition of transgenosis larch embryogenic cell line
（1）The preparation of recombinational agrobacterium
YEB fluid nutrient mediums（1L）：Beef extract 5g+ yeast extract 5g+ tryptone 5g+ sucrose 5g+MgSO4·
7H2O0.493g, solvent is water；pH7.0.
YEB solid mediums（1L）：Beef extract 5g+ yeast extract 5g+ tryptone 5g+ sucrose 5g+MgSO4·
7H2O0.493g+ agar powder 15g, solvent is water；pH7.0.
The recombinant expression carrier pSuper that step one is obtained::MIR166a imports Agrobacterium GV3101.Concrete operations are such as
Under：By 10 μ L recombinant expression carriers pSuper::MIR166a is added in 200 μ L Agrobacteriums GV3101 competent cell, gently
It is light to mix；Ice bath 30min, liquid nitrogen frozen 3~5min, 37 DEG C of water-bath 5min；Addition 1mL YEB fluid nutrient mediums, 28 DEG C,
180rpm shaken cultivations 4h；4000rpm, room temperature centrifugation 8min, abandon supernatant, add 200 μ L YEB fluid nutrient mediums and thalline is resuspended,
It is coated on YEB film solid medias（Containing rifampin, gentamicin and each 50mg/L of kanamycins）；28 DEG C, dark culturing 2d, picking
Single bacterium colony and PCR detection checkings, primer 1 and primer 2 in using primer for step one will obtain size about through PCR amplifications
GV3101/MIR166a is named for the recombinational agrobacterium of 525bp purpose bands.Set simultaneously and be transferred to pSuper1300+ empty carriers
Gained correspondence recombinational agrobacterium is named as GV3101/pSuper1300+ by Agrobacterium as control.
（2）The preparation of acceptor material and preculture
Larch cells,primordial is transferred to new culture medium S0（Specially " Application No. 200510090282.X is authorized
Notification number be CN100427586C patent " described in proliferated culture medium）After upper 15d, the vigorous embryo of picking superficial growth
About 1g is organized as the acceptor material of genetic transformation.
（3）It is prepared by agrobacterium liquid.
Culture medium Y0（1L）：Specially " Application No. 200510090282.X, Authorization Notice No. is CN100427586C's
Agar is not added with proliferated culture medium described in patent ".
Dip dyeing liquid for shell Y1（1L）：Culture medium Y0+AS10-40mg；PH5.75~5.85.
Taking-up is stored in -80 DEG C of glycerine Agrobacterium（Recombinational agrobacterium GV3101/MIR166a or recombinational agrobacterium
GV3101/pSuper1300+）, be placed in dissolves it on ice, then is placed in 4 DEG C and saves backup.Take out the restructuring agriculture bar thawed
Bacterium, in YEB solid mediums（Add rifampin mycin, each 50mg/L of gentamicin, kanamycins）It is upper to draw plate, 25 DEG C of dark trainings
Support.After about 48h, recombinational agrobacterium is grown.The picking single bacterium colony from flat board, is inoculated in YEB fluid nutrient mediums（Addition with it is above-mentioned
Identical antibiotic）In, 28 DEG C, dark, 180rpm concussion and cultivates.After about 18h, recombinational agrobacterium length to logarithmic phase, bacterium solution is dense
Degree about 0.6~0.8（OD600nm）When, transfer them in centrifuge tube, low-temperature and high-speed centrifuge be transferred to after trim,
4000rpm, 4 DEG C of centrifugation 10min.
Supernatant carefully is removed, by the bacterium solution being collected into dip dyeing liquid for shell Y1It is resuspended.It is transferred in sterilized triangular flask, and adjusts
To required concentration 0.4（OD600nm）.
（4）Infect, co-culture and resistance screening.
Culture medium S1（1L）：Culture medium S0+AS10-40mg；PH5.75~5.85.
Culture medium S2（1L）：Culture medium S0+ cephalosporin 200-700mg；PH5.75~5.85.
Culture medium S3（1L）：Culture medium S0+ cephalosporin 200-700mg+ hygromycin 3-20mg；PH5.75~5.85.
The cells,primordial being collected into is added to 20-30min in the above-mentioned recombinational agrobacterium liquid prepared.By triangular flask
Stand, remove supernatant liquid.Cells,primordial is transferred to solid medium S1On.Material after inoculation is placed in dark training room,
25 DEG C of co-cultivation 3-5d.After recombinational agrobacterium is grown around embryonal connective tissue block, with the careful picking of tweezers, it is transferred to sterilized
In triangular flask.Supernatant liquid is removed, with the fluid nutrient medium S containing cephalosporin in limpid clear to supernatant after cleaning repeatedly2
Clean 20min.Cells,primordial is collected, the culture medium S containing antibacterial antibiotic is transferred to2On, renewal cultivation 3-7d.By renewal cultivation
Callus be transferred to screening and culturing medium S3On, growing for resistant tissues is waited, resistant tissues are screened again, until resistance
Stable, acquisition is transferred to recombinant expression carrier pSuper::MIR166a transgenosis larch embryogenic cell line, and be transferred to
The transgenosis larch embryogenic cell line of pSuper1300+ empty carriers.Wherein, it is transferred to recombinant expression carrier pSuper::
MIR166a transgenosis larch embryogenic cell line obtains 5 altogether, and pSuper is designated as respectively::MIR166a1-5（Abbreviation a1-
a5）.
2nd, the identification of transgenosis larch embryogenic cell line
（1）PCR is detected
5 of the acquisition of extraction step 1 are transferred to recombinant expression carrier pSuper respectively::MIR166a transgenosis larch embryo
Property cell line a1-a5, and it is transferred to the genomic DNA of the transgenosis larch embryogenic cell line of pSuper1300+ empty carriers.Together
When using non-transformed larch embryogenic cell line as negative control, with recombinant expression carrier pSuper::MIR166a and restructuring agriculture
Bacillus GV3101/MIR166a bacterium solutions are positive control.Separately design target gene（On carrier, across target gene
LaMIR166a）Specific primer and hygromycin phosphotransferase gene（HPT）Specific primer.In addition, in order to exclude positive turn
Change the false positive that cell is polluted and occurred by Agrobacterium, design VirG genes（Outside T-DNA）Specific primer detects agriculture
Bacillus.
Target gene LaMIR166a specific primer, it is as follows：
Sense primer：5’-AGATACGCTGACACGCCAA-3’；
Anti-sense primer：5’-CCGGACACGCTGAACTTG-3’.
Hygromycin phosphotransferase gene（HPT）Specific primer, it is as follows：
Sense primer：5’-ATGGCATCTGCAACATTGTCCA-3’；
Anti-sense primer：5’-ATAGATACGCTGACACGCCA-3’.
VirG genes（Outside T-DNA）Specific primer, it is as follows：
Sense primer：5’-GATTTTATTGCCAAGCCTTTT-3’；
Anti-sense primer：5’-TACTTCTGTCATACACCTCCTCC-3’.
Using Ex-Taq enzymes（Takara）Enter performing PCR amplification, reaction system is as follows：Ex-Taq mix10 μ L, upstream and downstream is drawn
Each 1 μ L of thing, the μ L of template DNA 1, the benefit that adds water to 20 μ L.
1.2% agarose gel electrophoresis detects PCR results.As shown in Fig. 2 wherein target gene LaMIR166a（A in Fig. 2）
Specific primer is transferred to recombinant expression carrier pSuper at 5::MIR166a transgenosis larch embryogenic cell line a1-a5 and
The purpose band that size is about 800bp is amplified in positive control, and is fallen in the transgenosis for being transferred to pSuper1300+ empty carriers
Fail to expand purpose band in leaf pine embryogenic cell line and non-transgenic larch embryogenic cell line, it is consistent with expected results；Tide
Neomycin phosphotransferase gene（HPT）（B in Fig. 2）Specific primer is transferred to recombinant expression carrier pSuper at 5::MIR166a
Transgenosis larch embryogenic cell line a1-a5, be transferred to pSuper1300+ empty carriers transgenosis larch embryogenic cell line and
The purpose band that size is about 500bp is amplified in positive control, and is failed in non-transgenic larch embryogenic cell line
Expand purpose band.Thus 5 are proved and is transferred to recombinant expression carrier pSuper::MIR166a transgenosis larch cells,primordial
It is that a1-a5 carries target gene and hygromycin phosphotransferase gene fragment.Agrobacterium virulent gene VirG（C in Fig. 2）
The amplification of specific primer show only to amplify purpose band in Agrobacterium, therefore 5 are transferred to recombinant expression carrier
pSuper::The pollution that MIR166a transgenosis larch embryogenic cell line a1-a5 is not remained by Agrobacterium, so as to arrange
Except false positive.
（2）QRT-PCR is detected
5 obtained with step 1 are transferred to recombinant expression carrier pSuper::MIR166a transgenosis larch cells,primordial
It is a1-a5, is transferred to the transgenosis larch embryogenic cell line of pSuper1300+ empty carriers, and non-transformed larch embryo
Cell line is experiment material.Extract the total serum IgE of each experiment material.In order to exclude interference of the genomic DNA to follow-up test, in 1 μ
1 μ L DNase are added in g total serum IgEs（Fermentas, Canada）Carry out digestion and remove the genomic DNA remained in RNA.Again plus
Enter 1 μ L buffer, the benefit that adds water to 10 μ L；37 DEG C of reaction 30min；1 μ L EDTA are added, 65 DEG C are incubated 10min and lose DNase enzymes
Live with terminating reaction.
Use reverse transcription reagent box（Fermentas）, by RNA reverse transcriptions into cDNA.Add in the RNA that previous step has digested
Enter following components：1μL oligo（dT）Primer, 4 μ L Buffer, 1 μ L RNase Inhibitor, 2 μ L dNTPmix, 1 μ L are anti-
Transcriptase.42 DEG C of reaction condition, 60min；72 DEG C, 5min；Be stored in after the completion of reaction -80 DEG C it is standby.
QRT-PCR uses kit SYBR Premix EX Taq Kit（Precious bioengineering）, according to kit specification
Operated.After cDNA is diluted, 2 μ L are taken in 7500Real-time PCR System（Applied Biosystems,USA）
Upper progress qRT-PCR augmentation detection LaMIR166a gene expression amounts.Set up 20 μ L reaction systems as follows：SYBR Premix Ex
TaqTM10 μ L, forward primer（10 μM of concentration）0.4 μ L, reverse primer（10 μM of concentration）0.4 μ L, ROX Reference Dye
II0.4 μ L, cDNA2.0 μ L, dH2O6.8μL。
Forward primer：5’-CTTACGCCGCCGTCTATTCA-3’（38-57 of sequence 3）；
Reverse primer：5’-GCCAACCATTCCCAAGCTAT-3’（The reverse complemental sequence of 193-212 of sequence 3
Row）.
Response procedures：95 DEG C, 30s；95 DEG C, 5s, 60 DEG C, 34s repeats 40 circulations.
Reference gene uses LaEF1A1（GenBank：JX157845）.The amplimer of reference gene be 5 '-
GACTGTACCTGTTGGTCGTG-3 ' and 5 '-CCTCCAGCAGAGCTTCAT-3 '.
Data are calculated and counted using relative quantitation method, each sample is repeated 4 times, and is averaged.After PCR
Analyzed by solubility curve（Using machine default program）With the analysis of 1.5% agarose gel electrophoresis, checking PCR results are special
Property amplification.
The relative expression quantity of LaMIR166a genes（Using qRT-PCR technologies, using 2-ΔΔCtMethod is calculated）QRT-PCR
Testing result is as shown in figure 3,5 of step 1 acquisition are transferred to recombinant expression carrier pSuper::MIR166a transgenosis larch
The expression of LaMIR166a genes is used for the non-transgenosis larch cells,primordial of control in embryogenic cell line a1-a5
System's significantly rise, and the expression highest in a4.Transgenosis larch embryo for being transferred to pSuper1300+ empty carriers
Cell line, the expression of its LaMIR166a gene and basically identical, no significance difference as the non-transgenosis larch compareed
It is different.
3rd, transgenosis is larchen obtains
Expression quantity highest through above-mentioned identification LaMIR166a genes is transferred to recombinant expression carrier pSuper::
MIR166a transgenosis larch embryogenic cell line a4, is transferred to maturation medium（Specially " Application No.
200510090282.X, Authorization Notice No. be CN100427586C patent " described in maturation medium）Upper inducing somatic
The generation of embryo and ripe, the well-developed mature somatic embryo of acquisition, are placed on root media a17（1/2MS+
IBA1.0mg/L+ sucrose 20g/L+ agar 5.5g/L）Until rooting.By well-grown tissue-cultured seedling, be transplanted to greenhouse after
Continuous culture, final obtain is transferred to recombinant expression carrier pSuper::MIR166a transgenosis larch a4.
The larchen Function Identification of embodiment 2, transgenosis
Recombinant expression carrier pSuper is transferred to what embodiment 1 was obtained::MIR166a transgenosis larch a4, it is transferred to
The transgenosis larch of pSuper1300+ empty carriers, and the larch of non-transgenosis is experiment material.By each experiment material
Mature somatic embryo is placed in root media a17（1/2MS+IBA1.0mg/L+ sucrose 20g/L+ agar 5.5g/L）On, in 4 DEG C,
7d is handled under dark surrounds, 25 DEG C, illumination is subsequently placed in（16h/8h）Under the conditions of cultivate, observe and record the plant of each experiment material
Plant shape state, plant growth rate, root system development situation etc.., will be well-grown after the plant of each experiment material roots
Tissue-cultured seedling, which is transplanted to greenhouse, to be continued to cultivate.Experiment is in triplicate.
As a result show, when being cultivated 4 months on root media a17, the larchen tissue-cultured seedling of non-transgenosis only has master
Root extends, and does not have lateral root, plant strain growth is slow；Comparatively speaking, it is transferred to recombinant expression carrier pSuper::MIR166a's turns
Gene larch a4 tissue-cultured seedling lateral root largely occurs, and plant growth rate is significantly faster than the adjoining tree of non-transgenosis.Its shape
State is specifically as shown in Figure 4.And for the transgenosis larch for being transferred to pSuper1300+ empty carriers, its plant forms, plant
The adjoining tree with non-transgenosis such as growth rate, root system development situation is basically identical, no significant difference.Result above is said
Bright, the generation of larch tissue-cultured seedling lateral root can be promoted by being overexpressed MIR166a, so that beneficial to its transplanting survival rate of raising.
Claims (6)
1. application of the nucleic acid molecules shown in any in following (1)-(4) in growth and development of plants is promoted：
(1) RNA molecule in sequence table shown in sequence 1；
(2) RNA molecule in sequence table shown in sequence 2；
(3) DNA molecular in sequence table shown in sequence 3；
(4) DNA molecular in sequence table shown in 27-539 of sequence 3；
The promotion growth and development of plants is embodied in (A)-(C) as follows：
(A) development of plant lateral roots is promoted；
(B) plant growth rate is improved；
(C) plant survival rate is improved；
The plant is gymnosperm；The gymnosperm is larch.
2. nucleic acid molecules the answering in the plant variety that seed selection has following (I)-(III) shown in any in following (1)-(4)
With：
(1) RNA molecule in sequence table shown in sequence 1；
(2) RNA molecule in sequence table shown in sequence 2；
(3) DNA molecular in sequence table shown in sequence 3；
(4) DNA molecular in sequence table shown in 27-539 of sequence 3；
(I) lateral root well developed root system；
(II) plant growth rate is improved；
(III) survival rate of plant is improved；
The plant is gymnosperm；The gymnosperm is larch.
3. cultivate the method for the genetically modified plants with following (I)-(III), including it is following a) and b) the step of：
A) any shown nucleic acid molecules in following (1)-(4) are imported into purpose plant, obtain expressing the nucleic acid molecules
Genetically modified plants；
(1) RNA molecule in sequence table shown in sequence 1；
(2) RNA molecule in sequence table shown in sequence 2；
(3) DNA molecular in sequence table shown in sequence 3；
(4) DNA molecular in sequence table shown in 27-539 of sequence 3；
B) obtained from genetically modified plants obtained by step a) compared with the purpose plant, base is turned with following (I)-(III)
Because of plant：
(I) lateral root well developed root system；
(II) plant growth rate is improved；
(III) survival rate of plant is improved；
The plant is gymnosperm；The gymnosperm is larch.
4. method according to claim 3, it is characterised in that：DNA molecular in the sequence table shown in sequence 3, is logical
The form for crossing recombinant expression carrier is transferred in the purpose plant.
5. method according to claim 4, it is characterised in that：Start sequence in the sequence table on the recombinant expression carrier
The promoter of DNA molecular transcription shown in row 3 is Super promoters.
6. method according to claim 5, it is characterised in that：The recombinant expression carrier is in pSuper1300+ carriers
Multiple cloning sites at the recombinant plasmid that obtains after DNA molecular in insetion sequence table shown in 27-539 of sequence 3.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201310452485.3A CN104450770B (en) | 2013-09-25 | 2013-09-25 | Applications of the larch miR166a in regulating development of plants |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201310452485.3A CN104450770B (en) | 2013-09-25 | 2013-09-25 | Applications of the larch miR166a in regulating development of plants |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN104450770A CN104450770A (en) | 2015-03-25 |
| CN104450770B true CN104450770B (en) | 2017-08-15 |
Family
ID=52897520
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201310452485.3A Expired - Fee Related CN104450770B (en) | 2013-09-25 | 2013-09-25 | Applications of the larch miR166a in regulating development of plants |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN104450770B (en) |
Families Citing this family (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106047887B (en) * | 2016-05-04 | 2019-06-14 | 天津农学院 | Larch LkANT gene, albumen and application |
| 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 |
Family Cites Families (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US9562235B2 (en) * | 2009-12-06 | 2017-02-07 | A.B. Seeds Ltd. | MicroRNA compositions and methods for enhancing plant resistance to abiotic stress |
| BR112012033544A2 (en) * | 2010-06-30 | 2015-09-08 | Basf Plant Science Co Gmbh | isolated nucleic acid molecule, construct, vector, plant, plant cell or plant seed, methods, use and process |
| CN102888396B (en) * | 2012-09-06 | 2014-06-04 | 中国热带农业科学院橡胶研究所 | Method for separating low-molecular weight ribonucleic acid (RNA) of plant |
-
2013
- 2013-09-25 CN CN201310452485.3A patent/CN104450770B/en not_active Expired - Fee Related
Non-Patent Citations (2)
| Title |
|---|
| Genome-wide identification of microRNAs in larch and stage-specific modulation of 11 conserved microRNAs and their targets during somatic embryogenesis;Junhong Zhang et al.;《Planta》;20120413;第236卷;第647-657页 * |
| 日本落叶松小RNA文库构建及其microRNA鉴定;吴涛等;《林业科学研究》;20121231;第25卷(第6期);第677-684页 * |
Also Published As
| Publication number | Publication date |
|---|---|
| CN104450770A (en) | 2015-03-25 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN105132425B (en) | For enhancing the regulatory nucleic acid molecules that constitutive gene is expressed in plant | |
| CN103649314A (en) | Regulatory nucleic acid molecules for enhancing constitutive gene expression in plants | |
| AU2012238601B2 (en) | Seed - specific promoter in cotton | |
| US9957514B2 (en) | Transformation of duckweed and uses thereof | |
| CN113549635B (en) | Application of verticillium dahliae VdPRMT1 gene in improving disease resistance of crops or vegetables | |
| CN107937411A (en) | Chinese white poplar PtoWRKY40 genes, its expression vector and construction method and application | |
| CN105087640B (en) | Adjust gene and its application of vegetable seeds development | |
| CN115820686B (en) | Citrus CsGSTU18 gene and application thereof | |
| CN110938617B (en) | Lilium regale LrPAL-1 gene and application thereof | |
| CN115820685A (en) | Citrus CsGSTF1 gene and application thereof | |
| CN107828816A (en) | One primary yeast Agrobacterium shuttle vector and construction method and application | |
| CN107338264A (en) | The VIGS carriers of virus-mediated gene silencing | |
| CN102465114A (en) | Identification and application of specific expression promoter of plant root | |
| CN104450770B (en) | Applications of the larch miR166a in regulating development of plants | |
| CN106191075A (en) | A kind of Populus deltoides Bartr.cv. extensin gene and encoding proteins thereof and application | |
| CN110106269A (en) | Bna-miR156d is in the control developmental application of cabbage type rape branch | |
| CN103205456B (en) | A kind of plant binary expression vector | |
| BR112020008016A2 (en) | resistance to housing in plants | |
| Hai et al. | Characterization of the Populus PtrCesA4 promoter in transgenic Populus alba× P. glandulosa | |
| CN104109682B (en) | A kind of pectin lyase BnPL gene and promoter thereof and application | |
| CN106434692A (en) | Applications of rice OsPCF7 gene in culturing high-tillering rice varieties | |
| CN103966220B (en) | The root-specific promoter of synthetic and application thereof | |
| CN104140461B (en) | The LTP albumen relevant to plant cold tolerance and encoding gene thereof and application | |
| Zheng et al. | Sonication-assisted Agrobacterium-mediated transformation of the ACC gene to interfere the production of ethylene in spring Dendrobium cv.‘Sanya’ | |
| CN114480483B (en) | Method for silencing or knocking down gene expression in young fructus forsythiae fruits |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant | ||
| CF01 | Termination of patent right due to non-payment of annual fee | ||
| CF01 | Termination of patent right due to non-payment of annual fee |
Granted publication date: 20170815 Termination date: 20200925 |