CN109536515A - A kind of bean plant glycine-rich protein gene and its application - Google Patents
A kind of bean plant glycine-rich protein gene and its application Download PDFInfo
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Abstract
The present invention relates to field of biotechnology, specifically a kind of bean plant glycine-rich protein gene and its application, the nucleotide sequence of the bean plant glycine-rich protein gene is as shown in SEQ ID NO.1, the present invention is to further appreciate that the molecular mechanism of the Response to stress such as GRP gene participation arid is laid a good foundation, turn GRP over-express vector plant has stronger drought-resistant ability compared with CK, and fresh weight and the conspicuousness of ground dry weight become apparent from the ground, pass through plant height, overground part fresh weight, underground part fresh weight, overground part dry weight, the variation of this 5 indexs of underground part dry weight, it can be seen that, the drought-resistant ability of soybean is improved significantly.
Description
Technical field
The present invention relates to field of biotechnology, specifically a kind of bean plant glycine-rich protein gene and its application.
Background technique
Soybean is the very important cereal crops in China and oil crops, and cultivated area is only second to rice, corn and wheat,
Occupy the 4th.
Plant glycine-rich protein (GRP) is a kind of egg being made of in composed structure glycine highly repetitive sequence
White matter.Research in 1986 so far, separates and identifies more than 150 kinds of plants from the different plants such as corn, rice, arabidopsis, tobacco
Object glycine-rich protein, 1986, Codit and Meagher isolated gene PtGRP.1 from petunia.Reddy in 1987
GRP is extracted from strawberry with Poovaiah.Research finds that plant is rich in sweet all comprising the gene protein in almost all creatures
The expression of propylhomoserin protein gene GRP has stringent developmental regulation characteristic, and by the inducing expression volume production of various environmental factors
Raw different variations.Plant glycine-rich protein gene GRP albumen in soybean is positioned at all lignifying cell such as basts
Fiber, primary xylem and secondary xylem cell wall.Existing research shows arid, damages to plants caused by sudden drop in temperature, mechanical damage and abscisic acid
(ABA) etc. factors may induce GRPs gene expression dose to improve.
Therefore, bean plant glycine-rich protein (GRP) gene is studied using molecular biology method, in soybean gram
It is grand go out GRP gene, and establish plant expression vector, continue to study under Drought Stress stress conditions expression quantity in different tissues
Variation has great significance.
Soybean high temperature resistant, drought-resistant ability are weaker, and meeting with the abiotic stress such as arid, high temperature, saline and alkaline in breeding time is
Cause the important factor in order of the soybean underproduction.Arid is one of the main environmental factor for causing soybean yields to reduce, general
Time can cause the production loss of 10%-15%, and the serious time can reach 30%-50%.Therefore, the drought resisting of soybean varieties is improved
Ability, the new high-yield soybean variety for cultivating strong drought resistance are particularly significant.
Summary of the invention
It is above-mentioned to solve the purpose of the present invention is to provide a kind of bean plant glycine-rich protein gene and its application
The problem of being proposed in background technique.
To achieve the above object, the invention provides the following technical scheme:
A kind of bean plant glycine-rich protein gene, the nucleotide of the bean plant glycine-rich protein gene
Sequence is as shown in SEQ ID NO.1.
As a further solution of the present invention: used primer is antiweed in offspring's detection of transgenic plant
(bar), 35s promoter, noss terminator, target gene (GRP).
As a further solution of the present invention: the nucleotide sequence such as SEQ ID NO.2 of the antiweed (bar) and
Shown in SEQ ID NO.3.
As a further solution of the present invention: the nucleotide sequence of the 35s promoter such as SEQ ID NO.4 and SEQ ID
Shown in NO.5.
As a further solution of the present invention: the nucleotide sequence of the noss terminator such as SEQ ID NO.6 and SEQ
Shown in ID NO.7.
As a further solution of the present invention: the nucleotide sequence such as SEQ ID NO.8 of the target gene (GRP) and
Shown in SEQ ID NO.9.
A kind of bean plant glycine-rich protein gene is improving the application in soybean drought resisting ability.
Compared with prior art, the beneficial effects of the present invention are: the present invention is to further appreciate that GRP gene participates in arid etc.
The molecular mechanism of Response to stress is laid a good foundation, and turn GRP over-express vector plant has stronger drought-resistant ability compared with CK, and
Fresh weight and the conspicuousness of ground dry weight become apparent from the ground, by plant height, overground part fresh weight, underground part fresh weight, overground part dry weight,
The variation of this 5 indexs of underground part dry weight, it is seen then that the drought-resistant ability of soybean is improved significantly.
Detailed description of the invention
Fig. 1 is GRP phylogenetic analysis；
Fig. 2 is the hydrophobicity result figure of GRP gene protein；
Fig. 3 is that the transmembrane region of GRP gene protein analyzes result figure；
Fig. 4 is plant over-express vector pCAMBIA-3301-GRP-over expression structure chart；
Fig. 5 is plant interference expression vector pCAMBIA-3301-GRP-RNAi structure chart；
Fig. 6 is electrophoresis result figure (the pcr production of A:GRP gene of bean plant glycine-rich protein gene (GRP) segment
The pcr product of object B:PMD18-T-GRP)；
Fig. 7 is recombinant plasmid proof diagram (A:pCAMBIA-3301-35s-GRP-nos；B:pcr product)；
Fig. 8 is recombination plasmid PCR product proof diagram (A:1-2:pCAMBIA3301-GRP-RNAi digestion products；B:1,4,7
Water；2-3 sense fragment；5-6 antisense fragments；8-9 introne)；
Fig. 9 is PCR detection figure (the A:35s promoter for turning GRP over-express vector gene；B: terminator Nos；C: screening mark
Remember bar)；
Figure 10 is PCR detection figure (the A:35s promoter for turning GRP interference expression vector gene；B: terminator Nos；C: screening
Mark bar)；
Figure 11 is the Southern blot detection figure for turning GRP over-express vector gene plant；
Figure 12 is the Southern blot detection figure for turning GRP interference expression vector gene plant；
Figure 13 is the qRT-PCR qualification figure for turning GRP over-express vector gene；
Figure 14 is the qRT-PCR qualification figure for turning GRP interference expression vector gene.
Specific embodiment
Following will be combined with the drawings in the embodiments of the present invention, and technical solution in the embodiment of the present invention carries out clear, complete
Site preparation description, it is clear that described embodiments are only a part of the embodiments of the present invention, instead of all the embodiments.It is based on
Embodiment in the present invention, it is obtained by those of ordinary skill in the art without making creative efforts every other
Embodiment shall fall within the protection scope of the present invention.
Embodiment 1
1. bioinformatic analysis
The building of the chadogram of 1.1 plant glycine-rich protein genes: pass through in NCBI net with phytozome12
Blast carries out the access of sequence homology, selects to contain GRP base containing GRP gene and arabidopsis, corn, tobacco etc. in soybean
The homologous sequence of cause is downloaded, with MEGA7.0 software building phylogenetic evolution tree, as shown in Figure 1.Analysis learns, clone's
GRP gene is closer with arabidopsis affiliation, farther out with other affiliations.
The physicochemical property of 1.2 GRP gene proteins is predicted and analysis
Basic protein physicochemical property point is carried out to GRP gene with the ProtParam tool on ExPASy server
Analysis.As the result is shown: the molecular formula of GRP gene is C933H1530N254O416S10, relative molecular mass 23282.46, total atom number
Mesh is 3143, and theoretical isoelectric point (theoretical pI) is 9.06, amino acid number 236, wherein 4 positively charged amino
Sour (Arg+Lys), 1 negatively charged amino acid (Asp+Glu), unstability index 63.85 is labile protein.Half-life period
For 30h.Show from the composition analysis of amino acid: the content of serine is 36.0%, and the content of threonine is 31.8%, glycine
Content be 1.3%, the content of isoleucine and proline is 3.4%.Fat coefficient is 27.80.Hydrophily average be-
0.376。
The hydrophobicity analysis of 1.3 GRP gene proteins
Studies have shown that the amino acid of higher positive have stronger hydrophobicity, and the amino acid of lower negative value then have compared with
Strong hydrophily.The hydrophobicity analysis result of GRP gene is as shown in Fig. 2, minimum -1.7, hydrophily is most strong；Peak 1.25,
Hydrophobicity is most strong.From first and last, hydrophilic amino acid is more than hydrophobic amino acid in GRP gene amino acid sequence, thus
Know that GRP gene is hydrophilic protein.
The transmembrane region of 1.4 GRP genes is analyzed
Protein sequence contains transmembrane region, shown in complex chart 2,3, as the result is shown: the amino acid whole position of GRP gene protein
In cell membrane surface, there is no typical transmembrane helical regions, almost the same with the water repellent region analysis result of protein.Show
GRP gene protein is not a membrane receptor protein related with cellular signal transduction.
2. MATERIALS METHODS
2.1 material
Soya seeds mutant M18, Ji Nong 18, bacillus coli DH 5 bacterial strain, Agrobacterium EHA105 bacterial strain, clone's recombination carry
Body PMD18-T-GRP, pCAMBIA3301, cloning vector PMD18-T are provided by Jilin Agriculture University's Plant Biotechnology center
With preservation.
2.2 method
2.2.1 the acquisition of target fragment
By in previous research work, screening soybean drought resisting mutant M18.To the research of M18 discovery Root Characteristics with
Wild type Ji agriculture 18 has notable difference.Difference expression gene is screened using RNA-seq, obtains an expression piece homologous with GRP
Section.By the genome of extraction soybean drought resisting mutant M18 seedling leaf, target fragment is obtained using primer amplified,
Clone is carried out to target gene by homologous pcr amplification technique to obtain the gene sequence that purpose piece degree size is 848bp or so
Column, pcr amplification system are 25 μ l, comprising: amplification condition are as follows: 94 DEG C of initial denaturation 10min；94 DEG C of denaturation 45s, 60 DEG C of renaturation 45s,
72 DEG C of extension 45s, 42 circulations；Last 72 DEG C of extension 10min again, 4 DEG C of preservations.
Pcr product recovery product is sequenced, just by sequencing result after 1% agarose gel electrophoresis recycling
True recovery product is connected on cloning vector pMD18T, is obtained recombinant vector pMD18-T-GRP and is converted Escherichia coli impression
In state cell, picking single colonie extracts plasmid, and sample presentation is sequenced to Jilin provincial treasury U.S. company.
2.2.2 the building of plant over-express vector
Bgl II, Bste are carried out to recombinant cloning vector pMD18-T-GRP and plant expression vector pCAMBIA3301 respectively
II double digestion recycles pMD18-T-GRP small fragment and pCAMBIA3301 large fragment, utilizes T4DNA ligase connection, is used
GUS segment in the small fragment replacement pCAMBIA-3301 of PMD18-T-GRP, constructs recombinant plant over-express vector
PCAMBIA3301-GRP-over-expression.PCR detection and double digestion identification are carried out to the carrier, vector construction figure is such as
Shown in Fig. 4.
2.2.3 the building of RNAi interference expression vector
Pcr amplification has the positive antisense fragments of introne, and amplified production passes through 1% agarose gel electrophoresis, recycles pure
Change target fragment and by sense fragment and basal expression carrier pCAMBIA3301, while (restricted interior with Bgl- II and BstE II
Enzyme cutting is Products) double digestion is carried out, carrier and sense fragment are recycled, is attached by volume for 1:3, linked system is such as
Under: 12 μ L target fragments, 4uL carrier, 4 ligase of 1uLT, 2 μ L T4Buffer, 1 μ L ddH2O.22 DEG C of reactions are overnight.At this time
Obtained carrier is named as pCAMBIA-zy.Antisense fragments are connected with II double digestion of Bgl II and BstE using same method
It accesses in pCAMBIA-zy carrier, obtains using antiweed (bar) base as the RNAi interference expression vector of selection markers
PCAMBIA3301-GRP-RNAi.PCR detection and double digestion identification are carried out to the carrier.Vector construction figure is as shown in Figure 5.
2.2.4 the genetic transformation of soybean
The method of soybean cotyledon node is infected for recombinant plasmid PCAMBIA-GRP- using Agrobacterium herein
RNAi DNA and PCAMBIA-GRP-over expression DNA is transferred to receptor soybean varieties " Ji Nong 18 " respectively
In, and obtain transformed plant.
2.2.5 offspring's detection of transgenic plant
2.2.5.1 PCR is detected
With primer5.0 software design following primer: antiweed (bar), 35s promoter, noss terminator, purpose base
Because of the gene order of (GRP).BarS/BarAS, 35S/35AS, nossS/nossAS, target gene GRP primer sequence be shown in Table
1。
The tender soybean transformation plant leaf of children, the NuClean provided with Jilin provincial treasury U.S. Biotechnology Co., Ltd are provided
Plant Genomic DNA Kit kit extracts genome, carries out pcr detection, and with unconverted receptor soybean (Ji Nong
18) blade genome is as negative control.The expansion of antiweed (bar), 35s gene, noss terminator and target gene (GRP)
Increasing system is 25 μ l systems, PCR amplification condition such as table 2.
1. primer sequence of table
Table 2
PCR amplification condition
2.2.5.2 the southern blotting of transgenic plant is detected
Extract the T through PCR test positive2For the genomic DNA of soybean leaves, carried out with restriction enzyme Hind III
Digestion uses DIG DNALabeling andDetection Kit I kit (Roche Holding Ag using the bar of purifying as probe
Product), content-label prepares probe to specifications, prepares sample, transferring film, prehybridization, hybridizes, washes the journeys such as film, dyeing development
Sequence carries out Southern blotting detection.
2.2.5.3 the drought stress test of transgenic plant
Pot-culture method is a kind of mature mode for the experiment of plant water physiology Ecological Control.Potting is used in this test
Method controls moisture Drought stress simulation, and to 3 parts of transgenic lines: (unconverted recipient plant is as negative control by Ji Nong 18
CK), recombinant expression carrier PCAMBIA-GRP-over expression plant, recombinant expression carrier PCAMBIA-GRP-RNAi
The Identification of Drought in plant progress seedling stage.Pass through plant height, overground part fresh weight, underground part fresh weight, overground part dry weight, underground part dry weight
The variation of this 5 indexs carries out significance difference analysis, to compare the drought resisting degree of 3 parts of materials.
2.2.5.4 the fluorescence quantitative PCR detection of transgenic plant
It extracts in southern test and the RNA of the transgenic plant of hybridization signal and receptor soybean (Ji Nong 18) occurs,
And utilizing reverse transcription reagent box by its reverse transcription at cDNA, 5 times of dilutions are spare.And using the cDNA after 5 times of dilutions as template, choose
β-action (Genbank accession number: NM001252731.2) gene is the reference gene of qRT-PCR experiment, is utilized
Primer5.0 designs quantitative pcr primer QNCAS and QNCS, fluorescent quantitation pcr primer QCGRP and QACGRP primer sequence simultaneously,
Then quantitative fluorescent PCR is carried out.Primer sequence needed for fluorescent quantitation is shown in Table 3.
RNA is extracted: its step is produced RNAisoPlus kit operating procedure according to TaKaRa company and is carried out.
QPCR reaction: it is carried out according to the operation in the q RT-PCRmix specification of GeneCopoeia production, reactant
System is 20 μ L, wherein 2 × All-in-oneqRT-PCRmix10 μ L；Each 1.0 μ L of corresponding primer (20umol/L)；cDNA2.0μL；
Nuclease-free water6.0μL；Each sample and the mRNA detection for compareing receptor are repeated 3 times, amplification condition 95
℃3min；95℃40s；55℃40s；35 circulations, to obtain corresponding Ct value, after response procedures, output data
And it is saved, then pass through 2 in relative quantification-△△CtThe calculating of method progress quantitative result.
Final calculation formula: 1. Δ Δ Ct=(Ct target gene-Ct internal reference) experimental group-(Ct target gene-Ct internal reference) is right
According to group.
3. primer sequence of table
3 results and analysis
The detection and verifying of 3.1 soybean glycine-rich protein gene (GRP) cloning vectors
As shown in fig. 6, carrying out PCR amplification by template of 18 plant tender leaf total DNA of mutant Ji agriculture, amplifying size is
848bp high specificity, single amplified band result are consistent with expection.It will be connected into cloning vector after the recycling of PCR product electrophoresis.Root
According to cloning vector PMD18T-GRP PCR the result shows that, the fragment length of acquisition is consistent with target fragment length scale, illustrates gram
Grand segment is successfully connected into cloning vector.
The detection and verifying of 3.2 soybean glycine-rich protein gene (GRP) expression vectors
Double digestion test is carried out using two kinds of enzymes of BglII and BstEII, and carries out gel electrophoresis, to guarantee the plasmid of recombination
Carrier is correct.As shown in fig. 7, plant over-express vector pCAMBIA3301-GRP-over expression double digestion segment is big
It is small consistent with expected fragments size 900bp (summation comprising homology arm segment), tentatively illustrate expression vector establishment success, so
Afterwards using the above-mentioned expression vector plasmid built as template, Standard PCR detection is carried out, finds GRP target gene fragment and expection
Clip size position is identical, illustrates that plant over-express vector constructs successfully.
As shown in figure 8, plant RNA i expression vector pCAMBIA3301-GRP-RNAi double digestion clip size with it is expected that piece
Duan great little 1170bp (comprising positive and negative adopted segment and include the summation of sub-piece) is consistent, tentatively illustrate expression vector establishment at
Function carries out Standard PCR detection then using the above-mentioned expression vector plasmid built as template, finds the just piece of GRP gene
Section, antisense fragments with include sub-piece with expection clip size position it is identical, illustrate that plant interference expression vector constructs successfully.
3.3 T2For the acquisition and detection of transgenic plant
Over-express vector PCAMBIA3301-GRP-over expression Plasmid DNA is passed through into Agrobacterium infestation method point
It does not imported into soybean receptor kind " Ji Nong 18 ".It is detected through pcr and obtains 4 plants of T0For positive plant, receive T0For positive seeds 30
Grain.Added with outdoor continuous plantation for processing by indoor, obtains 11 plants of T1For positive plant, receive T1For positive seeds 60
Grain continues through indoor plant with outdoor continuous and is added for processing, obtains T2For 14 plant of positive plant, blade gene is extracted
Group DNA, is utilized respectively antiweed (bar), 35s promoter, noss terminator, the gene-specific primer of target gene GRP
It is detected one by one, using recombinant expression carrier PCAMBIA3301-GRP-over expression Plasmid DNA as positive control,
Unconverted receptor soybean plant strain " Ji Nong 18 " is negative control and water is blank control, and pcr testing result is as shown in Figure 9.
By interference carrier PCAMBIA3301-GRP-RNAi Plasmid DNA by Agrobacterium infestation method be directed respectively into soybean by
In body kind " Ji Nong 18 ".It is detected through pcr and obtains 3 plants of T0For positive plant, T is harvested0For positive seeds 22.Equally pass through room
It is interior to be added with outdoor continuous plantation for processing, obtain T1For 9 plants of positive plant, T is obtained1For positive seeds 53, continue through
Indoor and outdoor, which is continuously planted, to be added for processing, and T is obtained2For 12 plants of positive plant, leaves genomic DNA is extracted, sieve is utilized respectively
Choosing label bar, the gene-specific primer of 35s promoter, noss terminator, target gene GRP is detected one by one, with recombination
Expression vector PCAMBIA3301-GRP-RNAi Plasmid DNA be positive control, unconverted receptor soybean plant strain " Ji Nong 18 " and
Water is negative control, and pcr testing result is as shown in Figure 10.
By Fig. 9 and Figure 10 it is found that from T2For the band and mesh amplified in transgenic plant by gene-specific primer
Gene it is in the same size, preliminary proof plant glycine-rich protein (GRP) gene has successfully been integrated into the positive of conversion
In plant.
3.4 T2For the southern blotting detection of transgenic plant
The leaves genomic DNA by the transgenic plant that pcr testing result is the positive is extracted in order respectively, with limitation
Property restriction endonuclease Hind III carry out digestion, use selection markers bar as probe, respectively with recombinant expression carrier PCAMBIA3301-
GRP-over expression Plasmid DNA and interference carrier PCAMBIA3301-GRP-RNAi Plasmid DNA are positive control, non-
Plant receptor (Ji Nong 18) is negative control in soybean transformation, carries out southern blotting detection.
As shown in figure 11, the plant only converted just will appear hybridization signal, and detection turns the plant of GRP over-express vector gene
There are 2 plants apparent hybridization signal occur in strain, obtains 7 plants of transgenosis through southern blotting test positive altogether and plant
Strain.Function element is integrated into receptor soybean genome as we know from the figure in the form singly copied, and integration site is not identical.
As shown in figure 12, the plant only converted just will appear hybridization signal, and detection turns GRP interference carrier gene plant
In there is 1 plant apparent hybridization signal occur, obtain 6 plants of transgenic plants through southern blotting test positive altogether.
Function element is integrated into receptor soybean genome as we know from the figure in the form singly copied, and integration site is not identical.
The drought resistance of seedling testing inspection of 3.5 transgenic plants
With Nutrition Soil (microbial matrices) for cultivation medium, container selects unified flowerpot (diameter 12cm × height
10cm), unified before sowing to match soil, fill basin, irrigate bottom water, electronic scale weighs, it is ensured that each flowerpot is consistent with weight after soil, respectively
Weight is consistent after flowerpot irrigates bottom water.Every part of material plants 6 basins, and 5, every basin, after emergence, every basin was every 3 days watering 550ml, in three
The leaf phase starts to carry out processing of cutting off the water supply to stress group.According to the observation the case where stress group soybean plant strain, setting Stress treatment 5 days, 8
It, 11 days, 15 days 4 drought stress groups of cutting off the water supply.Each drought stress component of cutting off the water supply are as follows: CK processing, overexpression processing, RNAi are dry
Disturb processing.With control group (normally every 3 days watering 600ml): CK is untreated, overexpression is untreated, RNAi is untreated is contrasted.
Each stress group continues the indices for measuring stress group plant and control group plant after respective handling number of days, rehydration 3 days.
The measurement index after drought stress is handled 15 days, rehydration three days, as can be seen from Table 4: GRP is overexpressed gene processing
Occur significant difference with CK treated ground fresh weight, ground dry weight afterwards, and reach extremely significant level.Base is interfered with GRP
Because after processing compared to CK treated plant height, ground fresh weight, underground fresh weight, ground dry weight, to there is conspicuousness in underground dry weight poor
It is different, and reach extremely significant level；The result shows that turning GRP over-express vector gene plant under drought stress after drought stress processing
Drought-resistant ability more stronger than CK can be shown by gene expression.
The significance analysis of table 4 stress group and control group
3.6 T2For the fluorescent quantitation pcr detection of transgenic plant
It is that positive transgenic plant continues fluorescent quantitation pcr detection to southern blotting testing result.
GRP is overexpressed respectively and GRP interference gene is in T2For transformed plant and unconverted plant (Ji Nong 18CK) in different number of days
The expression quantity situation of drought stress is analyzed.Shown in result figure 13,14: in 15 days of simulating drought processed soybeans plant,
GRP in soybean leaves is overexpressed the trend subtracted after gene expression amount Cheng Xianzeng, GRP at Osmotic treatment 8 days, in soybean leaves
Gene expression amount highest is 5.2 times of control.The expression quantity of GRP gene has no significant change in Soybean Root and stem.GRP interference
Gene handles to obtain in 15 days in simulating drought, and the expression quantity in root, stem and leaf is at trend is gradually reduced, in Osmotic treatment the 15th
It when, the expression quantity in root is minimum.
There is the presence of GRP gene in many unifacial leaves and dicotyledonous middle plant.The present invention passes through in mutant (M18)
GRP gene has been cloned, with bioinformatic analysis, and has successfully constructed plant expression vector (plant over-express vector and plant
Interference carrier), function element is transferred in receptor soybean (Ji Nong 18) by agrobacterium-mediated transformation, southern blotting
Detection functions original part is integrated into soybean genome in the form of single copy.By being learnt in the test of plant drought resistance of seedling
The drought resistance that GRP is overexpressed gene is better than recipient plant Ji agriculture 18 (CK), and shows significant；GRP interferes gene in stress tests
Under do not show drought resistance better than recipient plant Ji agriculture 18 (CK)；GRP is overexpressed gene, GRP interference gene and CK difference
There is erratic behavior and unstability, therefore the detection of its Identification of Drought should carry out comprehensive analysis in conjunction with more multi objective.
Detected and found by fluorescent quantitation pcr: GRP is overexpressed expression quantity highest of the gene in soybean leaves, with dry
Drought coerces the increase of number of days, the trend subtracted after the expression quantity Cheng Xianzeng in blade；Expression quantity in root, stem is lower, expression
The variation of amount is unobvious.GRP interferes expression quantity of the gene in Soybean Root, stem, leaf less, with the increasing of drought stress number of days
Add, expression quantity gradually decreases.
By can be seen that in the environment of drought stress to the Biology identification for turning GRP gene plant, turn GRP overexpression
Carrier plant has stronger drought-resistant ability compared with CK, and fresh weight and the conspicuousness of ground dry weight become apparent from the ground, this with
In fluorescence quantitative PCR detection result, GRP gene higher result of expression quantity in stem, leaf is consistent.
The present invention is to further appreciate that the molecular mechanism of the Response to stress such as GRP gene participation arid is laid a good foundation, simultaneously
Condition is provided to study GRP gene in soybean.
It is obvious to a person skilled in the art that invention is not limited to the details of the above exemplary embodiments, Er Qie
In the case where without departing substantially from spirit or essential attributes of the invention, the present invention can be realized in other specific forms.Therefore, no matter
From the point of view of which point, the present embodiments are to be considered as illustrative and not restrictive, and the scope of the present invention is by appended power
Benefit requires rather than above description limits, it is intended that all by what is fallen within the meaning and scope of the equivalent elements of the claims
Variation is included within the present invention.Any reference signs in the claims should not be construed as limiting the involved claims.
In addition, it should be understood that although this specification is described in terms of embodiments, but not each embodiment is only wrapped
Containing an independent technical solution, this description of the specification is merely for the sake of clarity, and those skilled in the art should
It considers the specification as a whole, the technical solutions in the various embodiments may also be suitably combined, forms those skilled in the art
The other embodiments being understood that.
Sequence table
<110>Jilin Agriculture University
<120>a kind of bean plant glycine-rich protein gene and its application
<160> 13
<170> SIPOSequenceListing 1.0
<210> 1
<211> 711
<212> DNA
<213>soybean (Glycine max)
<400> 1
ttaatcgatg gctttgaagg agatggtttt ggcggtggtg gaggtggatt tggtggtggt 60
gctggaggag gtgcaggtgg tgggtttgga ggtggacacg ggagtggtgt aggaggagga 120
gctggcggag gtggtggagc tgggggaggc tatggaggtg gtgttggtgg aggttatgga 180
ggcgataagg gtggaggtgc tggaggtggc atagaaggtg gacatggtgg tattggagga 240
gaaggtggtg gtgttggagg aggagctgga ggaggtggtg ctggtgggat aggaggagga 300
catgctggtg gcattggagg aggagggggt ggaggtggtg gagccggaag tggtgctgca 360
ggcaacgtag gaggtggaca tggtggtggc attggagggg gagctggagg tgctggtgga 420
ggtggtcaag ccggaggtgg acatggtggt ggcattggag ggggagctgg aggtgctggt 480
ggaggtggtc aagccggagg tggacatggt ggtggcattg gaggtggaca tggtagtgga 540
attggaggag gagctggtgg tggtagtggg gctggaggca acataggagg tggacatggt 600
ggtggtgttg gaggaggagg agttggtgtt ggtggcggtt atggaggtgg taaaggagga 660
ggagctggtg gatgtggtgg aattaataat ggtggcatgg gaggtggaca t 711
<210> 2
<211> 18
<212> DNA
<213>soybean (Glycine max)
<400> 2
tagaggacct aacagaac 18
<210> 3
<211> 18
<212> DNA
<213>soybean (Glycine max)
<400> 3
ccgtgttctc tccaaatg 18
<210> 4
<211> 21
<212> DNA
<213>soybean (Glycine max)
<400> 4
gaatcctgtt gccggtcttt g 21
<210> 5
<211> 20
<212> DNA
<213>soybean (Glycine max)
<400> 5
ttatcctagt ttgcgcgcta 20
<210> 6
<211> 19
<212> DNA
<213>soybean (Glycine max)
<400> 6
tcaaatctcg gtgacgggc 19
<210> 7
<211> 18
<212> DNA
<213>soybean (Glycine max)
<400> 7
atgagcccag aacgacgc 18
<210> 8
<211> 20
<212> DNA
<213>soybean (Glycine max)
<400> 8
cccccagtgt caccttcagc 20
<210> 9
<211> 22
<212> DNA
<213>soybean (Glycine max)
<400> 9
gttcagtgat gggaaaatta cg 22
<210> 10
<211> 24
<212> DNA
<213>soybean (Glycine max)
<400> 10
atcttgactg agcgtggtta ttcc 24
<210> 11
<211> 19
<212> DNA
<213>soybean (Glycine max)
<400> 11
gctggtcctg gctgtctcc 19
<210> 12
<211> 19
<212> DNA
<213>soybean (Glycine max)
<400> 12
tgttggagga ggaggagtt 19
<210> 13
<211> 21
<212> DNA
<213>soybean (Glycine max)
<400> 13
cctcccatgc caccattatt a 21
Claims (7)
1. a kind of bean plant glycine-rich protein gene, which is characterized in that the bean plant glycine-rich protein base
The nucleotide sequence of cause is as shown in SEQ ID NO.1.
2. bean plant glycine-rich protein gene according to claim 1, which is characterized in that in transgenic plant
Used primer is antiweed (bar), 35s promoter, noss terminator, target gene (GRP) in offspring's detection.
3. bean plant glycine-rich protein gene according to claim 2, which is characterized in that the antiweed
(bar) nucleotide sequence is as shown in SEQ ID NO.2 and SEQ ID NO.3.
4. bean plant glycine-rich protein gene according to claim 2, which is characterized in that the 35s promoter
Nucleotide sequence as shown in SEQ ID NO.4 and SEQ ID NO.5.
5. bean plant glycine-rich protein gene according to claim 2, which is characterized in that the noss terminator
Nucleotide sequence as shown in SEQ ID NO.6 and SEQ ID NO.7.
6. bean plant glycine-rich protein gene according to claim 2, which is characterized in that the target gene
(GRP) nucleotide sequence is as shown in SEQ ID NO.8 and SEQ ID NO.9.
7. a kind of bean plant glycine-rich protein gene according to claim 1 is in improving soybean drought resisting ability
Using.
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Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US9029636B2 (en) * | 2008-02-05 | 2015-05-12 | Monsanto Technology Llc | Isolated novel nucleic acid and protein molecules from soy and methods of using those molecules to generate transgenic plants with enhanced agronomic traits |
| CN105985418A (en) * | 2015-01-30 | 2016-10-05 | 中国农业科学院作物科学研究所 | Application of growth-related protein GRP4 in regulation of plant growth |
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Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US9029636B2 (en) * | 2008-02-05 | 2015-05-12 | Monsanto Technology Llc | Isolated novel nucleic acid and protein molecules from soy and methods of using those molecules to generate transgenic plants with enhanced agronomic traits |
| CN105985418A (en) * | 2015-01-30 | 2016-10-05 | 中国农业科学院作物科学研究所 | Application of growth-related protein GRP4 in regulation of plant growth |
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| Title |
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| ANONYMOUS: "Sequence 10347 from patent US9029636", 《GENBANK》 * |
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