CN106399326A - Soybean bHLH transcription factor gene GmFER and encoded protein and application thereof - Google Patents
Soybean bHLH transcription factor gene GmFER and encoded protein and application thereof Download PDFInfo
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Abstract
The invention discloses a soybean bHLH transcription factor gene GmFER and encoded protein and application thereof. The nucleotide sequence of the gene GmFER is as shown in SEQ ID No.1, and the amino acid sequence of the protein encoded by the gene is as shown in SEQ ID No. 2. A semi-quantitative detecting result shows that the gene GmFER is mainly expressed in soybean roots; the expression quantity of the gene GmFER is increased evidently after Cd stress, that is to say, the gene GmFER is induced by Cd; by overexpressing the gene GmFER in yeast and plants, the Cd tolerance of the yeast and plants can be increased, and the gene GmFER is high in application value.
Description
Technical field
The invention belongs to plant genetic engineering field, it is related to plant FER gene and its encoding proteins and application, particularly to
Semen sojae atricolor bHLH transcription factor gene GmFER, its encoding proteins, and its answering in improving the yeast and Nicotiana tabacum L. toleration to cadmium
With.
Background technology
With social growing, non-agricultural construction land used is expanded rapidly, and waste discharge amount and agrochemical make
Consumption increases, and heavy metal pollution of soil is increasingly becoming worldwide environmental problem.Cadmium (Cd) is a kind of very strong huge sum of money of bio-toxicity
Belong to, the half-life is very long, excess intake can lead to kidney and skeleton pathological changes (referring to Nawrot T., Plusquin M.,
Hogervorst J.,et al.,Environmental exposure to cadmium and risk of cancer:a
prospective population-based study[J],Lancet Oncology,2006,7(2):119-126).
Semen sojae atricolor is the crops in agricultural production with higher economic worth, containing rich in protein and oils and fatss, with
The life of the mankind is closely bound up.But, Semen sojae atricolor easily accumulates heavy metal (referring to Wolnik K.A., Fricke F.L., Capar
S.G.,et al.,Elements in major raw agricultural crops in the United
States.1.Cadmium and lead in lettuce,peanuts,potatoes,soybeans,sweet corn,and
wheat[J],J.Agric.Food Chem.,1983,31(6):1240-1244), a large amount of accumulation in plant body for the heavy metal,
Not only have a strong impact on the g and D of plant, and the health of the mankind can be jeopardized by food chain.
BHLH (basic/Helix-Loop-Helix, alkalescence/helix-loop-helix) transcription factor is plant transcription factor
One of middle maximum family.Between two alpha-helixs in same bHLH transcription factor or different bHLH transcription factor α-spiral shell
Can interact between rotation, form homology or heterodimer, be combined with target gene promoters, the transcription (ginseng of controlling gene
See Ma P.C.M., Rould M.A., Weintraub H., et al., Crystal structure of MyoDbHLH
domain-DNA complex:Perspectives on DNA recognition and implications for
transcriptional activation[J],Cell,1994,77(3):451-459).BHLH is widely present in plant not
In organizing, the growth promoter regulation process of plant plays particularly important effect.Arabidopsiss bHLH gene (FIT,
AtbHLH38 and AtbHLH39) take part in the response that plant is coerced to Cd, start some participation heavy metals composition and separate
The expression of the gene (as HMA3, MTP3, IREG2 and IRT2) changed, thus by Cd the separating of major part of absorption in root, reduce
To overground part transhipment (referring to Wu HL, Chen CL, Du J, et al., Co-Overexpression FIT with
AtbHLH38 or AtbHLH39 inArabidopsis-Enhanced Cadmium Tolerance via
IncreasedCadmium Sequestration in Roots and Improved Iron Homeostasis of
Shoots[J],Plant Physiology,2012,158:790-800), but not yet have been reported that disclosure Semen sojae atricolor bHLH transcription so far
The related application of factor gene.
Content of the invention
For above-mentioned situation, the present invention, according to the method for homologous clone, clones one kind from the root system of the cultivated soybean
BHLH transcription factor gene GmFER, this gene pairs Cd stress has response.Separate and not only contribute to clone's Semen sojae atricolor GmFER gene
Disclose the anti-Cd mechanism of Semen sojae atricolor, the anti-Cd molecular breeding of microorganism or plant can also be carried out by the means of genetic manipulation, with
Just improve anti-Cd.
Specifically, the invention provides Semen sojae atricolor bHLH transcription factor gene GmFER, total length 972bp, its nucleotide sequence
As SEQ ID NO:Shown in 1.
Present invention also offers by the albumen of above-mentioned Semen sojae atricolor bHLH transcription factor gene GmFER coding, comprising 323 amino
Acid (conserved domain being wherein bHLH transcription factor from the aminoacid sequence of N-terminal the 129th to the 185th), its aminoacid
Sequence such as SEQ ID NO:Shown in 2.
Additionally, comprising as SEQ ID NO:Nucleotide sequence shown in 1 and encoding as SEQ ID NO:Amino shown in 2
The polynucleotide sequence of acid sequence and will be as SEQ ID NO:One or more of aminoacid sequence shown in 2 is (not more than
Ten) amino acid residue replaced and/or lacked and/or add and have transcriptional activation function, regulation and control stress resistance of plant
Aminoacid sequence also within protection scope of the present invention.
Present invention also offers comprising the expression vector of above-mentioned Semen sojae atricolor bHLH transcription factor gene GmFER, described expression carries
Body by above-mentioned Semen sojae atricolor bHLH transcription factor gene GmFER insertion vector is obtained, described carrier be selected from p424GPD, pBI121,
Any one in pCXSN, pCAMBIA2301, pCAMBIA1301, pAHC25.
When using above-mentioned Semen sojae atricolor bHLH transcription factor gene GmFER construction of expression vector, in its transcription initiation nucleotide
Before can be plus any composing type, organizing specific type, induction type or enhancement mode promoter.For the ease of to transgenic microorganism
Or plant identified and screen, expression carrier used thereof can be processed, such as add and there is the antibiotic marker thing of resistance (such as
Kanamycin label, Hygromycin marker thing etc.), anti-chemical reagent marker gene (as antiweed bar gene etc.) or can produce
The enzyme of raw color change or albumen (as gus gene, GFP gene etc.) etc..
Present invention also offers comprising the engineering bacteria of above-mentioned Semen sojae atricolor bHLH transcription factor gene GmFER, in described engineering bacteria
Host be escherichia coli or yeast, the method being mediated by PEG/LiAc is by above-mentioned Semen sojae atricolor bHLH transcription factor gene
GmFER proceeds to.
Present invention also offers comprising the transgenic plant cells system of above-mentioned Semen sojae atricolor bHLH transcription factor gene GmFER, institute
Stating plant is Nicotiana tabacum L., is proceeded to above-mentioned Semen sojae atricolor bHLH transcription factor gene GmFER by agriculture bacillus mediated method.
Present invention also offers being used for for a pair expanding the primer of above-mentioned Semen sojae atricolor bHLH transcription factor gene GmFER, wherein just
Nucleotide sequence such as SEQ ID NO to primer:Shown in 3, the nucleotide sequence such as SEQ ID NO of reverse primer:Shown in 4.
Present invention also offers above-mentioned Semen sojae atricolor bHLH transcription factor gene GmFER and its encoding proteins and comprise its expression
Application in improving the toleration to Cd for the yeast for the carrier.By Semen sojae atricolor bHLH transcription factor gene GmFER is imported in yeast,
Improve the anti-Cd of yeast.Additionally, above-mentioned Semen sojae atricolor bHLH transcription factor gene GmFER and its encoding proteins and comprise its table
Reach application in improving the toleration to Cd for the plant (preferably unifacial leaf and dicotyledon) for the carrier also in the protection of the present invention
Within the scope of.
The Semen sojae atricolor bHLH transcription factor gene GmFER that the present invention provides is derived from Semen sojae atricolor (Williams 82 kind), sxemiquantitative
PCR testing result shows that GmFER gene is mainly expressed in the root of Semen sojae atricolor.After Cd stress, its expression significantly improves, explanation
This gene is induced by Cd.In yeast, overexpression GmFER gene can improve the toleration to Cd for the yeast, and can improve turn
The toleration to Cd for the GmFER genetic tobacco.Therefore, GmFER gene can as genes of interest import plant (include unifacial leaf and
Dicotyledon), thus improving the toleration to Cd for the plant, there is higher using value.
Brief description
Fig. 1 is the aminoacid sequence of Semen sojae atricolor bHLH transcription factor gene GmFER encoding proteins and other plant bHLH domain
Row multiple alignment result, wherein：GmFER-like is derived from Semen sojae atricolor, and LjbHLH21 is derived from Radix Loti Corniculati, and MtbHLH is derived from M. truncatula,
AtFER is derived from arabidopsiss, and AtbHLH is derived from arabidopsiss.
Fig. 2 is the phylogenetic analysis figure of GmFER, wherein：GmFER-like(Glycine max,XP_003541980.1)；
LjbHLH21(Lotus japonicus,ACN21647.1)；MtbHLH(Medicago truncatula,XP_
003606331.1)；AtFER-like(Arabidopsis thaliana,NP_850114.1)；AtbHLH(Arabidopsis
thaliana,AAM10938.1)；VvFER-like(Vitis vinifera,XP_002272647.1)；SlbHLH(Solanum
lycopersicum,NP_001234654.1)；ZmbHLH(Zea mays,DAA38198.1)；BnbHLH(Brassica
napus,CAD54298.1)；VvbHLH35(Vitis vinifera,XP_002263999.1).
Fig. 3 is the semiquantitive PCR testing result of GmFERmRNA gene expression abundance in leaf, stem and root.
Fig. 4 is the CdCl using 100 μM2After processing different time, the semiquantitive PCR of GmFERmRNA expression impact is detected
Result.
Fig. 5 is the structure schematic diagram of Yeast expression carrier, wherein：A is p424GPD empty carrier physical map；B is
p424GPD::GmFER plant expression vector physical map.
Fig. 6 is the testing result of GmFER gene impact of the toleration to Cd for yeast.
Fig. 7 is pCXSN::GmFER plant expression vector schematic diagram.
Fig. 8 is transgene tobacco in CdCl2The long growing state of root under stress, wherein：WT represents wild-type tobacco, T table
Show transgene tobacco.
Specific embodiment
Below with reference to the drawings and specific embodiments, technical scheme to be described.It should be appreciated that these are real
Apply example to be only used for illustrating the present invention, rather than limit the scope of the present invention by any way.Unless otherwise stated, following enforcement
Term used in example has the implication that those of ordinary skill in the art are generally understood that, not marked experimental technique all can be according to
Conventional method is carried out, and writes referring for example to J. Pehanorm Brooker (Sambrook J.) and D.W. Russell (Russell D.W.)《Point
Sub- cloning experimentation guide》(Science Press, 2005) or according to the bar described in the operation instruction of biological reagent production firm
Part.In addition, instrument used in the following example, material, reagent all can be obtained by routine business means.
Embodiment 1：The clone of Semen sojae atricolor bHLH transcription factor gene GmFER.
The method clone's Semen sojae atricolor GmFER gene being combined using bioinformatics and Conventional polymerase chain formula reaction (PCR)
DNA sequence, concrete grammar is as described below：
Using the root system of Williams 82 kind Semen sojae atricolor as experiment material, using SV Total RNAlysis test kit
(Promega company, U.S.) extracts total serum IgE.The method providing according to TaKaRa company, using PrimeScriptTMReverse transcriptase
Synthesize cDNA, and performing PCR amplification is entered as template using it, used in PCR amplification procedure, pair of primers P1 is (as SEQ ID
NO:Shown in 3, by Shanghai, bio-engineering corporation synthesizes) and P2 (as SEQ ID NO:Shown in 4, by Shanghai, bio-engineering corporation closes
Become) then it is designed according to GmFER sequence known in NCBI and synthesize：
P1：5’-ATGGATGTTCACGAAGACACACTCA-3’；
P2：5’-TCAAGCAGGAAAAGATGCCACGAAT-3’.
Expanded by RT-PCR method, obtain complete open reading frame (ORF) fragment of GmFER gene.25 μ lPCR reactions
System is as follows：Containing MgCl210 × PCR buffer 2.5 μ l, positive, each 1.0 μ l of reverse primer (10 μM), dNTP (10mM) 1.0
μ l, cDNA sample 2.0 μ l, Ex-Taq enzyme (precious biological engineering (Dalian) company limited) 0.25 μ l, distilled water 17.5 μ l.PCR is anti-
Answer condition：95 DEG C of denaturations 5min；95 DEG C of degeneration 30s, 58 DEG C of renaturation 45s, 72 DEG C of extension 1min, after 30 circulations, 72 DEG C are prolonged
Stretch 10min.The DNA fragmentation of the about 800bp that amplification is obtained enters row agarose gel electrophoresis and separates, using gel reclaim reagent
Box (Hangzhou love pursue progress Bioisystech Co., Ltd) is simultaneously reclaimed according to the scheme providing in test kit, and be cloned into pEASY-
On T1 (Clontech) carrier, carry out sequencing analysis (invitrogen company completes by Shanghai), in sequencing result and NCBI
GmFER gene order is identical.
Embodiment 2：The impact to GmFER gene expression of the spatial and temporal distributions of GmFER transcript and Cd stress.
Using the method for semiquantitive PCR, detect tissue distribution patterns in soybean plant strain for the GmFER, and take following arranging
Apply the reliability ensureing testing result：Using a small amount of base that may be present in the total serum IgE that amplification rank DNase I digestion is extracted
Because organizing the pollution of DNA, in order to detect whether genomic DNA eliminates totally, the total of a part of DNase I digestion can be taken out
RNA sample carries out Standard PCR reaction.When finding not having amplified band to produce, then carry out reverse transcription step, simultaneously in order to enter one
When step confirms, whether the band of quantitative pcr amplification is exactly GmFER, PCR primer is cut glue reclaim and is sequenced.Concrete grammar
For：The material extraction total serum IgE such as Semen sojae atricolor different growing stage root, stem, leaf, flower and pod are taken to be template, the primer of semiquantitive PCR is P1
And P2.
Using Semen sojae atricolor Actin (GmActin) cDNA as internal reference, semiquantitive PCR primer P3 is (as SEQ ID NO:5 institutes
Show, by Shanghai, bio-engineering corporation synthesizes) and P4 (as SEQ ID NO:Shown in 6, by Shanghai, bio-engineering corporation synthesizes) core
Nucleotide sequence is as follows：
P3(actin-F)：5’-CGGTGGTTCTATCTTGGCATC-3’；
P4(actin-R)：5’-GTCTTTCGCTTCAATAACCCTA-3’.
First take 1 μ g total serum IgE, add amplification rank DNase I (Sigma company, the U.S.), place 30min to remove in room temperature
Go the pollution of genomic DNA, be subsequently adding stopping buffer (50mM EDTA), in 70 DEG C heating 10min degeneration DNase I and
RNA, then adopts the reverse transcription reagent box of precious biological engineering (Dalian) company limited and according to description record in test kit
Method carries out reverse transcription, and each sample takes 1 μ g total serum IgE, reacts 1h in 42 DEG C, takes out, be placed in ice after 70 DEG C of heating 10min
On so that reverse transcription inactivation, finally take 1 μ l reverse transcription product to carry out semiquantitive PCR amplification, testing result as shown in figure 3, from
In it can be seen that GmFER gene mainly in soybean root system express.
In order to study the response of GmFER gene pairss Cd stress, detect GmFER gene in Cd using the method for semiquantitive PCR
Expression pattern under stress.By tri-leaf period Semen sojae atricolor with 100 μM of CdCl2Process 0h, 1h, 3h and 24h, not do any process
Tri-leaf period Semen sojae atricolor, as comparison, then adopts method same as described above to detect the expression of GmFER gene in soybean root system,
Same using actin (Actin) cDNA of Semen sojae atricolor as internal reference, using P1 and P2 as primer, testing result as shown in figure 4, from
In as can be seen that Cd stress after, after in soybean root system, the expression of GmFER shows and falls before rise trend.
Embodiment 3：The structure comprising the Yeast expression carrier of GmFER gene and transgenic yeast are examined to the toleration of Cd
Survey.
(1) structure of Yeast expression carrier：
With GmFERcDNA full length sequence as template, in the forward primer P5 containing restriction enzyme site (as SEQ ID NO:7 institutes
Show, by Shanghai, bio-engineering corporation synthesizes) and reverse primer P6 (as SEQ ID NO:Shown in 8, by Shanghai, bio-engineering corporation closes
Become) guiding under, PCR amplification 5 ' end add BamHI recognition sites, 3 ' end add SmaI recognition sites GmFERcDNA total length
Sequence, the nucleotide sequence of primer is as follows：
P5：5′-CGCGGATCC(base with underscore is to limit inscribe to ATGGATGTTCACGAAGACACACTCA-3 '
Enzyme BamHI recognition site)；
P6：5′-TCCCCCGGG(base with underscore is to limit inscribe to TCAAGCAGGAAAAGATGCCACGAAT-3 '
Enzyme SmaI recognition site).
After reaction terminates, pcr amplification product is carried out with 1.0% agarose gel electrophoresiies detection, reclaim and purification 972bp
Purpose fragment, after being used BamHI and SmaI enzyme action, adopts T with the carrier p424GPD through same enzyme double digestion4DNA ligase
In 4 DEG C of connections, overnight (20 μ l reaction systems contain 1 × T4DNA ligase buffer, p424GPD fragment 0.03pmol, GmFER piece
Section 0.3pmol, T4DNA ligase 350U).
Connection product is converted (the method for transformation reference of bacillus coli DH 5 alpha competent cell《Molecular Cloning:A Laboratory guide》The
Two editions, 1992, Science Press, page 55～56), by the e.colidh5αcell suspension after conversion be coated on containing
On the flat board of 80mg/L ampicillin, the positive colony of antagonism ampicillin is expanded and extracted plasmid, is taken enzyme
The correctness that the method validation cut and be sequenced connects, obtains Yeast expression carrier p424GPD::GmFER (as shown in Figure 5).
(2) yeast conversion of PEG/LiAc method mediation and Screening and Identification：
Using PEG/LiAc method transformed yeast cell W303B, it is coated on the SD solid medium of scarce tryptophan (- Trp),
Cultivate 3d in 30 DEG C, picking monoclonal enters performing PCR detection from culture plate, using primer P1 and P2,25 μ l PCR reactants
System is as follows：Containing MgCl210 × PCR buffer 2.5 μ l, positive, each 1.0 μ l of reverse primer (10 μM), dNTP 1.0 μ l
(10mM), genomic DNA template 1.0 μ l, distilled water 18.5 μ l, and Ex-Taq enzyme (precious biological engineering (Dalian) company limited)
0.25μl.Amplification reaction condition is as follows：95 DEG C of denaturations 4min, 95 DEG C of degeneration 30s, 62 DEG C of renaturation 50s, 72 DEG C of extension 1min,
After 35 circulations, 72 DEG C of extension min.Amplified production is carried out gel electrophoresiss, has 3 positives to turn in 9 monoclonals of identification
p424GPD::The monoclonal of GmFER, obtains containing p424GPD::The yeast strain of GmFER.
(3) gene function analysis：
Containing p424GPD::The yeast of GmFER expression vector and the yeast with empty carrier p424GPD are respectively with 1%
Inoculum concentration be inoculated in 100ml and lack overnight incubation in the SD fluid medium of tryptophan (- Trp), until OD600=1, with SD (-
Trp) fluid medium is diluted to OD600=0.1,0.01,0.001, drops in respectively containing 50 μM of CdCl2With do not contain
CdCl2SD (- Trp) solid medium on, cultivate 3d in 30 DEG C, observe the growing state of bacterium colony, its result is as shown in Figure 6.
From fig. 6 it can be seen that containing 50 μM of CdCl2Culture medium on, containing p424GPD::GmFER expression vector
Yeast substantially must be good than the yeast growth with empty carrier p424GPD, the yeast strain with empty carrier p424GPD is containing 50
μM CdCl2Culture medium on can hardly grow.As can be seen here, GmFER gene can improve the toleration to Cd for the yeast.Will
GmFER gene imports plant (including unifacial leaf and dicotyledon) as genes of interest and can also improve the tolerance to Cd for the plant
Property, there is higher using value.
Embodiment 4：The structure comprising the plant expression vector of GmFER gene and transgene tobacco are examined to the toleration of Cd
Survey.
(1) structure of plant expression vector：
With GmFERcDNA full length sequence as template, performing PCR amplification is entered by P and P2.After reaction terminates, PCR is expanded and produces
Thing carries out 1.0% agarose gel electrophoresiies detection, reclaim and purification 972bp purpose fragment, the method cloned by TA,
The ORF of GmFER is connected to plant expression vector pCXSN.
Connection product is converted (the method for transformation reference of bacillus coli DH 5 alpha competent cell《Molecular Cloning:A Laboratory guide》The
Two editions, 1992, Science Press, page 55～56), by the e.colidh5αcell suspension after conversion be coated on containing
On the flat board of 80mg/L that syphilis of card, the positive colony of that syphilis of card is expanded and is extracted plasmid, the method taking sequencing
The correctness that checking connects, obtains plant expression vector pCXSN::GmFER (as shown in Figure 7).Using freeze thawing conversion method, this is carried
Body converts Agrobacterium tumefaciems EH105, obtains containing pCXSN::The agrobacterium strains of GmFER.
(2) agriculture bacillus mediated Transformation of tobacco and Screening and Identification：
Picking single Agrobacterium bacterium colony from culture plate, 2ml YEB culture medium (containing 50mg/L kanamycin,
30mg/L rifampicin) in overnight incubation；(containing 50mg/L card, that is mould to be inoculated in 100ml YEB culture medium with 1% inoculum concentration
Element, 30mg/L rifampicin) in overnight incubation, 6000rpm is collected by centrifugation thalline, with infecting culture medium Eddy diffusion, bacterial concentration
Adjust to OD600It is about 0.5, as infecting bacterium solution.Using leaf-disc transformation, with sterile razor blade by aseptic tobacco plantlets in vitro leaf
Piece is cut into the leaf dish of 4～6mm, and leaf dish explant is in OD600About 0.5 pCXSN::GmFER Agrobacterium infects infection in solution
10min, blots surface liquid with aseptic filter paper after taking-up, puts into that (it is fast that MS minimal medium adds 6- benzyl amino containing co-culture media
Purine 1.0mg/L, naphthalene acetic acid 0.1mg/L, sucrose 30g/L, pH=5.8) filter paper under dark condition, 25 DEG C co-culture 2 days.Warp
Co-culture the leaf dish Carbenicillin solution washing containing 500mg/L 3 times of 2 days, blot surface liquid with aseptic filter paper
Proceed in screening division culture medium, 25 DEG C of illumination cultivation.In screening division culture medium, (MS minimal medium adds for the screening of transformant
6-benzyl aminopurine 1.0mg/L, naphthalene acetic acid 0.1mg/L, kanamycin 80mg/L, Carbenicillin 400mg/L, sucrose 30g/L,
PH=5.8 carry out in).When blade edge grows regeneration bud of growing thickly to 2～3cm, with dissecting knife, regeneration bud is cut, and proceed to
Culture 25 days in root media (MS minimal medium adds kanamycin 50mg/L, sucrose 20g/L, pH=5.8).Transformed cell
The plant of regeneration has kalamycin resistance, for normal green plant, obtains transfer-gen plant.
Extract pCXSN::GmFER transgene tobacco genomic DNA, enters performing PCR reaction with genomic DNA for template.To expand
Increase production thing and carry out gel electrophoresiss, and reclaim PCR primer and be sequenced.In order to identify transfer-gen plant further, extract and pass through base
Because group test positive turn pCXSN::GmFER tobacco leaf total serum IgE, (precious raw through reverse transcription reagent box with 1 μ g blade total serum IgE
Thing engineering (Dalian) company limited) cDNA that obtains by the method reverse transcription of test kit is template, PCR primer is in example 4
Primer P1 and P2,25 μ l PCR reaction systems contain 2.5 μ l and contain MgCl210 × PCR buffer, forward and reverse 10 μM of primer each
1.0 μ l, the dNTP (deoxynucleotide mixture) of 1.0 μ l 10mM, 1.0 μ l cDNA templates and 18.5 μ l distilled waters, and
0.25 μ l rTaq enzyme (precious biological engineering (Dalian) company limited).Amplification reaction condition is as follows：95 DEG C of denaturations 4min, 95 DEG C
30s, 55 DEG C of 50s, 72 DEG C of 1min, 33 circulations, 72 DEG C of extension 10min.Amplified production is carried out gel electrophoresiss, obtains GmFER
Gene carries out the transfer-gen plant of normal expression.
(3) gene function analysis：
Transgene tobacco T0 is sprouted for seed and wild type seeds on filter paper, is trained in 28 DEG C of illumination box
Support, after 10 days, take out size and grow consistent turning GmFER gene masculine tobacco seedling and compare tobacco seedling, it is transferred to respectively
Containing 50 μM of CdCl2Filter paper on, in 28 DEG C of illumination box cultivate, process 4 days after, measurement root long, its result as scheme
Shown in 8.Result shows, overexpression in Nicotiana tabacum L. for the GmFER gene can improve the anti-of Transgenic Tobacco plant pair Cd stress
Property.
Claims (10)
1. Semen sojae atricolor bHLH transcription factor gene GmFER, its nucleotide sequence such as SEQ ID NO:Shown in 1.
2., by the albumen of Semen sojae atricolor bHLH transcription factor gene GmFER according to claim 1 coding, its aminoacid sequence is such as
SEQ ID NO:Shown in 2.
3. comprise the expression vector of Semen sojae atricolor bHLH transcription factor gene GmFER according to claim 1.
4. expression vector according to claim 3 it is characterised in that：
Described expression vector is by by Semen sojae atricolor bHLH transcription factor gene GmFER insertion vector according to claim 1
, described carrier is selected from any one in p424GPD, pBI121, pCXSN, pCAMBIA2301, pCAMBIA1301, pAHC25
Kind.
5. comprise the engineering bacteria of Semen sojae atricolor bHLH transcription factor gene GmFER according to claim 1.
6. engineering bacteria according to claim 5 it is characterised in that：
Host in described engineering bacteria is escherichia coli or yeast, and the method being mediated by PEG/LiAc will be according to claim
Semen sojae atricolor bHLH transcription factor gene GmFER described in 1 proceeds to.
7. comprise the transgenic plant cells system of Semen sojae atricolor bHLH transcription factor gene GmFER according to claim 1.
8. transgenic plant cells system according to claim 7 it is characterised in that：
Described plant is Nicotiana tabacum L., by agriculture bacillus mediated method by Semen sojae atricolor bHLH transcription factor according to claim 1
Gene GmFER proceeds to.
9. it is used for for a pair expanding the primer of Semen sojae atricolor bHLH transcription factor gene GmFER according to claim 1, wherein positive
The nucleotide sequence of primer such as SEQ ID NO:Shown in 3, the nucleotide sequence such as SEQ ID NO of reverse primer:Shown in 4.
10. Semen sojae atricolor bHLH transcription factor gene GmFER according to claim 1, albumen according to claim 2 with
And expression vector according to claim 3 application in improving the yeast and plant toleration to Cd.
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN112813079A (en) * | 2021-04-09 | 2021-05-18 | 西南大学 | Danbo black soybean GmFER84 gene and application thereof in soybean aluminum stress improvement |
| CN113373160A (en) * | 2021-07-21 | 2021-09-10 | 云南中烟工业有限责任公司 | Tobacco bHLH transcription factor gene NtFAMA and application thereof |
| CN113999856A (en) * | 2021-11-09 | 2022-02-01 | 江苏省农业科学院 | Soybean seed vigor regulation gene GmSV1 and application thereof |
| CN114717245A (en) * | 2022-04-12 | 2022-07-08 | 湖南农业大学 | Application of MsbHLH35 gene and protein coded by MsbHLH35 gene in regulation and control of alfalfa yield and stain resistance |
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Cited By (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN112813079A (en) * | 2021-04-09 | 2021-05-18 | 西南大学 | Danbo black soybean GmFER84 gene and application thereof in soybean aluminum stress improvement |
| CN112813079B (en) * | 2021-04-09 | 2022-02-11 | 西南大学 | Danbo black soybean GmFER84 gene and application thereof in soybean aluminum stress improvement |
| CN113373160A (en) * | 2021-07-21 | 2021-09-10 | 云南中烟工业有限责任公司 | Tobacco bHLH transcription factor gene NtFAMA and application thereof |
| CN113373160B (en) * | 2021-07-21 | 2022-07-29 | 云南中烟工业有限责任公司 | Tobacco bHLH transcription factor gene NtFAMA and application thereof |
| CN113999856A (en) * | 2021-11-09 | 2022-02-01 | 江苏省农业科学院 | Soybean seed vigor regulation gene GmSV1 and application thereof |
| CN113999856B (en) * | 2021-11-09 | 2024-04-16 | 江苏省农业科学院 | Soybean seed vitality regulation gene GmSV1 and application thereof |
| CN114717245A (en) * | 2022-04-12 | 2022-07-08 | 湖南农业大学 | Application of MsbHLH35 gene and protein coded by MsbHLH35 gene in regulation and control of alfalfa yield and stain resistance |
| CN114717245B (en) * | 2022-04-12 | 2023-08-11 | 湖南农业大学 | MsbHLH35 gene and application of encoding protein thereof in regulation and control of alfalfa yield and stain resistance |
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