CN100419080C - Soyabean thermal excited transcryption factor, its coding gene and application - Google Patents
Soyabean thermal excited transcryption factor, its coding gene and application Download PDFInfo
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Abstract
The present invention discloses a heat shock transcription factor of a soybean, a coded gene thereof and an application thereof. The heat shock transcription factor of a soybean is a protein with one of the following amino acid residue sequences: 1) SEQ ID No. 2 in a sequence list, and 2) a protein which is used for using substituting, deleting or adding one to ten amino acid residues of an amino acid residue sequence of the SEQ ID No. 2 in the sequence list and has the function of regulating the heat resistant related gene. Any one carrier which can be used for guiding a foreign gene to express in a plant is used for transferring the coded gene of the heat shock transcription factor of a soybean into the plant, and thus, the heat resistance capability of the plant is enhanced. The heat shock transcription factor of a soybean and the coded gene of the present invention play an important role in breeding plants (such as soybeans.)with strong heat resistance.
Description
Technical field
The present invention relates to a kind of soyabean thermal excited transcryption factor and encoding gene thereof and application, particularly this thermal excited transcryption factor and encoding gene thereof strengthen application in the plant such as soybean with them cultivating thermotolerance.
Background technology
Since Wu etc. at first cloned fruit bat (Drosophila melanogaster) thermal excited transcryption factor (heat shock transcriptional factor in 1987, HSF) since, the HSF of schizosaccharomyces pombe (Schizosaccharomycespombe), chicken, tomato, people, paddy rice, soybean, mouse, chimpanzee (Pantroglodytes), marine alga (Guillardia theta), Africa xenopus (Xenopus Laevis) etc. is cloned in succession.Although (heat shock elements HSEs) shows extremely strong conservative property to the heat stress element in all organisms, its trans-acting factor HSF has very big difference.The relative molecular mass of HSF has bigger difference in the different genera organism, does not also have tangible antibody cross reaction between each HSF.Find in many higher eucaryotes, to exist two or more HSFs simultaneously.The multiple HSFs that is existed jointly in higher eucaryote by the different physiological roles that a kind of HSF undertook in the unicellular lower eukaryote shares, several different HSFs can participate in respectively continuing or of short duration activation heat shock or heat shock protein(HSP) (heat shock protein, HSP) expression of gene shows composition or inducibility transcripting regulating activity.
Play a very important role aspect the relevant information that HSFs coerces in delivery context, HSFs is attached on the heat stress element heat of activation and coerces the gene of reaction, thereby induces heat shock response.The HSFs of different tissues comprises following important structural domain: DNA in conjunction with territory (DNA binding domain, DBD), oligomerization territory (oligmerization domain, OD), nuclear localization signal (nuclear localization signal, NLS) and activation domain (C-terminalactivation domain, CTAD).Wherein DBD is the structural domain of high conservative.Conservative property between the DBD of plant HSFs is higher than animal and yeast or other higher eucaryotes.The key distinction of the DBD of plant HSFs and yeast, Mammals and fruit bat DBD is to have lacked 11 to 12 amino acid between 67 to 68.OD is made up of 2 hydrophobic heptad repeat region territory A and B (HR-A/B).Zone A comprises seven hydrophobic peptide tumor-necrosis factor glycoproteinss of 5-6 group, and area B is made up of 2 seven peptide tumor-necrosis factor glycoproteinss that overlap mutually.Think that at present OD is that 3 strands of alpha-helixs form the homotrimer configuration of a kind of HSF by coiled coil type alpha-helix (α-helical coiled-coil) structure.In higher eucaryote, the trimerical formation of HSF needs heat-inducible.Under non-hot shock condition, the HSF trimerizing is suppressed.Studies show that, have another hydrophobic 7 peptide tumor-necrosis factor glycoproteinss (HR-C) at the C of HSF end.This zone is relevant with the adjusting of animal HSF trimerizing.The HR-C sudden change can cause a kind of HSF trimerizing and dna binding activity of composing type.Under the normal growth condition, interact by intramolecular coiled coil type alpha-helix between HR-A/B and the C, suppress the trimerical formation of HSF.Owing to contain more proline(Pro) and glycine in the HR-C of plant HSF, the potential that forms the α spiral is very low, so the function of the HR-C of plant HSF is not clear at present.
Having reported has 21 HSFs in the Arabidopis thaliana, found the HSFs more than 16 kinds in the tomato, has also found the HSFs more than 15 kinds in the soybean.CDNA:GmHSF5, GmHSF21, the GmHSF34 of 3 total lengths have been cloned in the soybean.Find that in animal and plant a large amount of HSFs shows that the HSF gene may specificity work to different coercing, or in growth, play a part different.Most HSF gene is to be subjected to thermoinduciblely, and some expression of gene are coerced because of calcium chloride and strengthened.Soybean HSF gene promptly has constitutive expression, and abduction delivering is also arranged.The expression amount of soybean HSF genome moulding is lower than the thermal induction type, and tomato LpHSF8 (being LpHSF A1) is a constitutive expression, and its expression amount also is lower than the HSFs of its two thermal induction types.
Summary of the invention
The purpose of this invention is to provide a kind of soyabean thermal excited transcryption factor and encoding gene thereof.
Soyabean thermal excited transcryption factor provided by the present invention, name is called GmHSF8, derives from Glycine soybean (Glycinemax L.), and its amino acid residue sequence is shown in SEQ ID NO:2.
Wherein, the sequence 2 in the sequence table is made up of 510 amino-acid residues, is the DBD of GmHSF8 from the 28th-122 amino acids residue of aminoterminal of sequence 2; The OD of GmHSF8 is made up of HR-A (from the 142nd-172 amino acids residue of aminoterminal of sequence 2) and HR-B (from the 200th-208 amino acids residue of aminoterminal of sequence 2); The 225th-239 amino acids residue of aminoterminal from sequence 2 is the NLS of GmHSF8; The CTAD of GmHSF8 is made up of AHA1 (IDFDSISPE is from the 434th-442 amino acids residue of aminoterminal of sequence 2) and AHA2 (NPHFWDDILRT is from the 451st-461 amino acids residue of aminoterminal of sequence 2).
The encoding gene of above-mentioned soyabean thermal excited transcryption factor (GmHSF8) also belongs to protection scope of the present invention.Its base sequence can be shown in SEQ ID NO:1.
SEQID № in the sequence table: 1 by 1781 based compositions, and this cDNA comprises the opening code-reading frame (from 5 of sequence 1 ' the 142nd-1674 bit base of end) of 1533bp.
Contain expression carrier of the present invention, clone and host bacterium and all belong to protection scope of the present invention.
In the GmHSF8 gene different plant tissues under normal operation expression is arranged all, multiple adverse circumstance is coerced as heat, salt etc. its expression amount is increased, and promotes relevant anti-contrary expression of gene, improves the resistance of reverse of plant to environment.
Utilize any carrier that can guide foreign gene to express in plant, in GmHSF8 gene transferred plant of the present invention (particularly soybean), the temperature capacity of plant strengthens.
GmHSF8 gene of the present invention can add any enhancing promotor or inducible promoter in being building up to plant expression vector the time before its transcription initiation Nucleotide.For the ease of transgenic plant cells or plant being identified and screening, can process employed carrier, as the antibiotic marker thing (gentamicin, kantlex etc.) that adds the alternative mark (gus gene, luciferase genes etc.) of plant or have resistance.By the plant transformed host both can be monocotyledons, also can be dicotyledons, as: soybean, paddy rice, wheat, corn, cucumber, tomato, willow, turfgrass, lucerne place etc.Carry that GmHSF8 expression carrier of the present invention can Ti-plasmids, Ri plasmid, plant viral vector, directly DNA conversion, microinjection, electricity be led, conventional biological method transformed plant cells or tissue such as agriculture bacillus mediated by using, and plant transformed is become plant through tissue culture.
Soyabean thermal excited transcryption factor of the present invention and encoding gene thereof will play an important role in cultivating thermotolerance enhanced plant (as soybean).
Description of drawings
The GmHSF8 gene PCR product electrophoretogram that Fig. 1 obtains for RACE method clone
Fig. 2 is the GmHSF8 schematic arrangement
Fig. 3 is GmHSF8 a 5 ' non-translational region heat shock element
Fig. 4 is that the influence to GmHSF8 genetic expression in leaf, root and the hypocotyl of soybean is handled in 150mM NaCl and 42 ℃ of heat shocks
Fig. 5 is the physical map of pCAMBIA2300-GmHSF8
Fig. 6 is that the PCR of transformed plant detects
Fig. 7 A is the GmHSF8 expression conditions of changeing GmHSF8 gene plant and non-transgenic plant under the normal temperature condition
Fig. 7 B is the HSP70 expression conditions of changeing GmHSF8 gene plant and non-transgenic plant under the normal temperature condition
Fig. 7 C is the HSP70 expression conditions of changeing GmHSF8 gene plant and non-transgenic plant under the hot shock condition
Fig. 8 is for changeing the reaction to heat stress of GmHSF8 gene plant and non-transgenic plant
Embodiment
The acquisition of embodiment 1, GmHSF8 and encoding gene thereof:
Soybean (Glycine max L.Merr.) sowing is cultivated in the greenhouse in vermiculite, with the seedling do experiment material in growth 2-3 week.From soybean seedling, extract total RNA with Trizol test kit (GIBCO-BRL).With the synthetic first chain cDNA of reverse transcription test kit (Promega).In order to separate full-length gene, according to the segmental est sequence design of discontinuous HSF8 cDNA in soybean est database upstream and downstream primer, 3 of isolated genes ' end, 5 ' end.GSPs (gene specific primer) is as follows with NGSPs (nested gene specific primer) sequence: 5 ' RACE:GSP1,5 '-TGAACAGCCTTTGCAAGGAATGACATC-3 '; NGSP1,5 '-CTCTGCAGCTGGTTATCAGTAGCCTG-3 '; 3 ' RACE:GSP2,5 '-GCGCCTTCAAGGAATGGAGCAACGACAGC-3 '; NGSP2,5 '-GATGTCATTCCTTGCAAAGGCTGTTCA-3 '.The result obtains containing cDNA3 ' end sequence (1041bp) and the 5 ' end sequence (695bp) of thermal excited transcryption factor 8 (HSF8) as shown in Figure 1 with the terminal rapid amplifying method of cDNA (GIBCO-BRL).Fig. 1, swimming lane M are DNA marker, DL2000; 1,2 is 3 ' RACE product; 3,4 is 5 ' RACE product).
According to ordinary method this 3 ' RACE product and 5 ' RACE product are checked order, utilize DNAMAN software that the end sequence of intermediate segment (twice amplified production overlap) with twice RACE acquisition spliced, obtain the cDNA (sequence 1) of a total length 1781bp soyabean thermal excited transcryption factor 8.This cDNA comprises the 3 ' non-translational region (from 5 of sequence 1 ' the 1675th-1781 bit base of end) of the 5 ' non-translational region (from 5 of sequence 1 ' the 1st-141 bit base of end) of the opening code-reading frame (from 5 of sequence 1 ' the 142nd-1674 bit base of end) of 1533bp, 141 bases, 107 bases.In 5 ' non-translational region+102 positions (from 5 of sequence 1 ' the 37th-39 bit base of end, " " part among Fig. 3) terminator codon (TAG) is arranged, and it is consistent with the reading frame of coding region, illustrate and do not have other more forward initiator codon, show that institute's cloned sequence is a full-length cDNA, with its called after GmHSF8.These cDNA coding one section 510 amino acid whose protein (sequence 2), name is called GmHSF8, and the supposition molecular weight is 56.2KD.
Utilize BLAST to carry out sequence alignment, carry out the amino acid sequence homology analysis with DNAMAN software, the result shows the amino acid sequence homology higher (52.46%) of GmHSF8 and tomato thermal excited transcryption factor (LpHSFA1), illustrates that they belong to same class thermal excited transcryption factor.The DBD homology of the DBD of GmHSF8 and LpHSFA1 is up to 90.43%.19 amino acid are arranged before the N-terminal of the hydrophobic heptad repeat region of GmHSF8 territory A (HR-A), and (from the 123rd-141 amino acids residue of aminoterminal of sequence 2, and LpHSFA1 and Arabidopis thaliana thermal excited transcryption factor (AtHSFA1) have 30 and 19 amino acid respectively.HR-A/B functional domain (from the aminoterminal 142-208 of sequence 2 amino acids residue) is conservative fully between GmHSF8 and LpHSFA1,4 place's differences is just arranged (from 1 57 Arg → Ile of aminoterminal of sequence 2,178 Ser → Ala, 185 Gly → Ser, 195 Leu → Gln) on amino acid is formed; The NLS of GmHSF8 is double base structure SRR-5aa-KKRRLKQ (from the 225th-239 amino acids residue of aminoterminal of sequence 2); The CTAD of GmHSF8 is made up of AHA1 (IDFDSISPE) and AHA2 (NPHFWDDILRT), this conservative domain the poorest (Fig. 2).This explanation GmHSF8 is a new thermal excited transcryption factor.Among Fig. 2, the insertion sequence between insert-HR-A and the HR-B, H2-spirane structure 2, H3-spirane structure 3, T-corner structure.
As shown in Figure 3, GmHSF8 gene 5 ' end non-translational region (141bp) can find 6 HSE response elements [A (and G, T, C) GAAn or A (G, T, C) GnAn or A (G, T, C) GAnn] (use underscore
Represent) and 13 (annotate: top among Fig. 3 "
6 bases" be 2 HSE response rear elements) HSE response rear element [nTTCt (A, G, C) or nTnCT (A, G, C) or nnTCT (A, G, C)] (with underscore " _ " expression).Although this 5 ' end non-translational region is shorter, 19 heat shock response elements are arranged, illustrate that existing complicated mutual work between thermal excited transcryption factor GmHSF8 and its encoding gene regulates relation.5 ' end non-translational region at this gene is not found the TAAT box, illustrates that these heat shock response elements are positioned at the downstream of TAAT box.
Soybean (Glycine max L.Merr.) sowing is cultivated in the greenhouse in vermiculite, with the seedling do experiment material in growth 2-3 week.
Seedling is placed on cultivates 3,6,9 and 18 hours under 42 ℃ respectively, control group is cultivated down at 24 ℃.Place 150mM NaCl solution to handle 3,6,9 and 18 hours seedlings root.
Extract total RNA from blade, root and the hypocotyl of the seedling of different treatment respectively.Analyze of the expression of GmHSF8 gene with quantitative RT-PCR method at the soybean different tissues.With OligodT
15For the synthetic cDNA of primer reverse transcription, carry out pcr amplification, analyze of the expression of GmHSF8 gene with quantitative RT-PCR method at the soybean different tissues.The special primer that PCR uses is forward primer P1:5 '-CTTCTCCAGCTTCGTTCGCC '-3 '; Reverse primer P2:5 '-TACCCTCCTGCTTAAGCCGG-3 '.
With the concentration of GmACTIN (Accession No.V00450) quantitative templates cDNA, the primer is: forward primer: 5 '-GGTGATGGTGTGAGTCACACTGTACC-3 '; Reverse primer: 5 '-GTGGACAATGGATGGGCCAGACTC-3 '.
PCR is reflected on the eppendorf type amplification instrument and carries out, the reaction cumulative volume is 25ul, contain cDNA 200ng, forward primer 10pM, reverse primer 10pM, 1 U Taq archaeal dna polymerase (TaKara company), 2.5 μ l 10 * damping fluid (200mM Tri s-HCl, pH 8.8,100mM KCl, 100mM (NH
4)
2SO
4, 20mM MgSO
4, 1%Triton X-100,1mg ml
-1Nuclease-free BSA and 0.2mM dNTPs).Amplification program is 94 ℃ of pre-sex change 3min, enters circulation then: 94 ℃ of sex change 30s, 60 ℃ of renaturation 30s, 72 ℃ are extended 90s, totally 30 circulations, at last again 72 ℃ extend 10min.The result shows that high temperature has different influences with high-salt stress to GmHSF8 genetic expression in the soybean different tissues.Influenced by pyritous not obvious for the GmHSF8 expression of gene in the blade, but be subjected to the inhibition (and 150mM NaCl handles the 9h exception) of high-salt stress; High temperature has slight restraining effect to GmHSF8 genetic expression in the hypocotyl, and this expression of gene then has slight inducing action in the high salt pair hypocotyl; The GmHSF8 expression of gene is subjected to the obvious inducing action of high temperature and salt in the root, this expression of gene amount all increases (but 150mM NaCl handles the 3h exception), this variation tendency of pyroprocessing more obvious (Fig. 4) with two kinds of lasting treatment time prolongations of coercing the factor.In a word, the GmHSF8 gene in Soybean Leaves, hypocotyl and the root all has expression (for constructive expression's type) under normal condition (42 ℃ of i.e. 24 ℃ of cultivations of 0h), and high temperature or high-salt stress can make its expression amount increase, and is especially true in root system.Among Fig. 4, LH represents GmHSF8 expression of gene in the blade, and LA represents the expression of actin gene in the blade; HH represents GmHSF8 expression of gene in the hypocotyl, and HA represents the expression of actin gene in the hypocotyl; RH represents GmHSF8 expression of gene in the root, and RA represents the expression of actin gene in the root.Swimming lane 1,2,3,4 and 5 represent respectively to handle 0,3,6,9 and 18 hours.
The pCAMBIA2300 carrier is available from the CABIA center.Taq DNA Polymerase, SmaI and ScaI are available from Takara company.6-BA, IBA, cephamycin C ef, kantlex Kanamycin (Kan) and Syringylethanone (AS) etc. are available from Beijing ancient cooking vessel state biotech company.
1, the structure of plant expression vector
Cut carrier pCAMBIA2300 with Restriction enzyme Sma I and SacI enzyme, carry out the GmHSF8 gene that pcr amplification obtains with primer P1:CCCGGGATGGACGGAAGAGC and P2:TTAAGAAAACCGTAGTCT (containing SmaI recognition site CCCGGG and SacI recognition site GAGCTC), use T
4Dna ligase connects, and will connect product transformed into escherichia coli JM109, and screening has the bacterium colony of kalamycin resistance on the X-gal flat board, carries out PCR and identifies, obtains containing the positive bacterium colony of plant expression vector pCAMBIA2300-GmHSF8.Extract the plasmid in the positive bacterium colony, obtain plant expression vector pCAMBIA2300-GmHSF8 (Fig. 5).
2, soybean heredity transforms
Soybean transformation receptor kind is new No. 3 of section.
Transform Agrobacterium LBA4404 with plant expression vector pCAMBIA2300-HSF8, containing the enterprising row filter of YEB substratum of Streptomycin sulphate 25mg/L and kantlex 50mg/L, and the picking mono-clonal extracts plasmid and carry out enzyme and cut evaluation, obtain positive colony, 4 ℃ of preservations.The single bacterium colony of Agrobacterium that picking contains expression vector from the flat board of 4 ℃ of preservations inserts the YEB substratum that contains 50mg/L Kan, 25mg/L Rifampin, 25mg/L Streptomycin sulphate, about 28 ℃, 200r/min shaking culture 12-14h to logarithmic phase, at 4 ℃, the centrifugal 10min of 8000rpm, remove supernatant liquor, with liquid division culture medium (MS+1.6mg/L 6-BA+0.2mg/L IBA+500mg/L Cef with volume, pH5.4), it is standby to add the resuspended back of 100 μ mol/L AS.
The soybean seeds of selecting full seed by Zhang etc. (1999, Plant Cell, Tissue, and OrganCulture, method 56:37-46) carries out disinfection.Full dry seeds single flat is laid in the culture dish, and culture dish opens wide it is positioned in the moisture eliminator, and the beaker of 100ml is positioned in the moisture eliminator, the saturated clorox that adds 50ml, add the 2ml concentrated hydrochloric acid again, cover the moisture eliminator lid immediately, place spend the night in the ventilating kitchen (14-16h).Seed after the sterilization is inoculated on the germination medium MSO (pH5.8), cultivates in 25 ℃ the greenhouse, and the dark 8h of light 16h/ obtains aseptic seedling behind the 5-6d.
The cotyledonary node of getting aseptic seedling is an explant, draw map cracking with scalper at the cotyledonary node position, with its be immersed in infect 30min in the resuspended liquid of Agrobacterium after, place the dark place to cultivate 3d altogether after, change the induced bundle substratum MS+1.6mg/L 6-BA+0.2mg/L IBA+500mg/L Cef of sprouting over to; Wait to grow and grow thickly behind the bud, it is inoculated in screening culture medium MS+1.6mg/L 6-BA+0.2mg/L IBA+50mg/L kan+500mg/L Cef, per 2 all subcultures 1 time, obtained the conversion seedling of a collection of anti-kantlex (Kan), the indefinite bud of screening is connected to elongation medium MS+0.8mg/L 6-BA+0.2mg/L IBA+50mg/L kan+500mg/L Cef when growing to 0.5-1cm; The seedling that some buds of growing thickly are extracted stem out and grown up to 3-4cm after about 2 weeks moves into root media MS+1.5mg/L IBA with it, is transplanted in the flowerpot after waiting to grow healthy and strong root system.Obtain 13 strain transformed plants at last.
3, transformed plant PCR detects
Choose resistance transplant survival plant through the Kan screening, every strain clip 1-2 sheet leaf, according to Edwards etc. (1991, Nucleic Acids Res., 19:1349) method of describing is extracted genomic dna, with it is template, adopts the 35s promoter primer of no homologous sequence in the plant materials to carry out pcr amplification.It is synthetic that the PCR primer is given birth to the worker by Shanghai.Primer sequence is respectively: F:5-GGGTCTTGCGAAGGATAG-3, R:5-GCTTACGCAGCAGGTCTC-3.PCR is reflected on the PTC-100 type PCR instrument and carries out, and carries out amplification cycles by following condition behind 94 ℃ of pre-sex change 5min: 94 ℃ of sex change 1min, 50 ℃ of annealing 1min, 72 ℃ of extension 1min, follow-up 30 same circulations.Reaction finishes back 72 ℃ and extends 10min.Amplified production detects with 1% agarose gel electrophoresis.With the positive contrast of pCAMBIA2300 plasmid, the negative contrast of non-transgenic regrowth that section is new No. 3.The PCR detected result as shown in Figure 6, showing has 9 strains to amplify the DNA band of 830bp in the 13 strain transformed plants, and negative control does not amplify corresponding fragment (Fig. 6).Proof GmHSF8 gene has been integrated in the genome of this 9 strain, and actual conversion is 2.39%.Among Fig. 6,1: non-transgenic stock plant (negative control); 2:pCAMBIA2300-GmHSF8 (positive control); 3-15: transformed plant; M:DNA marker (DL2000).
4, transfer-gen plant Northern detects
Extract the commentaries on classics 6mHSF8 gene soybean plant strain of cultivation under the normal temperature (24 ℃) and the total RNA of blade of non-transgenic soybean plant strain respectively, (with plasmid pCAMBIA2300-GmHSF8 DNA is template with one section conserved sequence of GmHSF8 cDNA, the dna fragmentation that PCR obtains under the guiding of primers F: 5-GGGTCTTGCGAAGGATAG-3 and R:5-GCTTACGCAGCAGGTCTC-3) is probe, detects the GmHSF8 expression of gene by the Northern method.The result shows that changeing the GmHSF8 of GmHSF8 gene plant gene expression amount at normal temperatures is significantly higher than non-transgenic plant (a small amount of expression) shown in Fig. 7 A.Among Fig. 7 A, 1 is the non-transgenic plant, and 2 for changeing the GmHSF8 gene plant.
The seedling that changes 3 ages in week of GmHSF8 gene soybean plant strain and non-transgenic soybean plant strain is cultivated down and 42 ℃ of heat shocks 3 hours/day (handling continuously 3 days) at normal temperature (24 ℃) respectively, extract the total RNA of blade that changes GmHSF8 gene soybean plant strain and non-transgenic soybean plant strain respectively, (with the new No. 3 soybean plant strain genomic dnas of section is template with one section conserved sequence of HSP70 cDNA, the dna fragmentation that PCR obtains under the guiding of primer P1:CATAGGCATCGATCTCGGCAC and P2:GAAGACGGTGTTCTGCGGG) is probe, detects the HSP70 expression of gene by the Northern method.The result shows that the HSP70 gene expression amount that changes the GmHSF8 gene plant under normal temperature (24 ℃) condition is apparently higher than the non-transgenic plant (shown in Fig. 7 B) of expressing hardly; Change the HSP70 gene overexpression of GmHSF8 gene plant under heat shock (42 ℃) condition, its expression amount is significantly higher than non-transgenic plant (a certain amount of expression is arranged) (shown in Fig. 7 C).Among Fig. 7 B and Fig. 7 C, 1 is the non-transgenic plant, and 2 for changeing the GmHSF8 gene plant.
5, transfer-gen plant heat stress experiment
For whether the plant of verifying overexpression GmHSF8 gene has improved thermotolerance, transfer-gen plant and non-transgenic plant are carried out high temperature (heat shock) processing experiment.The result show normal temperature (28-30 ℃ cultivate 6h) down for all blades of examination plant all be equatorial (A among Fig. 8, B); When temperature rises to 38-40 ℃, when cultivating 40min, the leaflet tablet of non-transgenic plant begins inwardly to curl (a C-left side, a D-left side among Fig. 8), at 42-45 ℃, curl in when cultivating 40min and show comparatively obviously (an E-left side, a F-left side among Fig. 8), at 42-48 ℃, interior amount of crimp raises gradually on obviously (an E-left side, a F-left side, a G-left side, a H-left side among Fig. 8) with temperature when cultivating 40min, and the leaflet tablet of transfer-gen plant curls in occurring.After 15 minutes, the top vane of non-transgenic plant begins here to wither (an I-left side among Fig. 8) 52 ℃ of cultivations, and the leaflet tablet of transfer-gen plant is slightly curl (the I-right side among Fig. 8).Illustrate that the soybean plant strain that changes the GmHSF8 gene shows stronger temperature capacity (improving 8-10 ℃) than non-transgenic adjoining tree, GmHSF8 gene provided by the present invention has the function that activates heat-resisting genes involved.Among Fig. 8, the left side plant among the A-I is that non-transgenic plant, right side plant are for changeing the GmHSF8 gene plant.
Sequence table
<160>2
<210>1
<211>1781
<212>DNA
＜213〉Glycine soybean (Glycine max L.)
<400>1
tctttcacta?ctccccatct?gttaaatttc?gaacactagg?gttcaatttt?ctccaacaat 60
gaccctcact?ctattcctct?gacttcccat?agaccacaat?acactcaccc?caggcaaaaa 120
atttcgtttc?aaaattgtga?tatggacgga?agagcgagca?gcagcgtggg?cggagaagca 180
tcgccagctc?cagcaccggt?gccgataacg?aatgcgaacg?cgccgccgcc?tttcttgagc 240
aagacatacg?agatggtaga?ggacccttcg?acggactcga?tagtgtcgtg?gagtccaacg 300
aacaacagtt?tcgtggtttg?gaaccctccc?gagttcgcca?gagatctctt?gcccaaacac 360
ttcaagcaca?acaacttctc?cagcttcgtt?cgccaattaa?acacctacgg?atttaggaag 420
gtagatccag?atcgctggga?atttgcaaat?gagggatttt?tgaggggtca?aaagcacttg 480
cttaagacta?taactcggcg?gaaacctgcc?catggtcata?atcaacaggc?acagcaagca 540
catggacaga?gttcatctgt?tggggcttgt?gttgaagttg?ggaagtttgg?acttgaggaa 600
gaggttgaga?ttctcaagag?agataagaat?gtgctcatgc?aagagcttgt?gagattgagg 660
cagcagcaac?aggctactga?taaccagctg?cagagtatgg?ttcagcgcct?tcaaggaatg 720
gagcaacgac?agcaacaaat?gatgtcattc?cttgcaaagg?ctgttcagag?tcctggtttt 780
ttagctcaat?ttgtacaaca?gcaaaatgag?agtagtagac?gcataacgga?ggcaaataaa 840
aaacgccggc?ttaagcagga?gggtattggt?gaaatggaac?atactgctgc?ttctgatggc 900
caaattgtta?aatatcaacc?tctgataaat?gaagcagcaa?aagcaatgct?gaggcaaatg 960
atgaaattgg?atacttctcg?actagaatct?tttagtaata?acgctgataa?ttacttgatt 1020
ggtgatcatt?catcatcatc?cggtgcaacg?gacaggggaa?actctttgag?ccggacttct 1080
ggagtaacac?ttcaagtggt?ccctctgact?acaatccagt?cttctcacat?tccatctgca 1140
acggggatag?gggatgaccc?ttcaacagga?aaatctgaga?ttctatctac?tcctcaagtt 1200
gtagcctgtg?atgaagttac?gaaagctcag?tactctaatg?taaatgtttc?ggttggagaa 1260
tctaatgcac?ctgctatccc?tgctactcaa?acagatgaaa?tcatgcggga?cctctctaca 1320
ataccagaca?tagtggcagg?aaatattctt?gatattcctc?aagaaaatta?tatggcacct 1380
gagacaggcg?gtgaaggata?tatggatcct?acttcatttg?gagtgaatgt?gtcattgccc 1440
attgattttg?atagtatttc?acctgaagca?gacattgatg?atttgttgaa?caatcctcac 1500
ttttgggatg?atattttgcg?aactccagtg?tcagaggaga?ttgatacgaa?tgatgctgaa 1560
gtattcaagg?agaatgaggt?gcagccaatg?gaaaatggat?tggacgaatc?acaaaatatg 1620
gaccaactta?ctgagcagat?gggcctactt?tcttctgatg?ccaaaagaat?ttgagtgtat 1680
tatgaaagtg?tacattatta?atatttcata?ttgtgcaagg?taaccttagc?ctagacaatg 1740
ggttatgttg?ttttagttaa?aaaaaaaaaa?aaaaaaaaaa?a 1781
<210>2
<211>510
<212>PRT
＜213〉Glycine soybean (Glycine max L.)
<400>2
Met?Asp?Gly?Arg?Ala?Ser?Ser?Ser?Val?Gly?Gly?Glu?Ala?Ser?Pro?Ala
1 5 10 15
Pro?Ala?Pro?Val?Pro?Ile?Thr?Asn?Ala?Asn?Ala?Pro?Pro?Pro?Phe?Leu
20 25 30
Ser?Lys?Thr?Tyr?Glu?Met?Val?Glu?Asp?Pro?Ser?Thr?Asp?Ser?Ile?Val
35 40 45
Ser?Trp?Ser?Pro?Thr?Asn?Asn?Ser?Phe?Val?Val?Trp?Asn?Pro?Pro?Glu
50 55 60
Phe?Ala?Arg?Asp?Leu?Leu?Pro?Lys?His?Phe?Lys?His?Asn?Asn?Phe?Ser
65 70 75 80
Ser?Phe?Val?Arg?Gln?Leu?Asn?Thr?Tyr?Gly?Phe?Arg?Lys?Val?Asp?Pro
85 90 95
Asp?Arg?Trp?Glu?Phe?Ala?Asn?Glu?Gly?Phe?Leu?Arg?Gly?Gln?Lys?His
100 105 110
Leu?Leu?Lys?Thr?Ile?Thr?Arg?Arg?Lys?Pro?Ala?His?Gly?His?Asn?Gln
115 120 125
Gln?Ala?Gln?Gln?Ala?His?Gly?Gln?Ser?Ser?Ser?Val?Gly?Ala?Cys?Val
130 135 140
Glu?Val?Gly?Lys?Phe?Gly?Leu?Glu?Glu?Glu?Val?Glu?Ile?Leu?Lys?Arg
145 150 155 160
Asp?Lys?Asn?Val?Leu?Met?Gln?Glu?Leu?Val?Arg?Leu?Arg?Gln?Gln?Gln
165 170 175
Gln?Ala?Thr?Asp?Asn?Gln?Leu?Gln?Ser?Met?Val?Gln?Arg?Leu?Gln?Gly
180 185 190
Met?Glu?Gln?Arg?Gln?Gln?Gln?Met?Met?Ser?Phe?Leu?Ala?Lys?Ala?Val
195 200 205
Gln?Ser?Pro?Gly?Phe?Leu?Ala?Gln?Phe?Val?Gln?Gln?Gln?Asn?Glu?Ser
210 215 220
Ser?Arg?Arg?Ile?Thr?Glu?Ala?Asn?Lys?Lys?Arg?Arg?Leu?Lys?Gln?Glu
225 230 235 240
Gly?Ile?Gly?Glu?Met?Glu?His?Thr?Ala?Ala?Ser?Asp?Gly?Gln?Ile?Val
245 250 255
Lys?Tyr?Gln?Pro?Leu?Ile?Asn?Glu?Ala?Ala?Lys?Ala?Met?Leu?Arg?Gln
260 265 270
Met?Met?Lys?Leu?Asp?Thr?Ser?Arg?Leu?Glu?Ser?Phe?Ser?Asn?Asn?Ala
275 280 285
Asp?Asn?Tyr?Leu?Ile?Gly?Asp?His?Ser?Ser?Ser?Ser?Gly?Ala?Thr?Asp
290 295 300
Arg?Gly?Asn?Ser?Leu?Ser?Arg?Thr?Ser?Gly?Val?Thr?Leu?Gln?Val?Val
305 310 315 320
Pro?Leu?Thr?Thr?Ile?Gln?Ser?Ser?His?Ile?Pro?Ser?Ala?Thr?Gly?Ile
325 330 335
Gly?Asp?Asp?Pro?Ser?Thr?Gly?Lys?Ser?Glu?Ile?Leu?Ser?Thr?Pro?Gln
340 345 350
Val?Val?Ala?Cys?Asp?Glu?Val?Thr?Lys?Ala?Gln?Tyr?Ser?Asn?Val?Asn
355 360 365
Val?Ser?Val?Gly?Glu?Ser?Asn?Ala?Pro?Ala?Ile?Pro?Ala?Thr?Gln?Thr
370 375 380
Asp?Glu?Ile?Met?Arg?Asp?Leu?Ser?Thr?Ile?Pro?Asp?Ile?Val?Ala?Gly
385 390 395 400
Asn?Ile?Leu?Asp?Ile?Pro?Gln?Glu?Asn?Tyr?Met?Ala?Pro?Glu?Thr?Gly
405 410 415
Gly?Glu?Gly?Tyr?Met?Asp?Pro?Thr?Ser?Phe?Gly?Val?Asn?Val?Ser?Leu
420 425 430
Pro?Ile?Asp?Phe?Asp?Ser?Ile?Ser?Pro?Glu?Ala?Asp?Ile?Asp?Asp?Leu
435 440 445
Leu?Asn?Asn?Pro?His?Phe?Trp?Asp?Asp?Ile?Leu?Arg?Thr?Pro?Val?Ser
450 455 460
Glu?Glu?Ile?Asp?Thr?Asn?Asp?Ala?Glu?Val?Phe?Lys?Glu?Asn?Glu?Val
465 470 475 480
Gln?Pro?Met?Glu?Asn?Gly?Leu?Asp?Glu?Ser?Gln?Asn?Met?Asp?Gln?Leu
485 490 495
Thr?Glu?Gln?Met?Gly?Leu?Leu?Ser?Ser?Asp?Ala?Lys?Arg?Ile
500 505 510
Claims (6)
1. the encoding gene of a soyabean thermal excited transcryption factor, its base sequence is shown in SEQ ID NO:1.
2. soyabean thermal excited transcryption factor, its amino acid residue sequence is shown in SEQ ID NO:2.
3. the encoding gene of the described soyabean thermal excited transcryption factor of claim 2.
4. the expression vector that contains the described soyabean thermal excited transcryption factor encoding gene of claim 3.
5. the host bacterium that contains the described soyabean thermal excited transcryption factor encoding gene of claim 3.
6. the application of the encoding gene of the described soyabean thermal excited transcryption factor of claim 3 in cultivating thermotolerance enhancing soybean.
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| CN109722441B (en) * | 2019-01-22 | 2020-06-16 | 广东省农业科学院蔬菜研究所 | Cucumber small heat shock protein Cu-sHSP gene and application thereof |
| CN110903364B (en) * | 2019-11-12 | 2021-02-19 | 中国农业大学 | Application of CsHSFA1d protein and coding gene thereof in regulation and control of cold resistance of plants |
| CN112251448A (en) * | 2020-10-26 | 2021-01-22 | 上海植物园 | Clematis CvHSFB2a gene and application of protein coded by same in high temperature stress resistance |
| CN112251462B (en) * | 2020-10-26 | 2022-05-17 | 南京农业大学 | Application of soybeans GmHSFA2 and GmHSP20a in enhancing heat resistance of plants in flowering period |
| CN113024648B (en) * | 2021-03-07 | 2023-07-21 | 安徽农业大学 | Corn heat shock transcription factor ZmHsf05 and application thereof |
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| WO2004007660A2 (en) * | 2002-07-10 | 2004-01-22 | Consejo Superior De Investigaciones Cientificas | Novel hsf transcription factors and use thereof in transgenic plants |
| CN1583789A (en) * | 2003-08-21 | 2005-02-23 | 中国科学院遗传与发育生物学研究所 | Soybean transcripting factor, its coding gene and use thereof |
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2005
- 2005-02-25 CN CNB2005100087557A patent/CN100419080C/en not_active Expired - Fee Related
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2004007660A2 (en) * | 2002-07-10 | 2004-01-22 | Consejo Superior De Investigaciones Cientificas | Novel hsf transcription factors and use thereof in transgenic plants |
| CN1583789A (en) * | 2003-08-21 | 2005-02-23 | 中国科学院遗传与发育生物学研究所 | Soybean transcripting factor, its coding gene and use thereof |
Non-Patent Citations (1)
| Title |
|---|
| 农杆菌介导法将热激转录因子8基因转入大豆. 尹青女等.分子植物育种，第2卷第6期. 2004 * |
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| CN1824779A (en) | 2006-08-30 |
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