CN1789280A - PHD transcription factor of soybean and its coding gene and usage - Google Patents
PHD transcription factor of soybean and its coding gene and usage Download PDFInfo
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Abstract
The invention discloses a soybean PHD transcription factor, its encoding gene and use, wherein the soybean PHD transcription factor is protein having one of the following amino acid residue sequences: (1) SEQ ID No.2 in the sequence table, (2) protein with plants resistance regulation of PHD transcription activation function obtained through subjecting SEQ ID No.2 amino acid sequences in the sequence table to one or several amino acid sequences substitution and/or deletion and/or addition. The soybean PHD transcription factor and its encoding genes can be used for the cultivation of abiotic stress tolerant plant varieties, especially abiotic stress tolerant soybean varieties, such as salt resistant soybean.
Description
Technical field
The present invention relates to the PHD transcription factor relevant and encoding gene and application in the plant genetic engineering field, particularly derive from the PHD transcription factor relevant and encoding gene and its application in cultivating the plant with adverse resistance kind of soybean with resistance of reverse with resistance of reverse.
Background technology
The variation of physics, chemical factor in the environment, the factors of coercing such as for example arid, saline and alkaline, low temperature have material impact to growth and development of plant, can cause the extensive underproduction of farm crop when serious, therefore cultivating the high crop of resistance of reverse is one of major objective of plant husbandry.At present, using gene engineering technique carries out breeding and has become one of important method that improves the crop resistance of reverse.Higher plant cell has many approach and replys various environment stresses in the environment, and wherein transcription factor plays a part the regulation and control effector of anti-the retrocorrelation and expresses.Now in plant, found the multiclass transcription factor relevant, for example: the DREB class among the EREBP/AP2, bZIP, MYB or the like with plant stress tolerance.PHD (Plant Homodomain) class transcription factor extensively is present in animals and plants and bacterium, the viral protein, such as mammiferous CBP proteinoid, and people's INGl family, yeast Yng2p albumen, viral MIRl albumen.PHD proteinoid structural domain is the zinc fingers of C4HC3 class, and its function is mainly following a few class: (1) participates in the relevant transcriptional control of chromatin as acetylize or deacetylase; (2) participate in proteolytic degradation as the E3 ubiquitin ligase; (3) participate in the relevant transcriptional control of chromatin as PIPs acceptor perception PIPs signal.Found the PHD proteinoid in Arabidopis thaliana first, such proteic functional study is still few in the plant.
Soybean is one of China five big crops, understands fully its anti-abiotic stress molecule mechanism, and then provides the basis for improving its resistance of reverse, has important theory and realistic meaning.
Summary of the invention
An object of the present invention is to provide a kind of soybean PHD transcription factor and encoding gene and application.
Soybean PHD transcription factor provided by the present invention, name is called GmPHD2, derives from Glycine soybean (Glycinemax (L.)), is the protein with one of following amino acid residue sequences:
1) the SEQ ID № in the sequence table: 2;
2) with SEQ ID № in the sequence table: 2 amino acid residue sequence is through the replacement of one or several amino-acid residue and/or disappearance and/or interpolation and have the protein of the regulation and control stress resistance of plant of transcriptional activation function.
Wherein, the sequence 2 in the sequence table is made up of 252 amino-acid residues, is the PDH conserved domain of PDH class transcription factor from the 199th-252 amino acids residue sequence of aminoterminal (N end).
The replacement of described one or several amino-acid residue and/or disappearance and/or interpolation are meant replacement and/or the disappearance and/or the interpolation of no more than ten amino-acid residues.
The encoding gene (GmPHD2) of above-mentioned soybean PHD class transcription factor also belongs to protection scope of the present invention.
The cDNA gene of above-mentioned soybean PHD class transcription factor can have one of following nucleotide sequence:
1) SEQ ID № in the sequence table: 1 dna sequence dna;
2) SEQ ID № in the code sequence tabulation: the polynucleotide of 2 protein sequences;
3) under the rigorous condition of height can with SEQ ID № in the sequence table: the nucleotide sequence of the 1 dna sequence dna hybridization that limits.
The rigorous condition of above-mentioned height can be 0.1 * SSPE (or 0.1 * SSC), in the solution of 0.1%SDS, under 65 ℃, hybridize and wash film.
Wherein, the SEQ ID № in the sequence table: 2 are made up of 759 deoxynucleotides, and this sequence is the reading frame of GmPHD2 gene, and coding has SEQ ID № in the sequence table: the protein of 1 amino acid residue sequence; From 5 ' end the 595th to the 756th bit base is the encoding sequence of PHD structural domain conservative among the GmPHD2.
Contain expression carrier of the present invention, clone and host bacterium and all belong to protection scope of the present invention.
Amplification GmPDH2 one segmental primer is to also within protection scope of the present invention.
Utilize plant expression vector, the encoding gene importing vegetable cell with GmPDH2 of the present invention can obtain environment stress tolerance enhanced transgenic cell line and transfer-gen plant.
When using GmPDH2 to make up plant expression vector, before its transcription initiation Nucleotide, can add any enhancement type promotor or inducible promoter.For the ease of transgenic plant cells or plant being identified and screening, can process used plant expression vector, as adding selected marker's (gus gene, luciferase genes etc.) that can in plant, express or antibiotic marker thing (gentamicin marker, kantlex marker etc.) with resistance.From the security consideration of transgenic plant, can not add any selected marker, directly with adverse circumstance screening transformed plant.
Carry GmPDH2 of the present invention plant expression vector 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 the plant transformed tissue cultivating is become plant.By the plant transformed host both can be monocotyledonss such as paddy rice, wheat, corn, also can be dicotyledonss such as cucumber, tomato, willow, turfgrass, lucerne place.
The expression of GmPHD2 is not subjected to inducing of high salt and arid, but is subjected to inducing of dormin ABA.It is relevant that known ABA and plant abiotic stress are replied, so GmPHD2 may be relevant with the regulation and control that plant is replied abiotic stress.Import the Arabidopis thaliana of GmPHD2 and compare with transgenic arabidopsis not, its salt tolerance significantly improves, and is not subjected to high salt, drought-induced although the GmPHD2 expression of gene is described, the regulation and control that its involved in plant is replied abiotic stress.
GmPDH2 of the present invention is to cultivating the particularly anti-contrary soybean varieties of plant with adverse resistance kind, as the salt tolerant soybean, particularly the output of soybean is significant to improve farm crop, and proved the regulation and control that the certain involved in plant of some members the PDH family is replied abiotic stress from the molecular biology angle, it is expressed and the anti-abiotic stress of plant is proportionate.
The present invention will be further described below in conjunction with drawings and Examples.
Description of drawings
Fig. 1 is the expression characterization figure of GmPDH2 under abiotic stress is coerced
Fig. 2 is the part-structure synoptic diagram that contains the plant expression vector of GmPDH2
Fig. 3 A is G2-3, G2-6, G2-8 and the wild-type plant growth comparison diagram under salt stress and under the normal growth condition for changeing the strain of GmPDH2 Arabidopis thaliana
Fig. 3 B is transfer-gen plant TG-1, TG-2, TG-3 and wild-type plant growing state photo under normal operation
Fig. 3 C is transfer-gen plant TG-1, TG-2, and TG-3 and wild-type plant are handled the growing state photo of replying again after 14 days after 14 days with 150mM NaCl
Embodiment
Method therefor is ordinary method if no special instructions among the following embodiment.
The screening of embodiment 1, soybean GmPDH2 and encoding gene thereof and the clone of cDNA thereof
Knitting (available from Shanxi Province Academy of Agricultural Sciences) with soybean drought resisting kind Shanxi beans 23 and non-irrigated sensitive varieties ash cloth is material, a difference expression gene surplus method screening by cDNA-AFLP obtains 60, a 296bp fragment is wherein arranged, this fragment shows that through BLAST soybean est database it is the zinc finger protein that comprises the PHD structural domain, finds to have in the drought, salt cDNA storehouse at a plurality of species the homologous sequence of this gene in comparison.Obtain 6 these family members through comparison in the soybean est database, respectively called after GmPHD1-6.Extend the full length sequence that splicing obtains 4 GmPHD genes through EST, wherein GmPHD2 has the nucleotide sequence of sequence 1 in the sequence table, the amino acid residue sequence that coding has sequence 2 in the sequence table.Amino acid sequence analysis shows, sequence 2 is made up of 252 amino-acid residues in sequence table, from the 199th-252 amino acids residue sequence of aminoterminal (N end) is the PDH structural domain of PDH class transcription factor, PDH structural domain and very conservative from N-terminal the 1st to 114 amino acids residue, and the sequence variations of middle portion is bigger.
Design primer according to the GmPHD2 gene order: GmPHD2 ORF sense:5 '-ATGGACGGTGGTGGAGTGA-3 ', GmPHD2 ORF antisense:5 '-TCAAGGCCGTGCTCTCTTAT-3 ' uses the RT-PCR method, amplification GmPHD2 gene from the total RNA of soybean.Get Shanxi beans 23 blades, place liquid nitrogen to grind, be suspended from the 4mol/L sulphur hydracid guanidine, and, in supernatant, add dehydrated alcohol and precipitate, will precipitate total RNA that obtains soluble in water at last with acid phenol, chloroform extracting.Getting the total RNA of 5 μ g and carry out reverse transcription with reverse transcription test kit (Promega company) by the method for test kit, is that template is carried out pcr amplification reaction with the cDNA fragment that obtains.20 μ l PCR reaction systems are: 1 μ l, one chain cDNA (0.05 μ g), 1 μ l primer (20 μ M), 2 μ l, 10 * PCR damping fluid, 0.4 μ l dNTP (10mM) and 1U Taq archaeal dna polymerase, supply 20 μ l with ultrapure water, and liquid level covers Witco 70.Be reflected on the PE9600 type PCR instrument and carry out, its program is 94 ℃ of sex change 5min; 94 ℃ of 1min again, 56 ℃ of 1min, 72 ℃ of 1min, 30-32 circulation altogether; 72 ℃ are extended 10min then; 4 ℃ of preservations.The sequential analysis after reclaiming of PCR product, the result shows the nucleotide sequence that this PCR product has sequence 1 in the sequence table, clones then in the multiple clone site of pMD18-T plasmid, obtains recombinant vectors pMDGmPHD2.
Soybean drought resisting kind Shanxi beans 23 are carried out arid, NaCl, ABA, damage to plants caused by sudden drop in temperature to handle and be used to analyze the expression of soybean GmPHD2 under abiotic stress.The seed kind of Shanxi beans 23 in basin, after 2 weeks of growing, is carried out the following processing of coercing respectively to seedling:
Arid is handled: soybean seedling is taken out the moisture that blots on the root from soil, place on the exsiccant filter paper, arid is cultivated sampling after 0 hour, 1 hour, 3 hours, 6 hours and 12 hours under illumination condition respectively.
Salt is handled: the root system of soybean seedling is placed 150mM NaCl solution, take a sample after 0 hour, 1 hour, 3 hours, 6 hours and 12 hours in illumination cultivation respectively.
ABA handles: the root system of soybean seedling places 20 μ M ABA, takes a sample after 0 hour, 1 hour, 3 hours, 6 hours and 12 hours in illumination cultivation respectively.
Subzero treatment: wheat seedling is placed 4 ℃ of incubators, take a sample after 0 hour, 1 hour, 3 hours, 6 hours and 12 hours in illumination cultivation respectively.
The extraction of total RNA is with embodiment one.Be template with different soybean samples of handling with aforesaid method and the RNA that does not have process to coerce the soybean sample (contrast) of processing respectively, with
32The GmPHD2cDNA of P-CTP mark is a probe, carry out Northern according to a conventional method and analyze, the result as shown in Figure 1, the GmPHD2 expression of gene is not subjected to high salt, drought-induced, ABA handle GmPHD2 down be expressed in 12 hours the time rising arranged.The expression of GmPHD2 is reduction trend during subzero treatment, not induced by high salt and osmotic stress though show the expression of GmPHD2, suppressed by low temperature (4 ℃), induce but be subjected to conduct closely-related plant hormone ABA, so GmPHD2 possibility involved in plant is to the responsing reaction of abiotic stress with plant abiotic stress signal.
The Function Identification of embodiment 3, GmPHD2 proteins encoded
The GmPHD2 forward is inserted pBin438 (Li Taiyuan, Yingchuan, field, Qin Xiaofeng etc., the research of efficient insect-resistant transgenic tobacco, Chinese science (B collects), 1994,24 (3): 276-282) between the BamHI of plant expression vector and the Kpn I restriction enzyme site, the plant expression vector that structure obtains containing GmPHD2 as shown in Figure 2, called after pBGmPHD2.PBGmPHD2 is by the mediated transformation Arabidopis thaliana of agrobacterium tumefaciens GV3101, the PCR detected result shows 11 transformed plants of acquisition, the Northern analysis revealed wherein at least 3 strains expression amount of GmPHD2 very high, three strains that expression amount is high are called after G1-3, G1-6, G1-8 respectively.The seed kind of G1-3, G1-6, G1-8 is gone into the MS substratum, with seedling difference called after G2-3, G2-6, the G2-8 that obtains, after 5 days and transgenic arabidopsis (WT) seedling is not transplanted to respectively in the 1/2MS substratum that contains 50mM, 100mM, 150mM and 200mM NaCl and cultivated 14 days, the result as shown in Figure 3A, show in 100mM NaCl and 150mM NaCl, the contrast blade obviously curls, and that transfer-gen plant G2-3, G2-6, G2-8 change is not obvious, but in 200mM NaCl, contrast and transfer-gen plant are all obviously withered.
With the seed of transfer-gen plant G2-3, G2-6, G2-8 and not the seed of transgenic arabidopsis (contrast) plant in the MS substratum, cultivate 14 days under the same conditions after, handle 14 days with 150mM NaCl after, placed under the normal condition growth again 14 days.Under normal condition (nothing is coerced), but the equal normal growth of transfer-gen plant and wild-type plant, and phenotype does not have significant difference, as Fig. 3 B.And 150mM NaCl handled after 14 days, and contrast can not recover growth, and changeed pBGmPHD2 Arabidopis thaliana TG-1, and TG-2, TG-3 can recover growth.As seen under hypersaline environment, the growing way of changeing pBGmPHD2 Arabidopis thaliana plant obviously is better than the wild-type plant, and also grows (Fig. 3 C) than the easier recovery of wild-type Arabidopis thaliana behind salt stress, illustrates that GmPHD2 is relevant with salt tolerance, can improve the salt tolerance of plant.TG-1 among Fig. 3 A, 3B and the 3C, TG-2, TG-3 are by G2-3, G2-6, the plant that the seed of G2-8 grows up to contrasts and is transgenic arabidopsis not.
Sequence table
<160>2
<210>1
<211>759
<212>DNA
＜213〉Glycine soybean (Glycine max (L.))
<400>1
atggacggtg?gtggagtgaa?ctacaaccct?cgcaccgtgg?aacaggtttt?ccgggacttc 60
aagggccgta?gagctggcat?gatcaaggct?ctcaccactg?atgttgaaga?atttttccag 120
cagtgcgatc?ctgaaaagga?taatctttgt?ctgtacggat?tccctaatga?gcaatgggaa 180
gttaatttac?ctgcggaaga?agttcctccg?gagcttcctg?agcctgcatt?gggcataaac 240
tttgctaggg?atgggatgca?agacaaggac?tggctgtctt?tggttgccgt?tcacagcgat 300
gcatggttac?ttgcagtggc?tttctacttt?ggggcacgat?ttggttttga?taatgctgac 360
aggaaacgcc?tgttctctat?gattaatgat?ttaccaacaa?tatttgagat?tgtgactgga 420
agcgcaaaaa?aacagacgaa?ggaaaaatca?tccatttcaa?accacagcag?taacaaatca 480
aaatctggtt?caaaagggcg?aggatctgaa?tcagggaagt?attcaaagga?aacaaaggac 540
gaggaggaag?aggtactgga?tgaagaagat?gacgaggagc?atgaggagac?cttgtgtggg 600
gcatgtgggg?agcactatgc?atccgatgag?ttctggattt?gttgcgacat?atgtgagaag 660
tggttccatg?gcaagtgtgt?gaagatcact?ccagccaggg?ccgaacacat?caagcagtat 720
aagtgcccct?catgcagcaa?taagagagca?cggccttga 759
<210>2
<211>252
<212>PRT
＜213〉Glycine soybean (Glycine max (L.))
<400>2
Met?Asp?Gly?Gly?Gly?Val?Asn?Tyr?Asn?Pro?Arg?Thr?Val?Glu?Gln?Val
1 5 10 15
Phe?Arg?Asp?Phe?Lys?Gly?Arg?Arg?Ala?Gly?Met?Ile?Lys?Ala?Leu?Thr
20 25 30
Thr?Asp?Val?Glu?Glu?Phe?Phe?Gln?Gln?Cys?Asp?Pro?Glu?Lys?Asp?Ash
35 40 45
Leu?Cys?Leu?Tyr?Gly?Phe?Pro?Asn?Glu?Gln?Trp?Glu?Val?Asn?Leu?Pro
50 55 60
Ala?Glu?Glu?Val?Pro?Pro?Glu?Leu?Pro?Glu?Pro?Ala?Leu?Gly?Ile?Asn
65 70 75 80
Phe?Ala?Arg?Asp?Gly?Met?Gln?Asp?Lys?Asp?Trp?Leu?Ser?Leu?Val?Ala
85 90 95
Val?His?Ser?Asp?Ala?Trp?Leu?Leu?Ala?Val?Ala?Phe?Tyr?Phe?Gly?Ala
100 105 110
Arg?Phe?Gly?Phe?Asp?Asn?Ala?Asp?Arg?Lys?Arg?Leu?Phe?Ser?Met?Ile
115 120 125
Asn?Asp?Leu?Pro?Thr?Ile?Phe?Glu?Ile?Val?Thr?Gly?Ser?Ala?Lys?Lys
130 135 140
Gln?Thr?Lys?Glu?Lys?Ser?Ser?Ile?Ser?Asn?His?Ser?Ser?Asn?Lys?Ser
145 150 155 160
Lys?Ser?Gly?Ser?Lys?Gly?Arg?Gly?Ser?Glu?Ser?Gly?Lys?Tyr?Ser?Lys
165 170 175
Glu?Thr?Lys?Asp?Glu?Glu?Glu?Glu?Val?Leu?Asp?Glu?Glu?Asp?Asp?Glu
180 185 190
Glu?His?Glu?Glu?Thr?Leu?Cys?Gly?Ala?Cys?Gly?Glu?His?Tyr?Ala?Ser
195 200 205
Asp?Glu?Phe?Trp?Ile?Cys?Cys?Asp?Ile?Cys?Glu?Lys?Trp?Phe?His?Gly
210 215 220
Lys?Cys?Val?Lys?Ile?Thr?Pro?Ala?Arg?Ala?Glu?His?Ile?Lys?Gln?Tyr
225 230 235 240
Lys?Cys?Pro?Ser?Cys?Ser?Asn?Lys?Arg?Ala?Arg?Pro
245 250
Claims (10)
1, a kind of soybean PHD transcription factor is the protein with one of following amino acid residue sequences:
1) the SEQ ID № in the sequence table: 2;
2) with SEQ ID № in the sequence table: 2 amino acid residue sequence is through the replacement of one or several amino-acid residue and/or disappearance and/or interpolation and have the protein of the regulation and control stress resistance of plant of PHD transcriptional activation function.
2, protein according to claim 1 is characterized in that: described protein has the SEQID № in the sequence table: 2 amino acid residue sequence.
3, the encoding gene of claim 1 or 2 described soybean PHD transcription factors.
4, gene according to claim 3 is characterized in that: the cDNA gene of described soybean PHD class transcription factor has one of following nucleotide sequence:
1) SEQ ID № in the sequence table: 1 dna sequence dna;
2) SEQ ID № in the code sequence tabulation: the polynucleotide of 2 protein sequences;
3) under the rigorous condition of height can with SEQ ID № in the sequence table: the nucleotide sequence of the 1 dna sequence dna hybridization that limits.
5, contain claim 3 or 4 described expression carrier.
6, the clone that contains claim 3 or 4 described genes.
7, the host bacterium that contains claim 3 or 4 described genes.
8, the primer of amplification claim 3 or 4 described genes.
9, claim 1 or 2 described soybean PHD class transcription factors and encoding gene thereof the application in cultivating the resistance of reverse plant.
10, application according to claim 9 is characterized in that: described resistance of reverse is a salt tolerance.
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| CNB2005101237570A CN100349916C (en) | 2005-11-22 | 2005-11-22 | PHD transcription factor of soybean and its coding gene and usage |
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| Application Number | Priority Date | Filing Date | Title |
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| CNB2005101237570A CN100349916C (en) | 2005-11-22 | 2005-11-22 | PHD transcription factor of soybean and its coding gene and usage |
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| CN1789280A true CN1789280A (en) | 2006-06-21 |
| CN100349916C CN100349916C (en) | 2007-11-21 |
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102146130A (en) * | 2011-04-29 | 2011-08-10 | 中国科学院植物研究所 | Plant stress tolerance associated protein SeVP2 as well as coding gene and application thereof |
| CN101456909B (en) * | 2009-01-13 | 2012-07-04 | 南京农业大学 | Soja bean HKT protein and coding gene thereof and application |
| CN103102402A (en) * | 2011-11-14 | 2013-05-15 | 中国科学院遗传与发育生物学研究所 | Soybean transcription active protein GmPHD5, and coding gene and application thereof |
| CN111118036A (en) * | 2020-03-02 | 2020-05-08 | 东北林业大学 | PHD3 transcription factor encoding gene of Tamarix hispida and application thereof |
| CN112694524A (en) * | 2021-02-03 | 2021-04-23 | 浙江省农业科学院 | Anti-fusarium wilt PHD transcription factor ClPHD23, gene, expression vector, transformant and application thereof |
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| CN1247228A (en) * | 1999-07-27 | 2000-03-15 | 中国医学科学院基础医学研究所 | Human pluripotent leukosis cell differentiated correlative gene |
| CN1216904C (en) * | 2002-08-14 | 2005-08-31 | 清华大学 | Soybean ethylene response protein transcription factor and its coded gene and application |
| CN1216906C (en) * | 2002-08-19 | 2005-08-31 | 清华大学 | Transcription factor capable of regulating and controlling soybean adverse resistance, its coding gene and application |
| CN1286849C (en) * | 2003-08-21 | 2006-11-29 | 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
- 2005-11-22 CN CNB2005101237570A patent/CN100349916C/en not_active Expired - Fee Related
Cited By (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101456909B (en) * | 2009-01-13 | 2012-07-04 | 南京农业大学 | Soja bean HKT protein and coding gene thereof and application |
| CN102146130A (en) * | 2011-04-29 | 2011-08-10 | 中国科学院植物研究所 | Plant stress tolerance associated protein SeVP2 as well as coding gene and application thereof |
| CN102146130B (en) * | 2011-04-29 | 2012-06-13 | 中国科学院植物研究所 | Plant stress tolerance associated protein SeVP2 as well as coding gene and application thereof |
| CN103102402A (en) * | 2011-11-14 | 2013-05-15 | 中国科学院遗传与发育生物学研究所 | Soybean transcription active protein GmPHD5, and coding gene and application thereof |
| CN111118036A (en) * | 2020-03-02 | 2020-05-08 | 东北林业大学 | PHD3 transcription factor encoding gene of Tamarix hispida and application thereof |
| CN111118036B (en) * | 2020-03-02 | 2023-01-24 | 东北林业大学 | PHD3 transcription factor coding gene of Tamarix hispida and application thereof |
| CN112694524A (en) * | 2021-02-03 | 2021-04-23 | 浙江省农业科学院 | Anti-fusarium wilt PHD transcription factor ClPHD23, gene, expression vector, transformant and application thereof |
| CN112694524B (en) * | 2021-02-03 | 2022-04-19 | 浙江省农业科学院 | Anti-fusarium wilt PHD transcription factor ClPHD23, gene, expression vector, transformant and application thereof |
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| Publication number | Publication date |
|---|---|
| CN100349916C (en) | 2007-11-21 |
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