CN100465190C - Plant anti-reverse related protein, and its coding gene and use - Google Patents
Plant anti-reverse related protein, and its coding gene and use Download PDFInfo
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- CN100465190C CN100465190C CNB2006100834507A CN200610083450A CN100465190C CN 100465190 C CN100465190 C CN 100465190C CN B2006100834507 A CNB2006100834507 A CN B2006100834507A CN 200610083450 A CN200610083450 A CN 200610083450A CN 100465190 C CN100465190 C CN 100465190C
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Abstract
The invention discloses a plant anti-versus relative albumen and the coding gene and the application. The albumen contains one of the following amino acid residue sequences: SEQ ID No:1 in the sequence table; the albumen that take replacement and/or lacking and/or adding one or plural amino acid residue of SEQ ID No:1 in sequence table. The invention could enhance the anti-versus of paddy.
Description
Technical field
The present invention relates to a plant resistance relevant protein and encoding gene thereof and application.
Background technology
Whole world arid, semiarid zone account for 36% of the soil total area, account for 43% of cultivated area.The arid of China, semiarid zone mainly are distributed in areas such as North China, northwest, the Inner Mongol and Qinghai-Tibet Platean, and its area accounts for 1/2nd of national land area.Along with population increase, socio-economic development and natural climate condition change, the shortage of water resources situation is increasingly sharpened, and has a strong impact on agriculture production.Drought stress usually influences growth and development of plants, causes the serious underproduction of crop.Arid accounts for the first place to being lost in all abiotic stress of causing of farm crop, is only second to biology and coerces the loss that disease and pest causes.In order to solve these difficult problems, the crop drought resistance Study on Mechanism is received much concern in the world, drought resistance has become one of important indicator of estimating crop varieties.Carry out the crop drought resistance Study on Mechanism, the drought resistance that improves crop is the important selection that solves shortage of water resources, realization agricultural water conservation, ensures agricultural sustainable development.The crop drought resistance Study on Mechanism can disclose the life quintessence that plant adapts to drought environment, is the final artificial basis of handling plant vital activity of realizing, therefore, also is a great problem in science in the life science.Carry out the crop drought resistance Study on Mechanism can improve China's life science the fundamental research level, strengthen China's science and technology competition power.
Summary of the invention
An object of the present invention is to provide a plant resistance relevant protein and encoding gene thereof.
Plant adversity resistance related protein provided by the present invention, name is called TCTP albuminoid (translationallycontrolled tumor family protein similar to translationally controlled tumorprotein, be abbreviated as TCTP), it derives from paddy rice, is the protein with one of following amino acid residue sequences:
1) the SEQ ID № in the sequence table: 1;
2) with SEQ ID № in the sequence table: 1 amino acid residue sequence is through replacement and/or disappearance and/or the interpolation and the protein relevant with stress resistance of plant of one or several amino-acid residue.
Wherein, the sequence in the sequence table 1 is made up of 168 amino-acid residues.
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.
Above-mentioned plant adversity resistance related protein encoding gene (TCTP) and promotor thereof also belong to protection scope of the present invention.
The cDNA gene of above-mentioned plant adversity resistance related protein can have one of following nucleotide sequence:
1) SEQ ID № in the sequence table: 2 dna sequence dna;
2) SEQ ID № in the code sequence tabulation: the polynucleotide of 1 protein sequence;
3) with sequence table in SEQ ID №: 2 dna sequence dna has 90% above homology, and the identical function protein DNA sequence of encoding;
4) under the rigorous condition of height can with SEQ ID № in the sequence table: the nucleotide sequence of the 2 dna sequence dnas hybridization that limit.
The rigorous condition of above-mentioned height can be at 0.1 * SSPE (or in the solution of 0.1 * SSC), 0.1% SDS, hybridizes under 65 ℃ and washes film.
Wherein, the SEQ ID № in the sequence table: 1 is made up of 970 deoxynucleotides, is encoding sequence from the 113rd to 619 deoxynucleotides of 5 ' end.
The genomic gene of plant adversity resistance related protein provided by the present invention can have one of following nucleotide sequence:
1) SEQ ID № in the sequence table: 3 nucleotide sequence;
2) SEQ ID № in the code sequence tabulation: the DNA of 1 protein sequence;
3) with sequence table in SEQ ID №: 3 dna sequence dna has 90% above homology, and the identical function protein DNA sequence of encoding;
4) under the rigorous condition of height can with the SEQ ID № in the sequence table: the nucleotide sequence of the 3 dna sequence dnas hybridization that limit.
Wherein, sequence 3 is made up of 4245 deoxynucleotides in the sequence table, contains 5 exons and 4 introns.
Contain expression carrier of the present invention, clone and host bacterium and all belong to protection scope of the present invention.
Environment stress experimental results show that the encoding gene of plant adversity resistance related protein of the present invention changes in the paddy rice, can strengthen the resistance of paddy rice, particularly strengthens salt, cold and non-irrigated resistance.
Description of drawings
Fig. 1 is the structure diagram of TCTP albuminoid encoding gene
Fig. 2 is the structure diagram of TCTP overexpression carrier TCTP-pHOS
Fig. 3 is that 30% PEG6000 simulating drought is coerced the back and changeed photo after TCTP-pHOS rice plant and contrast (in the spend 11) rehydration
Fig. 4 A is that cold is coerced the photo of spending 11 rice varieties (contrast) in the back
Fig. 4 B is cold photo of coercing back commentaries on classics TCTP-pHOS rice plant
Fig. 5 A is a photo of spending 11 rice varieties (contrast) in behind the salt stress
Fig. 5 B is the photo that changes the TCTP-pHOS rice plant behind the salt stress
Embodiment
Method among the embodiment is ordinary method if no special instructions.
The acquisition and the functional verification thereof of embodiment 1, paddy rice anti contravariance associated protein and encoding gene thereof
One, the acquisition of paddy rice anti contravariance associated protein and encoding gene thereof
By yeast-two hybrid technique, screening obtains paddy rice anti contravariance associated protein TCTP.Yeast two-hybrid system (CLONGTECH company) is a kind of molecular biology method of research protein-protein interaction, it is based on transcription factor can be divided into independently district, be DNA-in conjunction with territory and transcription activating territory, and realize the natural process that the manual simulation transcribes.The bait protein that adopts is the DREB transcription factor, promptly arid response element binding protein matter, and it can contain the DRE/CRT cis element in the specific combination promotor, activates the expression of many adversity inducible genes, strengthens the endurance of plant to adverse circumstance.By the albumen of yeast two-hybrid screening and DREB transcription factor interaction, infer that these albumen may be the same with the DREB transcription factor, with degeneration-resistant relevant.By experiment, screening has obtained TCTP albuminoid (translationally controlled tumor family protein similar totranslationally controlled tumor protein).Tctp gene is positioned on the paddy rice o.11 karyomit(e).Tctp gene total length 4245bp is promoter region 1990bp wherein, gene 2255bp.Tctp gene contains 5 exons and 4 introns.Exon is respectively 143bp, 71bp, 130bp, 157bp and 469bp.Exons 1 comprises mRNA5 ' end non-translational region and coding methionine(Met) initiation codon (translation starting point), 48 amino acid whose codons, and exon 2 is made up of a coding TCTP protein 24 amino acid whose Nucleotide.Exon 3 comprises that proteic 43 the amino acid whose nucleotide sequences of coding TCTP constitute.Exon 4 comprises that proteic 52 the amino acid whose nucleotide sequences of encoding mature TCTP constitute.Exon 5 comprises terminator codon and 3 ' end non-coding region.At 3 ' non-coding region 2 poly a-signals are arranged.Intron is respectively 312bp, 687,79bp and 191bp.Its structure diagram is seen Fig. 1.
Show that through order-checking this albumen has the aminoacid sequence of sequence 1 in the sequence table, its cDNA gene has the nucleotide sequence of sequence 2 in the sequence table.
The special primer TCTP-F:5 '-CACCCTCTCTCTCCGTCCGTCCGCGCC-3 ' and the TCTP-R:5 '-CAATACAAAGGTTGCTTCTAAAG-3 ' of the cDNA gene of the encoding gene TCTP of amplification TCTP albuminoid, added " CACC " joint at 5 of TCTP-F primer ' end, as the joint of TOPO clone.As template, amplify the cDNA gene fragment of TCTP with spend 11 in the paddy rice cDNA of (industry is planted by the inferior China in Hunan) with PCR method (Invitrogen company); Wherein, amplification system is: 10 * pfu buffer (contains Mg
2+) 5ul, 10mM dNTPs 1ul, 10uM primer TCTP-F 1ul, 10uM primer TCTP-R 1ul, rice cDNA 5ul, pfu archaeal dna polymerase 2ul adds water to 50ul.Amplification program is: 95 ℃ of pre-sex change 5 minutes; 95 ℃ 20 seconds, 58 ℃ 30 seconds, 72 ℃ of 30 circulations in 1 minute; 72 ℃ were extended 7 minutes.Amplification obtains the 974bp fragment, is connected to pENTR/D-TOPO carrier (Invitrogen company) and obtains recombinant vectors, shows that through order-checking this amplified fragments has the nucleotide sequence of sequence 2 in the sequence table, and this amplified fragments is the cDNA gene of TCTP.113-619 at 5 of sequence 2 ' end is the encoding sequence of the cDNA gene of TCTP in sequence table, the aminoacid sequence of sequence 1 in the code sequence tabulation.With this recombinant vectors called after pENTR/D-TCTP.
Two, tctp gene functional verification
1, the structure of overexpression carrier
By LR reaction site attL1 and attL2 on the pENTR/D-TOPO carrier, through LR reaction (Invitrogen company) scale being in the cDNA gene fragment reorganization of the TCTP among the recombinant vectors pENTR/D-TCTP reaches on the carrier pHOS (Invitrogen company), transformed into escherichia coli, carry out enzyme and cut evaluation, will identify the correct plasmid called after TCTP-pHOS (structure diagram as shown in Figure 2) that contains tctp gene with bacterium colony PCR.
2, the acquisition and the resistance thereof of changeing the TCTP-pHOS paddy rice is crossed detection of expression
TCTP-pHOS is changed in the middle flower ride on Bus No. 11 paddy rice by the mediation of agrobacterium tumefaciens (Agrobacterium tumefaciens) LBA 4404 (TIANGEN company), screen with 50mg/L Hygmycin, then to showing the plant of Hygmycin resistance, detect the hpt gene with PCR method, experimental result is positive, the proof gene has been incorporated in the rice genome, the paddy rice of the commentaries on classics TCTP-pHOS that obtains for screening, and promptly T0 is for transfer-gen plant.The seed that sowing TO ties for transfer-gen plant (T1 generation), screen with 50mg/LHygmycin, then to showing the plant of Hygmycin resistance, detect the hpt gene with PCR method, the result shows that screening 93 strains in the 100 grain germination seeds changes the positive seedling of TCTP-pHOS, this positive seedling carried out the experiment of drought resisting, cold-resistant, anti-salt aspect respectively.
(1) drought resisting experiment
Come the environment of simulating drought with 30% PEG6000.Seed (T1 generation) with T0 ties for transfer-gen plant is placed directly in the water that contains selective agent Hygmycin 50mg/L and screened ten days.Transgenic seed can resist Hygmycin because of carrying the hpt gene.Select the resistance seedling and forward in the water and cultivate, carry out the drought resisting test after 3 days.Spend 11 waters to send out seed in the wild-type paddy rice, grow to 13 days seedling, as the contrast of this experiment.The T1 that screening is obtained for change the TCTP-pHOS paddy rice and in spend 11 (contrasts) with 30% PEG6000 solution-treated.After coercing 4 days, in spend 11 serious dehydration to become dry, be that partial blade curls and change the TCTP-pHOS rice plant.Rehydration after 4 days forwards in the water after the PEG that is about to processed seedling root cleans and cultivated 3 days.In can not recover death after spending 11 rehydrations.Recover immediately after changeing TCTP-pHOS rice plant rehydration, the result is (a figure left side is contrast, and the figure right side is a transfer-gen plant) as shown in Figure 3.The transgenic drought-resistant plant changes in the nutrition soil and normally cultivates.
(2) cold-resistant experiment
Seed (T1 generation) with T0 ties for transfer-gen plant is placed directly in the water that contains selective agent Hygmycin 50mg/L and screened ten days.Select the resistance seedling and forward in the water and cultivate, carry out cold-resistant test after the week.Spend in the wild-type paddy rice 11 in the same period water send out seed, as the contrast of this experiment.With in spend 11 kinds (contrast) and the T1 that obtains of screening to move in the 4 degree freezers and cultivate for changeing the TCTP-pHOS paddy rice.The back adjoining tree death of two weeks, it is very little to change the suffered influence of TCTP-pHOS rice plant.The result is (Fig. 4 A is an adjoining tree, and Fig. 4 B is for changeing the TCTP-pHOS rice plant) shown in Fig. 4 A and Fig. 4 B
(3) anti-salt experiment
With the seed (T1 generation) that TO ties for transfer-gen plant, put into the water that contains selective agent Hygmycin 50mg/L and screened ten days.Select the resistance seedling and forward in the water and cultivate, carry out anti-salt test after the week.Spend in the wild-type paddy rice 11 in the same period water send out seed, as the contrast of this experiment.With in spend 11 kinds (contrast) and the T1 that obtains of screening to put into 100mM NaCl solution and cultivate for changeing the TCTP-pHOS rice seedling, through one month processing, obviously upgrowth situation was good to change TCTP-pHOS paddy rice relative comparison plant (in spend 11).The results are shown in Figure 5A and Fig. 5 B (Fig. 5 A is an adjoining tree, and Fig. 5 B is a transfer-gen plant).
More than experiment proves that this tctp gene has the biological function of abilities such as improving plant drought, cold-resistant, anti-salt, can strengthen the endurance of plant to adverse circumstance.
Sequence table
<160>3
<210>1
<211>168
<212>PRT
＜213〉Oryza paddy rice (Oryza sativa var.Lansheng)
<400>1
<210>2
<211>970
<212>DNA
＜213〉Oryza paddy rice (Oryzasativa var.Lansheng)
<400>2
<210>3
<211>4245
<212>DNA
＜213〉Oryza paddy rice (Oryza sativa var.Lansheng)
<400>3
Claims (8)
1, a plant resistance relevant protein is that amino acid residue sequence is the SEQ ID № in the sequence table: 1 protein.
2, the application of the encoding gene of the described plant adversity resistance related protein of claim 1 in cultivating the resistance plant.
3, application according to claim 2 is characterized in that: described encoding gene is the cDNA gene of described plant adversity resistance related protein.
4, application according to claim 3 is characterized in that: described encoding gene has SEQ ID №: 2 nucleotide sequence.
5, application according to claim 2 is characterized in that: described encoding gene is the genomic gene of described plant adversity resistance related protein.
6, application according to claim 5 is characterized in that: described encoding gene has SEQ ID №: 3 nucleotide sequence.
7, according to the described application of the arbitrary claim of claim 2～6, it is characterized in that: described resistance is salt resistance, drought resistance and winter resistance.
8, application according to claim 7 is characterized in that: described plant is a paddy rice.
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| CN101139385B (en) * | 2007-07-31 | 2010-06-09 | 中国科学院植物研究所 | Vegetable stress-resistant related protein and its coding gene and application |
| CN101333250B (en) * | 2008-08-06 | 2012-06-20 | 中国农业科学院生物技术研究所 | Plant stress related protein MASTER applications thereof for encoding gene |
| CN103014017B (en) * | 2012-11-28 | 2014-07-02 | 上海市农业生物基因中心 | OsSRP14 gene related to rice stress resistance and coding protein as well as application thereof |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20040016027A1 (en) * | 2001-08-07 | 2004-01-22 | Hirohiko Hirochika | Novel rice gene controlling tolerance to salt stress |
| CN1478893A (en) * | 2002-08-29 | 2004-03-03 | 中国科学院遗传与发育生物学研究所 | Paddy rice anti-reverse transcripfactor and its coding gene and application |
| CN1772899A (en) * | 2005-05-09 | 2006-05-17 | 中国科学院植物研究所 | Wild rice drought-resisting gene and its coded protein and application |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20040016027A1 (en) * | 2001-08-07 | 2004-01-22 | Hirohiko Hirochika | Novel rice gene controlling tolerance to salt stress |
| CN1478893A (en) * | 2002-08-29 | 2004-03-03 | 中国科学院遗传与发育生物学研究所 | Paddy rice anti-reverse transcripfactor and its coding gene and application |
| CN1772899A (en) * | 2005-05-09 | 2006-05-17 | 中国科学院植物研究所 | Wild rice drought-resisting gene and its coded protein and application |
Non-Patent Citations (2)
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| . GENBANK, AK105453. 2003 |
| . GENBANK, AK105453. 2003 * |
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