CN105504032B - A GmSIZ1a/b protein related to plant stress resistance and its encoding gene and application - Google Patents
A GmSIZ1a/b protein related to plant stress resistance and its encoding gene and application Download PDFInfo
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Abstract
本发明公开了一种与植物抗逆性相关的蛋白GmSIZ1a/b及其编码基因与应用。该蛋白质，是如下a)或b)的蛋白质：a)由序列表中序列2/4所示的氨基酸序列组成的蛋白质；b)将序列表中序列2/4所示的氨基酸序列经过一个或几个氨基酸残基的取代和/或缺失和/或添加且与植物抗逆性相关的蛋白质。实验结果证明：抑制大豆中的GmSIZ1a/b表达的转基因株系，对大豆疫霉菌的抗性明显提高。The invention discloses a protein GmSIZ1a/b related to plant stress resistance, its encoding gene and application. The protein is the protein of the following a) or b): a) a protein consisting of the amino acid sequence shown in the sequence 2/4 in the sequence listing; b) the amino acid sequence shown in the sequence 2/4 in the sequence listing through one or Proteins with substitutions and/or deletions and/or additions of several amino acid residues that are associated with plant stress resistance. The experimental results show that the transgenic lines that inhibit the expression of GmSIZ1a/b in soybean have significantly improved resistance to Phytophthora sojae.
Description
Technical field
The present invention relates to field of biotechnology, and in particular to a kind of GmSIZ1a/b albumen relevant to stress resistance of plant and
Its encoding gene and application.
Background technique
Soybean is one of most important commercial crops in the world, however its serious various pathogen of infection.Soybean epidemic disease
Mould (Phytophthora sojae) category Oomycete class leads to the phytopathogen of root rot, becomes most destructive
One of soybean diseases lead to hundred million dollars of 10-20 of loss every year.Soybean plant strain resists soybean at least through two different mechanism
Phytophthora: the partial resistance of immune response (ETI) and polygenes regulation that the effect that single-gene (Rps gene) mediates causes
(partial resistance), this may defend (basal defense) and weak ETI associated with background.Currently, there are about
20 Rps genes have been positioned on four chromosomes.In these Rps genes only have Rps1k, RpsYD29, Rps10 and
RpsJS is by finely positioning.However the molecular mechanism that Rps gene regulation soybean resists soyabean phytophthora is still unclear.Due to Rps base
Because the anti-soyabean phytophthora of mediation has specificity, the resistance of single Rps gene mediated can only maintain about 8-15.Therefore,
There is an urgent need to select the soybean varieties for being able to maintain that permanent resistance.Improving soybean is an effectively way to phytophthora partial resistance
Diameter, because partial resistance can maintain permanent and resistance of wide spectrum.
SUMO (small ubiquitin-related modifier) change is a kind of protein post-translational modification, passes through E1
(SUMO kinase), E2 (SUMO desmoenzyme), E3 (SUMO ligase) are connected to SUMO in the lysine SUMOization site of substrate.
It has now been found that SUMOization modification is closely related with the interaction of plant and pathogen, but whether unclear its regulates and controls Genes For Plant Tolerance
Phytophthora root rot reaction.
Summary of the invention
It is an object of the present invention to provide a kind of GmSIZ1a protein.
Protein described above is following protein a) or b):
A) protein that the amino acid sequence shown in sequence 2 in sequence table forms；
B) by amino acid sequence shown in sequence 2 in sequence table by one or several amino acid residues substitution and/or
Deletion and/or addition and protein relevant to stress resistance of plant.
It is a further object to provide a kind of GmSIZ1b protein.
Protein described above is following protein a) or b):
A) protein that the amino acid sequence shown in sequence 4 in sequence table forms；
B) by amino acid sequence shown in sequence 4 in sequence table by one or several amino acid residues substitution and/or
Deletion and/or addition and protein relevant to stress resistance of plant.
Biomaterial relevant to protein described above also belongs to protection scope of the present invention.
Biomaterial described above is any one of following B1) to B3):
B1 the nucleic acid molecules of albumen described above) are encoded；
B2) contain B1) recombinant vectors of the nucleic acid molecules；
B3) contain B1) recombinant bacteriums of the nucleic acid molecules, contain B2) recombinant bacterium of the recombinant vector.
In above-mentioned biomaterial, B1) described in coding GmSIZ1a albumen nucleic acid molecules such as sequence 1 in sequence table the
Shown in 1-2643 nucleic acid molecules；
B1 the 1-2640 nucleotide point of sequence 3 in the nucleic acid molecules of the coding GmSIZ1b albumen described in) such as sequence table
Shown in son.
It is a still further object of the present invention to provide protein described above or the relevant biological material to improve Genes For Plant Tolerance
Application in inverse property.
In above-mentioned application, the resistance is the resistance to soyabean phytophthora.
The last one purpose of the invention is to provide a kind of method for improving stress resistance of plant.
The method of raising stress resistance of plant described above, includes the following steps: to inhibit GmSIZ1a albumen or GmSIZ1b
The encoding gene of albumen is expressed in the plant that sets out, and then improves the resistance of plant.
In the above method, the encoding gene for inhibiting GmSIZ1a albumen or GmSIZ1b albumen is expressed in the plant that sets out
Method are as follows: the interference piece for inhibiting the encoding gene expression of GmSIZ1a albumen or GmSIZ1b albumen is imported into the plant that sets out
Section.
In the above method, the sequence of the interference fragment is as shown in the 1894-2249 nucleic acid molecules of sequence 1.
In the above method, the interference fragment is imported by interference carrier.
In the above method, the construction method of the interference carrier, include the following steps: with restriction enzyme SacI and
PFGC1008 digestion is taken small fragment by PmeI, is denoted as intermediate segment；The intermediate segment is connected to and uses restriction enzyme
In pCambia3300 carrier after SacI and PmeI digestion, pCambia3300-RNAi carrier is obtained；Again by 1 1894- of sequence
Interference fragment shown in 2249 nucleic acid molecules is positive and anti-by restriction enzyme A scI/SwaI and BamHI/SpeI
To being connected into pCambia3300-RNAi carrier, final acquisition pCambia3300-GmSIZ1RNAi recombinant vector is as interfered
Carrier.
In the above method, the resistance is the resistance to soyabean phytophthora.
In the above method, the plant that sets out is monocotyledon or dicotyledon；The dicotyledon is specially
Soybean.
Interference fragment shown in 1894-2249 nucleic acid molecules also belongs to protection scope of the present invention in sequence 1.
It is a still further object of the present invention to provide a kind of interference carriers.
The construction method of interference carrier described above: with restriction enzyme SacI and PmeI by pFGC1008 digestion,
Small fragment is taken, intermediate segment is denoted as；The intermediate segment is connected to after restriction enzyme SacI and PmeI digestion
In pCambia3300 carrier, pCambia3300-RNAi carrier is obtained；Again by the 1894-2249 nucleic acid molecule institutes of sequence 1
The interference fragment shown is connected into pCambia3300- by restriction enzyme A scI/SwaI and BamHI/SpeI, forward and reverse
It is final to obtain pCambia3300-GmSIZ1RNAi recombinant vector, as interference carrier in RNAi carrier.
The present invention provides a kind of Protein G mSIZ1a/b relevant to stress resistance of plant and its encoding gene and applications.Knot
Fruit shows: the adjustable soybean of Protein G mSIZ1a/b and soyabean phytophthora interact, and provide a kind of strategy to improve soybean
The resistance of anti-soyabean phytophthora.
Detailed description of the invention
Fig. 1 is the structural domain schematic diagram of soybean GmSIZ1a/b and arabidopsis AtSIZ1 albumen.
Fig. 2 is the Dwarfing phenotypes figure of GmSIZ1a and GmSIZ1b complement Arabidopsis siz1-2 mutant.
Fig. 3 is that the SUMOization modification level of heat stress induction in GmSIZ1a/b complementation siz1-2 is restored.
Fig. 4 is Resistance detecting and GmSIZ1a/b the expression quantity detection of GmSIZ1RNAi transgenic plant and WT lines
Comparison diagram.
Fig. 5, which is that the SUMOization modification of heat stress induction in GmSIZ1RNAi transgenic plant is horizontal, reduces phenotype.
Fig. 6 is the phenotype of the anti-Phytophthora sojae of GmSIZ1RNAi transgenic plant.
Specific embodiment
Experimental method used in following embodiments is conventional method unless otherwise specified.
The materials, reagents and the like used in the following examples is commercially available unless otherwise specified.
This research vegetable material used is soybean (Glycine max L.Merr.Cv), and kind is middle beans 32, in document
" Guo Bingfu, Jiang Lingxue, Li Maiquan care for sea blue, Jin Longguo, Qiu Lijuan.Tolerance of the different soybean varieties to contact killing type herbicide
Property, Chinese oil crops journal, 2012,34 (5): being disclosed in 551-555 ", and the public can be from crop section of the Chinese Academy of Agricultural Sciences
Research institute is learned to obtain.It after soya seeds are sprouted 1 day on the filter paper of deionized water, is then transferred into soil, in 100 μm of ol
m-2 s-1Light under (16 hours illumination/8 hour dark), temperature be 25 DEG C and relative humidity be 70% under conditions of grow.
Arabidopsis siz1-2 mutant and AtSIZ1 promoter document " Jin JB, Jin YH, Lee J, Miura K,
Yoo CY,Kim WY,Van Oosten M,Hyun Y,Somers DE,Lee I,Yun DJ,Bressan RA,Hasegawa
PM.The SUMO E3ligase,AtSIZ1,regulates flowering by controlling a salicylic
acid-mediated floral promotion pathway and through affects on FLC chromatin
It is disclosed in structure.2008, Plant J 53:530-540 ", the public can obtain from Institute of Botany, Chinese Academy of Sciences.
PsJS2 strain is soyabean phytophthora (Phytophthora sojae) " Zhang J, Sun S, Wang in the literature
G,Duan C,Wang X,Wu X,Zhu Z.Characterization of Phytophthora resistance in
In soybean cultivars/lines bred in Henan province.Euphytica, 2014,196:375-384 "
It is disclosed, the public can obtain from Institute of Crop Science, Chinese Academy of Agricultural Science.
The acquisition of embodiment 1, GmSIZ1a/b gene
1, primer: F1 (forward primer): 5 '-CGGGATCCATGGATTTGGTACCGAGCG-3 '；R1 (reverse primer):
5’-CGGGATCCTCTCTGAATCTGAATCAATAGAA-3’。
2, the RNA of soybean leaves is extracted, and reverse transcription obtains cDNA.
3, using above-mentioned cDNA as template, the primer pair designed using step 1, PCR amplification GmSIZ1a/b gene.
4, amplified production is sequenced, sequencing result shows: GmSIZ1a albumen is compiled as shown in the sequence 2 of sequence table
Code gene order is as shown in the sequence 1 of sequence table；GmSIZ1b albumen is as shown in the sequence 4 of sequence table, and coding gene sequence is such as
Shown in the sequence 3 of sequence table.
The acquisition of embodiment 2, transgenic plant
1, the building of carrier
With restriction enzyme HindIII and NcoI enzyme respectively by the GmSIZ1a/b full-length cDNA obtained in embodiment 1 and
PCambia1302 carrier carries out double digestion, and connection obtains recombinant vector.AtSIZ1 promoter region is connected into recombination respectively again
Carrier obtains pCambia1302-ProAtSIZ1:GmSIZ1a:GFP carrier and pCambia1302-ProAtSIZ1:
GmSIZ1b:GFP carrier, and sequence verification is carried out to it.
Sequencing result shows: in HindIII the and NcoI enzyme of pCambia1302-ProAtSIZ1:GmSIZ1a:GFP carrier
The GmSIZ1a gene order being inserted between enzyme site shows that carrier is correct as shown in 1-2643 nucleotide in sequence 1；?
The GmSIZ1b base being inserted between HindIII the and NcoI restriction enzyme site of pCambia1302-ProAtSIZ1:GmSIZ1b:GFP carrier
Because sequence is as shown in 1-2640 nucleotide in sequence 3, show that carrier is correct.1-2643 nucleotide are compiled in sequence 1
The 1-2640 encoded albumen of nucleotide are such as shown in 1-880 amino acid in sequence 2, in sequence 3 for the albumen of code
In sequence 4 shown in 1-879 amino acid.
2, arabidopsis thaliana transformation siz1-2 mutant
Using freeze-thaw method, respectively by pCambia1302-ProAtSIZ1:GmSIZ1a:GFP carrier and pCambia1302-
ProAtSIZ1:GmSIZ1b:GFP carrier is transferred in Agrobacterium tumefaciems GV3101, converts quasi- south respectively by inflorescence infestation method
Mustard siz1-2 mutant, obtains the GmSIZ1a transgenosis of the 1-2643 encoded albumen of nucleotide in overexpression sequence 1
The GmSIZ1b transgenic plant of the 1-2640 encoded albumen of nucleotide in plant and overexpression sequence 3.
The result shows that: compared with arabidopsis siz1-2 mutant and the only plant of conversion empty carrier, convert GmSIZ1a/b's
Plant part is restored to the plant size of wild type Col-0, illustrate GmSIZ1a/b can complement Arabidopsis siz1-2 mutant,
As shown in Figure 2 A and 2 B.
The SUMOization that heat stress induction is detected in embodiment 3, GmSIZ1a/b complementation transgenic plant modifies level
Under conditions of the transgenic arabidopsis sample that embodiment 2 obtains is placed in 37 DEG C after 30min, total protein is extracted.Egg
White extracting solution includes: NaCl, 5% (w/v) SDS, 0.5% (v/v) NP40,6mM EDTA, 3mM DTT, 1mM PMSF and 30%
(v/v) glycerol.
It is obtained using Bradford Protein Assay Reagent (Cat:CW0013, CWBIO, Beijing, China) measurement embodiment 2
The protein concentration of the transgenic arabidopsis sample obtained is 20g/L.Polyacrylamide gel electrophoresis is carried out to the albumen obtained is extracted
It separates, after transferring film, carries out western blot detection with anti-AtSUMO1 antibody.
The result shows that: compared with arabidopsis siz1-2 mutant and the only plant of conversion empty carrier, convert GmSIZ1a/b's
Plant SUMO conjugates content increases, and is restored to the level of wild type Col-0, illustrates that GmSIZ1a/b can promote quasi- south
SUMOization modification is horizontal in mustard, illustrates that GmSIZ1a/b has SUMO E3 connection enzymatic activity, as shown in Figure 3.
The acquisition of experimental example 4, GmSIZ1RNAi transgenic plant
1, the acquisition of the RNAi segment of GmSIZ1a
Soybean cDNA is extracted, using GmSIZ1RNAi-F and GmSIZ1RNAi-R primer, the RNAi of PCR amplification GmSIZ1a
Segment.Primer sequence is as follows: GmSIZ1RNAi-F:AAGGCCTCCATGGGCTACAGATGGTTACTCTGTA；GmSIZ1RNAi-
R:CCTTAATTAAGGCGCGCCAATGGATTGTCCACCAAACCA.1894- in the sequence such as sequence table 1 of amplified production
Shown in 2249 nucleic acid molecules.
2, the building of carrier
The intermediate segment that will be obtained after pFGC1008 (ABRC, CD3-446) digestion with restriction enzyme SacI and PmeI；
The intermediate segment of above-mentioned acquisition is connected to in the pCambia3300 carrier after restriction enzyme SacI and PmeI digestion,
Obtain pCambia3300-RNAi carrier；It again will be with the GmSIZ1a's of GmSIZ1RNAi-F/GmSIZ1RNAi-R primer amplification
RNAi segment is connected into pCambia3300-RNAi by restriction enzyme A scI/SwaI and BamHI/SpeI, forward and reverse
It is final to obtain pCambia3300-GmSIZ1RNAi recombinant vector in carrier.
3, it converts
Using freeze-thaw method, the recombinant vector that step 2 is constructed is transferred in Agrobacterium tumefaciems GV3101, is situated between by Agrobacterium
The cotyledonary node method soybean transformation led, acquisition turn GmSIZ1RNAi gene plant.
4, the Resistance detecting of GmSIZ1RNAi transgenic plant
GmSIZ1RNAi transgenic plant is subjected to anti-glufocinate (glufosinate-ammonium) identification, by 200mg/L's
Glufocinate is applied to respectively on the blade of GmSIZ1RNAi transgenic plant and WT lines, is observed after 4 days.
The result shows that: compared with wild type, the resistance of GmSIZ1RNAi transgenic plant is improved, as shown in Figure 4 A.
5, the GmSIZ1a/b detection of expression of GmSIZ1RNAi transgenic plant.
We are from anti-glufocinate plant, by the primer sequence of table 1 to the GmSIZ1RNAi of above-mentioned acquisition
Transgenic plant carry out GmSIZ1a/b qRT-PCR detection, as a result screened GmSIZ1a/b transcription be suppressed turn base
Because of strain (35A3,38A2,45B3), as shown in Figure 4 B.
Table 1, primer sequence
The SUMOization that heat stress induction is detected in embodiment 5, GmSIZ1RNAi transgenic plant modifies level.
Under conditions of the GmSIZ1RNAi genetically engineered soybean sample that embodiment 4 obtains is placed in 45 DEG C after 30min, extract total
Albumen.Protein extract includes: NaCl, 5% (w/v) SDS, 0.5% (v/v) NP40,6mM EDTA, 3mM DTT, 1mM PMSF
With 30% (v/v) glycerol.Polyacrylamide gel electrophoresis separation is carried out to the albumen obtained is extracted, after transferring film, uses anti-
GmSUMO1 antibody carries out western blot detection.
The result shows that: compared with wild type, the heat side of body in GmSIZ1RNAi Transgenic soybean plants (35A3,38A2,45B3)
The SUMO conjugates content for compeling induction is reduced, and illustrates the SUMO E3 ligase in GmSIZ1RNAi Transgenic soybean plants
Activity reduces, as shown in Figure 5.
The soyabean phytophthora Resistance detecting of embodiment 6, GmSIZ1RNAi transgenic plant.
We utilize PsJS2 strain (soyabean phytophthora), turn base to wild type and GmSIZ1RNAi by bacterium layer infestation method
Because soybean is identified.The case where statistics soybean is infected after two weeks.Susceptible degree is subjected to 1 to 9 grades of divisions, be respectively as follows: 1,
There is no a root-rot, 2, faint root-rot, 3, bottom 1/3 have a root-rot, 4, bottom 2/3 have a root-rot, 5, all plant there is root-rot+10%
Young plant is dead, and 6,50% young plant death+some growth inhibits, and 7,75% young plant death+growth seriously inhibits, and 8,90% young plant is dead
It dies, 9, whole young plant death.
The result shows that: the susceptible degree of wild type is 6, and the susceptible degree of GmSIZ1RNAi transgenic line is 4, to big
The resistance of beans phytophthora improves, as shown in Fig. 6 and table 2.
Table 2, GmSIZ1RNAi transgenic line have the disease-resistant phenotype statistical form of soyabean phytophthora
Claims (10)
1.GmSIZ1a protein, the protein that the amino acid sequence shown in sequence 2 in sequence table forms.
2.GmSIZ1b protein, the protein that the amino acid sequence shown in sequence 4 in sequence table forms.
3. it is following B1 biomaterial relevant to protein as claimed in claim 1 or 2) any one of to B3):
B1 the nucleic acid molecules of albumen as claimed in claim 1 or 2) are encoded；
B2) contain B1) recombinant vectors of the nucleic acid molecules；
B3) contain B1) recombinant bacteriums of the nucleic acid molecules, contain B2) recombinant bacterium of the recombinant vector.
4. relevant biological material according to claim 3, it is characterised in that: B1) nucleic acid point of the coding claim 1
Son is as shown in 1-2643 nucleic acid molecules of sequence 1 in sequence table；B1) it is described coding claim 2 nucleic acid molecules such as
In sequence table shown in 1-2640 nucleic acid molecules of sequence 3.
5. a kind of method for improving stress resistance of plant includes the following steps: the coding for inhibiting the protein of claim 1 and 2
Gene is expressed in the plant that sets out, and then improves the resistance of plant；
The resistance is the resistance to soyabean phytophthora.
6. according to the method described in claim 5, it is characterized by: the coding for inhibiting the protein of claim 1 and 2
The method that gene is expressed in the plant that sets out are as follows: the coding for inhibiting the protein of claim 1 and 2 is imported into the plant that sets out
The interference carrier of gene expression；
The interference carrier constructs as follows: with restriction enzyme SacI and PmeI by pFGC1008 digestion, taking small
Segment is denoted as intermediate segment；The intermediate segment is connected to after restriction enzyme SacI and PmeI digestion
In pCambia3300 carrier, pCambia3300-RNAi carrier is obtained；Again by the 1894-2249 nucleic acid molecule institutes of sequence 1
The DNA fragmentation shown is connected into pCambia3300- by restriction enzyme A scI/SwaI and BamHI/SpeI, forward and reverse
It is final to obtain pCambia3300-GmSIZ1RNAi recombinant vector, as interference carrier in RNAi carrier.
7. according to method described in claim 5 or 6, it is characterised in that: the plant that sets out is monocotyledon or dicotyledonous plant
Object.
8. according to the method described in claim 7, it is characterized by: the dicotyledon is soybean.
9. DNA fragmentation shown in 1894-2249 nucleic acid molecules in sequence 1.
10. a kind of interference carrier, construct as follows: with restriction enzyme SacI and PmeI by pFGC1008 digestion,
Small fragment is taken, intermediate segment is denoted as；The intermediate segment is connected to after restriction enzyme SacI and PmeI digestion
In pCambia3300 carrier, pCambia3300-RNAi carrier is obtained；Again by the 1894-2249 nucleic acid molecule institutes of sequence 1
The DNA fragmentation shown is connected into pCambia3300- by restriction enzyme A scI/SwaI and BamHI/SpeI, forward and reverse
It is final to obtain pCambia3300-GmSIZ1RNAi recombinant vector, as interference carrier in RNAi carrier.
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