CN102134273A - Application of oryza sativa heat stress transcription factors OsHsfA2d in cultivating species - Google Patents
Application of oryza sativa heat stress transcription factors OsHsfA2d in cultivating species Download PDFInfo
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Abstract
The invention discloses an application of oryza sativa heat stress transcription factors OsHsfA2d in cultivating species which have storage stability and seeding time cold damage resistance. The inventor establishes an oryza sativa mutant strain excessively expressing the OsHsfA2d and then discovers the seed germination, the germination delay and even the germinating capacity losing of the oryza sativa mutant strain. An available new gene resource is provided for cultivating new plant species which have the storage stability and the seeding time cold damage resistance through the result.
Description
Technical field
The present invention relates to plant breeding, be specifically related to paddy rice heat stress transcription factor OsHsfA2d and cultivating the anti-storage of seed and resisting the application of damaging to plants caused by sudden drop in temperature sowing time in the plant variety.
Background technology
Most of regional rice growing phase of northern China is in early summer, meets with chilling injury easily, and it is impaired that rice paddy seed is sprouted, and causes the agriculture underproduction impaired.But if postpone sowing, miss soil moisture content easily, increase peasant's production cost.The soil moisture content phase that the new rice variety of cultivation delay seed germination can well neither make the peasant miss, can effectively avoid the germination period chilling injury again, protect the normal percentage of germination of positive rice paddy seed, keep normally carrying out of agriculture production.
Can cause the loss in the production and then influence grain security because of going mouldy or germinateing of some seeds in the foodstuff seed storage process.So provide a safety control if can find the gene that suppresses seed germination to make the impaired or forfeiture germinating power of seed germination can be seed storage.
So illustrate the molecular mechanism that paddy rice sprouts and germinates, separate, identify a collection of gene that paddy rice sprouts and germinates that influences as soon as possible, be that we utilize genetic engineering means to accelerate one of key factor of improvement of crop cultivar process.
The heat stress environmental factors that to be many plants all must face at high temperature every day in summer, though paddy rice is the warm crop of happiness, paddy rice is very responsive to high temperature in flowering period and filling stage, forms easily " high temperature is forced ripe ", and output is fallen sharply.Discover, to pyritous tolerance otherness is the decision of kind inherent hereditary property, the parental combination that contains the japonica rice gene all shows as non-refractory heat evil with the parental combination that contains precocious gene, and the paddy rice of conventional rice, medium fringe type, its elevated temperature heat evil is obviously light to be sent out, and major cause is a japonica rice gene non-refractory, precocious gene non-refractory, large spike is to the nutritional requirement height, and is tight to environmental requirement, also non-refractory.
Land plant is in the evolution of long period of time process, produced a series of measure so that the quick variation that conforms, because terrestrial plant can not be moved, plant is for the extreme environment of low temperature, high temperature, salt stress, heavy metal and extreme lack of water arid, can make a response rapidly, make them be survived and multiply.Wherein, heat stress albumen (heat stress proteins, Hsps) extensively be present in all eukaryotes and the prokaryotic organism, they all play an important role various coercing in the reaction: the heat stress albumen of constitutive expression is molecule companion (Molecular Chaperones), and their participate in keeping processes such as proteinic synthetic, folding, transportation, function and degraded.The proteic expression of heat stress directly is heated, and (these transcription factors are the crucial regulatory factors in the heat stress reaction to stress transcription factor for heat stress transcriptionfactors, regulation and control Hsfs).
According to the protein structure of heat stress transcription factor, the heat stress transcription factor of higher plant is divided three classes, A, B and C class.The same with other transcription factor, the size and the sequence of heat stress transcription factor have nothing in common with each other, but they have the certain structure pattern: N-terminal has DNA binding domains (DBD), it can discern specifically sequence on the heat stress protein promoter (heat stress promoter elements, HSE).HSE is made up of the tumor-necrosis factor glycoproteins with eighty percent discount symmetrical structure (5 '-AGAAnnTTCT-3 '), is present in the upstream that is subjected to thermoinducible gene promoter TATABox.Adjacent with the DNA binding domains is HR-A/B district and nuclear localization signal (NLS) and nuclear export signal (NES) and activating peptide structural domain (AHA).
In higher plant, only comparatively systematic research is arranged for the heat stress transcription factor at Arabidopis thaliana and tomato, for discovering of more than 20 Hsfs of tomato, HsfA1, A2 and B1 have represented three most important families, the three phases that they take place at heat stress respectively, bring out, keep and recover in play a different role.In tomato, HsfA1a is a topmost adjusting albumen in the heat stress reaction, and it participates in inducing the thermal induction albumen that comprises HsfA2 and HsfB1; The transgenic Fructus Lycopersici esculenti of overexpression HsfA1a increases than wild-type contrast thermotolerance; And reduce the tomato that HsfA1a expresses, for the high temperature stress hypersensitization, and the delaying of minimizing of transcribing with Hsp70 and fruit maturation, so HsfA1a is absolutely necessary in the reaction high temperature resistant coercing; HsfA2 only expresses after thermal induction, is topmost Hsf, in participation in the summer of sweltering heat heat stress and recovery process repeatedly; The activity of HsfA2 is subjected to a series of albumen, and its solvability, intracellular location and mobilizing function realize by regulating and control; Hsp17-CII can interact with the HsfA2 C-terminal, form special common inhibition, both form big particulate polymers in kytoplasm, this polymkeric substance can in the presence of the Hsp17-CI or HsfA2 and HsfA1 form heterodimer to enter nucleus after depolymerization, the depolymerization process need depends on the activity of Hsp70 and the Hsp101 molecular chaperones of ATP; HsfB1 and HsfA1 synergy, recruitment comprises that acetylation of histone enzyme HAC1 transcribes co-activation.
Summary of the invention
The purpose of this invention is to provide a kind of heat stress transcription factor of paddy rice OsHsfA2d is cultivating the anti-storage of seed and is resisting the application of damaging to plants caused by sudden drop in temperature sowing time in the breeding
Heat stress transcription factor provided by the present invention derives from paddy rice (Oryza sativa L.), and name is called OsHsfA2d, is the protein of following (a):
(a) protein of forming by the aminoacid sequence shown in the sequence in the sequence table 1;
Wherein, the sequence in the sequence table 1 is made up of 379 amino-acid residues.
The proteic encoding gene of described heat stress transcription factor OsHsfA2d specifically can be following 1) or 2) gene:
1) its nucleotide sequence is the sequence 2 in the sequence table;
2) under stringent condition with 1) the dna sequence dna hybridization and the proteic dna molecular of coding heat stress transcription factor OsHsfA2d that limit.
Described stringent condition can be 0.1 * SSPE (or 0.1 * SSC), in the solution of 0.1%SDS,, and wash film with this solution 65 ℃ of hybridization down.
Sequence 2 in the sequence table is made up of 1140 deoxyribonucleotides, is the CDS gene order of OsHsfA2d.
The recombinant expression vector that contains above-mentioned heat stress transcription factor OsHsfA2d protein coding gene specifically can be pCAMBIA2300-Ubiqutin-FLAG-OsHsfA2d as shown in Figure 1.
Described recombinant plant expression vector can be by cell or tissue such as conventional biological method rice transformation such as agriculture bacillus mediated grade, and the rice tissue that transforms is cultivated into plant.
Experiment showed, that the pCAMBIA2300-Ubiqutin-FLAG-OsHsfA2d transgenic paddy rice seed is sprouted is suppressed even is lost germinating power.Illustrate that the OsHsfA2d gene can influence seed germination and germination, therefore can be used for cultivating seed and store and resist sowing time chilling injury.
The present invention will be further described below in conjunction with drawings and Examples.
Description of drawings
Fig. 1 is the physical map of pCAMBIA2300-Ubiqutin-FLAG-OsHsfA2d
Fig. 2 is that pCAMBIA2300-Ubiqutin-FLAG-OsHsfA2d transgenic line seed germination postpones even forfeiture germinating power (A:28 ℃, B:24 ℃)
Embodiment
The structure of embodiment 1, plant expression vector
With pCAMBIA1302-FLAG (XF0349) is template, go out the full length fragment of FLAG with following primer amplification, singly cut this PCR product and pCAMBIA2302-Ubiqutin-ocs (XF0422) respectively with KpnI and BamHI, connection obtains pCAMBIA2302-Ubiqutin-FLAG (XF0426) paddy rice and crosses expression vector after reclaiming.The sequence:GGTACCATGGACTACAAGGACGATGATGACAAGGGCGACTATAAGGA TGACGACGATAAGACTAGTCCCGGGACGCGTGGATCC that contains FLAG after the amplification
From rice cDNA, amplify corresponding osHsfA2d full-length cDNA gene with primer CX01078:5 '-CGCTCTAGA ATGATTAAGAGGAGGAGACCA-3 ' and CX01079:5 '-CGTGGATCCCTACTTTTGGCTACTATTGC-3 ' (containing XbaI and BamHI recognition site), cut carrier XF0426 with XbaI and BamHI enzyme, link with the T4DNA ligase enzyme, to connect product and be transformed into e. coli jm109, screening resistance bacterium colony on the corresponding resistant panel, carry out PCR and identify, obtain containing the positive bacterium colony of plant expression vector pCAMBIA2300-Ubiqutin-FLAG-OsHsfA2d.Extract the plasmid in the positive bacterium colony, obtain plant expression vector
Embodiment 2, transgenic line seed germination and germination are suppressed
Choose wild-type and change each about 40 rice paddy seed of OsHsfA2d gene and be placed in the culture dish that is covered with double-deck filter paper of water-soaked, cover with gauze, place 24 ℃ and 28 ℃ of incubators to cultivate respectively, sooner or later change water with ready prepd clear water every day.Observe the seed germination situation after 9 days, it is slow even suppressed to change OsHsfA2d gene seed germination as shown in Figure 6, and the inhibition degree that the pure and mild seed germination of OsHsfA2d gene is subjected to is stronger.First is the wild-type rice paddy seed on the left of each row among Fig. 6, and second for changeing OsHsfA2d gene T1 for the heterozygosis seed, and the 3rd for changeing OsHsfA2d gene T1 for pure and mild rice paddy seed.
SEQUENCE?LISTING
＜110〉Chinese Academy of Sciences's heredity and developmental biology institute
＜120〉application of a kind of paddy rice heat stress transcription factor in breeding
<130>CXF4
<160>1
<170>PatentIn?version?3.5
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Claims (8)
1. heat stress transcription factor and encoding gene thereof are being cultivated the application of supporting in the anti-stored seed breeding, and its base sequence is shown in SEQID NO:2, and its amino acid residue sequence is shown in SEQ ID NO:1.
2. heat stress transcription factor and encoding gene thereof are resisted the application of damaging to plants caused by sudden drop in temperature sowing time in the plant variety in cultivation, and its base sequence is shown in SEQ ID NO:2, and its amino acid residue sequence is shown in SEQ ID NO:1.
3. according to the described application of claim 1, it is characterized in that described plant is monocotyledons or dicotyledons.
4. according to the described application of claim 2, it is characterized in that described plant is monocotyledons or dicotyledons.
5. according to the described application of claim 1, it is characterized in that described plant is paddy rice, corn, wheat, soybean and tomato.
6. according to the described application of claim 2, it is characterized in that described plant is paddy rice, corn, wheat, soybean and tomato.
7. the plant expression vector that contains the described paddy rice heat stress of claim 1 transcription factor encoding gene.
8. the host bacterium that contains the described paddy rice heat stress of claim 1 transcription factor encoding gene.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103288940A (en) * | 2012-02-29 | 2013-09-11 | 中国科学院上海生命科学研究院 | Heat-resistance related protein of plant maintaining stability of thylakoid membrane and application thereof |
| CN108017697A (en) * | 2017-12-25 | 2018-05-11 | 中国科学院遗传与发育生物学研究所 | Plant tillering angle GAP-associated protein GAP HSFA2D and its encoding gene and application |
Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1796559A (en) * | 2004-12-21 | 2006-07-05 | 华中农业大学 | Using gene of transcriptional factor OSNACX of paddy to increase drought resistance and salt tolerant abilities of plants |
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2010
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| CHUANG WANG ET AL.: "Identification and expression analysis of OsHsfs in rice", 《 浙江大学学报B(生物医学、生物工程版)(英文版)》 * |
| DOMINIQUE GAGLIARDI ET AL.: "Expression of heat shock factor and heat shock protein 70 genes during maize pollen development", 《PLANT MOLECULAR BIOLOGY》 * |
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103288940A (en) * | 2012-02-29 | 2013-09-11 | 中国科学院上海生命科学研究院 | Heat-resistance related protein of plant maintaining stability of thylakoid membrane and application thereof |
| CN103288940B (en) * | 2012-02-29 | 2015-08-05 | 中国科学院上海生命科学研究院 | A kind ofly maintain the stable Heat Resistance of Plant associated protein of thylakoid membrane and application thereof |
| CN108017697A (en) * | 2017-12-25 | 2018-05-11 | 中国科学院遗传与发育生物学研究所 | Plant tillering angle GAP-associated protein GAP HSFA2D and its encoding gene and application |
| CN108017697B (en) * | 2017-12-25 | 2020-06-30 | 中国科学院遗传与发育生物学研究所 | Plant tillering angle related protein HSFA2D, and coding gene and application thereof |
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