CN102226192B - Gene associated with low temperature resistance of plant and application thereof - Google Patents
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Abstract
The invention discloses a gene associated with low temperature resistance of plant, which has responsibility to low temperature stress of plants, and proves that the plant low temperature resistance-associated gene has a tight connection with cold resistance of plants, thereby, the present conventional gene engineering method is used for obtaining transgenic plants with high cold resistance, and the invention has significant meaning for raising crop output.
Description
Technical field
The present invention relates to gene engineering technology field, especially a kind of gene relevant to plant frigostabile and application thereof.
Background technology
Soybean is important in the world albumen and oil crops.Low temperature causes and has a strong impact on soybeans they grow, and particularly at later stages, cold often coercing does great damage to soybean yields, becomes the important factor that the restriction soybean yields improves.For a long time, mainly by the method for traditional genetic breeding, the winter resistance to soybean is improved people, yet this method has the shortcoming that efficiency is low, periodically long, and along with its effect of long-term use is more and more not obvious.Utilizing transgenic technology that cold-resistant genes involved is transformed in soybean is to improve soybean cold resistant more direct effectively approach and an inexorable trend.
At present, cold resistance of plant mechanism is studied more in Arabidopis thaliana, wherein research is clear that CBFs (CRT-binding factor1) transcription factor the most, comprise cold signal path (Jaglo-Ottosen et al., 1998 that each gene of the upstream and downstream performance function of CBFs mediates; Gilmour et al., 2000).In addition, except the cold signal path of CBFs transcription factor mediation, other researchs that are independent of the cold signal path outside the CBFs transcription factor also have made some progress (Zhu et al., 2004; Xin et al., 2007; Zhu et al., 2008).
The research of the cold mechanism of Soybean Resistance is relatively less, and clone and the functional study of cold genes involved relatively lag behind.At present, mainly by the method for homologous clone, some cold relevant genes have been cloned in soybean.These genes mainly comprise the transcription factor of the families such as some Ap2, bZIP, MYB, WRKY.Chen etc. search for the soybean est database by homology and obtain a gene GmDREB3 who contains the Ap2 structural domain, its can with arid response element DRE combination, this gene is caught a cold and is induced rise, simultaneously the mistake of this gene transformation Arabidopis thaliana is expressed plant and can be strengthened the resistance (Chen et al., 2008) to stress conditions such as cold, arid, salt.Liao etc. take Arabidopis thaliana bZIP structural domain as probe to soybean est sequence search, splicing, finally identified the gene of 4 bZIP families, its expression is subject to the adjusting of Different stress condition, can express plant with the mistake of ABRE combination of elements and its arabidopsis thaliana transformation has strengthened freezing and resistance (Liao et al., 2008) condition of salt stress.The method that Zhou etc. compare by homology has been identified the gene of the several WRKY of containing structural domains, can be with the combination of W-box functional element, its expression is subject to the adjusting of salt, arid, the stress conditions such as cold, its transgenosis is crossed and is expressed the resistance that the Arabidopis thaliana plant coerces salt, arid, cold etc. and increase (Zhou et al., 2008).Cheng etc. utilize the cDNA-AFLP technology to obtain a gene of catching a cold and raising in soybean plumular axis, and it is crossed expression Arabidopis thaliana plant and has strengthened resistance (Cheng et al., 2009) cold, Drought and salt.Xie etc. utilize the RACE technology to obtain the transcription factor of 2 trihelix families, and its albumen is decided to be in nucleus expresses, and it is crossed expression Arabidopis thaliana plant and has strengthened resistance (Xie et al., 2009) cold, Drought and salt.
Summary of the invention
The technical problem to be solved in the present invention is to provide a kind of gene relevant to plant frigostabile and application thereof.
For solving the problems of the technologies described above, the technical solution used in the present invention is as follows.
A kind of gene relevant to plant frigostabile, it has the nucleotide sequence shown in SEQ ID NO:1 in sequence table.
The recombinant expression vector that contains said gene, empty carrier used is PEGAD.
The restriction endonuclease sma I and the BamH I enzyme that goal gene are inserted into to PEGAD are cut between recognition site, obtain recombinant expression vector.
The recombinant expression vector that contains said gene, empty carrier used is pTCK303.
Restriction enzyme Kpn I and BamH I enzyme that goal gene is connected on pTCK303 are cut between recognition site, and SpeI and SacI enzyme cut between recognition site, obtain recombinant expression vector.
The application of said gene in cultivating low temperature resistant transgenic plant.
At first build the recombinant expression vector that contains gene shown in SEQ ID NO:1, then utilize the gained recombinant expression vector to build transformant, recycling gained transformant transforms the purpose plant, the screening positive plant, comparing with normal plants of obtaining isozygotying through three generations's screening has high cold resistant transgenic plant.
Described purpose plant is soybean.
The beneficial effect that adopts technique scheme to produce is: the contriver proves that by a large amount of scientific researches the low temperature stress of gene pairs plant of the present invention has responsiveness, the winter hardiness that proves itself and plant has and is closely connected, based on this, utilize existing conventional gene engineering method can access the transgenic plant with high winter hardiness, the output that improves crop is significant.
The accompanying drawing explanation
Fig. 1 be under low temperature stress quantitative PCR detection to the expression characteristic of GmICT1 in Soybean Root and leaf.
Fig. 2 is the expression pattern analysis of GmICT1 in Soybean Root during with hairly root system anlysis low temperature stress.
Embodiment
Following examples describe the present invention in detail.Various raw material used in the present invention and items of equipment are conventional commercially available prod, all can buy directly and obtain by market.Quantitative test in following examples, all arrange repeated experiments three times, results averaged.
The protein of gene of the present invention and coding thereof, derive from Glycine soybean (Glycine max L.), by its called after GmICT1.
The adverse circumstance of embodiment 1, GmICT1 gene and tissue expression pattern
Utilize the RT-PCR technology to carry out analysis verification to adverse circumstance and the tissue expression pattern of GmICT1 gene;
(1), Stress treatment: the experiment material therefor is WILLIAMS-DARLING Ton 82 (being called for short W82); Seedling Stage is coerced material and is carried out according to following flow process: 70% the smart sterilizing 30S that spills for soybean seeds, and dH2O is seeded in sterile petri dish after rinsing 6 times, puts 2 aseptic filter papers at the bottom of ware, and dH2O soaks, 28 ℃ of dark sproutings 3 days.When bud grows to 2cm, low N water planting, 10,000lux, temperature is 26 ℃, relative humidity is 70%, cultivates 15 days, carries out the Seedling Stage Stress treatment, the root of drawing materials, leaf; 1. the processing of contrast: the soybean material of directly getting the home cultivation respectively is organized in-80 ℃ frozen (0 hour) in contrast; 2.) subzero treatment: Seedling Stage or strain phase material 4 ℃ process respectively 0.5,3,6,12 and 24 hour after, get its each tissue, in liquid nitrogen flash freezer ,-80 ℃ save backup;
(2), the separation of mRNA: adopt the Trizol method to extract the total RNA of soybean, 1. first tissue shear is cut into small pieces, put into grinding liquid nitrogen grinding 3 times, by ground, to organize 50-100mg to add in the 1.5mL centrifuge tube then to add 1ml RNAVzol, split product should be the transparent thick liquid of clarification. room temperature is placed 5 minutes; 2. add 200 μ l chloroforms, vibration mixes, the standing 5min of room temperature, 12,000r/min, 4 ℃ of centrifugal 10min; 3. get 500 μ l supernatants in another centrifuge tube, add the equal-volume Virahol, room temperature is placed 10min, 12,000r/min, 4 ℃ of centrifugal 10min; 4. add 1ml 75% ethanol to wash precipitation, 8,000r/min, 4 ℃ of centrifugal 5min, repeat twice; The air-dry 10min of room temperature left and right, add RNase Free water (DEPC water) dissolution precipitation that 20 μ l left and right DEPC processed;
(3), reverse transcription is cDNA: by the mRNA of extraction, with the ThermoScript II AMV reverse transcription of Promega company, be cDNA;
Synthesizing of cDNA the first chain:
Configuration the first mixed solution: total RNA, 7 μ l (2 μ g); Oligo dT (10 μ M), 3 μ l; Cumulative volume 10 μ l, place 8min for 70 ℃ on the PCR instrument, is placed in immediately on ice;
Configuration the second mixed solution: 5 * Buffer, 4 μ l; DNTPs (dATP, dGTP, dTTP, dCTP 2.5mmol/L each), 4 μ l; RNase inhibitor (40U/ μ l), 0.5 μ l; RNase-free water, 0.5 μ l; M-MLV (200U/ μ l) ThermoScript II, 1 μ l; Mix, the second mixed solution is joined in the first mixed solution, every pipe 10 μ l, hatch 90min for 42 ℃ on the PCR instrument, and then 70 ℃, the 10min termination reaction;
(4), utilize the RT-PCR technology to carry out analysis verification to adverse circumstance and the tissue expression pattern of GmICT1 gene: to using above-mentioned cDNA as template, the design primer, take 18srRNA as internal reference, and the SYBR Premix Ex TagTM that uses TaKaRa company to produce carries out quantitative PCR analysis.Add cDNA template 2 μ l, forward and reverse primer each 0.4 μ l, SYBRPrimix Ex taqTM (2 *) 10 μ l, ROX Reference Dye II (50 *) 0.4 μ l, ddH2O 6.8 μ l in 20 μ l systems.Amplification program is: 95 ℃ of 30s; 95 ℃ of 5s, 60 ℃ of 34s, 45cycles; 95 ℃ of 15s, 60 ℃ of 1min, 95 ℃ of 15s;
Primer sequence is respectively:
qICT1-F：5’-AACTGGGTAATAAGTCGTTG-3’(SEQ?ID?NO：3)；
qICT1-R：5’-CCGTAAACTGGTTGAGATC-3’(SEQ?ID?NO：4)；
18srRNA-F：5’-CCTTGCTTGTTGCTTTACTAAAT-3’(SEQ?ID?NO：5)；
18srRNA-R：5’-ATGCACCTTTTCGTTTGTTTCGGAG-3’(SEQ?ID?NO：6)；
(5), result:
Result as shown in Figure 1, the time point that X-coordinate is 4 ℃ of subzero treatment, the relative expression quantity that ordinate zou is the GmICT1 gene; The expression of subzero treatment GmICT1 gene after 0.5 hour is significantly induced at Soybean Root and Ye Zhongjun, and the subzero treatment time lengthening can obviously be lowered the expression of GmICT1 gene; Result shows, the GmICT1 gene has participated in the response process of soybean to low temperature stress.Like this, the recombinant expression vector that can utilize the structures such as empty carrier PEGAD or pTCK303 to contain gene shown in SEQ ID NO:1, then utilize the gained recombinant expression vector to build transformant, recycling gained transformant transforms the purpose plant, the screening positive plant, comparing with normal plants of obtaining isozygotying through three generations's screening has high cold resistant transgenic plant.
The adverse circumstance of embodiment 2, GmICT1 gene and tissue expression pattern
Utilize the GUS staining technique to carry out analysis verification to the adverse circumstance expression pattern of GmICT1 gene;
(1), the clone of GmICT1 promotor
According to the sequence of the Promoter of ICT1, (ATG gets 1473bp in upstream, referring to SEQ IDNO:2) the design primer pair, according to carrier pCAMBIA3301 multiple clone site, the primer end introduces respectively EcoRI and the NcoI enzyme is cut recognition site, the DNA of the soybean order-checking kind Williams82 that the CTAB method of take is extracted is template, the promotor of pcr amplification ICT1 gene;
Primer sequence is:
Pro-ICT1-F-EcoRI：CGGAATTC?GAATGAACGGTTTGAAGGTG(SEQ?IDNO：7)；
Pro-ICT1-F-NcoI：CATGCCATGG?TAGTTGCGACAAGAAGGGAG(SEQID?NO：8)；
Pcr amplification product carries out 1% agarose gel electrophoresis, adopts Shanghai to give birth to work glue and reclaims the band that test kit reclaims purifying 1473bp left and right; Connect T carrier (pMD19-T), choose positive colony, order-checking;
(2), the structure of recombinant expression vector
1. extract the T vector plasmid of the ICT1Promoter sequence that contains correct order-checking, cut with restriction enzyme EcoRI and NcoI enzyme, reclaim enzyme and cut product;
2. cut empty carrier pCAMBIA3301 with restriction enzyme EcoRI and NcoI enzyme, reclaim carrier framework;
3. the carrier framework of with the T4 ligase enzyme, the enzyme of step 1 being cut to product and step 2 is connected;
4. by the connection product heat shock transformed competence colibacillus DH5 α bacterial strain of step 3,37 ℃ of incubated overnight, the picking positive colony is checked order; Sequencing result shows, has obtained recombinant plasmid pCAMBIA3301-P
gmICT1:: GUS;
(3) soybean material is prepared and subzero treatment:
Take the W82 soybean varieties as material, material, is sprouted and carried out the hairly root conversion in 4 days after 10 hours with disinfection by chlorine on B5 medium, utilize recombinant expression vector to transform Agrobacterium GV3101, recycling Agrobacterium-mediated Transformation body transforms the purpose plant, obtains transgenic plant; After explant goes on common training substratum and grows 2 days, go to again on the MS/2 substratum and cultivate, after 15 days, hairly root grows, select the approaching hairly root of the about 1-2 of length centimetre of etap respectively 4 ℃ process 0,3,6,12,24 hour after, GUS dyeing is carried out in sampling, and dyeing time is defined as 3 hours;
(4), the preparation of GUS dye liquor
At first prepare the GUS reaction buffer: Tris Buffer, comprising 100mM Tris and 50mM NaCl, adjust pH to 7.4 with concentrated hydrochloric acid.Then with Tris buffer preparation 0.5mM K
3[Fe (CN)
6] solution.Concentration by 1mg/ml takes x-glu (the chloro-3-indoles of the bromo-4-of 5-glucuronide), according to every mg, adds the ratio of 10 μ l DMSO (dimethyl sulfoxide (DMSO)) to dissolve x-glu, adds the 0.5mM K of Tris buffer preparation
3[Fe (CN)
6] the solution constant volume, can add several TritonX-100 to strengthen Color.After packing, in-20 ℃, keep in Dark Place rapidly;
(5), GUS dyeing
Hairly root to be determined is got to plant sample and put into the GUS reaction buffer, submergence, 37 ℃ of insulations in the dark.Dissecting the Microscopic observation color developing effect every half an hour.Dye after 3 hours, sucking-off GUS reaction buffer, add 70% ethanol termination reaction and, 37 ℃ of decolourings, change once every a few hours;
(6), result
See Fig. 2, wherein scheme A for the accumulation volume that transformed GUS in the soybean hairly root of pCAMBIA3301 (Control) and the PGmICT1::GUS contrast picture before and after 4 ℃ of subzero treatment 0.5 hour, figure B is that the hairly root to there being GUS to accumulate accounts for the result that the ratio of total hairly root number is analysed and compared.The result of Fig. 2 shows, in the conversion root of empty carrier pCAMBIA3301 (Control), the GUS accumulation is not subject to the impact of 0.5 hour of 4 ℃ of subzero treatment, and in the conversion root of PGmICT1::GUS, the accumulation volume of GUS has occurred obvious increase 4 ℃ of processing after 0.5 hour; Result shows, the response of GmICT1 participation soybean root system to low temperature stress.Like this, the recombinant expression vector that can utilize the structures such as empty carrier PEGAD or pTCK303 to contain gene shown in SEQ ID NO:1, then utilize the gained recombinant expression vector to build transformant, recycling gained transformant transforms the purpose plant, the screening positive plant, comparing with normal plants of obtaining isozygotying through three generations's screening has high cold resistant transgenic plant.
Foregoing description only proposes as the enforceable technical scheme of the present invention, not as the Single restriction condition to its technical scheme itself.
Claims (5)
1. a gene relevant to plant frigostabile, it is characterized in that: its nucleotide sequence is as shown in SEQ ID NO:1 in sequence table.
2. contain the recombinant expression vector of the described gene of claim 1, it is characterized in that: empty carrier used is PEGAD.
3. recombinant expression vector according to claim 2, it is characterized in that: the restriction endonuclease sma I and the BamH I enzyme that goal gene are inserted into to PEGAD are cut between recognition site, obtain recombinant expression vector
.
4. contain the recombinant expression vector of the described gene of claim 1, it is characterized in that: empty carrier used is pTCK303.
5. recombinant expression vector according to claim 4, it is characterized in that: restriction enzyme Kpn I and BamH I enzyme that goal gene is connected on pTCK303 are cut between recognition site, and SpeI and SacI enzyme cut between recognition site, obtain recombinant expression vector.
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| KR20100113909A (en) * | 2009-04-14 | 2010-10-22 | 전남대학교산학협력단 | Novel cold stress resistance kinase promoter from cucumis sativas and method for enhancing the cold stress resistance of plants using the same |
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|---|---|---|---|---|
| CN101037696A (en) * | 2006-03-16 | 2007-09-19 | 华中农业大学 | Paddy cool injury gene and application |
| CN101503467A (en) * | 2009-02-27 | 2009-08-12 | 中国科学院遗传与发育生物学研究所 | Plant stress tolerance related transcription factor GmNAC20, coding gene and use thereof |
| KR20100113909A (en) * | 2009-04-14 | 2010-10-22 | 전남대학교산학협력단 | Novel cold stress resistance kinase promoter from cucumis sativas and method for enhancing the cold stress resistance of plants using the same |
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