CN103173462A - Cloning of related Heilongjiang vitis amurensis seedling cold resisting gene VaERD15 - Google Patents
Cloning of related Heilongjiang vitis amurensis seedling cold resisting gene VaERD15 Download PDFInfo
- Publication number
- CN103173462A CN103173462A CN2012105788553A CN201210578855A CN103173462A CN 103173462 A CN103173462 A CN 103173462A CN 2012105788553 A CN2012105788553 A CN 2012105788553A CN 201210578855 A CN201210578855 A CN 201210578855A CN 103173462 A CN103173462 A CN 103173462A
- Authority
- CN
- China
- Prior art keywords
- gene
- vaerd
- vaerd15
- primer
- expression vector
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
Images
Landscapes
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
According to the invention, an ERD (early response to dehydration) gene is cloned from the wild vitis amurensis which has extremely strong cold resistance and a plant expression vector of the ERD gene is constructed. A VaERD15 gene is cloned from the wild vitis amurensis seedling which has extremely strong cold resistance and is produced in China by adopting the degenerated PCR (polymerase chain reaction) and the RACE (rapid amplification of cDNA ends), and a plant expression vector of the VaERD15 gene is constructed on the basis of the pC3301. The full length of the ERD early dewatering response gene is 685bp, the molecular weight is 16.2kDa, and the pI is 4.80. The length of the ORF (open reading frame) sequence of the ERD gene is 423bp, the size of the intron is 88bp, and the ERD gene can code 140 amino acids. The accession number of the nucleotide sequence of the ORF on the GenBank is JQ687321. The plant expression vector of the VaERD15 gene can be used for the genetic engineering breeding of the cold-resisting wild vitis amurensis to culture a transgenic plant with strong cold resistance.
Description
One, technical field
The invention belongs to plant biotechnology field or fruit tree crop breeding field, specially refer to the grow directly from seeds structure of the early stage dehydration response gene cloning of coding and expression vector thereof of Vitis Amurensis Heilungkiang.
Two, background technology
The cultivated area of China's grape and output are all at the forefront in the world, aspects such as being widely used in eating raw, making wine, juice processed, system are done.In production, the grape variety of cultivation belongs to Eurasian more and plants, and is best in quality but winter resistance is poor.No matter be damaging to plants caused by sudden drop in temperature after winter freeze calamity or breaking dormancy, all can damage the tree body, bring huge financial loss.Cold damage is ubiquitous outstanding problem in grape production, when being subject to environment-stress, effectively regulate and control the expression of correlation function gene in body by signal transduction path, and then cause a series of physiology, biochemical reaction, form the signals-modulating network of effective, with the harm that reduces or the elimination environment stress brings to the tree body.China is one of country of origin of vitis spp, and the distribution of Wild Grape is arranged from Heilungkiang to Hainan Island, and resource is very abundant, disease-resistant, strong stress resistance.Wherein, Vitis Amurensis (Vitis amurensis) is kind the most cold-resistant in vitis spp, is very important cold-resistant genetic resources.Therefore, utilize the winter resistance of Wild Grape resource, it is the effective ways that solve the production problem to cultivate the cold-resistant grape new variety that cold-resistant gene is changed in vitis vinifera by the transgenosis means.
ERD is a kind of gene that can the rapid answer environment stress, except having the feature of rapid answer drought stress, also has the feature of the multiple environment stress signals such as the cold of replying, salt, aging, ABA.Existing 16 ERD genes are being found in Arabidopis thaliana so far, these gene actions are in different pathways metabolisms, coerce by perception and transmission of signal respectively, produce functional protein, participate in important metabolic reaction, strengthen Arabidopis thaliana to the functions such as resistance of water stress by different modes such as degraded toxin.The Arabidopis thaliana ERDl5 little acidic protein of encoding, its expression can various abiotic stress be instantaneous induces by arid, low temperature, high salt, mechanical trauma, ABA, SA and phytopathogen etc.This gene is coerced with replying of abiotic stress multiple biology fast and show that it may play an important role in the various environment stress of plant.This gene may be the transhipment station of multi-signal regulation and control, plays a significant role in the multiple biology of response and abiotic stress.The present invention studies from improving the expression of cold-resistant genes involved grape the winter resistance that how to improve grape, and studies the effect of genes involved in plant materials by gene clone and expression thereof.
Three, summary of the invention
The object of the invention is to, Vitis Amurensis (Vitis amurensis) Heilungkiang grow directly from seeds early stage dehydration response response gene VaERD sequence and corresponding aminoacid sequence and the application aspect the improvement plant cold resistance thereof are provided.
To achieve these goals, the present invention adopts the expression amount of real-time quantitative PCR side roof part different sites VaERD gene, and the cloning vector that contains VaERD pC/VaERD is provided.Utilize achievement of the present invention, can build early stage dehydration response gene VaERD genetic engineering bacterium, be used for the conversion of plant, reach the purpose of improving plant resistance to environment stress.
Key step of the present invention comprises: grow directly from seeds as vegetable material take Vitis Amurensis Heilungkiang, extract the total RNA of plant, reverse transcription becomes cDNA, utilize biosoftware design primer, method by degenerate pcr and RACE has obtained the early stage dehydration response response gene of VaERD full length cDNA sequence, with early stage dehydration response response gene VaERD and the gus reporter gene fusion of growing directly from seeds of clone's Heilungkiang, in growing directly from seeds, Heilungkiang carried out the low temperature induction expression, prove the characteristic of the cold stress regulatory of this gene tool, and take pC3301 as fundamental construction plant expression vector.
Four, description of drawings
The total RNA electrophorogram of Fig. 1
Fig. 2 goal gene RACE electrophorogram
Fig. 3 goal gene total length electrophorogram
Fig. 4 goal gene full length nucleotide and coding protein sequence
Fig. 5 VaERD gene is subjected to the expression under different induction times in root after low temperature induction
Fig. 6 VaERD gene is subjected to the expression under different induction times in stem after low temperature induction
Fig. 7 VaERD gene is subjected to the expression under different induction times in leaf after low temperature induction
Fig. 8 VaERD genophore design of graphics
Fig. 9 VaERD genophore builds enzyme and cuts detection figure
Five, embodiment
1. adopt to improve SDS/ phenol method (Journal of Fruit Science, Zhang Jinjin etc. 2003,20(3): 178-181), with 4 ℃ of deepfreezes 0,2,6,12, blade, tender stem and fibrous root are grown directly from seeds for trying material, extraction, separation, purifying Yeast Nucleic Acid (RNA) in the Heilungkiang of 24,48 hours;
2. the reverse transcription product of the total RNA equal amount of mixture that is grown directly from seeds in above-mentioned Heilungkiang is template, and design GSP primer carries out the RACE amplification.
VaERD-3:5’-GTTCGTTCCGTTGGCATATCGGACGGT-3’
VaERD-5：5’-CTCAAACTACCAGGAAACCCTAACTCGC-3’。
2.1 the 5'-RACE of goal gene amplification
2.1.1First-Strand cDNA's is synthetic
A. add following reagent in 0.2ml thin-walled PCR pipe:
RNA Sample 3μL
5’-CDS Primer 1μL
SMART II A oligo 1μL
B. mixing and centrifugal after, in 70 ℃ of incubation 2min, then in cooled on ice 2min;
C. add following material in above-mentioned reaction system:
D. react 1.5h in 42 ℃ after mixing on the PCR instrument;
E. with reaction product with 100 μ L TE[10mM Tris-Cl(pH8.0), 1mM EDTA(pH8.0)] solution dilution;
Reaction product after dilution is heated 7min at 70 ℃;
F. final reaction product saves backup in-20 ℃.
2.1.2 the pcr amplification of reverse transcription product
A. add following reagent in 0.2ml thin-walled PCR pipe:
B. after the mentioned reagent mixing is centrifugal, add 2 mineral oil in the surface, be placed on the PCR instrument and react, the PCR program is: 5cycles:94 ℃ of 30s, 72 ℃ of 3min → 5cycles:94 ℃ of 30s, 70 ℃ of 30s, 72 ℃, 3min → 25cycles:94 ℃ of 30s, 68 ℃ of 30s, 72 ℃ of 3min.The PCR product is carried out agarose gel electrophoresis, after blob of viscose cutting-out with purpose band place, reclaim test kit with gel and reclaim the purpose band, then will reclaim product and be connected and import in the TOP10 competent cell with the pGEM-Teasy carrier and clone, by selecting positive colony to check order after blue hickie screening.
2.2. the 3'-RACE of goal gene amplification
2.2.1 First-Strand cDNA's is synthetic
A. add following reagent in 0.2ml thin-walled PCR pipe:
RNA Sample 4μL
3’-CDS Primer A 1μL
B. mixing and centrifugal after, in 70 ℃ of incubation 2min, then in cooled on ice 2min;
C. add following material in above-mentioned reaction system:
A. react 1.5h in 42 ℃ after mixing on the PCR instrument;
B. with reaction product with 100 μ LTE(10 mM Tris-Cl(pH 8.0), 1 mM EDTA(pH8.0)) solution dilution;
Reaction product after dilution is heated 7min at 70 ℃;
C. final reaction product saves backup in-20 ℃.
2.2.2 the pcr amplification of reverse transcription product
A. add following reagent in 0.2ml PCR thin-walled tube:
B. after the mentioned reagent mixing is centrifugal, add 2 mineral oil in the surface, be placed on the PCR instrument and react, the PCR program is: 5 cycles:94 ℃ 30s, 72 ℃ of 3min → 5 cycles:94 ℃ 30s, 70 ℃ of 30s, 72 ℃ of 3min → 25 cycles:94 ℃ 30s, 68 ℃ of 30s, 72 ℃ of 3min.The PCR product is carried out agarose gel electrophoresis, after blob of viscose cutting-out with purpose band place, reclaim test kit with gel and reclaim the purpose band, then will reclaim product and be connected and import in the TOP10 competent cell with the pGEM-Teasy carrier and clone, by selecting positive colony to check order after blue hickie screening.
3. the acquisition of full-length cDNA
3.1 design 5 ' terminal specific primer before 5 ' the end ATG initiator codon that obtains designs 3 ' sequence conserved regions because sequence is as follows after 3 ' end TAG:
VaERID-F：5’-ATGGCTATGGAGGTAATTTCACGTAC-3’
VaERD–R：5’-TTACCGCGGCTGCTGAATCG-3’
3.2PCR reaction system:
3.3PCR reaction conditions:
95℃3min，94℃30s，58℃30s，72℃2min→go to2，30cycles，72℃10min，4℃Forever。
3.4PCR the recovery of product and order-checking.
4.VaERD the Real time PCR of gene mRNA expression
4.1 real-time fluorescence quantitative PCR is used
Premix Ex TaqTM II (Takara Biotechnology) carries out.
4.2 the cDNA that obtains take reverse transcription carries out respectively pcr amplification as template, uses 18S rRNA as internal reference.
The PCR primer of 18S rRNA is: 18S rRNA-F:5'-TGGCCTTCGGGATCGGAGTAA-3', 18S rRNA-R:5'-ATCCCTGGTCGGCATCGTTTAT-3', the PCR primer of VaERD is: VaERD-F:5'-ATGGCTATGGAGGTAATTTCACGTAC-3', VaERD-R:5'-TTACCGCGGCTGCTGAATCG-3'.
4.3PCR system is:
4.4PCR response procedures:
Reaction is carried out on BIO-RAD iQ5 quantitative real time PCR Instrument, and the PCR program is: 95 ℃ of 3min, and 95 ℃ of 30s then, 58 ℃ of 1min circulate 35 times.
5. expression vector establishment
5.1 take carrier pC3301 as the basis, hold at 5 ' of upstream primer and add Bgl II restriction enzyme site, 5 ' end of downstream primer adds Bst II restriction enzyme site design Auele Specific Primer:
VaERD-BglⅡ：5’-GGGagatctATGGCTATGGAGGTAATTTCACGTAC-3’
VaERD-BstⅡ：5’-GGGggtgaccTTACCGCGGCTGCTGAATCG-3’
5.2PCR amplifying target genes VaERD
5.2.1PCR the preparation of reaction solution:
5.2.2PCR reaction conditions:
95℃3min，94℃30s，58℃30s，72℃2min→go to 2，30cycles，72℃10min，4℃Forever。
5.2.3PCR recovery and the enzyme of product are cut evaluation
Method is carried out according to the test kit specification sheets in strict accordance with sky, Beijing glue recovery test kit specification sheets use pMD-19-T Vector of root company, and in 16 ℃ of connection 4h, reaction system is as follows:
5.2.4 enzyme is cut evaluation
Enzyme is cut identification system:
37 ℃ of reaction 3h.1% agarose gel electrophoresis detects.
5.3 the structure of Overexpression vector
5.3.1PCR the double digestion of product
The enzyme system of cutting is:
Instantaneous centrifugal after sample blending, cut 3h in 37 ℃ of enzymes.
5.3.2 the double digestion of Overexpression vector pCAMBIA-3301
It is the same that enzyme is cut system
5.3.3 goal gene is connected connection with expression vector
After goal gene and expression vector enzyme are cut, identify with 1% agarose gel electrophoresis.Reclaim respectively the purpose segment.
Linked system is:
The recombinant cloning vector enzyme is cut the goal gene fragment 6 μ L of rear recovery
The pCAMBIA-3301 that the prokaryotic expression enzyme is cut rear recovery carries 2 μ L body fragments
Solution I 8μL
After the mixing sample, connect 3h in 16 ℃.
5.3.4 the enzyme of recombinant expression vector is cut evaluation
It is the same that enzyme is cut authentication method.
6. the pcr amplification of genomic deoxyribonucleic acid (DNA)
6.1 adopt the DNA in CTAB method extraction healthy leaves tissue, with above-mentioned early stage dehydration response gene primer amplifying genom DNA.
6.2 amplified production reclaims in strict accordance with sky root test kit operation steps; Send and check order and carry out sequential analysis.
Claims (6)
1. early stage dehydration response gene ERD, clone and its expression vector of (Vitisamurensis) early stage dehydration response gene VaERD15 grow directly from seeds in the Vitis Amurensis Heilungkiang of it is characterized in that encoding:
(1) clone's primer
(2) goal gene
(3) expression vector.
2. according to claim 1, it is characterized in that, RACE (Rapid Amplification of cDNA Ends) clone primer comprises 3 ' end primer, for VaERD-3:5 '-GTTCGTTCCGTTGGCATATCGGACGGT ' and 5 ' end primer, be VaERD-5:5 '-CTCAAACTACCAGGAAACCCTAACTCGC-3 '.
3. according to claim 1, it is characterized in that, clone's primer also comprises the total length primer, is VaERD – F:5 '-ATGGCTATGGAGGTAATTTCACGTAC-3 ' and VaERD – R:5 '-TTACCGCGGCTGCTGAATCG-3 '.
4. according to claim 1, it is characterized in that, goal gene total length 685bp, open reading frame (ORF) sequence is 423bp, the intron size is 88bp, 140 amino acid of encoding, its molecular weight is 16.2kDa, pI is 4.80.
5. according to claim 1-4 is described, and the nucleotide sequence of early stage dehydration response gene VaERD15 and the protein sequence of coding grow directly from seeds for Vitis Amurensis Heilungkiang:
1GTCATTTTCGTTATCACAACCAGCTCGAACTTCTATCTTGAGATATTTTTTCAGCTTCTT
61AGCGAA
ATGGCTATGGAGGTAATTTCACGTACAGCACCGTCATCGTCGACGTTGAATCCG
M A M E V I S R T A P S S S T L N P
121AACGCACCTTTGTTCGTTCCGTTGGCATATCGGACGGTGGAGGACTTTTCCGACCAATGG
N A P L F V P L A Y R T V E D F S D Q W W
181TGGGCCCTGGTACAGTCCTCTCCCTGGTTTCGCGATTACTGGCTTCAAGAATGCTTCCAA
A L V Q S S P W F R D Y W L Q E C F Q D
241GATCCCGAAACTGACCCTTAaagtagcgcttttccaatctattgactctctcagttccaa
P E T D P Y
301ctcgatacctcgtccgattcgattcagtttgtccttctttgtgcaggCTGTTACGATATT
C Y D I
361GACGATCCTGCCCTCCCCGACATCGACGCTCTCTTCGACGACTGCACCGGAAGCAAAGCG
D D P A L P D I D A L F D D C T G S K A
421GAGGAGGAGAAGGAGCATCAGAGGGAGTTGGTGTCGCTGGGGGCGTTGGTGGCGGAAGAC
E E E K E H Q R E L V S L G A L K W R K
481TCGAGATCCGGTGAAGTGACGCCTAGGCACTTCGAGAAGGCTCCGAAGATTGTGAACGTG
T R D P V V T P R H F E K A P K I V N V
541AAGGTGAGTCCGAGGCCGATTCAGCAGCCGCGG
TAGAGCGACTCCGATGCTTGTGGCGTC
K V S P R P I Q Q P R*
601AATGAGTTCGAGGCCGGCGAGTTAGGGTTTCCTGGTAGTTTGAGTTTATTTGTGCTTTTT
661TTTCCCTCTTTGAATGTGTACATAGAGATCACTCTAACTCGATGCATAGATCTTCAAAAT
721CGCGGTCATGTTCTGTGTATGTGTTTTTAATAAATAAAATTTGTTCAAAACC。
6. according to claim 1, it is characterized in that, with pCAMBIA-3301 basis introducing Bgl II and Bst II restriction enzyme site, built the pC3301/VaERD expression vector, can be used for genetic engineering breeding, to cultivate cold-resistant transfer-gen plant.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN2012105788553A CN103173462A (en) | 2012-12-27 | 2012-12-27 | Cloning of related Heilongjiang vitis amurensis seedling cold resisting gene VaERD15 |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN2012105788553A CN103173462A (en) | 2012-12-27 | 2012-12-27 | Cloning of related Heilongjiang vitis amurensis seedling cold resisting gene VaERD15 |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN103173462A true CN103173462A (en) | 2013-06-26 |
Family
ID=48633651
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN2012105788553A Pending CN103173462A (en) | 2012-12-27 | 2012-12-27 | Cloning of related Heilongjiang vitis amurensis seedling cold resisting gene VaERD15 |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN103173462A (en) |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1373222A (en) * | 2000-11-22 | 2002-10-09 | 理化学研究所 | Environment stress effect promoter |
| CN101831430A (en) * | 2010-04-27 | 2010-09-15 | 华中农业大学 | Identification and use of rice drought-inducible promoter Oshox24P |
-
2012
- 2012-12-27 CN CN2012105788553A patent/CN103173462A/en active Pending
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1373222A (en) * | 2000-11-22 | 2002-10-09 | 理化学研究所 | Environment stress effect promoter |
| CN101831430A (en) * | 2010-04-27 | 2010-09-15 | 华中农业大学 | Identification and use of rice drought-inducible promoter Oshox24P |
Non-Patent Citations (6)
| Title |
|---|
| DONGDONG YU ET AL.: "VaERD15, a Transcription Factor Gene Associated with Cold-Tolerance in Chinese Wild Vitis amurensis", 《FRONTIERS IN PLANT SCIENCE》 * |
| TARJA KARIOLA ET AL.: "EARLY RESPONSIVE TO DEHYDRATION 15, a Negative Regulator of Abscisic Acid Responses in Arabidopsis", 《PLANT PHYSIOL.》 * |
| TOMOHIRO KIYOSUE ET AL.: "Characterization of two cdnas (erd11 and erd13)for dehydration inducible genes that encode putative glutathione S-transferase in arabidapsis thaliana L.", 《FEBS LETTERS》 * |
| ZHANG,J ET AL.: "Vitis amurensis ERD protein(ERD)mRNA, complete cds", 《GENBANK》 * |
| 刘颖慧等: "拟南芥脱水诱导早期应答基因研究进展", 《中国农业大学学报》 * |
| 张剑侠等: "中国野生山葡萄VaERD15基因的原核表达及多克隆抗体制备", 《西北林学院学报》 * |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN110845590B (en) | Wild grape VyPPR gene and application of encoding protein thereof in drought stress | |
| CN104561025A (en) | Tomato slml1 gene and application | |
| CN112481294A (en) | Application of persimmon WRKY transcription factor gene in improvement of persimmon anthracnose resistance | |
| CN104928318A (en) | Application of rice OsLEA5c-1 gene in heavy metal resisting rice variety cultivation | |
| CN110964740B (en) | Preparation method and application of tobacco with high flavonol content | |
| CN110452917B (en) | Wild grape VyGOLS gene and application of encoding protein thereof in drought stress | |
| CN105111295A (en) | Cultivation method of genetically modified WMYB-R wheat resistant to root rot and sheath blight and related biological material thereof | |
| CN102477435A (en) | Method for improving plant drought resistance using Poncirus trifoliata transcription factor gene PtrABF | |
| CN102206260B (en) | Drought resistant and salt tolerant associated protein TaAP2 of plant, encoding gene and application thereof | |
| CN114292856B (en) | Gene PeCLH2 for regulating salt tolerance of populus euphratica and application thereof | |
| CN116064436A (en) | Loquat EjFADC1 gene, protein coded by same and application thereof | |
| CN108864264B (en) | Corn OXS2a gene, and encoding protein and application thereof | |
| CN106222171B (en) | Method for improving soybean yield by using RNAi technology | |
| CN103173462A (en) | Cloning of related Heilongjiang vitis amurensis seedling cold resisting gene VaERD15 | |
| CN104611345A (en) | Gene capable of improving quality of plant fruit as well as encoding product and application thereof | |
| CN104120134B (en) | The application in cultivating resistance of reverse transgenic plant of the GsHSFB2b albumen | |
| CN104371991B (en) | Atriplex canescens cysteine protease gene clone and its application | |
| CN106929518A (en) | A kind of rubber tree HbAG genes and its application | |
| CN109234289B (en) | Method for creating stress-resistant transgenic alfalfa | |
| CN110964729A (en) | Cloning method, application and application method of common wheat gene TaSNX1 | |
| CN104497112A (en) | Atriplex canescens[Puesh]Nute photosystem II oxygen-evolving complex-containing protein gene cloning and use thereof | |
| CN105695462B (en) | Soybean MicroRNA172 is cultivating the application in plant with adverse resistance | |
| Abdullah-Al-Emran et al. | Cloning and transformation of the transcription factor SNAC1 from rice (Oryza sativa L.) Landrace Pokkali | |
| CN115851812B (en) | Dragon fruit HuC3H35 gene and encoding protein and application thereof | |
| CN103088036B (en) | Soybean gmmads2 gene and application thereof |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| WD01 | Invention patent application deemed withdrawn after publication |
Application publication date: 20130626 |
|
| WD01 | Invention patent application deemed withdrawn after publication |