CN105452453A - Thellungiella halophila calcineurin b-like protein cbl-4, coding gene of same, and application thereof - Google Patents
Thellungiella halophila calcineurin b-like protein cbl-4, coding gene of same, and application thereof Download PDFInfo
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- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
- C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
- C12N15/8241—Phenotypically and genetically modified plants via recombinant DNA technology
- C12N15/8261—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield
- C12N15/8271—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance
- C12N15/8273—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance for drought, cold, salt resistance
Abstract
Provided are a Thellungiella halophila-sourced calcineurin B-like protein ThCBL-4, a coding gene thereof, and an application of the gene in cultivating a transgenic plant of increased drought tolerance.
Description
A kind of small salt mustard calcium adjusting phosphatase B analogy albumen CBL-4 and its encoding gene and application
The present invention relates to vegetable protein and its encoding gene and application, the more particularly to one calcium adjusting phosphatase B analogy albumen CBL-4 and its encoding gene from small salt mustard, and its application in the genetically modified plants that drought tolerance is improved are cultivated for technical field.The environment stresses such as background technology temperature, salt marsh and arid can cause to seriously endanger to growing for higher plant, cause crop yield to reduce, quality decline is serious to threaten agricultural production and natural environment.Wherein influence of the arid to crop yield, first place is accounted in many natural adverse circumstances, and it is endangered equivalent to other disaster sums, be many areas be agricultural development bottleneck.According to statistics, world's arid, semiarid zone account for the 34% of land area;China's arid, semiarid zone account for the 52% of area, year area suffered from drought up to 200-270 ten thousand hectares, the national billion cubic meter of the annual water shortage in irrigation district about 30 receives 350-400 hundred million kilograms of grain because of water shortage less；Particularly China relationship and primary grain producing such as North China, northeast and northwest, is the area of China's water shortage most serious, and spring drought frequently reaches 10 years nine chances.
Drought resistance in plants belongs to the quantitative character of controlled by multiple genes mostly, is lacked using the drought resistance of conventional breeding methods Crop Improvement by cycle length, quality germplasm and is limited.The Preliminary Study of transcription group in recent years, protein science and gene expression regulation discloses the effect molecule mechanism of plant drouhgt stress.At present, the drought-resistant ability of plant is improved using drought stress related gene, the study hotspot and the important research direction of plant stress-resistance genetic engineering of plant stress-resistance molecular biology is had become.
Plant is by that can produce corresponding responsing reaction during environment stress, to reduce or eliminate the harm that environment stress comes to vegetational zone.This responsing reaction of plant is a complex process for being related to polygenes, multi signal approach and polygenes product.But for current research situation, because its mechanism is sufficiently complex, many plants still need to be studied to the biochemistry under adverse circumstance and physiological response mechanism.In the research in terms of the function and expression regulation of degeneration-resistant response gene important basis is provided by the research that network system is transmitted in the contact between the signaling pathways related to plant stress-resistance and whole signal.Content of the invention the present inventor utilizes SSH (Subtractive hybridizations）With RACE (cDNA ends rapid amplifyings）The side being combined
Method has cloned a kind of calcium adjusting phosphatase B analogy albumen of small salt mustard（The encoding gene being named as herein, and determine its DNA sequence dna.And it was found that being conducted into after plant overexpression, the drought tolerance of transfer-gen plant is can obviously improve, and these characters can stablize heredity.
First aspect present invention provides a kind of calcium adjusting phosphatase B analogy albumen gene ThCBL-4 of small salt mustard encoding gene (being named as ThCBL-4 herein)；Preferably, its sequence is SEQ ID NO: 2.
Second aspect of the present invention provides a kind of recombinant expression carrier, it contains the gene described in first aspect present invention, its be by by the gene be inserted into it is a kind of be used to building the carrier is carrier of the recombinant expression carrier and obtain, and the nucleotide sequence of the gene is operably connected with the expression control sequence of the carrier is carrier；Preferably, the carrier is carrier is pCAMBIA2300;Preferably, the recombinant expression carrier is the 35S-7 J-$-2300 carriers shown in accompanying drawing 2.
Third aspect present invention provides a kind of recombinant cell, and it contains the recombinant expression carrier described in gene or second aspect of the present invention described in first aspect present invention；Preferably, the recombinant cell is restructuring agrobatcerium cell.
Fourth aspect present invention provides a kind of method of improvement drought resistance in plants, including：Recombinant expression carrier described in gene or second aspect of the present invention described in first aspect present invention is imported into plant or plant tissue and makes the gene expression；Preferably, the plant is arabidopsis.
Fifth aspect present invention provides a kind of method of prepare transgenosis plant, including：The plant containing the recombinant expression carrier described in the gene or second aspect of the present invention described in first aspect present invention or plant tissue are cultivated under conditions of plant is effectively produced；Preferably, the plant is arabidopsis.
Gene, the recombinant expression carrier described in second aspect of the present invention or the recombinant cell described in third aspect present invention that sixth aspect present invention is provided described in first aspect present invention are used to improve drought resistance in plants and the purposes for plant breeding;Preferably, the plant is arabidopsis.
Seventh aspect present invention provides the protein of the gene code described in first aspect present invention, its amino acid sequence such as SEQ ID NO:Shown in 1.Brief description of the drawings Fig. 1 is ThCBL-4 plant expression vector C35S-7 J-$-2300;>Structure flow（Scheme la-lb).
Fig. 2 is ThCBL-4 plant expression vector C35S-7 J-$-2300) plasmid figure.
Fig. 3 is ThCBL-4 T1 for transgenic Arabidopsis plants（In figure, T1K2) and it is used as the non-transgenic Arabidopsis plant of control（In figure, CK) drought-enduring simulated experiment result.（Fig. 3 a are the normal growth Arabidopsis plant of 20 days；Fig. 3 b are normal growth Osmotic treatment Arabidopsis plant of 14 days after 20 days）.
Fig. 4 is protein expression the results of the transgenosis Tl for Arabidopsis plant and non-transgenic reference plant on transcriptional level.M is DNALadderMarker (DL2000, TakaRa), and 1-7 is drought-enduring transgenic arabidopsis Tl for plant（It is followed successively by：The Κ 2 of T1K1, Τ 1, the Κ 3 of Τ 1, the Κ 4 of Τ 1, the Κ 5 of Τ 1, the Κ 6 of Τ 1, T1K7), 8-11 be not drought-enduring transgenic arabidopsis Tl for plant, 12-15 is the control of non-transgenic arabidopsis.
The present invention is further described with reference to non-limiting example for embodiment.The embodiment is not intended to limit the scope of the present invention merely for exemplary purpose.
The restriction enzyme in the unreceipted source mentioned in example below is purchased from New England Biolabs companies.Slight salt mustard SSH library constructions under the drought stress of embodiment 1
Specific method is：
Subtracted library is built by Subtractive hybridization method using the method shown in the PCR-select cDNA Subtraction Kit of Clontech companies.Using the mRNA of the blade of the small salt mustard seedling of Osmotic treatment as sample (Tester) in experimentation, control is used as using the mRNA of the blade of untreated slight salt mustard seedling（Driver).Specific steps are summarized as follows：
Small salt mustard（T ellungiella halophila, purchased from inner mongolia Ba Yan Nor City carex meyeriana green plants garden halophytes Breeding Center）, it is seeded on sterilized vermiculite, in 25 °C, 16 hours photoperiods illumination/8 hour dark（The Lx of light intensity 2000-3000) under the conditions of cultivate, 1/2MS culture mediums are poured weekly（ 9.39 mM KN03, 0.625 mM KH2P04, 10.3 mMNH4N03, 0.75 mM MgS04, 1.5 mMCaCl2, 50 μ Μ Κ Ι, 100 μ Μ Η3ΒΟ3, 100 MMnSO4, 30 μ Μ ZnS04, 1 μΜΝα2Μο04, 0.1 μΜ CoCl2, 100 μΜ Na2EDTA, 100 MFeSO4)-secondary.It is used to test when seedling strain is cultivated 1 month or so.
(2) material process：
2 groups, every group of 4 basins, per 1 plant of basin will be divided into for examination seedling.First group is control group, is cultivated under 25 °C, 16 hours photoperiods illumination/8 hour dark condition, normal to pour.Second group is Osmotic treatment group, 25 °C, cultivate under 16 hours photoperiods illumination/8 hour dark condition, stops pouring, processing 10 days, the blade of two groups of seedling apicals 1/3 of timely clip after being disposed, after liquid nitrogen quick freeze, is preserved in -70 °C of refrigerators.
(3) Total RNAs extraction：
Control group and the small g of salt mustard blade 0.1 of Osmotic treatment group are taken respectively, use plant RNA extraction kit（Purchased from Invitrogen) extract the total serum IgE of small salt mustard blade.With the ultraviolet specrophotometer U-2001 of HITACHI companies
Total serum IgE is determined in 260 nm and 280 nm absorbance, OD260/OD280Ratio is 1.8-2.0, shows that total serum IgE purity is higher；The integrality of total serum IgE is detected with 1.0% agarose gel electrophoresis, the brightness of 28S bands is about 2 times of 18S bands, shows that RNA integrality is good.MRNA is separated using the Oligotex mRNA purification kits (purification of poly A+ RNA from total RNA) of Qiagen companies.
(4) Subtractive hybridization：
By the PCR-select of Clontech companiesTMMethod shown in cDNA Subtraction Kit kits carries out Subtractive hybridization.Driver mRNA and Tester mRNA are first distinguished into reverse transcription, double-strand cDNA, then the progress subtractive hybridization using the g Driver cDNA of 2 Tester cDNA standing grain P 2 as parent material is obtained.Then the Tester cDNA after digestion are divided into joints different in two equal portions, connection by Tester cDNA and Driver cDNA Rsa I digestions 1.5 hours respectively under 37 °C of water-baths, and Driver cDNA are not connected to head.Two kinds of Tester cDNA for being connected with different joints are mixed with excessive Driver cDNA respectively, carry out positive subtractive hybridization for the first time.The product of two kinds of first time subtractives hybridization is mixed, then second of positive subtractive is carried out with the Driver cDNA that are newly denatured and is hybridized, the fragment of differential expression is then expanded by inhibition PCR twice, it is enriched with.
In order to have increased access to EST（Expressed sequence tag, EST) the validity of (Unigene), avoid gene without restriction enzyme site and obtained sequence in non-translational region, digestion is carried out to Tester cDNA and Driver cDNA by above-mentioned steps with restriction endonuclease Haelll simultaneously for this experiment and priority carries out positive subtractive hybridization and inhibition PCR amplifications twice twice, finally merges second of inhibition PCR primer that two groups of positive subtractives hybridize cDNA fragments.
(5) structure of cDNA subtracted libraries and preliminary screening, clone, identification
According to the program of pGEM-T Easy kits, the positive subtractive of above-mentioned merging is hybridized to second of PCR primer of cDNA fragments（Purified using QIAquick PCR Purification Kit, purchased from Qiagen) it is connected with pGEM-T Easy (being purchased from Promega kits) carrier, it is comprised the following steps that：Following ingredients are sequentially added with 200 μ PCR pipes：The positive subtractive of purifying hybridizes the ligase buffer solutions 5 of second of PCR primer, 31,2 χ Τ of μ 4 of cDNA fragments, and pGEM-T Easy carriers 1 μ 1, the μ of T4 DNA ligases 1 are stayed overnight in 4 °C of connections.10 coupled reaction products are taken, are added in 100 competence e. coli jm109s (being purchased from TAKARA), ice bath 30 minutes, heat shock 60 seconds, ice bath 2 minutes separately add 250 μ L LB nutrient solutions（1% Tryptone is purchased from OXOID, and 0.5% Yeast Extract are purchased from OXOID, and 1% NaCl is purchased from traditional Chinese medicines）Put in 37 °C of water-baths, with 225 revs/min of shaken cultivations 30 minutes, take 200 μ bacterium solutions to be coated on containing 50 g/mL ampicillins（Purchased from TIANGEN Biotech (Beijing) Co., Ltd.）LB (ibid）(X-gal/IPTG is purchased from TAKARA to/X-gal/IPTG, and lg packagings are configured to 20 mg/ml mother liquors, and its working concentration is：The ml LB culture mediums of 200 μ of above mother liquor 1/100；IPTG is purchased from TaKaRa, and 5g packs the mother liquor for being configured to 100 mM concentration, and its working concentration is：Above mother liquor Ι Ο Ο μ Ι/lOOml LB culture mediums）On solid culture flat board, 37 °C are cultivated 18
Hour.Count diameter in culture plate>1 mm clear white and blue colonies number, random 198 white colonies of picking (numbering：Gh-B001 to Gh-B198).All white colonies are inoculated in 96 porocyte culture plates (CORNING) of the LB fluid nutrient mediums containing 50 g/mL ampicillins respectively, glycerol adding is saved backup to final concentration 20% (v/v) in -80 °C after 37 °C of overnight incubations.With the standing grain P Primer 2R of nest-type PRC primer Primer 1, (the PCR-select cDNA Subtraction Kit kits of Clontech companies are carried）Bacterium solution PCR amplification checkings are carried out, 166 positive colonies is obtained, is then sending Invitrogen by all positive colonies（Shanghai）Trade Co., Ltd is sequenced.
(6) the cDNA sequencing analysis of differential cloning：
After the cDNA that DNA sequencing results are removed to carrier and indefinite sequence and redundancy, 123 EST (Unigene are obtained；).There are 22 contigs through analysis, there are 101 single sequences.Find that wherein 53 EST (Unigene) have homologous sequence in GenBank through BlastN, 21 EST Unknown Functions are hypothesis albumen, separately there are 27 not obtain homologous matching, thus it is speculated that to be probably the shorter sequence in 3' or 5' ends non-translational region.The small salt mustard calcium adjusting phosphatase B analogy albumen encoding gene ThCBL-4 of embodiment 2 clone
Clone YLS-66 removes after redundant DNA, and sequence is SEQ ID No:3, sequence analysis shows that the protein of the coding of the sequence belongs to calcium adjusting phosphatase B analogy albumen, the corresponding total length encoding genes of clone YLS-66 is named as into ThCBL-4 herein, its corresponding albumen is named as CBL-4.
SEQ ID No: 3
1 TTCCATCCAG ATGCATCTGT CCATGAAAAA ATCAAATTTG CITTCAAGTT GTACCATTTA 61 CGGCAAACTG GATTCATGGA GCGGGAAGAA CTGAAAGAGA TGGTAATCGC TCTTCTTCAC 121 GAATCCGAAC TATACTTTC CGAAGATATG ATTGAAGTAA TGG GGATAA GGCTITTATC 181 GAAGCAGATC GCAAGAACGA TGGGAGAATT GATATAGA G AATGGAAAGA T TCGTGTCC 241 AAGAATCCGT CGCTCATCAA AAACATGACT T GCCGTATC TAAAGGACAT AAACGGGGCG 301 TTTCCAAGTT TGITTCATC TTG GAAGAC GATGAATTGG AGTTGCAAAA ACTAAATTTT 361 ΤΑΆ
The clone of CBL-4 total length encoding genes
According to the SEQ ID No obtained:3 sequence analyses, SEQ ID No:3 be encoding gene CBL-4 3 terminal sequences.According to the SEQ ID NO obtained:3 sequences, design following three specific primers, are used as reverse transcription primer and 5 ' RACE specific primer.
YLS-66GSP1 ( SEQ ID NO: 4) ：
GGACACGAAATCTTTCCATTC YLS-66GSP2 ( SEQ ID NO: 5 ) ：
TCGTTCTTGCGATCTGCTTC
YLS-66GSP3 ( SEQ ID NO:6) ：
TCCATCCAGATGCATCTGTC kits carry universal primer：
AAP ( SEQ ID NO: 7 ) ：
GGCCACGCGTCGACTAGTACGGGIIGGGIIGGGIIG AUAP ( SEQ ID NO: 8 ) ：
GGCCACGCGTCGACTAGTAC experimental procedures are operated by kit specification（5'RACE System for Rapid Amplification of cDNA Ends kits are purchased from Invitrogen companies）.
With YLS-66GSP1CSEQ ID NO:4) it is reverse transcription primer, reverse transcription is carried out by template of slight salt mustard mRNA, obtain cDNA templates, then Poly C tails are added according to the step in above-mentioned 5'RACE kit specifications, the amplification of first round PCR is carried out by template of the product after tailing, the primer is SEQ ID NO:4 and universal primer SEQ ID NO:7 (kit is carried, and I is a, c, g or t) that Hypoxanthine is modified, and is comprised the following steps that：
50 μ PCR reaction systems：5 μ Ι Ο Ε χ Buffer, the 3 μ 2.5 mM μ Ex of dNTP, the cDNA of 2.0 μm of RNA reverse transcriptions, 1.0 Taq (being purchased from TAKARA), 10 μ Μ primer SEQ ID NO:4 and SEQ ID NO:7 each 2.0 μ 1, and 35 μ 1 distilled water.PCR reaction conditions：94 °C of pre-degenerations 5 minutes, 33 circulations（94 °C are denatured 45 seconds, and 58 °C are annealed 45 seconds, and 72 °C extend 45 seconds）, 72 °C extend 10 minutes.
The PCR primer of gained takes 2.0 μ as template after diluting 50 times with distilled water, with SEQ ID NO:5 and universal primer SEQ ID NO:8, which carry out second, takes turns PCR amplifications, comprises the following steps that：
50 y l PCR reaction systems：The first round PCR primer of 5 μ 1 10 X Ex Buffer, 3 μ 1 2.5 mM dNTP, 2.0 μ 1 dilution, the Ex Taq of 1.0 μ 1,10 μ Μ primer SEQ ID NO:5 and SEQ ID NO:8 each 2.0 μ 1, and 35 μ 1 distilled water.PCR reaction conditions：94 °C of pre-degenerations 5 minutes, 33 circulations（94 °C are denatured 45 seconds, and 58 °C are annealed 45 seconds, and 72 °C extend 45 seconds）, 72 °C extend 10 minutes.Reclaim in second of PCR product be about 500bp sizes band（Gel Extraction Kit are purchased from OMEGA), and pGEM-T Easy Vector are connected to, being then transformed into JM109, (specific method is ibid）, random 6 white colonies of picking are inoculated in the LB fluid nutrient mediums containing 50 g/mL ampicillins and cultivate respectively, and glycerol adding is saved backup under -80 °C to final concentration 20% (v/v) after 37 °C of overnight incubations.With primer SEQ ID NO:5 and 3' ends primer SEQ ID NO:6 carry out bacterium solution PCR amplifications（Reaction system and reaction condition are ibid）, 6 positive colonies are obtained, Invitrogen is sent（Shanghai）Trade Co., Ltd's sequencing sequencing, obtains the cDNA of the gene one section of 5' terminal sequence.
It is SEQ ID NO that the sequencing of 5'RACE product clonings 04 of gained, which obtains sequence,: 9：
1 GGGGGGGGGG ATGGGCTGCT CTCCGTCGAA GAGGAAGAAG GCAACACGAC CGCCGGGATA
61 TGAGGATCGT AACCTTCTCG CCTCCGCTAC GCCATTCAGG GTAGCAGAAG TGGAGGCTTT
121 GTATGAACTG TTCATGAAGC TAAGCAGTTC AA TATCGAC GACGGTC TA TTCATAAGGA
181 AGAATTTCAG CTGGCI TAT TCAGAAATAG GAATCGGAAG AAGCTTTTCG CTGATCGGAT
241 ATTTGA GTA TTTGATGTGA AGCGAAATGG AGTGATCGAG TTTGGGGAAT T GTCCGGTC
301 TTTAGG GTC TTCCATCCAG ATGCATCTGT CCATGAAAAA ATCAAATTTG CITTCAAGTT
361 GTACCATTTA CGGCAAACTG GAT CATCGA GCGGGAAGAA CTGAAAGAGA TGGTAATCGC
421 TC TCTTCAC GAATCCGAAC TTATACTTTC CGAAGATA G ATTGAAGTAA TGGTGGATAA
481 GGCTITTATC GAAGCAGATC GCAAGAACGA
The sequence SEQ ID NO that the RACE of If 5 ' are obtained:9, the sequence SEQ ID NO with acquisition:3 splicings, obtain SEQ
ID NO: 10:
1 GGGGGGGGGG ATGGGCTGCT CTCCGTCGAA GAGGAAGAAG GCAACACGAC CGCCGGGATA
61 TGAGGATCGT AACCTTCTCG CCTCCGCTAC GCCATTCAGG GTAGCAGAAG TGGAGGCTTT
121 GTATGAACTG TTCATGAAGC TAAGCAGTTC AA TATCGAC GACGGTC TA TTCATAAGGA
181 AGAATTTCAG CTGGCITTAT TCAGAAATAG GAATCGGAAG AAGCTTTTCG CTGATCGGAT
241 ATTTGA GTA TTTGATGTGA AGCGAAATGG AGTGATCGAG TTTGGGGAAT TTGTCCGGTC
301 TTTAGGTGTC TTCCATCCAG ATGCATCTGT CCATGAAAAA ATCAAATTTG CITTCAAGTT
361 GTACCATTTA CGGCAAACTG GAT CATCGA GCGGGAAGAA CTGAAAGAGA TGGTAATCGC
421 TC TCTTCAC GAATCCGAAC TTATACTTTC CGAAGATA G ATTGAAGTAA TGGTGGATAA
481 GGCTITTATC GAAGCAGATC GCAAGAACGA TGGGAGAATT GATATAGATG AATGGAAAGA
541 T TCGTGTCC AAGAATCCGT CGCTCATCAA AAACATGACT T GCCGTATC TAAAGGACAT
601 AAACGGGGCG TTTCCAAGTT TTGTTTCATC TTGTGAAGAC GATGAATTGG AGTTGCAAAA
661 ACTAAAT TT Τ Α are according to SEQ ID NO:10 sequence retrievals are analyzed, SEQ ID NO:10 be ThCBL-4 full length sequence.According to
SEQ ID NO:10 sequences Design pair of primers are as follows：
ThCBL-4F: SEQ ID NO: 1 1：
ATGGGCTGCTCTCCGTCGAAG
ThCBL-4R:SEQ ID NO: 12：
TTAAAAATTTAGTTTTTGCAAC AP: SEQ ID NO: 13：
GGCCACGCGTCGACTAGTACTTTTTTTTTTTTTTTTT passes through SEQ ID NO:11 and SEQ ID NO:12 clone ThCBL-4 complete encoding sequences.
Slight salt mustard RNA is extracted, with primer SEQ ID NO:13 be reverse transcription primer, obtains the PfuUltra II Fusion HS DNA Polymerase that slight salt mustard cDNA use Stratagene, and performing PCR reaction is entered by template of the cDNA of slight salt mustard.50 μ PCR reaction systems：5 μ 1 10 X PfuUltra II reaction Buffer, 0.5 μ 1 25 mM dNTP, 10 μM of the PfuUltra II Fusion HS DNA Polymerase of 2.0 μ 1 cDNA, 1.0 μ 1 primer SEQ ID NO:11 and SEQ ID NO:12 each 2.0 μ 1, and 37.5 μ distilled water.PCR is anti-
Answer condition：95 °C of pre-degenerations 2 minutes, 35 circulations（95 °C are denatured 25 seconds, and 51 °C are annealed 25 seconds, and 72 °C extend 30 seconds), 72 °C extend 5 minutes.
Pcr amplification product adds A tails：PCR primer moisturizing first takes out one time with chloroform and removes removing protein to 400 μ 1, draws supernatant and adds the μ 1 of 3 Μ sodium acetate solutions 40, plus 2 times of absolute ethyl alcohol, -20 °C are placed 10 minutes, centrifugation, supernatant is removed, is dried, is dissolved with the distilled waters of 21 μ 1.Add 2.5 μ 1 Ι Ο Χ Ε χ Buffer, 0.5 μ 15 mM dATP, 1.0 μ Ex Taq.Reaction condition：70 °C are reacted 30 minutes.The DNA fragmentation for obtaining about 2.8Kbp is reclaimed（Omega QIAquick Gel Extraction Kits）, it is connected on pGEM T-easy carriers（Obtain 7 d-$-pGEM plasmids）, JM109 is then converted, random 6 white colonies of picking are inoculated in the LB fluid nutrient mediums containing 50 g/mL ampicillins and cultivated respectively, and glycerol adding is to final concentration 20% after 37 °C of overnight incubations, and -80 °C save backup.With primer SEQ ID NO:11 and SEQ ID NO:12 carry out bacterium solution PCR amplifications（Reaction system and reaction condition are ibid）, 6 positive colonies are obtained, Invitrogen is delivered to（Shanghai）Trade Co., Ltd is sequenced, and sequence is SEQ ID NO:2, the amino acid sequence of its albumen encoded is SEQ ID NO: 1.
The amino acid sequence of CBL-4 albumen： SEQ ID NO: 1
1 MGCSPSKRKK ATRPPGYEDP NLLASATPFT VAEVEALYEL FMKLSSSIID DGLIHKEEFQ 61 LALFRNRNR NLFADRIFDV FDVKRNGVIE FGEFVRSLGV FHPDASVHEK IKFAF LYDL 121 RQTGFIEREE LKE VIALLH ESELILSEDM IEVMVDKAFI EADRKNDGRI DIDEWKDFVS 181 KNPSLIKNMT LPYLKDIKGA FPSFVSSCED DELELQKL F
The nucleotide sequence of 7M L-$ encoding genes： SEQ ID NO: 2 1 ATGGGCTGCT CTCCGTCGAA GAGGAAGAAG GCAACACGAC CGCCGGGATA TGAGGATCCT
61 AACCTTCTCG CCTCTGCTAC GCCAT CACG GTAGCAGAAG TGGAGGCTTT GTATGAACTG
121 TTCATGAAGC TAAGCAGTTC AA TATCGAC GACGGTC TA TTCATAAGGA AGAATTTCAG
181 CTGGCTTTAT TCAGAAATAG GAATCGGAAG AACC I TCG CTGATCGGAT ATTTGATGTA
241 TTTGATGTGA AGCGAAATGG AGTGATCGAG TTTGGGGAAT T GTCCGGTC TTTAGGTGTC 301 TTCCATCCAG ATGCATCTGT CCATGAAAAA ATCAAATTTG CITTCAAGTT GTACGAT TA
361 CGGCAAACTG GAT CATCGA GCGGGAAGAA CTGAAAGAGA TGGTAATCGC TCTTCTTCAC
421 GAATCCGAAC TTATACTTTC CGAAGATATG ATTGAAGTAA TGGTGGATAA GGCTITTATC
481 GAAGCAGATC GCAAGAACGA TGGGAGAATT GATATAGATG AATGGAAAGA T TCG GTCC
541 AAGAATCCGT CGCTCATCAA AAACA GACT T GCCGTATC TAAAGGACAT AAAGGGGGCG 601 TTTCCAAGTT TTGTTTCATC TTGTGAAGAC GATGAATTGG AGTTGCAAAA ACTAAATTTT
661 ΤΑΆ
(^-$ gene plant the expression vector establishments of embodiment 37
Plant binary expression vector pCAMBIA2300 is selected (to be purchased from Beijing DingGuo ChangSheng Biology Technology Co., Ltd）As plant expression vector, the 35S promoter that Ν Ρ Τ Π genes contain double enhancers is replaced with Pnos promoters, to reduce expression of the Ν Ρ Τ Π albumen in plant.Selection contains 35S promoter and terminator the Tnos promoter and terminator respectively as ThCBL-4 genes of double enhancers.
With primer SEQ ID NO:14 and SEQ ID NO:15 (are purchased from ocean Science and Technology Ltd. of Beijing China with plant expression vector pBI121）For template amplification Pnos, using TaKaRa PrimeSTAR HS archaeal dna polymerases.The Ρ Ο reaction systems of 50 μ 1：10 μ 5 xPS Buffer, 3 μ 2.5 mM dNTP, the μ PrimeSTAR of 1.0 μ ρ Β Ι 121,1.0,10 μ Μ primer SEQ ID Ν Ο:14 and SEQ ID NO:15 each 2.0 μ 1, and 31 μ distilled water.PCR reaction conditions：94 °C of pre-degenerations 5 minutes, 33 circulations（94 °C are denatured 30 seconds, and 56 °C are annealed 30 seconds, and 72 °C extend 30 seconds）, 72 °C extend 10 minutes.PCAMBIA2300 (Promega, T4 ligase box) is connected to as the PCR primer by obtained by after EcoRI, Bglll digestion and obtains pCAMBIA2300-l.
SEQ ID NO: 14 ：
GCACGAATTCATACAAATGGACGAACGGAT SEQ ID NO: 15：
ATCCAGATCTAGATCCGGTGCAGATTATTTG
SEQ ID NO:16 standing grain P SEQ ID NO:17 using pBI121 as template amplification Tnos, using TaKaRa PrimeSTAR HS archaeal dna polymerases.50 μ PCR reaction systems： 10 μΐ 5 ><PS Buffer, 3 μ 2.5 mM dNTP, 1.0 μ pBI121,1.0 μ Prime STAR, 10 μ Μ primer SEQ ID NO:16 standing grain P SEQ ID NO:17 each 2.0 μ 1, and 31 μ distilled water.PCR reaction conditions：94 °C of pre-degenerations 5 minutes, (94 °C are denatured 30 seconds, and 58 °C are annealed 30 seconds, and 72 °C extend 30 seconds for 33 circulations）, 72 °C extend 10 minutes.PCAMBIA2300-lCPromega T4 ligase boxes are connected to as the PCR products by obtained by after Kpnl, EcoRI digestion）Obtain pCAMBIA2300-2.
SEQ ID NO: 16：
CGGGGTACCGAATTTCCCCGATCGTTCAAA SEQ ID NO: 17：
TCAGAATTCCCAGTGAATTCCCGATCTAGTA
SEQ ID NO:18 and SEQ ID NO:19 using pCAMBIA2300 plasmids as template amplification arabidopsis 35S promoters.Using TaKaRa PrimeSTAR HS archaeal dna polymerases.50 μ Ι Ρ Ο reaction systems：10 μ 5 xPS Buffer, 3 μ 2.5 mM dNTP, 1.0 μ dilute 50 times of pCAMBIA2300 plasmids, 1.0 μ PrimeSTAR, 10 μ Μ primer SEQ ID NO:18 standing grain P SEQ ID NO:19 each 2.0 μ 1, and 31 μ distilled water.PCR reaction conditions：94 °C of pre-degenerations 5 minutes, (94 °C are denatured 30 seconds, and 50 °C are annealed 30 seconds, and 72 °C extend 30 seconds for 33 circulations；), 72 °C extend 10 minutes.It is connected to as the PCR primer by obtained by after HindIII, Xbal digestion（Connection method is ibid）PCAMBIA2300-2 obtains pCAMBIA2300-3 SEQ ID NO: 18：
ACTAAGCTTATGGTGGAGCACGACACTCT
SEQ ID NO : 19：
GCTCTAGAAGAGATAGATTTGTAGAGAGAGAC
SEQ ID NO:20 and SEQ ID NO:(template is the positive ThCBL-4-pGEM plasmids that embodiment 2 is obtained to 21 amplification ThCBL-4）, using Stratagene PfuUltra II Fusion HS DNA Polymerase.50 μ PCR reaction systems：5 μ l O PfuUltra II reaction Buffer, 0.5 μ 1 25 mM dNTP, 2.0 μ ThCBL-4-pGEM plasmids, the μ Μ of 1.0 μ PfuUltra II Fusion HS DNA Polymerase 10 primer SEQ ID NO:20 and SEQ ID NO:21 each 2.0 μ 1, and 37.5 μ distilled water.PCR reaction conditions：95 °C of pre-degenerations 2 minutes, (95 °C are denatured 25 seconds, and 52 °C are annealed 25 seconds, and 72 °C extend 30 seconds, and 72 °C extend 5 minutes for 35 circulations.Connected as the PCR primer by obtained by after Xbal, Kpnl digestion（Connection method is ibid）Arrive
PCAMBIA2300-3, obtains the S-7 CAL-$-2300 of plant expression vector 35.
SEQ ID NO: 20：
AATCTAGAATGGGCTGCTCTCCGTCGAAG SEQ ID NO : 21：
The 35S-ThCBL-4-23 expression vectors of GCGGTACCTTAAAAATTTAGTTTTTGCAAC embodiments 4 convert Agrobacterium
The preparation of Agrobacterium LBA4404 (being purchased from Biovector Science Lab, Inc) competent cell：Agrobacterium LBA4404 was drawn to single spot inoculation on the LB solid mediums containing 50 g/ml rifampins and 50 g/ml streptomysins in 1-2 days in advance, 28 °C are cultivated 1 to 2 day.Picking single bacterium colony is inoculated in 5 ml containing 50 μ§The rifampins of/ι η 1 and 50 μ§Overnight incubation (about 12-16 hours) is shaken in the LB fluid nutrient mediums of the streptomysins of/ι η 1, under 28 °C to OD6(K)It is worth for 0.4, formation seed bacterium solution.Take the bacterium solution after 5 ml activation（1 :20 ratio）In the LB fluid nutrient mediums for being inoculated in the same concentration antibiotic of 100 ml, 28 °C of shakes cultivate 2-2.5 hours to OD6(K)=0.8.Ice bath bacterium solution 10 minutes, shook up once every 3 minutes, made bacterium even into resting state.Centrifuged 10 minutes in 4000 g under 4 °C, abandon supernatant；Add 4000 g under 10% (v/v) glycerine resuspension thalline of 10 ml ice precoolings, 4 °C to centrifuge 10 minutes, collect precipitation；Ice-cold 10% (v/v) glycerine repeats to wash 3-4 times；Adding 10% (v/v) glycerine of 4 ml ice bath precoolings, suspended bacterial is precipitated again, is dispensed, is saved backup in -70 °C with 40 μ/pipe.
Convert Agrobacterium：Melt competent cell on ice, the positive 35S-7 d-$-2300 plasmids of gained in 1 μ embodiments 3, ice bath about 10 minutes after mixing are added into 40 μ competent cell.The mixture of the competent cell and DNA is transferred in the electric shock of ice precooling cup with liquid-transfering gun, rapping makes suspension reach bottom, has been careful not to bubble.To be shocked by electricity cup（Purchased from Bio-Rad) it is put on the slideway of electroporation chamber, promote slideway to put electric shock cup to electroporation chamber
At base electrode.Using the electric shock cup of 0.1 cm specifications, MicroPuMUer (being purchased from Bio-Rad) program is set to " Agr ", and electric shock is once.Electric shock cup is taken out immediately, adds the l ml LB culture mediums of 28 °C of preheatings.It is quickly soft to be beaten cell with liquid-transfering gun.Suspension is transferred to 1.5 ml centrifuge tube, under 28 °C, 225 rpm are cultivated 1 hour.100-200 μ bacterium solution is taken to be coated on corresponding resistance screening culture medium flat plate（LB solid mediums, containing 50 μ§The rifampins of/ι η 1,50 μ§The streptomysins of/ι η 1,50 μ§The kanamycins of/ι η 1）, 28 °C of cultures.Positive transformants clone is screened, and its bacterium solution is saved backup in -70 °C.Embodiment 5 obtains transgenic arabidopsis using Agrobacterium-medialed transformation method
Plant culture to be transformed：Arabidopsis seed（Colombia's type, the arabidopsis Biological Resource Center from Ohio State Univ-Columbus USA）Sowing is in peat soil, after 4 °C of low-temperature treatments 3 days, is placed in 23 °C, germinates in the dark incubator in 16 hours illumination/8 hour.It is transplanted to after 7-10 days equipped with peat soil and vermiculite（Volume ratio 3:1) bore is in 7.5 cm polypots, every 6 plants of pot culture kind is placed in 23 °C, the dark incubator in 16 hours illumination/8 hour and grown.The ml of nutrient solution 40 is poured before transplanting per alms bowl, backsight soil moisture is transplanted and keeps the skin wet in time.In the appropriate nutrient solution of growth period.On demand per 3-4 weeks once（Or the time is longer）.In order to obtain more bud on each plant, first inflorescence is cut off after most of first inflorescence of plant are formed, apical dominance is released, promotes the synchronous appearance of multiple secondary inflorescences.When the cm of most of inflorescences about 1-10 is high（Cut off after first inflorescence 4-8 days）When prepare contaminate.
The culture of Agrobacterium：Take out after the bacterium liquid activation of Agrobacterium positive transformants clone of conservation in embodiment 4, picking Agrobacterium single bacterium colony is inoculated into the sterile LB fluid nutrient mediums of 10 mL（Containing 75 mg/ L rifampins, 100 mg/ L streptomysins and 100 mg/ L kanamycins）, the lower 250 revs/min of shakings incubated overnight of 28 °C of constant temperature.Resulting bacterium solution is inoculated into the 200 mL equally LB fluid nutrient mediums containing above-mentioned antibiotic in 1% -2% (v/v) ratio again, 28 °C of constant temperature shakings make the concentration of Agrobacterium reach OD6(K)=1.8, then centrifuged 15 minutes at 4 °C lower 3000 revs/min, with dip-dye culture medium after abandoning supernatant（The dip-dye culture medium contains 5.0% (w/v) sucrose and 0.05% (500 μ Ι Τ) Silwet L-77) suspend Agrobacterium again, is suspended into OD6QQAbout 0.80.
The dip-dye of inflorescence：The above-mentioned dip-dye culture medium containing Agrobacterium is added in big mouth container, the dip-dye culture medium containing Agrobacterium described in 200-300 mL is added in each cm of bore 9 container to be used to contaminate.Plant is inverted, aerial tissues is fully immersed in agrobacterium suspension 3-5 seconds, and to be gently agitated for.There should be one layer of liquid film after infiltration on plant.The plant contaminated is placed in vinyl disc, with clean plastics or preservative film covering with moisturizing, is then placed within dim light or dark place is stayed overnight, note carefully preventing direct sunlight plant.Remove covering within about 12-24 hours after processing.Normal culture plant, the week of plant further growth 3-5, until silique browning is dried.Seed is harvested, and by seed centrifuge tube in 4 °C of lower dry storages.
Transgenic seed is screened:The aqueous solution containing 1/4 MS a great number of elements is prepared, 0.8 % agar powders is added, is heated to agar with micro-wave oven and dissolves completely, to be cooled to 50 °C or so, the desired amount of final concentration of 50 η ^ are added!/1Kanamycins, per 25 mL are poured into culture dish after shaking up, putting can sow after experimental bench cooled and solidified.Load weighted seed is poured on a plain copying paper, use finger tap copy paper, seed is equably sowed on agaropectin, cover culture dish lid, after putting 4 °C of refrigerator cold treatments 72 hours, move to 23 °C, germinate in the dark incubator in 16 hours illumination/8 hour, resistance seedling is transplanted in Nutrition Soil by periodic statistical germination and growth of seedling situation in time.Backsight soil moisture is transplanted to keep the skin wet in time.In the appropriate nutrient solution of growth period.The g of Arabidopsis leaf 0.1 of growth 20 days is taken, DNA is extracted, with SEQ ID NO: 11：Standing grain P SEQ ID NO:12 amplification ThCBL-4:(50 μ PCR reaction systems：5 μ Ι Ο χ Ε χ Buffer, 3 μ 2.5 mM dNTP, 2.0 μ DNA, 1.0 μ Ex Taq, 10 μ Μ primer SEQ ID NO:11 standing grain P SEQ ID NO:12 each 2.0 μ 1, and 35 μ distilled water.PCR reaction conditions：94 °C of pre-degenerations 5 minutes, 33 circulations（94 °C are denatured 45 seconds, and 51 °C are annealed 45 seconds, and 72 °C extend 30 seconds）, 72 °C extend 7 minutes）, the PCR plant for being accredited as the positive are numbered（), T1K1-T1K12 and preserve.Embodiment 6 is overexpressed the sterilized vermiculite of drought-enduring simulated experiments and Function Identification of the ThCBL-4 transgenic arabidopsis T1 for plant and is impregnated with 1/2MS culture mediums.T1K1-T1K6 and control arabidopsis seed are sowed on vermiculite respectively, and 10 seeds, 25 °C, optical culture/14 hour light culture circulation in 10 hours are sowed per basin, pour a 1/2MS within every 7 days, after culture 20 days, per 4 more consistent seedlings of basin reservation size, for arid experiment.Transgenic arabidopsis, control arabidopsis arid 14 days（Do not water), 25 °C, optical culture/14 hour light culture circulation in 10 hours.T1 is for the transfer-gen plant (plant that T0 grows up to for the seed of transfer-gen plant）Identification of Drought show that adjoining tree is all wilted seriously, and totally 26 plants of six strains of T1K1, T1K2, T1K3, T1K4, T1K5, T1K6（Per each 4-5 of strain）25 plants can survive and continued growth shows obvious drought tolerance in arabidopsis（Referring to Fig. 3 a and 3b, by taking T1K2 as an example, T1K1, T1K3, T1K4, T1K5, T1K6 result are similar with T1K2, are not shown here）.Embodiment 7 verifies 7 CB-$ protein expressions on transcriptional level
Control Arabidopsis plant, drought-enduring transgenic arabidopsis T1 are taken respectively for plant（Be belonging respectively to the Κ 2 of T1K1, Τ 1,
The Κ 3 of Τ 1, the Κ 4 of Τ 1, the Κ 5 of Τ 1, six strains of T1K6）Not drought-enduring transgenic arabidopsis Tl uses plant RNA extraction kit for each 0.05 g of the arid blade of 10 days of plant（Invitrogen) the total serum IgE extracted.Total serum IgE is determined in 260 nm and 280 nm absorbance with the ultraviolet specrophotometer U-2001 of HITACHI companies, calculates each RNA concentration.Method shown in neat Ll boxes Superscript III Reverse Transcriptase, which is tried, according to Invitrogen reverse transcriptions carries out reverse transcription（2 total serum IgEs are used as template, reverse transcription primer SEQ ID NO: 13 ).Pass through SEQ ID NO:11 and SEQ ID NO:12 amplification T CBL-4, detect albumen relative expression's situation.
Using TaKaRa PrimeSTAR HS archaeal dna polymerases, performing PCR reaction is entered by template of the cDNA of reverse transcription.The Ρ Ο reaction systems of 50 μ 1：10 μ 5xPS Buffer, 3 μ 2.5 mM dNTP, 2.0 μ cDNA, 1.0 μ PrimeSTAR 10 μ Μ primer SEQ ID NO:16 standing grain Ρ SEQ ID NO:17 each 2.0 μ 1, and
30 μ distilled water.PCR reaction conditions：94 °C of pre-degenerations 5 minutes, 29 circulations（94 °C are denatured 45 seconds, and 51 °C are annealed 45 seconds, and 72 °C extend 45 seconds）, 72 °C extend 10 minutes.
Product electrophoresis result is as shown in Figure 4：M is DNA Ladder Marker (DL2000, TakaRa), and 1-7 is drought-enduring transgenic arabidopsis Tl for plant（It is followed successively by：The Κ 1 of Τ 1, the Κ 2 of Τ 1, the Κ 3 of Τ 1, the Κ 4 of Τ 1, the Κ 5 of Τ 1, the Κ 6 of Τ 1, T1K7), 8-11 is not drought-enduring 1 generations of transgenic arabidopsis Τ plant, and 12-15 compares for non-transgenic arabidopsis.The size of PCR primer electrophoretic band shown in figure is in the same size with 7 CAL-$'s（About 660bp).As a result show, control arabidopsis does not have J J-$ transcriptions, drought-enduring transgenic arabidopsis T1 is stronger for the transcription of ThCBL-4 in plant, not drought-enduring transgenic arabidopsis T1 transcribes very weak for plant.
Claims (1)
- Claims1. a kind of calcium adjusting phosphatase B analogy albumen of small salt mustard, its sequence is SEQ ID NO: 1.2. encode SEQ ID NO:The gene of 1 calcium adjusting phosphatase B analogy albumen, preferably its sequence are SEQ ID NO: 2.3. a kind of recombinant expression carrier, it contains the gene described in claim 2, and the nucleotide sequence of the gene is operably connected with the expression control sequence of the carrier is carrier for building the recombinant expression carrier；Preferably, the carrier is carrier is pCAMBIA2300.4. the recombinant expression carrier described in claim 3, it is 35 S-J J-$-2300 carriers shown in accompanying drawing 2.5. a kind of recombinant cell, it contains the recombinant expression carrier described in gene or Claims 2 or 3 described in claim 2；Preferably, the recombinant cell is restructuring agrobatcerium cell.6. a kind of method of improvement drought resistance in plants, including：Recombinant expression carrier described in gene or claim 3 or 4 described in claim 2 is imported into plant or plant tissue and makes the gene expression；Preferably, the plant is arabidopsis.7. a kind of method of prepare transgenosis plant, including：The plant containing the recombinant expression carrier described in the gene or claim 3 or 4 described in claim 2 or plant tissue are cultivated under conditions of plant is effectively produced.8. the method described in claim 7, wherein the plant is arabidopsis.9. the recombinant expression carrier described in gene, claim 3 or 4 described in claim 2 or the recombinant cell described in claim 4 are used to improve drought resistance in plants and the purposes for plant breeding.10. the purposes described in claim 9, wherein the plant is arabidopsis.
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