CN105838726B - A kind of Salt Tolerance Gene in Alfalfa gene M sCDPK and its coding albumen and application - Google Patents
A kind of Salt Tolerance Gene in Alfalfa gene M sCDPK and its coding albumen and application Download PDFInfo
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Abstract
The invention discloses a kind of Salt Tolerance Gene in Alfalfa gene M sCDPK and its coding albumen and applications, belong to field of plant genetic project technology.MsCDPK gene open reading frame overall length 1650bp disclosed by the invention, encode 549 amino acid, nucleotide sequence is as shown in SEQ.ID.NO.1, utilize nucleotide sequence information, obtain the gene, construct the cloning vector and plant expression vector of the gene, and the gene is transferred in model plant tobacco by agrobacterium-mediated transformation, by transgene tobacco culture on the MS culture medium containing salt component, the result shows that transgenic plant has stronger salt resistance ability compared with the control, illustrate that the expression of MsCDPK gene improves the resistance of plant pair salt, imply that the gene can participate in the salt resistance process of plant.MsCDPK gene in the present invention provides theoretical foundation for research clover salt resistance molecular mechanism and breeding.
Description
Technical field
The invention belongs to field of plant genetic project technology, are related to the acquisition of alfalfa salt resistance related gene MsCDPK,
More particularly to a kind of Salt Tolerance Gene in Alfalfa gene M sCDPK and its coding albumen and application.
Background technique
A kind of important leguminous forage of alfalfa (Medicago sativa L.) as China or even in the world, because of it
Yield is high, herbaceous stem is excellent, it is full of nutrition, adaptable, can be the well-grown on slight salt lick many advantages, such as and by day
Benefit is paid attention to.Zhaodong clover belongs to tetraploid alfalfa, and the main cultivated orages as Northeast Area of China are widely cultivated, and eastern
The backlands area soil salinization is unfavorable for the normal growth and development of crop, seriously constrains the development of farming and animal husbandry.Therefore, it excavates pale reddish brown
Resistant gene of salt in clover furthers investigate the Salt-resistance mechanism of alfalfa, for overcoming the natural conditions of the soil salinization to cause
Breeding issue, be of great significance.
The plant anti-salt research carried out currently with plant gene engineering technology has obtained many progress.Studies have shown that will
In plant or other biological in gene transferred plant relevant to salt resistance, it can be obtained significantly with resistance of the render transgenic plant to salt
It improves.
CDPK(Ca2+- dependent, calmodulin independent protein kinase) it is plant Ca2+ oscillations
Crucial protein kinase in conductive process plays an important role during environment stress signal transduction.CDPKs is widely present
In plant, and each organ is distributed widely in plant.Wherein, there are 34 CDPK family members, rice in arabidopsis
In have 29 CDPK family members, have 20 family members in wheat.Protein structure between each member of CDPK family compares guarantor
It keeps, contains 4 structural domains: the end N variable domain (N-terminal variable domain), serine/threonine kinase domain
(protein kinase domain), inhibit domain (auto-inhibitory domain) and class calmodulin binding domain certainly
(calmodulin-like domain).CDPKs is the important receptor of plant cell Ca2+ oscillations transduction, a variety of by acting on
The interaction substrate of multiplicity participates in adaptation of the plant to drought stress, salt stress and low temperature stress.On a molecular scale, CDPKs
By the activity of the different substrate protein of phosphorylated regulation, and then realize gene expression regulation, ionic equilibrium regulation and active oxygen
Balance regulation, to play respective biological function.When plant is by biology or abiotic stress, a series of signal can be passed through
Transduction causes the physiological reaction of plant.During the signal transduction of plant, CDPK and second messenger's calcium ion (Ca2+) combine,
And then Ca2+Signal is converted into phosphorylation signal, this process does not need the participation of other oroteins.CDPK is signal transduction
Important conservative carrier in access, adverse circumstance signal cause Ca in cytoplasm2+The Ca that concentration variation generates2+Signal passes through activation
Specific CDPK component corresponding with respective signal is experienced, the substrate for acting on CDPK occur phosphorylation, cause various Ca2+Signal
It is downstream transmitted by respective specific access, and then regulates and controls downstream gene expression, regulation biochemical metabolism, ion and moisture cross-film
Transport etc. participates in the biological process responded to signal.
Summary of the invention
The purpose of the present invention is to provide a kind of Salt Tolerance Gene in Alfalfa gene M sCDPK and its coding albumen and applications.
The present invention is to be achieved through the following technical solutions:
The invention discloses a kind of Salt Tolerance Gene in Alfalfa gene M sCDPK, and the nucleotide sequence of resistant gene of salt MsCDPK is such as
Shown in SEQ.ID.NO.1.
The invention also discloses the albumen by above-mentioned Salt Tolerance Gene in Alfalfa gene M sCDPK coding, the amino acid of the albumen
Sequence is as shown in SEQ.ID.NO.2.
The invention also discloses the expression vector for containing above-mentioned Salt Tolerance Gene in Alfalfa gene M sCDPK, which is
Plant expression vector pCBM-MsCDPK.
The invention also discloses the primers for expanding above-mentioned Salt Tolerance Gene in Alfalfa gene M sCDPK overall length or its any segment
It is right.
The invention also discloses application of the above-mentioned Salt Tolerance Gene in Alfalfa gene M sCDPK in plant anti-salt stress.
Construct the plant expression vector of above-mentioned Salt Tolerance Gene in Alfalfa gene M sCDPK, then by constructed plant expression vector
Through Agrobacterium-mediated transformation into Tissues of Tobacco, screening obtains the plant of salt resistance enhancing.
It is coerced the invention also discloses the albumen by above-mentioned Salt Tolerance Gene in Alfalfa gene M sCDPK coding in plant anti-salt
In application.
Compared with prior art, the invention has the following beneficial technical effects:
The present invention obtains the overall length CDS of MsCDPK gene, such as sequence table of the nucleotide sequence of the gene from alfalfa
Shown in SEQ ID NO:1, the amino acid sequence of coding is as shown in sequence table SEQ ID NO:2.MsCDPK provided by the invention
Gene structure specificity analysis shows: MsCDPK gene C DS overall length is 1650bp, which encodes 549 amino acid.By its amino
Acid sequence Blast analysis in NCBI, discovery and MtCDPK (M. truncatula, Sequence ID:ref | XP_003618125.1)
It compares, amino acid identity 95%.MsCDPK gene disclosed by the invention is that research clover salt resistance molecule mechanism and breeding mention
For theoretical foundation.
The invention discloses the cloning vectors for containing above-mentioned alfalfa salt stress responsive genes MsCDPK.And then it constructs
Plant expression vector containing said gene MsCDPK, then constructed plant expression vector is passed through into Agrobacterium-mediated transformation
Into Tissues of Tobacco, screening obtains the plant of salt resistance enhancing.Transgene tobacco is cultivated on the MS culture medium containing salt component,
The result shows that transgenic plant has stronger salt resistance ability compared with the control, genetically modified plants are significantly higher than open country to the resistance of salt
Raw type illustrates that the overexpression of MsCDPK gene promotes transgenic plant to the resistance of salt, illustrates that the gene participates in the anti-of plant
Salt process.The Function Identification of MsCDPK gene helps to disclose the Mechanisms of Salt Resistance of alfalfa, to the genetic breeding of clover, product
Kind improvement is of great significance.
Detailed description of the invention
Fig. 1 is the protein structure domain of MsCDPK；
Fig. 2 is the albumen clustering tree of MsCDPK and other species；
Fig. 3 is that plant expression vector pCBM-CDPK constructs schematic diagram:
Fig. 4 is to turn MsCDPK genetic tobacco seedling leaves PCR detection figure；Wherein, "+" is positive control, and "-" is negative right
According to, " 0 " is blank control, and 1-4 is the purpose band that PCR is obtained, it is consistent with positive band, it is transgenic tobacco plant；
Fig. 5 is the relative expression quantity result that qRT-PCR analysis turns MsCDPK genetic tobacco strain；
Phenotype of the leaf disk that Fig. 6 is MsCDPK transgene tobacco L4 and wild-type tobacco WT under salt stress processing is real
It tests；Wherein, the leaf of CK is 600mmol L-1NaCl Stress treatment 0 day MsCDPK transgene tobacco L4 and wild-type tobacco WT
Disk, the leaf circle for the MsCDPK transgene tobacco L4 and wild-type tobacco WT that 6d is 600mmol L-1NaCl Stress treatment 6 days
Piece；
Fig. 7 is MsCDPK transgene tobacco L4 and wild-type tobacco WT in 600mmol L-1NaCl Stress treatment 0 day and 6
The relative chlorophyll content of tobacco leaf after it；
Fig. 8 is MsCDPK transgene tobacco L4 and wild-type tobacco WT in 600mmol L-1NaCl Stress treatment 0 day and 6
The relative conductivity of tobacco leaf after it.
Specific embodiment
The new resistant gene of salt MsCDPK of one kind disclosed by the invention, the gene are the specific phases obtained in the alfalfa
It is as follows to close experiment:
1, the acquisition of alfalfa MsCDPK gene
(http://www.ncbi.nlm.nih.gov/) is searched for and is downloaded known wheat in ncbi database
TaCDPK sequence text file, from M. truncatula Genome Project website (http://medicago.jcvi.org/
Medicago/ the database for) downloading M. truncatula is applied and is carried out in the ncbi-blast-2.2.30+ software of the website NCBI downloading
Local Blast obtains M. truncatula CDPK gene order.Utilize the primers, primer are as follows:
MtCDPK-F:5'-CAGAGGCGATGGGCAATACA-3'；
MtCDPK-R:5'-GCCACGAGACTAATGAGCAC-3'.
Detailed process is as follows:
1) extract RNA: it is total to extract alfalfa spire using RNAprep Pure plant total RNA extraction reagent box (Tiangeng)
RNA；
2) it obtains cDNA: using the RNA of extraction as template, obtaining cDNA using high efficiency Reverse Transcriptase kit (TOYOBO),
And MsCDPK full length sequence is expanded by template RT-PCR of this cDNA；
3) PCR reacts: carrying out PCR reaction, response procedures with Ex Taq archaeal dna polymerase (TaKaRa) are as follows:
4) PCR product is connected to V site EcoR in pMD18-T (TaKaRa) carrier；
5) (Bo Shi biotech firm) is sequenced into Escherichia coli TOP10 in recombinant plasmid transformed；
6) final identification obtains the full length sequence of MsCDPK gene ORF.MsCDPK full length gene 1650bp, the gene are compiled
549 amino acid of code.By its amino acid sequence, Blast is analyzed in NCBI, is found compared with MtCDPK, amino acid identity is
95%, have the conserved domain of CDPK, as shown in Figure 1.Fig. 2 is the albumen clustering tree of MsCDPK and other species.
2, the building of alfalfa MsCDPK gene plant expression vector
1) with the CDS sequence of MsCDPK in the present invention, design primer is as follows:
MsCDPK-F:5'-ATGGGCAATACATGTCGTGG-3'；
MsCDPK-R:5'-CTAATGAGCACTTGATGCGTCCC-3'.
Using alfalfa cDNA as template, MsCDPK gene order is expanded, response procedures are as follows:
PCR product low-temperature preservation is spare.
2) it is produced with TaKaRa MiniBEST Agarose Gel DNA Extraction Kit (TaKaRa) recycling PCR
The PCR product of recycling is connect by object with pMD18-T carrier, and connection product converts competent escherichia coli cell TOP10, picking
Conversion positive colony extracts plasmid and send sequencing, determines that correct positive colony is used for subsequent experimental by sequencing.
3) double digestion carried out to pCBM vector plasmid with BamH I and Pst I simultaneously, and with TaKaRa MiniBEST
Agarose Gel DNA Extraction Kit (TaKaRa) recycles pCBM carrier segments, while with I pair of enzyme of BamH I and Pst
The carrier T plasmid for being connected with MsCDPK gene is cut, and recycles MsCDPK gene.The MsCDPK genetic fragment of recycling and pCBM are carried
The connection of body segment, then converts competent escherichia coli cell TOP10 afterwards, obtains recombinant clone.It detects and mentions by bacterium solution PCR
It by correct clone designation is pCBM-MsCDPK after taking the digestion of plasmid to identify, carrier figure is as shown in Figure 3.
3, Agrobacterium-mediated transformation tobacco
1) culture of tobacco
Nicotiana gossei SR-1 30 to 40, seed be soaked in 75% alcohol 3min, later with sterilizing distilled water flushing 5
To 6 times, then seed is soaked in 10% sodium hypochlorite stoste 15min and is planted seed with distilled water flushing 5 to 6 times of sterilizing
It trains in MS solid medium.When seed germination and growth to seedling, transplants seedlings into the big bottle equipped with MS solid medium and continues to cultivate,
Its stand-by blade carries out infecting experiment.
2) preparation of Agrobacterium bacterium solution
- 80 DEG C of picking freeze containing pCBM-MsCDPK plasmid Agro-Bacterium EHA105 glycerol stock in YEB+50mg/L Rif
Activation culture on the solid medium of+50mg/L Kan+50mg/L Str, and culture 36-48h is inverted in 28 DEG C of incubators,
The Agrobacterium single colonie that activation obtains is inoculated in the YEB fluid nutrient medium of identical component, 28 DEG C of shaken cultivation about 16-
24h.As OD600=0.4-0.6, takes out in YEB fluid nutrient medium of the culture solution according to 1:10 inoculation identical component and carry out two
Bacterium solution is poured into the centrifuge tube of sterile 50ml as OD600=0.4-0.6 by secondary activation, under the conditions of 4 DEG C, 5000rpm
It is centrifuged 10min, discards supernatant liquid, thallus is resuspended with MS culture solution, is used for subsequent transformation.
3) tobacco is infected
In superclean bench, the Agrobacterium bacterium solution of resuspension is poured into sterile petri dish, by the tobacco SR-1 leaf of culture
Piece is switched to the rectangular of 0.8mm, is placed in bacterium solution as explant and impregnates 8min, therebetween constantly concussion.It takes out explant and is placed in nothing
Attachment bacterium solution is sucked on bacterium filter paper, blade back is inoculated in the culture medium MS of evoked callus formation up1(MS+2.0mg/L
6-BA+0.2mg/L NAA), under the conditions of 25 ± 2 DEG C after dark culture 48-72h, by MS1In blade move to MS2(MS+2.0mg/
L 6-BA+0.2mg/L NAA+1.1mg/L PPT+500mg/L Cef) screening and culturing is carried out on culture medium, every 20d replacement is primary
Induced medium.When the blade edge infected grows new young shoot, cuts young shoot and put to MS3(MS+ 1.3mg/L PPT+
500mg/L Cef) culture of rootage is carried out in culture medium, to subsequent Molecular Identification.
4) extraction of transgene tobacco DNA
1. weighing plant sample 0.1g, it is placed in 1.5ml centrifuge tube；
2. liquid nitrogen grinding is added, 2 × CTAB Buffer buffer of 65 DEG C of 700 μ l preheatings is added into pipe, it is soft mixed
Even, 65 DEG C of water-baths 45min, every 5min are soft to be mixed by inversion；
3. centrifuge tube taking-up is cooled to room temperature, 13000rpm room temperature is centrifuged 10min；
4. take supernatant, be added with the isometric Tris- balance phenol of supernatant and chloroform mixed liquor (Tris- balance phenol and
Chloroform volume ratio is 1:1) it is mixed by inversion, 4 DEG C, 13000rpm is centrifuged 10min；
5. taking supernatant, the chloroform-isoamyl alcohol isometric with supernatant is added into pipe, and (chloroform and isoamyl alcohol volume ratio are
It 24:1) mixes gently, 4 DEG C, 13000rpm is centrifuged 10min；
6. taking supernatant, the anhydrous second of 1/10 volume 3mol/L sodium acetate (pH5.2) of supernatant and the pre-cooling of 2 times of volumes is added
Alcohol (or isometric isopropanol), gently shakes up to there is flocculent deposit, is placed in -20 DEG C of alcohol precipitation 30min；
7. 4 DEG C, 13000rpm is centrifuged 15min, liquid is discarded supernatant；
8. 70% ethanol washing of 500 μ l pre-cooling is added, 4 DEG C, 13000rpm is centrifuged 10min, discards supernatant liquid, and air is dry
It is dry；
9. 20 μ l deionized waters are added sufficiently to dissolve.
4, turn the acquisition of MsCDPK genetic tobacco transformant and the PCR detection of transformant
MsCDPK gene internal primer needed for PCR identification is as follows:
MsCDPK GI plant F:5 '-TGCTGTTCTTTCTCGTCTCA-3 '
MsCDPK GI plant R:5 '-TCAATCCTTCCATCGTTATCT-3 '
Response procedures are as follows:
Referring to fig. 4, PCR amplification is carried out with MsCDPK gene internal primer, the template of 1-4 is the PPT resistance screening extracted
The DNA of tobacco plant out, agarose gel electrophoresis results show that number 1-4 is that the purpose band that PCR is obtained is consistent with the positive
(the target dna band that can amplify about 500bp) is transgenic plant.Wherein, "+" is positive control (i.e. with pCBM-
MsCDPK expression vector plasmid is the PCR testing result of template), "-" is negative control (i.e. with WT lines genomic DNA
For the PCR testing result of template), " 0 " is blank control, and " M " is marker (DL2000).
5, turn the qRT-PCR detection of MsCDPK genetic tobacco strain
To probe into transcription situation of the target gene in transgenic line, extract the total serum IgE of transgenic plant, reverse transcription at
CDNA is subsequently used for qRT-PCR analysis, the transcriptional level of detection MsCDPK gene in transgenic plants.Referring to Fig. 5, as a result
Prove that transgenic plant has the corresponding transcription product of purpose gene, and WT lines are then without transcription product.
6, the verifying of transgenic tobacco plant function
1) 600mmol L is prepared-1NaCl culture solution
2) the identical wild type of growth conditions and transgenic tobacco leaf are taken, is punched with the punch of 7mm diameter, uses NaCl
Culture solution Stress treatment 6 days, observation blade variation was recorded and is taken pictures.No matter transgene tobacco or wild-type tobacco, salt coerce
Under compeling, with the increase of NaCl processing number of days, it is also gradually serious that blade is damaged degree, but on the whole, transgene tobacco leaf
Piece is damaged degree and is weaker than control.Through 600mmol L-1After NaCl is handled 6 days, WT strain blade bleaches, transgenic line
Blade bleach it is several less, referring to Fig. 6.This illustrates that expression of the MsCDPK gene in tobacco can improve tobacco pair to a certain extent
The resistance of salt stress.
3) above-mentioned 6 days tobacco leafs of Stress treatment are taken, chlorophyll is extracted, measures its chlorophyll content.
Experimental method is as follows:
1. 0.1g tobacco leaf is taken to be put into the mortar of pre-cooling, into mortar plus 80% acetone of 1ml pre-cooling, addition are a small amount of
Quartz sand is ground；
2. the liquid that grinding obtains is transferred in big centrifuge tube, it is with 80% acetone washing mortar until colourless；
3. 4 DEG C, 10000rpm, being centrifuged 5min；
4. taking supernatant and the record amount of collecting；
5. 80% acetone is added into precipitating, washing precipitating, then be centrifuged, until precipitating colourless (the record amount of collecting).Note: it receives
Take the centrifuge tube dark treatment of supernatant.
6. sample measures: 3ml supernatant being taken to survey light absorption value (zeroing of 80% acetone) at 645nm and 663nm；
7. result calculates:
Ca=12.72A663-2.59A645
Cb=22.88A645-4.67A663
CTen b of a=20.21A645+8.02A663
Chlorophyll content (mgg-1Or mgdm-2)=C × V/A × 1000
In formula: C is chlorophyll concentration (mgL-1)；V is extracting solution total volume (ml)；A is sampling fresh weight (g).
Transgenic plant and WT lines after 600mM NaCl Stress is handled 6 days, chlorophyll content all substantially under
It adjusts.However, the fall of transgenic plant chlorophyll content is significantly less than the fall of WT lines chlorophyll content,
Referring to Fig. 7.
4) it takes the same period and the identical wild-type tobacco of growth conditions and the blade at the same position of transgene tobacco is placed in
In 600mmol L-1NaCl culture solution, Stress treatment 6 days, it is as follows to survey its relative conductivity experimental method:
1. stand-by beaker, graduated cylinder and triangular flask are scrubbed clean, then distilled water and deionized water flushing 3 times are used respectively, most
Deionized water balance 12h is used afterwards；
2. treated blade is rinsed 3 times with deionized water, filter paper sucks blade surface moisture, then with 9mm diameter
Punch punches on blade, and the leaf disk of acquisition is put into the triangular flask that deionized water balance is crossed, and 20ml deionization is added
Water, each processing set 3 repetitions；
3. being vacuumized 15min, its conductivity is surveyed with conductivity meter, record numerical value is S1, is then put into triangular flask
20min is heated in boiling water bath, is cooled to room temperature, and surveys its conductivity with conductivity meter, record numerical value is S2.
Relative conductivity (L) calculation formula is as follows: L=S1/S2 × 100%
After 600mM NaCl Stress is handled 6 days, relative conductivity is all obviously mentioned for transgenic plant and WT lines
It is high.However, the ascensional range of transgenic plant relative conductivity is significantly less than the ascensional range of WT lines relative conductivity,
Referring to Fig. 8.
In conclusion the invention discloses alfalfa salt stress responsive genes MsCDPK and its coding albumen and application,
Belong to gene engineering technology field.Gene open reading frame overall length 1650bp disclosed by the invention, encodes 549 amino acid.Core
Nucleotide sequence is as shown in SEQ ID NO:1, using nucleotide sequence information, obtains the gene, constructs the cloning vector of the gene
And plant expression vector, and the gene is transferred in model plant tobacco by agrobacterium-mediated transformation, transgene tobacco is trained
It supports on the MS culture medium containing salt component, the results showed that transgenic plant has stronger salt resistance ability, explanation compared with the control
The expression of MsCDPK gene improves the resistance of plant pair salt, implys that the gene can participate in the salt resistance process of plant.This hair
Bright MsCDPK gene provides theoretical foundation for research clover salt resistance molecular mechanism and breeding, and the Function Identification of MsCDPK gene has
Help disclose the Mechanisms of Salt Resistance of alfalfa, to the genetic breeding of clover, breed improvement is of great significance.
Claims (3)
1. a kind of application of Salt Tolerance Gene in Alfalfa gene M sCDPK in plant anti-salt stress, which is characterized in that the pale reddish brown lucerne
The nucleotide sequence of Mu resistant gene of salt MsCDPK is compiled as shown in SEQ ID NO:1 by the Salt Tolerance Gene in Alfalfa gene M sCDPK
The amino acid sequence of the albumen of code is as shown in SEQ ID NO:2.
2. application as described in claim 1, which is characterized in that building contains Salt Tolerance Gene in Alfalfa gene described in claim 1
The plant expression vector of MsCDPK, then constructed plant expression vector is passed through into Agrobacterium-mediated transformation to Tissues of Tobacco
In, with 600mmol L-1NaCl culture solution Stress treatment 6 days, screening obtain the plant of salt resistance enhancing.
3. a kind of application of albumen of Salt Tolerance Gene in Alfalfa gene M sCDPK coding in plant anti-salt stress, which is characterized in that
The nucleotide sequence of the Salt Tolerance Gene in Alfalfa gene M sCDPK is as shown in SEQ ID NO:1, the Salt Tolerance Gene in Alfalfa gene
The amino acid sequence of the albumen of MsCDPK coding is as shown in SEQ ID NO:2.
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| CN109628466A (en) * | 2019-01-07 | 2019-04-16 | 西北农林科技大学 | A kind of alfalfa salt tolerance and drought resistance gene MsCKX and its application |
| CN110106184A (en) * | 2019-04-11 | 2019-08-09 | 西北农林科技大学 | The clone of alfalfa salt tolerance and drought resistance gene MsBBX a kind of and application |
| CN114790449B (en) * | 2022-05-11 | 2023-08-01 | 新疆农业科学院核技术生物技术研究所（新疆维吾尔自治区生物技术研究中心） | Application of calpain gene GhCPK4 in verticillium resistance of plants |
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| CN102108364A (en) * | 2009-12-24 | 2011-06-29 | 上海市农业科学院 | Salt-resistant and drought-resistant CPK (creatine phosphokinase) protein kinase gene derived from arabidopsis thaliana |
Non-Patent Citations (3)
| Title |
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| CDPK gene expression in salt tolerant rolB and rolC transformed cell cultures of panax ginseng;Konstantin V Kiselev et al.;《Biologia Plantarum》;20101231;第54卷(第4期);第621-630页 * |
| Functional characterisation of OsCPK21, a calcium-dependent protein kinase that confers salt tolerance in rice;Takayuki Asano et al.;《Plant Mol Biol》;20101207;第75卷;摘要 * |
| XM_003618077.1;Young,N.D.et al.;《GenBank》;20111117;FEATURES，Origin * |
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