CN103555740A - Wheat CBL-CIPK (CBL-interacting protein kinase) stress tolerance regulatory factor as well as encoding gene and application thereof - Google Patents
Wheat CBL-CIPK (CBL-interacting protein kinase) stress tolerance regulatory factor as well as encoding gene and application thereof Download PDFInfo
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Abstract
The invention relates to a wheat CBL-CIPK (CBL-interacting protein kinase) stress tolerance regulatory factor as well as an encoding gene and an application thereof. According to the wheat CBL-CIPK stress tolerance regulatory factor, protein kinase genes which are consciously induced by salt and drought when being expressed in roots and leaves are selected according to expression profile chip information of salt and drought stress of wheat, and a cDNA (complementary deoxyribonucleic acid) sequence with a whole-length encoding area is cloned, wherein the nucleotide sequence is as shown in SEQ ID NO.1. Analysis shows that the gene can encode CBL-CIPK serine/threonine protein kinase, the amino acid sequence is as shown in SEQ ID NO.2, and is named as TaCIPK33. Transgenic function analysis shows that the gene has the functions of improving plant salt tolerance and drought resistance. Basis is provided for further understanding molecular mechanisms of wheat in responding to drought and salt stress, and meanwhile the gene can be applied to genetic improvement of novel stress tolerance germplasms of crops such as wheat.
Description
Technical field
The present invention relates to the degeneration-resistant regulatory factor of grow wheat CBL-CIPK class and encoding gene and an application, belong to Protocols in Molecular Biology and gene engineering technology field.
Background technology
Wheat is the main food of about 35% world population.The adverse circumstances such as arid, salinification are the important environmental factorss of restriction yield and quality of wheat, and easily cause infecting disease and pest, cause larger financial loss.But wheat cannot be escaped adverse circumstance environment, therefore, clone drought resisting, resistant gene of salt, study the molecular mechanism of its drought resisting, salt tolerant, cultivate drought resistance and salt tolerance new variety and there is great theory and practice meaning.
The responsing reaction of serine/threonine protein kitase involved in plant to different adverse circumstances, can play a role aspect raising stress resistance of plant.Serine/threonine protein kitase has the function of " central processing unit (CPU) ", the input messages such as plant hormone or other extraneous factors that reception passes over from acceptor, change into suitable output information, as the variation of the aspects such as metabolism, genetic expression, Growth of Cells and division.
Similarity according to serine/threonine protein kitase catalysis territory, is divided into: calcium dependent kinases (CDPK) subfamily, SNF1 related protein kinase (SnRK) subfamily, receptoroid kinases (RLKs) subfamily, MAPK/MAPKK/MAPKKK subfamily, cyclin dependent kinase (CDK) subfamily, GSK3/SHAGGY subfamily etc.Various environment stresses act on vegetable cell, first Ca in trigger cell
2+the change of concentration.Calcium signal is by various calcium ion susceptor perception in cell and transmit calcium signal, has two class Ca in plant
2+dependent form susceptor, a class is calmodulin (CaM), another kind of is the distinctive calcium adjusting phosphatase B analogy albumen of plant (CBL).CBL susceptor and protein kinase C IPK(CBL-Interacting Protein Kinases) interacting forms Ca
2+-CBL-CIPK complex body, CBL-CIPK complex body is activated, and in the CBL-CIPK of state of activation complex body phosphorylation modification downstream object target protein, the calcium signal of the various dynamic changes of decoding, regulates and controls relevant physiological process.CIPK is considered to belong to one of plant snf 1-related protein kinase family three large subtribes SnRK3 subtribe.
Find at present, in Arabidopis thaliana, have 26 CIPKs family members, paddy rice CIPKs family member has 30.Plant SOS signal transduction under CBL-CIPK signal pathway wide participation plant high-salt stress.Plant under salt stress, Ca in cell
2+concentration increases, the Ca on cytolemma
2+susceptor SOS3/CBL4 is in conjunction with Ca
2+and acting on kinases SOS2/CIPK24, SOS3-SOS2 complex body is activated and phosphorylation cytolemma Na
+/ H
+counter transport carrier S OS1, has improved Na
+/ H
+antiport ability, by Na excessive in cell
+discharge is to maintain Na in cell
+balance.Potassium (K in plant growth and development process
+) play an important role.Plant is to K
+absorption and transhipment be mainly to pass through K
+transporter.CBL-CIPK signal transduction system plays an important role in the low potassium stress response of regulating plant.In Arabidopis thaliana, CIPK23 and CBL1 or CBL9 interact, the K in phosphorylation downstream
+passage AKT1, activates K
+transhipment.CBL1 and CBL9 can also interact with CIPK6, CIPK16, transmit low potassium signal, regulation and control AKT1.CBL-CIPK also participates in relying on the signal transduction of ABA and non-dependence ABA.CBL1 afunction mutant cbl1 to arid, damage to plants caused by sudden drop in temperature with the susceptibility of the multiple abiotic stress such as salt stress and do not rely on ABA, with the CBL9 afunction mutant cbl9 of CBL1 height homology, the susceptibility of abiotic stress is highly relied on ABA.In addition, except the mediated plant response of environment to external world, other physiological process of the CBL-CIPK system process that also involved in plant development is relevant and regulating plant.
Summary of the invention
The present invention is directed to the deficiencies in the prior art, the degeneration-resistant regulatory factor of grow wheat CBL-CIPK class and encoding gene and an application is provided.
Summary of the invention
The present invention according to early stage wheat salt, drought coerce the chip of expression spectrum information of acquisition, be chosen in root and leaf and express and be obviously subject to salt and non-irrigated protein kinase gene (probe) of inducing, and cloned its cDNA sequence containing full length coding region, nucleotide sequence is as shown in SEQ ID NO.1.Analysis shows, this genes encoding CBL-CIPK class serine/threonine protein kitase, and aminoacid sequence, as shown in SEQ ID NO.2, is named as TaCIPK33.By transgenosis functional analysis, show, this gene has the function that improves plant salt endurance and drought resistance.This provides the foundation for deeply understanding the molecule mechanism of wheat response arid, salt stress, and this gene can be applicable to the genetic improvement of the degeneration-resistant new germ plasms of crop such as wheat simultaneously.
Detailed Description Of The Invention
The encoding gene TaCIPK33 of the degeneration-resistant regulatory factor of one grow wheat CBL-CIPK class, nucleotide sequence is as shown in SEQ ID No.1.1463 bases of encoding gene TaCIPK33 sequence total length of the degeneration-resistant regulatory factor of wheat CBL-CIPK class, there are 1332 bases coding region.
The degeneration-resistant regulatory factor of above-mentioned wheat CBL-CIPK class, aminoacid sequence is as shown in SEQ ID No.2.The described degeneration-resistant regulatory factor of wheat CBL-CIPK class is comprised of 443 amino-acid residues.
A kind of recombinant vectors that inserts nucleotide sequence shown in above-mentioned SEQ ID No.1.
, contain above-mentioned recombinant vectors.
, contain above-mentioned recombinant vectors.
The application of encoding gene TaCIPK33, recombinant vectors, transgenic cell line or the transfer-gen plant of the degeneration-resistant regulatory factor of above-mentioned wheat CBL-CIPK class in cultivating salt tolerant, drought-resistant plant.
Preferred according to the present invention, described plant is wheat or tobacco.
Beneficial effect
The present invention has been cloned into wheat TaCIPK33 gene first, this genes encoding CBL-CIPK class serine/threonine protein kitase, and proved that by transgenosis functional analysis described gene has the function that improves plant salt endurance and drought resistance.The approach that the present invention mediates by agrobacterium tumefaciens proceeds to overexpression in tobacco by this gene, compares with wild-type tobacco plant, and the transgene tobacco offspring plant that contains TaCIPK33 gene of the present invention has more obvious salt tolerant, drought-resistant ability.Gene of the present invention can be the degeneration-resistant new variety of crop such as cultivating wheat theoretical foundation and genetic resources is provided.
Accompanying drawing explanation
Fig. 1, be the pcr amplification electrophoresis result photo of TaCIPK33 full length gene cDNA sequence;
Wherein: M, DNA molecular amount standard Marker(Trans2k plus), 1, negative control, 2, PCR product;
After Fig. 2, NaCl, PEG Stress treatment, TaCIPK33 gene melts the electrophoresis result photo that the RT-PCR in No. 3 seedling roots and leaf analyzes on wheat mountain;
Wherein: Actin is internal reference; Leaf is leaf; Root is root;
The expression of TaCIPK33 gene under 200mM NaCl Stress treatment in A, root;
In B, root, TaCIPK33 gene is the expression under 18%PEG6000 Stress treatment in volumetric concentration;
The expression of TaCIPK33 gene under 200mM NaCl Stress treatment in C, leaf;
In D, leaf, TaCIPK33 is the expression under 18%PEG6000 Stress treatment in volumetric concentration;
The enzyme of the plant expression vector pROK II-TaCIPK33 of Fig. 3, structure is cut checking electrophoresis result photo;
Wherein: M, DNA molecular amount standard Marker(Trans2k plus), 1, pROKII plasmid, 2, BamH I and the KpnI double digestion result of pROK II-TaCIPK33;
The regenerative process photo of Fig. 4, TaCIPK33 transgene tobacco;
Wherein: induction and the screening in inducing culture of A, kalamycin resistance bud, B, kalamycin resistance bud are grown in screening culture medium; C, transgenic tobacco plant are at greenhouse normal growth;
The PCR of Fig. 5, transgene tobacco identifies electrophoresis result photo;
Wherein: M, DNA molecular amount standard Marker(Trans2k plus), 1-11, transfer-gen plant, 12, positive control, 13, water contrast, 14, wild-type non-transgenic tobacco plant;
The phenotypic evaluation photo of Fig. 6, transgene tobacco strain;
Wherein: A, two weeks long growing states of seedling root of vertical cultivation on the MS substratum that contains 100mM NaCl;
B, on the substratum that contains 100mM N.F,USP MANNITOL, vertically cultivate two weeks long growing states of seedling root;
In A, B figure, upper left is the wild strain WT of contrast, and upper right is transgenic line S/T-8, and lower-left is transgenic line S/T-9, and bottom right is transgenic line S/T-10;
C, on the MS substratum that contains 100mM NaCl seed germination situation;
D, on the MS substratum that contains 100mM N.F,USP MANNITOL seed germination situation;
In C, D figure, top is the wild strain WT of contrast, and below is transgenic line S/T-10.
Embodiment
Below in conjunction with specification drawings and specific embodiments, technical scheme of the present invention is described further, but institute of the present invention protection domain is not limited to this.
Biological material source
Bacillus coli DH 5 alpha, Beijing Quanshijin Biotechnology Co., Ltd is on sale;
Agrobacterium LBA4404, Invitrogen company is on sale;
Plant expression vector pROK II, Biovector China's plasmid vector strain gene storehouse is on sale;
Melt No. 3 on wheat mountain, and Lu Nong examines word [2004] No. 030, common commercially available prod;
In following examples, if no special instructions, the method for use is this area ordinary method, and substratum is this area conventional medium.
The clone of embodiment 1TaCIPK33 gene cDNA sequence
Clone and the sequencing of TaCIPK33 full length gene cDNA sequence
1. primer sequence
According to the SSH of wheat and chip of expression spectrum data results design gene-specific primer, the masterplate that wheat seedlings blade cDNA article one chain of take is amplifying target genes, the full-length cDNA of amplification gene, primer sequence is:
TaCIPK-S：5′CGACCTACCTCCTACCCTC3′，SEQ ID NO.3
TaCIPK–A：5′GCAAGATAGTTCCATTCCG3′。SEQ ID NO.4
2.PCR reaction system (20 μ l)
Add successively 10 * PCR damping fluid (containing Mg
2+) 2.0 μ L, 2.5mM dNTP1.6 μ L, reverse transcription cDNA the first chain product 1 μ L, forward primer (TaCIPK-S) 0.5 μ L, reverse primer (TaCIPK-A) 0.5 μ L, 5U/ μ L Taq archaeal dna polymerase 0.2 μ L, adds water to 20 μ L.
3.PCR response procedures is: 94 ℃ of denaturation 5min; 94 ℃ of sex change 30Sec, 62 ℃ of renaturation 30Sec, 72 ℃ are extended 1min, 30cycles; 72 ℃ are extended 10min, 4 ℃ of preservations.
4.1wt% agarose gel electrophoresis
Pcr amplification product detects with 1wt% agarose gel electrophoresis, and finding has the band of an entry at 1463bp place, as shown in Figure 1.
5. the recovery of amplification object fragment, be cloned into pMD18-T carrier
Amplified fragments is adopted after 1wt% agarose gel electrophoresis, and using TIANgel Midi Purification kit test kit to reclaim goal gene fragment is PCR product, and concrete operation step carries out according to test kit specification sheets.PCR product is connected with the pMD18-T of TaKaRa company carrier, obtains and connects product, and linked system is:
1uL pMD18-T Vector, 4uL PCR product, 5uL Ligation Mix;
Condition is 16 ℃ and connects 30 minutes.
6. reclaim clone and the order-checking of fragment
The connection product of full dose is transformed in bacillus coli DH 5 alpha competent cell, and picking white bacterial plaque is shaken bacterium in 37 ℃, extracts the plasmid that has inserted gene fragment.
(1) double digestion detection validation: Hind III and EcoR I endonuclease digestion with TaKaRa company, operate as follows: enzyme is cut system 20 μ L, comprise 2 μ L damping fluids, 14 μ L have inserted the plasmid of gene fragment, 1 μ L HindIII restriction endonuclease, 1 μ L EcoRI restriction endonuclease, moisturizing 2 μ L.In 37 ℃ of water-baths 4 hours, electrophoresis detection enzyme was cut result.
(2) the bacterium liquid of the positive colony of connection carrier is sent to the order-checking of order-checking company, guarantee that correctly sequencing result is as shown in SEQ ID No.1, by 1463 based compositions, called after TaCIPK33.
The expression analysis of embodiment 2 genes
The expression analysis of TaCIPK33 gene under NaCl, PEG Stress treatment condition
1. material processing
Choose full wheat mountain and melt seed No. 3, normally sprout.
Select grow to two leaves wholeheartedly, unanimous on the whole, the healthy and strong mountain of situation melts No. 3 wheats and carries out following Stress treatment: after 200mM NaCl bubble root is processed 48h, recover 48h; Concentration expressed in percentage by volume is to recover 48h after 18%PEG6000 solution soaks root processing 48h, contrasts as healthy plant under normal condition.Respectively in 0h, 0.5h, 1h, 3h, 6h, 12h, 24h, 48h, recovery 24h and recovery 48h sampling ,-70 ℃ of preservations.Get root and leaf that the Stress treatment different time points of No. 3 wheat plants is melted on mountain, Trizol method is extracted the total RNA of wheat.
2.Trizol method is extracted the total RNA of wheat
The seedling leaves that wheat mountain melts No. 3 10 days seedling ages of take is material
(1) organization material is put into the mortar of Liquid nitrogen precooler, abundant grind into powder in liquid nitrogen, powder is packed in 1.5mL centrifuge tube, every 100mg material adds rapidly the TRIzol extracting solution of the Invitrogen company of 1ml, concuss 15 seconds, mix sample, make the abundant cracking of sample, room temperature is placed 5 minutes;
(2) add 100uL2M sodium-acetate (PH4.0), incubation 2-3 minute;
(3) add 0.2ml chloroform (chloroform), thermal agitation 15 seconds, incubation 2-3 minute;
(4) 4 ℃, centrifugal 15 minutes of 12000rpm, gets supernatant liquor and moves in new 1.5mL centrifuge tube;
(5) add 800 μ L chloroforms, with hand, acutely rocked for 15 seconds, incubation 2-3 minute;
(6) 4 ℃, centrifugal 15 minutes of 12000rpm, gets supernatant liquor and moves in new 1.5mL centrifuge tube;
(7) add 500 μ L Virahols, put upside down and mix, standing 10 minutes of room temperature;
(8) 4 ℃, centrifugal 10 minutes of 12000rpm, absorbs supernatant liquor;
(9) add the ethanolic soln that the volume percent of 1m L precooling is 75%, spiral concussion.4 ℃, centrifugal 5 minutes of 7500rpm, abandons supernatant, collecting precipitation;
(10) the ethanolic soln washing RNA precipitation that to repeat by volume percent be 75% once;
(11) remove supernatant, RNA is deposited in and on aseptic operating platform, dries about 10-15 minute, it is transparent that RNA shows slightly, and adds appropriate DEPC water (being generally 20 μ L) dissolution precipitation (or to preserve (put in-80 ℃ of refrigerators and save backup) in the volume percent ethanolic soln that is 75%;
(12) get 1 μ L RNA sample, 1wt% agarose gel electrophoresis detects, and imaging in ultraviolet gel imaging system identifies whether RNA degrades and rough concentration.
3. the first chain cDNA's is synthetic
Adopt the RevertAid First Strand cDNA Synthesis Kit test kit of Fermentas company to carry out, reactions steps is as follows:
First, mRNA reverse transcription is become to the first chain cDNA, ThermoScript II used is the RevertAid First Strand cDNA Synthesis Kit of Fermentas company, and reaction system is 20 μ L.Add successively 1 μ L oligo (dT)
18primer(100uM), 1 μ g Total RNA and DEPC water to 10 μ L, 65 ℃ of water-bath sex change 5min, chilling, slightly centrifugal on ice; Then add successively 1 μ L RNase Inhabitor (20U/uL), 4 μ L5x Reaction Buffer, 2 μ L dNTP Mixture(10mM) 1 μ L M-MuLV ReverseTranscriptase (200U/uL), 4 μ L RNase Free ddH
2o, mixes;
Then on PCR instrument according to " 42 ℃, 60min; 70 ℃, 5min; 4 ℃, preserve " program operation; Reverse transcription obtains article one chain of wheat cDNA, in-20 ℃ of preservations.
4.PCR reaction system
With mountain, melting No. 3 wheat cDNA is template, usings TaActin-S and TaActin-A as amplimer, obtains the actin internal reference product fragment of 558bp left and right.According to the amplification situation of internal reference Actin, determine the cycle number of PCR, adjust the masterplate amount of cDNA.
(1) internal reference Actin primer sequence:
TaActin-S5′AGCCATACCGTGCCAATC3′
TaActin-A5′AGAGCCTCCAATCCAGAC3′
(2) reaction system is as follows: TaqMix10uL, and template x uL, TaActin-S0.5, TaActin-A0.5, moisturizing is to total system 20 μ L.
(3) PCR response procedures: 94 ℃ of 5min; 94 ℃ of 30Sec, 58 ℃ of 30Sec, 72 ℃ of 1min, 30cycles; 72 ℃ of 10min; 4 ℃ of preservations.
1wt% agarose gel electrophoresis detects expression analysis, and result as shown in Figure 2.
The structure of the overexpression plant expression vector of embodiment 3CaMV35S promoters driven
Plant expression vector pROK II is the binary vector that contains CaMV35S promotor and NPTII gene, contains restriction enzyme BamH I and Kpn I site in its multiple clone site.According to the cDNA coding region sequence of gene TaCIPK33, design packet is containing the gene-specific primer of complete ORF, and primer sequence: TaCIPK-S1 adds BamH I(GGATCC) site, TaCIPK-A1 adds Kpn I(GGTACC) site,
TaCIPK-S15′CGC
GGATCCCGACCTACCTCCTACCCTC3′，SEQ ID NO.5
TaCIPK-A15′CGG
GGTACCGCAAGATAGTTCCATTCCG3′，SEQ ID NO.6
CDNA sequence with this to primer amplification gene, amplifies the gene fragment of 1463bp, and is cloned on the pMD18-T carrier of TaKaRa company.Then by restriction enzyme BamH I and Kpn I double digestion empty carrier pROK II and carry the pMD18-T carrier of goal gene fragment respectively, reclaim respectively carrier large fragment and TaCIPK33 gene fragment, 16 ℃ of connections of spending the night, obtain recombinant plasmid plant expression vector pROK II-TaCIPK33.
(1) empty carrier pROK II and carry T-carrier B amH I restriction endonuclease and the Kpn I enzymes double zyme cutting of goal gene fragment
Endonuclease reaction system (20 μ L):
BamH I restriction endonuclease 1 μ L
Kpn I restriction endonuclease 1 μ L
Empty carrier pROK II
(or goal gene fragment) 5 μ L
10×Buffer 2μL
Mend ddH
2o to 20 μ L,
37 ℃ of thermostat water bath incubations 2 hours;
(2) enzyme is cut product electrophoresis and recovery
After double digestion has reacted, enzyme is cut to product and carry out 0.8wt% agarose gel electrophoresis, sepharose test kit reclaims carrier large fragment and TaCIPK gene fragment;
(3) connect
By carrying out 16 ℃ through the carrier large fragment of double digestion and goal gene fragment according to the ratio of volume ratio 1:4, spend the night and is connected, reaction system is (25 μ L) as follows: TaCIPK gene fragment 10 μ L, pROK II carrier large fragment 4 μ L, 10 * T
4dNA Ligase buffer2.5 μ L, T
4dNA Ligase1 μ L, moisturizing to 25 μ L; 16 ℃ of connections are spent the night;
(4) transform
To connect product and transform bacillus coli DH 5 alpha competent cell by heat shock method, transformed bacteria drop on containing on the LB solid plate of Kan50 μ g/ml 37 ℃ cultivate 16 hours;
(5) enzyme of positive recombinant is cut evaluation
Picking white bacterial plaque is shaken bacterium in 37 ℃, extract plasmid, BamH I restriction endonuclease and Kpn enzymes double zyme cutting detection validation, endonuclease reaction system is with (1) step in embodiment 3, enzyme is cut product through 0.8wt% agarose gel electrophoresis, goal gene band and the carrier segments band of suitable size detected, illustrate that TaCIPK gene has successfully been connected to pROK II carrier, makes recombinant expression vector plasmid DNA.Result as shown in Figure 3.The recombinant vectors connecting is checked order, guarantee correct.
The embodiment competent preparation of 4 Agrobacterium and conversion
1. the preparation of Agrobacterium competent cell
(1) from the picking Agrobacterium LBA4404 list bacterium colony of YEP dull and stereotyped (containing 50 μ g/ml Rifampins), be inoculated in and contain in the YEP liquid nutrient medium of 50 μ g/ml Rifampins, 200rpm, overnight incubation at 28 ℃;
(2) get 2ml incubated overnight liquid and be inoculated in 50ml containing in identical antibiotic YEP liquid nutrient medium, under the same terms, be cultured to OD
600to 0.5;
(3) bacterium liquid ice bath 30min, 4 ℃, the centrifugal 10min of 5000rpm, collects thalline;
(4) thalline is resuspended in the NaCl of 10ml0.15mol/L of ice bath, and 4 ℃ of centrifugal 10min of 5000rpm collect thalline;
(5) bacterium liquid Eddy diffusion is in the CaCl of 1ml20mmol/L ice precooling
2in solution, bacterium liquid is divided in 1.5ml Eppendorf pipe with every pipe 200 μ L, liquid nitrogen flash freezer 1min ,-70 ℃ save backup.
2. freeze-thaw method transforms Agrobacterium LBA4404
(1) melt Agrobacterium competent cell on ice, add 1 μ g recombinant expression vector plasmid DNA, mix rear ice bath 30min;
(2) liquid nitrogen flash freezer 1min, moves to rapidly 37 ℃ of insulation 3min;
(3) add the liquid YEP800 μ L of antibiotic-free, 4hr is cultivated in 28 ℃ of concussions;
(4) the centrifugal 30s of 7000rpm, collects thalline, is applied on the YEP flat board that contains 50 μ g/ml Rifampins, 50 μ g/ml Kan, is inverted dark cultivation 2～3 days for 28 ℃.
3. positive bacterium colony PCR identifies
Bacterium colony PCR the primer is with embodiment 1, and method and program are with the step 2 in embodiment 1.
Embodiment 5 transgenosis functional verifications----Transformation of tobacco, screening and phenotype analytical
1. leaf disc transformation method transformation of tobacco
(1) the ethanolic soln sterilization 1min that tobacco seed is 75% in volume percent, aseptic water washing 3～5 times; Mass ratio dilution by clorox and water with 1:4, to seed disinfection 10min, aseptic water washing 5 times, uniform spreading spills in 1/2MS
0on minimum medium, make its germination.
(2) after seed germination (approximately two weeks), transferred to (containing 1/2MS) in independent bottle, to grow 4～5 weeks, plant to be planted grows enough leaves for conversion.
(3) picking Agrobacterium (carrying the single bacterium colony of Agrobacterium of recombinant expression vector plasmid) is inoculated in and contains 50 μ g/ml kantlex, and in the YEP liquid nutrient medium of 50 μ g/ml Rifampins, 28 ℃, the about 24h of 200rpm shaking culture, to logarithmic phase.
(4), by the centrifugal 6min of bacterium liquid 6000rpm, collect thalline, MS
0liquid nutrient medium is resuspended.
(5) get tobacco leaf, be cut into small pieces (0.5 * 0.5cm), is put in 1.5min in resuspended bacterium liquid by the tobacco leaf shearing, constantly vibration, uses aseptic filter paper suck dry moisture after taking out, and it is neatly closely arranged on MS division culture medium, 28 ℃, secretly cultivate 2d.
(6) dark cultivation two days later, is arranged in MS by blade loosely and selects on substratum, and 28 ℃, light application time 16h/d, changed a subculture every two weeks.
(7) when it grows Multiple Buds, cut and put in elongation medium, make its elongation.
(8) when Multiple Buds grows to 3～5cm, transfer to root media, short its taken root.
(9), after root system development is good, seedling is washed to root immigration and fill in the flowerpot of sterile soil, lid mulch film 3d.
(10) under light, cultivate, grow to and to a certain degree move on in large basin, greenhouse Routine Management, results T
0for seed.Result as shown in Figure 4.
2. the screening of transgene tobacco positive plant
T
0after sterilizing with 7.5wt% chlorine bleach liquor (comprising 7.5wt% clorox and 0.01wt%Triton-X100) for seed, sowing is selected on substratum (50mg/L kantlex) at MS, in culturing room, cultivate 10 days, select kalamycin resistance plant and (grow true leaf 1-2 couple, root is stretched in substratum) and be transplanted in nutrition pot, cultivate until seed maturity is adopted the screening T that uses the same method
1for seed, obtain T
2for plant, and at T
1for selecting resistance in plant, than the single copy for 3:1, insert strain, at T
2in generation, is selected the homozygous lines of resistance part, that is: T isozygotys
2for strain, T will isozygoty
2for strain, carry out Molecular Detection and the phenotypic evaluation of transgene tobacco.
The acquisition of the homozygous lines that single copy inserts is this area conventional steps: by the T of results
1for planting seed, be added with on the MS substratum of kantlex, meeting albefaction, death after non-transgenic seed germination, transgenic seed is normal green seedling after sprouting, by the side of card (x
2) test, calculate the ratio of the dead seedling of green seedling and albefaction, if being single copy, foreign gene is inserted in acceptor tobacco, the ratio of the dead seedling of so green seedling and albefaction is exactly 3:1, the transgenic line that single copy inserts, otherwise be not just satisfactory transgenic progeny (meeting mendelian inheritance).The seed of the transgenic line that the single copy of results inserts, continues to screen being added with on the MS substratum of kantlex, be completely green seedling be homozygote, the T isozygotying
2for strain.Transgenosis T
3for strain, it is single copy homozygous lines selfing acquisition of going down to posterity.
3. the PCR of transgene tobacco identifies
(1) CTAB method is extracted tobacco leaf genomic dna
1. get the fresh blade of 100mg left and right, put into 1.5ml centrifuge tube, liquid nitrogen flash freezer, grinds, and adds 600 μ L to be preheated to 2 * CTAB Extraction buffer of 65 ℃, turns upside down and mixes, and is placed in 65 ℃ of standing 30min of water-bath; Continuous jog, fully mixes CTAB extracting solution and vegetable material therebetween;
2. mixture adds isopyknic phenol/chloroform/primary isoamyl alcohol after being chilled to room temperature, mixes the standing 15min of room temperature, 4 ℃, the centrifugal 10min of 12000rpm;
3. get supernatant, add isopyknic chloroform/primary isoamyl alcohol, mix, 4 ℃, the centrifugal 10min of 12000rpm;
4. get supernatant, add the 3mol/L NaAc(pH5.3 of 1/10 volume) and the Virahol of 0.7 times of volume, mix the standing 15min of room temperature, 4 ℃, the centrifugal 15min of 12000rpm, precipitation DNA;
5. 75% ethanol is washed precipitation twice.Abandon supernatant, dry several minutes of super clean bench
6. precipitation is dissolved in appropriate TE damping fluid, in-20 ℃ of preservations.
(2) pcr amplification of transgene tobacco
The tobacco gene group DNA of said extracted of take is template, with gene-specific primer (with embodiment 1), carries out pcr amplification.
PCR reaction system is with the step 2 in embodiment 1, and PCR response procedures is: 94 ℃ of denaturation 5min; 94 ℃ of sex change 30Sec, 62 ℃ of renaturation 30Sec, 72 ℃ are extended 1min, 30cycles; 72 ℃ are extended 10min, 4 ℃ of preservations.PCR product detects the object band that amplifies 1463bp left and right in transgenic tobacco plant through 1.0% agarose gel electrophoresis, in turning the plant of empty carrier, have no amplified band with the negative contrast of aqua sterilisa, and result as shown in Figure 5.
4. the phenotypic evaluation of transgene tobacco
(1) plantation of tobacco
T
3in generation,, single seed that copies the tobacco strain of isozygotying was sterilized 10 minutes with 7.5wt% chlorine bleach liquor (comprising 7.5wt% clorox and 0.01wt%Triton-X100), then use rinsed with sterile water 5～6 times, point is sowed on MS flat board, then be transplanted to (Nutrition Soil mixes by equal proportion with vermiculite) in nutrition pot 25 ℃ of cultivations.
(2) NaCl and drought stress are processed
The long statistics of root
The tobacco seedling (contrast WT and transgenic line S/T-8, S/T-9, S/T-10) of sprouting 2 days is carefully moved in the MS culture dish that contains 100mM NaCl, 100mM N.F,USP MANNITOL, vertically cultivate and within two weeks, observe the long length of statistics root, it is kept burning day and night aobvious than the root of wild-type adjoining tree long (as shown in Fig. 6 A, 6B) that result shows to turn the root of tobacco plant of TaCIPK33 gene.
Germination rate statistics
By transgenic progeny tobacco seed (S/T-10) and wild-type tobacco seed (WT) with clorox sterilization after 10 minutes, rinsed with sterile water 5 times, select and be sowed in the MS culture dish that contains respectively 100mM NaCl, 100mM N.F,USP MANNITOL, cultivate after two weeks, add up respectively the germination rate of seed, found that and turn TaCIPK33 genetic tobacco offspring's seed germination rate higher than the germination rate (as shown in Fig. 6 C, 6D) of wild type seeds.
Interpretation of result
By above-mentioned experimental result, can be drawn gene TaCIPK33 coding CBL-CIPK class serine/threonine protein kitase described in the application, the response of involved in plant to arid, high salt adverse circumstance.After this gene TaCIPK33 overexpression, transfer-gen plant can produce the characteristic of salt-tolerant drought-resistant.
Claims (7)
1. the encoding gene TaCIPK33 of the degeneration-resistant regulatory factor of a grow wheat CBL-CIPK class, nucleotide sequence is as shown in SEQ ID No.1.
2. the degeneration-resistant regulatory factor of wheat CBL-CIPK class described in claim 1, aminoacid sequence is as shown in SEQ ID No.2.
3. a recombinant vectors that inserts nucleotide sequence shown in SEQ ID No.1.
4. a transgenic cell line, contains recombinant vectors claimed in claim 3.
5. a transfer-gen plant, contains recombinant vectors claimed in claim 3.
6. the application of transfer-gen plant in cultivating salt tolerant, drought-resistant plant described in transgenic cell line or claim 5 described in recombinant vectors, claim 4 described in the encoding gene TaCIPK33 of the degeneration-resistant regulatory factor of wheat CBL-CIPK class, claim 3 described in claim 1.
7. application as claimed in claim 6, is characterized in that, described plant is wheat or tobacco.
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