CN106244595A - Lignum seu Ramulus Cunninghamiae Lanceolatae phytosulfokine-α CLPSK1 gene and application thereof - Google Patents
Lignum seu Ramulus Cunninghamiae Lanceolatae phytosulfokine-α CLPSK1 gene and application thereof Download PDFInfo
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/415—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from plants
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- 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)
- C12N15/8216—Methods for controlling, regulating or enhancing expression of transgenes in plant cells
- C12N15/8222—Developmentally regulated expression systems, tissue, organ specific, temporal or spatial regulation
- C12N15/8223—Vegetative tissue-specific promoters
- C12N15/8227—Root-specific
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- 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
- C12N15/8242—Phenotypically and genetically modified plants via recombinant DNA technology with non-agronomic quality (output) traits, e.g. for industrial processing; Value added, non-agronomic traits
- C12N15/8259—Phytoremediation
Abstract
The invention discloses Lignum seu Ramulus Cunninghamiae Lanceolatae phytosulfokine-αCLPSK1Gene and application thereof, Lignum seu Ramulus Cunninghamiae Lanceolatae phytosulfokine-αCLPSK1The DNA sequence of gene is as shown in SEQ ID NO.1.The Lignum seu Ramulus Cunninghamiae Lanceolatae phytosulfokine-α of the present inventionCLPSK1Gene, is confirmed by test, functionally can promote the growth of root, can promote again callus proliferation, will have a wide range of applications in plant breeding has grown.
Description
Technical field
The invention belongs to gene technology field, be specifically related to Lignum seu Ramulus Cunninghamiae Lanceolatae phytosulfokine-α (PSK)CLPSK1Gene and application thereof.
Background technology
Lignum seu Ramulus Cunninghamiae Lanceolatae is one of south China peculiar evergreen needle-leaved tree kind, and its growth is fast, and form is logical straight satisfactory, corrosion-resistant, timber
Excellent, there is the important ecological value and economic worth.Lignum seu Ramulus Cunninghamiae Lanceolatae has had the cultivation history of more than 3000 year, cultivated area in China
Reach 853.86Mha, account for China stand abreast savings area 25 ~ 30%, merchantable timber provide account for about the 25% of China artificial forest.20 generation
Having recorded since the fifties, China starts the planned Lignum seu Ramulus Cunninghamiae Lanceolatae genetic improvement that carries out, and by a series of breeding technique, selects big
Criticize excellent Chinese fir kind source, family and superior clone.At present, Breeding Strategies is developed into the tight phase of molecular biology by traditional breeding method
Connect, and the selection and use new stage based on genomic information.
At present, provide the breeding mode of seed by Chinese Fir Seed Orchards, easily by natural environment influence, easily occur that biennial bearing is existing
As thus cause elite seed yield unstable, it addition, sexual propagation progeny character is unstable, this affects China fir to a certain extent
Wood grow directly from seeds forest land development.For the difficult problem in terms of meeting the market demand and solving breeding, one excellent Chinese fir fast and effectively is asexual
Reproduction technique, i.e. blastula tire generation technique arise at the historic moment.It is many that the Development of Somatic Embryogenesis has reproductive number, and speed is fast, then
Raw rate advantages of higher, is to realize the important means that good seed medelling produces.There are some researches show that PSK-α can promote somatic cell
The formation of embryo, and have been set up Lignum seu Ramulus Cunninghamiae Lanceolatae body embryo generation system, but must body embryo generation synchronization degree the highest, poor stability.
Therefore understand in depthPSKGene function and Regulation Mechanism thereof are significant.
Summary of the invention
Goal of the invention: for the deficiencies in the prior art, it is an object of the invention to provide a kind of Lignum seu Ramulus Cunninghamiae Lanceolatae plant sulphur peptide
ElementCLPSK1Gene, meets use demand.Another ground of the present invention is to provide a kind of above-mentioned Lignum seu Ramulus Cunninghamiae Lanceolatae phytosulfokine-αCLPSK1
The application of gene.
Technical scheme: in order to realize foregoing invention purpose, the technical solution used in the present invention is:
Lignum seu Ramulus Cunninghamiae Lanceolatae phytosulfokine-αCLPSK1Gene, its DNA sequence is as shown in SEQ ID NO.1.
Described Lignum seu Ramulus Cunninghamiae Lanceolatae phytosulfokine-αCLPSK1The expressing protein of gene, its aminoacid sequence such as SEQ ID NO.2 institute
Show.
Containing described Lignum seu Ramulus Cunninghamiae Lanceolatae phytosulfokine-αCLPSK1The expression vector of gene or host cell.
Described Lignum seu Ramulus Cunninghamiae Lanceolatae phytosulfokine-αCLPSK1Gene application in promoting plant establishment.
Described Lignum seu Ramulus Cunninghamiae Lanceolatae phytosulfokine-αCLPSK1Gene is promoting that plant promotes the application in callus proliferation.
Beneficial effect: compared with prior art, the Lignum seu Ramulus Cunninghamiae Lanceolatae phytosulfokine-α of the present inventionCLPSK1Gene, is demonstrate,proved by test
Real, functionally can promote the growth of root, callus proliferation can be promoted again to have wide in plant breeding has grown
General application.
Accompanying drawing explanation
Fig. 1 is transgenic kalamycin resistance plant result figure；
Fig. 2 is transfer-gen plant PCR result electrophoretogram；In figure: M-DL2000；A1, B1, C1,2,4,6,8,10,12,14,16-
Use primer 35S-F and CLPSK1-R；A2, B2, C2,1,3,5,7,9,11,13,15-use primer CLPSK1-F and
CLPSK1-R；A1, A2-template is ddH2O；B1, B2-template is wildtype Arabidopsis thaliana DNA；C1, C2-template is recombiant plasmid；1
~ 16-template is the DNA of arabidopsis Preliminary screening positive plant；
Fig. 3 isCLPSK1What gene pairs arabidopsis wound healing was bred affects result figure；In figure: WT-wild type, P1-turnsCLPSK1Strain
System；
Fig. 4 is to turnCLPSK1 T2 is for the fresh weight result figure of plant Yu wild-type Arabidopsis plants induced synthesis wound healing；In figure: 1-
Wildtype Arabidopsis thaliana；2 ~ 4 turnsCLPSK1The different strains in gene T2 generation；
Fig. 5 is overexpressionCLPSKGene pairs arabidopsis root length affect result figure；In figure: 1 ~ 3-wild type Col, 4 ~ 5-CLPSK1Transgenic line；
Fig. 6 is to turnCLPSK T2 is for the comparative result figure of plant Yu wild-type Arabidopsis plants root length；In figure: 1-wild type Col；
2 ~ 4-turnsCLPSK1The different strains in gene T3 generation；
Fig. 7 is overexpressionCLPCKResult figure is affected on arabidopsis is organogenetic；In figure: A, B, C, D-are liquid suspension training
Arabidopsis wound healing state before supporting, arabidopsis wound healing state when E, F, G, H-are suspension culture 7d, I, J, K, L-are suspension culture 14d
Arabidopsis callus state；A, E, I-compare, and B, F, J-0.1mg/L PSK process, C, G, K-turnCLPSK1Gene；
Fig. 8 is the kenel result figure that under inverted microscope, Arabidopsis callus forms adventitious root；In figure: during suspension culture 14d,
The kenel of arabidopsis adventitious root, A-0.1mg/L PSK-α process, B-turnsCLPSK1Gene；
Fig. 9 is Cell cycle-related genes semi-quantitative results figure；In figure: A-non-transgenic calli suspension material, B-
The non-transgenic calli suspension material that 0.1mg/L PSK-α processes, C-turns CLPSK1 callus suspension material；
Figure 10 is Cell cycle-related genes relative expression quantity result figure；
Figure 11 is that root morphology builds up related gene semi-quantitative results figure；In figure: what A ~ E was corresponding in turn to is organized as: root, stem, leaf, embryo
Property callus, ripe cotyledon embryo；
Figure 12 is that root morphology builds up gene relative expression quantity result figure.
Detailed description of the invention
Below in conjunction with specific embodiment, the present invention is described further.
Embodiment 1
Lignum seu Ramulus Cunninghamiae Lanceolatae phytosulfokine-α geneCLPSK1Clone, specifically comprise the following steps that
1) CTAB method is used to extract Lignum seu Ramulus Cunninghamiae Lanceolatae wound healing RNA, it is thus achieved that the RNA that purity is higher, OD260/OD280Value is at 1.8-2.0, OD260/
OD230Value is 2.0.
2) using POCHE company reversal agents box, template is said extracted RNA, to specifications step synthesis cDNA the
One chain, gained cDNA is put in-20 DEG C of preservations.
3) PCR expands purpose fragment: with cDNA as template, uses the high-fidelity enzyme KOD-Fx of TOYOBO company to carry out PCR
Amplification, 25 μ L PCR amplification system are: 12.5 μ L 2 × PCR Buffer, 5 μ L dNTP(2 mM), 0.75 μ L Forward
Primer, 0.75 μ L Reverse Primer, 1.0 μ L cDNA, 0.5 μ L KOD-Fx polymerase, 4.5 μ L ddH2O。
Concrete primer sequence is as shown in the table:
CLPSK1-F:5'-ATGGCGAAAATCTGTAGAAGTTCAGTG-3'；
CLPSK1-R:5'-TGGGCTCTTGTGTTGTTGTGTATAGATG-3'.
PCR reaction condition: 94 DEG C of 2min；98 DEG C of 10s, 50 DEG C of 30s, 36 cycles；68℃1min；4℃forever.
After PCR reaction terminates, carrying out 1.5% detected through gel electrophoresis, stripe size is consistent with expection.
4) the purpose fragment being consistent with expection size that will obtain, uses the DNA gel of AXYGEN company to reclaim test kit
Reclaim through row, reclaim product after purification, take 2 μ L 1.5% agaroses and carry out detected through gel electrophoresis recovering effect.
5) reclaiming purpose fragment tailing, high-fidelity enzyme KOD-Fx pcr amplification product is flat end, it is necessary to add polyA tail
Rear just can carry out TA clone, 20 L reaction systems are: 2 L 10 × PCR Buffer, 0.4 L 10mMdNTP, 1.6 L 25mM
MgCl2, 10 L DNA, 0.2 L rTaq polymerase, 5.8 L ddH2O.PCR reaction system is: 72 DEG C, 30min.
6) Ligature: the cloning vehicle of connection provides for PMD19-T Vector(Takara company), 10 L chain junctors
System: 5 L Solution I, 1 L PMD19-T Vector, 4 L PCR primer, by link system 0.2mL EP pipe, afterwards
It is placed in 16 DEG C of water-baths, Ligature 4-5h.
7) will connect product to convert to JM109 competent escherichia coli cell, picking positive colony carries out PCR checking
After, will determine that the monoclonal bacterial strain having expection band is delivered to the handsome company in Shanghai and checked order.
The gene order of gained as shown in SEQ ID NO.1, named phytosulfokine-α geneCLPSK1.Its aminoacid sequence
Row are as shown in SEQ ID NO.2.
Embodiment 2
Phytosulfokine-α geneCLPSK1Overexpression vector build and convert, step is as follows:
1) add restriction enzyme site: owing in embodiment 1, the genes of interest both sides of clone do not have restriction enzyme site, need to add enzyme action position
Point could be connected with PBI121 carrier.With the cloning vehicle PMD19-T positive recombiant plasmid with purpose fragment as template, pass through
Rebuild primer, add in purpose fragment both sidesXba I andBam Two restriction enzyme sites of HI.
2) plasmid extraction: use SDS alkaline lysis method of extracting plasmid.
3) 20 L PCR reaction system: 1.0 L 10 × PCR Buffer, 0.4 L 10mMdNTP, 1.2 L 25mM
MgCl2, 1.0 L Forward Primer, 1.0 L Reverse Primer, 1.0 L plasmids, 0.1 L rTaq
Polymerase, 13.3 L ddH2O.The primer:
CLPSK1Xba I-F:5'-TCTAGAATGGCGAAAATCTGTAG-3'；
CLPSK1 Bam HI-R:5'-GGATCCTGGGCTCTTGTGTTG-3'；
Reaction condition: 94 DEG C of 5 min；95 DEG C of 30s, 57 DEG C of 30s, 32cycles；72℃1min30s；72℃7min；4℃
forever。
Take 5 L PCR primer and carry out 1.5% agarose gel electrophoresis detection, reclaim test kit with AXYGENA gel and carry out pure
Change purpose fragment.
4) build overexpression vector: the purpose fragment with restriction enzyme site that will separate, provide with Takara company
Restricted enzyme (Xba I andBam HI) enzyme action, by the purpose fragment reclaimed after endonuclease reaction and PBI121 zero load fragment,
The T4 link enzyme provided with New England Biolabs company is through row link, carrier segments: purpose fragment=3:1-5:1.Chain
Jointing temp is 16 DEG C, links 4-6h.Construct overexpression vector.
5) screening of recombiant plasmid: purpose fragment and PBI121 link product, and to proceed to the telegraphy of e. coli jm109 thin
In born of the same parents.By the positive single colony inoculation that obtains in the LB culture medium containing antibiotic (containing 50mg/ml kanamycin), it is placed in
On constant-temperature table, 37 DEG C, 250rpm cultivates 12-16h.With bacterium solution as template, use universal primer 35S forward primer and purpose bar
Band downstream primer and purpose band upstream and downstream primer, by bacterium solution PCR, screening positive clone.35S-F sequence is: 5'-
TGAAGATAGTGGAAAAGGAAGGTG-3'.Positive colony bacterium solution is delivered to the order-checking of the handsome company in Shanghai, analyzes sequencing result,
See if there is gene mutation phenomenon.
6) recombinant plasmid transformed Agrobacterium: the correct escherichia coli of sequencing result carry out plasmid extraction, the restructuring that will extract
Plasmid proceeds in Agrobacterium receptor cell, screens positive strain with antibiotic (kanamycin and gentamycin), and band is correct
Positive colony bacterium solution and 50% sterile glycerol, after mixing with 2:1 volume ratio, be put in-80 DEG C of preservations.
7) agrobacterium mediation converted arabidopsis: by the Agrobacterium GV3101 containing overexpression vector, the side contaminated with titbit
Formula proceeds in wildtype Arabidopsis thaliana Col.Key step includes: select the arabidopsis that grows fine, removes pod and before converting
Bloom pollination flower；Arabidopsis titbit is immersed in the Agrobacterium bacterium solution prepared, soaks 30-40s；The plan that will soak
South mustard square is in pallet, and lucifuge places 24h；Arabidopsis is taken out, is put in 23 DEG C of 16h illumination, photoperiod dark for 8h cultivation
Case is cultivated.
8) screening of transfer-gen plant: after the arabidopsis transgenic T1 of collection is carried out sterilization treatment for seed, sowing is extremely
On 1/2MS solid medium (containing 50mg/ml kanamycin and 100mg/ml cephamycin) containing antibiotic, purification 24h
After, it is placed in 23 DEG C of illumination cultivation.Selecting the arabidopsis positive plant of anti-kanamycin, transplant to Nutrition Soil, condition of culture is
It is placed in 23 DEG C of 16h illumination, photoperiod incubator dark for 8h.
In the screening culture medium containing Ka Na, screening positive plant, transgenic positive plant has card to receive chloramphenicol resistance, will
Can survive, as shown in Figure 1 in the culture medium of mycin receiving containing card.
9) qualification of arabidopsis transfer-gen plant: CTAB method extracts Arabidopsis leaf DNA, to extract Arabidopsis leaf DNA
For template, with universal primer 35S forward primer and purpose band downstream primer and purpose band upstream and downstream primer, carry out PCR
Reaction.
From Preliminary screening resistant plant choose 8 and carry out PCR detection, with resistant plant DNA as template, with general
Primer 35S forward primer and purpose band downstream primer and purpose band upstream and downstream primer, carry out PCR reaction, and PCR result is such as
Shown in Fig. 2.As shown in Figure 2, after agarose gel electrophoresis separates, all there is specific band in resistant plant, and with purpose sheet
Section is in the same size, illustrates to exist in transfer-gen plantCLPSK1Gene.
Embodiment 3
CLPSK1The application of gene, main process is as follows:
1, transfer-gen plant tissue culture
1) culture medium prescription: minimal medium: MS+20g/l sucrose, agar 6.8g/l, pH5.8.Calli induction media: MS+
2.0g/l 2,4D+0.2g/l 6-BA+0.5g/l caseinhydrolysate+30g/l sucrose, agar 6.8g/l, pH5.8.Regeneration body
Somatic embryo inducing culture: MS+30g/l sucrose, pH5.8.
2) callus induction: by wild type Col seed and transgenic arabidopsis T2 strain P1-1, P1-5, P1-21, P2-
2, the Seed sterilization of P2-4, P2-12, puts in the A culture medium of minimal medium and Jia Kana mycin respectively, is placed in 23 DEG C of illumination
Cultivate.When arabidopsis grows four true leaves, carry out outer implant and draw materials and wound healing inducing culture.
By wild type Col and transgenic positive plant (verifying through PCR, containing purpose fragment), scratch blade with dissecting knife
And be inoculated in calli induction media behind rhizome portion, wild type is comparison, and comparison and each transgenic line respectively process 30.
It is placed in light culture in 23 DEG C of incubators, obtains callus.
3) induction organ occurs: by wild type Col and transgenic positive plant induction embryo callus turn respectively
Entering to regenerate in somatic embryo inducement culture medium, wild type is comparison, 23 DEG C, 85rpm, dark fluid suspension culture, three groups of repetitions,
Microexamination cell state.
Arabidopsis is inoculated on calli induction media, after cultivating 30d, finds that transgenic line wound healing growth rate is bright
Aobvious faster, as shown in Figure 3 than comparison.Fresh weight and dry weight to callus carry out data statistics, as shown in Figure 4, find transgenic
The fresh weight of strain formation callus and dry weight numerical value are apparently higher than matched group, and diversity is notable.
4) transfer-gen plant root length is observed
By wild type Col seed and the pure and mild seed of transgenic arabidopsis T3, put in A culture medium after sterilizing, culture dish is vertically put
Put, be placed in 23 DEG C of illumination cultivation.Observe the change of arabidopsis Root Morphology.
The transgenic positive plant T3 cultivating 15d is carried out root measurement for plant, finds that transgenic line root is the longest
In ratio comparison, as shown in Figure 5.Wild type and transgenic line root length are carried out data statistics, result as shown in Figure 6, transgenic
Strain root length is considerably longer than matched group, and diversity is notable.
Arabidopsis callus induction is inoculated in basic MS fluid medium cultivation, the wound healing group that wildtype Arabidopsis thaliana is formed
Knitting through fluid suspension culture wound healing surface without significant change, 0.1mg/L PSK-α processes the wound healing that wildtype Arabidopsis thaliana is formed
Tissue is through suspension culture, and during 7d, there is protruding generation (Fig. 7 F) on surface, and during 14d, callus surface has adventitious root to produce (figure
7J)；Transgenic calli is through suspension culture, and during 7d, there is protruding generation (Fig. 7 G, H) on transgenic calli surface, and 14d sends out
Existing transgenic wound healing surface has adventitious root to produce (Fig. 7 K, L).Observing with inverted microscope, the projection that wound healing surface is formed has bright
The feature (Fig. 7-8) of aobvious root.
5) gene expression analysis
Take wild type and wound healing that transgenic line T2 system is formed extracts RNA, and invert formation cDNA, by sxemiquantitative and qRT-
The method detection Cell cycle-related genes expression of PCR.The callus taking liquid culture 7d extracts RNA, and inverts shape
Become cDNA, detected by the method for sxemiquantitative and qRT-PCR and build up related gene expression level with root type state.Primer sequence is such as
Under:
AtActin-F:5'-GGCTCCTCTTAACCCAAAGGC-3'
AtActin-R:5'-CACACCATCACCAGA ATCCAGC-3'；
AtCYCD4-F:5'-GATGAGGGCATGArTGTTGACG-3'；
AtCYCD4-R:5'-CCAAACTGGTGTACTTCACAAGC-3'；
AtCYCB1-F:5'-CTCAAAATCCCACGCTTCTTGTGG-3'；
AtCYCB1-R:5'-CACGTCTACTACCTTTGGTTTCCC-3'；
AtCDKB1-F:5'-GGTGGTGACATGTGGTCTGTTGG-3'；
AtCDKB1-R:5'-CGCAGTGTGGAAACACCCGG-3'；
AtRBR-F:5'-CTCATAAGTCGCCTGCTGCTAAG-3'；
AtRBR-R:5'-TTGCTGTGCTCACTGGTGTTG-3'；
SCR-F:5'-TGGTCGAGGAGGArGAGGAATAG-3'；
SCR-R:5'-CGCTTGTGTAGCTGCATTTCC-3'；
SHR-F:5'-ACACTGTACCATCGACCAAACACC-3'；
SHR-R:5'-ACGAAAACCAATCCAACCAC-3'；
PLT1-F:5'-CCTAGACTGGCCTTCCCTTC-3'；
PLT1-R:5'-GTTACCTACAGTCGTCACTTGTGC-3'；
PLT2-F:5'-ACTCTTGTCTCGTCATGTTTTTCA-3'；
PLT2-R:5'-TAGCGGTTGGAGGACCATCG-3'；
WOX5-F:5'-AAGCTTGCGAAGAAGATTGTCAAGAGG-3'；
WOX5-R:5'-GATATCCGTGGTGGTCTCTCGAATATA-3'.
Semiquantitive PCR: with cDNA as template, prepares PCR reaction mixture, 20 L sxemiquantitative systems: 2 L 10 × PCR
Buffer, 0.4 L 10mM dNTPs, 1.2 L MgSO4 (25mM), 0.5 L Forward Primer, 0.5 L Reverse
Primer, 2 L cDNA, 0.1 L rTaq, 12.3 L ddH2O.In test, the amount of each template is identical, PCR system and journey
Sequence is the most identical, and test is repeated 3 times.
PCR condition: 94 DEG C of 2min；94 DEG C of 15s, 54 DEG C of 30s, 28-30cycles；72℃1min；72℃10min；4℃
forever。
Real-time fluorescence quantitative PCR: real-time quantitative PCR uses LightCycler 480 SYBR of Roche company
Green I Master test kit, completes on LightCycler 480 II (Roche), three weights of each example reaction
Multiple, data extraction and analysis uses LightCycler 480 software (Roche).Real-time quantitative reaction system is: 10 L
SYBR Green PCR Master Mix, 0.5 L Forward Primer, 0.5 L Reverse Primer, 3 L cDNA
(10ng/ L), ddH2O up to 20µL。
Response procedures: 95 DEG C of 5min；95 DEG C of 10s, 60 DEG C of 15s, 40cycles；72℃15s.
Take the callus suspension material of suspension culture 7d, carry out Cell cycle-related genesCYCB1;1、CYCD4;1、CDKB1;1、RBRThe relative analysis of expression, result is such as Figure 10, shown in 11.CYCB1;1、CYCD4;1、CDKB1;1、RBRTurningCLPSK1Callus and in the callus that 0.1mg/L PSK-α processes equal up-regulated expressions, turnCLPSK1Callus table
The amount of reaching is slightly above the callus that 0.1mg/L PSK-α processes, and overexpression is describedCLPSK1Promote cell division.
Take the callus suspension material of suspension culture 7d, carry out root development related genePLT1、PLT2、SHR、SCR、 WOX5The relative analysis of expression, result is as shown in Figure 11,12.PLT1、PLT2、SHR、SCR、WOX5TurningCLPSK1Wound healing group
Knit and equal up-regulated expression in the callus that PSK-α processes, turnCLPSK1Callus expression is slightly above what PSK-α processed
Callus.Overexpression is describedCLPSK1Promote cell differentiation and the morphogenesis of root.
Heterogenous expression in arabidopsisCLPSK1Gene can be obviously promoted the elongation of arabidopsis root, with arabidopsis overexpressionAtPSK4Phenotype is consistent, explanationCLPSK1Gene functionally can promote the growth of root.
OverexpressionCLPSK1The callus growth speed that arabidopsis is formed can be made considerably beyond matched group, explanationCLPSK1Callus proliferation can be promoted.
SEQUENCE LISTING
<110>Nanjing Forestry University
<120>Lignum seu Ramulus Cunninghamiae Lanceolatae phytosulfokine-α CLPSK1 gene and application thereof
<130> 100
<160> 27
<170> PatentIn version 3.3
<210> 1
<211> 303
<212> DNA
<213> Cunninghamia lanceolata
<400> 1
atggcgaaaa tctgtagaag ttcagtgcaa cagggaaggc tgtggatttt tagcatatgc 60
agtatctttc ttgtggttac agtcatggct gcacgcccac atccaagagg tgtcctgaat 120
gcttctgtgt tgttcacaaa agatcaaatg gagataaagc acacttttgc tagcaaggtg 180
gaaaaagaag agtcaaaggt agggaatgaa tgtggcaatg atatgaatga agaagaatgc 240
ttagatcgga gaacattagc tgctcagaca gattacatct atacacaaca acacaagagc 300
cca 303
<210> 2
<211> 101
<212> PRT
<213> Cunninghamia lanceolata
<400> 2
Met Ala Lys Ile Cys Arg Ser Ser Val Gln Gln Gly Arg Leu Trp Ile
1 5 10 15
Phe Ser Ile Cys Ser Ile Phe Leu Val Val Thr Val Met Ala Ala Arg
20 25 30
Pro His Pro Arg Gly Val Leu Asn Ala Ser Val Leu Phe Thr Lys Asp
35 40 45
Gln Met Glu Ile Lys His Thr Phe Ala Ser Lys Val Glu Lys Glu Glu
50 55 60
Ser Lys Val Gly Asn Glu Cys Gly Asn Asp Met Asn Glu Glu Glu Cys
65 70 75 80
Leu Asp Arg Arg Thr Leu Ala Ala Gln Thr Asp Tyr Ile Tyr Thr Gln
85 90 95
Gln His Lys Ser Pro
100
<210> 3
<211> 27
<212> DNA
<213> Artificial
<220>
<223>CLPSK1-F sequence
<400> 3
atggcgaaaa tctgtagaag ttcagtg 27
<210> 4
<211> 28
<212> DNA
<213> Artificial
<220>
<223>CLPSK1-R sequence
<400> 4
tgggctcttg tgttgttgtg tatagatg 28
<210> 5
<211> 23
<212> DNA
<213> Artificial
<220>
<223>CLPSK1Xba I-F sequence
<400> 5
tctagaatgg cgaaaatctg tag 23
<210> 6
<211> 21
<212> DNA
<213> Artificial
<220>
<223> CLPSK1 Bam HI-R
<400> 6
ggatcctggg ctcttgtgtt g 21
<210> 7
<211> 24
<212> DNA
<213> Artificial
<220>
<223>35S-F sequence
<400> 7
tgaagatagt ggaaaaggaa ggtg 24
<210> 8
<211> 21
<212> DNA
<213> Artificial
<220>
<223>AtActin-F sequence
<400> 8
ggctcctctt aacccaaagg c 21
<210> 9
<211> 22
<212> DNA
<213> Artificial
<220>
<223>AtActin-R sequence
<400> 9
cacaccatca ccagaatcca gc 22
<210> 10
<211> 22
<212> DNA
<213> Artificial
<220>
<223> AtCYCD4-F
<400> 10
gatgagggca tgartgttga cg 22
<210> 11
<211> 23
<212> DNA
<213> Artificial
<220>
<223>AtCYCD4-R sequence
<400> 11
ccaaactggt gtacttcaca agc 23
<210> 12
<211> 24
<212> DNA
<213> Artificial
<220>
<223>AtCYCB1-F sequence
<400> 12
ctcaaaatcc cacgcttctt gtgg 24
<210> 13
<211> 24
<212> DNA
<213> Artificial
<220>
<223>AtCYCB1-R sequence
<400> 13
cacgtctact acctttggtt tccc 24
<210> 14
<211> 23
<212> DNA
<213> Artificial
<220>
<223>AtCDKB1-F sequence
<400> 14
ggtggtgaca tgtggtctgt tgg 23
<210> 15
<211> 20
<212> DNA
<213> Artificial
<220>
<223>AtCDKB1-R sequence
<400> 15
cgcagtgtgg aaacacccgg 20
<210> 16
<211> 23
<212> DNA
<213> Artificial
<220>
<223>AtRBR-F sequence
<400> 16
ctcataagtc gcctgctgct aag 23
<210> 17
<211> 21
<212> DNA
<213> Artificial
<220>
<223> AtRBR-R
<400> 17
ttgctgtgct cactggtgtt g 21
<210> 18
<211> 23
<212> DNA
<213> Artificial
<220>
<223> SCR-F
<400> 18
tggtcgagga ggargaggaa tag 23
<210> 19
<211> 21
<212> DNA
<213> Artificial
<220>
<223>SCR-R sequence
<400> 19
cgcttgtgta gctgcatttc c 21
<210> 20
<211> 24
<212> DNA
<213> Artificial
<220>
<223>SHR-F sequence
<400> 20
acactgtacc atcgaccaaa cacc 24
<210> 21
<211> 20
<212> DNA
<213> Artificial
<220>
<223> SHR-R
<400> 21
acgaaaacca atccaaccac 20
<210> 22
<211> 20
<212> DNA
<213> Artificial
<220>
<223>PLT1-F sequence
<400> 22
cctagactgg ccttcccttc 20
<210> 23
<211> 24
<212> DNA
<213> Artificial
<220>
<223>PLT1-R sequence
<400> 23
gttacctaca gtcgtcactt gtgc 24
<210> 24
<211> 24
<212> DNA
<213> Artificial
<220>
<223>PLT2-F sequence
<400> 24
actcttgtct cgtcatgttt ttca 24
<210> 25
<211> 20
<212> DNA
<213> Artificial
<220>
<223>PLT2-R sequence
<400> 25
tagcggttgg aggaccatcg 20
<210> 26
<211> 27
<212> DNA
<213> Artificial
<220>
<223>WOX5-F sequence
<400> 26
aagcttgcga agaagattgt caagagg 27
<210> 27
<211> 27
<212> DNA
<213> Artificial
<220>
<223>WOX5-R sequence
<400> 27
gatatccgtg gtggtctctc gaatata 27
Claims (5)
1. Lignum seu Ramulus Cunninghamiae Lanceolatae phytosulfokine-αCLPSK1Gene, its DNA sequence is as shown in SEQ ID NO.1.
2. the Lignum seu Ramulus Cunninghamiae Lanceolatae phytosulfokine-α described in claim 1CLPSK1The expressing protein of gene, its aminoacid sequence such as SEQ ID
Shown in NO.2.
3. contain the Lignum seu Ramulus Cunninghamiae Lanceolatae phytosulfokine-α described in claim 1CLPSK1The expression vector of gene or host cell.
4. the Lignum seu Ramulus Cunninghamiae Lanceolatae phytosulfokine-α described in claim 1CLPSK1Gene application in promoting plant establishment.
5. the Lignum seu Ramulus Cunninghamiae Lanceolatae phytosulfokine-α described in claim 1CLPSK1Gene is promoting that plant promotes answering in callus proliferation
With.
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN114158481A (en) * | 2021-12-27 | 2022-03-11 | 浙江宜格企业管理集团有限公司 | Preparation method of magnolia callus culture |
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| WO2012017067A1 (en) * | 2010-08-06 | 2012-02-09 | Genoplante-Valor | Plants resistant to pathogens and methods for production thereof |
| CN103070074A (en) * | 2013-01-30 | 2013-05-01 | 南京林业大学 | Somatic embryogenesis method for cunninghamia lanceolata |
| CN103088058A (en) * | 2013-01-30 | 2013-05-08 | 南京林业大学 | Genetic transformation method of Chinese fir |
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2016
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| WO2012017067A1 (en) * | 2010-08-06 | 2012-02-09 | Genoplante-Valor | Plants resistant to pathogens and methods for production thereof |
| CN103237893A (en) * | 2010-08-06 | 2013-08-07 | 加诺普兰特-维勒公司 | Plants resistant to pathogens and methods for production thereof |
| CN103070074A (en) * | 2013-01-30 | 2013-05-01 | 南京林业大学 | Somatic embryogenesis method for cunninghamia lanceolata |
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| CN114158481A (en) * | 2021-12-27 | 2022-03-11 | 浙江宜格企业管理集团有限公司 | Preparation method of magnolia callus culture |
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Application publication date: 20161221 Assignee: Nanjing jianhucheng Trading Co., Ltd Assignor: Nanjing Forestry University Contract record no.: X2019320000289 Denomination of invention: Cunninghamia lanceolata phytosulfokine CLPSK1 gene and applications thereof Granted publication date: 20190507 License type: Common License Record date: 20191120 |
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Application publication date: 20161221 Assignee: Fujian Jinshuo Biotechnology Co., Ltd. Assignor: Nanjing Forestry University Contract record no.: X2019320000321 Denomination of invention: Cunninghamia lanceolata phytosulfokine CLPSK1 gene and applications thereof Granted publication date: 20190507 License type: Common License Record date: 20191202 Application publication date: 20161221 Assignee: Sichuan Beidou Ecological Forestry Co., Ltd Assignor: Nanjing Forestry University Contract record no.: X2019320000320 Denomination of invention: Cunninghamia lanceolata phytosulfokine CLPSK1 gene and applications thereof Granted publication date: 20190507 License type: Common License Record date: 20191202 |
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