CN108660143A - Carrier and the application of a kind of cabbage type rape BnKCS1-2 genes and its structure - Google Patents
Carrier and the application of a kind of cabbage type rape BnKCS1-2 genes and its structure Download PDFInfo
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Abstract
The invention discloses carrier and the applications of a kind of 2 genes of cabbage type rape BnKCS1 and its structure, the present invention has cloned the key gene BnKCS1 2 that cabbage type rape wax synthesis precursor substance over-long chain fatty acid is formed, and the nucleotides sequence of the gene is classified as SEQ ID NO:Shown in 1.The rapeseed plants blade wax content for being transferred to 2 genes of BnKCS1 increases, and when promoting plant epidermis wax to synthesize using the overexpression of 2 genes of BnKCS1, can be used for developing bio-fuel.And the variation for turning the rape leaf wax content and component of 2 genes of BnKCS1 makes plant resistance be enhanced, and this provides new effective selection and feasible method to improve stress resistance of plant.
Description
Technical field
The invention belongs to rapeseed gene applied technical field, it is related to a kind of cabbage type rape BnKCS1-2 genes and its structure
Carrier and application.
Background technology
Fossil fuel is the major part of world's primary energy.Tellurian fossil fuel is extremely limited, and in the short time
It is unable to Natural re generation, therefore potential the crisis of fossil fuel energy shortage.Meanwhile the mankind constantly combustion of fossil fuels and arrange
Carbon dioxide is put, this is also to speed up one of the factor of global warming.How to solve the problems, such as the energy shortage of facing mankind and subtracts
Few influence of the fossil fuel to environment, will be the significant problem put in face of the mankind.To solve this problem, it is being possible to
In the case of use regenerative resource more, such as biological energy source, this can be helped to increase that needed for the energy in the whole world.In addition, biology combustion
Carbon dioxide composition in material comes from atmosphere, therefore development bio-fuel can reduce the carbon dioxide on atmosphere, from
And lower greenhouse effects.
Rape is important one of the oil crops in China, and economic value is high.Rapeseed oil is other than being used for edible oil, also
It can carry out substitute fossil fuels as the raw materials for production of biodiesel.However, rape can often encounter during growth
Influence the adverse environmental factors of yield, such as arid, low temperature, accumulated water.These extraneous factors are difficult to overcome, and it is serious to easily cause rape
Lodging, sclerotiniose largely occur.Therefore, gene traits kind drought-enduring, disease-resistant, resistant to lodging is filtered out, for realizing yield of Brassica napus L
Stable yields is of great significance.Plant can generate environment stress certain response, when the stimulation of external environment is transmitted to plant
After body, a series of response signals are will produce in vivo, to adjust its adaptability to environmental stress factor.For example, stomata is closed
With the adjusting of infiltration coefficient etc..With the development of molecular biology, China's rape resistance research direction no longer only rests on biography
It unites in the research level of breeding, researchers start the novel highly resistance of exploration genetic engineering means selection and breeding or how anti-plant variety.
Plant epidermis wax is covered in the surface of aboveground vegetation part, is the first layer barrier of plant and external environment, dredges
The wax coat of water can prevent loss of water in plant, surface waterproofing, reduces shortwave radiation and protect the blade from accumulation ash
The influence of dirt and the lethal spore of mushroom.Plant surface wax is mainly a kind of aliphatic compound, cyclic compound and sterol
The organic mixture general name of class compound.Therefore high wax plant also can be used as one of bioenergy material.Up to the present, it closes
It is also indefinite to the contribution of the degeneration-resistant mechanism of cabbage type rape in epicutile wax, also created by transgenic molecules breeding without any
The report of high wax cabbage type rape.
KCS gene families are the second largest gene families in arabidopsis, there is 21 members ,-CoA condensations of coding β -one acyls
Enzyme is the rate-limiting enzyme in over-long chain fatty acid biosynthetic process.Verify FAE1 (KCS18) gene tune in arabidopsis before this
The synthesis of erucic acid is controlled, KCS6 genes participate in the synthesis of arabidopsis wax, primary alcohol, aldehyde in arabidopsis KCS1 mutant wax components
Class content is reduced.In cabbage type rape KCS gene families may at least 46 members, at present only KCS18 genes are carried out
Relatively go deep into systematic research.
Invention content
Present invention solves the problem in that providing the carrier of a kind of cabbage type rape BnKCS1-2 genes and its structure and answering
With BnKCS1-2 genes can improve Plant cuticle wax content and resistance.
The present invention is to be achieved through the following technical solutions：
A kind of cabbage type rape BnKCS1-2 genes, the gene are the key enzymes that cabbage type rape over-long chain fatty acid is formed
Gene：β -one acyl-CoA synthase genes, nucleotide sequence is as shown in SEQ.ID.NO.1.
A kind of carrier that cabbage type rape BnKCS1-2 is gene constructed is by BnKCS1-2 bases shown in SEQ.ID.NO.1
Because being cloned into the multiple cloning sites of plant expression vector.
Further, which is pCambia-35S-BnKCS1-2 expression vectors, passes through BamHI and SacI restriction enzyme sites
BnKCS1-2 gene clonings are entered into plant expression vector pCambia2301M1DPB.
A kind of preparation method of the transgenic brassica napus based on BnKCS1-2 gene overexpressions, including following operation：
1) BnKCS1-2 gene clonings are entered by plant expression vector by BamHI and SacI restriction enzyme sites
PCambia2301M1DPB builds pCambia-35S-BnKCS1-2 expression vectors；
2) the plant overexpression vector pCambia-35S-BnKCS1-2 built is imported into Agrobacterium；
3) and then using agriculture bacillus mediated Regenerated from Hypocotyl Explants method rape is imported：
Sterile Brassica campestris L seedling hypocotyl is cut into segment, is uniformly inoculated in MSp culture mediums, 28 DEG C of illumination cultivations 2-3 days
Carry out preculture；The hypocotyl of preculture is immersed into OD600For 5-8min in 0.4 or so resuspension Agrobacterium bacterium solution, remove later
Extra Agrobacterium bacterium solution, hypocotyl is inoculated on MSc culture mediums, 28 DEG C of dark culturings 48 hours, is inoculated in MSi trainings later
It supports on base, illumination cultivation 14 days or so at 28 DEG C, until explant both ends grow apparent kanamycin-resistant callus tissue；
Explant with kanamycin-resistant callus tissue is inoculated on MSd culture mediums, illumination cultivation at 28 DEG C, replaces one within 15 days or so
Secondary MSd culture mediums, until kanamycin-resistant callus tissue differentiation budding；
Will differentiation budding explant switching on MSe culture mediums, illumination cultivation at 28 DEG C, until growing stem and blade；
The seedling for growing flourishing stem and leaf is moved on MSr culture mediums, illumination cultivation at 28 DEG C, until growing flourishing root
Until；
Stem, leaf, root all grow normal seedling in MSr culture mediums, open wide culture tank, grow 2-3 days between culture, then
0.1MS fluid nutrient mediums are moved into, wait for its length to 5-6 leaves.
Application of the cabbage type rape BnKCS1-2 genes in preparing the increased transgene rape of epidermis wax content.
Application of the cabbage type rape BnKCS1-2 genes in improving stress resistance of plant.
The application is that cabbage type rape BnKCS1-2 genes are transfected into rape to make its overexpression, changes rape leaf
The variation of wax content improves drought resistance, the disease resistance of rape.
The cabbage type rape BnKCS1-2 genes are in preparing the transgenic brassica napus for bio-fuel
Using.
Compared with prior art, the present invention has technique effect beneficial below：
Cabbage type rape BnKCS1-2 genes provided by the invention are to clone to close from the β -one acyls-CoA of cabbage type rape
Enzyme gene KCS1, nucleotide sequence length 1587bp；The gene mainly cabbage type rape aerial part organ stem, leaf,
It is expressed in flower, silique, the expression quantity highest in blade, this knits the distribution characteristics phase of organ with wax in rape overground part each group
Unanimously, show that this gene may take part in the synthesis of cuticular wax of each histoorgan of cabbage type rape aerial part；
Expression quantity of the BnKCS1-2 in the stem and leaf for having wax powder material is all remarkably higher than wax powder-free material, shows the transcription of BnKCS1-2
Level synthesizes closely related with wax.
The present invention utilizes the gene constructed plant expression vector pCambia-35S-BnKCS1-2 of cabbage type rape BnKCS1-2,
Agrobacterium LBA4404 bacterial strain is imported, then rape is imported using agriculture bacillus mediated Regenerated from Hypocotyl Explants method, after to recombination
Plant be detected, experiments have shown that the rapeseed plants blade wax total amount for being transferred to BnKCS1-2 genes dramatically increases, wax
In component, acids, level-one alcohols, aldehydes, alkanes, secondary alcohols and ketone also dramatically increase.Therefore BnKCS1-2 bases are utilized
When the overexpression of cause promotes the synthesis of plant epidermis wax, it can be used for developing bio-fuel.Experimental result of the present invention, which is also shown that, to be turned
The variation of the rape leaf wax content and component of BnKCS1-2 genes so that plant resistance is enhanced, this is raising plant
Resistance provides new effective selection and feasible method, has a good application prospect.Therefore, cabbage type rape BnKCS1-2
Gene is improving Plant cuticle wax content with resistance with good Utilization prospects.
Description of the drawings
Fig. 1 is expression pattern figures of the BnKCS1-2 in cabbage type rape different tissues organ.
Fig. 2 be BnKCS1-2 genes in having wax powder material double 11 with the differential expression in wax powder-free material NoWax stems, leaf
Ideograph.
Fig. 3 is plant over-express vector structure chart.Wherein, A is pCambia2301M1DPB carrier schematic diagrames；B is
PCambia-35S-BnKCS1-2 carrier schematic diagrames.
Fig. 4 is the PCR qualification result schematic diagrames of BnKCS1-2 transgene rapes.Wherein, 1 is blank control；2 be negative right
According to；3 be positive control；4-22 is the test strip of regeneration plant.
Fig. 5 is that the GUS of BnKCS1-2 transgene rapes dyes qualification figure.
Fig. 6 is that BnKCS1-2 gene overexpressions are analyzed with adjoining tree gene quantification.
Fig. 7 is BnKCS1-2 gene overexpressions and adjoining tree leaf epicuticular wax sedimentation analysis.
Fig. 8 is the signal compared with treated in drought stress with adjoining tree of BnKCS1-2 gene overexpressions rapeseed plants
Figure.
Fig. 9 is BnKCS1-2 gene overexpressions rapeseed plants and adjoining tree blade be inoculated with sclerotinite treated compared with
Schematic diagram.
Specific implementation mode
In order to which clear cabbage type rape epicutile wax synthesis mechanism and its effect in resistance, the present invention have cloned wax
Matter synthesizes the key gene BnKCS1-2, the nucleotide sequence such as SEQ ID of the gene that precursor substance over-long chain fatty acid is formed
NO:Shown in 1.Further the gene is had detected in rape different tissues organ and wax using Real-Time Fluorescent Quantitative PCR Technique
Expression pattern in difference material, and confirm that the gene is promoting wax biosynthesis, providing to resist by rape genetic transformation
Effect in inverse property and function.
With reference to specific embodiment, the present invention is described in further detail, it is described be explanation of the invention and
It is not to limit.Reagent chemicals in present example do not do illustrate be it is common commercially available, MATERIALS METHODS is not done specifically
Bright refers to《Molecular Cloning:A Laboratory guide》(Sambrook and Russell,2000).
Embodiment 1：The clone of rape BnKCS1-2 genes
1, the extraction of rape RNA
Choose double 11 (Southwestern University's rape Industry Technology Center preserves) fresh blade 100mg, liquid nitrogen in cabbage type rape
The extraction of grinding, total serum IgE is extracted using TRIzol reagent methods, and operating procedure refers to the kit explanation of TransGen biotech firms
Book carries out.
2, the synthesis of the first chains of cDNA, utilizes PrimeScriptTMRT reagent Kit (TaKaRa) are synthesized,
Operation illustrates to carry out with reference to used kit.
3, PCR amplification
It is probe with arabidopsis KCS1 gene orders (AT1G02205), is carried out in cabbage type rape genome database
The search of homology sequence obtains relevant 4 homology sequences (SEQ ID NO:2、SEQ ID NO:3、SEQ ID NO:4、
SEQ ID NO:5).According to SEQ ID NO:The following primer of 2 sequence designs.
Forward primer is：5’-CGGATCCATGGAGAGAACAAACAGCATAGAGAT-3’
Wherein, underscore is identified as the sites BamHI of introducing；
Reverse primer is：5’-TGAGCTCTCACTGCGTAATCTTAACCGGATA-3’
Wherein, underscore is identified as the sites SacI of introducing.
Double 11 leaf cDNAs are expanded as template in, obtain the band that size is 1587bp.The amplification system of 50ul
It is as follows：
PCR amplification program is as follows：94 DEG C of pre-degenerations 2min, 94 DEG C of denaturation 30s, 57 DEG C of renaturation 30s, 72 DEG C of extension 1min,
After 30 cycles, 72 DEG C extend 10min, 4 DEG C of 10min.
PCR product carries out glue recycling after purification, to target fragment, is connected to pMD-19T carriers, finally converts Escherichia coli
Competent cell is selected positive clone molecule and is sequenced, and the coding region sequence of β -one acyl-CoA synthase genes KCS1 is obtained
(SEQ ID NO:1), it is named as BnKCS1-2.An equal amount of sequence is obtained by template amplification of genomic DNA, is shown
BnKCS1-2 is free of introne.
Sequence analysis shows that the nucleotide sequence homology of BnKCS1-2 and the code areas arabidopsis KCS1 is 90.5%, ammonia
Base acid sequence homology is 93.6% (having 34 amino acid of differences).
Embodiment 2：The expression analysis of rape BnKCS1-2 genes
1, expression analysis of the BnKCS1-2 genes in each histoorgan of rape
The total serum IgE of double 11 each histoorgan in cabbage type rape is extracted, then reverse transcription cDNA (the same embodiments of method
1), using the first chains of the cDNA of synthesis as template, expression analysis is carried out using real-time quantitative kit (Bio-Rad).
Primer used in quantitative fluorescent PCR is as follows：
Sense primer is：5 '-AGTAAACTGCAGCTTATTCAATCCGAC-3 ',
Downstream primer is：5’-TGTTGGCGAGATCGATTGAGATTAGT-3’.
Reaction system is as follows：
Quantitative fluorescent PCR response procedures are：95℃30s；95 DEG C of 10s, 54 DEG C of 30s, 45 cycles；65 DEG C of 5s, 95 DEG C
30s.Reference genes of the Actin7 (AT5G09810) as detection expression quantity, upstream internal control primer are：5’-
GTGACAATGGAACTGGAATGGTGA-3 ', downstream internal control primer are：5’-GTGCCTAGGACGACCAACAATACTC-3’.
Fluorescent quantitative PCR result (as shown in Figure 1) shows BnKCS1-2 mainly in cabbage type rape aerial part organ
It is expressed in stem, leaf, flower, silique, the expression quantity highest in blade, this knits the distribution of organ with wax in rape overground part each group
Feature is consistent, shows that this gene may take part in the conjunction of cuticular wax of each histoorgan of cabbage type rape aerial part
At.
2, expression analysis of the BnKCS1-2 genes in rape wax difference material
Stem, the leaf RNA of double 11 and NoWax in cabbage type rape are extracted, then reverse transcription cDNA (method is with embodiment 1),
Using the first chains of cDNA of synthesis as template, expression analysis is carried out using real-time quantitative kit (Bio-Rad).Fluorescent quantitation
Primer, PCR response procedures used in PCR are same as above.
Display (as shown in Figure 2) to the gene expression analysis result in wax sedimentary difference material, BnKCS1-2 are having wax
Expression quantity in the stem and leaf of powder material is all remarkably higher than wax powder-free material, shows that the transcriptional level of BnKCS1-2 is synthesized with wax
It is closely related.
This result further illustrates that BnKCS1-2 genes play an important roll in rape wax deposition.
Embodiment 3：The structure of BnKCS1-2 gene plant overexpression vectors
The above-mentioned BnKCS1-2 sequences after sequence verification are scaled off from pMD-19T carriers with BamHI and SacI,
With BamHI and SacI cleaving plant expression vectors pCambia2301M1DPB, (Southwestern University's rape Industry Technology Center is protected simultaneously
Deposit), then it is attached using T4 ligases.The plant expression vector contains 1 set of CaMV35S promoters control NPTII gene
Plant expression elements, the plant expression elements of 1 set of CaMV35S promoter control report genes GUS, 1 set of MAS promoters control
The plant expression elements of the plant expression elements of BAR genes and a set of CaMV35S promoters control targe gene, it can be achieved that Kan,
Triple label screenings of GUS and Basta.It is inserted into foreign gene in multiple cloning sites, the overexpression of foreign gene may be implemented.
The recombinant plant overexpression vector for building successful BnKCS1-2 genes is named as pCambia-35S-BnKCS1-2 (Fig. 3).
Embodiment 4：The identification of the genetic transformation and transgenic positive plant of rape
The plant overexpression vector pCambia-35S-BnKCS1-2 built is imported into Agrobacterium LBA4404 with freeze-thaw method
Bacterial strain, concrete operations are as follows：
The Agrobacterium LBA4404 competent cell that freezen protective is taken out from -80 DEG C, is placed into ice bath and thaws up to just
State.5 μ L recombinant expression carriers pC2301M1DPB-BnKCS1-1 are gently added in 100 μ L Agrobacterium competent cells, are used
Gently mixing, ice-water bath 5min are immersed in cold shock 8min in liquid nitrogen to liquid-transfering gun later, then place it in 37 DEG C of water-baths again
5min, the LB liquid mediums that 900 μ L4 DEG C are preserved are added, and (final concentration of Rif containing antibiotic and Str, the two are respectively 40mg
L-1With 20mg L-1), 28 DEG C of 180rpm shaking table recoveries 3h.By LB solid medium tablets (Kan containing antibiotic, Rif and Str, it
Final concentration be respectively 100mg L-1、40mg L-1With 20mg L-1) be inverted in 28 DEG C of incubators and preheat 1h.Recovery terminates
Bacterium solution 4000rmp is centrifuged into 6min afterwards.900 μ L supernatants are abandoned in suction, remaining bacterium solution liquid-transfering gun gently mixing, then uniform to apply
It is distributed on tablet, 28 DEG C are inverted culture until the bacterium colony for growing suitable size (about 48h).Monoclonal on picking tablet is used
BnKCS1-2 amplimers carry out PCR amplification detection, and the primer, reaction system and PCR programs are the same as embodiment 1.It is detected as sun
Property bacterium solution add sterile glycerol (final volume of glycerine be 40%), -80 DEG C save backup.
Then rape is imported using agriculture bacillus mediated Regenerated from Hypocotyl Explants method.The specific method is as follows：
(1) aseptic seedling is sent out：With clear water impregnate it is full in double 11 seed 3h, 75% ethyl alcohol sterilizes 5min, 10% secondary chlorine
Sour sodium rinses 10min, and rinsed with sterile water 5-6 times is seeded on MSg culture mediums, 28 DEG C of illumination cultivations 6 days.
(2) preculture：The aseptic seedling hypocotyl of previous step is cut into the segment of 0.5cm or so, is uniformly inoculated in MSp trainings
Support base, 28 DEG C of illumination cultivations 2-3 days.
(3) it infects and co-cultures：(Kan 100mg L are added in YEB fluid nutrient mediums-1、Str40mg L-1、Rif 60mg L-1) in inoculation Agrobacterium (1% volume inoculum concentration), 28 DEG C, 250rmp cultivate 2 days or so activate；It is (same to YEB fluid nutrient mediums
On) in the Agrobacterium that newly activates of inoculation, 28 DEG C, 250rmp cultivate 6-8 hours to OD600Value be 0.8 or so, room temperature 5000rmp from
Heart 9min removes supernatant, and MSm fluid nutrient mediums, resuspended bacterium solution to OD is added600It is 0.4 or so.By the hypocotyl of preculture
8min in above-mentioned resuspended bacterium solution is immersed, extra bacterium solution is sucked, hypocotyl is positioned on MSc culture mediums, 28 DEG C of dark culturings
48 hours.
(4) Agrobacterium and induction of resistance callus are killed：Hypocotyl of the previous step in MSc culture mediums after dark culturing is uniform
Neat is placed on MSi culture mediums, illumination cultivation 14 days or so at 28 DEG C, until explant both ends grow apparent kanamycin-resistant callus tissue
Until tissue.
(5) differentiation culture：Hypocotyl with kanamycin-resistant callus tissue on MSi culture mediums is transferred on MSd culture mediums, at 28 DEG C
Illumination cultivation replaces a MSd culture medium in 15 days or so, until kanamycin-resistant callus tissue differentiation budding.
(6) long shoot：The explant for breaking up budding on MSd culture mediums is transferred in MSe culture mediums, illumination cultivation at 28 DEG C,
To growing stem and blade.
(7) it takes root：The seedling that flourishing stem and leaf are grown on MSe culture mediums moves in MSr culture mediums, illumination training at 28 DEG C
It supports, until growing flourishing root.
(8) domestication and transplanting：Stem, leaf, root all grow normal seedling in MSr culture mediums, culture tank are opened wide, between culture
Growth 2-3 days, then moves into 1/10MS fluid nutrient mediums, waits for its length to 5-6 leaves.If positive season, then transplant to solarium just
It is frequently grown；If not positive season then needs vernalization 14-24 days in growth cabinet, then moves into phjytotron.
Culture medium used in the present embodiment is as follows：
Send out MSg culture mediums used in aseptic seedling：MS powder 41.74g L-1, 1L, 121 DEG C of height are settled to after adjusting pH value to 5.8-6.0
It is spare after temperature sterilizing 25min.
MSp culture mediums used in preculture：MS powder 41.74g L-1、2,4-D 1mg L-1、6-BA 1mg L-1, adjust pH value extremely
It is settled to 1L after 5.8-6.0, it is spare after 121 DEG C of high-temperature sterilization 25min.
It infects and uses MSm fluid nutrient mediums：MS powder (without agar and sucrose) 4.74g L-1, sucrose 30g L-1、2,4-D
1mg L-1、6-BA 1mg L-1, it is standby to be down to 100 μM of AS of room temperature addition by pH=5.8-6.0,121 DEG C of autoclave sterilization 25min
With.
Co-culture MSc culture mediums used：MS powder 41.74g L-1、2,4-D 1mg L-1、6-BA 1mg L-1, 50 μM of AS,
It is settled to 1L, 121 DEG C of high-temperature sterilization 25min after adjusting pH value to 5.8-6.0, it is standby that 50 μM of AS are added after being down to 70 DEG C in culture medium temperature
With.
MSi culture mediums used in induction of resistance callus：MS powder 41.74g L-1、2,4-D 1mg L-1、6-BA 1mg L-1、Kan
50mg L-1、Cef500mg L-1, 1L, 121 DEG C of high-temperature sterilization 25min, culture medium temperature are settled to after adjusting pH value to 5.8-6.0
50mg L are added after being down to 70 DEG C-1Kan and 500mg L-1Cef is spare.
Differentiation culture MSd culture mediums used：MS powder 41.74g L-1、ZT 2mg L-1、6-BA4mg L-1、Kan 50mg L-1、Cef500mg L-1、AgNO35mg L-1, 1L, 121 DEG C of high-temperature sterilization 25min are settled to after adjusting pH value to 5.8-6.0, are cultivated
50mg L are added after being down to 70 DEG C in base temperature-1Kan、5mg L-1AgNO3With 500mg L-1Cef is spare.
MSe culture mediums used in long shoot：MS powder 41.74g L-1、6-BA 0.05mg L-1、Kan 50mg L-1、Cef300mg
L-1, it is settled to 1L, 121 DEG C of high-temperature sterilization 25min after adjusting pH value to 5.8-6.0,50mg is added after being down to 70 DEG C in culture medium temperature
L-1Kan and 300mg L-1Cef is spare.
MSr culture mediums used in taking root：MS powder 41.74g L-1、IBA 5mg L-1、Cef300mg L-1, adjust pH value to 5.8-
1L, 121 DEG C of high-temperature sterilization 25min are settled to after 6.0,300mg L are added after being down to 70 DEG C in culture medium temperature-1Cef is spare.
YEB fluid nutrient mediums：Yeast 1g L-1, peptone 5g L-1, sucrose 5g L-1、MgSO4H2O 5g L-1, pH=
7.0,121 DEG C of autoclave sterilization 25min, 4 DEG C save backup.
Carrying out positive identification to the transfer-gen plant of acquisition can take histochemical stain identification to reflect with PCR amplification respectively
It is fixed.PCR amplification is identified：It takes rotaring gene plant blade to extract genomic DNA, template is done with the DNA of extraction, utilizes sense primer
5 '-TAAAGGGACCACCTATGA-3 ' and downstream primer 5 '-GGACGCAGAAGGCAAT-3 ' carry out selectable marker gene
(NPTII) it expands, in double 11 gDNA makees negative control, using water as blank control, with pCambia2301M1DPB plasmids
As positive control.PCR programs are as follows:94 DEG C of pre-degenerations 5min, 94 DEG C of denaturation 30s, 57 DEG C of renaturation 30s, 72 DEG C of extension 1min,
After 30 cycles, 72 DEG C extend 10min, 4 DEG C of 10min.Agarose electrophoresis testing result shows that most of transformed plants can
Amplify the band (527bp) (Fig. 4) of expected size.
Histochemical stain is identified：It takes a fritter blade of overexpression regeneration plant to be put into 1.5mL centrifuge tubes, is added
The GUS dye liquors of 200 μ L dye 24 hours in 37 DEG C, then with being observed after 75% ethanol decolorization, the results are shown in Figure 5, and transgenosis is planted
The GUS dyeing of strain blade is the positive.
Embodiment 5：Detect expression variation of the BnKCS1-2 genes in transgene rape
According to the method for extracting rape RNA in embodiment 1, extraction transgene rape and the total serum IgE for compareing rape leaf, and
Synthesize mono- chains of cDNA.The expression of target gene in transgene rape is analyzed with fluorescence quantifying PCR method.PCR system and amplification journey
Sequence is same as Example 2.Quantitative result is as shown in fig. 6, the expression quantity of BnKCS1-2 genes is notable in overexpression transfer-gen plant
Up-regulation.This result illustrates the transgene rape plant for having obtained overexpression BnKCS1-2.
Embodiment 6：The blade wax of transgene rape plant and adjoining tree is analyzed
BnKCS1-2 overexpression strains are taken to carry out waxy component, content analysis with adjoining tree blade, concrete operations are as follows：
Plant leaf is acquired in Boiling tube, 5ml chloroforms are added, in being extracted on turbula shaker twice, each 30s collects extracting solution.
A concentration of 1mg mL of 10ul are previously added in chloroform-124 alkane as internal standard.Leaching liquor is dried up with nitrogen evaporator (in 45 DEG C),
With 20 μ L BSTFA (N, O-Bis (trimethylsily) Trifluoroa-cetamide, BSTFA) and 20 μ L at 70 DEG C
Pyridine derived 45min dries up derivating agent in nitrogen evaporator, and product is dissolved in 50 μ L chloroforms.Carry out gas-chromatography and mass spectrum point
Analysis.
Gas-chromatography (GC) capillary column length 30m, diameter 0.32mm, 0.25 μm of thickness of liquid film；Nitrogen is as carrier gas；Sample introduction
Amount is 1.0 μ l.The temperature of column film and fid detector is respectively 300 DEG C and 320 DEG C.Temperature-programmed mode：80 DEG C of initial temperature,
Constant temperature time 0.1s rises to 245 DEG C with 15 DEG C per minute, keeps 5min；Then 325 DEG C are warming up to 2 DEG C per minute, kept
12min.Wax quantization is based on FID peak values, goes out peak position according to what the standard that mass spectrum is identified determined various chemical compositions in chromatography
It sets, the practical wax content of plant units area is calculated with internal standard, unit is μ g cm–2.Leaf area utilizes digital scanner
(EPSON V750) and WinFOLIA blade specialized images analysis software (Regent Instrument Inc, Canada) carry out
It measures and records.
GC-MS analysis results are (as shown in Figure 7) to be found, the wax composition of transgenosis and adjoining tree mainly by alkanes,
Primary alcohol, secondary alcohols, acids, ketone and aldehydes composition, but wax total amount and the content of each component have differences.BnKCS1-2's
Overexpressing plant compared with the control, each component content dramatically increases, and wherein acids, level-one alcohols increase 28% -90%,
Aldehydes, alkanes, secondary alcohols, ketone and wax total amount increase 28% -91% compared with the control.
Embodiment 7：The resistance of transgene rape plant is identified
Drought stress processing is carried out to turning BnKCS1-2 rapeseed plants and adjoining tree respectively, as seen from Figure 8, is coerced in arid
After compeling processing 7d, it is overexpressed plant and slightly wilts, and adjoining tree is seriously wilted, and after drought stress is handled 11 days, compares seedling
It has been stopped growing that, all blades have dried up wilting, and are overexpressed plant shoots and remain to grow, and keep a piece of greenery.Arid
After coercing rehydration, it is overexpressed plant restoration ecosystem, and adjoining tree seedling is thoroughly withered dead.
Take be overexpressed in plant and control double 11 blades respectively its vein both sides respectively one nuclear fungal hyphae block of inoculation (
Cover with to be beaten with the sterilization punchers of diameter 0.7cm on the PDA plate of nuclear fungal hyphae and take mycelia block), the results showed that, it is planted with compareing
Strain blade is compared, and is overexpressed the blade morbidity evening of plant, scab is small, and Development process is slow.
The variation for the rape leaf wax content and component that result above prompt turns BnKCS1-2 genes so that plant is degeneration-resistant
Property enhanced, this provides new effective select and feasible method to improve stress resistance of plant.
Therefore the present invention proposes following application：
Application of the cabbage type rape BnKCS1-2 genes in preparing the increased transgene rape of epidermis wax content.
Application of the cabbage type rape BnKCS1-2 genes in improving stress resistance of plant.
Specifically, being that cabbage type rape BnKCS1-2 genes are transfected into rape to make its overexpression, change rape leaf
The variation of wax content improves drought resistance, the disease resistance of rape.
The cabbage type rape BnKCS1-2 genes are in preparing the transgenic brassica napus for bio-fuel
Using.
Example given above is to realize the present invention preferably example, and the present invention is not limited to the above embodiments.This field
Technical staff any nonessential addition, the replacement made according to the technical characteristic of technical solution of the present invention, belong to this
The protection domain of invention.
Sequence table
<110>Southwestern University
<120>Carrier and the application of a kind of cabbage type rape BnKCS1-2 genes and its structure
<160> 5
<170> SIPOSequenceListing 1.0
<210> 1
<211> 1587
<212> DNA
<213>Cabbage type rape (Brassica napus L.)
<400> 1
<210> 2
<211> 1587
<212> DNA
<213>Cabbage type rape (Brassica napus L.)
<400> 2
<210> 3
<211> 1586
<212> DNA
<213>Cabbage type rape (Brassica napus L.)
<400> 3
<210> 4
<211> 1584
<212> DNA
<213>Cabbage type rape (Brassica napus L.)
<400> 4
<210> 5
<211> 1586
<212> DNA
<213>Cabbage type rape (Brassica napus L.)
<400> 5
Claims (8)
1. a kind of cabbage type rape BnKCS1-2 genes, which is characterized in that the gene is cabbage type rape over-long chain fatty acid shape
At key gene：β -one acyl-CoA synthase genes, nucleotide sequence is as shown in SEQ.ID.NO.1.
2. a kind of carrier that cabbage type rape BnKCS1-2 is gene constructed, which is characterized in that being will be shown in SEQ.ID.NO.1
BnKCS1-2 gene clonings enter the multiple cloning sites of plant expression vector.
3. carrier gene constructed cabbage type rape BnKCS1-2 as claimed in claim 2, which is characterized in that the carrier is
BnKCS1-2 gene clonings are entered plant by pCambia-35S-BnKCS1-2 expression vectors by BamHI and SacI restriction enzyme sites
Expression vector pCambia2301M1DPB.
4. a kind of preparation method of the transgenic brassica napus based on BnKCS1-2 gene overexpressions, which is characterized in that including
It operates below：
1) BnKCS1-2 gene clonings are entered by plant expression vector by BamHI and SacI restriction enzyme sites
PCambia2301M1DPB builds pCambia-35S-BnKCS1-2 expression vectors；
2) the plant overexpression vector pCambia-35S-BnKCS1-2 built is imported into Agrobacterium；
3) and then using agriculture bacillus mediated Regenerated from Hypocotyl Explants method rape is imported：
Sterile Brassica campestris L seedling hypocotyl is cut into segment, is uniformly inoculated in MSp culture mediums, 28 DEG C of illumination cultivations carry out for 2-3 days
Preculture；The hypocotyl of preculture is immersed into OD600For 5-8min in 0.4 or so resuspension Agrobacterium bacterium solution, it is extra to remove later
Agrobacterium bacterium solution, hypocotyl is inoculated on MSc culture mediums, 28 DEG C of dark culturings 48 hours are inoculated in MSi culture mediums later
On, illumination cultivation 14 days or so at 28 DEG C, until explant both ends grow apparent kanamycin-resistant callus tissue；
Explant with kanamycin-resistant callus tissue is inoculated on MSd culture mediums, illumination cultivation at 28 DEG C, is replaced within 15 days or so primary
MSd culture mediums, until kanamycin-resistant callus tissue differentiation budding；
Will differentiation budding explant switching on MSe culture mediums, illumination cultivation at 28 DEG C, until growing stem and blade；
The seedling for growing flourishing stem and leaf is moved on MSr culture mediums, illumination cultivation at 28 DEG C, until growing flourishing root and being
Only；
Stem, leaf, root all grow normal seedling in MSr culture mediums, open wide culture tank, grow 2-3 days, then move between culture
0.1MS fluid nutrient mediums wait for its length to 5-6 leaves.
5. cabbage type rape BnKCS1-2 genes described in claim 1 are in preparing the increased transgene rape of epidermis wax content
Application.
6. application of the cabbage type rape BnKCS1-2 genes described in claim 1 in improving stress resistance of plant.
7. application as claimed in claim 6, which is characterized in that be that cabbage type rape BnKCS1-2 genes are transfected into rape
Make its overexpression, changes the variation of rape leaf wax content, improve drought resistance, the disease resistance of rape.
8. cabbage type rape BnKCS1-2 genes described in claim 1 are preparing the transgenic cabbages type oil for bio-fuel
Application in dish.
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Cited By (1)
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