CN104988176A - Method for increasing gel content of eucommia ulmoides - Google Patents
Method for increasing gel content of eucommia ulmoides Download PDFInfo
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Abstract
The invention relates to a method for increasing the gel content of eucommia ulmoides. The method includes the following steps: firstly, phloem of eucommia ulmoides bark is peeled off to form multiple cells, and cambium on the surface is exposed out in an interval cell shape; secondly, the dip dyeing operation is conducted, wherein bacterium liquid cultivated through single agrobacterium colonies containing EuTIDS5 recombinant expression vectors is picked to infect the cambium on the surface of peeled eucommia ulmoides; and thirdly, the peeled face is wrapped with a plastic film after peeling and infecting are conducted, and moisture preservation and sun protection growth is achieved. The infecting method is simple, feasible and excellent in gel content increasing effect.
Description
Technical field
The present invention relates to a kind of method improving bark of eucommia gel content, be specifically related to imported by specific gene and improve the method for bark of eucommia gel content.
Background technology
The bark of eucommia (Eucommia ulmoides Oliv.) is the distinctive famous and precious Medicinal species of China, and all containing a large amount of white rope-Chinese gutta percha (Eucommia ulmoides Rubber in the medium each tissue of bark, root, stem, leaf, flower and fruit, EUR), therefore the bark of eucommia is described as the natural rubber seeds of high-quality.Rubber is the important strategic material resource of China, and rubber industry is one of most important basic industry of Chinese national economy.Para rubber is very narrow in territory, China normal region, and production capacity reaches the limit.Domestic natural rubber demand is considerably beyond output in domestic, and the scarcity of natural rubber resource restricts the development of China's Rubber Industry, therefore develops the second natural rubber resource extremely urgent.Chinese gutta percha is the terpene macromolecular compound that bark of eucommia secondary metabolism approach produces, different from para rubber, the structure of Chinese gutta percha is using trans-polyisoprene, exclusive " rubber-plastic duplicity ", its low-temp plastic, thermo-elasticity, blended property, high-insulativity and erosion resistance aspect are considerably beyond para rubber, with the rubber combination of Chinese gutta percha modification, there is the advantages such as wear-resisting, corrosion-resistant, energy-conservation, multiple fields such as rubber industry, aerospace, military project, boats and ships, chemical industry, medical treatment can be widely used in.But because Eucommia ulmoides Oliv. leaves gel content is low, cause production cost to remain high, become the bottleneck of Chinese gutta percha industrialization.
The most basic approach improving Chinese gutta percha output is exactly disclose the gene regulating mechanism in the biosynthetic process of Chinese gutta percha.Chinese gutta percha is as a kind of high molecular using trans-polyisoprene, under a kind of special prenyltransferase effect, continuous for lower molecular weight trans-isoprene condensation is formed, and the biosynthesizing of lower molecular weight isoprene is continuous condensation by isopentenyl pyrophosphate (IPP) and dimethyl propylene thiazolinyl tetra-sodium (DMAPP) and forms (Nishibe-S.Bioactive lignans and flavonoids from traditional medicines [J] .Polyphenols the International Conference, 1995, 113-122.).At present, identify the gene that several participate in trans rubber biosynthesis, as important hevein encoding gene (MLP), rubber elongation factor (RFF), rubber grain membranin (RPMP), small-sized rubber grain albumen (SRPP), (the Michel Rohmer.The discovery of a mevalonate-independent pathway for isoprenoid biosynthesis in bacteria such as trans-prenyl pyrophosphate synthase gene (TIDS), algae and higher plants [J] .Nat.Prod.Rep, 1999, 16:565-574.Lytle BL etc., Structures of two Arabidopsis thaliana major latex proteins represent novel helix grip folds [J] .Proteins, 2009, 76:237-243.Nessler CL etc., Organization of the major latex protein gene family in opium poppy [J] .Plant Mol Biol, 1992, 20:749-752.Nobuaki Suzuki etc., Construction and analysis of EST libraries of the transpolyisoprene producing plant, Eucommia ulmoides Oliver.Planta [J] .2012, DOI10.1007/s00425-012-1679-x.).Although carried out clone and gene transformation to Chinese gutta percha synthesis related gene, not yet disclose the application method of genes involved.The present invention has filtered out the key gene of Chinese gutta percha synthesis, and devises genetically modified exhaust processes, increases substantially Chinese gutta percha content from genetic level.The method is simple, improves the gel content of newborn perithelium, and to reduction Chinese gutta percha production cost, promote the fast development of Chinese gutta percha industry, the demand meeting China's Rubber Industry has great importance.
Summary of the invention
In order to solve the problem to a certain extent, the object of the invention is to utilize genetic expression rule and Chinese gutta percha synthesising law, propose a kind of Chinese gutta percha key gene, and pass through genetic transformation exhaust processes by this gene transformation in the bark of eucommia, obtain transfer-gen plant and pass through biological function verification, show that EuTIDS5 gene that the present invention clones significantly increases the gel content of the bark of eucommia, namely the invention provides a kind of method improving bark of eucommia gel content.
In order to realize above object, the present invention by the following technical solutions:
According to Gene Clone in Plant clone EuTIDS5 gene, Real-Time PCR is utilized to detect the expression of this gene at different times different tissues, Soxhlet extraction method is utilized to extract Chinese gutta percha, analyze Chinese gutta percha synthesising law, thus determine that the key gene that Chinese gutta percha synthesizes is EuTIDS5.Utilize the Agrobacterium-mediated genetic transformation bark of eucommia, and by the transfer-gen plant that obtains through biological function verification, show that EuTIDS5 gene that the present invention clones has the function of regulation and control Chinese gutta percha synthesis.Case study on implementation part of the present invention, will describe the separating clone of the key gene EuTIDS5 gene of rubber synthesis in detail, the method for expression rule and functional verification.
EuTIDS5 gene can derive from the common bark of eucommia kind of China, such as, can be kind listed in Table:
One aspect of the present invention there is provided a kind of method improving bark of eucommia gel content, comprises the following steps:
1) peeling of eucommia bark phloem is become multiple grid, at interval every the form layers of shape exposing surface;
2) carry out dip-dye operation: the bacterium liquid that the single bacterium colony of Agrobacterium picking being contained EuTIDS5 recombinant expression vector is cultivated, infect the surperficial form layers of barked eucommia;
3) peeling is infected rear plastic film wrapped and is shelled face, the sun-proof growth of moisturizing.
Further, single lattice area of described grid is 2-10cm
2, be more preferably 3-6cm
2, described grid can be different shape, is 2cm × 2cm grid as a preferred embodiment.
Further, step 2) described in the OD of bacterium liquid
600value is 0.5 ~ 0.9.
Further, step 3) the plastic film wrapped stripping face time is 20 ~ 100 days, can be such as 20 days, 40 days or 50 days or 60 days or 70 days or 80 days or 90 days or 100 days, be more preferably 30 ~ 90 days, more preferably 40 ~ 80 days.
Further, step 2) dip-dye operation carry out 2-5 time, every minor tick 2-4 days, preferred dip-dye operates carries out 3 times, every minor tick 3 days.
Further, the single bacterium colony of the described Agrobacterium containing recombinant expression vector obtains by the following method: added by the plasmid containing EuTIDS5 in band Agrobacterium tumefaciens strain GV3101 competent cell, join after softly mixing carry out in the electroporated cup of precooling electroporated; After electroporated, the competent cell matter with plasmid is added substratum, cultivate; Get liquid, be evenly coated in containing on corresponding antibiotic solid medium, be inverted and cultivate; Picking list bacterium colony shakes bacterium, shakes bacterium to growing logarithmic phase.A preferred embodiment is: added by the plasmid that 1 μ L contains EuTIDS5 in band Agrobacterium tumefaciens strain GV3101 competent cell, join after soft mixing carry out in the electroporated cup of precooling electroporated; After electroporated, the competent cell matter with plasmid is added 200 μ L liquid nutrient mediums, 28 DEG C, 180rpm, cultivate 2h; Get 100 μ L bacterium liquid even, even spread, to containing on corresponding antibiotic solid medium, is inverted for 28 DEG C and is cultivated 48h; Picking list bacterium colony shakes bacterium, 28 DEG C, 180rpm, shakes bacterium 24h to growing logarithmic phase.
Further, the preparation method of the described plasmid containing EuTIDS5 is: by the method amplification EuTIDS5 of PCR, connect Plastid transformation cultivation and extract the plasmid obtained containing EuTIDS5 afterwards.A preferred embodiment is: cloned by the method for PCR, and the PCR system of described clone sees the following form:
PCR cloning reaction condition is: 98 DEG C of denaturation 1min; 98 DEG C of sex change 10s, 55 DEG C of annealing 15s, 68 DEG C extend 2min, 8 circulations; 98 DEG C of sex change 10s, 68 DEG C extend 2min, 32 circulations; 68 DEG C extend 10min;
Ligation product conversion to intestinal bacteria, and extracts plasmid.
Further, the encoding sequence of EuTIDS5 is as shown in SEQ ID NO:2.
Further, the upstream primer (5'-3') of pcr amplification EuTIDS5 and downstream primer (5'-3') are respectively as shown in SEQ ID NO:3 and 4.
Advantage of the present invention is: can significantly improve bark of eucommia gel content.
Accompanying drawing explanation
Fig. 1: different times Eucommia ulmoides Oliv. leaves and fruit gel content.
Fig. 2: Eucommia ulmoides Oliv. leaves and fruit are at the expression amount of different times EuTIDS5.
Fig. 3: EuTIDS5 relative expression quantity and the rule containing glue rate of rise in different times Eucommia ulmoides Oliv. leaves.
Fig. 4: EuTIDS5 relative expression quantity and the rule containing glue rate of rise in different times Eucommia Fruit.
Fig. 5: transgenic tobacco plant PCR detects electrophorogram.
Fig. 6: the relative expression quantity of the different strain 35s::EuTIDS5 of transgene tobacco.
Fig. 7: the conversion of eucommia bark.
Fig. 8: transgenic trees epidermis type analysis.
Embodiment
Below in conjunction with embodiment, the specific embodiment of the present invention is described, so that those skilled in the art better understand the present invention, and when not departing from spirit and scope of the invention, various change and amendment can be made to the present invention, use different purposes and condition to make it.
The bark of eucommia kind that following examples relate to is the 12001X deriving from Shennongjia, Hubei Province.
For the ease of better understanding the present invention, provide the embodiment of relevant rudimentary experiment below in the lump together with embodiments of the invention, the core content that the present invention relates to refers to embodiment 8-10, and embodiment 1-7 is that the present invention may be better understood in relevant rudimentary test.The rubber content of embodiment 1 different times Eucommia ulmoides Oliv. leaves and fruit
The present invention adopts the method for soxhlet extraction (referring to Overexpression of an isopentenyl diphosphateisomerase gene to enhance trans-polyisopreneproduction in Eucommia ulmoides Oliver [J] .2012, the 12:78 such as Ren Chen) to measure the content of the polyisoprene of the different tissue (fruit and blade) of different times [lasting 5 months to fruit maturation (late August) from after pollination (at the beginning of 4 months)].Result shows, and As time goes on and constantly the gel content of different times Eucommia ulmoides Oliv. leaves and fruit all increases, and illustrates that the synthesis of Chinese gutta percha is a process of increasing accumulation gradually.After pollination 50d, fruit rubber content increases sharply the bark of eucommia this (mid-April) to mid-May in period, and the rubber content subsequently in fruit slowly increases, and gel content tends to be steady gradually.Relative to Eucommia Fruit, the gel content in Eucommia ulmoides Oliv. leaves is always in time in increasing more stably.Contemporaneity, the rubber content of Eucommia Fruit is 1.2-3.2 times (referring to Fig. 1) of rubber content in blade.
The separating clone of embodiment 2 EuTIDS5 gene
Utilize bark of eucommia transcript profile database, obtain about EuTIDS5 mono-section of sequence.The present invention adopt the method for RACE utilize 5 ' of Taraka company and 3 '-RACE test kit (TaKaRa, Dalian, China) amplify the complete sequence of EuTIDS5.RNA extracts and adopts QiAgen company RNA to extract test kit (RNeasy Plant mini Kit, QiAgen, Hilden, Germany), and the synthesis of cDNA first chain is according to the SuperScript of Inviterogen company
tMiII First-Strand Synthesis System (Life technologies, Carlsbad, California, U.S.) Reverse Transcription box, all operates according to test kit specification sheets step.
The primer adopting PrimerPremier 5.0 to design EuTIDS5 sequence increases, and primer is as follows:
Core fragment forward (SEQ ID NO:5):
5’-AAGGTTGGGATGATTGCGAT-3’
Core fragment reverse (SEQ ID NO:6):
5’-TTTCACGACTAACCAAGTGC-3’
5’RACE OuterPrimer(SEQ ID NO:7):
5’-CATGGCTACATGCTGACAGCCTA-3’
5’RACE InterPrimer(SEQ ID NO:8):
5’-CGCGGATCCACAGCCTACTGATGATCAGTCGATG-3’
5 ' RACE reverse (SEQ ID NO:9):
5’-TGTCCTTCATGCCACTATGAGTTTCTAG-3’
3 ' RACE forward (SEQ ID NO:10):
5’-GAGGAAGCATTTTCGGACCAAACCTT-3’
3’RACE OuterPrimer(SEQ ID NO:11):
5’-TACCGTCGTTCCACTAGTGATTT-3’
3’RACEInterPrimer(SEQ ID NO:12):
5’-CGCGGATCCTCCACTAGTGATTTCACTATAGG-3’
Pcr amplification reaction system is as follows:
Core fragment: Primer 1 (10 μm of olL of the Taq PCR Mastermix of 12.5 μ L, 1 μ L
-1) and Primer 2 (10 μm of olL
-1), the template DNA of 2 μ L, the sterilizing ddH of 8.5 μ L
2o.
3 ' RACE and 5 ' RACE adopts nest-type PRC to increase.
3 ' the RACE Outer Primer (10 μMs) of 1 × cDNA Dilution Buffer II of the cDNA of 3 ' RACE:2 μ L, 8 μ l, 2 μ L, 10 × LA PCR Buffer II (Mg2 of 3 ' the RACE forward Primer of 2 μ L, 4 μ L
+free), the MgCl of 3 μ L
2(25mM), the TaKaRa LA of 0.25 μ L
(5U/ μ l), the ddH of 28.75 μ L
2o.5 ' the RACE Outer Primer (10 μMs) of 1 × cDNA Dilution Buffer II of the cDNA of 5 ' RACE:2 μ L, 8 μ L, 2 μ L, 10 × LA PCR Buffer II (Mg2 of the reverse Primer of 5 ' RACE of 2 μ L, 4 μ L
+free), the MgCl2 (25mM) of 3 μ Ll, the TaKaRa LA of 0.25 μ L
(5U/ μ l), the ddH of 28.75 μ L
2o.
Pcr amplification condition is:
Core fragment: 94 DEG C of denaturation 3min; 94 DEG C of sex change 30s, 50 DEG C of annealing 30s, 72 DEG C extend 1min, 30 circulations, 72 DEG C of total elongation 5min.
3’RACE：
1,94 DEG C of denaturation 3min; 94 DEG C of sex change 30s, 52 DEG C of annealing 30s, 72 DEG C extend 1min, 30 circulations, 72 DEG C of total elongation 5min.
2, obtain PCR primer described in 1 and dilute 10 times as masterplate
94 DEG C of denaturation 3min; 94 DEG C of sex change 30s, 55 DEG C of annealing 30s, 72 DEG C extend 1min, 30 circulations, 72 DEG C of total elongation 5min.
5’RACE：
1,94 DEG C of denaturation 3min; 94 DEG C of sex change 30s, 55 DEG C of annealing 30s, 72 DEG C extend 1min, 30 circulations, 72 DEG C of total elongation 5min.
2, obtain PCR primer described in 1 and dilute 10 times as masterplate
94 DEG C of denaturation 3min; 94 DEG C of sex change 30s, 58 DEG C of annealing 30s, 72 DEG C extend 1min, 30 circulations, 72 DEG C of total elongation 5min
Pcr amplification product reclaims and operates according to the Taraka MiniBEST Agarose Gel DNA Extraction Kit test kit specification sheets step of Taraka company.The object band obtained increasing links with pMD18-T carrier after running gel reclaims, and reaction system is as follows: the md18-T Vertor of 0.5 μ L, the sert DNA of 2 μ L, the Solution of 2.5 μ L I, 4 DEG C, link of spending the night.Get 1 μ L connection product and add intestinal bacteria (Escherichia coil) DH5 α competent cell, after leaving standstill 30min on ice, after 42 DEG C of thermal shock 50s, put into rapidly 2min on ice, then 200 μ L liquid nutrient mediums (not containing microbiotic) are added, 37 DEG C of shaking culture 1h (180r/min), get the bacterium liquid uniform application of 100 μ L shaking culture on the LB solid medium containing penbritin, be placed in 37 DEG C and be inverted overnight incubation (12-16h), random picking list bacterium colony send company (U.S. calm and peaceful Bioisystech Co., Ltd) to check order.Spliced by sequencing result, shown by comparison, it is the goal gene needed that the present invention clones EuTIDS5 gene, and full length sequence is as shown in SEQ ID NO:1.EuTIDS5 gene coding region is 43-1092, and total length is 1050bp (as shown in SEQ ID NO:2), and 349 amino acid (see sequence table 2) of encoding, molecular weight is 40.29kD, and iso-electric point is 5.40, is the hydrophilic protein of instability.The aminoacid sequence of the EuTIDS5 gene that the display of Multiple Sequence Alignment result is derived and known plant sequence very high homology.
The expression rule analysis of embodiment 3 EuTIDS5
In order to analyze EuTIDS5 gene in the different expression of tissue of different times and the relation of rubber content, the present invention adopts real-time quantitative PCR (Real-Time PCR), reacts the SYBR Premix Ex Taq according to TaKaRa company
tMtest kit specification sheets operates, and adopts two-step approach (amplification curve and solubility curve curve) to increase.Analyze Eucommia ulmoides Oliv. leaves and the fruit relative expression quantity at different times EuTIDS5, the RNA of Eucommia ulmoides Oliv. leaves and fruit extract and cDNA synthesis with embodiment 1.The primer is house-keeping gene ACTIN forward (SEQ ID NO:13):
5’-TGAGATGCACCACGAAGCTC-3’
Oppositely (SEQ ID NO:14):
5’-CCAACATTGTCACCAGGAAGTG-3’,
And gene-specific primer forward (SEQ ID NO:15):
5’–ACTTGGTTAGTCGTGAAAGC-3’，
Oppositely (SEQ ID NO:16): 5 '-TAAAGCTCCTTCACTTTTGC-3 '.
Result shows: EuTIDS5 has expression (referring to Fig. 2) in the Eucommia ulmoides Oliv. leaves and fruit of different times, wherein mid-April to mid-May EuTIDS5 expression amount apparently higher than other periods, and the same period fruit EuTIDS5 gene expression amount exceed more than 90% than at blade, EuTIDS5 gene is highly expressed at young fruit period, meet the phenomenon that the gel content of the fruit same period is higher than blade, therefore infer that the expression of EuTIDS5 gene is synthesized relevant to Chinese gutta percha.EuTIDS5 gene relative expression quantity in different times Eucommia ulmoides Oliv. leaves and fruit is analyzed containing the Changing Pattern of glue rate of rise in conjunction with different times Eucommia ulmoides Oliv. leaves and fruit, the expression rule of EuTIDS5 gene in Eucommia ulmoides Oliv. leaves and fruit of different times all fits like a glove with Eucommia ulmoides Oliv. leaves and fruit rubber synthesising law and (refers to Fig. 3, Fig. 4), and the expression amount of EuTIDS5 gene when rubber Fast back-projection algorithm period in fruit is higher than blade, the gel content meeting the fruit same period is greater than the rule of blade, therefore specify that EuTIDS5 gene is the key gene of Chinese gutta percha synthesis.
The structure of embodiment 4 plant expression vector
The present invention adopts Gateway method to build rubber synthesis key gene Overexpression vector 35S::EuTIDS5 and rna interference vector RNAi-EuTIDS5.Structure according to Gateway carrier (it has been that the circulation of major research mechanism of China uses, is given here by the secondary research institute of China Forestry Science Research Institute Chen Jun) requires to build: the clone of object fragment, entry vector builds, the structure of expression vector.
1, the clone of object fragment
Utilize Primer Premier 5.0 to design primer and add following (underscore part is Gateway joint sequence) the overexpression primer of sequence with Gateway joint at primer two ends:
Forward (SEQ ID NO:17):
5’–
GGGGACAACTTTGTACAAAAAAGTTGGAATGGCGGAAACGACCCA-3’
Oppositely (SEQ ID NO:18):
5’–
GGCGGCCGCACAACTTTGTACAAGAAAGTTGGGTATCAATAATGCCTCCGATAGATCTT-3’。
RNAi primer:
Forward (SEQ ID NO:19):
5’-
GGGGACAACTTTGTACAAAAAAGTTGGAATGGCGGAAACGACCCA-3’
Oppositely (SEQ ID NO:20):
5’-
GGCGGCCGCACAACTTTGTACAAGAAAGTTGGGTACGGACCAAACCTTATTATCT-3’，
The clone of gene object fragment is carried out by pcr amplification.
Gateway PCR reaction system: 25 μ L's
primer 1 (10 μm of olL of DNA Polymerase, 1 μ L
-1) and Primer 2 (10 μm of olL
-1), the template DNA of 1 μ L, the sterilizing ddH of 22 μ L
2o.
Gateway PCR cloning reaction condition is: 98 DEG C of denaturation 1min; 98 DEG C of sex change 10s, 55 DEG C of annealing 15s, 68 DEG C extend 2min, 8 circulations; 98 DEG C of sex change 10s, 68 DEG C extend 2min, 32 circulations; 68 DEG C extend 10min.
2, entry vector builds
The structure carrying out entry vector according to Gateway BP Clonase Enzyme Mix test kit connects the attB-PCR product that entry vector reaction system is 2 μ L, the pDONR222.1 of 0.4 μ L, the BP Clonase of 0.6 μ L, mix gently, of short duration centrifugal, make at the bottom of collection reaction solution to centrifuge tube, 25 DEG C, link of spending the night.Ligation product conversion intestinal bacteria, get 1 μ L plasmid and add intestinal bacteria (Escherichia coil) DH5 α competent cell, after leaving standstill 30min on ice, after 42 DEG C of thermal shock 50s, put into rapidly 2min on ice, then 200 μ L liquid nutrient mediums (not containing microbiotic) are added, 37 DEG C of shaking culture 1h (180r/min), the bacterium liquid uniform application getting 100 μ L shaking culture, to containing on the LB solid medium of penbritin, is placed in 37 DEG C and is inverted overnight incubation (12-16h) until single bacterium colony grows.Picking list bacterium colony, illustrates according to Axygen company plasmid extraction kit and extracts recombinant plasmid.
3, the structure of expression vector
Need to carry out linearizing after entry vector successfully constructs and just can be connected to expression vector.By the entry vector plasmid Mlu I digestion with restriction enzyme successfully constructed, it is as follows that enzyme cuts system: the Mlu I enzyme of 0.5 μ L, and the BP of 10 × Buffer 3,2 μ L of μ L reacts plasmid, the sterilizing ddH of 4.5 μ L
2o.Digestion products is carried out LR recombining reaction according to Gateway LR Clonase Enzyme Mix reagent specification sheets, system is as follows: 2 μ L linearizing entry vectors, 0.4 μ L expression vector, 0.6 μ L LR Clonase, 1.8 μ L TE Buffer (PH=8.0), thus object fragment subclone is entered expression vector.
The genetic transformation of embodiment 5 tobacco
1 contaminates: in Bechtop, get appropriate tobacco tests for sterility, evenly mark several roads wound with sterile razor blade vertical main lobe arteries and veins direction on blade, and the single bacterium colony of Agrobacterium that picking contains recombinant expression vector is cultured to OD
600=0.6-0.8, is then placed on blade in fresh bacterium liquid and contaminates 15min, therebetween rotational oscillation gently, blade is fully contacted with Agrobacterium, is blotted by blade after infecting with aseptic thieving paper, prevents later experiments Agrobacterium from growing too much;
2 Dual culture: the explant contaminated is placed in division culture medium (MS+ agar 5g/L+ sucrose 30g/L+6-BA0.5mg/L
+ NAA0.05mg/L), around blade, there is a small amount of Agrobacterium in light culture (28 DEG C, about 3d);
3 selectivity are cultivated: be placed in by the tobacco leaf of dip-dye in screening culture medium (MS+ agar 5g/L+ sucrose 30g/L+6-BA0.5mg/L+NAA0.05mg/L+200mg/L Ticarcillin/Clavulanate Acid+50mg/L Totomycin) and carry out selection cultivation (24 DEG C, 16h/8h);
4 root culture: when indefinite bud grows to about 2cm, indefinite bud is transferred to root media (1/2MS+ agar 5g/L+ sucrose 30g/L+6-BA0.5mg/L+NAA0.05mg/L+200mg/L Ticarcillin/Clavulanate Acid+50mg/L Totomycin) and carries out root culture (24 DEG C, 16h/8h).
The qualification of embodiment 6 transfer-gen plant
1, the PCR of transfer-gen plant detects
Traditional CT AB method is adopted to extract tobacco gene group DNA (referring to the Rapid isolation of high molecular weight plant DNA.Nucleic Acids Research such as Murray M, 1980,8:4321-4326.).With tobacco gene group DNA for template, EuTIDS5 gene upstream and downstream special primer is adopted to detect, forward primer: 5 ’ – AGAGCTAACTGATCATAA
AAAAGGG-3 ' reverse primer: 5 ’ – TCAATAATGCCTCCGATAGATCTT-3 '.And adopt non-transgenosis wild-type tobacco to be negative control (-), with the expression vector plasmid successfully constructed for positive control (+).
PCR response procedures is: 94 DEG C of denaturation 4min; 94 DEG C of sex change 30s, 60 DEG C of annealing 30s, 72 DEG C extend 30s 30 circulation; 72 DEG C extend 7min; Pcr amplification product detects (referring to Fig. 5) through 1.0% agarose gel electrophoresis, filters out transgenic positive plant.
2, Real-time PCR detects the transcriptional expression of transfer-gen plant
Real-time PCR is utilized to detect the above-mentioned relative expression quantity of tobacco positive plant overexpression on transcriptional level through qualification, the RNA of transgenic tobacco leaf extracts, cDNA synthetic method with example 2, Real-time PCR operate and the primer see example 3.Result shows, compared with wild-type tobacco, and EuTIDS5 genetic expression all high than wild-type (referring to Fig. 6) in transgene tobacco.
The gel content of embodiment 7 transfer-gen plant measures
Gather above-mentioned transgenic tobacco plant after measured, utilize not genetically modified wild-type tobacco to contrast, carry out trans-isoprene assay.Measuring method is see embodiment 1.Result shows, the content of trans-isoprene is not detected in wild-type tobacco, and detect that the content of trans-isoprene is 0.28% turning in the positive tobacco of 35S::EuTIDS5, show that EuTIDS5 participates in the synthesis of trans-isoprene, the expression improving EuTIDS5 gene is conducive to the synthesis of trans-isoprene.
Embodiment 8 expression vector establishment
1) clone of object fragment
This research adopts Gateway method to build Subcellular Localization, overexpression and rna interference vector.According to the requirement of Gateway method vector construction, utilize Primer Premier 5.0 to design primer and add the sequence with Gateway joint at primer two ends, being carried out the clone of gene object fragment by pcr amplification.Primer refers to table 2, and underscore part is Gateway joint sequence.
Table 2 object fragment cloning primer
The PCR system of Gateway clone is as following table 3
The PCR system of table 3:Gateway clone
Gateway PCR cloning reaction condition is: 98 DEG C of denaturation 1min; 98 DEG C of sex change 10s, 55 DEG C of annealing 15s, 68 DEG C extend 2min, 8 circulations; 98 DEG C of sex change 10s, 68 DEG C extend 2min, 32 circulations; 68 DEG C extend 10min.
2) entry vector builds
Operate according to Gateway BP Clonase Enzyme Mix test kit specification sheets, connect entry vector reaction system as follows:
Mix gently, of short duration centrifugal, make at the bottom of collection reaction solution to centrifuge tube, 25 DEG C, link of spending the night.
3) ligation product conversion intestinal bacteria
Get 1 μ L plasmid and add intestinal bacteria (Escherichia coil) DH5 α competent cell, after leaving standstill 30min on ice, after 42 DEG C of thermal shock 50s, put into rapidly 2min on ice, then 200 μ L liquid nutrient mediums (not containing microbiotic) are added, 37 DEG C of shaking culture 1h (180r/min), get the bacterium liquid uniform application of 100 μ L shaking culture on the LB solid medium containing penbritin, be placed in 37 DEG C and be inverted overnight incubation (12-16h), picking list bacterium colony is in LB (containing 50mg/L penbritin) liquid nutrient medium, 37 DEG C of shaken overnight, carry out bacterium liquid PCR to detect, PCR reaction system is as following table 4.
Table 4:PCR reaction system
Pcr amplification condition is, 94 DEG C of 4min denaturations, 94 DEG C of 30s, 60 DEG C of 30s, 72 DEG C of 60s, 30 circulations, and 72 DEG C extend 10min.Through the positive list bacterium colony Jun Yesong company order-checking that PCR detects.Positive colony bacterium liquid adds sterile glycerol, and-80 DEG C save backup.
4) recombinant plasmid is extracted
According to the plasmid extraction kit specification sheets step operation of Axygen company.
5) structure of expression vector
Need to carry out linearizing after entry vector successfully constructs and just can be connected to expression vector.By the corresponding digestion with restriction enzyme of entry vector plasmid successfully constructed, digestion products is carried out LR recombining reaction according to Gateway LR Clonase Enzyme Mix reagent specification sheets, thus object fragment subclone is entered expression vector.Expression vector establishment mainly comprises the structure of rubber synthesis relative enzyme gene Subcellular Localization carrier pEarleyGate 101-EuGene, pEarleyGate104-EuGene, and rubber synthesizes key gene Overexpression vector pMDC32-EuGene and composes the structure of key gene rna interference vector pH7G-EuGene.Enzyme is cut with LR recombining reaction system and reaction conditions as following table 5.
Table 5: reaction system and reaction conditions
Mlu I endonuclease reaction system is as following table 6.
37 DEG C of enzymes cut 10h, 65 DEG C, 20min termination reaction.
Xba I endonuclease reaction system is as following table 6.
Table 6: endonuclease reaction system
37 DEG C of enzymes cut 10h, 65 DEG C, 20min termination reaction.
LR reaction system is as following table 7.
Table 7:LR reaction system
37 DEG C of link 5h.
Embodiment 9 expression vector transformation Agrobacterium
1 μ L plasmid is added in band Agrobacterium tumefaciens strain GV3101 competent cell, join after soft mixing carry out in the electroporated cup of precooling electroporated.After electroporated, the competent cell matter with plasmid is added 200 μ L liquid nutrient mediums, 28 DEG C, 180rpm, cultivate 2h; Get 100 μ L bacterium liquid even, even spread, to containing on corresponding antibiotic solid medium, is inverted for 28 DEG C and is cultivated 48h; Picking list bacterium colony shakes bacterium, 28 DEG C, 180rpm, shakes bacterium 24h to growing logarithmic phase.
Embodiment 10 transient expression transforms
1), after Agrobacterium activation, picking mono-clonal adds to be cultivated containing 28 DEG C of concussions in 50mg/L Gen, 17mg/L Rif and the antibiotic liquid nutrient medium of 50mg/L Ka;
2) above-mentioned Agrobacterium 500uL is got, after detecting bacterial concentration, with re-suspension liquid (MgCl
2/ Syringylethanone) resuspended bacterium liquid;
3) under the condition of faint light, utilize syringe to draw agrobacterium liquid resuspended on a small quantity, bacterium liquid is injected in the lower epidermis cell of tobacco leaf, under being put into normal illumination after 6h is cultivated in dark lower placement, cultivate (22 DEG C, illumination 16h, dark 8h);
4) normal cultivation is after the 3rd day, gets tobacco leaf injection areas, about 5mm × 5mm size with blade, places slide glass central authorities, instills a clear water, covered, avoids producing bubble, utilizes laser copolymerization to hand over fluorescent microscope detection fluorescent signal.
Embodiment 11 barked eucommia transforms and performance is observed
Utilize the bark regeneration principle of the bark of eucommia, set up eucommia bark genetic conversion system.Eucommia bark (phloem) peeling is become multiple 2cm × 2cm grid, the form layers of exposing surface, the single bacterium colony of Agrobacterium picking being contained recombinant expression vector is cultured to OD
600value is the bacterium liquid of 0.6 ~ 0.8, repeatedly infects the surperficial form layers of barked eucommia, makes bacterium liquid be distributed in form layers surface uniformly, detects with different time, and different number of times of contaminating is tested.Owing to being exposed in air by the form layers of peeling, in order to prevent trees dehydration withered dead, rear application plastic film wrapped stripping face is infected in peeling, and moisturizing is sun-proof, the efficiency that raising is infected and promotion prematurity tracheid accelerate division under favorable conditions, form new perithelium.
Result shows: with contrast (same tree body peeling, but unconverted) compare, after having infected for 3 times, the form layers of contrast in the 5th day and conversion is without obvious change, after about 30 days, contrast and the form layers transformed all grow green callus, and after about 90 days, bark grows completely (referring to Fig. 7).Peel bark and carry out rubber content observation, compared with the control, turn institute's inthegum showed increased in the bark of the tree body of 35S::EuTIDS5, show that the gel content in bark increases, and the new bark gel content of the bark of eucommia behind new bark place again peeling still keeps high level; Turn institute's inthegum in the bark of the tree body of RNAi-EuTIDS5 obviously to reduce, show that the gel content in bark reduces (referring to Fig. 8).Therefore demonstrate again the content that EuTIDS5 is conducive to improving Chinese gutta percha, the decline of the expression amount of EuTIDS5, has restraining effect to the synthesis of Chinese gutta percha.Result is see table 8.
Table 8
Above-described embodiment is only for the invention example is clearly described, and the restriction not to the invention embodiment.For those of ordinary skill in the field, can also make other changes in different forms on the basis of the above description.Here exhaustive without the need to also giving all embodiments.All within the spirit and principles in the present invention any apparent change of extending out or variation be still among the protection domain of the invention claim.
Claims (10)
1. improve a method for bark of eucommia gel content, it is characterized in that, comprise the following steps:
1) peeling of eucommia bark phloem is become multiple grid, at interval the form layers of trellis exposing surface;
2) carry out dip-dye operation: the bacterium liquid that the single bacterium colony of Agrobacterium picking being contained EuTIDS5 recombinant expression vector is cultivated, infect the surperficial form layers of barked eucommia;
3) peeling infects rear application plastic film wrapped stripping face, the sun-proof growth of moisturizing.
2. the method for raising bark of eucommia gel content according to claim 1, is characterized in that, step 1) described in single lattice area of grid be 2-10cm
2.
3. the method for raising bark of eucommia gel content according to claim 1, is characterized in that, step 2) described in the OD of bacterium liquid
600value is 0.5 ~ 0.9.
4. the method for raising bark of eucommia gel content according to claim 1, is characterized in that, step 2) dip-dye for repeatedly to contaminate, make bacterium liquid be distributed in form layers surface uniformly.
5. the method for raising bark of eucommia gel content according to claim 1, is characterized in that, step 3) the plastic film wrapped time is 20 days ~ 100 days.
6. the method for raising bark of eucommia gel content according to claim 1, is characterized in that, step 2) dip-dye operation carry out 2-5 time, every minor tick 2-4 days.
7. the method for raising bark of eucommia gel content according to claim 1, it is characterized in that, the single bacterium colony of the described Agrobacterium containing recombinant expression vector is by being added by the plasmid containing EuTIDS5 in band Agrobacterium tumefaciens strain GV3101 competent cell, join after softly mixing carry out in the electroporated cup of precooling electroporated; After electroporated, the competent cell matter with plasmid is added substratum, cultivate; Get liquid, be evenly coated in containing on corresponding antibiotic solid medium, be inverted and cultivate; Picking list bacterium colony shakes bacterium, shakes bacterium to growing logarithmic phase.
8. the method for raising bark of eucommia gel content according to claim 7, is characterized in that, the preparation method of the described plasmid containing EuTIDS5 is:
By the method amplification EuTIDS5 of PCR, connect Plastid transformation cultivation and extract the plasmid obtained containing EuTIDS5 afterwards.
9. the method for raising bark of eucommia gel content according to claim 1, is characterized in that, the encoding sequence of EuTIDS5 is as shown in SEQ ID NO:2.
10. the method for raising bark of eucommia gel content according to claim 8, is characterized in that, the upstream primer (5'-3') of pcr amplification EuTIDS5 and downstream primer (5'-3') are respectively as shown in SEQID NO:3 and 4.
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| CN113122569B (en) * | 2021-04-26 | 2022-07-08 | 北京林业大学 | Construction method of eucommia transgenic plant regeneration system |
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