CN109913475A - A kind of gene, mediation carrier and the application of enhancing kok-saghyz antiweed - Google Patents
A kind of gene, mediation carrier and the application of enhancing kok-saghyz antiweed Download PDFInfo
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- CN109913475A CN109913475A CN201910248552.7A CN201910248552A CN109913475A CN 109913475 A CN109913475 A CN 109913475A CN 201910248552 A CN201910248552 A CN 201910248552A CN 109913475 A CN109913475 A CN 109913475A
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- saghyz
- kok
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- antiweed
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Abstract
A kind of gene, mediation carrier and the application of enhancing kok-saghyz antiweed.The present invention enhances the nucleotide sequence of Genes For Plant Tolerance Herbicide resistance genes as shown in SEQ ID NO:1；Mediation carrier and its application in kok-saghyz genetic improvement containing present invention enhancing kok-saghyz anti-herbicide gene.The gene that the present invention enhances kok-saghyz antiweed is used in kok-saghyz genetic transformation, and kok-saghyz antiweed performance improves after conversion.
Description
Technical field
The present invention relates to a kind of gene, mediate carrier and application.
Background technique
Rubber is important the raw material of industry and strategic materials, although kok-saghyz is a kind of natural lactiferous plant, yield
Industrial requirement can not be unable to satisfy compared with Brazilian gum, it is difficult to draw attention, 20th century mid-term, since rubber raw materials are in great shortage,
Once the Soviet Union, the U.S., China etc. was caused to study its morphosis, cultivation technique and tissue physiology etc., but molecule is raw
Research in terms of object is less.
Summary of the invention
The present invention provides a kind of gene for enhancing kok-saghyz antiweed, mediate carrier and application.
The present invention enhances the nucleotide sequence of kok-saghyz anti-herbicide gene as shown in SEQ ID NO:1.
Mediation carrier containing above-mentioned enhancing kok-saghyz anti-herbicide gene.Wherein mediating carrier is Agrobacterium.
The gene of above-mentioned enhancing kok-saghyz antiweed is used for the genetic improvement of kok-saghyz.
Chrysothamnus is in sprawl growth, and rubber is contained in root, common weeding by machine's mode, can hurt rubber grass roots and
The growth of leaf, gene of the invention is stronger for what is in kok-saghyz genetic transformation, expressed on bud, rubber after conversion
The antiweed performance of grass is good, while removing weeds, the growth of kok-saghyz is not influenced, conducive to the accumulation of root rubber.
Detailed description of the invention
Fig. 1 is the result figure of the DNA extracted in freeze-thaw method conversion Agrobacterium.
Specific embodiment
The technical solution of the present invention is not limited to the following list, further includes between each specific embodiment
Any combination.
Specific embodiment 1: the nucleotide sequence such as SEQ ID of present embodiment enhancing kok-saghyz anti-herbicide gene
Shown in NO:1.
The extracting method of present embodiment gene is as follows:
1, enhance the extraction of kok-saghyz anti-herbicide gene total DNA:
(1) the CTAB Extraction buffer of 5~20mL is taken, 68 DEG C preheat, wherein CTAB Extraction buffer: 2%CTAB,
100mmol/L Tris-Cl, 20mmol/LEDTA, 1.4mol/L NaCl, 2% volume mercaptoethanol, pH8.0；
(2) 0.5g agrobacterium rhizogenes (Agrobacterium Rhizogenes, R1601) is taken to be ground into a powder with liquid nitrogen, to the greatest extent
It measures finely ground, is transferred in Eppendorf pipe, the CTAB Extraction buffer of 700 μ L preheating is added, shakes vigorously and mix well；
(3) plus the 20%SDS of 50 μ L, the mercaptoethanol of the 10%PVP of 50 μ L and 20 μ L are gently mixed by inversion, are placed in 68
30min in DEG C water-bath；
(4) it is primary that 300 μ LCI extracting is added, 12000rpm centrifugation 5min；
(5) supernatant is taken, 300 μ LCI are added and extract again once, 12000rpm is centrifuged 5min；
(6) supernatant is taken, isometric isopropanol is added, 4 DEG C of ice baths precipitate 20~30min；
(7) 12000rpm is centrifuged 5min, goes supernatant, precipitating plus 600 μ L, 70% ethanol washing；
(8) 8000rpm is centrifuged 5min, removes supernatant, and precipitating is set volatilizees to ethyl alcohol completely at room temperature；
(9) 30~50 μ L distilled waters dissolution precipitating is added, is stored in -20 DEG C.
2, enhance the clone of kok-saghyz anti-herbicide gene
Using the total DNA for enhancing kok-saghyz anti-herbicide gene as template, using specific primer, added in design primer
Two restriction enzyme sites of XbaI and SmaI, wherein specific primer are as follows:
EPSPS-1:5'-ACGTCTAGAATGAACAGGACCATGGCTCCATTG-3'XbaI
EPSPS-2:5'-CCCGGGTTACTTCCTGGCGAGACA-3 ' SmaI
PCR reaction system (20 μ L of total volume):
PCR reaction condition:
PCR product is detected with 0.8% agarose gel electrophoresis
3, enhance the monoclonal sequencing of kok-saghyz anti-herbicide gene
The PCR product being cloned into is connect with pMD18-T vector cloning vector, then converts bacillus coli DH 5 alpha.It chooses
Selected episode expands the monoclonal in 1.5kb or so, delivers three rich Radix Polygalae sequencings.
Linked system (7 μ L of total volume):
Specific embodiment 2: present embodiment contains the enhancing kok-saghyz anti-herbicide gene of specific embodiment one
Mediate carrier.
The mediation carrier of present embodiment is Agrobacterium LBA4404.
Specific embodiment 3: the gene of the enhancing kok-saghyz antiweed as described in specific embodiment one is used for rubber
The genetic improvement of grass.
Embodiment 1: application of the gene of enhancing kok-saghyz antiweed of the invention in kok-saghyz genetic transformation, specifically
Method is as follows:
One, gene expression is into Agrobacterium:
(1) preparation of E. coli competent
E.coli competent cell prepares (CaCl2Method).
(1) picking bacillus coli DH 5 alpha single colonie, in 10mL LB liquid medium, 37 DEG C, 180rpm cultivates 12h；
(2) it is inoculated in 60mL LB liquid medium by 1% inoculum concentration, 37 DEG C, 180rpm cultivates 2~2.5h, until OD600
=0.3~0.5；
(3) 15~25min of bacterium solution ice bath is sub-packed in 1.5mL sterile eppendorf tubes, and 4 DEG C, 5000rpm centrifugation
5min；
(4) supernatant is abandoned, cell overhangs the 0.1M CaCl of 800 μ L ice pre-cooling2In, set 20~30min on ice；
(5) 4 DEG C, 5000rpm is centrifuged 5min, recycles cell；
(6) the 0.1M CaCl of 100 μ L ice pre-cooling is added2Cell is resuspended；
(7) it dispenses, 4 DEG C of preservations 12~for 24 hours, or -70 DEG C save backup.
CaCl2Preparation: anhydrous CaCl20.66g, PIES 0.3g, glycerol 15mL, Jia Shui are settled to 100mL, use NaOH
Adjust PH up to 7.0, high pressure sterilization is saved backup in 4 DEG C of refrigerators.
(2) conversion of Escherichia coli
(1) take competent escherichia coli cell in the 15min that thaws on ice；
(2) gene (1~2 μ L) of enhancing plant antiweed is added, tip-tap mixes, ice bath 30min；
(3) 42 DEG C of 60~90s of water bath heat shock；
(4) it takes out rapidly and is placed in 2min on ice；
(5) 1mLLB fluid nutrient medium, 37 DEG C of culture 1h are added；
(6) 5000rpm is centrifuged 3min, leaves appropriate supernatant, and mixing is opened in precipitating vibration, about 100 μ L bacterium solutions are transferred to
On the plate of the LB solid medium of additional Sm, Rif and Kan；Wherein add strepto- in the LB solid medium of Sm, Rif and Kan
Element: Streptomycin, Sm are 50mg/L using concentration；Rifampin: Rifampicin, Rif are 50mg/ using concentration
L；Kanamycins: Kanamycin, Kan are 100mg/L using concentration.
(7) 37 DEG C of insulating box 10~12h of culture, picking resistance hickie；
(8) PCR amplification identification and analysis is carried out after shaking bacterium.
(3) identification of recombinant plasmid
(1) PCR is detected: picking single bacterium is fallen in antibiotic LB liquid medium, 37 DEG C of overnight incubations, using alkali cracking
Solution rapidly extracting Plasmid DNA carries out pcr amplification reaction with specific primer with this plasmid as template, solidifying by agarose
Gel electrophoresis has detected whether desired purpose band, primarily determines recon successful connection；
Specific primer:
EPSPS-1:5'-ACGTCTAGAATGAACAGGACCATGGCTCCATTG-3'XbaI
EPSPS-2:5'-CCCGGGTTACTTCCTGGCGAGACA-3 ' SmaI
(2) digestion detects: picking restriction enzyme carries out endonuclease reaction, observes DNA item by agarose gel electrophoresis
Band size, so that it is determined that whether exogenous sequences are inserted into and whether the size of Insert Fragment correct, and then judge recombinant clone at
Whether function.
(4) preparation and conversion of Agrobacterium competent cell
(1) preparation of Agrobacterium (LBA4404) competent cell
1) from the single colonie of picking Agrobacterium LBA4404 on the plate of the YEB containing 50mg/L Sm, 50mg/L Rif；
2) it is inoculated in the YEB fluid nutrient medium that 5mL contains Sm, Rif, 28 DEG C, 180rpm, 1~2d of shaken cultivation；YEB
It is 50mg/L that the concentration of Sm, which is the concentration of 50mg/L, Rif, in fluid nutrient medium.
3) bacterium solution is taken to be reinoculated in the YEB fluid nutrient medium containing Sm, Rif of 50mL in the ratio of 1:10；
4) shaken cultivation 6-8h, until logarithmic growth phase OD600≈0.5；
5) bacterium solution is placed in 20~30min on ice, is sub-packed in autoclaved 1.5mL eppendorf pipe；
6) 4 DEG C, 5000rpm is centrifuged 10min, abandons supernatant；
7) the 0.05M CaCl of 800 μ L ice pre-cooling is added in precipitating2Suspension cell, 20~30min of ice bath；
8) 4 DEG C, 5000rpm is centrifuged 10min, abandons supernatant；
9) 0.05MCaCl of 100 μ L ice pre-cooling is added in precipitating2Cell is resuspended；
10) 4 DEG C are placed in save backup.
(2) freeze-thaw method converts Agrobacterium LBA4404
1) Agrobacterium competent cell is taken, 3~4 μ L, which are added, enhances kok-saghyz anti-herbicide gene, mixes gently, sets on ice
30min；
2) 3~5min of liquid nitrogen frozen moves to rapidly 2~3min in 37 DEG C of water-baths placements and melts；
3) 800 μ L YEB, 28 DEG C of 4~5h of shaken cultivation are added；
4) 8000rpm is centrifuged 5min, removes supernatant, leaves appropriate supernatant, and precipitating is mixed, 100 μ L is taken to be applied to containing Sm, Rif
YEB plate on, 28 DEG C of inversion dark culture 48h.
5) the carry out DNA extraction of the Agrobacterium LBA4404 after converting, specific extracting method are as follows:
A, the CTAB Extraction buffer of 5~20mL is taken, 68 DEG C preheat, wherein CTAB Extraction buffer: 2%CTAB,
100mmol/L Tris-Cl, 20mmol/LEDTA, 1.4mol/L NaCl, 2% volume mercaptoethanol, pH8.0；
B, the Agrobacterium LBA4404 after taking 0.5g to convert is ground into a powder with liquid nitrogen, finely ground as far as possible, is transferred to Eppendorf
Guan Zhong is added the CTAB Extraction buffer of 700 μ L preheating, shakes vigorously and mix well；
C, plus the 20%SDS of 50 μ L, the mercaptoethanol of the 10%PVP of 50 μ L and 20 μ L are gently mixed by inversion, are placed in 68 DEG C
30min in water-bath；
D, it is primary that 300 μ LCI extracting is added, 12000rpm centrifugation 5min；
E, supernatant is taken, 300 μ LCI are added and extract again once, 12000rpm is centrifuged 5min；
F, supernatant is taken, isometric isopropanol is added, 4 DEG C of ice baths precipitate 20~30min；
G, 12000rpm is centrifuged 5min, goes supernatant, precipitating plus 600 μ L, 70% ethanol washing；
H, 8000rpm is centrifuged 5min, removes supernatant, and precipitating is set volatilizees to ethyl alcohol completely at room temperature；
I, 30~50 μ L distilled waters dissolution precipitating is added, is stored in -20 DEG C.
The extraction result of DNA is as shown in Figure 1, M:marker in figure；1: enhancing kok-saghyz anti-herbicide gene；2: positive right
According to the size of enhancing kok-saghyz anti-herbicide gene group pcr amplification product is about 1.5kb, it can be seen from the figure that big with compareing
Small consistent band, illustrating, which enhances kok-saghyz anti-herbicide gene, is successfully transferred in Agrobacterium, can be used for subsequent infect
?.
Two, the activation of strain: the competent cell that step 1 obtains is inoculated in the LB solid culture of additional Sm, Rif and Kan
In base, 36~48h of dark culture in 28 DEG C of incubators；Picking single colonie is inoculated in the LB liquid training that 10mL adds Sm, Rif and Kan
It supports in base, 28 DEG C, 80rpm shaken cultivation；It is long to logarithmic growth phase to bacterium solution, i.e. when OD600=0.4~0.6, again by bacterium solution
It is inoculated in the LB liquid medium of additional Sm, Rif and Kan and carries out re-activation；Wherein add the LB solid of Sm, Rif and Kan
Culture medium streptomycin: Streptomycin, Sm are 50mg/L using concentration；Rifampin: Rifampicin, Rif make
It is 50mg/L with concentration；Kanamycins: Kanamycin, Kan are 100mg/L using concentration.
Three, the preparation of kok-saghyz explant: the seed of kok-saghyz K445 is with after 2~3h of water logging kind, with 75% alcohol to kind
Son carries out 30~60s of surface sterilization, then sterilizes 3~5min with 0.1% mercuric chloride, aseptic water washing 3~10 times, is then inoculated in
MS0In culture medium；When blade grows to 2~3cm, blade is cut, and blade is cut into the fritter of 1cm × 1cm, is then inoculated with
In MS0On culture medium, dark culture 2~3 days, obtains kok-saghyz explant in 28 DEG C of constant incubators；
Four, infect: the kok-saghyz explant of step 3 is put into the bacterium solution after activating in step 1, impregnates 10~15min,
Jog 2~10 times therebetween；It takes out material and then sucks extra liquid with aseptic filter paper with aseptic water washing 3~8 times；
Five, co-culture: the kok-saghyz explant after infecting is inoculated into MS0In culture medium, 28 DEG C of 1~2d of dark culture；
Six, squamous subculture: explant is transferred into differential medium MS after co-culturing1In, 28 DEG C of optical cultures, point
Change callus, about 10d or so subculture is primary；
Seven, screening and culturing: after squamous subculture 2~3 weeks, explant callus starts to differentiate resistant buds, by resistant buds
It is transferred to screening and culturing medium MS2On, and continue squamous subculture；
Eight, when bud it is long to 3~4cm when, cut and be transferred to root media MS3On, root induction, 15~20d long
Adventitious root out；Complete genetic transformation of the gene of present invention enhancing plant antiweed in plant.
Above-mentioned MS0、MS1、MS2、MS3Specific ingredient:
MS0(germination culture medium): (MS+1.0mg/L 6-BA+0.1mg/L NAA), 0.8% agar, pH5.8.
MS1(differential medium): MS+2.0mg/L 6-BA+0.1mg/LNAA, pH5.8.
MS2(screening and culturing medium): MS+Cb+PPT
MS3(root media): 1/2MS, pH5.8.
Gene of the invention is used in kok-saghyz genetic transformation, and that expresses on bud is stronger, by antiweed
Service check, with the present invention conversion after kok-saghyz antiweed performance it is good, while removing weeds, without influence rubber
The growth of grass, conducive to the accumulation of root rubber.
Sequence table
<110>Xiao Yu
Heilungkiang Wa Weiluojia development in science and technology Co., Ltd
<120>a kind of gene for enhancing kok-saghyz antiweed, mediation carrier and application
<160> 3
<170> SIPOSequenceListing 1.0
<210> 1
<211> 1584
<212> DNA
<213>agrobacterium rhizogenes (Agrobacterium rhizogenes)
<400> 1
atggcacaaa ttaacaacat ggcacaaggg atacaaaccc ttaatcccaa ttccaatttc 60
cataaacccc aagttcctaa atcttcaagt tttcttgttt ttggatctaa aaaactgaaa 120
aattcagcaa attctatgtt ggttttgaaa aaagattcaa tttttatgca aaagttttgt 180
tcctttagga tttcagcatc agtggctaca gcctgcatgc ttcacggtgc aagcagccgg 240
cccgcaaccg cccgcaaatc ctctggcctt tccggaaccg tccgcattcc cggcgacaag 300
tcgatctccc accggtcctt catgttcggc ggtctcgcga gcggtgaaac gcgcatcacc 360
ggccttctgg aaggcgagga cgtcatcaat acgggcaagg ccatgcaggc catgggcgcc 420
aggatccgta aggaaggcga cacctggatc atcgatggcg tcggcaatgg cggcctcctg 480
gcgcctgagg cgccgctcga tttcggcaat gccgccacgg gctgccgcct gaccatgggc 540
ctcgtcgggg tctacgattt cgacagcacc ttcatcggcg acgcctcgct cacaaagcgc 600
ccgatgggcc gcgtgttgaa cccgctgcgc gaaatgggcg tgcaggtgaa atcggaagac 660
ggtgaccgtc ttcccgttac cttgcgcggg ccgaagacgc cgacgccgat cacctaccgc 720
gtgccgatgg cctccgcaca ggtgaagtcc gccgtgctgc tcgccggcct caacacgccc 780
ggcatcacga cggtcatcga gccgatcatg acgtgcgatc atacggaaaa gatgctgcag 840
ggctttggcg ccaaccttac cgtcgagacg gatgcggacg gcgtgcgcac catccgcctg 900
gaaggccgcg gcaagctcac cggccaagtc atcgacgtgc cgggcgaccc gtcctcgacg 960
gccttcccgc tggttgcggc cctgcttgtt ccgggctccg acgtcaccat cctcaacgtg 1020
ctgatgaacc ccacccgcac cggcctcatc ctgacgctgc aggaaatggg cgccgacatc 1080
gaagtcatca acctgcgcct tgccggcggc gaagacgtgg cggacctgcg cgttcgctcc 1140
tccacgctga agggcgtcac ggtgccggaa gaccgcgcgc ctccgatgat cgacgaatat 1200
ccgattctcg ctgtcgccgc cgccttcgcg gaaggggcga ccgtgatgaa cggtctggaa 1260
gaactccgcg tcaaggaaag cgaccgcctc tcggccgtcg ccaatggcct caagctcaat 1320
ggcgtggatt gcgatgaggg cgagacgtcg ctcgtcgtgc gtggccgccc tgacggcaag 1380
gggctcggca acgcctcggg cgccgccgtc gccacccatc tcgatcaccg catcgccatg 1440
agcttcctcg tcatgggcct cgtgtcggaa aaccctgtca cggtggacga tgccacgatg 1500
atcgccacga gcttcccgga gttcatggac ctgatggccg ggctgggcgc gaagatcgaa 1560
ctctccgata cgaaggctgc ctga 1584
<210> 2
<211> 33
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 2
acgtctagaa tgaacaggac catggctcca ttg 33
<210> 3
<211> 24
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 3
cccgggttac ttcctggcga gaca 24
Claims (3)
1. a kind of gene for enhancing kok-saghyz antiweed, it is characterised in that the nucleotides sequence of enhancing kok-saghyz anti-herbicide gene
Column are as shown in SEQ ID NO:1.
2. containing the mediation carrier of gene described in claim 1.
3. the application of the gene of enhancing kok-saghyz antiweed described in claim 1, enhances the gene of kok-saghyz antiweed
Genetic improvement for kok-saghyz.
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| CN201910248552.7A CN109913475A (en) | 2019-03-29 | 2019-03-29 | A kind of gene, mediation carrier and the application of enhancing kok-saghyz antiweed |
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Cited By (1)
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| CN110669783A (en) * | 2019-10-30 | 2020-01-10 | 中国热带农业科学院热带生物技术研究所 | Genetic transformation method for kokstroemia indica |
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| CN103013938A (en) * | 2012-12-25 | 2013-04-03 | 北京大北农科技集团股份有限公司 | Herbicide resistance protein, coding gene and application thereof |
| CN103074351A (en) * | 2012-10-23 | 2013-05-01 | 中国农业大学 | Synthetic EPSPS gene for transgenic herbicide resistant plant |
| US20160340689A1 (en) * | 2012-09-06 | 2016-11-24 | Nanjing Agricultural University | Glyphosate-tolerant gene and use thereof |
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2019
- 2019-03-29 CN CN201910248552.7A patent/CN109913475A/en active Pending
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| US20160340689A1 (en) * | 2012-09-06 | 2016-11-24 | Nanjing Agricultural University | Glyphosate-tolerant gene and use thereof |
| CN103074351A (en) * | 2012-10-23 | 2013-05-01 | 中国农业大学 | Synthetic EPSPS gene for transgenic herbicide resistant plant |
| CN103013938A (en) * | 2012-12-25 | 2013-04-03 | 北京大北农科技集团股份有限公司 | Herbicide resistance protein, coding gene and application thereof |
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110669783A (en) * | 2019-10-30 | 2020-01-10 | 中国热带农业科学院热带生物技术研究所 | Genetic transformation method for kokstroemia indica |
| CN110669783B (en) * | 2019-10-30 | 2021-07-02 | 中国热带农业科学院热带生物技术研究所 | Genetic transformation method for kokstroemia indica |
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