CN107858372A - A kind of agriculture bacillus mediated cotton transient transformation methods - Google Patents
A kind of agriculture bacillus mediated cotton transient transformation methods Download PDFInfo
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Abstract
The invention belongs to cotton gene field of engineering technology, and in particular to a kind of foundation of efficiently quick agriculture bacillus mediated cotton instantaneous conversion system.The steps such as this method specifically includes and prepares instantaneous conversion solution, cultivates cotton seedling and carries out hypertonic pretreatment, instantaneous conversion processing.In cotton transient transformation methods provided herein, mainly Agrobacterium is infected seedling using osmosis, so as to which foreign gene is carried into cotton, and then the gene being transferred to is expressed in cotton.In general, this method has the advantages that cost is cheap, easy to operate, transformation efficiency is high, can be that gene functional research establishes methodology basis in cotton, simultaneously or Subcellular Localization of the cotton functional gene in cotton provides reference method with interaction, thus is worth with preferable practicality and research application.
Description
Technical field
The invention belongs to cotton gene field of engineering technology, and in particular to a kind of efficiently quick agriculture bacillus mediated cotton
The foundation of instantaneous conversion system.
Background technology
Cotton is the important source material of edible oil, albumen and fiber, therefore is a kind of important industrial crops.The whole world has super
Cross 80 national plant cottons, including the U.S., China, India and many other developing countries.Although China is planted extensively
Cotton, but as China increasingly increases the demand of raw cotton, only rely on and bring more land under cultivation that to increase the total yield be unpractical.
Cotton is a kind of crops of very easy infection pest and disease damage, and existing research generally believes that cultivating transgene cotton is
Solve that yield and ecological environment problem be most basic and most effective way.Since eighties of last century, transgenic pest-resistant cotton-wadded quilt is extensive
Plant in all over the world.
Since the Chinese Academy of Agriculture Science and Technologys Cotton Research Institute in 2015 discloses the whole genome sequence of upland cotton, to cotton work(
The research of energy gene enters new climax.And with the extensive use of gene editing technology, clone is expected to disclosing many cottons
The function of flower gene.But in existing transgene cotton breeding mode, the agriculture bacillus mediated transgene cotton of traditional dependence turns
It is very low to change efficiency.It is considerably less relative to other crops such as tobacco and soybean, the quantity of the transgene cotton successfully obtained.Thus
The low-conversion of existing transgenic technology largely limits the announcement and further investigation of gene function.
Existing agriculture bacillus mediated transgenosis method for transformation, its main development course are：Price and Smith in 1979
Report obtains embryoid by Gossypium klotzschianumAnderss cell suspension cultures；1986, the Umbeck of Agracetus companies of the U.S. passed through
Agrobacterium-mediated transformation, the in the world successful cotton by Bt channel genes first；And China just will be outer early in 1991
Report in the Bt channel genes cotton plants of source.
In general, the existing method for obtaining transgene cotton mainly has agrobacterium-mediated transformation, particle bombardment, pollen tube to lead
Enter method etc..Wherein converted using agrobacterium-mediated transformation, transformation efficiency is relatively low, and transformed cells are directly divided into plant and compared
It is difficult；And the characteristics of particle bombardment maximum is exactly not influenceed by recipient genotypes, but easily forms multicopy and cause gene silencing
Or chimera is easily formed, application is not extensive.The method for obtaining transgene cotton is also a lot, such as PEG mediated methods, fat
Plastid mediated method, ultrasonic-mediated method, electrization, injection, infusion method etc., but the efficiency of transgenosis is by the shadow of many factors
Ring, including plant variety, growth period, growing environment and method for transformation etc..Further, since the special tectonic of cotton floral organ and
Agrobacterium-mediated Transformation is limited by genotype, and some domestic research institutions also have cultivates transgene cotton using pollen-tube pathway method
Trial.In a word, either agricultural production, or the research to cotton gene function, be required for establishing it is a kind of it is stable, efficiently,
The method of simple cotton transgenic, so as to be agricultural production and theoretical research aspect provider's Fundamentals of Science of Law.
The content of the invention
Present invention aims at provide a kind of side of efficient, simple and quick agriculture bacillus mediated cotton instantaneous conversion
Method, so as to which the expression for homologous gene or heterologous gene in cotton provides methodological study basis, while can also be cotton
Subcellular Localization and repercussion study of the functional gene in cotton establish methodology basis.
Details are as follows for the technical scheme that the application is taken.
A kind of agriculture bacillus mediated cotton transient transformation methods, specifically comprise the following steps：
（1）Prepare instantaneous conversion solution
Gene to be transformed will be contained（Testing gene）Recombinant plasmid transformed Agrobacterium after, by the Agrobacterium of restructuring in YEP liquid
Shaking culture in culture medium, after culture terminates, centrifugation, collects thalline, and thalline is resuspended with re-suspension liquid, adjusts OD600=0.8 ~ 1.0 make
For instantaneous conversion solution；
The re-suspension liquid is 1/2 MS nutrient solutions（pH=5.8）, wherein containing 150 ~ 170 μM of acetosyringones, 2 ~ 4%（w/v）Sugarcane
Sugar and 0.01 ~ 0.02%（v/v）Tween20（It is preferably in a proportion of：165 μM of acetosyringones, 3%（w/v）Sucrose and 0.01%
（v/v）Tween20）；
For by taking objectives gene and plasmid as an example：
Respectively willpGhGPX1pro-GUS、p35S-GhGPX1-GFP、pGhGPX8pro-GUSWithp35S-GhGPX8-GFPPlasmid
After Transformed E HA105 Agrobacteriums, by the EHA105 agrobacterium strains recombinated after conversion respectively in YEP fluid nutrient mediums（Containing 50 mg/
L cards receive mycin and 50 mg/L rifampins）Middle culture, 28 DEG C, the h of 220 rpm Shaking cultures 16 ~ 17 or so；
Then, the bacterium solution for taking 2 mL to cultivate, is added to YEP fluid nutrient mediums, cultivates to bacterial concentration OD600About 1.2 ~ 1.5,
2000 g centrifuge 10 min, collect Agrobacterium；
Use re-suspension liquid（Optimization formula is：Contain 165 μM of acetosyringones, 3%（w/v）Sucrose and 0.01%（v/v）Tween20
1/2 MS（pH=5.8））Agrobacterium concentration is adjusted to OD600=0.8 ~ 1.0 or so, the solution is instantaneous conversion solution；
（2）Cotton seedling is cultivated, and carries out hypertonic pretreatment
By cotton seeds after planting, the cotton seedling of growth 6 ~ 8 days or so is taken, after cleaning, sterilization, is placed in high sepage and carries out height
Ooze pretreatment；
The hypertonic formula of liquid is：The 1/2 MS solution containing acetosyringone and sucrose, it is specific to be, for example,：Containing 165 μM of acetyl fourths
Ketone musk and 3% sucrose, 1/2 MS solution of pH=5.8；
The osmotic pressure of the high sepage is preferably 0.7 ~ 0.75 kg/cm2, hypertonic pretreatment time is preferably 3 min or so；
The cotton material is, for example, specifically：CCRI 36；
（3）Instantaneous conversion is handled
By step（2）Seedling after middle and high infiltration processing is put into step（1）In instantaneous conversion solution in impregnate culture, preferably cultivate
Condition is：25℃、120 rpm（Preferably provide suitable illumination）Cultivate 5 h or so（More than under 5 h or non-illuminated conditions, seedling is held
It is easily dead）；
After culture terminates, the seedling after conversion is cleaned up, is placed in containing antibiotic and 0.8%（w/v）1/2 MS of agar powder
Continue culture growth in culture medium；Condition of culture is：The h illumination of photoperiod 16/8 h are dark, and light intensity is 80 ~ 90 μm of ol m-2 s-1, cultivation temperature is：20 DEG C of 25 DEG C/night of day；Culture can carry out gene expression detection or other analyses after 5 days, or continue to train
Support and can be used to transfer-gen plant cultivation；
It should be noted that it is fresh plant need to be moved to another if occurring Agrobacterium bacterium colony around seedling during culture
Cultivated in culture medium.
The agriculture bacillus mediated cotton transient transformation methods, it is understood that be a kind of transfer-gen plant construction method.
Application of the agriculture bacillus mediated cotton transient transformation methods in transfer-gen plant structure, is obtained for building
Transgene cotton new varieties.
The agriculture bacillus mediated cotton transient transformation methods are applied in cotton is studied, and are studied for gene expression pattern
With the Subcellular Localization research of protein.
In cotton transient transformation methods provided herein, mainly Agrobacterium is set to infect seedling using osmosis,
So as to which foreign gene is carried into cotton, and then the gene being transferred to is expressed in cotton.In general, this method has cost
The advantages that cheap, easy to operate, transformation efficiency is high, can be that gene functional research establishes methodology basis in cotton, while
The Subcellular Localization that can be cotton functional gene in cotton and interaction provide reference method, such as utilize bimolecular fluorescence mutual
Complement system（Yellow fluorescence protein, YFP）Or luciferase（LUC）, with reference to the transient transformation methods of the application, you can detection cotton
The Subcellular Localization of albumen in vivo and interaction etc., thus be worth with preferable practicality and research application.
Brief description of the drawings
Fig. 1 is the ideograph of agriculture bacillus mediated cotton instantaneous conversion flow provided herein, and wherein EHA105 is to turn
Agrobacterium used in change, GFP are green fluorescent protein, and GUS is beta-glucosidase, and kan+ is kanamycins；
Fig. 2 is the upland cotton photo in conversion process, wherein：A, growth are used for the cotton material converted for 7 days；B, to convert solution
The upland cotton converted；C, the upland cotton grown after conversion in screening and culturing medium；
Fig. 3 is overexpressionGhGPX1WithGhGPX8Different plants in, real-time fluorescence quantitative PCR detectionGhGPX1（A）WithGhGPX8（B）Expression quantity；Wherein error represents that experiment at least individually repeat three times, is divided with Student ' s t-test
Analyse data difference conspicuousness, * * P< 0.01；
Fig. 4 isGhGPX1WithGhGPX8Allelic expression is analyzed, wherein：DetectionGhGPX1（A）WithGhGPX8（B）Promoter
The beta-glucosidase of driving（GUS）Activity；A, b and c are respectively expression characteristic of the gene in leaf, stem and root；C and D points
The RNA of the same time cotton tip of a root, root, stem, cotyledon and true leaf Wei not be extracted, using in fluorescence quantitative PCR detection different tissuesGhGPX1（C）WithGhGPX8（D）Expression pattern；
Fig. 5 is in upland cottonGhGPX1（A）WithGhGPX8（B）Subcellular Localization；Wherein：A, GhGPX1 Subcellular Localization
In chloroplaset；B, GhGPX8 are positioned in cytoplasm；C, GFP albumen（Empty carrier is expressed）Subcellular Localization, positive control
Experimental group；Length of the scale：White is 5 microns（It is descending to scheme A）, red is 10 microns（It is up to scheme A, schemes B, two width figures of figure C left sides）,
Yellow is 100 microns（Scheme two width figures on the right side of C）.
Embodiment
Explanation is further explained to the application with reference to embodiment, before specific embodiment is introduced, with regard to following realities
The briefly introduction of involved part biological material in example, experiment reagent and experimental facilities situation is applied to be described as follows.
Biomaterial：
Strain：
EHA105 Agrobacterium strains, provided by China Agricultural University Ren Dongtao professor's friendship（It is of course also possible to use other commodity
The agrobacterium strains of change carry out related transgenic operation experiments）；
E.colistraindh5α used in conversion process, a kind of commercialization bacterial strain；
Cotton material：CCRI 36, is provided by the Chinese Academy of Agriculture Science and Technologys Cotton Research Institute；
Plasmid：
pCAMBIA1381Plasmid, provided by He'nan University Song Chunpeng professor's friendship；
p35S-GFP, provided by China Agricultural University Guo Yan professor's friendship；
pGhGPX1pro-GUS,pGhGPX1-GFP（p35S-GhGPX1-GFP）,pGhGPX8pro-GUSWithpGhGPX8-GFP （p35S-GhGPX8-GFP）It is briefly to be situated between in embodiment by the recombinant plasmid of both the above plasmid construction, specific construction step
Continue；
Experiment reagent
The reagent such as acetosyringone, kanamycins, rifampin, Tween20, X-Gluc is Solarbio Products；
Extract RNA and be Tiangeng by the kit that its reverse transcription is cDNA（Tiangen, China）Products；
Real-time fluorescence quantitative PCR kit is Pu Luomaige（Promega, the U.S.）Products；
Partial medium specific formula is：
YEP culture mediums：10 g/L peptones, 10 g/L yeast extracts, 5 g/L sodium chloride；
MS culture mediums（Containing 0.8%（w/v）Agar powder）：4.4 g/L MS salt（Murashige & Skoog Basal Medium
with Vitamins, Phyto Technology Laboratories）；3 g/L sucrose；6 g/L agar powders；1/2MS be by
The content of MS salt reduces half, and other compositions content is identical.
Experimental facilities：
PCR instrument, Biometra GmbH, EasyCycler 96, Germany；
Laser confocal microscope, Zeiss 710, Germany.
Embodiment 1
With recombinant plasmidpGhGPX1pro-GUS、p35S-GhGPX1-GFP、pGhGPX8pro-GUSWithp35S-GhGPX8-GFP
Exemplified by, the present embodiment mainly introduces the building process of related recombinant plasmid.
pGhGPX1pro-GUSWithpGhGPX8pro-GUSIt is to clone respectivelyGhGPX1WithGhGPX8Promoter region（It is
The bp of initiation codon ATG upstreams about 2000 sequence）, it is then attached topCAMBIA1381Recombinant plasmid；
pGhGPX1-GFP（p35S-GhGPX1-GFP）WithpGhGPX8-GFP（p35S-GhGPX8-GFP）It is to clone respectivelyGhGPX1WithGhGPX8Coded sequence, be connected top35S-GFPThe recombinant plasmid of plasmid construction；
Primer sequence and restriction enzyme site are with specific reference to as shown in the table used in the process of related construction of recombinant plasmid：
。
Specific building process refers to existing Protocols in Molecular Biology associative operation, carries out gene cloning first, then carries out
Digestion and with corresponding plasmid connect after, conversion, Screening and Identification, for details, reference can be made to《The Glutathione
Peroxidase Gene Family in Gossypium hirsutum: Genome-Wide Identification,
Classification, Gene Expression and Functional Analysis》（Chen et al., 2017,Scientific Reports, 7: 44743. doi: 10.1038/srep44743）Middle associative operation introduction.
Embodiment 2
Agriculture bacillus mediated cotton transient transformation methods provided herein, specifically include following operating procedure, its operation stream
Journey schematic diagram is as shown in Figure 1.
（One）Instantaneous conversion solution is prepared, is comprised the following steps that：
Respectively by prepared by embodiment 1pGhGPX1pro-GUS、p35S-GhGPX1-GFP、pGhGPX8pro-GUSWithp35S- GhGPX8-GFPEHA105 agrobacterium strains after the conversion of recombinant plasmid, resistance screening is carried out, and carry out bacterium colony PCR checkings, chosen
The correct colony inoculation of checking is taken in YEP fluid nutrient mediums（Mycin and 50 mg/L rifampins are received containing 50 mg/L cards）Middle culture,
28 DEG C, the h of 220 rpm Shaking cultures 16 ~ 17 or so.
Then cultivated again：The bacterium solution for taking 5 mL to cultivate goes to 300 mL fresh liquid YEP culture mediums（Without antibiosis
Element）In, 28 DEG C, 220 rpm Shaking cultures to OD600About 1.2 ~ 1.5, then 2000 g centrifugations, 10 min, collect Agrobacterium bacterium
Body is standby.
Thalline collected after above-mentioned centrifugation is resuspended, re-suspension liquid is specially：Contain 165 μM of acetosyringones, 3%
（w/v）Sucrose and 0.01%（v/v）Tween20 1/2 MS（pH=5.8）Solution, Agrobacterium concentration is adjusted extremely with re-suspension liquid
OD600=0.8 ~ 1.0 or so, this is instantaneous conversion solution.It should be noted that the change of each reagent concentration is directed in conversion fluid
During different plasmid Transformation Applications, its transformation efficiency has Different Effects, and above-mentioned concentration is only the preferred concentration for the present embodiment.
（Two）Cotton seedling is cultivated, and carries out hypertonic pretreatment, operation in detail is as follows：
The cotton variety seed of CCRI 36 is after planting cultivated, condition of culture is：The h illumination of photoperiod 16/8 h are dark, light
Strong is 150 ~ 200 μm of ol m-2 s-1, cultivation temperature is 20 DEG C of 26 DEG C/night day；
Take the cotton seedling of growth 7 days or so（Before true leaf does not occur, Fig. 2 left figures are seen）, first washed away and sticked on seedling with clear water
Nutrient solution and other impurity, slightly spray alcohol（75%, v/v）After be put into superclean bench, with 0.1%（w/v）HgCl2Enter
Row full scale wash, then seedling is soaked in ddH25 min in O, weak vibrations wash away the HgCl of residual2, ddH is changed repeatedly2O, clearly
4 ~ 5 times are washed to ensure to clean up；
Cotton seedling after cleaning and sterilizing is placed in high sepage and carries out 3 min of hypertonic pretreatment, high sepage is specially：Containing 165 μM
Acetosyringone and 3% sucrose, 1/2 MS solution of pH=5.8, the osmotic pressure of high sepage is 0.7 ~ 0.75 kg/cm2Left and right
（It should be noted that too high osmotic pressure easily damages seedling, but it is too low when can not then effectively facilitate genetic transformation）.
（Three）Instantaneous conversion is handled, specifically：
By step（2）Seedling after middle and high infiltration processing is put into step（1）In instantaneous conversion solution in, 25 DEG C, 120 rpm（Carry
For suitable illumination, about 80 μm of ol m of intensity of illumination-2 s-1）Cultivate 5 h（See Fig. 2 intermediate pictures, incubation time is long and nothing
Cultivated under illumination condition, seedling is easily dead）.
By the seedling ddH after conversion2O is washed twice, and after removing the Agrobacterium being stained with seedling, is placed in containing antibiotic
（50 mg/L cards receive mycin and 50 mg/L rifampins）With 0.8%（w/v）The 1/2 MS culture basal growths of agar powder；Condition of culture
For：The h illumination of photoperiod 16/8 h are dark, and light intensity is 80 ~ 90 μm of ol m-2 s-1, cultivation temperature is：20 DEG C of 26 DEG C/night of day；
Culture can carry out gene expression detection or other analyses after 5 ~ 7 days（See Fig. 2 right figures）；
It should be noted that if occurring Agrobacterium bacterium colony around seedling during culture, need to be by plant to avoid Agrobacterium from polluting
Move in another fresh culture medium and cultivated.
Embodiment 3
Based on the upland cotton plant of the transgenosis transient overexpression constructed by embodiment 2, the table available for detection upland cotton gene
Expression patterns and Subcellular Localization etc. are tested, and related experiment application is briefly discussed below.
（One）Transient transformation methods can be used for the upland cotton that structure gene overexpresses in short term
Transgenosis conversion operation method based on embodiment 2, constructs instantaneous conversionp35S-GhGPX1-GFPWithp35S- GhGPX8-GFPUpland cotton, respectively take 3 plants of conversions respectivelyp35S-GhGPX1-GFPWithp35S-GhGPX8-GFPUpland cotton, carry
It is cDNA to take RNA and reverse transcription, is detected with real-time fluorescence quantitative PCR in instantaneous conversion plantGhGPX1WithGhGPX8Gene table
Up to amount（UBQ7For reference gene, gene searching number is DQ116441）.
When real-time fluorescence quantitative PCR detects, primer sequence is specifically listed as follows as shown in SEQ ID NO.1 ~ 6：：
。
Testing result is shown, in transient expressionGhGPX1Plant in, highestGhGPX1Expression quantity is wild type（WT）
More than 10 times of upland cotton；And in transient expressionGhGPX8Plant in, highestGhGPX8Expression quantity is wild type（WT）Land
More than 5 times of ground cotton（Referring to Fig. 3）.The result shows that the transient transformation methods of the application can express land in upland cotton
Cotton gene, obtain the upland cotton of gene transient overexpression.Real-time fluorescence quantitative PCR is shown in Table 2. using primer
（Two）Transient transformation methods are used for the gene expression pattern for analyzing upland cotton
Based on the instantaneous conversion constructed by embodiment 2pGhGPX1pro-GUSWithpGhGPX8pro-GUSUpland cotton, take respectively
The materials such as root, stem and the leaf of instantaneous conversion upland cotton, detected using the method for chemical stainingGhGPX1WithGhGPX8Promoter
The expression of the gus gene of driving（Associative operation can be found in《AIK1, A Mitogen-Activated Protein
Kinase, Modulates Abscisic Acid Responses through the MKK5-MPK6 Kinase
Cascade》Li et al., 2017, Plant Physiology, 173(2): 1391-1408. doi: 10.1104/
pp.16.01386）, so that it is determined thatGhGPX1WithGhGPX8The expression pattern of gene.
Experimental data shows,GhGPX1There is expression in root, stem and Ye Zhongjun（Fig. 4 A）, with expression quantity highest in cotyledon（Figure
4C）；GhGPX8There is expression in root, stem and Ye Zhongjun（Fig. 4 B）, with expression quantity highest in stem（Fig. 4 D）.
（Three）Transient transformation methods are used for the Subcellular Localization for analyzing upland cotton protein
Based on the instantaneous conversion constructed by embodiment 2p35S-GhGPX1-GFPWithp35S-GhGPX8-GFPUpland cotton, respectively
Take blade table leather strap and the root of instantaneous conversion material, with laser confocal scanning microscope observation GhGPX1-GFP and
GhGPX8-GFP green fluorescence distribution situation（Laser confocal scanning microscope detection operation referring to《AIK1, A
Mitogen-Activated Protein Kinase, Modulates Abscisic Acid Responses through
the MKK5-MPK6 Kinase Cascade》Li et al., 2017, Plant Physiology, 173(2): 1391-
1408. doi: 10.1104/pp.16.01386）.
As a result show, the self-luminous of GhGPX1-GFP green fluorescence and chloroplaset is completely superposed, and shows GhGPX1-GFP
It is positioned in the chloroplaset of guard cell（Chloroplast auto-fluorescence is red fluorescence, is yellow after being superimposed with GFP green fluorescence
Fluorescence）（Fig. 5 A）；And GhGPX8-GFP is positioned in the cytoplasm of guard cell and root cells（Fig. 5 B）；Fig. 5 C arep35S-GFP
GFP distribution in empty carrier instantaneous conversion upland cotton, it is the control group of experiment.
It should be added that based on bimolecular fluorescence complementary system, using the instantaneous conversion side in the application
Method, two upland cotton genes of cotransformation, it can be used for the interaction for detecting protein in upland cotton.
Also need to illustrate, above-described embodiment is prepared for recombinant plasmid only by taking GUS, GFP gene as an example, and by its
Converting cotton seedling has carried out preliminary test, on the basis of above-mentioned technical thought, selects other genes and plasmid, can equally obtain
Similar technique effect is obtained, is not repeated to describe herein.
SEQUENCE LISTING
<110>He'nan University
<120>A kind of agriculture bacillus mediated cotton transient transformation methods
<130> none
<160> 6
<170> PatentIn version 3.5
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Claims (7)
1. a kind of agriculture bacillus mediated cotton transient transformation methods, it is characterised in that specifically comprise the following steps：
（1）Prepare instantaneous conversion solution
After the recombinant plasmid transformed Agrobacterium containing gene to be transformed, the Agrobacterium of restructuring is shaken in YEP fluid nutrient mediums
Bottle culture, after culture terminates, centrifugation, collects thalline, and thalline is resuspended with re-suspension liquid, adjusts OD600=0.8 ~ 1.0 are used as instantaneous conversion
Solution；
The re-suspension liquid is 1/2 MS solution, wherein containing 150 ~ 170 μM of acetosyringones, 2 ~ 4%（w/v）Sucrose and 0.01 ~
0.02%（v/v）Tween20；
（2）Cotton seedling is cultivated, and carries out hypertonic pretreatment
By cotton seeds after planting, the cotton seedling of growth 6 ~ 8 days or so is taken, after cleaning, sterilization, is placed in high sepage and carries out height
Ooze pretreatment；
The hypertonic formula of liquid is：The 1/2 MS solution containing acetosyringone and sucrose；
（3）Instantaneous conversion is handled
By step（2）Seedling after middle and high infiltration processing is put into step（1）In instantaneous conversion solution in impregnate culture；
After culture terminates, after the seedling after conversion is cleaned up, it is placed in containing antibiotic and 0.8%（w/v）The 1/2 of agar powder
Continue to cultivate in MS solution.
2. agriculture bacillus mediated cotton transient transformation methods as claimed in claim 1, it is characterised in that step（1）In, re-suspension liquid
For 1/2 MS nutrient solutions, wherein containing 165 μM of acetosyringones, 3%（w/v）Sucrose and 0.01%（v/v）Tween20.
3. agriculture bacillus mediated cotton transient transformation methods as claimed in claim 1, it is characterised in that step（3）In, by step
（2）Seedling after middle and high infiltration processing is put into step（1）In instantaneous conversion solution in when impregnating culture, cultivate under illumination condition,
25 DEG C, 120 rpm cultures, 5 h.
4. agriculture bacillus mediated cotton transient transformation methods as claimed in claim 1, it is characterised in that step（2）In, the height
Sepage is containing 165 μM of acetosyringones and 3% sucrose, 1/2 MS solution of pH=5.8；The osmotic pressure of the high sepage is 0.7
~0.75 kg/cm2, hypertonic pretreatment time is 3 min.
5. agriculture bacillus mediated cotton transient transformation methods as claimed in claim 1, it is characterised in that step（1）In, it is described heavy
Organizing plasmid ispGhGPX1pro-GUS、p35S-GhGPX1-GFP、pGhGPX8pro-GUSOrp35S-GhGPX8-GFP。
6. any one of the claim 1 ~ 5 agriculture bacillus mediated cotton transient transformation methods answering in transfer-gen plant structure
With, it is characterised in that for building the cotton seedling of gene instantaneous conversion.
7. any one of the claim 1 ~ 5 agriculture bacillus mediated cotton transient transformation methods are applied in cotton is studied, it is special
Sign is, for gene expression pattern research and the Subcellular Localization research of protein.
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109402167A (en) * | 2018-12-07 | 2019-03-01 | 北京林业大学 | A method of carrying out gene transient expression in Chinese pine hypocotyl |
| CN112852863A (en) * | 2021-02-03 | 2021-05-28 | 中国林业科学研究院林业研究所 | Xanthoceras sorbifolia petal instantaneous conversion method and application thereof |
| CN113355353A (en) * | 2021-06-04 | 2021-09-07 | 郑州大学 | Application and construction method of four-component BSMV (B-cell-mediated isothermal amplification) overexpression cotton gene vector |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1351668A (en) * | 1999-05-19 | 2002-05-29 | 阿温提斯作物科学公司 | Improved method for agrobacterium mediated transformation of cotton |
| CN105177039A (en) * | 2015-10-26 | 2015-12-23 | 东北林业大学 | Method for transferring exogenous genes into betula platyphylla Suks. seedlings for transient expression |
-
2017
- 2017-10-31 CN CN201711045105.9A patent/CN107858372A/en active Pending
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1351668A (en) * | 1999-05-19 | 2002-05-29 | 阿温提斯作物科学公司 | Improved method for agrobacterium mediated transformation of cotton |
| CN105177039A (en) * | 2015-10-26 | 2015-12-23 | 东北林业大学 | Method for transferring exogenous genes into betula platyphylla Suks. seedlings for transient expression |
Non-Patent Citations (5)
| Title |
|---|
| BAOHONG ZHANG: "Agrobacterium -Mediated Transformation of Cotton", 《METHODS IN MOLECULAR BIOLOGY》 * |
| 尤民生: "《提高农业综合生产能力的理论与实践》", 30 September 2005, 海风出版社 * |
| 徐清锋: "龙眼体胚发生系统的优化及其在种质保存与遗传转化上的应用", 《中国优秀硕士学位论文全文数据库 农业科技辑》 * |
| 李妮娜等: "棉花叶肉原生质体分离及目标基因瞬时表达体系的建立", 《作物学报》 * |
| 王蕾: "棉花子叶瞬时表达系统的建立及应用研究", 《中国优秀硕士学位论文全文数据库 农业科技辑》 * |
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109402167A (en) * | 2018-12-07 | 2019-03-01 | 北京林业大学 | A method of carrying out gene transient expression in Chinese pine hypocotyl |
| CN112852863A (en) * | 2021-02-03 | 2021-05-28 | 中国林业科学研究院林业研究所 | Xanthoceras sorbifolia petal instantaneous conversion method and application thereof |
| CN113355353A (en) * | 2021-06-04 | 2021-09-07 | 郑州大学 | Application and construction method of four-component BSMV (B-cell-mediated isothermal amplification) overexpression cotton gene vector |
| CN113355353B (en) * | 2021-06-04 | 2023-03-10 | 郑州大学 | Application and construction method of four-component BSMV (B-cell-mediated isothermal amplification) overexpression cotton gene vector |
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