CN105441470A - Gene mapping expression vector for detecting plant autophagy phenomenon and application - Google Patents

Gene mapping expression vector for detecting plant autophagy phenomenon and application Download PDF

Info

Publication number
CN105441470A
CN105441470A CN201610017008.8A CN201610017008A CN105441470A CN 105441470 A CN105441470 A CN 105441470A CN 201610017008 A CN201610017008 A CN 201610017008A CN 105441470 A CN105441470 A CN 105441470A
Authority
CN
China
Prior art keywords
gene
ntatg8
expression carrier
location expression
phenomenon
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Pending
Application number
CN201610017008.8A
Other languages
Chinese (zh)
Inventor
周会娜
陈千思
刘萍萍
陈霞
金立锋
翟妞
郑庆霞
张慧
林福呈
徐国云
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Zhengzhou Tobacco Research Institute of CNTC
Original Assignee
Zhengzhou Tobacco Research Institute of CNTC
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Zhengzhou Tobacco Research Institute of CNTC filed Critical Zhengzhou Tobacco Research Institute of CNTC
Priority to CN201610017008.8A priority Critical patent/CN105441470A/en
Publication of CN105441470A publication Critical patent/CN105441470A/en
Pending legal-status Critical Current

Links

Classifications

    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/66—General methods for inserting a gene into a vector to form a recombinant vector using cleavage and ligation; Use of non-functional linkers or adaptors, e.g. linkers containing the sequence for a restriction endonuclease
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
    • G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
    • G01N21/64—Fluorescence; Phosphorescence
    • G01N21/6402—Atomic fluorescence; Laser induced fluorescence

Landscapes

  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Chemical & Material Sciences (AREA)
  • Physics & Mathematics (AREA)
  • Engineering & Computer Science (AREA)
  • Organic Chemistry (AREA)
  • Biomedical Technology (AREA)
  • Biotechnology (AREA)
  • General Engineering & Computer Science (AREA)
  • Molecular Biology (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • General Health & Medical Sciences (AREA)
  • Wood Science & Technology (AREA)
  • Zoology (AREA)
  • Biochemistry (AREA)
  • Biophysics (AREA)
  • Microbiology (AREA)
  • Plant Pathology (AREA)
  • Optics & Photonics (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • Analytical Chemistry (AREA)
  • General Physics & Mathematics (AREA)
  • Immunology (AREA)
  • Pathology (AREA)
  • Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)

Abstract

The invention belongs to the technical field of biology, and particularly relates to construction of a gene vector for detecting a cell autophagy phenomenon. A protein of an Atg8 gene mapping expression vector is expressed in a suspension cell after being fused with a green fluorescence protein (GFP), and subcellular localization is performed by detecting the position of a fused protein through a laser scanning confocal microscope. Through a simple, convenient and accurate method for locating in living cells, a reference basis is laid for the research of the molecular mechanism of the tobacco cell autophagy phenomenon.

Description

A kind of gene location expression carrier and application detecting plant autophagy phenomenon
Technical field
The invention belongs to biological technical field, be specifically related to the structure of the genophore detecting cell autophagy phenomenon, and utilize described gene location expression study on the carrier cell autophagy phenomenon.
Background technology
Autophagy (Autophagy) is an important degradation mechanism in cell, senescence protein in cell can be transported to lysosome degrade by autophagy.Cell autophagy phenomenon is extensively present in most of eukaryote and comprises in the cell of yeast, fruit bat, plant and human body, being that cell cycle utilizes and self damaged a kind of important way of the nutritive substances such as the amino acid comprised in protein, organoid, is also a kind of adaptation to environment formed in eukaryote evolutionary process.
The process of cell autophagy can be roughly divided into four-stage: one, and cell receives as after the autophagy inducement signals such as hunger, nutritional factor shortage or disease and pest invasion and attack, produces a certain amount of flat free phospholipid bilayer membrane structure, be centered around and treat around degradation product in endochylema; Two, membrane structure constantly extends, and endochylema, organoid is waited for that degradation product holds, forms the autophagosome closed; Three, autophagosome is transported in lysosome also to merge with it by cytoskeletal microtubule network system and forms autophagy lysosome; Four, after forming autophagy lysosome, the membrane structure of autophagosome is degraded, and degraded product is transported in endochylema to be waited to re-use, and residue is trapped in endochylema or is transported out cell.Research in recent years shows in plant pest course of infection, and autophagy has played very large evil effect, even without the existence of any paraprotein, loses the generation that autophagocytosis just can cause disease.The expression of some autophagy related genes (Autophagyrelatedgene, Atg) plays an important role to the maturation that autophagy regulates and controls, and in the different steps that autophagosome is formed, different Atg carries out different regulation and control to it.
Atg8 extremely guards in yeast, Arabidopis thaliana.Have experiment to show, in eukaryote, the protein complexes of Atg5-Atg12 coding take part in the formation of autophagosome; Atg8-phosphatidylethanolamine complex body controls the expansion of autophagosome.Have and show that Atg8 and homologous gene thereof can be located autophagosome clearly and therefore become the marker gene of autophagy phenomenon about yeast and mammiferous research.In plant, is also no lack of the research about Atg8 gene.Su etc. have cloned OsAtg8 from paddy rice, and the joint access function demonstrating Atg8 is by experiment conservative in paddy rice, therefore infer that this gene has probably played vital role in paddy rice autophagy system.Certainly, Atg8 gene has played important effect in autophagy process, but wants clear and definite Atg8 to participate in a series of molecule mechanisms of the formation of autophagy, still lacks relevant Identification and detection material at present.
Summary of the invention
The invention provides a kind of gene location expression carrier detecting eukaryotic cells autophagy phenomenon and cytolysosome, described genophore comprises a relevant autophagy phenomenon found in tobacco, participates in the gene GFP of conservative homologous gene NtAtg8 and the mark fluorescent albumen that autophagosome is formed as reporter gene.
For achieving the above object, the present invention is by the following technical solutions:
A kind of construction process detecting the gene location expression carrier of plant autophagy phenomenon, with the large gold dollar tobacco bred of safflower for material, extract RNA, reverse transcription obtains cDNA, take NtAtg8-F/NtAtg8-R as primer pair, carry out pcr amplification NtAtg8 gene, double digestion system process DNA fragmentation also reclaims, and adopts same double digestion system process pFF19-GFP plasmid and reclaims; NtAtg8 gene containing same sticky end is connected with pFF19-GFP carrier, connects product conversion in DH5 α competent cell, select positive bacterium colony sequence verification.
The system that described NtAtg8 gene is connected with pFF19-GFP carrier is: enzyme cuts each 1 μ l of rear plasmid, Buffer1 μ l, T4 ligase enzyme 1 μ l, and moisturizing to 10 μ l, 16 DEG C of connections are spent the night.
Described NtAtg8-F/NtAtg8-R primer pair sequence is as shown in SEQIDNO:1/SEQIDNO:2, and wherein GGTACC is KpnI restriction enzyme site, and GGATCC is BamHI restriction enzyme site.
The fragment of described NtAtg8 gene is as shown in SEQIDNO:3.
The fusion rotein aminoacid sequence of described NtAtg8 gene translation is as shown in SEQIDNO:4.
The reaction conditions of described pcr amplification is as follows: 94 DEG C of denaturation 5min, 94 DEG C of 30s, 62 DEG C of 30s, 72 DEG C of 40s, 35 circulations, then 72 DEG C of 10min, 16 DEG C of 10min, 4 DEG C of preservations.
Described double digestion system is: BufferK2.5 μ L, BamHI2.5 μ L, SphI2.5 μ L, template 20 μ L, adds aqua sterilisa to 50 μ L.
Detect an application for the gene location expression carrier of plant autophagy phenomenon, NtAtg8-pFF19-GFP plasmid is proceeded to tobacco suspension cell, by confocal laser scanning microscope suspension cell, location autophagosome.
Described method plasmid being proceeded to tobacco suspension cell is Biolistic mediated transformation method.
Described gene location expression carrier is analyzing the application of vegetable cell autophagy molecule mechanism.
Beneficial effect of the present invention is: utilize the carrier that the present invention builds, express in suspension cell after the protein fusion that its albumen and goal gene are marked, the Subcellular Localization of being undertaken by the position of laser confocal microscope detection fusion albumen, a kind of easy and method of positioning in viable cell accurately, for the molecule mechanism of research tobacco cell autophagy phenomenon provides reference frame.
Accompanying drawing explanation
Fig. 1 is NtAtg8-pFF19-GFP carrier digestion verification figure, and wherein M is 1KbPlusDNALadder, and 1 is NtAtg8-pFF19-GFP plasmid enzyme restriction figure;
Fig. 2 is NtAtg8-pFF19-GFP genophore location map.
Embodiment
A kind of construction process detecting the gene location expression carrier of plant autophagy phenomenon, with the large gold dollar tobacco bred of safflower for material, extract RNA, reverse transcription obtains cDNA, take NtAtg8-F/NtAtg8-R as primer pair, carry out pcr amplification NtAtg8 gene, double digestion system process DNA fragmentation also reclaims, and adopts same double digestion system process pFF19-GFP plasmid and reclaims; NtAtg8 gene containing same sticky end is connected with pFF19-GFP carrier, connects product conversion in DH5 α competent cell, select positive bacterium colony sequence verification.
The system that described NtAtg8 gene is connected with pFF19-GFP carrier is: enzyme cuts each 1 μ l of rear plasmid, Buffer1 μ l, T4 ligase enzyme 1 μ l, and moisturizing to 10 μ l, 16 DEG C of connections are spent the night.
Described NtAtg8-F/NtAtg8-R primer pair sequence is as shown in SEQIDNO:1/SEQIDNO:2, and wherein GGTACC is KpnI restriction enzyme site, and GGATCC is BamHI restriction enzyme site.
The fragment of described NtAtg8 gene is as SEQIDNO:3.
The reaction conditions of described pcr amplification is as follows: 94 DEG C of denaturation 5min, 94 DEG C of 30s, 62 DEG C of 30s, 72 DEG C of 40s, 35 circulations, then 72 DEG C of 10min, 16 DEG C of 10min, 4 DEG C of preservations.
Described double digestion system is: BufferK2.5 μ L, BamHI2.5 μ L, SphI2.5 μ L, template 20 μ L, adds aqua sterilisa to 50 μ L.
Detect an application for the gene location expression carrier of plant autophagy phenomenon, NtAtg8-pFF19-GFP plasmid is proceeded to tobacco suspension cell, by confocal laser scanning microscope suspension cell, location autophagosome.
Described method plasmid being proceeded to tobacco suspension cell is Biolistic mediated transformation method.
Described gene location expression carrier is analyzing the application of vegetable cell autophagy molecule mechanism.
Below in conjunction with specific embodiment, set forth the present invention further, these embodiments are only not used in for illustration of the present invention and limit the scope of the invention.The test method of unreceipted actual conditions in the following example, usually conveniently condition.
(1) NtAtg8-pFF19-GFP plasmid construction
Use DNAMAN6.0, in conjunction with the relevant tobacco Atg8 gene coded sequence design primer pair sequence of including in NCBI as shown in SEQIDNO:1 and SEQIDNO:2.
Use Tris-EDTApH=8.0 solution dilution to working concentration 10 μMs after synthetic.
Adopt the method for PCR, template is tobacco Hongda cDNA, and amplification NtAtg8 gene, PCR reaction kit is purchased from Beijing Quanshijin Biotechnology Co., Ltd.Reaction system is: 5 μ L10 × IIbuffer, cDNA are 2 μ L, and each 2.5 μ L of upstream and downstream primer, TransTaqHiFiDNA polysaccharase 0.3 μ L, dNTP4 μ L, add aqua sterilisa to 50 μ L.
Amplification condition is: 94 DEG C of denaturation 5min; 94 DEG C of 30s; 62 DEG C of 30s; 72 DEG C of 40s, 35 circulations; 72 DEG C of 10min; 16 DEG C of 10min.Amplified production is by electrophoresis detection in 1% sepharose and recovery obtains the double chain DNA fragment containing KpnI and BamHI restriction enzyme site.
TaKaRa Bioisystech Co., Ltd KpnI and BamHI enzyme system of cutting is used to digest the fragment obtaining having sticky end, BufferK2.5 μ L, BamHI2.5 μ L, SphI2.5 μ L, template 20 μ L, aqua sterilisa 22.5 μ L, and adopt same double digestion system digestion pFF19-GFP plasmid.
The SolutionI ligase enzyme of TaKaRa Bioisystech Co., Ltd is adopted to be connected with the carrier pFF19-GFP (purchased from addgene company) containing same sticky end by the NtAtg8 gene containing sticky end.Linked system is: NtAtg8 gene 4 μ L, pFF19-GFP plasmid 1 μ L, SolutionI1 μ L.Mixed solution is placed in 16 DEG C after 2 hours and connection product is transformed into bacillus coli DH 5 alpha competent cell with heat shock method, on the LB substratum containing Amp (50 μ g/mL), screening has the bacterial strain of resistance and carries out bacterium colony PCR inspection.
Thus, select positive bacterium colony and can obtain NtAtg8-pFF19-GFP plasmid after sequence verification.
(2) preparation of tobacco suspension cell
Preparing reagent required in the liquid nutrient medium of tobacco suspension cell is macroelement, CaCl 2, trace element, molysite, VB1, sucrose, 2,4-D, desired concn is as follows:
Mother liquor one (10 ×): macroelement comprises KNO 319g/L, NH 4nO 316.5g/L, MgSO 47H 2o3.7g/L, KH 2pO 41.7g/L.
Mother liquor two (100 ×): CaCl 233.2g/L.
Mother liquor three (200 ×): trace element comprises KI33.2mg/200ml, H 3bO 3248mg/200ml, MnSO 4h 20676mg/200ml, ZnSO 47H 2o344mg/200mL, Na 2moO 42H 2o10mg/200mL, CuSO 45H 2o1mg/200mL, CoCl 26H 2o1mg/200mL.
Mother liquor four (200 ×): molysite comprises Na 2eDTA3.73g/500mL, FeSO 47H 2o2.78g/500mL.
Mother liquor five (1000 ×): VB 14mg/ml.
Sucrose: 30g/L.
2，4-D(1000×)：2mg/mL。
According to above concentration preparation BY-2 liquid suspension cell culture medium, adjust its pH to 5.8-6.0, add in another liquid medium within volume be 0.8% agar be configured to BY-2 solid medium.Sterilizing in high-pressure steam sterilizing pan (if produce white precipitate in substratum after sterilizing, rocking rear disappearance, then without impact).
Cultivate and after configuring, get the callus of incubation growth on solid medium in advance, crush with the spoon of sterilizing, the liquid nutrient medium that rear access newly configures.26 DEG C, 130rpm shaking table is cultivated about seven days, callus is placed in 26 DEG C of cultivations.
After seven days, observe the BY-2 cell in liquid medium within, if suspension cell becomes glassy yellow, namely can be used to carry out Biolistic mediated transformation.Callus can grow 2-3 month in solid medium, repeats for preparation BY-2 suspension cell.
Suspension cell need be transferred once in 7 days again, and draw ripe suspension cell with liquid-transfering gun (cutting off rifle head most advanced and sophisticated) and add in new substratum, general proportions is 1:10, if need urgent need, suitably can improve inoculative proportion to shorten the cell maturation time.Callus needs a switching in month once, gets a fritter and puts into new culture dish, can put 3 fritters in each culture dish during switching with rifle choicest.
(3) by Biolistic mediated transformation method, NtAtg8-pffmGFP Plastid transformation is entered tobacco suspension cell
First need to prepare bronze suspension.Getting 60mg bronze joins in 1.5mL import centrifuge tube; In centrifuge tube, add the ethanolic soln that 1mL70% newly prepares, vortex 3-5min, leave standstill the centrifugal 5s of 15min, 3000rpm, carefully remove supernatant; Repeat previous step three times, add 1mL sterilized water, vibration 1min, leaves standstill the centrifugal 2s of 1min, 3000rpm, removes supernatant; Add 50% glycerine 1mL (bronze concentration 60 μ g/ μ L) ,-20 DEG C of preservations.
Next needs to prepare micro-bullet (6 secondary amounts).Get ready bronze vortex 5min; Get 50 μ L bronzes and put into 1.5mL sterile centrifugation tube respectively; Under vorticity, in centrifuge tube, first put into appropriate NtAtg8-pFF19-GFP genophore (1 μ g/ μ L), after be sequentially added into 50 μ LCaCl 2(2.5M, autoclaving), 20 μ L spermidines (0.1M, filtration sterilization); Abundant vibration 3min, leaves standstill 1min to ensure that DNA molecular is behind bronze surface fully sedimentation, the centrifugal 2s of 3000rpm; As far as possible fully supernatant discarded, adds 140 and adds 140 μ L70% washing with alcohol bronzes, do not touch bronze; Abandon supernatant, add 140 μ L absolute ethanol washing bronzes; Abandon supernatant, add 48 μ L dehydrated alcohols and flick tube wall, bronze is mixed, and the micro-bullet in each centrifuge tube can for 6 rifles.
Again, the preparation work of carrying out particle gun is needed.Particle gun is placed in Bechtop to carry out aseptic technique; 70% ethanol is used to carry out disinfection to vacuum chamber; Disk can be split, stop net and micro-bullet film 70% ethanol sterilizing 30min, be placed in aseptic filter paper dries for subsequent use; Get outstanding even genophore-bronze 6 μ L and evenly put middle section in aseptic micro-bullet carrier film, dry up for subsequent use on Bechtop; Disk loading fixed cap can be split screw.To micro-missile-borne body of micro-bullet be housed and stop that net loads micro-bullet launching device.
Finally, with particle gun, NtAtg8-pFF19-GFP genophore is bombarded.Open voltage stabilized source, vacuum pump and nitrogen valve switch; Acceptor material is put into sample chamber, range 9cm; Exhaust, takes out sample, seals up with sealed membrane; The suspension cell room temperature lucifuge of bombarding is placed, spends the night.
(4) autophagosome is located by confocal laser scanning microscope suspension cell
First the preparation of slide will be carried out.Draw appropriate ultrapure water droplet with suction pipe and bombard position (slightly rubescent) in bronze, pressure-vaccum several times gently, and cell is fully suspended; Draw appropriate cell suspension drop on slide glass, covered gently.
Then observed by laser confocal microscope.Being inverted by slide is put on Stage microscope, focuses by bright field, finds the cell sending bright green fluorescence after getting a clear view by dark-field; The z-axis of setting laser copolymerization Jiao is taken pictures to cell.
Fluorescent places in figure, in the distribution of cell internal specific, form size structure conforms to the cytolysosome of vegetable cell, therefore, this carrier can be utilized as the gene positioning carrier altogether of cell autophagy corpusculum.

Claims (10)

1. one kind is detected the construction process of the gene location expression carrier of plant autophagy phenomenon, it is characterized in that: with the large gold dollar tobacco bred of safflower for material, extract RNA, reverse transcription obtains cDNA, take NtAtg8-F/NtAtg8-R as primer pair, carry out pcr amplification NtAtg8 gene, double digestion system process DNA fragmentation also reclaims, and adopts same double digestion system process pFF19-GFP plasmid and reclaims; NtAtg8 gene containing same sticky end is connected with pFF19-GFP carrier, connects product conversion in DH5 α competent cell, select positive bacterium colony sequence verification.
2. the construction process detecting the gene location expression carrier of plant autophagy phenomenon as claimed in claim 1, it is characterized in that: the system that described NtAtg8 gene is connected with pFF19-GFP carrier is that enzyme cuts each 1 μ l of rear plasmid, Buffer1 μ l, T4 ligase enzyme 1 μ l, moisturizing to 10 μ l, 16 DEG C of connections are spent the night.
3. the construction process detecting the gene location expression carrier of plant autophagy phenomenon as claimed in claim 1, it is characterized in that: described NtAtg8-F/NtAtg8-R primer pair sequence is as shown in SEQIDNO:1/SEQIDNO:2, wherein GGTACC is KpnI restriction enzyme site, and GGATCC is BamHI restriction enzyme site.
4. the construction process detecting the gene location expression carrier of plant autophagy phenomenon as claimed in claim 1, is characterized in that: the fragment of described NtAtg8 gene is as shown in SEQIDNO:3.
5. the construction process detecting the gene location expression carrier of plant autophagy phenomenon as claimed in claim 1, is characterized in that: the fusion rotein aminoacid sequence of described NtAtg8 gene translation is as shown in SEQIDNO:4.
6. the construction process detecting the gene location expression carrier of plant autophagy phenomenon as claimed in claim 1, it is characterized in that: the reaction conditions of described pcr amplification is as follows: 94 DEG C of denaturation 5min, 94 DEG C of 30s, 62 DEG C of 30s, 72 DEG C of 40s, 35 circulations, then 72 DEG C of 10min, 16 DEG C of 10min, 4 DEG C of preservations.
7. the construction process detecting the gene location expression carrier of plant autophagy phenomenon as claimed in claim 1, is characterized in that: described double digestion system is: BufferK2.5 μ L, BamHI2.5 μ L, SphI2.5 μ L, template 20 μ L, adds aqua sterilisa to 50 μ L.
8. detect an application for the gene location expression carrier of plant autophagy phenomenon, it is characterized in that: NtAtg8-pFF19-GFP plasmid is proceeded to tobacco suspension cell, by confocal laser scanning microscope suspension cell, location autophagosome.
9. the application detecting the gene location expression carrier of plant autophagy phenomenon as claimed in claim 6, is characterized in that: described method plasmid being proceeded to tobacco suspension cell is Biolistic mediated transformation method.
10. the application detecting the gene location expression carrier of plant autophagy phenomenon as claimed in claim 6, is characterized in that: described gene location expression carrier is analyzing the application of vegetable cell autophagy molecule mechanism.
CN201610017008.8A 2016-01-08 2016-01-08 Gene mapping expression vector for detecting plant autophagy phenomenon and application Pending CN105441470A (en)

Priority Applications (1)

Application Number Priority Date Filing Date Title
CN201610017008.8A CN105441470A (en) 2016-01-08 2016-01-08 Gene mapping expression vector for detecting plant autophagy phenomenon and application

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
CN201610017008.8A CN105441470A (en) 2016-01-08 2016-01-08 Gene mapping expression vector for detecting plant autophagy phenomenon and application

Publications (1)

Publication Number Publication Date
CN105441470A true CN105441470A (en) 2016-03-30

Family

ID=55552101

Family Applications (1)

Application Number Title Priority Date Filing Date
CN201610017008.8A Pending CN105441470A (en) 2016-01-08 2016-01-08 Gene mapping expression vector for detecting plant autophagy phenomenon and application

Country Status (1)

Country Link
CN (1) CN105441470A (en)

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN107058373A (en) * 2017-04-07 2017-08-18 中国烟草总公司郑州烟草研究院 One carry NtRBSC1 genes transient expression vector
CN109191467A (en) * 2018-08-01 2019-01-11 华中科技大学鄂州工业技术研究院 A kind of prediction technique and device of cell autophagy phenotype

Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN104535551A (en) * 2015-01-05 2015-04-22 南京农业大学 Method for detecting available nitrogen in soil based on duration for Atg8 completely entering vacuole
US20150299719A1 (en) * 2011-08-30 2015-10-22 Korea University Research And Business Foundation Transgenic tree induced by rabg3bca and use thereof

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20150299719A1 (en) * 2011-08-30 2015-10-22 Korea University Research And Business Foundation Transgenic tree induced by rabg3bca and use thereof
CN104535551A (en) * 2015-01-05 2015-04-22 南京农业大学 Method for detecting available nitrogen in soil based on duration for Atg8 completely entering vacuole

Non-Patent Citations (3)

* Cited by examiner, † Cited by third party
Title
K. TOYOOKA ET AL.: "Autophagy and Non-Classical Vacuolar Targeting in Tobacco BY-2 Cells", 《BIOTECHNOLOGY IN AGRICULTURE AND FORESTRY》 *
KOHKI YOSHIMOTO ET AL.: "Processing of ATG8s, Ubiquitin-Like Proteins, and Their Deconjugation by ATG4s Are Essential for Plant Autophagy", 《THE PLANT CELL》 *
孙建生等: "烟草NtGGPPS3基因的亚细胞定位和组织表达分析", 《贵州农业科学》 *

Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN107058373A (en) * 2017-04-07 2017-08-18 中国烟草总公司郑州烟草研究院 One carry NtRBSC1 genes transient expression vector
CN109191467A (en) * 2018-08-01 2019-01-11 华中科技大学鄂州工业技术研究院 A kind of prediction technique and device of cell autophagy phenotype
CN109191467B (en) * 2018-08-01 2021-06-01 华中科技大学鄂州工业技术研究院 Method and device for predicting autophagy phenotype of cell

Similar Documents

Publication Publication Date Title
JP4343430B2 (en) Method for recovering polypeptides from plants and plant parts
CN1876819B (en) Genetically engineered duckweed
CN105861332B (en) A kind of bacterial strain of the promotion with leaf pocket orchid plant strain growth and its application
CN102994401A (en) Method for preparing apple tree valsa ceratosperma transformant and GFP (Green Fluorescent Protein) labelled strain
CN105441470A (en) Gene mapping expression vector for detecting plant autophagy phenomenon and application
CN106755080A (en) It is a kind of quickly and efficiently in grape instantaneous conversion gene method for transformation
CN110468150A (en) RGS1 gene is improving widow according to the application under environment in tomato bacterial leaf spot resistance as negative regulatory factor
CN107858372A (en) A kind of agriculture bacillus mediated cotton transient transformation methods
CN108841748A (en) Sinorhizobium nitrogen-fixing bacteria strain H6 and its application
CN104131021A (en) Antibacterial peptide coexpression vector, construction and expression method thereof
CN113481234B (en) Method and device for transient expression of genes in tomato plants
CN116463374A (en) Application of GhSTP18 gene in regulation and control of salt tolerance of plants
CN114085854B (en) Drought-resistant and salt-tolerant gene OsSKL2 for rice and application thereof
CN102533809B (en) Jujube glutathione peroxidase gene
CN102120997A (en) Method for hydrolyzing RNA by grape pathogenesis-related (PR) protein PR10-1 and improving bacteriostatic activity
CN108124873A (en) Application of the insecticidal proteins in scarabs are prevented
CN102746391A (en) Arsenic-resistance related protein PvArrp1, and coding gene and application thereof
CN103468724A (en) Peanut salt resistance gene AhSOS2, clone method and application
CN107459564A (en) The application of rice ZYGO1 albumen and its encoding gene in pollen fertility is regulated and controled
CN103233039A (en) Agrobacterium tumefaciens-mediated rice seedling gene transformation method
CN109265553A (en) A kind of fusion protein of cytoglobin and Sipunculus nudus plasmin
CN105524930A (en) Arabidopsis thaliana disease resistance-related gene AtIAA17 and preparation method and application thereof
CN105505943A (en) Pregnancy-specific glycoprotein 3 standard substance, preparation method thereof and recombinant strain used for preparation
CN117264999B (en) Application of NtUGT A1L1 gene in tobacco cold resistance
CN114317467B (en) Eucommia laccase EuLAC1 gene and application thereof

Legal Events

Date Code Title Description
C06 Publication
PB01 Publication
C10 Entry into substantive examination
SE01 Entry into force of request for substantive examination
RJ01 Rejection of invention patent application after publication

Application publication date: 20160330

RJ01 Rejection of invention patent application after publication