CN107267507A - A kind of method for suppressing Brassica plants style nadph oxidase gene expression - Google Patents

A kind of method for suppressing Brassica plants style nadph oxidase gene expression Download PDF

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Publication number
CN107267507A
CN107267507A CN201710224342.5A CN201710224342A CN107267507A CN 107267507 A CN107267507 A CN 107267507A CN 201710224342 A CN201710224342 A CN 201710224342A CN 107267507 A CN107267507 A CN 107267507A
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gene expression
style
complementary nucleotide
nucleotide chain
nadph oxidase
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CN201710224342.5A
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王春雷
黄旭
宋平平
孙娅
刘正阳
张盼盼
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Yangzhou University
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Yangzhou University
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    • 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
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/113—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing
    • C12N15/1137—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing against enzymes
    • 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
    • C12N15/87—Introduction of foreign genetic material using processes not otherwise provided for, e.g. co-transformation
    • 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
    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/50—Physical structure
    • C12N2310/53—Physical structure partially self-complementary or closed
    • C12N2310/531—Stem-loop; Hairpin

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  • Health & Medical Sciences (AREA)
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  • General Health & Medical Sciences (AREA)
  • Plant Pathology (AREA)
  • Virology (AREA)
  • Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)

Abstract

The invention discloses a kind of method for suppressing Brassica plants style nadph oxidase gene expression.Utilize known nadph oxidase gene (the Genbank numbers of logging in：XM_009114548.2 complementary nucleotide chain) is designed at the possibility binding site of its secondary structure, added in culture medium.Brassica plants style is put into culture medium and cultivated 4 hours, allows it to fully absorb complementary nucleotide chain.Style RNA is extracted, reverse transcription goes out nadph oxidase gene expression amount in style by fluorescence quantitative PCR detection and substantially reduced, it was demonstrated that the complementary nucleotide chain can suppress the gene expression into after cDNA.

Description

A kind of method for suppressing Brassica plants style nadph oxidase gene expression
Technical field
The present invention relates to a kind of method for suppressing Brassica plants style nadph oxidase gene expression, it is related to complementary core The design and application of thuja acid chain, belong to biology field.
Background technology
Nadph oxidase gene forms nadph oxidase after transcription and translation.Nadph oxidase is located on plasma membrane, It is a class to generate enzyme of the levels of reactive oxygen species as major function.It can be formed it into by electro transfer to oxygen molecule It is transformed into superoxide anion, further forms the active chalcogen of a classes such as hydroxyl radical free radical, Both peroxyl radical.Because cell is deposited Response to oxidative stress, the height of active chalcogen concentration can adjust the dynamic equilibrium of cell quantity.
At present it is known that the mode of cryptiogene expression is mainly RNA interference, gene knockout, the suppression of activating genes of interest Gene etc..But the experimental cost of these methods is higher, the cycle is long, and the requirement to technology is high, the targeting of part way Property is poor, and experiment effect can not be fully up to expectations.
The content of the invention
It is an object of the invention to provide a kind of method for suppressing Brassica plants style nadph oxidase gene expression, profit Suppress nadph oxidase gene expression, nadph oxidase gene (the Genbank numbers of logging in complementary nucleotide chain：XM_ 009114548.2)。
By extracting the RNA of the Brassica plants column cap treated through complementary nucleotide chain, reverse transcription is utilized into after cDNA Quantitative fluorescent PCR determines the expression quantity of its nadph oxidase.
The technical solution adopted in the present invention is：
A kind of method for suppressing Brassica plants style nadph oxidase gene expression, is comprised the steps of：
1) complementary nucleotide chain and primer synthesis：According to the sequence of target gene, fluorescence quantification PCR primer is designed；And root According to its secondary structure, one section of complementary nucleotide chain is designed at its stem ring；The target gene is nadph oxidase gene；
2) culture medium is configured：10%MS culture mediums, 1.0% agarose, 80mM sucrose, add complementary nucleotide chain, make it Final concentration reaches 400 μM；
3) style culture：Brassica plants style is cut, is put into culture medium and cultivates, after 4 hours, extract its style RNA, and reverse transcription is into cDNA.
4) gene expression amount is determined：Using the cDNA of reverse transcription as template, its NADPH oxygen is determined by quantitative fluorescent PCR Change enzyme gene expression amount.
Further, the complementary nucleotide chain is in the target gene hair fastener ring, inner loop, expansion loop and multi-branched On the equiprobable binding site of ring, complementary nucleotide chain is designed, and carry out thiosulfates modification and HPLC purifying.
Further, the complementary nucleotide chain (5 ' -3 ') is：ATTCTTGTCCACTATGTC.
Further, according to the nadph oxidase gene (number of logging in during gene expression amount is determined：XM_009114548.2) Gene order is designed：Primer sequence (5 ' -3 ') is：It is positive：GAACAGCACAGGAAGCAACA；Reversely： AGCTCTGCCACTTCGTTCAT。
Further, reaction system is：Positive, each 1 μ L of reverse primer (10 μM)；The μ L of template 1；SsoFast EvaGreen supermix (the kit name that quantitative fluorescent PCR is determined) 10 μ L；Without the μ L of enzyme water 7.
Beneficial effects of the present invention：
1) this method can quickly, efficiently suppress expression of the nadph oxidase gene in Brassica plants style；
2) this method has the high feature of the degree of accuracy, can be used for the functional characteristic identification of target gene, before good Scape.
3) the time-consuming shorter, cost of this method is relatively low, and has important meaning for the functional characteristic identification of target gene.
Brief description of the drawings
Fig. 1 is the experiment flow figure of the method for the invention.
Fig. 2 is style culture schematic diagram of the present invention.
Fig. 3 is nadph oxidase gene expression amount of the present invention.
Embodiment
It is not to present invention protection with reference to the step and method of concrete scheme embodiment, further the explaination present invention The limitation of scope.
This example is using wild cabbage as experiment material, and operation is as follows, refers to Fig. 1：
1) nadph oxidase gene order is obtained from Genebank, and (number of logging in is：XM_009114548.2), sent out at it On snap ring, inner loop, expansion loop and the equiprobable binding site of multi-branched ring, complementary nucleotide chain is designed, and carry out thio Sulfuric ester is modified and HPLC purifying.
The complementary nucleotide chain (5 ' -3 ')：ATTCTTGTCCACTATGTC
2) culture medium is configured：10%MS culture mediums, 1.0% agarose, 80mM sucrose.Complementary nucleotide chain is added, makes it Final concentration reaches 400 μM.
3) Btassica style is cut, insertion is cultivated in the culture medium containing 400 μM, and sets blank control group (ck) (Fig. 2).After 4 hours, its style RNA is extracted, and reverse transcription is into cDNA.
4) using the cDNA of transcription as template, fluorescent quantitation pcr is carried out.Reaction system is：Positive, reverse primer (10 μM) is each 1μL；The μ L of template 1；SsoFast EvaGreen supermix10μL；Without the μ L of enzyme water 7.
5) primer sequence (5 ' -3 ') is：
It is positive：GAACAGCACAGGAAGCAACA；
Reversely：AGCTCTGCCACTTCGTTCAT
6) collated analysis is drawn：The expression quantity of nadph oxidase gene after treatment substantially reduces that (Fig. 3, ck are The blank test not dealt with).

Claims (5)

1. a kind of method for suppressing Brassica plants style nadph oxidase gene expression, it is characterised in that：Include following step Suddenly：
1) complementary nucleotide chain and primer synthesis：According to the sequence of target gene, fluorescence quantification PCR primer is designed；And according to it Secondary structure, designs one section of complementary nucleotide chain at its stem ring；The target gene is nadph oxidase gene；
2) culture medium is configured：10%MS culture mediums, 1.0% agarose, 80mM sucrose, add complementary nucleotide chain, make it dense eventually Degree reaches 400 μM；
3) style culture：Brassica plants style is cut, is put into culture medium and cultivates, after 4 hours, extract its style RNA, and Reverse transcription is into cDNA.
4) gene expression amount is determined：Using the cDNA of reverse transcription as template, its nadph oxidase is determined by quantitative fluorescent PCR Gene expression amount.
2. the method for gene expression according to claim 1, it is characterised in that：The complementary nucleotide chain is in the mesh Gene hair fastener ring, inner loop, expansion loop and the equiprobable binding site of multi-branched ring on, design complementary nucleotide chain, and Carry out thiosulfates modification and HPLC purifying.
3. the method for gene expression according to claim 2, it is characterised in that：The complementary nucleotide chain (5 ' -3 ') is： ATTCTTGTCCACTATGTC 。
4. the method for gene expression according to claim 1, it is characterised in that：According to NADPH in gene expression amount measure Oxidase gene (the number of logging in：XM_009114548.2 gene order design)：Primer sequence (5 ' -3 ') is：
It is positive：GAACAGCACAGGAAGCAACA；Reversely：AGCTCTGCCACTTCGTTCAT.
5. the method for gene expression according to claim 4, it is characterised in that：The positive, reverse primer (10 μM) each 1 μL；The μ L of template 1；SsoFast EvaGreen supermix10μL；Without the μ L of enzyme water 7.
CN201710224342.5A 2017-04-07 2017-04-07 A kind of method for suppressing Brassica plants style nadph oxidase gene expression Withdrawn CN107267507A (en)

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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN108611313A (en) * 2018-05-09 2018-10-02 扬州大学 A method of detaching papilla cell from turnip column cap

Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN1671847A (en) * 2002-07-22 2005-09-21 巴斯福植物科学有限公司 Method for obtaining the pathogenic resistance in plants
CN102181482A (en) * 2011-01-25 2011-09-14 中山大学 Method for rapidly regulating activity of endogenous miRNA in rice
CN105358695A (en) * 2013-01-01 2016-02-24 A.B.种子有限公司 Methods of introducing dsRNA to plant seeds for modulating gene expression

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN1671847A (en) * 2002-07-22 2005-09-21 巴斯福植物科学有限公司 Method for obtaining the pathogenic resistance in plants
CN102181482A (en) * 2011-01-25 2011-09-14 中山大学 Method for rapidly regulating activity of endogenous miRNA in rice
CN105358695A (en) * 2013-01-01 2016-02-24 A.B.种子有限公司 Methods of introducing dsRNA to plant seeds for modulating gene expression

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
SOO JIN WI等: "Synergistic Biosynthesis of Biphasic Ethylene and Reactive Oxygen Species in Response to Hemibiotrophic Phytophthora parasitica in Tobacco Plants", 《PLANT PHYSIOL.》 *
张万年: "《现代药物设计学》", 31 May 2006, 北京：中国医药科技出版社 *

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN108611313A (en) * 2018-05-09 2018-10-02 扬州大学 A method of detaching papilla cell from turnip column cap

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