CN107312845A - Differentiate primer, kit and the discrimination method of wax powder-free Chinese cabbage - Google Patents
Differentiate primer, kit and the discrimination method of wax powder-free Chinese cabbage Download PDFInfo
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- CN107312845A CN107312845A CN201710549794.0A CN201710549794A CN107312845A CN 107312845 A CN107312845 A CN 107312845A CN 201710549794 A CN201710549794 A CN 201710549794A CN 107312845 A CN107312845 A CN 107312845A
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
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- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
- C12Q1/6888—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms
- C12Q1/6895—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms for plants, fungi or algae
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- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
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Abstract
The invention provides primer, kit and the discrimination method for differentiating wax powder-free Chinese cabbage.The primer is as shown in SEQ ID NO.1 and SEQ ID NO.2.Present invention also offers the kit of the discriminating wax powder-free Chinese cabbage including above-mentioned primer.The primer of the present invention can effectively identify the Chinese cabbage of wax powder-free, and the quality trait breeding for distinct Chinese characteristics vegetables Chinese cabbage provides theoretical foundation and material source, to realize that quality trait breeding provides theoretical foundation.
Description
Technical field
The present invention relates to biology field, more particularly, to primer, the reagent for differentiating wax powder-free Chinese cabbage
Box and discrimination method.
Background technology
Plant wax refers to that be covered in extracellular one layer of plant epidermis is more than 18 carbon atoms, masters by lipophilicity is a series of
What over-long chain fatty acid (the very-long-chain fatty acids) compound to be made up of 24 to 34 carbon atoms was constituted
There is wax covering on the surface of hydrophobic layer, the organ such as leaf, stem, flower and the fruit of plant and tissue.Plant wax is satisfied by long-chain
With the material composition (Kunst and Samuels 2003) such as aliphatic acid, alcohols, alkanes, aldehydes, ketone.It is distributed in Plant Cuticular
The epicutile wax layer of layer surface is first of barrier that plant resistant external environment is stimulated, and is lost with non-gas porosity moisture is prevented
Lose, the invasion of resistance germ and the effect such as prevent insect from nibbling, moreover it is possible to resist the influence of the abiotic stresses such as ultraviolet radioactive and frost.
Different floristics its wax composition and content have a very big difference, or even the different parts of same plant its wax contents is all
There is very big difference.By being found to the research of model plant arabidopsis and some other plant, the aliphatic of epicutile wax into
Part is synthesized by chain saturated fatty acids (VLCFA) in epidermal cell.The formation of VLCFA wax precursors is one complicated
Process, it particularly may be divided into two steps, i.e., by being synthesized in two stages of different sub-cellular locations：C16 and C18 in plastid
The outer C16 or C18 saturated fatty acids extension of the de novo formation and plastid of acyl group chain fatty acid and the synthesis of various derivatives.It is most of
The synthesis of plant wax has two Basic Ways：Decarbonylation approach and acyl group reduction approach.Decarbonylation approach generation aldehyde, alkane, secondary
Alcohol and ketone, and the acyl group primary alcohol of reduction approach generation and wax ester.
Chinese cabbage is according to the presence or absence of epicutile wax of blade and a kind of sedge stem, and being divided into has wax powder and wax powder-free two types,
Generally, there is the vigorous hair a kind of sedge ability of Chinese cabbage growth potential of wax powder type strong, yield height, strong stress resistance, wax powder-free
The Chinese cabbage growth potential of type is weaker, and yield is more relatively low, and its advantage is wax powder-free on tender flower stalk epidermis, light, commodity
It is good.Wide research is had at present for brassica plant wax powder-free Heredity, but it is deposited between different materials
In diversity.Therefore, how effectively to differentiate the Chinese cabbage of wax powder-free character, preferably meet the difference of agricultural production
One of the problem of demand is current urgent need to resolve.And the research in terms of Chinese cabbage wax powder-free trait molecular marker, mesh
It is preceding that there is not been reported.
The content of the invention
In order to solve the Chinese cabbage for how effectively differentiating wax powder-free character, it is used to differentiate the invention provides one kind
The primer of wax powder-free Chinese cabbage blade, the primer includes：
BrCER1-SF：5 '-CTACCATTCCCACCACCAC-3 ', as shown in SEQ ID NO.1；
BrCER1-SR：Shown in 5 '-GAAAGACGCTATGGATGCCG-3 ', SEQ ID NO.2.
The present invention builds F by two Chinese cabbage self-mating systems for having wax powder-free character1And F2Segregating population, in analysis
In the hereditary basis of wax powder character, by the control wax powder-free character gene to having been cloned in Brassica Crops in two parents
Gene order and promoter sequence compare, design gene specific primer is lost according to 39-bp sequences and (codominant marker) and tested
Demonstrate,prove the mark to isolate with wax powder-free character, draw candidate base of the BrCER1 genes for control Chinese cabbage wax powder-free character
Cause.Obtained gene-specific primer (codominant marker) BrCER1-SF and BrCER1-SR can effectively identify wax powder-free
The Chinese cabbage of shape.
Present invention also offers a kind of kit containing above-mentioned primer.
Present invention also offers a kind of identification reagent containing above-mentioned primer.
Further, Tris-HCl, (NH are also included in kit or identification reagent4)2SO4、MgCl2、Triton X-
100th, dNTPs, Taq enzyme and template DNA.
The kit or identification reagent include being used to differentiate the primer of the Chinese cabbage of wax powder-free character, pass through primer knot
Mentioned reagent is closed, the Chinese cabbage of wax powder-free character can be efficiently identified.
In order that differentiating more rapidly and efficiently, 10mmol/L Tris-HCl, 10mmol/L are also included in the kit
(NH4)2SO4、10mmol/L MgCl2, 0.1%Triton X-100,0.2mmol/L dNTPs, 0.5U Taq enzymes and 100ng moulds
Plate DNA.
Present invention also offers a kind of method for differentiating wax powder-free Chinese cabbage, including：
(1) sample DNA is extracted；
(2) DNA using step (1) extraction utilizes Chinese cabbage plant described in primer pair described in claim 1 as template
Sample enters performing PCR amplification；Wherein, the primer includes：
BrCER1-SF：5 '-CTACCATTCCCACCACCAC-3 ', as shown in SEQ ID NO.1；
BrCER1-SR：Shown in 5 '-GAAAGACGCTATGGATGCCG-3 ', SEQ ID NO.2；
(3) PCR primer is analyzed.
Further, differentiate in order to more accurate, step (1) is specially：
Chinese cabbage Plant samples are sowed in hole tray, DNA is extracted using CTAB methods in seedling stage.
CTAB methods in the embodiment of the present invention are CTAB methods commonly used in the art.Wherein, CTAB concentration is preferably
0.8wt%.
In a preferred embodiment of the invention, in order to be able to preferably expand, make result more accurate, in step (2),
PCR amplification system includes：In terms of 10 μ L, 10mmol/L Tris-HCl, 10mmol/L (NH4)2SO4、10mmol/L MgCl2、
0.1%Triton X-100,0.2mmol/L dNTPs, 0.5U Taq enzymes, 100ng template DNAs, 10ng primers BrCER1-SF and
10ng BrCER1-SR。
Wherein, BrCER1-SF and BrCER1-SR nucleotide sequence is respectively such as SEQ ID NO.1 and SEQ ID NO.2 institutes
Show.
Further, in the step (2), PCR reaction conditions are：After 94 DEG C of pre-degeneration 1min, 94 DEG C are reacted 30s, 55
DEG C reaction 30s, 72 DEG C reaction 1min, totally 30 circulation；Last 72 DEG C of extensions 8min.
In a preferred embodiment of the invention, step (3) is specially：By pcr amplification product through Ago-Gel electricity
After swimming, EB dyeing, recorded using gel image analysis system；Banding pattern is counted, aa genotype wax powder-frees character not balling is identified
Chinese cabbage.
Wherein, agarose gel electrophoresis is usually 1.5% agarose gel electrophoresis, and EB dyeing can use this area
In common agarose gel electrophoresis and EB staining procedures.
Wherein, gel image analysis system is GDS-7600 (UVP).
After wax powder-free Chinese cabbage is identified, filtered out, and corresponding plant is transplanted to big field in good time.
The present invention uses Protocols in Molecular Biology, and F is built by two Chinese cabbage self-mating systems for having wax powder-free character1
And F2Segregating population, obtains pair of primers BrCER1-SF and BrCER1-SR, nothing can be effectively identified using above-mentioned primer
The Chinese cabbage of wax powder, the quality trait breeding for distinct Chinese characteristics vegetables-Chinese cabbage provides theoretical foundation and material comes
Source, to realize that quality trait breeding provides theoretical foundation, material source linked marker assisting sifting is favorably improved quality breeding
The efficiency of method, is more conducive to cultivate required kind according to actual conditions.
Brief description of the drawings
Fig. 1 is amplification of the gene specific primer (codominant marker) in F2 individual plants in the embodiment of the present invention 1.
Fig. 2 has the field character table of cured powder self-mating system and wax powder-free self-mating system for Chinese cabbage in the embodiment of the present invention 1
Type figure；
Fig. 3 is the Technology Roadmap in the embodiment of the present invention 1.
Embodiment
With reference to embodiment, the embodiment to the present invention is described in further detail.Following examples are used for
Illustrate the present invention, but be not limited to the scope of the present invention.
Unless otherwise specified, the routine techniques hand that technological means used in embodiment is well known to those skilled in the art
Section.Unless otherwise specified, reagent used in embodiment is commercially available.
Embodiment 1
The Technology Roadmap of the present embodiment is as shown in Figure 3.
The selection of primer
(1) Juvenile stage
Parent material 13S126 (wax powder-free) for green light the not affine system of inbreeding of more generation, in it is ripe, plant height is medium, plant
Development degree is smaller, and leaf is smaller, and petiole is long, yields poorly, quality better；Another self incompatible line parent material 13S106 (has wax
Powder), in it is ripe, plant height is medium, and plant development degree is smaller, and leaf is larger, and petiole is long, and yield is high, inferior quality.
(2) parent and F1、F2The phenotype investigation of offspring field
In Hua Zhong Agriculture University's vegetables improvement multi-center trial field, two parents, F are planted within -2016 years 20141And bagging is certainly
Hand over, individual plant harvest seed broadcasts into cell, maturity period investigation parent and offspring's phenotype.F2Approached for wax powder plant and wax powder-free plant
3:1.Wherein, Fig. 2 is the field trait phenotypes figure that Chinese cabbage has cured powder self-mating system and Chinese cabbage wax powder-free self-mating system.
The genetic development of wax powder-free character see the table below 1 in Chinese cabbage.
The genetic development information of wax powder-free character in the Chinese cabbage of table 1
(3) exploitation and checking of linked marker
According to the control wax powder-free character gene cloned in Brassica Crops, Chinese cabbage wax powder-free character is verified
In whether by same loci control, CGL1 and BrWAX1 are carried out to two Chinese cabbage parents and carry out full-length genes and promoter
Clone, by sequence alignment analysis find wax powder-free parent there occurs 39- in BrCER1 genes (with CGL1 DNA homologs)
Bp loss.1 pair of codominant marker's (gene-specific primer) is devised according to lost regions to screen wax powder-free plant,
It was found that the mark is isolated with wax powder-free character.Codominant marker, also referred to as gene-specific primer, its information see the table below 2.
BrCER1-SF the and BrCER1-SR sequences of table 2
Wax powder-free does not tie the discriminating and seed selection of Chinese cabbage
(4) sowing Chinese cabbage plant is in hole tray, and seedling stage uses CTAB methods (CTAB concentration is 0.8%) to extract DNA.
(5) DNA obtained with step (4), with the codominant marker's (gene specific primer) obtained in step (3) to plant
Enter performing PCR amplification；
Wherein, pcr amplification reaction cumulative volume is 10 μ L, includes 10mmol/L Tris-HCL pH 9.0,10mmol/L
(NH4)2SO4、10mmol/L KCL、2.0mmol/L MgCL2, 0.1%Triton X-100,0.2mmol/L dNTPs, gene
Each 10ng of special primer BrCER1-SF and BrCER1-SR, Taq enzyme 0.5U, template DNA 100ng, the amplification instrument used is
EASTWIN(ETC811)。
Wherein, after PCR amplification programs are 94 DEG C of pre-degeneration 1min, 94 DEG C are reacted 30s, 55 DEG C of reaction 30s, 72 DEG C of reactions
1min reacts, altogether 30 circulations, finally extends 8min at 72 DEG C.
(6) amplified production is after 1.5% agarose gel electrophoresis, EB dyeing, using gel image analysis system GDS-
7600 (UVP) are recorded.
(7) banding pattern is counted, corresponding plant is transplanted to big field by screening aa genotype (wax powder-free) in good time.Obtain
Wax powder-free on the Chinese cabbage tender flower stalk epidermis of wax powder-free, light, commodity is good.
Wherein, Fig. 1 is the PCR amplifications of obtained codominant marker's (gene specific primer) in F2 individual plants in step (3)
As a result.It can be seen that using codominant marker's (gene specific primer) of the present invention respectively in two parent 13S106
With pcr amplification product difference in size is shown in 13S126 and F2 segregating populations, the result and the conclusion drawn in theory
That is product is small consistent after 13S126 wax powder-frees parent loss 39bp.Meanwhile, obtained PCR stable amplification results.
Finally, method of the invention is only preferably embodiment, is not intended to limit the scope of the present invention.It is all
Within the spirit and principles in the present invention, any modification, equivalent substitution and improvements made etc. should be included in the protection of the present invention
Within the scope of.
Sequence table
<110>Hua Zhong Agriculture University
<120>Differentiate primer, kit and the discrimination method of wax powder-free Chinese cabbage
<130> KHP171113871.0
<160> 2
<170> PatentIn version 3.5
<210> 1
<211> 19
<212> DNA
<213>Artificial sequence
<400> 1
ctaccattcc caccaccac 19
<210> 2
<211> 20
<212> DNA
<213>Artificial sequence
<400> 2
gaaagacgct atggatgccg 20
Claims (10)
1. a kind of primer for being used to differentiate wax powder-free Chinese cabbage, it is characterised in that including：
BrCER1-SF：5’-CTACCATTCCCACCACCAC-3’；
BrCER1-SR：5’-GAAAGACGCTATGGATGCCG-3’.
2. the kit containing primer described in claim 1.
3. kit according to claim 2, it is characterised in that also include Tris-HCl, (NH in the kit4)2SO4、MgCl2, Triton X-100, dNTPs, Taq enzyme and template DNA.
4. kit according to claim 2, it is characterised in that also include 10mmol/L Tris- in the kit
HCl、10mmol/L(NH4)2SO4、10mmol/L MgCl2, 0.1%Triton X-100,0.2mmol/L dNTPs, 0.5U
Taq enzyme and 100ng template DNAs.
5. a kind of method for differentiating wax powder-free Chinese cabbage, it is characterised in that including：
(1) DNA of Chinese cabbage Plant samples is extracted；
(2) DNA using step (1) extraction utilizes Chinese cabbage Plant samples described in primer pair described in claim 1 as template
Enter performing PCR amplification；
(3) PCR primer is analyzed.
6. method according to claim 5, it is characterised in that the step (1) is specially：
Chinese cabbage Plant samples are sowed in hole tray, DNA is extracted using CTAB methods in seedling stage.
7. method according to claim 6, it is characterised in that CTAB concentration is 0.8% in the CTAB methods.
8. the method according to claim 5 or 6, it is characterised in that in the step (2), PCR amplification system includes：With
10 μ L are counted, 10mmol/L Tris-HCl, 10mmol/L (NH4)2SO4、10mmol/L MgCl2, 0.1%Triton X-100,
0.2mmol/L dNTPs, 0.5U Taq enzyme, 100ng template DNAs, 10ng primer BrCER1-SF and 10ng BrCER1-SR.
9. the method according to any one of claim 5-8, it is characterised in that PCR reaction conditions in the step (2)
For：After 94 DEG C of pre-degeneration 1min, 94 DEG C of reactions 30s, 55 DEG C of reactions 30s, 72 DEG C of reaction 1min, totally 30 circulations；Finally 72
DEG C extension 8min.
10. the method according to any one of claim 5-9, it is characterised in that the step (3) is specially：
By pcr amplification product after agarose gel electrophoresis, EB dyeing, recorded using gel image analysis system；Count banding pattern,
Identify aa genotype wax powder-free Chinese cabbages.
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| CN201710549794.0A CN107312845B (en) | 2017-07-07 | 2017-07-07 | Primer, kit and method for identifying wax powder-free non-heading Chinese cabbage |
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110724756A (en) * | 2019-11-22 | 2020-01-24 | 荆州农业科学院 | Molecular marker closely linked with non-nodulation cabbage stalk brilliant green character and application thereof |
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| WO2006111512A1 (en) * | 2005-04-19 | 2006-10-26 | Basf Plant Science Gmbh | Improved methods controlling gene expression |
| CN1936021A (en) * | 2006-09-06 | 2007-03-28 | 南京农业大学 | Brassica campestris ssp. Chinensis male sterile molecular marker-assisted selection method |
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110724756A (en) * | 2019-11-22 | 2020-01-24 | 荆州农业科学院 | Molecular marker closely linked with non-nodulation cabbage stalk brilliant green character and application thereof |
| CN110724756B (en) * | 2019-11-22 | 2023-08-04 | 荆州农业科学院 | Molecular marker closely linked with bright green character of non-heading Chinese cabbage stalk and application thereof |
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| CN107312845B (en) | 2020-11-03 |
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