CN1624153A - Laver DNA fingerprint graphic and its forming process and application - Google Patents
Laver DNA fingerprint graphic and its forming process and application Download PDFInfo
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- CN1624153A CN1624153A CN 200310105268 CN200310105268A CN1624153A CN 1624153 A CN1624153 A CN 1624153A CN 200310105268 CN200310105268 CN 200310105268 CN 200310105268 A CN200310105268 A CN 200310105268A CN 1624153 A CN1624153 A CN 1624153A
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Abstract
A DNA fingerprint map of laver is created through extracting and purifying the DNA from the laver to be tested, using it as template, RAPD analyzing, choosing the representative amplified polymorphic RAPD bands, and configuring the DNA fingerprint map of the laver to be tested. Said DNA fingerprint map can be used to discriminate the quality of laver variety.
Description
Technical field
The present invention relates to the seed authenticate technology, specifically a main laver dna fingerprinting and establishment method and application.
Background technology
Laver (Porphyra) is one of most important marine algae resource of China, and annual value of production occupies first of the various economical algas, and China's laver ultimate production occupies first place in the world.But the laver profile characterizes simple, and the feature that can be used for Idioplasm identification is limited, and therefore, only according to morphological specificity thallus of porphyra being carried out Idioplasm identification has certain difficulty.The free conchocelis of laver is the diplophase, be to carry out the heredity and the main raw of breeding research, but free conchocelis can't identify from outward appearance, form, with the free conchocelis be specially investigation of materials laver Idioplasm identification article also seldom.Fast development along with the molecular biology research level, molecular marking technique has been widely used in the genetic research of plant, molecular mark is applied in the research of important cash crop such as paddy rice, wheat and cotton with in producing, and obtains tangible economic benefit.Wherein: the RAPD technological operation is convenient, it is a kind of practical and effective molecular marking technique, the analysis of genetic diversity that has been used for algae, existing report in researchs such as sea-tangle, laver, wakame and fragrant plant mentioned in ancient texts has had not yet to see the research report but build dna fingerprinting and utilize the dna fingerprinting technology to carry out Idioplasm identification.In addition, the dna fingerprint analytical technology has been considered to the strongest method in legal medical expert's evaluation, also widespread use aspect the evaluation of food crop seed, but be not employed as yet aspect the marine alga Idioplasm identification.
Summary of the invention
The purpose of this invention is to provide a kind of laver dna fingerprinting and the establishment method and application that can be used for Idioplasm identification.
To achieve these goals, technical solution of the present invention is as follows:
The laver dna fingerprinting, the sentence that serve as reasons " 1 " and " 0 " is formed is by digital 10011000001,10010101001,10111101000,10111001101,10011000101,10111101100,10110010100,10010000000,10010100100,10110000000,10010101010,11011000001,01001001001,10010011010,10011001011,10000100000,10010100000,10010001000,10010110000,10010111000,10110100010,00100000000,10111100000,00010100000,10000101100,10011001001 and 000110001011 show with array format;
Wherein: have amplified band to exist on certain position in described numeral " 1 " the expression collection of illustrative plates, digital " 0 " is illustrated in does not have amplified band to exist on certain position.
The establishment method of its laver dna fingerprinting, operation as follows: 1) extraction and the purifying of the DNA of laver cell system; 2) be that template is carried out the RAPD analysis with obtaining high-purity laver DNA; 3) select 11 bands of representational polymorphism RAPD amplification, having, not having and make up according to these 11 bands for trying the material dna fingerprinting;
Wherein: each clone of described laver is got its free conchocelis stage; Each laver material all has its special dna fingerprint in the dna fingerprinting, can make a distinction one by one for the examination material with other; The primer that adopts in the described RAPD amplification is OPJ-18, OPC-04 and OPX-06;
Described laver cell is to be preferably yezoensis laver (P.yezoensis), porphyra haitanensis (P.haitanensis), few smart laver (P.oligospermatangia), laver (P.tenera), half leaf laver (P.katadai.var.hemiphylla), the shell laver (P.tenuipedalis) that the row laver (P.seriata) or the life history are special; Described laver free conchocelis can be yezoensis laver (P.yezoensis), porphyra haitanensis (P.haitanensis), few smart laver (P.oligospermatangia), laver (P.tenera), half leaf laver (P.katadai.var.hemiphylla), row laver (P.seriata), shell laver (P.tenuipedalis) that the life history is special or wild laver (P.oligospermatangia and P.katadai var.hemiphylla).
The laver dna fingerprinting is used for the Idioplasm identification of laver.
The present invention has following advantage:
1. the present invention has created the laver dna fingerprinting, discloses with the dna fingerprint analytical technology laver is carried out the result of study of Idioplasm identification, and its establishment method can carry out Idioplasm identification to laver from inheritance, therefore accurately, reliable, has legal effect.
2. employing the present invention, after in a single day dna fingerprinting is set up, when certain sample is truly detected, extract the DNA of testing sample with quick, the minim DNA extraction method of present widespread use,, carry out RAPD and analyze as template with the DNA that extracts, in several hrs, just can know this dna fingerprint, therefore, just can judge its verity rapidly.
3. adopting the present invention to make up the used sample of this dna fingerprinting can constantly increase as required, and fingerprint is upgraded, to satisfy the needs of development.
4. the present invention has built the dna fingerprinting of laver, and utilizes this dna fingerprinting technology to carry out Idioplasm identification to laver, and this application facet in the marine alga Idioplasm identification has been opened a precedent.
5. because dna fingerprinting of the present invention is the form tabular form with figure, seem more directly perceived, understandable; And, be convenient to computer recognition and analysis owing to converted digital form to.
Description of drawings
The dna fingerprinting that Fig. 1 for laver cell of the present invention is; Wherein: its X-coordinate is the numbering (seeing table 1 for details) that is respectively 27 laver cell systems that adopt in the one embodiment of the invention; Ordinate zou is for making up 11 used band numberings of this dna fingerprint figure.
Embodiment
Below in conjunction with drawings and Examples the present invention is described in further detail.
It is for the examination material that present embodiment adopts 27 of collecting to have extensive representational laver cell, free conchocelis with each clone extracts DNA, carrying out RAPD analyzes, set up their dna fingerprinting, be used further to these 27 materials are carried out Idioplasm identification, and for the Idioplasm identification of other marine alga provide one can be for effective new way of using for reference.The concrete operations of setting up the laver dna fingerprinting are as follows:
1, the experiment material of selecting for use:
Present embodiment adopts 27 laver materials as shown in table 1, wherein yezoensis laver (P.yezoensis) is 15,6 of porphyra haitanensis (P.haitanensis), 2 of few smart lavers (P.oligospermatangia), 1 of laver (P.tenera), 1 of half leaf laver (P.katadai.var.hemiphylla), 1 of the row laver (P.seriata) and the life history 1 of special shell laver (P.tenuipedalis), selection is based on the high-quality laver of widespread use in producing, also comprise some wild lavers (P.oligospermatangia and P.katadai var.hemiphylla) and some laver materials, have representative preferably from external introduction.Present embodiment is material extraction DNA with the free conchocelis of 27 laver cell systems, has set up the dna fingerprinting that carries out Idioplasm identification for examination laver cell system to these 27.
The source and the characteristic of table 1 laver cell system
Kind numbering characteristic source
The Y9101 high yield, high temperature resistant, sea area, Lianyun Harbour, the strong Jiangsu of disease resistance
The green mutant Jiangsu marine fishery institute of Ymg9602 Y9602
Y9430 is of fine quality, and the filament maturation is difficult, the few sea area, Nantong of monospore
Y9502 is high temperature resistant, strong stress resistance, the few sea area, Fujian of monospore
Sea area, Y9830 maintenance line Nantong
The Y92-1 research material is abroad introduced
The Y94-1 research material is abroad introduced
Yezoensis laver
The Y95-1 research material is abroad introduced
P.yezoensis
Sea area, Yqd-2 research material Jiangsu Rudong
The Ynt-6 high yield, thermophilic is wide, sea area, Lianyun Harbour, strong stress resistance Jiangsu
Ynt-8 is of fine quality, and thermophilic is wide, strong stress resistance, and frond approaches the sea area, Jiangsu
Sea area, Yqd-6 research material Jiangsu Rudong
Yqd-8 research material Qingdao sea area
Seawater is big on the green mutant of Ysh-6 Y92-1
The Y92-3 research material is abroad introduced
The H9208 high-quality, Nan Ridao sea area, the ripe Fujian of filament
The sea area, cultivation germplasm Fujian of H9161 wild species
Sea area, porphyra haitanensis H182 hybrid Fujian
Sea area, Zhoushan, P.haitanensis Hqd-1 research material Zhejiang
Sea area, Liaanjiang county, Hqd-5 research material Fujian
H02-1 research material Zhejiang sea
Few sea area, Liaanjiang county, smart laver O9406 research material Fujian
Sea area, Liaanjiang county, P.oligospermatangia O9301 research material Fujian
Laver
The T94-2 research material is abroad introduced
P.tenera
The row laver
The S02-1 research material is abroad introduced
P.seriata
Half leaf laver
Sea area, K9401 research material Qingdao
P.katadai
The shell laver
The Tsh-8 research material is abroad introduced
P.tenuipedalis
2, laver cell is extraction and the purifying of DNA:
By following program (referring to Wang Bin etc., the acquisition of corn inbred line 8112 special SCAR marks, hi-tech communication, 1999,9 (3): 45-47.) extract the genomic dna of each laver free conchocelis material:
Get flesh tissue 2g, in liquid nitrogen, pulverize, place the 50ml centrifuge tube, add 8ml extracting solution (100mM Tris pH8.0,50mM EDTA, 500mM NaCl, 1.5%SDS), following 30 minutes of 50 ℃ of situations are put upside down mixing frequently, add 2.5ml 5M potassium acetate (KAC), ice bath 10 minutes, add the 2.5ml chloroform, put upside down mixing, centrifugal 8 minutes of 10000rmp, get supernatant and move into new pipe, add the pre-cold isopropanol of 2/3 volume, put upside down mixing, put 1-2 hour for-20 ℃, go out cotton-shaped white DNA, 70% ethanol is washed 1-2 time, dries up, and is dissolved among 100 μ l (or an amount of) TE, and on 1% sepharose electrophoresis, compare with standard λ DNA, the concentration of estimation laver DNA sample obtains high-purity laver DNA.
3, be that template is carried out the RAPD analysis with obtaining high-purity laver DNA:
RAPD is reflected on the MJ Research PTC-100 type PCR instrument and finishes, and primer is respectively OPJ-18, OPC-04 and OPX-06 (product of U.S. Operon company).The RAPD-PCR reaction system is 25 μ l, wherein contains 10mM Tris-HCl (pH9.0), 50mM KCl, 2.0mM Mg
2+, 0.15mM dNTP, 15ng primer, 20ng template DNA, 1 Taq of unit archaeal dna polymerase; Response procedures is 94 ℃ of pre-sex change 4 minutes, then by 94 ℃ 45 seconds, 37 ℃ 1 minute, 72 ℃ 1.5 minutes, 42 circulations of increase, last 72 ℃ extensions 5 minutes; Amplified production is electrophoresis on 1.0% sepharose, observes in Bio-Rad DOC-1000 type gel imaging system after EB dyeing and the record result.
4, select representational polymorphism RAPD amplified band to make up for examination material dna fingerprinting:
When making up collection of illustrative plates in line with minimum primer, minimum tape, the principle that again all confession test agents made a distinction one by one, present embodiment is to have chosen 11 stability wherein and repeatability is good, polymorphism is high again band in 254 bands being amplified of 3 primers (OPJ-18, OPC-04 and OPX-06) of filtering out from the primer of 420 Operon companies, is used to make up the dna fingerprinting of these 27 laver materials.According to the having, do not have of this 11 bands, draw out 27 dna fingerprint mode chart (see figure 1)s for the examination materials, in this dna fingerprinting, each material all has its special dna fingerprint, can distinguish one by one with other material.Because this dna fingerprinting is the form tabular form with figure, seems very directly perceived.But it is not too convenient to narrate, express, exchange, therefore, the present invention is ad hoc to be decided with having amplified band to exist on certain position in " 1 " expression collection of illustrative plates, and being illustrated in " 0 " does not have amplified band to exist on certain position, and dna fingerprint has just become the sentence (referring to table 2) that " 1 " and " 0 " is formed like this.
As shown in Figure 1, in the dna fingerprinting of 27 laver cell systems of present embodiment, the numbering 1-27 of figure top is respectively 27 the laver cell systems (seeing table 1 for details) for examination, and the numeral on a figure left side is for making up the used 11 bands numbering of this dna fingerprint figure.
The digital dna fingerprint of 27 laver materials of table 2
| The laver material | The dna fingerprint of numberization |
| ????Y9101 | ????10011000001 |
| ????Ymg9602 | ????10010101001 |
| ????Y9430 | ????10111101000 |
| ????Y9502 | ????10111001101 |
| ????Y9830 | ????10011000101 |
| ????H9208 | ????10111101100 |
| ????H9161 | ????10110010100 |
| ????Y92-1 | ????10010000000 |
| ????Y94-1 | ????10010100100 |
| ????Y95-1 | ????10110000000 |
| ????Yqd-2 | ????10010101010 |
| ????Ynt-6 | ????11011000001 |
| ????Ynt-8 | ????01001001001 |
| ????Yqd-6 | ????10010011010 |
| ????Yqd-8 | ????10011001011 |
| ????Ysh-6 | ????10000100000 |
| ????Y92-3 | ????10010100000 |
| ????H182 | ????10010001000 |
| ????Hqd-1 | ????10010110000 |
| ????Hqd-5 | ????10010111000 |
| ????H02-1 | ????10110100010 |
| ????T94-2 | ????00100000000 |
| ????S02-1 | ????10111100000 |
| ????P.tenuipedalis?sh-8 | ????00010100000 |
| ????O9406 | ????10000101100 |
| ????O9301 | ????10011001001 |
| ????K9401 | ????00011000101 |
Laver dna fingerprinting of the present invention is used for the Idioplasm identification of laver:
Suppose for the yezoensis laver Y9101 in the examination material it is to produce to go up the improved seeds of using, an existing batch of material can not determine whether be real yezoensis laver Y9101, therefore, as follows its dna fingerprint is identified.
The use of laver dna fingerprinting: after the standard DNA finger printing is set up, in the time of will carrying out the verity detection to certain sample, with present widespread use fast, the minim DNA extraction method is extracted the DNA of testing sample, with the DNA that extracts as template, use OPJ-18, three primers of OPC-04 and OPX-06 carry out the RAPD amplification to it, amplified production carries out electrophoretic analysis on 1% agarose, according to having of 11 selected amplified bands, do not have, draw out the dna fingerprint of this testing sample, compare with the standard DNA finger printing, just can know that it is to be used for any in 27 main lavers of constructed dna finger printing.
The result shows: if its dna fingerprint is " 10011000001 ", this with the standard DNA finger printing in the fingerprint of yezoensis laver Y9101 be consistent, therefore, this batch of material is exactly real yezoensis laver Y9101.If the dna fingerprint of testing sample is not " 10011000001 ", this batch of material is not real yezoensis laver Y9101 just so.
Claims (6)
1. a main laver dna fingerprinting is characterized in that: the sentence that serve as reasons " 1 " and " 0 " is formed, by digital 10011000001,10010101001,10111101000,10111001101,10011000101,10111101100,10110010100,10010000000,10010100100,10110000000,10010101010,11011000001,01001001001,10010011010,10011001011,10000100000,10010100000,10010001000,10010110000,10010111000,10110100010,00100000000,10111100000,00010100000,10000101100,10011001001 and 00011000101 show with array format.
2. by the described laver dna fingerprinting of claim 1, it is characterized in that: have amplified band to exist on certain position in described numeral " 1 " the expression collection of illustrative plates, digital " 0 " is illustrated in does not have amplified band to exist on certain position.
3. the establishment method of a main laver dna fingerprinting is characterized in that operating as follows: 1) extraction and the purifying of the DNA of extraction and purifying laver cell system; 2) be that template is carried out the RAPD analysis with obtaining high-purity laver DNA; 3) select representational polymorphism RAPD amplified band, having, not having and make up according to selected band for trying the material dna fingerprinting.
4. by the establishment method of the described laver dna fingerprinting of claim 3, it is characterized in that: each clone of described laver is got its free conchocelis stage; Each laver material all has its special dna fingerprint in the dna fingerprinting, can make a distinction one by one for the examination material with other.
5. by the establishment method of the described laver dna fingerprinting of claim 3, it is characterized in that: the primer that adopts in the described RAPD amplification is OPJ-18, OPC-04 and OPX-06.
6. the application of a main laver dna fingerprinting is characterized in that: the Idioplasm identification that is used for laver.
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| Application Number | Priority Date | Filing Date | Title |
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| CN 200310105268 CN1624153A (en) | 2003-12-05 | 2003-12-05 | Laver DNA fingerprint graphic and its forming process and application |
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN 200310105268 CN1624153A (en) | 2003-12-05 | 2003-12-05 | Laver DNA fingerprint graphic and its forming process and application |
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| CN1624153A true CN1624153A (en) | 2005-06-08 |
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106701907A (en) * | 2015-11-18 | 2017-05-24 | 中国检验检疫科学研究院 | Primer, probe, method and kit for detecting cassava-derived ingredients |
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2003
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106701907A (en) * | 2015-11-18 | 2017-05-24 | 中国检验检疫科学研究院 | Primer, probe, method and kit for detecting cassava-derived ingredients |
| CN106701907B (en) * | 2015-11-18 | 2022-08-09 | 中国检验检疫科学研究院 | Primer probe, method and kit for cassava-derived component detection |
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