CN102134587B - Kit and method for identifying seed coat color of rape seed in very early development stage - Google Patents
Kit and method for identifying seed coat color of rape seed in very early development stage Download PDFInfo
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- CN102134587B CN102134587B CN2010105262229A CN201010526222A CN102134587B CN 102134587 B CN102134587 B CN 102134587B CN 2010105262229 A CN2010105262229 A CN 2010105262229A CN 201010526222 A CN201010526222 A CN 201010526222A CN 102134587 B CN102134587 B CN 102134587B
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Abstract
The invention relates to an identifying kit, and particularly discloses a method for quickly identifying the seed coat color of a rape seed in the very early development stage. The kit and the identifying method disclosed by the invention are created on the basis that a primer pair designed according to the sequence of differential expression genes in the very early development stage of a yellow/black rape seed is used as the main body. By using the reverse transcription-polymerase chain reaction (RT-PCR) technology to carry out qualitative detection on specific RNA nucleic acid segments of the seed coat (with the hilum removed) of the rape in the early development stage (4-5 days after blooming), the invention has the advantages of convenient and fast process, good specificity and high sensitivity, and can be widely used for breeding selection based on the seed coat color in the very early stage, thereby improving the breeding efficiency, accelerating the breeding process, saving the breeding cost, improving the scientific management efficiency and having high use value.
Description
Technical field
The present invention relates to biological field, specifically relate to a kind of Semen Brassicae campestris polus animalium early stage kernel seed coat colour Rapid identification test kit and authentication method thereof.
Background technology
Big both at home and abroad quantity research shows, under identical genetic background, yellow seed coat rape (abbreviation yellow seed rape) compare with black kind skin rape (being called for short black seed rape) have that kind of skin approaches, oleaginousness is high, pigment and advantage such as impurity is few, content of lignin is low in the oil.Rape yellow seed is the problem that the rapeseed breeding men are concerned about most always, to the selection (blooming back 10 days in) extremely in early days of the yellow seed proterties of seed, is just can display but the Semen Brassicae campestris color will arrive maturation especially, and previous method is difficult to accomplish this point.What no matter be yellow seed or black seed in seed development is extremely early stage, and it all is green planting skin, and the breeder can not select kernel seed coat colour seed development extremely in early days, need behind seed maturity, could select the kernel seed coat colour proterties.Simultaneously, in rape heterosis utilizes,, extremely in early days parent of cross-fertilize seed and the purity of cross-fertilize seed are identified in seed development according to the apparent recessive relation of the kernel seed coat colour between the hybrid strain.Usually to identify rape seed skin color; Could judge after all will waiting until seed maturity; Though we once used chemical identification method (Vanillin staining) can identify the color of rape kind skin, to could judge after 10 days in pollination at least, if can just can identify in back 10 days blooming to kernel seed coat colour; That can significantly reduce the workload of rape yellow seed breeding; Can accelerate the process of breeding like this, reduce the breeding cost, so early stage Rapid identification test kit and the authentication method of Semen Brassicae campestris kernel seed coat colour polus animalium there is extremely important meaning for rapeseed breeding.
Polymerase chain reaction (polymerase chain reaction; Be PCR); It is the technology of present most popular amplification in vitro dna fragmentation; Have fast, characteristics such as sensitivity and high specificity, if can this technology be used for identifying extremely in early days of rape seed skin color, with having great importance and wide application prospect.
Summary of the invention
The objective of the invention is to overcome existing kernel seed coat colour and identify the deficiency that exists extremely in early days; The test kit of a kind of Semen Brassicae campestris polus animalium quick, that specificity is good, highly sensitive early stage (blooming back 4-5 days) Rapid identification kernel seed coat colour is provided, so that be applied to the breeding of rape; Another object of the present invention provides the method for utilizing the mentioned reagent box to identify the kernel seed coat colour that the rape polus animalium is early stage.
The early stage kernel seed coat colour identification kit of a kind of Semen Brassicae campestris polus animalium is characterized in that said test kit is made up of following parts:
(1) RNA extracts A liquid, interior dress Trizol liquid;
(2) RNA extracts B liquid, interior dress phenol-chloroform, and the volume ratio of phenol, chloroform is 25:24;
(3) RNA extracts C liquid, interior dress Virahol;
(4) RNA extracts D liquid, in adorn 75% ethanol;
(5) RNA extracts E liquid, and interior dress DEPC handles the ultrapure water of back high-temperature sterilization;
(6) RT reaction F liquid, interior reaction cartridge buffer B uffer, dNTP, oligo dT primer, RNA enzyme suppress and ThermoScript II M-MLV, and the volume proportion of each component is:
(5 * Bbuffer) 2 parts of reaction buffers
1 part of dNTP
1 part of oligo dT primer
The RNA enzyme suppresses 0.3 part
0.5 part of ThermoScript II M-MLV
4.2 parts of the ultrapure waters that DEPC handles;
(7) RT-PCR reaction G1 liquid, interior dress contains Mg
2+Reaction buffer Buffer, dNTP, Taq enzyme, forward primer F and reverse primer R, the volume proportion of each component is following:
Contain mg
2+(10 * Bbuffer) 2 parts of reaction buffers
0.3 part of dNTP
1 part of forward primer F
1 part of reverse primer R
13.3 parts of ultrapure waters
0.4 part of Taq enzyme;
(8) RT-PCR reaction G2 liquid, interior dress contains Mg
2+Reaction buffer, the forward primer ACTF of dNTP, Taq enzyme, internal control gene Actin and the reverse primer ACTR of internal control gene Actin, the volume proportion of each component is following:
Contain mg
2+(10 * Bbuffer) 2 parts of reaction buffers
0.3 part of dNTP
1 part of forward primer ACTF
1 part of reverse primer ACTR
13.3 parts of ultrapure waters
0.4 part of Taq enzyme;
(9) positive control H1 liquid, interior dress is deceived the seed rape positive template;
(10) negative control H2 liquid, the negative template of interior dress yellow seed rape.
Said forward primer F sequence is: 5'-TCAGCTGCTGCGGTTTCTATC-3', and said reverse primer R sequence is: 5'-CAGATAGCTTCTGCATTTCCTTG-3'; Said forward primer ACTF sequence is: 5'-GACATTCAACCTCTTGTTTGC-3', said reverse primer ACTR sequence is: 5'-CTGCTCGTAGTCAAGAGCAATG-3'.
A kind of method of utilizing said test kit to identify the early stage kernel seed coat colour of rape polus animalium, carry out according to the following steps:
(1) get the kind skin sample that removes hilum after to be measured the blooming 4-5 days, the RNA that adds 5 times of volumes extracts A liquid, ice bath homogenate, the static 2-5min of room temperature, homogenate;
(2) RNA that in above-mentioned homogenate, adds 0.5 times of volume extracts B liquid, mixing, static 2-5min;
(3) 4-10 ℃, the centrifugal 5min of 12000g;
(4) get supernatant, the RNA that adds 1 times of volume extracts C liquid, and mixing leaves standstill 5-10 min;
(5) 4-10 ℃, the centrifugal 5min of 12000g;
(6) abandon supernatant, extract the washing of D liquid with RNA after, drying at room temperature or in super clean bench, dry up adds RNA and extracts the dissolving of E liquid, RNA solution;
(7) get gained RNA solution, add the RT reaction F liquid of 4 times of volumes, 42 ℃ of insulation 30 min handle 5 min for 99 ℃ then, get the template of RT-PCR;
(8) template, positive control H1 liquid, the negative control H2 liquid of RT-PCR is added to respectively in RT-PCR reaction G1 liquid and the RT-PCR reaction G2 liquid, behind the mixing as on the PCR appearance;
(9) carry out pcr amplification: 94 ℃ of 3min of elder generation, 94 ℃ again, 30s; 58 ℃, 30s; 72 ℃, 30 min, after 30 circulations, 72 ℃ prolong 6 min, after reaction finishes, get amplified production.
(10) get amplified production, add the sample-loading buffer (containing tetrabromophenol sulfonphthalein) of 1/5 times of volume, behind 1% agarose gel electrophoresis 15-30min, on ultraviolet device, observe; If bright band occurs with 319 bp places of positive control same position; Then positive for detecting, explain that planting skin behind this testing sample seed maturity is black, otherwise negative; Explain that it is transparent planting skin behind this testing sample seed maturity, seed shows the yellow of embryo.
The present invention has following beneficial effect: the present invention is the basis according to the gene order with differential expression in the yellow black seed kind skin of rape, designs a pair of primer, has set up reverse transcription---gather PCR (RT-PCR system); Be optimized on this basis; Set up the early stage qualitative authentication method of rape seed skin color polus animalium, this method fast, sensitivity and high specificity, especially can identify the extremely kind skin of early stage (blooming 4-5 days) that Semen Brassicae campestris is grown; Shifted to an earlier date the time that rape seed skin color is identified greatly; Practice thrift the time for the selection of rape seed skin color breeding and yellow seed breeding, improved the efficient that breeding is selected, had very high practical value.
Description of drawings
Accompanying drawing is the bloom RT-PCR qualification result figure of 4-5 days kind skin samples of different kernel seed coat colour rape materials.
Among the figure: ACT: β-Actin gene is confidential reference items; B: black seed kind skin positive control; Y: yellow seed kind skin negative control; Lane 1-8: the swede type rape kind skin sample of evaluation (1,2,4,6 is yellow seed behind the seed maturity, and 3,5,7,8 are black seed); Lane 9-16: the mustard type rape kind skin sample of evaluation (14 is yellow seed behind the seed maturity, and 9,10,11,12,13,15,16 are black seed); Lane 17-18: the turnip type rape kind skin sample of evaluation (17 is yellow seed behind the seed maturity, and 18 are black seed).
Embodiment
Semen Brassicae campestris is grown early stage kernel seed coat colour identification kit and is made up of following each several part:
(1) RNA extracts A liquid, interior dress Trizol liquid, 2 pipes, 5ml/ pipe.
(2) RNA extracts B liquid, interior dress phenol-chloroform, and the volume ratio of phenol, chloroform is 25:24,1 pipe, 5ml/ pipe.
(3) RNA extracts C liquid, interior dress Virahol, 2 pipes, 5ml/ pipe.
(4) RNA extracts D liquid, in adorn 75% ethanol, 2 pipes, 5ml/ pipe.
(5) RNA extracts E liquid, and interior dress DEPC handles the ultrapure water of back high-temperature sterilization, 1 pipe, 1ml/ pipe.
(6) RT reaction F liquid, interior reaction cartridge damping fluid (5 * Buffer), dNTP, oligo dT primer, RNA enzyme suppress, ThermoScript II M-MLV and DEPC handle ultrapure water, 1 pipe, 0.2ml/ pipe
(7) RT-PCR reaction G1 liquid, interior dress contains Mg
2+Reaction buffer (10 * Buffer), dNTP, Taq enzyme, forward primer F, reverse primer R and ultrapure water, 1 pipe, 0.4ml/ pipe; Said forward primer F sequence is: 5'-TCAGCTGCTGCGGTTTCTATC-3', and said reverse primer R sequence is: 5'-CAGATAGCTTCTGCATTTCCTTG-3';
(8) RT-PCR reaction G2 liquid, interior dress contains Mg
2+Reaction buffer (10 * Buffer), the forward primer ACTF of dNTP, Taq enzyme, internal control gene Actin, reverse primer ACTR and the ultrapure water of internal control gene Actin, 1 pipe, 0.4ml/ pipe; Said forward primer ACTF sequence is: 5'-GACATTCAACCTCTTGTTTGC-3', and reverse primer ACTR sequence is: 5'-CTGCTCGTAGTCAAGAGCAATG-3';
(9) positive control H1 liquid, interior dress is deceived the seed rape positive template, 1 pipe, 0.02ml/ pipe.
(10) negative control H2 liquid, the negative template of interior dress yellow seed rape, 1 pipe, 0.02ml/ pipe.
(11) rectangle box, size are 8 ㎝ * 6 ㎝ * 5cm; Including a piece size is 8 ㎝ * 6 cm fiber boards, on the aperture is arranged is 8 in the hole of 1.5cm, on the aperture is arranged is 6 in the hole of 0.75cm, above-mentioned each tubule is positioned over respectively in the corresponding hole.
The component of the RT reaction F liquid of 10 μ L systems and the volume of each component are following:
Reaction buffer (5 * Bbuffer) 2 μ L
dNTP 1μL
The RNA enzyme suppresses 0.3 μ L
ThermoScript II M-MLV 0.5 μ L
The ultrapure water 4.2 μ L that DEPC handles
The component of the RT-PCR reaction G1 liquid of 20 μ L systems and the volume of each component are following:
Contain mg
2+Reaction buffer (10 * Bbuffer) 2 μ L
dNTP 0.3μL
Ultrapure water 13.3 μ L
Taq enzyme 0.4 μ L
The component of the RT-PCR reaction G2 liquid of 20 μ L systems and the volume of each component are following:
Contain mg
2+Reaction buffer (10 * Bbuffer) 2 μ L
dNTP 0.3μL
Ultrapure water 13.3 μ L
Taq enzyme 0.4 μ L
Semen Brassicae campestris is grown the authentication method of early stage kernel seed coat colour, uses the test kit among the embodiment 1, follows these steps to carry out:
(1) remove fresh kind of skin 0.05 ~ 0.1g after the hilum part, the RNA that adds 500 μ L extracts A liquid, ice bath homogenate, the static 2 ~ 5min of room temperature.
(2) RNA that in above-mentioned homogenate, adds 250 μ L extracts B liquid, mixing, static 2-5min.
(3) 4-10 ℃, the centrifugal 5min of 12000g.
(4) get supernatant 500 μ L, the RNA that adds 500 μ L extracts C liquid, and mixing leaves standstill 5-10 min.
(5) 4-10 ℃, the centrifugal 5min of 12000g.Remove supernatant, extract D liquid washing 2 times with the RNA of 250 μ L after, drying at room temperature or in super clean bench, dry up, the RNA that adds 15 ~ 30 μ L extracts the dissolving of E liquid, RNA solution.
(6) get the above-mentioned RNA solution of 1 μ L, add the RT reaction F liquid of 9 μ L, 42 ℃ of insulation 30 min handle 5 min for 99 ℃ then, the template of RT-PCR.
(7) respectively template, positive control H1 liquid and the negative control H2 liquid of the RT-PCR of 2 μ L is added in the RT-PCR reaction G1 liquid and RT-PCR reaction G2 liquid of 18 μ L, mixing is placed on the PCR appearance.
(8) carry out pcr amplification: 94 ℃ of 3min of elder generation, 94 ℃ again, 30s; 58 ℃, 30s; 72 ℃, 30 min, after totally 30 circulations, 72 ℃ prolong 6 min, after reaction finishes, amplified production.
(9) get 8-10 μ L amplified production, add 2 μ L sample-loading buffers (containing tetrabromophenol sulfonphthalein), mixing; Behind 1% agarose gel electrophoresis 15-30min, on ultraviolet device, observe; If bright band (with the positive control same position) to occur then positive for detecting with 319 bp places, explain that the kind skin is a black behind this testing sample seed maturity, otherwise negative; Explain that it is transparent planting skin behind this testing sample seed maturity, seed shows yellow (see figure 1).
Claims (2)
1. early stage kernel seed coat colour identification kit of Semen Brassicae campestris polus animalium is characterized in that said test kit is made up of following parts:
(1) RNA extracts A liquid, interior dress Trizol liquid;
(2) RNA extracts B liquid, interior dress phenol-chloroform, and the volume ratio of phenol, chloroform is 25:24;
(3) RNA extracts C liquid, interior dress Virahol;
(4) RNA extracts D liquid, in adorn 75% ethanol;
(5) RNA extracts E liquid, and interior dress DEPC handles the ultrapure water of back high-temperature sterilization;
(6) RT reaction F liquid, the ultrapure water that interior reaction cartridge buffer B uffer, dNTP, oligo dT primer, the inhibition of RNA enzyme, ThermoScript II M-MLV and DEPC handle, the volume proportion of each component is:
Reaction buffer, 2 parts of 5 * Buffer
1 part of dNTP
1 part of oligo dT primer
The RNA enzyme suppresses 0.3 part
0.5 part of ThermoScript II M-MLV
4.2 parts of the ultrapure waters that DEPC handles;
(7) RT-PCR reaction G1 liquid, interior dress contains Mg
2+Reaction buffer Buffer, dNTP, Taq enzyme, forward primer F, reverse primer R and ultrapure water, the volume proportion of each component is following:
Contain mg
2+Reaction buffer, 2 parts of 10 * Buffer
0.3 part of dNTP
1 part of forward primer F
1 part of reverse primer R
13.3 parts of ultrapure waters
0.4 part of Taq enzyme;
(8) RT-PCR reaction G2 liquid, interior dress contains Mg
2+Reaction buffer, the forward primer ACTF of dNTP, Taq enzyme, internal control gene Actin, reverse primer ACTR and the ultrapure water of internal control gene Actin, the volume proportion of each component is following:
Contain mg
2+Reaction buffer, 2 parts of 10 * Buffer
0.3 part of dNTP
1 part of forward primer ACTF
1 part of reverse primer ACTR
13.3 parts of ultrapure waters
0.4 part of Taq enzyme;
(9) positive control H1 liquid, interior dress is deceived the seed rape positive template;
(10) negative control H2 liquid, the negative template of interior dress yellow seed rape;
Said forward primer F sequence is: 5'-TCAGCTGCTGCGGTTTCTATC-3', and said reverse primer R sequence is: 5'-CAGATAGCTTCTGCATTTCCTTG-3'; Said forward primer ACTF sequence is: 5'-GACATTCAACCTCTTGTTTGC-3', and said reverse primer ACTR sequence is: 5'-CTGCTCGTAGTCAAGAGCAATG-3';
Said extremely in early days for blooming back 4-5 days.
2. a method of utilizing the said test kit of claim 1 to identify the early stage kernel seed coat colour of rape polus animalium is characterized in that, carries out according to the following steps:
(1) get the rape kind skin sample that removes hilum after to be measured the blooming 4-5 days, the RNA that adds 5 times of volumes extracts A liquid, ice bath homogenate, the static 2-5min of room temperature, homogenate;
(2) RNA that in above-mentioned homogenate, adds 0.5 times of volume extracts B liquid, mixing, static 2-5min;
(3) 4-10 ℃, the centrifugal 5min of 12000g;
(4) get supernatant, the RNA that adds 1 times of volume extracts C liquid, and mixing leaves standstill 5-10 min;
(5) 4-10 ℃, the centrifugal 5min of 12000g;
(6) abandon supernatant, extract the washing of D liquid with RNA after, drying at room temperature or in super clean bench, dry up adds RNA and extracts the dissolving of E liquid, RNA solution;
(7) get gained RNA solution, add the RT reaction F liquid of 4 times of volumes, 42 ℃ of insulation 30 min handle 5 min for 99 ℃ then, get the template of RT-PCR;
(8) template, positive control H1 liquid, the negative control H2 liquid of RT-PCR is added to respectively in RT-PCR reaction G1 liquid and the RT-PCR reaction G2 liquid, behind the mixing as on the PCR appearance;
(9) carry out pcr amplification: 94 ℃ of 3min of elder generation, 94 ℃ again, 30s; 58 ℃, 30s; 72 ℃, 30 min, after 30 circulations, 72 ℃ prolong 6 min, after reaction finishes, get amplified production;
(10) get amplified production, add the sample-loading buffer of 1/5 times of volume, contain tetrabromophenol sulfonphthalein; Behind 1% agarose gel electrophoresis 15-30min, on ultraviolet device, observe, bright band occurs, then for detecting the positive as if 319 bp places with the positive control same position; Explain that planting skin behind this testing sample seed maturity is black; Otherwise negative, explain that it is transparent planting skin behind this testing sample seed maturity, seed shows the yellow of embryo.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN2010105262229A CN102134587B (en) | 2010-11-01 | 2010-11-01 | Kit and method for identifying seed coat color of rape seed in very early development stage |
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN2010105262229A CN102134587B (en) | 2010-11-01 | 2010-11-01 | Kit and method for identifying seed coat color of rape seed in very early development stage |
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| Publication Number | Publication Date |
|---|---|
| CN102134587A CN102134587A (en) | 2011-07-27 |
| CN102134587B true CN102134587B (en) | 2012-08-22 |
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|---|---|---|---|
| CN2010105262229A Expired - Fee Related CN102134587B (en) | 2010-11-01 | 2010-11-01 | Kit and method for identifying seed coat color of rape seed in very early development stage |
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Families Citing this family (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102318512B (en) * | 2011-09-05 | 2012-11-28 | 湖南科技大学 | Rape seed husk color regulation and control method |
| CN102634511B (en) * | 2012-04-13 | 2013-03-13 | 淮阴师范学院 | Extraction method for RNA (ribonucleic acid) of secondary dormancy seeds of mature rapes |
| CN105510319A (en) * | 2015-12-07 | 2016-04-20 | 湖南科技大学 | Method for quickly identifying color of seed coats of rape seeds in early development period |
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