CN108077063A - A kind of arabidopsis double base is overexpressed the technical method of strain structure - Google Patents
A kind of arabidopsis double base is overexpressed the technical method of strain structure Download PDFInfo
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- CN108077063A CN108077063A CN201810106829.8A CN201810106829A CN108077063A CN 108077063 A CN108077063 A CN 108077063A CN 201810106829 A CN201810106829 A CN 201810106829A CN 108077063 A CN108077063 A CN 108077063A
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- 241000219194 Arabidopsis Species 0.000 title claims abstract description 64
- 238000000034 method Methods 0.000 title claims abstract description 13
- 241000196324 Embryophyta Species 0.000 claims abstract description 38
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 claims abstract description 24
- 230000008774 maternal effect Effects 0.000 claims abstract description 13
- 230000010152 pollination Effects 0.000 claims abstract description 13
- 239000011780 sodium chloride Substances 0.000 claims abstract description 12
- 238000012216 screening Methods 0.000 claims abstract description 10
- 239000007921 spray Substances 0.000 claims abstract description 6
- 238000005507 spraying Methods 0.000 claims abstract description 3
- 238000009331 sowing Methods 0.000 claims description 6
- 235000013399 edible fruits Nutrition 0.000 claims description 4
- 239000001963 growth medium Substances 0.000 claims description 4
- 230000017260 vegetative to reproductive phase transition of meristem Effects 0.000 claims description 4
- 238000003306 harvesting Methods 0.000 claims description 2
- 239000000203 mixture Substances 0.000 claims description 2
- 238000009738 saturating Methods 0.000 claims description 2
- 230000002018 overexpression Effects 0.000 abstract description 11
- 238000005516 engineering process Methods 0.000 abstract description 5
- 108090000623 proteins and genes Proteins 0.000 abstract description 4
- 210000001672 ovary Anatomy 0.000 abstract description 2
- 230000008775 paternal effect Effects 0.000 abstract description 2
- 238000012545 processing Methods 0.000 abstract description 2
- 102000004169 proteins and genes Human genes 0.000 abstract description 2
- 241000219195 Arabidopsis thaliana Species 0.000 description 7
- 230000001105 regulatory effect Effects 0.000 description 6
- 238000011160 research Methods 0.000 description 5
- 238000013518 transcription Methods 0.000 description 5
- 230000035897 transcription Effects 0.000 description 5
- 230000034303 cell budding Effects 0.000 description 4
- 230000000694 effects Effects 0.000 description 3
- 238000002474 experimental method Methods 0.000 description 3
- 238000005286 illumination Methods 0.000 description 3
- 230000008827 biological function Effects 0.000 description 2
- 230000014509 gene expression Effects 0.000 description 2
- 230000003712 anti-aging effect Effects 0.000 description 1
- 238000009395 breeding Methods 0.000 description 1
- 230000001488 breeding effect Effects 0.000 description 1
- 238000011161 development Methods 0.000 description 1
- 230000018109 developmental process Effects 0.000 description 1
- 238000002224 dissection Methods 0.000 description 1
- 230000006870 function Effects 0.000 description 1
- 230000002068 genetic effect Effects 0.000 description 1
- 238000009396 hybridization Methods 0.000 description 1
- 239000007788 liquid Substances 0.000 description 1
- 239000000463 material Substances 0.000 description 1
- 230000035479 physiological effects, processes and functions Effects 0.000 description 1
- 238000006467 substitution reaction Methods 0.000 description 1
- 238000012360 testing method Methods 0.000 description 1
- 230000009466 transformation Effects 0.000 description 1
Classifications
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01H—NEW PLANTS OR NON-TRANSGENIC PROCESSES FOR OBTAINING THEM; PLANT REPRODUCTION BY TISSUE CULTURE TECHNIQUES
- A01H1/00—Processes for modifying genotypes ; Plants characterised by associated natural traits
- A01H1/02—Methods or apparatus for hybridisation; Artificial pollination ; Fertility
-
- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01H—NEW PLANTS OR NON-TRANSGENIC PROCESSES FOR OBTAINING THEM; PLANT REPRODUCTION BY TISSUE CULTURE TECHNIQUES
- A01H1/00—Processes for modifying genotypes ; Plants characterised by associated natural traits
- A01H1/02—Methods or apparatus for hybridisation; Artificial pollination ; Fertility
- A01H1/027—Apparatus for pollination
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- Life Sciences & Earth Sciences (AREA)
- Health & Medical Sciences (AREA)
- General Health & Medical Sciences (AREA)
- Genetics & Genomics (AREA)
- Botany (AREA)
- Developmental Biology & Embryology (AREA)
- Environmental Sciences (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
The invention discloses the technical methods that arabidopsis double base is overexpressed strain structure, this method is to select arabidopsis after incubator culture to start the single plant buddingged, closed environment is placed after removing the flower buddingged, the flower of arabidopsis is sprayed with 1% sodium chloride solution after removing the stamen as maternal single plant with tweezers, sodium chloride solution pollinates to target single plant for use as the flower of male parent after spraying.The present invention sprays processing due to having been carried out before pollination with sodium chloride solution, reduce rejection of the maternal single plant column cap protein to external pollen, column cap is passed through so as to improving paternal pollen and enters ovary, effectively increases the setting percentage that arabidopsis double base is overexpressed strain.Simultaneously because the setting percentage that arabidopsis double base is overexpressed strain is higher, follow-up filial generation screening becomes to be more prone to, and greatly reduces the workload of filial generation screening.And entirely arabidopsis double base overexpression strain building process is easy to operate, and technology requirement degree is low.
Description
Technical field
The present invention relates to biotechnology research field, particularly a kind of arabidopsis double base is overexpressed the technical side of strain structure
Method.
Technical background
Arabidopsis is one mode plant, is eucaryon higher organism, and since its breeding time is short, genome is relatively easy,
Plant science research field has very extensive purposes, in research phytomorph dissection, Physiology and biochemistry, molecular genetic, gene
Expression regulation, biological information etc. have played very important effect.
With the progress of plant science, the functional gene of arabidopsis is excavated gradually deeply, substantial amounts of functional gene
And transcription regulatory factor is found and has carried out detailed parsing.Transcription regulatory factor is widely present in arabidopsis, to arabidopsis
Growth and development, physiological and biochemical activity, anti-aging and anti-environment stress etc. play very important biological function, common turns
Record regulatory factor has MYB, NAC, NAM, WRKY etc..And plant scientist is gradually sent out in the research process to transcription regulatory factor
It is existing, transcription regulatory factor when its function is played often 2 or 2 or more common coordinated expressions so as to completing a certain item
Specific biological function, structure double base overexpression strain, which just seems, when being further investigated transcription regulatory factor very must
Will, and when actual implementation arabidopsis double base is overexpressed strain, often complicated for operation, technology requirement degree height, and build double
It is relatively low that member is overexpressed strain cross setting percentage, it is difficult to meet the needs of scientific research reality.
More than reason is based on, gropes the technical method that a set of technical solution arabidopsis double base is overexpressed strain structure, to carry
High arabidopsis double base is overexpressed the setting percentage of strain structure hybridization, is to have very great meaning.
The content of the invention
The technical problems to be solved by the invention are：It is overexpressed for arabidopsis double base in the prior art miscellaneous when strain is built
The problem of setting percentage is low, the screening of target single plant is difficult, technology requirement degree is high is handed over, a kind of arabidopsis double base is provided and is overexpressed strain
The technical method of structure, this method is low to operator's technology requirement degree, double base is overexpressed Cross fertile rate height when strain is built,
The screening of target single plant is easy.
In order to solve the above technical problems, the technical solution adopted in the present invention is：
Arabidopsis starts the single plant buddingged, removes closed loop after the flower buddingged in incubator culture 30 days or so, selection
When border placement 2 is small, the flower of arabidopsis is sprayed with 1% sodium chloride solution after removing the stamen as maternal single plant with tweezers
Apply, sodium chloride solution spray 2 it is small when after pollinate for use as the flower of male parent to target single plant.
Said program implement the step of be：1st, arabidopsis is selected firm from maternal strain in incubator culture 30 days or so
Just there is first colored 10 single plant and formed first group, the flower buddingged all is removed, then with transparent polybag
To at the top of arabidopsis and surrounding covers, external pollen being prevented to be passed to, incubator place 2 it is small when after with 1% sodium chloride
Solution uniformly sprays the flower of arabidopsis.
2nd, 3 days sowing arabidopsis male parent strains are shifted to an earlier date compared with first group, in incubator culture 30 days or so, from male parent strain
Select first second group of colored 10 single plant composition just occurred in system, the flower buddingged all is removed, then with saturating
Bright polybag covers arabidopsis top and surrounding, and external pollen is prevented to be passed to, and places 3-5 days, treats in the incubator
There are a large amount of flowers and carry out next step experiment.
3rd, the stamen prepared in first group of i.e. maternal strain on pollination single plant is removed with tweezers, from second group i.e. male parent strain
Flower on the middle single plant for selecting flowering more has gently smeared the column cap that pollination single plant is ready in first group
Into pollination.
4th, continue to cover arabidopsis top and surrounding with polybag after pollinating, prevent what external pollen was passed to
Pay attention to keeping 70% or so humidity simultaneously, polybag is removed after 7 days or so, harvest fruit pod is in the sowing of resistance screening culture medium
Arabidopsis double base can be obtained and be overexpressed strain.
Compared with prior art, the advantageous effect of present invention is that：
1st, due to having been carried out spraying processing with sodium chloride solution before pollination, it is external to reduce maternal single plant column cap protein
The rejection of boundary's pollen passes through column cap so as to improving paternal pollen and enters ovary, effectively increases arabidopsis double base
It is overexpressed the setting percentage of strain.
2nd, since the setting percentage of arabidopsis double base overexpression strain is higher, follow-up filial generation screening becomes to be more prone to,
Greatly reduce the workload of filial generation screening.
3rd, entire arabidopsis double base overexpression strain building process is easy to operate, and technology requirement degree is low.
Specific embodiment
With reference to embodiment, the invention will be further described, but not as any limitation of the invention.
Embodiment 1：
1st, on June 10th, 2017 carries out under the conditions of 4 DEG C at vernalization col Arabidopsis thaliana ecotypes male parent strain OEWR26-1
Reason, arabidopsis seed starts to germinate after 3 days, starts to budding after 28 days in tissue culture room culture arabidopsis and bloom, from male parent strain
Just has there is first colored 10 single plant and has formed first group in system's selection, the flower of appearance is removed with tweezers, then with transparent
Polybag covers arabidopsis top and surrounding, and external pollen is prevented to be passed to, places 5 days, treats out in tissue culture room
Now a large amount of flowers carry out next step experiment.
2nd, on June 13rd, 2017 carries out under the conditions of 4 DEG C at vernalization col Arabidopsis thaliana ecotypes female parent strain OEWR71-3
Reason, arabidopsis seed starts to germinate after 3 days, starts to budding after 30 days in tissue culture room culture arabidopsis and bloom, from female parent strain
Just has there is first colored 10 single plant and has formed second group in system's selection, the flower of appearance is removed with tweezers, then with transparent
Polybag at the top of arabidopsis and surrounding covers, external pollen being prevented to be passed to, place 2 it is small when after it is molten with 1% sodium chloride
Liquid uniformly sprays the flower of arabidopsis.
3rd, for step 2 hero prepared in second group of arabidopsis female parent strain OEWR71-3 on pollination single plant is removed with tweezers
Stamen, the flower on single plant for selecting flowering more from first group of arabidopsis male parent strain OEWR26-1, to maternal strain
The column cap that pollination single plant is ready in OEWR71-3 carries out gently smearing completion pollination.
4th, continue after pollinating with polybag to having pollinated in maternal second group of strain OEWR71-3 at the top of single plant and
Surrounding is covered, and prevents from paying attention to the humidity for keeping 70% or so while external pollen is incoming, polybag is removed after 7 days, receive
It obtains fruit pod and obtains arabidopsis double base overexpression strain in the sowing of resistance screening culture medium.
Explanation：Above embodiment has selected arabidopsis to be overexpressed strain OEWR26-1 and has been overexpressed strain OEWR71-3 structures
It builds double base and is overexpressed strain, it is actual to be applicable in for all overexpression strains.
Embodiment 2：
1st, ler Arabidopsis thaliana ecotypes is selected to be overexpressed strain OESE-10 as male parent, in August in 2017 10 days to male parent strain
It is that OESE-10 carries out vernalization treatment under the conditions of 4 DEG C, ler Arabidopsis thaliana ecotypes seed starts to germinate after 3 days, in illumination cultivation
Case culture arabidopsis starts to budding after 30 days blooms, and first colored 15 has just occurred from strain OESE-10 selections are overexpressed
Single plant forms first group, flower is gently removed with tweezers, next with transparent polybag to the top of arabidopsis flowerpot and four
Zhou Jinhang is covered, and is prevented the incoming of extraneous pollen, is placed 7 days in illumination box, and a large amount of flowers to appear are for real in next step
Test use.
2nd, ler Arabidopsis thaliana ecotypes is selected to be overexpressed female parents of the strain OEWR35-8 as experiment, take appropriate overexpression strain
It is that the seed of OEWR35-8 carries out vernalization treatment 3 days in August in 2017 13 days under the conditions of 4 DEG C, treats that arabidopsis seed starts to send out
Bud is sowed, and is started to budding after 30 days in illumination box culture arabidopsis seed and be bloomed, and has just been gone out from the selection of maternal strain
Existing first colored 15 arabidopsis single plant forms second group, the flower of appearance is carefully removed with tweezers, then with transparent modeling
Material bag covers the top of arabidopsis flowerpot and surrounding, place 2 it is small when after with 1% sodium chloride solution to the flower of arabidopsis
Uniformly sprayed.
3rd, for 15 arabidopsis single plants in the overexpression strain OEWR35-8 in step 2, hero is gently removed with tweezers
Stamen, the flower for selecting flowering more from 15 single plants of arabidopsis male parent strain, to 15 in overexpression strain OEWR35-8
The column cap of a arabidopsis single plant gently smears completion pollination.
4th, 15 arabidopsis being overexpressed to maternal strain with polybag in strain OEWR35-8 are continued after pollinating
Single plant is covered, and prevents from paying attention to the humidity for keeping 70% or so while external pollen is incoming, polybag is removed after 7 days, receive
It obtains fruit pod and obtains arabidopsis double base overexpression strain in the sowing of resistance screening culture medium.
Explanation：Above embodiment has selected col Arabidopsis thaliana ecotypes to be overexpressed strain OEWR26-1, be overexpressed strain
OEWR71-3 and Ler Arabidopsis thaliana ecotypes OESE-10 is overexpressed strain, OEWR35-8 is overexpressed strain structure double base and is overexpressed strain
System, it is actual to be applicable in for all overexpression strains.
The concrete application example of the above simply present invention, the present invention also has other embodiments, all to use equivalent substitution
Or the technical solution that equivalent transformation is formed, it all falls within protection domain of the presently claimed invention.
Claims (2)
1. arabidopsis double base according to claim 1 is overexpressed the technical method of strain structure, it is characterised in that including such as
Lower step：Arabidopsis selects to start the single plant buddingged after incubator culture, and closed environment is placed after removing the flower buddingged, and is used
Tweezers spray the flower of arabidopsis with 1% sodium chloride solution after removing the stamen as maternal single plant, sodium chloride solution
It pollinates after spraying for use as the flower of male parent to target single plant.
2. arabidopsis double base according to claim 1 is overexpressed the technical method of strain structure, it is characterised in that including such as
Lower step：
Step 1, arabidopsis selects first colored 10 list just occurred in incubator culture 30 days or so from maternal strain
Strain forms first group, and the flower buddingged all is removed, and then arabidopsis top and surrounding are carried out with transparent polybag
Cover, external pollen prevented to be passed to, incubator place 2 it is small when after with 1% sodium chloride solution the flower of arabidopsis is carried out it is equal
It is even to spray；
Step 2,3 days sowing arabidopsis male parent strains are shifted to an earlier date compared with first group, in incubator culture 30 days or so, from male parent strain
Select first second group of colored 10 single plant composition just occurred in system, the flower buddingged all is removed, then with saturating
Bright polybag covers arabidopsis top and surrounding, and external pollen is prevented to be passed to, and places 3-5 days, treats in the incubator
There are a large amount of flowers to carry out in next step；
Step 3, the stamen prepared in first group of i.e. maternal strain on pollination single plant is removed with tweezers, from second group i.e. male parent strain
Flower on the middle single plant for selecting flowering more has gently smeared the column cap that pollination single plant is ready in first group
Into pollination；
Step 4, continue to cover arabidopsis top and surrounding with polybag after pollination, prevent what external pollen was passed to
The humidity of 60-80% is kept simultaneously, polybag is removed after 6-8 days, and harvest fruit pod can be obtained in the sowing of resistance screening culture medium
Arabidopsis double base is overexpressed strain.
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| CN201810106829.8A CN108077063A (en) | 2018-02-02 | 2018-02-02 | A kind of arabidopsis double base is overexpressed the technical method of strain structure |
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Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| KR100741255B1 (en) * | 2006-07-14 | 2007-07-19 | 고려대학교 산학협력단 | Arabidopsis atlej1 gene that negatively regulates on biosynthesis of jasmonic acid and ethlylene and production method of male-sterile plant using the same |
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Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| KR100741255B1 (en) * | 2006-07-14 | 2007-07-19 | 고려대학교 산학협력단 | Arabidopsis atlej1 gene that negatively regulates on biosynthesis of jasmonic acid and ethlylene and production method of male-sterile plant using the same |
Non-Patent Citations (3)
| Title |
|---|
| 何金环等: "活性氧不敏感型拟南芥突变体的遗传特性分析", 《安徽农业科学》 * |
| 侯瑞贤等: "不同NaCl浓度处理对不结球白菜结实能力的影响", 《上海农业学报》 * |
| 叶要妹: "百日草杂交亲本的选育与自交系间遗传多样性评价", 《中国博士学位论文全文数据库》 * |
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Application publication date: 20180529 |