CN108949811A - A kind of breeding method of the transgenic paddy rice sterile line based on C6 gene - Google Patents
A kind of breeding method of the transgenic paddy rice sterile line based on C6 gene Download PDFInfo
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- C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
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- C12N15/8261—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield
- C12N15/8287—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for fertility modification, e.g. apomixis
- C12N15/8289—Male sterility
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- C12N15/09—Recombinant DNA-technology
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Abstract
This application discloses a kind of breeding methods of transgenic paddy rice sterile line based on C6 gene, comprising the following steps: the amplification of A, Wheat Pollen lethal gene Ki；B, the amplification of Wheat Pollen specificity promoter Pg47；C, the acquisition of mCerulean expression casette；D, the acquisition of paddy gene expression cassette C6；E, the connection of each gene expression element.The connection of the step E, each gene expression element, include: to introduce mCerulean gene expression element on the pCAMBIA1300 carrier for having connected Wheat Pollen lethal gene Ki and Wheat Pollen specificity promoter Pg47, is finally connected to complete rice C6 gene on the complex carrier of first three step.F1 generation heterozygote of the present invention follows Mendel's law of segregation, the offspring of the generation existing heterozygote for being able to maintain three linked genes during self-fertility, and whether there is or not the sterile lines of fertility.
Description
Technical field
The invention belongs to genetic engineering genetic thremmatology technical fields, and in particular to a kind of transgenosis water based on C6 gene
The breeding method of rice sterile line.
Background technique
Rice originates in Tropical Asian, after China widely plants, gradually travels to all over the world.Rice is in the world nearly one
The staple food of half population can also make wine, refine sugar and big as the raw material of industry in addition to edible.Production practices table for many years
Bright, hybrid rice is generally than 20% or more conventional Rice volume increase, therefore hybrid rice shows huge yield potential.
The development of hybrid rice depends on the cultivation of sterile line of hybridized rice.The research of China hybrid rice starts from last generation
It records the sixties, starts the seventies to be planted on a large scale.First generation hybrid rice is with nucleo-cytoplasmic interreaction male sterility system for hereditary work
The three line method of tool, second generation hybrid rice be using photoperiod-temperature sensitive male sterility system as the two line method of genetic tool, " three line method " and " two
It is method " crossbreeding technology is huge to increases in grain production contribution, but there are also problems.It is available in three line method that there is excellent shape
Parent it is limited, combo not freely, it is that frequency is lower that holding is lost in open country.
Hereditary difference is small between existing sterile line in production, and cenospecies fertility stability is inadequate, and it is poor to resist adverse circumstance ability.
And the fertility of photoperiod-temperature sensitive male sterility system is controlled by ambient temperature in " two line method ", easily leads to photo-thermo-sensitive genetic male sterile line self-fertility,
Production of hybrid seeds failure.Therefore it is most important to the development of hybrid rice to develop sterile line of new generation.
Rice C6 gene is a member in bHLH transcription factor, and bHLH transcription factor largely participates in the hair of anther tapetum
It educates or microspore development.Anther tapetum is most important to the growth and development of pollen, and the callose enzyme of tapetum secretion can fit
When decompose the callose wall of pollen mother cell and tetrad, to guarantee that microspore is separated from each other.And rice AID1 gene mutation
After will lead to tapetal cell and cannot degrade in time, programmed death delay, so that the callose enzyme of tapetum secretion influences
The development and separation of microspore, are eventually exhibited as infertility.
Summary of the invention
The present invention is chain by rice Male sterile gene C6 and Wheat Pollen lethal gene Ki, while with blue-green fluorescent
Protein gene mCerulean carries out genetic modification as reporter gene, to rice C6 mutant, obtains F1 generation heterozygote.F1 generation
Heterozygote follows Mendel's law of segregation, the offspring of the generation existing heterozygosis for being able to maintain three linked genes during self-fertility
Body, but whether there is or not the sterile lines of fertility.
For achieving the above object, the present invention is achieved by the following scheme:
A kind of breeding method of the transgenic paddy rice sterile line based on C6 gene, it is characterised in that it the following steps are included:
A, the amplification of Wheat Pollen lethal gene Ki；
B, the amplification of Wheat Pollen specificity promoter Pg47；
C, the acquisition of mCerulean expression casette；
D, the acquisition of paddy gene expression cassette C6；
E, the connection of each gene expression element.
The step E, each gene expression element Connection Step include:
Wheat Pollen lethal gene Ki is passed through single endonuclease digestion by the first step, the Msel restriction enzyme site all introduced using upstream and downstream
Site is connected on pCAMBIA1300 binary vector；
Second step utilizes introducing Kpn I and Sma I restriction enzyme site and pCAMBIA1300 binary vector in upstream and downstream primer
On Kpn I and Sma I restriction enzyme site, Wheat Pollen specificity starting Pg47 is connected to the first step and has connected upper wheat flower
On the pCAMBIA1300 carrier of powder lethal gene Ki；
Third step utilizes TrulI the and Kpn1 restriction enzyme site and pCAMBIA1300 binary vector introduced in upstream and downstream primer
MCerulean gene expression element is connected to first two steps and has connected upper Wheat Pollen cause by upper TrulI and Kpn1 restriction enzyme site
On the pCAMBIA1300 of dead gene Ki and Wheat Pollen specificity promoter Pg47；
4th step connects C6, utilizes Smal the and Sau96I restriction enzyme site and pCAMBIA1300 introduced in upstream and downstream primer
The introducing of complete rice C6 gene has been connected with the lethal base of Wheat Pollen by Smal the and Sau96I restriction enzyme site on binary vector
Because on the pCAMBIA1300 carrier of Ki and Wheat Pollen specificity promoter Pg47 and mCerulean gene expression element.
The amplification of the step A, Wheat Pollen lethal gene Ki further comprise:
Using Wheat volatiles DNA as template, the amplification of wheat lethal gene Ki is carried out using F3/R3 as upstream and downstream primer,
Msel restriction enzyme site is introduced in upstream and downstream primer；Its lethal gene Ki expanded includes all exon and introne.
The amplification of the step B, Wheat Pollen specificity promoter Pg47 further comprise:
Using Wheat volatiles DNA as template, pollen specific promoter Pg47 expansion is carried out by upstream and downstream primer of F4/R4
Increase, introduces Kpn I and Sma I restriction enzyme site respectively in upstream and downstream primer.
The acquisition of step C, the mCerulean expression casette further comprises:
Using the plasmid containing mCerulean gene expressed intact box as template, expanded by upstream and downstream primer of F1/R1
Increase, introduces TrulI and Kpn I site, while the Kpn I introduced in downstream primer in amplification procedure in upstream and downstream primer respectively
Msel restriction enzyme site is added before site.
The acquisition of the step D, paddy gene expression cassette C6 further comprise:
The genomic DNA for extracting rice plant carries out complete using genomic DNA as template using F2/R2 as upstream and downstream primer
The amplification of C6 expression casette introduces Sma I and Sau96I digestion in amplification procedure in upstream primer and downstream primer respectively
The rice C6 gene in site, amplification includes the promoter of its upstream and the terminator in downstream, while including all exons
And introne.
The present invention provides a kind of transgenic paddy rice sterile lines based on C6 gene such as any one of aforementioned method breeding.
It is educated such as any one of aforementioned the method in the transgenic paddy rice heredity based on C6 gene the present invention also provides a kind of
Application in kind.
Beneficial effect of the present invention includes: that the present invention connects rice Male sterile gene C6 and Wheat Pollen lethal gene Ki
Lock, while using cyan fluorescent protein gene mCerulean as reporter gene, genetic modification is carried out to rice C6 mutant,
Obtain F1 generation heterozygote.F1 generation heterozygote follows Mendel's law of segregation, the offspring of generation existing energy during self-fertility
The heterozygote of three linked genes is kept, and whether there is or not the sterile lines of fertility.
Detailed description of the invention
Fig. 1: Wheat Pollen lethal gene Ki amplification detected through gel electrophoresis figure；
Fig. 2: Wheat Pollen specificity promoter Pg47 amplification detected through gel electrophoresis figure；
Fig. 3: mCerulean gene expressed intact box PCR amplification detected through gel electrophoresis figure；
Fig. 4: rice C6 expression casette PCR amplification detected through gel electrophoresis figure；
Fig. 5: plant expression vector gene linkage, transcriptional orientation and restriction enzyme site map；
Fig. 6: the spike of rice photo that transgenic plant is born.
Specific embodiment
The technical scheme of the present invention will be explained in further detail With reference to embodiment.
The present invention is that rice Male sterile gene C6 and Wheat Pollen lethal gene Ki is chain while glimmering with blue-green
Aequorin mCerulean carries out genetic modification as reporter gene, to rice C6 mutant, obtains F1 generation heterozygote.F1
Mendel's law of segregation is followed during self-fertility for heterozygote, the offspring of generation is existing to be able to maintain the miscellaneous of three linked genes
Zoarium, but whether there is or not the sterile line of fertility, i.e., so-called third generation sterile lines.
A kind of breeding method of the transgenic paddy rice sterile line based on C6 gene, it is characterised in that it the following steps are included:
A, the amplification of Wheat Pollen lethal gene Ki；
B, the amplification of Wheat Pollen specificity promoter Pg47；
C, the acquisition of mCerulean expression casette；
D, the acquisition of paddy gene expression cassette C6；
E, the connection of each gene expression element.
The step E, each gene expression element Connection Step include:
Wheat Pollen lethal gene Ki is passed through single endonuclease digestion by the first step, the Msel restriction enzyme site all introduced using upstream and downstream
Site is connected on pCAMBIA1300 binary vector；
Second step utilizes introducing Kpn I and Sma I restriction enzyme site and pCAMBIA1300 binary vector in upstream and downstream primer
On Kpn I and Sma I restriction enzyme site, Wheat Pollen specificity starting pPG47 is connected to the first step and has connected upper wheat
On the pCAMBIA1300 carrier of pollen lethal gene Ki；
Third step utilizes Tru1I the and Kpn1 restriction enzyme site and pCAMBIA1300 binary vector introduced in upstream and downstream primer
MCerulean gene expression element is connected to first two steps and has connected upper Wheat Pollen cause by upper Tru1I and Kpn1 restriction enzyme site
On the pCAMBIA1300 of dead gene Ki and Wheat Pollen specificity promoter Pg47；
4th step connects C6, utilizes Smal the and Sau96I restriction enzyme site and pCAMBIA1300 introduced in upstream and downstream primer
The introducing of complete rice C6 gene has been connected with the lethal base of Wheat Pollen by Smal the and Sau96I restriction enzyme site on binary vector
Because on the pCAMBIA1300 carrier of Ki and Wheat Pollen specificity promoter Pg47 and mCerulean gene expression element.
1, the amplification of Wheat Pollen lethal gene Ki
Using Wheat volatiles DNA as template, the amplification of wheat lethal gene Ki is carried out using F3/R3 as upstream and downstream primer,
Msel restriction enzyme site is introduced in upstream and downstream primer；Its lethal gene Ki expanded includes all exon and introne.
2, the amplification of Wheat Pollen specificity promoter Pg47
Using Wheat volatiles DNA as template, pollen specific promoter Pg47 expansion is carried out by upstream and downstream primer of F4/R4
Increase, introduces Kpn I and Sma I restriction enzyme site respectively in upstream and downstream primer.
3, the acquisition of mCerulean expression casette
Using the plasmid containing mCerulean gene expressed intact box as template, expanded by upstream and downstream primer of F1/R1
Increase, introduces Tru1I and Kpn I site, while the Kpn I introduced in downstream primer in amplification procedure in upstream and downstream primer respectively
Msel restriction enzyme site is added before site.
4, the acquisition of paddy gene expression cassette C6
The genomic DNA for extracting rice plant carries out complete using genomic DNA as template using F2/R2 as upstream and downstream primer
The amplification of C6 expression casette introduces Sma I and Sau96I digestion in amplification procedure in upstream primer and downstream primer respectively
The rice C6 gene in site, amplification includes the promoter of its upstream and the terminator in downstream, while including all exons
And introne.
The present invention provides a kind of transgenic paddy rice sterile lines based on C6 gene such as any one of aforementioned method breeding.
It is educated such as any one of aforementioned the method in the transgenic paddy rice heredity based on C6 gene the present invention also provides a kind of
Application in kind.
Table 1: the restriction enzyme site added in primer that each gene magnification is used, primer
Embodiment described above not does any type of limitation to the present invention, although the present invention, with preferable, embodiment is taken off
Show as above, however be not intended to limit the invention, any person skilled in the art, is not departing from technical solution of the present invention
In the range of, a little variation or modification are made using the technology contents of the disclosure above and is equal to equivalence enforcement case, are belonged to
In in technical solution of the present invention protection scope.
The present invention provides a kind of transgenic paddy rice sterile lines based on C6 gene such as any one of aforementioned method breeding.
It is educated such as any one of aforementioned the method in the transgenic paddy rice heredity based on C6 gene the present invention also provides a kind of
Application in kind.
Claims (8)
1. a kind of breeding method of the transgenic paddy rice sterile line based on C6 gene, it is characterised in that it the following steps are included:
A, the amplification of Wheat Pollen lethal gene Ki；
B, the amplification of Wheat Pollen specificity promoter Pg47；
C, the acquisition of mCerulean expression casette；
D, the acquisition of paddy gene expression cassette C6；
E, the connection of each gene expression element.
2. the breeding method of the transgenic paddy rice sterile line based on C6 gene according to claim 1, it is characterised in that described
Step E, the Connection Step of each gene expression element includes:
Wheat Pollen lethal gene Ki is passed through single endonuclease digestion site by the first step, the Msel restriction enzyme site all introduced using upstream and downstream
It is connected on pCAMBIA1300 binary vector；
Second step introduces on Kpn I and Sma I restriction enzyme site and pCAMBIA1300 binary vector using in upstream and downstream primer
Wheat Pollen specificity starting Pg47 is connected to the first step and has connected upper Wheat Pollen cause by Kpn I and Sma I restriction enzyme site
On the pCAMBIA1300 carrier of dead gene Ki；
Third step, using on Tru1I the and Kpn1 restriction enzyme site and pCAMBIA1300 binary vector introduced in upstream and downstream primer
Tru1I and Kpn1 restriction enzyme site, mCerulean gene expression element is connected to first two steps, and to have connected upper Wheat Pollen lethal
On the pCAMBIA1300 of gene Ki and Wheat Pollen specificity promoter Pg47；
4th step connects C6, utilizes Smal the and Sau96I restriction enzyme site and pCAMBIA1300 double base introduced in upstream and downstream primer
The introducing of complete rice C6 gene has been connected with Wheat Pollen lethal gene Ki by Smal the and Sau96I restriction enzyme site on carrier
On the pCAMBIA1300 carrier of Wheat Pollen specificity promoter Pg47 and mCerulean gene expression element.
3. the breeding method of the transgenic paddy rice sterile line based on C6 gene, feature exist according to claim 1, the step
The amplification of rapid A, Wheat Pollen lethal gene Ki further comprise:
Using Wheat volatiles DNA as template, the amplification of wheat lethal gene Ki is carried out using F3/R3 as upstream and downstream primer, upper and lower
It swims and introduces Msel restriction enzyme site in primer；Its lethal gene Ki expanded includes all exon and introne.
4. the breeding method of the transgenic paddy rice sterile line based on C6 gene, feature exist according to claim 1, the step
The amplification of rapid B, Wheat Pollen specificity promoter Pg47 further comprise:
Using Wheat volatiles DNA as template, pollen specific is carried out as upstream and downstream primer using F4/R4 and starts pPG47 amplification, upper
Kpn I and Sma I restriction enzyme site is introduced in downstream primer respectively.
5. the breeding method of the transgenic paddy rice sterile line based on C6 gene, feature exist according to claim 1, the step
The acquisition of rapid C, mCerulean expression casette further comprises:
It using the plasmid containing mCerulean gene expressed intact box as template, is expanded, is expanded by upstream and downstream primer of F1/R1
Introduce TrulI and Kpn I site, while the Kpn I site introduced in downstream primer in increasing process in upstream and downstream primer respectively
Msel restriction enzyme site is added in front.
6. the breeding method of the transgenic paddy rice sterile line based on C6 gene, feature exist according to claim 1, the step
The acquisition of rapid D, paddy gene expression cassette C6 further comprise:
The genomic DNA for extracting rice plant carries out complete C6 base by upstream and downstream primer of F2/R2 using genomic DNA as template
Because of the amplification of expression cassette, Sma I and Sau96I restriction enzyme site is introduced in upstream primer and downstream primer respectively in amplification procedure,
Its rice C6 gene expanded includes the promoter of its upstream and the terminator in downstream, while including all exon and include
Son.
7. the transgenic paddy rice sterile line based on C6 gene of any one of claim 1~6 the method breeding.
8. the breeding method of the transgenic paddy rice sterile line described in any one of claim 1~6 based on C6 gene rice not
Educating is application in breeding.
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Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102876711A (en) * | 2012-10-31 | 2013-01-16 | 湖南杂交水稻研究中心 | Cultivation method of rice engineering maintainer line and application thereof to breeding of rice genic male sterile line |
| CN102870670A (en) * | 2012-10-31 | 2013-01-16 | 湖南杂交水稻研究中心 | Universal type breeding method for rice engineering maintainer line, and application thereof in propagation of ordinary nucleic male sterility lines of rice |
| CN104837334A (en) * | 2013-05-23 | 2015-08-12 | 深圳市作物分子设计育种研究院 | Establishment of maintainer plant line and sterile line and use thereof |
| CN108148855A (en) * | 2017-12-31 | 2018-06-12 | 青岛袁策生物科技有限公司 | A kind of rice genetic engineering sterile line breeding method |
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2018
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Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102876711A (en) * | 2012-10-31 | 2013-01-16 | 湖南杂交水稻研究中心 | Cultivation method of rice engineering maintainer line and application thereof to breeding of rice genic male sterile line |
| CN102870670A (en) * | 2012-10-31 | 2013-01-16 | 湖南杂交水稻研究中心 | Universal type breeding method for rice engineering maintainer line, and application thereof in propagation of ordinary nucleic male sterility lines of rice |
| CN104837334A (en) * | 2013-05-23 | 2015-08-12 | 深圳市作物分子设计育种研究院 | Establishment of maintainer plant line and sterile line and use thereof |
| CN108148855A (en) * | 2017-12-31 | 2018-06-12 | 青岛袁策生物科技有限公司 | A kind of rice genetic engineering sterile line breeding method |
Non-Patent Citations (3)
| Title |
|---|
| DASHENG ZHANG ET AL.: "OsC6, Encoding a Lipid Transfer Protein, Is Required for Postmeiotic Anther Development In Rice", 《PLANT PHYSIOLOGY》 * |
| 张正斌编著: "《小麦遗传学》", 30 September 2001, 中国农业出版社 * |
| 麦景强等: "水稻雄性不育的分子机理", 《安徽农业科学》 * |
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