CN108949777A - A kind of breeding method of the transgenic paddy rice sterile line based on MIL1 gene - Google Patents
A kind of breeding method of the transgenic paddy rice sterile line based on MIL1 gene Download PDFInfo
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- CN108949777A CN108949777A CN201810728704.9A CN201810728704A CN108949777A CN 108949777 A CN108949777 A CN 108949777A CN 201810728704 A CN201810728704 A CN 201810728704A CN 108949777 A CN108949777 A CN 108949777A
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- 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
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Abstract
This application discloses a kind of breeding methods of transgenic paddy rice sterile line based on MIL1 gene, comprising the following steps: the acquisition of A, rice MIL1 expression casette；B, the acquisition of mCherry expression casette；C, the amplification of Wheat Pollen specificity promoter Pg47；D, the amplification of Wheat Pollen lethal gene Ki；E, the connection of each gene expression element.The connection of the step E, each gene expression element further comprise: complete rice MIL1 gene is connected on pCAMBIA1390 carrier by the first step；MCherry gene expression element is introduced into the first step on the pCAMBIA1390 carrier for having been incorporated into MIL1 gene by second 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 present invention relates to molecular biology nucleic acid chemistry field, in particular to a kind of rice genetic engineering sterile line
Preparation method.
Background technique
Rice is the staple food of the population of nearly half in the world, in addition to edible, can also make wine, refine sugar and as industry
Raw material, demand of the mankind to rice are very big.Production practices for many years show hybrid rice generally than conventional Rice volume increase 20%
More than, 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.
Summary of the invention
Technical problem to be solved by the present invention lies in provide a kind of transgenic paddy rice sterile line based on MIL1 gene
Breeding method.The present invention is chain by rice Male sterile gene MIL1 and Wheat Pollen lethal gene Ki, while with red
Fluorescin mCherry gene carries out genetic modification as reporter gene, to rice MIL1 mutant, obtains F1 generation heterozygote.
F1 generation heterozygote follows Mendel's law of segregation during self-fertility, and the offspring of generation is existing to be able to maintain three linked genes
Heterozygote, but whether there is or not the sterile lines of fertility.
In order to solve the above technical problems, the present invention provides a kind of trainings of transgenic paddy rice sterile line based on MIL1 gene
Educate method, comprising the following steps:
A, the acquisition of rice MIL1 expression casette；
B, the acquisition of mCherry expression casette；
C, the amplification of Wheat Pollen specificity promoter Pg47；
D, the amplification of Wheat Pollen lethal gene Ki；
E, the connection of each gene expression element.
The connection of the step E, each gene expression element, comprising:
Complete rice MIL1 gene is connected on pCAMBIA1390 carrier by the first step；
MCherry gene expression element is introduced into the first step pCAMBIA1390 for having been incorporated into MIL1 gene by second step
On carrier；
Wheat Pollen specificity starting Pg47 is connected into and has been connected with rice MIL1 gene and mCherry base by third step
Because on the pCAMBIA1390 binary vector of Expression element；
Wheat Pollen lethal gene Ki is connected to first three step and has connected upper rice MIL1 gene, mCherry by the 4th step
On the complex carrier of gene expression element and Wheat Pollen specificity starting Pg47.
The connection of the step E, each gene expression element further comprise:
The first step utilizes SmaI the and AceI restriction enzyme site and pCAMBIA1390 binary vector introduced in upstream and downstream primer
Upper SmaI and AceI restriction enzyme site, MIL1 gene is connected on pCAMBIA1390 carrier.
The connection of the step E, each gene expression element further comprise::
Second step connects mCherry expression casette, utilizes Msel the and KpnI restriction enzyme site introduced in upstream and downstream primer
With Msel the and KpnI restriction enzyme site on pCAMBIA1390 binary vector, complete mCherry gene expression element is introduced the
It has been connected in one step on the pCAMBIA1390 carrier of MIL1 gene.
The connection of the step E, each gene expression element further comprise::
Third step utilizes introducing Kpn I and Sma I restriction enzyme site and pCAMBIA1390 binary vector in upstream and downstream primer
On Kpn I and Sma I restriction enzyme site, Wheat Pollen specificity promoter Pg47 is connected into be connected with MIL1 gene and
On mCherry gene expression element pCAMBIA1390 binary vector.
The connection of the step E, each gene expression element further comprise::
Wheat Pollen lethal gene Ki is passed through single endonuclease digestion by the 4th step, the DraI restriction enzyme site all introduced using upstream and downstream
Site is connected to first three step and has connected MIL1 gene, mCherry gene expression element and Wheat Pollen specificity promoter
On the complex carrier of Pg47.
In order to solve the above technical problems, the present invention separately provides a kind of transgenic paddy rice sterile line based on MIL1 gene
Breeding method, which is characterized in that as described in aforementioned any one prepared by breeding method using the building of rice MIL1 expression casette
Rice sterile line.
In order to solve the above technical problems, the present invention separately provide it is a kind of if any one of aforementioned method is in Genetic and breeding in rice
Application.
Beneficial effect of the present invention includes: of the invention by rice Male sterile gene MIL1 and Wheat Pollen lethal gene Ki
It is chain, while using red fluorescent protein mCherry gene as reporter gene, genetic modification is carried out to rice MIL1 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: rice MIL1 expression casette PCR amplification detected through gel electrophoresis figure；
Fig. 2: mCherry gene expressed intact box PCR amplification detected through gel electrophoresis figure；
Fig. 3: Wheat Pollen specificity promoter Pg47 amplification detected through gel electrophoresis figure；
Fig. 4: Wheat Pollen lethal gene Ki 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 present invention is described in detail below with reference to embodiment.To keep the objectives, technical solutions, and advantages of the present invention clearer, bright
Really, the present invention is described in more detail as follows in conjunction with drawings and embodiments, but the invention is not limited to these embodiments.
The present invention is that rice Male sterile gene MIL1 and Wheat Pollen lethal gene Ki is chain, while with red glimmering
Photoprotein mCherry gene carries out genetic modification as reporter gene, to rice MIL1 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.
In order to solve the above technical problems, the present invention provides a kind of trainings of transgenic paddy rice sterile line based on MIL1 gene
Educate method, comprising the following steps:
A, the acquisition of rice MIL1 expression casette；
B, the acquisition of mCherry expression casette；
C, the amplification of Wheat Pollen specificity promoter Pg47；
D, the amplification of Wheat Pollen lethal gene Ki；
The acquisition of the step A, rice MIL1 box further includes:
The genomic DNA for extracting rice plant carries out complete using genomic DNA as template using F1/RI as upstream and downstream primer
The amplification of MIL1 expression casette introduces SmaI and AceI digestion position in amplification procedure in upstream primer and downstream primer respectively
The rice MIL1 gene of point, amplification includes the promoter of its upstream and the terminator in downstream, while including all exons
And introne.
The acquisition of step B, the mCherry expression casette further comprises:
Using the plasmid containing mCherry gene expressed intact box as template, expanded by upstream and downstream primer of F2/R2,
Introduce the site Msel and KpnI in amplification procedure in upstream and downstream primer respectively, while before the site KpnI that downstream primer introduces
DraI restriction enzyme site is added in face.
The amplification of the step C, Wheat Pollen specificity promoter Pg47 further comprise:
Using oryza sativa genomic dna as template, pollen specific is carried out as upstream and downstream primer using F3/R3 and starts Pg47 amplification,
Introduce Kpn I and Sma I restriction enzyme site respectively in upstream and downstream primer.
The amplification of the step D, Wheat Pollen lethal gene Ki further comprise:
Using oryza sativa genomic dna as template, the expansion of Wheat Pollen lethal gene Ki is carried out using F4/R4 as upstream and downstream primer
Increase, DraI restriction enzyme site is introduced in upstream and downstream primer；Its lethal gene Ki expanded includes all exon and introne.
The connection of the step E, each gene expression element, comprising:
Complete rice MIL1 gene is connected on pCAMBIA1390 carrier by the first step；
MCherry gene expression element is introduced into the first step pCAMBIA1390 for having been incorporated into MIL1 gene by second step
On carrier；
Wheat Pollen specificity starting Pg47 is connected into and has been connected with rice MIL1 gene and mCherry base by third step
Because on the pCAMBIA1390 binary vector of Expression element；
Wheat Pollen lethal gene Ki is connected to first three step and has connected upper rice MIL1 gene, mCherry by the 4th step
On the complex carrier of gene expression element and Wheat Pollen specificity starting Pg47.In order to solve the above technical problems, the present invention is again
A kind of utilization method of common Genetic Sterility MIL1 mutant of rice is provided, it is such as aforementioned using the building of rice MIL1 expression casette
The rice sterile line of any one method preparation.
In order to solve the above technical problems, the present invention separately provides a kind of transgenic paddy rice sterile line based on MIL1 gene
Breeding method, which is characterized in that as described in aforementioned any one prepared by breeding method using the building of rice MIL1 expression casette
Rice sterile line.
In order to solve the above technical problems, the present invention separately provide it is a kind of if any one of aforementioned method is in Genetic and breeding in rice
Application.
The connection of each gene expression element
The first step utilizes SmaI the and AceI restriction enzyme site and pCAMBIA1390 binary vector introduced in upstream and downstream primer
Upper SmaI and AceI restriction enzyme site, MIL1 gene is connected on pCAMBIA1390 carrier.Second step connects mCherry gene
Expression cassette utilizes the Msel on Msel the and KpnI restriction enzyme site and pCAMBIA1390 binary vector introduced in upstream and downstream primer
With KpnI restriction enzyme site, complete mCherry gene expression element is introduced into the first step and has been connected with MIL1 gene
On pCAMBIA1390 carrier.Third step, using in upstream and downstream primer introduce Kpn I and Sma I restriction enzyme site and
Kpn I and Sma I restriction enzyme site on pCAMBIA1390 binary vector, Wheat Pollen specificity promoter Pg47 is connected into
It has been connected on MIL1 gene and mCherry gene expression element pCAMBIA1390 binary vector.Final step, using upper and lower
The DraI restriction enzyme site all introduced is swum, Wheat Pollen lethal gene Ki is connected to first three step by single endonuclease digestion site and has been connected
On the complex carrier of MIL1 gene, mCherry gene expression element and Wheat Pollen specificity promoter Pg47.
Table 1: each gene magnification to primer, the restriction enzyme site that adds in primer
The above is only several embodiments of the present invention, not any type of limitation is done to the present invention, although this hair
It is bright to be disclosed as above with preferred embodiment, however be not intended to limit the invention, any person skilled in the art, it is not taking off
In the range of technical solution of the present invention, a little variation or modification are made using the technology contents of the disclosure above and is equal to
Case study on implementation is imitated, is belonged in technical solution of the present invention protection scope.
Claims (8)
1. a kind of breeding method of the transgenic paddy rice sterile line based on MIL1 gene, which comprises the following steps:
A, the acquisition of rice MIL1 expression casette；
B, the acquisition of mCherry expression casette；
C, the amplification of Wheat Pollen specificity promoter Pg47；
D, the amplification of Wheat Pollen lethal gene Ki；
E, the connection of each gene expression element.
2. a kind of breeding method of the transgenic paddy rice sterile line based on MIL1 gene, feature exist according to claim 1
In: the connection of the step E, each gene expression element, comprising:
Complete rice MIL1 gene is connected on pCAMBIA1390 carrier by the first step；
MCherry gene expression element is introduced into the first step pCAMBIA1390 carrier for having been incorporated into MIL1 gene by second step
On；
Wheat Pollen specificity starting Pg47 is connected into and has been connected with rice MIL1 gene and mCherry gene table by third step
Up on the pCAMBIA1390 binary vector of element；
Wheat Pollen lethal gene Ki is connected to first three step and has connected upper rice MIL1 gene, mCherry gene by the 4th step
On the complex carrier of Expression element and Wheat Pollen specificity starting Pg47.
3. a kind of breeding method of the transgenic paddy rice sterile line based on MIL1 gene, feature exist according to claim 1
In: the connection of the step E, each gene expression element further comprise:
The first step, using on SmaI the and AceI restriction enzyme site and pCAMBIA1390 binary vector introduced in upstream and downstream primer
MIL1 gene is connected on pCAMBIA1390 carrier by SmaI and AceI restriction enzyme site.
4. a kind of breeding method of the transgenic paddy rice sterile line based on MIL1 gene, feature exist according to claim 1
In: the connection of the step E, each gene expression element further comprise::
Second step connects mCherry expression casette, using Msel the and KpnI restriction enzyme site that is introduced in upstream and downstream primer and
Complete mCherry gene expression element is introduced first by Msel the and KpnI restriction enzyme site on pCAMBIA1390 binary vector
It has been connected in step on the pCAMBIA1390 carrier of MIL1 gene.
5. a kind of breeding method of the transgenic paddy rice sterile line based on MIL1 gene, feature exist according to claim 1
In: the connection of the step E, each gene expression element further comprise::
Third step introduces on Kpn I and Sma I restriction enzyme site and pCAMBIA1390 binary vector using in upstream and downstream primer
Kpn I and Sma I restriction enzyme site, Wheat Pollen specificity promoter Pg47 is connected into be connected with MIL1 gene and
On mCherry gene expression element pCAMBIA1390 binary vector.
6. a kind of breeding method of the transgenic paddy rice sterile line based on MIL1 gene, feature exist according to claim 1
In: the connection of the step E, each gene expression element further comprise::
Wheat Pollen lethal gene Ki is passed through single endonuclease digestion site by the 4th step, the DraI restriction enzyme site all introduced using upstream and downstream
It is connected to first three step and has connected MIL1 gene, mCherry gene expression element and Wheat Pollen specificity promoter Pg47
On complex carrier.
7. a kind of breeding method of the transgenic paddy rice sterile line based on MIL1 gene, which is characterized in that apply rice MIL1 base
The rice sterile line prepared by expression cassette building breeding method as described in any one of claim 1~6.
8. a kind of application of breeding method as described in any one of claim 1~6 in Genetic and breeding in rice.
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| CN201810728704.9A CN108949777A (en) | 2018-07-04 | 2018-07-04 | A kind of breeding method of the transgenic paddy rice sterile line based on MIL1 gene |
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Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| 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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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| 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 (2)
| Title |
|---|
| LILAN HONG等: "Somatic and Reproductive Cell Development in Rice Anther Is Regulated by a Putative Glutaredoxin", 《THE PLANT CELL》 * |
| 王鹏等: "小麦花粉致死基因Ki的SSR标记分析", 《中国农学通报》 * |
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