CN108949814A - A kind of breeding method of the transgenic paddy rice sterile line based on TDR gene - Google Patents
A kind of breeding method of the transgenic paddy rice sterile line based on TDR gene Download PDFInfo
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- CN108949814A CN108949814A CN201810729442.8A CN201810729442A CN108949814A CN 108949814 A CN108949814 A CN 108949814A CN 201810729442 A CN201810729442 A CN 201810729442A CN 108949814 A CN108949814 A CN 108949814A
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- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
- C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
- C12N15/8241—Phenotypically and genetically modified plants via recombinant DNA technology
- 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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- C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
- C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
- C12N15/8216—Methods for controlling, regulating or enhancing expression of transgenes in plant cells
- C12N15/8222—Developmentally regulated expression systems, tissue, organ specific, temporal or spatial regulation
- C12N15/823—Reproductive tissue-specific promoters
- C12N15/8231—Male-specific, e.g. anther, tapetum, pollen
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Abstract
This application discloses a kind of breeding methods of transgenic paddy rice sterile line based on TDR gene, comprising the following steps: the acquisition of A, TagRFP expression casette；B, the amplification of Wheat Pollen lethal gene Ki；C, the acquisition of rice TDR box: the amplification of D, pollen specific promoter Pg47；E, the connection of each gene expression element.The connection of the step E, each gene expression element, further comprise: TagRFP gene expression element is connected on pCAMBIA1390 carrier by the first step；Complete rice TDR gene is introduced into the first step on the pCAMBIA1390 carrier for having been incorporated into TagRFP gene expression element 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 TDR gene
Breeding method.The present invention is chain by rice Male sterile gene TDR and Wheat Pollen lethal gene Ki, while with orange glimmering
Photoprotein TagRFP gene carries out genetic modification as reporter gene, to rice TDR 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.
In order to solve the above technical problems, the present invention provides a kind of trainings of transgenic paddy rice sterile line based on TDR gene
Educate method, comprising the following steps:
A, the acquisition of TagRFP expression casette；
B, the amplification of Wheat Pollen lethal gene Ki；
C, the acquisition of rice TDR box；
D, the amplification of pollen specific promoter Pg47；
E, the connection of each gene expression element.
The acquisition of the step C, rice TDR box further includes:
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 TDR expression casette introduces Sma I and Nael digestion position in amplification procedure in upstream primer and downstream primer respectively
Point, while DraI restriction enzyme site is added at the rear Nael, the rice TDR gene of amplification includes promoter and the downstream of its upstream
Terminator, while including all exon and introne.
The acquisition of step A, the TagRFP expression casette further comprises:
It using the plasmid containing TagRFP gene expressed intact box as template, is expanded, is expanded by upstream and downstream primer of F1/RI
Introduce AauI and Kpn I site in increasing process in upstream and downstream primer respectively.
The amplification of the step B, 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 F3/R3 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 amplification of the step D, pollen specific promoter Pg47 further comprise:
Using oryza sativa genomic dna as template, pollen specific is carried out as upstream and downstream primer using F4/R4 and starts Pg47 amplification,
Introduce Kpn I and Sma I restriction enzyme site respectively in upstream and downstream primer.
The connection of the step E, each gene expression element further comprise:
TagRFP gene expression element is connected on pCAMBIA1300 carrier by the first step；
Complete rice TDR gene is introduced into the first step and has been incorporated into TagRFP gene expression element by second step
On pCAMBIA1390 carrier；
Wheat Pollen lethal gene Ki is connected into and has been connected with TagRFP gene expression element and rice TDR by third step
On the pCAMBIA1390 binary vector of gene；
Paddy pollen specificity starting Pg47 is connected to first three step and has connected upper TagRFP gene expression member by the 4th step
On the complex carrier of part, rice TDR gene and Wheat Pollen lethal gene Ki.
In order to solve the above technical problems, invention further provides a kind of utilization sides of the common Genetic Sterility TDR mutant of rice
Method, using the building of rice TDR expression casette such as the rice sterile line of any one of aforementioned method preparation.
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 TDR and Wheat Pollen lethal gene Ki
It is chain, while using orange fluorescent protein TagRFP gene as reporter gene, genetic modification is carried out to rice TDR mutant, is obtained
Obtain F1 generation heterozygote.F1 generation heterozygote follows Mendel's law of segregation during self-fertility, and the offspring of generation is existing to be protected
The heterozygote of three linked genes is held, and whether there is or not the sterile lines of fertility.
Detailed description of the invention
Fig. 1: TagRFP gene expressed intact box PCR amplification detected through gel electrophoresis figure；
Fig. 2: rice TDR expression casette PCR amplification detected through gel electrophoresis figure；
Fig. 3: Wheat Pollen lethal gene Ki amplification detected through gel electrophoresis figure；
Fig. 4: pollen specific promoter Pg47 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 TDR and Wheat Pollen lethal gene Ki is chain, while with orange glimmering
Photoprotein TagRFP gene carries out genetic modification as reporter gene, to rice TDR 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 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 TDR gene
Educate method, comprising the following steps:
A, the acquisition of TagRFP expression casette；
B, the amplification of Wheat Pollen lethal gene Ki；
C, the acquisition of rice TDR box；
D, the amplification of pollen specific promoter Pg47；
E, the connection of each gene expression element.
The acquisition of the step C, rice TDR box further includes:
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 TDR expression casette introduces Sma I and Nael digestion position in amplification procedure in upstream primer and downstream primer respectively
Point, while DraI restriction enzyme site is added at the rear Nael, the rice TDR gene of amplification includes promoter and the downstream of its upstream
Terminator, while including all exon and introne.
The acquisition of step A, the TagRFP expression casette further comprises:
It using the plasmid containing TagRFP gene expressed intact box as template, is expanded, is expanded by upstream and downstream primer of F1/R1
Introduce AauI and Kpn I site in increasing process in upstream and downstream primer respectively.
The amplification of the step B, 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 F3/R3 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 amplification of the step D, pollen specific promoter Pg47 further comprise:
Using oryza sativa genomic dna as template, pollen specific is carried out as upstream and downstream primer using F4/R4 and starts Pg47 amplification,
Introduce Kpn I and Sma I restriction enzyme site respectively in upstream and downstream primer.
The connection of the step E, each gene expression element further comprise:
TagRFP gene expression element is connected on pCAMBIA1300 carrier by the first step；
Complete rice TDR gene is introduced into the first step and has been incorporated into TagRFP gene expression element by second step
On pCAMBIA1390 carrier；
Wheat Pollen lethal gene Ki is connected into and has been connected with TagRFP gene expression element and rice TDR by third step
On the pCAMBIA1390 binary vector of gene；
Paddy pollen specificity starting Pg47 is connected to first three step and has connected upper TagRFP gene expression member by the 4th step
On the complex carrier of part, rice TDR gene and Wheat Pollen lethal gene Ki.
In order to solve the above technical problems, invention further provides a kind of utilization sides of the common Genetic Sterility TDR mutant of rice
Method, using the building of rice TDR expression casette such as the rice sterile line of any one of aforementioned method preparation.
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.
1, the acquisition of rice TDR expression casette
The genomic DNA for extracting rice force fortune No. 7 plant of round-grained rice draws using genomic DNA as template by upstream and downstream of F2/R2
Object carries out the amplification of complete TDR expression casette, introduced in upstream primer and downstream primer respectively in amplification procedure Sma I and
Nael restriction enzyme site, while DraI restriction enzyme site is added at the rear Nael, the rice TDR gene of amplification includes opening for its upstream
The terminator of mover and downstream, while including all exon and introne.
2, the acquisition of TagRFP expression casette
The plasmid containing TagRFP gene expressed intact box saved using laboratory draws as template by upstream and downstream of F1/R1
Object is expanded, and introduces AauI and Kpn I site in amplification procedure in upstream and downstream primer respectively.Expression cassette total length is
696bp is detected by PCR amplification and agarose gel electrophoresis, tentatively obtains TagRFP gene expressed intact box.
3, the amplification of Wheat Pollen lethal gene Ki
Using oryza sativa genomic dna as template, the expansion of Wheat Pollen lethal gene Ki is carried out using F3/R3 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.
4, the amplification of pollen specific promoter Pg47
Using oryza sativa genomic dna as template, pollen specific is carried out as upstream and downstream primer using F4/R4 and starts Pg47 amplification,
Introduce Kpn I and Sma I restriction enzyme site respectively in upstream and downstream primer.
5, the connection of each gene expression element
The first step utilizes AauI the and Kpn1 restriction enzyme site and pCAMBIA1390 binary vector introduced in upstream and downstream primer
Upper AauI and Kpn1 restriction enzyme site, TagRFP gene expression element is connected on pCAMBIA1390 carrier.Second step, connection
TDR gene utilizes the Smal on Smal the and Nael restriction enzyme site and pCAMBIA1390 binary vector introduced in upstream and downstream primer
With Nael restriction enzyme site, complete rice TDR gene is introduced into the first step and has been incorporated into TagRFP gene expression element
On pCAMBIA1390 carrier, while DraI restriction enzyme site is added at the rear Nael.Third step is all introduced using upstream and downstream
Wheat Pollen lethal gene Ki is connected by single endonuclease digestion site and has been connected with TagRFP gene expression member by DraI restriction enzyme site
On the pCAMBIA1390 binary vector of part and rice TDR gene.Final step, using in upstream and downstream primer introduce Kpn I and
Kpn I and Sma I restriction enzyme site on Sma I restriction enzyme site and pCAMBIA1390 binary vector, paddy pollen specificity is opened
Dynamic Pg47 is connected to first three step and has connected TagRFP gene expression element, rice TDR gene and Wheat Pollen lethal gene
On the complex carrier of Ki.
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 TDR gene, which comprises the following steps:
A, the acquisition of TagRFP expression casette；
B, the amplification of Wheat Pollen lethal gene Ki；
C, the acquisition of rice TDR box；
D, the amplification of Wheat Pollen specificity promoter Pg47；
E, the connection of each gene expression element.
2. a kind of breeding method of the transgenic paddy rice sterile line based on TDR gene, feature exist according to claim 1
In: the acquisition of the step C, rice TDR box further comprise:
The genomic DNA for extracting rice plant carries out complete TDR by upstream and downstream primer of F2/R2 using genomic DNA as template
The amplification of expression casette introduces Sma I and Nael restriction enzyme site in amplification procedure in upstream primer and downstream primer respectively,
DraI restriction enzyme site is added at the rear Nael simultaneously, the rice TDR gene of amplification includes promoter and the downstream of its upstream
Terminator, while including all exon and introne.
3. a kind of breeding method of the transgenic paddy rice sterile line based on TDR gene, feature exist according to claim 1
In: the acquisition of step A, the TagRFP expression casette further comprises:
Using the plasmid containing TagRFP gene expressed intact box as template, is expanded, expanded by upstream and downstream primer of F1/R1
Introduce AauI and Kpn I site in journey in upstream and downstream primer respectively.
4. a kind of breeding method of the transgenic paddy rice sterile line based on TDR gene, feature exist according to claim 1
In: the amplification of the step B, Wheat Pollen lethal gene Ki further comprise:
Using oryza sativa genomic dna as template, the amplification of Wheat Pollen lethal gene Ki is carried out using F3/R3 as upstream and downstream primer,
DraI restriction enzyme site is introduced in upstream and downstream primer；Its lethal gene Ki expanded includes all exon and introne.
5. a kind of breeding method of the transgenic paddy rice sterile line based on TDR gene, feature exist according to claim 1
In: the amplification of the step D, pollen specific promoter Pg47 further comprise:
Using oryza sativa genomic dna as template, pollen specific is carried out as upstream and downstream primer using F4/R4 and starts Pg47 amplification, upper
Kpn I and Sma I restriction enzyme site is introduced in downstream primer respectively.
6. a kind of breeding method of the transgenic paddy rice sterile line based on TDR gene, feature exist according to claim 1
In: the connection of the step E, each gene expression element further comprise:
TagRFP gene expression element is connected on pCAMBIA1300 carrier by the first step；
Complete rice TDR gene is introduced into the first step and has been incorporated into TagRFP gene expression element by second step
On pCAMBIA1390 carrier；
Wheat Pollen lethal gene Ki is connected into and has been connected with TagRFP gene expression element and rice TDR gene by third step
PCAMBIA1390 binary vector on；
4th step, by Wheat Pollen specificity starting Pg47 be connected to first three step connected upper TagRFP gene expression element,
On the complex carrier of rice TDR gene and Wheat Pollen lethal gene Ki.
7. a kind of breeding method of the transgenic paddy rice sterile line based on TDR gene, which is characterized in that apply rice TDR gene
The rice sterile line of expression cassette building such as any one of claim 1~6 the method preparation.
8. a kind of such as application of the method in Genetic and breeding in rice of any one of claim 1~6.
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Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| 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 |
| US20160255782A1 (en) * | 2013-09-16 | 2016-09-08 | Xingwang Investment Co., Ltd | Use of genic male sterility gene and mutation thereof in hybridization |
| 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
- 2018-07-04 CN CN201810729442.8A patent/CN108949814A/en active Pending
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| 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 |
| US20160255782A1 (en) * | 2013-09-16 | 2016-09-08 | Xingwang Investment Co., Ltd | Use of genic male sterility gene and mutation thereof in hybridization |
| 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 |
|---|
| 王东芳: "内置双重安全控制机制的水稻生物反应器表达药物蛋白质", 《中国博士学位论文全文数据库农业科技辑》 * |
| 王鹏等: "小麦花粉致死基因Ki的SSR标记分析", 《中国农业通报》 * |
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