Method for creating phenotypic variation transgenic plant by using DNA demethylase gene
阅读说明：本技术 一种利用dna去甲基化酶基因创造表型变异转基因植物的方法 (Method for creating phenotypic variation transgenic plant by using DNA demethylase gene ) 是由 张冰玉 苏晓华 常英英 吴晓娟 于 2019-11-01 设计创作，主要内容包括：本发明属于植物育种领域,尤其是植物生物技术育种领域,具体涉及一种利用DNA去甲基化酶基因创造表型变异转基因植物的方法。本发明利用农杆菌介导法,将化学诱导启动子与拟南芥DNA去甲基化酶基因AtDME导入84K杨基因组中,获得转AtDME 84K杨植株,利用雌激素分别诱导转基因植株无菌离体叶片AtDME表达3h、12h和24h后,诱导叶片再生获得表型变异植株。该研究利用化学诱导启动子人工启动外源去甲基化酶基因表达,创造表型变异种质,丰富了植物育种资源。(The invention belongs to the field of plant breeding, in particular to the field of plant biotechnology breeding, and particularly relates to a method for creating phenotypic variation transgenic plants by using DNA demethylase genes. The invention utilizes an agrobacterium-mediated method to introduce a chemically induced promoter and an arabidopsis DNA demethylase gene AtDME into a 84K poplar genome to obtain an AtDME 84K poplar plant, and after estrogen is utilized to respectively induce sterile in vitro leaves of the transgenic plant to express the AtDME for 3h, 12h and 24h, the leaves are induced to regenerate to obtain a phenotype variation plant. The research utilizes a chemical induction promoter to artificially start the expression of exogenous demethylase genes, creates phenotypic variation germplasm and enriches plant breeding resources.)
1. A method of creating a phenotypically variant transgenic plant using the arabidopsis thaliana AtDME gene, comprising the steps of:
(1) obtaining an arabidopsis AtDME gene;
(2) constructing a chemical inducible plant expression vector containing a chemical inducible promoter and an arabidopsis AtDME gene;
(3) transforming host cells by adopting the plant expression vector constructed in the step (2);
(4) genetically transforming plant cells by using the host cells obtained in the step (3) to obtain AtDME gene-transformed plants;
(5) treating the transgenic plant with a chemical reagent to obtain a plant with AtDME gene transferred phenotypic variation;
the plant is a plant capable of genetic transformation, preferably poplar, more preferably 84K poplar.
2. The method according to claim 1, wherein said chemically inducible plant expression vector is the plant expression vector pER 8-DME.
3. The method according to claim 1 or 2, wherein the host cell is an agrobacterium, preferably agrobacterium GV 3101.
4. A method according to any one of claims 1 to 3, wherein step (4) is carried out by genetically transforming an excised leaf of the plant with the obtained host cell, inducing regeneration of adventitious shoots and obtaining the AtDME gene-transferred plant.
5. The method according to any one of claims 1 to 4, wherein step (5) is carried out by treating the excised leaves of the plant with 100 μ M17- β -estradiol for 3h, 12h or 24h to induce regeneration of adventitious shoots and obtain plants with phenotypic variation of AtDME gene.
6. The method of any one of claims 1-5, further comprising determining phenotypic variation in the phenotypically varied plant.
7. The method according to claim 6, wherein the measured indicators include plant height, leaf length and width, plant diameter, chlorophyll content in functional leaves, photosynthetic parameters, soluble sugar content in leaves, soluble protein content in leaves, POD activity and SOD activity.
8. A chemically inducible plant expression vector containing an Arabidopsis thaliana AtDME gene is a plant expression vector pER 8-DME.
9. The method for preparing the plant expression vector of claim 8, comprising the steps of:
(1) using pET30b-DME plasmid as template, and using AtDME gene full-length primer to amplify the full-length sequence of AtDME gene;
(2) connecting the amplified full-length AtDME gene to a cloning Vector pMDTM19-T Vector to obtain a pMD19-T-DME recombinant plasmid;
(3) respectively carrying out enzyme digestion on pMD19-T-DME and pER8-GFP, recovering a D3 fragment and a pER8 linear fragment in pMD19-T-DME, connecting to obtain an intermediate vector pER8-D3, and carrying out enzyme digestion on the pER8-D3 vector to obtain a linearized pER8-D3 fragment;
(4) and (3) amplifying D1-1 and D1-2 fragments by using pET30b-DME plasmid as a template, and connecting the amplified fragments with linearized pER8-D3 fragments to obtain a pER8-DME plant expression vector.
10. A host cell, preferably Agrobacterium, more preferably Agrobacterium GV3101, transformed with the plant expression vector of claim 8.
Technical Field
The invention belongs to the field of plant breeding, in particular to the field of plant biotechnology breeding, and particularly relates to a method for creating phenotypic variation transgenic plants by using DNA demethylase genes.
Background
The poplar belongs to the genus Populus (Populus L.) of the family Salicaceae (Salicae), and is a perennial woody tree. The poplar is not only an important afforestation greening tree species and an industrial material tree species, but also a mode tree species of perennial forest genetic engineering.
Since the development of poplar crossbreeding in the 40 th of the 20 th century, China breeds a batch of poplar varieties with excellent growth performance, and popularizes and plants the poplar varieties in various domestic cultivation areas, thereby obtaining good social and economic benefits. With the development of economy, the social demand on the improved varieties of the poplar is increased day by day, but the effect and the speed of the selective breeding of the improved varieties of the poplar are difficult to meet the demand of the development of the modern economic society due to the limitation of the shortage of breeding resources.
The breeding of poplar should first have breeding resources that contain sufficiently abundant genetic variation. As a material basis of breeding work, the abundance of breeding resources directly determines the efficiency of improved variety breeding. At present, besides deeply excavating the specific poplar germplasm resources in China and introducing foreign excellent gene resources in a large scale, the creation of new germplasm resources by using artificial methods (radiation mutagenesis technology, transgenic technology and the like) is another effective means for solving the shortage of poplar breeding resources.
DNA methylation is one of the important epigenetic changes, and changes in DNA methylation levels can cause changes in the epigenetic characteristics of a plant, and such epigenetic changes can be transmitted to progeny. Research shows that the methylation state of plant genome can be changed by methods such as hybridization, environmental stress, methylation inhibitor treatment, genetic transformation and the like, so that new heritable phenotypic variation can be obtained. Finally, plant improvement can be targeted by selection for such heritable phenotypic variations.
In arabidopsis, the DME gene (DEMETER) is one of its four base excision repair enzyme (BER) glycosylase homologues, specifically excising 5-methylcytosine (5mC) instead of the damaged base, thereby catalysing active DNA demethylation, which plays a key role in the active demethylation process of plants.
Disclosure of Invention
In order to solve the problem of insufficient germplasm resources in plant breeding, the invention provides a method for creating phenotypic variation transgenic plants by using a DNA demethylase gene, and a large number of new plant germplasm with changed phenotypes can be obtained by using the method.
To this end, one aspect of the present invention provides a method for creating a phenotypically variant transgenic plant using the arabidopsis thaliana AtDME gene, comprising the steps of:
1. obtaining an arabidopsis AtDME gene;
2. constructing a chemical inducible plant expression vector containing a chemical inducible promoter and an arabidopsis AtDME gene;
3. transforming host cells by adopting the plant expression vector constructed in the
4. adopting the host cell obtained in the
5. treating the transgenic plant with a chemical reagent to obtain a plant with AtDME gene transferred phenotypic variation;
the plant is a plant capable of genetic transformation, preferably poplar, more preferably 84K poplar.
In a further preferred embodiment of the invention, said chemically inducible plant expression vector is the plant expression vector pER 8-DME.
In a further preferred embodiment of the invention, said host cell is an Agrobacterium, preferably Agrobacterium GV 3101.
In a further preferred embodiment of the present invention,
In a further preferred embodiment of the invention,
In a further preferred embodiment of the invention, the phenotypic variation is determined in plants having phenotypic variation.
In a more preferred embodiment of the present invention, the measured indicators include plant height, leaf length and width, plant ground diameter, chlorophyll content of functional leaves, photosynthetic parameters, soluble sugar content of leaves, soluble protein content of leaves, POD activity and SOD activity.
In another aspect, the present invention provides a chemically inducible plant expression vector containing the arabidopsis thaliana AtDME gene, which is a plant expression vector pet 8-DME.
In another aspect, the present invention provides a method for preparing the plant expression vector, comprising the following steps:
1. using pET30b-DME plasmid as template, and using AtDME gene full-length primer to amplify the full-length sequence of AtDME gene;
2. connecting the amplified full-length AtDME gene to a cloning Vector pMDTM19-T Vector to obtain a pMD19-T-DME recombinant plasmid;
3. respectively carrying out enzyme digestion on pMD19-T-DME and pER8-GFP, recovering a D3 fragment and a pER8 linear fragment in pMD19-T-DME, connecting to obtain an intermediate vector pER8-D3, and carrying out enzyme digestion on the pER8-D3 vector to obtain a linearized pER8-D3 fragment;
4. and (3) amplifying D1-1 and D1-2 fragments by using pET30b-DME plasmid as a template, and connecting the amplified fragments with linearized pER8-D3 fragments to obtain a pER8-DME plant expression vector.
In yet another aspect, the invention provides a host cell transformed with the plant expression vector, preferably Agrobacterium, more preferably Agrobacterium GV 3101.
The analysis shows that the invention creates a genetic engineering method for obtaining plant variant germplasm by using demethylase gene, and the method can generate positive promotion effect on breeding and production of crops and forest trees in China.
Drawings
FIG. 1: plant expression vector pER8-AtDME vector construction scheme.
FIG. 2: AtDME gene 84K transgenic poplar hygromycin resistant bud (A) and resistant plant (B).
FIG. 3: and (3) carrying out PCR detection on the hygromycin gene of the AtDME gene 84K poplar resistant plant.
M: DL2000 Marker; 1: negative control; 2: a positive control; 3-8: AtDME transgenic resistant plants.
FIG. 4: and (3) carrying out PCR detection on the ATDME gene transferred 84K poplar resistant plant HD primer.
M: DL2000 Marker; 1: negative control; 2: a positive control; 3-8: AtDME transgenic resistant plants.
FIG. 5: the estradiol induction expression condition of the exogenous gene AtDME of the transgenic 84K poplar leaf disc.
FIG. 6: the growth character and the photosynthetic parameter change condition of the regenerated plant after the induction of the AtDME 84K poplar estradiol.
FIG. 7: the change condition of the biochemical indexes of the regenerated plants after the induction of AtDME 84K poplar estradiol.
Detailed Description
The present invention is further illustrated by the following examples, which are intended to be illustrative, but not limiting, of the present invention. It should be noted that, for those skilled in the art, without departing from the principle of the present invention, several improvements and modifications can be made to the present invention, and these improvements and modifications also fall into the protection scope of the present invention.