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Novel selective marker and application thereof in plant genetic transformation

A technology of selection marker and plant expression vector, applied in the field of safe selection marker and its application in obtaining transgenic plants, can solve the problem of inability to play a role, inability to distinguish transformed and non-transformed cells well, delaying adventitious bud differentiation, etc. problems, to achieve the effect of addressing security concerns

Inactive Publication Date: 2011-04-20
JILIN AGRICULTURAL UNIV
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Problems solved by technology

Antibiotics or herbicides can kill non-transgenic cells, but there are also some problems in resistance selection marker genes: (1) The use of antibiotics or herbicides often negatively affects the development and differentiation of transformed cells, and may delay indefinitely bud differentiation; (2) For insensitive varieties, these selection reagents cannot distinguish transformed and non-transformed cells well, so they cannot play their due role (Ebinuma et al., 1997); (3) Selection markers are due to It is often integrated into the plant genome together with the target gene, causing a series of adverse effects on transgenic plants
Most of the selectable marker genes are constitutively expressed, which consumes a large amount of plant bioenergy, and has a negative impact on the growth and development of transgenic plants; (4) from the perspective of health and safety, the expression products of resistance selectable marker genes can be used in food. Intestinal parasites that may be transferred to humans or animals to produce drug resistance, thereby reducing or losing the therapeutic effect of certain antibiotics (Zechendorf B.1994.12: 870-875)

Method used

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  • Novel selective marker and application thereof in plant genetic transformation
  • Novel selective marker and application thereof in plant genetic transformation
  • Novel selective marker and application thereof in plant genetic transformation

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0045] Embodiment 1 Cloning of safe selection marker promoter and gene (cloning promoter and gene full-length fragment by fusion PCR)

[0046] 1) Select genes from Solexa sequencing data

[0047] ①Plant material and treatment

[0048] The plant material is saline-alkali tolerant soybean HJ-1, which is hydroponically cultivated with Hoagland basal culture medium, and treated with 2% PEG stress when the soybean seedling grows the first true leaf. The roots and leaves of the drought-treated material and the control material were collected after 48 hours of stress treatment, and three samples were taken for each sample. After sampling, the samples were wrapped in new tinfoil paper and immediately stored in liquid nitrogen at -80°C;

[0049] ②Solexa sequencing and data analysis

[0050] Solexa sequencing was completed by BGI Shenzhen; the differentially expressed genes in roots and leaves were analyzed, and the gene GmP5CS co-expressed in roots and leaves was found. The study con...

Embodiment 2

[0079] Example 2 Construction of the plant expression vector pCAMBIA1301-DP-P5CS with a safe selection marker

[0080] After the vector pCAMBIA1301 and fusion fragment DP-P5CS were cut with Xho I and Sac I, the fusion fragment DP-P5CS was inserted into pCAMBIA1301 to obtain pCAMBIA1301-DP-P5CS. The specific method is:

[0081] 1) Digest vector pCAMBIA1301 with Xho I and Sac I, and detect by 1% agarose gel electrophoresis (see Figure 4 , where the left side is DNA marker 10000, the arrow indicates the fragment size of the corresponding band), and the 9703bp vector fragment was recovered;

[0082] 2) Ligate the vector fragment with the promoter and gene fragment obtained by digestion, transform Escherichia coli, smear it on a 50 mg / L kanamycin LB plate, then select positive clones for PCR detection, and perform sequencing if the detection is correct, and Extract the corresponding positive clone plasmid and name it pCAMBIA1301-DP-P5CS;

[0083] 3) Digest the vector pCAMBIA130...

Embodiment 3

[0085] Example 3 Agrobacterium-mediated transformation of soybean cotyledon nodes and screening and detection of safe selection marker transgenic soybeans

[0086] 1) Agrobacterium co-transformation:

[0087] Using CaCl 2 Transform the plasmid pCAMBIA1301-DP-P5CS into Agrobacterium EHA105, and transform soybean Changnong 13 by Agrobacterium-mediated transformation of cotyledonary nodes;

[0088] 2) Screening of transgenic soybeans with safe selection markers:

[0089] When soybean seedlings grow the 2nd to 3rd pairs of true leaves, carry out stress treatment with 4% PEG for 5 days, the growth condition of transgenic plants is good, and the leaves of non-transgenic plants become yellow and wilted due to dehydration, so that soybean transformants can be screened, such as Figure 7 As shown, A is a transformed plant, and B is a non-transformed plant, which proves that the gene has the ability to resist drought so as to enhance the drought tolerance of the transgenic plant;

[...

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Abstract

The invention discloses a novel selective marker and an application thereof in plant genetic transformation. A PCR method is utilized to clone a P5CS gene and a promoter DP, and a safe selective marker DP-P5CS is obtained; a plant expression carrier containing the safe selective marker DP-P5CS is utilized to transform a target gene into a plant, and a drought stress screening transgenosis method is adopted, thus being applicable to genetic transformation of most plants; and safety and high efficiency are realized, the problem that people is worried about the safety of selectable marker gene is solved, and the obtained transgenic plant not only plays the function of the target gene but also has drought resistance, thus having great significance and attractive prospect and being a good way for producing selectable marker transgenic plants.

Description

technical field [0001] The invention belongs to the field of biotechnology, specifically a safety selection marker and its application in obtaining transgenic plants. Background technique [0002] At present, the efficiency of transgenic technology is low, and the frequency of exogenous gene integration into the genome of the target organism is usually a few thousandths to a few millionths. Therefore, the introduction of the target gene usually requires a series of easily identifiable marker genes for screening transformants. This marker gene is also called "selectable marker" or "selectable marker". Currently, the widely used selectable marker genes are often some dominant genes. Encodes antibiotic or herbicide resistance genes (Yoder and Goldsbrough, 1994). Antibiotics or herbicides can kill non-transgenic cells, but there are also some problems in resistance selection marker genes: (1) The use of antibiotics or herbicides often negatively affects the development and diff...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/11C12N15/10C12N15/82A01H5/00
Inventor 李海燕范秀朵陈静王南
Owner JILIN AGRICULTURAL UNIV
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