Recombinase gene, binary expression vector, construction method for recombinase gene and binary expression vector, and application of binary expression vector

A binary expression vector, recombinase technology, applied in application, recombinant DNA technology, botanical equipment and methods, etc., can solve the problems of changing nutrients, affecting the acquisition of resistant transgenic plants, low activity, etc.

Inactive Publication Date: 2012-05-09
SOUTHWEST UNIV
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Problems solved by technology

The inventors found in the research of heat-shock deletion marker genes using the above-mentioned heat-shock promoters: (1) Using the Cre / LxoP recombination system of heat-shock promoters such as HSP18.2 and HSP70m, deletion selection in Arabidopsis and tobacco The effect of the marker gene is good, but it takes nearly 1 month to process, and the activity is low in tomato, which requires longer heat shock deletion treatment
(2) The temperature environment control requirements in the tissue culture room are relatively strict. It is necessary to prevent power outages in high temperature seasons, causing the temperature in the culture room to reach the activation temperature of the heat shock promoter, thereby activating the Cre / LxoP recombination system integrated into the plant genome, thereby affecting Obtaining of resistant transgenic plants
(3) When using a heat shock temperature of 30-37°C to delete the selection marker gene of the resistant or visible transgenic plants, the medium is easily evaporated and the nutrient content is changed, which is unfavorable to the growth and differentiation of plants such as cucumbers and tomatoes

Method used

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  • Recombinase gene, binary expression vector, construction method for recombinase gene and binary expression vector, and application of binary expression vector
  • Recombinase gene, binary expression vector, construction method for recombinase gene and binary expression vector, and application of binary expression vector
  • Recombinase gene, binary expression vector, construction method for recombinase gene and binary expression vector, and application of binary expression vector

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Effect test

Embodiment 1

[0091] Example 1: Preparation of recombinase ntCre gene, construction of T-DNA of plant gene binary expression vector pVCT2221, transformation into Agrobacterium tumefaciens EHA105, transformation of plants and obtaining transgenic plants (tobacco) and application effects

[0092] 1. Preparation method of recombinase ntCre gene with nuclear localization signal coding sequence

[0093] A source of 1040 bp size was PCR amplified from the genomic DNA of Escherichia coli strain BM25.8 (Clontech, USA) using primers SEQ ID.4. and primers SEQ ID.NO.5, PrimStar HS DNA polymerase (TaKaRa, Japan) The Cre gene from P1 phage (GenBank accession X03453), the reaction conditions are: pre-denaturation at 95°C for 2 min, denaturation at 95°C for 10 sec, annealing at 50°C for 20 sec, extension at 72°C for 1 min 10 sec, 3 cycles, denaturation at 95°C 10 sec, annealing at 60°C for 20 sec, extension at 72°C for 1 min and 10 sec, 27 cycles, and finally extension at 72°C for 10 min. After adding th...

Embodiment 2

[0101] Example 2: Construction method of T-DNA of plant binary expression vector pVCT2224, transformation into Agrobacterium tumefaciens EHA105, transformation of tobacco and obtaining transgenic tobacco and application effect

[0102] 1. Construction method of T-DNA of plant binary expression vector pVCT2224 ( figure 2 , Figure 11 )

[0103] The vector pVCT2231 was digested with Sal I, filled with Klenow Fragment enzyme, passed through a DNA recovery column to remove the enzyme, then digested with Sac I, the 8779 bp fragment was recovered by electrophoresis, and the 1456 bp fragment was discarded. The vector pVCT2238 was digested with BstEII, filled with Klenow Fragment enzyme, passed through a DNA recovery column to remove the enzyme, and then digested with Sac I to recover the 3779 bp fragment and discard the 8233 bp fragment. The recovered fragments were purified and ligated with T4 DNA ligase, transformed into Escherichia coli XL1-Blue competent cells to obtain Kan-re...

Embodiment 3

[0108] Example 3: Functional verification of the RD29A promoter after transformation of tobacco with the binary expression vector of the present invention.

[0109] The vector containing RD29A::GUS reporter gene was mediated by Agrobacterium EHA105 to transgenic tobacco, and kanamycin-resistant plants integrated with RD29A::GUS gene were obtained. The transgenic and non-transgenic plants were treated at room temperature and low temperature, and the chemical staining analysis of GUS active tissue was carried out. The results showed that the leaves of a single transgenic plant that had not been treated with low temperature were fully decolorized with 70% ethanol after X-Gluc staining, and only a very small amount was observed. Few or no blue spots appear, indicating that the RD29A promoter does not have the function of promoting the expression of downstream genes at room temperature. The transgenic individual plants treated overnight at a low temperature of about 4°C were staine...

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Abstract

The invention provides a recombinase which is an ntCre gene and has a sequence as represented by SEQ ID No: 1, and a binary expression vector containing the recombinase; T-DNA structure of the binary expression vector contains an RD29A:: ntCre:: Tnos gene and a LoxP sequence, wherein RD29A is a cold induction specific promoter for the recombinase ntCre, and LoxP is an identification, cutting and recombination sequence for the recombinase ntCre. According to the invention, the recombinase ntCre gene provided in the invention expresses under low temperature induction of the induction type promoter RD29A; after the binary expression vector containing the recombinase ntCre gene transforms a plant, a selective marker gene and the RD29A:: ntCre:: Tnos gene are highly efficiently deleted under low temperature induction, and a transgenic plant without the selective marker gene is obtained.

Description

technical field [0001] The present invention relates to a recombinase gene, a binary expression vector and its construction method and application, in particular to a recombinase ntCre gene and a plant gene binary expression vector with low-temperature induction and deletion of a selectable marker gene and related construction methods and its application in Applications in transformed tobacco. Background technique [0002] In the process of plant transgenics, in order to separate transgenic cells from non-transgenic cells, a selection marker gene is often introduced, and the protein encoded by the selection marker gene can make plant cells have the ability to resist antibiotics, so that transgenic cells can survive in the environment of antibiotics survived. Since the selection marker gene has been integrated into the plant genome, the resistance gene may undergo horizontal transfer (gene drift) and vertical inheritance in the new ecological environment, and its potential h...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/00C12N15/52C12N15/63C12N15/82C12N15/66A01H5/00
Inventor 张兴国苏承刚杜小兵陈吉裕张香琴宋波
Owner SOUTHWEST UNIV
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