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Double-gene expression vector as well as construction method and application method thereof

An expression vector and construction method technology, applied in the field of plant genetic engineering, can solve problems such as complex methods, time-consuming and laborious, and achieve the effects of enhancing stress tolerance, solving dwarfing, and improving cold tolerance

Inactive Publication Date: 2015-09-09
HUNAN AGRICULTURAL UNIV
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Problems solved by technology

The method is complicated, time-consuming and labor-intensive

Method used

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  • Double-gene expression vector as well as construction method and application method thereof
  • Double-gene expression vector as well as construction method and application method thereof
  • Double-gene expression vector as well as construction method and application method thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0041] Embodiment 1, construct double expression vector rapidly with PCR method:

[0042] Materials: pGEM-T Easy Vector plasmid (Promega) cloning vector containing MtDREB1C and RcXET genes and pBI121 plasmid (AF485783) (Clontech).

[0043] 1. The first round of PCR: use 3 pairs of primers (a / b, c / d, e / f) to amplify three fragments of MtDREB1C, RcXET and -nos-35S-fragment respectively. Add to the 50 μl reaction system: 10×Buffer 5 μl; dNTP 2.5 μl; Pfu enzyme 0.25 μl; upstream and downstream primers 2.5 μl each; pBI121 expression vector) 15ng; ddH 2 O was added to 50 μl. The PCR reaction program was 94°C-40s, 50°C-1min, 72°C-1min, 30 cycles. The target band was recovered and purified by gel cutting, and was used for the next step of connection. See figure 1 .

[0044] 2. The second round of PCR: a / f is used as a primer pair, and three purified fragments are used as templates for PCR amplification. Add to the 50μl reaction system: 10×Buffer 5μl; dNTP 2.5μl; Pfu enzyme 0.2...

Embodiment 2

[0047] Example 2, the application of the double-gene expression vector based on the rapid construction of PCR in improving the two traits of Chinese rose by genetic engineering:

[0048] Materials: Agrobacterium GV3101, Chinese rose.

[0049] 1. Engineering strain construction: the MtDREB1C / RcXET double expression vector was used CaCl 2 The transformation method introduces Agrobacterium GV3101 to construct an engineering strain for transformation, which is used for gene transformation.

[0050] 2. Chinese rose transformation: using translucent callus as transformation material, transforming with engineering strain containing pBin438-MtDREB1C-nos-35S-RcXET, culturing on antibiotic-containing screening medium for 6 weeks to differentiate into more There are many globular embryos, but some of the globular embryos will brown, and some globular embryos even die after 12 weeks of culture, but there are also heart-shaped embryos produced. After about 16-20 weeks on the antibiotic-c...

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Abstract

The invention discloses a double-gene expression vector as well as a construction method and application method thereof. Co-expression of two target genes Mt DREB1C and RcXET is adopted to design a primer pair, the primer pair is adopted to perform PCR amplification on the target genes and an intercalary terminator promoter sequence -nos-35S-, and only one PCR is adopted to rapidly and integrally connect the two target genes and the intercalary terminator promoter sequence into an integral Mt DREB1C-nos-35S-RcXET structure sequence. The Mt DREB1C-nos-35S-RcXET structure sequence is inserted in a pBin438 expression vector and converts agrobacterium GV3101 to further convert Chinese rose to obtain regenerated plants. Six of the regenerated plants obtain PCR verification, and five of the six regenerated plants obtain verification of Southern hybridization. Physiological detection results show that the cold tolerance and drought tolerance of some transformation plants are better than those of non-transformation plants (MtDREB1C expression results), and the growth rate is remarkably increased (RcXET expression results). The construction method can be applicable to rapid construction of an expression vector requiring co-expression of two or more genes.

Description

Technical field: [0001] The invention belongs to the technical field of plant genetic engineering; relates to a double-gene expression carrier, a primer, and a construction and application method thereof, in particular to a method for quickly constructing a double-gene expression carrier by PCR and the double-gene expression carrier constructed by the method in Application in the breeding of new plant varieties. Background technique: [0002] With the continuous disclosure of the genome and the emergence of new functional genes, more and more breeding researchers have turned their attention to genetic engineering research and made rapid progress. However, most transformation technologies improve single traits with a single gene. Since most traits of organisms are often controlled by polygenes, that requires the coordinated expression of multiple related genes. Therefore, studies on the improvement of the same trait or multiple traits by double-gene or multi-gene aggregatio...

Claims

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Application Information

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IPC IPC(8): C12N15/84C12N15/11A01H5/00
Inventor 陈己任陈彦斌熊兴耀邓子牛
Owner HUNAN AGRICULTURAL UNIV
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