Plant expression vector facilitating connection of genes as well as construction method and applications thereof

A plant expression vector and expression vector technology, which is applied in the field of plant expression vectors for convenient connection of genes and its construction, can solve problems such as meaninglessness, and achieve the effects of improving work efficiency, simplifying operation steps, and reducing costs

Inactive Publication Date: 2013-06-26
TIANJIN UNIV
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

When transformed plants are selected, the presence of these antibiotic resistance genes in the genome is rendered meaningless, an

Method used

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  • Plant expression vector facilitating connection of genes as well as construction method and applications thereof
  • Plant expression vector facilitating connection of genes as well as construction method and applications thereof
  • Plant expression vector facilitating connection of genes as well as construction method and applications thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0042] Construction of vector pJWWD-12

[0043] pfu buffer (10×) 2.5 L dNTPs (2.5mmol / L) 2.0 L 1#F primer 0.5 L 1#R primer 0.5 L pfu enzymes 0.25 L pGreenII0229 1.0 L wxya 2 o 18.25 L

[0044] A total of 8 tubes were prepared for rubber cut recovery. Reaction conditions: 94°C, 3min; 94°C, 30sec; 55°C, 30sec; 72°C, 4.1min; 72°C, 10min, 30 cycles. According to the instructions of Takara Company's Agarose Gel DNA Recovery Kit, a 2.0 kb fragment was recovered.

[0045] pfu buffer (10×) 2.5 L dNTPs (2.5mmol / L) 2.0 L 2#F primer 0.5 L 2#R primer 0.5 L pfu enzymes 0.25 L pGreenII0229 1.0 L wxya 2 o 18.25 L

[0046] A total of 8 tubes were prepared for rubber cut recovery. Reaction conditions: 94°C, 3min; 94°C, 30sec; 55°C, 30sec; 72°C, 3.2min; 72°C, 10min, 30 cycles. The 1.6 kb fragment was recovered according to the instructions of the Takara company's...

Embodiment 2

[0055] Construction of vector pJWWD-123

[0056] pfu buffer (10×) 2.5 L dNTPs (2.5mmol / L) 2.0 L 3#F primer 0.5 L 3#R primer 0.5 L pfu enzymes 0.25 L pJWW0230 1.0 L wxya 2 o 18.25 L

[0057] A total of 8 tubes were prepared for rubber cut recovery. Reaction conditions: 94°C, 3min; 94°C, 30sec; 55°C, 30sec; 72°C, 2.5min; 72°C, 10min, 30 cycles. According to the instructions of the Takara company's agarose gel DNA recovery kit, the 1 kb fragment was recovered.

[0058] ② use Xho I The pJWWD-12 and 3# fragments were digested respectively, and the two fragments were purified with the common DNA product purification kit of Tiangen Company. The reaction system is:

[0059] Green Buffer (10×) 20 L FastDigest Xho I 2 L pJWWD-12 30 L wxya 2 o 148 L Green Buffer (10×) 20 L FastDigest Xho I 2 L 3# segment 30 L wxya 2 o 148 L

[0060] Reacti...

Embodiment 3

[0065] Construction of vector pJWWD-1234

[0066] pfu buffer (10×) 2.5 L dNTPs (2.5mmol / L) 2.0 L 4#F primer 0.5 L 4#R primer 0.5 L pfu enzymes 0.25 L pGH-X6145G 1.0 L wxya 2 o 18.25 L

[0067] A total of 8 tubes were prepared for rubber cut recovery. Reaction conditions: 94°C, 3min; 94°C, 30sec; 55°C, 30sec; 72°C, 1min; 72°C, 10min, 30 cycles. According to the instructions of Takara Company's Agarose Gel DNA Recovery Kit, a 0.3 kb fragment was recovered.

[0068] Green Buffer (10×) 20 L FastDigest Sac I 2 L pJWWD-123 30 L wxya 2 o 148 L Green Buffer (10×) 20 L FastDigest Sac I 2 L 4# segment 30 L wxya 2 o 148 L

[0069] Reaction condition: 200 L system, 37°C, 30min, cut the gel and recover.

[0070] pJWWD-123 1 L 4# segment 5 L T4 DNA Ligase buffer (5×) 2 L T4 DNA Ligase 1 L wxya 2...

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Abstract

The invention relates to a plant expression vector facilitating connection of genes as well as a construction method and applications thereof. The vector is prepared through the following steps: cloning 1#, 2#, 3#, 4# and 5# fragments from starting expression vectors pGreenII0229, pCAMBIA3300-35S-HAK, pGH-X6145G (purchased from Shanghai Sangon Biological Engineering Technology & Services Co., Ltd) and pCAMBIA2300 by using a PCR method; respectively carrying out enzyme digestion on the 1# and 2# fragments by using NotI and MIUI to be connected to the obtained product so as to obtain a vector pJWWD-12; respectively carrying out enzyme digestion on the pJWWD-12 and the 3# fragment by using XhoI to be connected to the obtained product so as to obtain a vector pJWWD-123; respectively carrying out enzyme digestion on the vector pJWWD-123 and the 4# fragment by using SacI to connected to the obtained product so as to obtain an expression vector pJWWD-1234; and respectively carrying out enzyme digestion on the pJWWD-1234 and the 5# fragment by using PstI to be connected to the obtained product so as to obtain an expression vector pJWWD-1718 finally. According to the invention, target genes can be conveniently and quickly inserted, and the target genes can be inserted in the vector through TA cloning, thereby greatly simplifying the operation steps, reducing the cost and improving the operating efficiency. The vector disclosed by the invention can provide a strong support for plant genetic engineering breeding, and lay the foundation for the safety evaluation and commercial planting of genetically modified plants.

Description

technical field [0001] The invention relates to a plant expression vector and its construction method, in particular to a plant expression vector for convenient gene connection, its construction method and application. Background technique [0002] Since the 20th century, cultivating new plant varieties through transgenic technology to improve yield and stress resistance is considered to be an effective way for directional transformation of plants. At present, genetically modified cotton, soybeans, corn, and rapeseed have been widely planted, bringing revolutionary reforms to the field of agricultural planting, and also bringing a new dawn for increasing agricultural income. [0003] Plant expression vector is a medium for the amplification and expression of exogenous genes in plant cells, so it is also called engineering vector. But it is not easy to construct an efficient plant expression vector in scientific research. In the middle, a series of steps such as double enzy...

Claims

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Application Information

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IPC IPC(8): C12N15/82C12N15/66C12N1/21A01H5/00
Inventor 季静王罡吴疆刁进进
Owner TIANJIN UNIV
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