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Construction and application of novel transgenic plant marker gene

A marker gene and transgene technology, applied in the field of plant biology, can solve the problems of weak gene translation initiation ability, large polycistronic expression structure, etc.

Inactive Publication Date: 2019-11-19
SHANGHAI ACAD OF AGRI SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The polycistronic expression structure mediated by IRES is large, and its application is often limited by the capacity of the vector. When IRES is placed in the middle of adjacent genes, it is helpful for the expression of bicistronic proteins, but the IRES pair is located behind it. Gene translation initiation ability is relatively weak

Method used

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  • Construction and application of novel transgenic plant marker gene
  • Construction and application of novel transgenic plant marker gene
  • Construction and application of novel transgenic plant marker gene

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0016] Embodiment 1: marker gene Hygfpbar construction

[0017] Hygromycin resistance gene (HPT), green fluorescent protein gene EGFP and glufosinate resistance gene Bar were serially connected by overlap extension PCR. The designed primers are: HPTZ1: 5'-CCGCTCGAGATG AAAAAGCCTGAACTCACCG-3' (shown in SEQ ID NO.3); HPTF1:5'-ACATCAC CAGCAAGCTTAAGAAGATCGAAATTCAACAGCTATTTCTTTGCCCTCGG ACG-3' (shown in SEQ ID NO.4); GFPZ: 5' -CTTAAGCTTGCTGGTGATGTTGA ATCCAACCCAGGTCCAATGGTGAGCAAGGGCGAGGAG-3'(SEQ ID NO.5所示);GFPF:5'-ACATCACCAGCAAGCTTAAGAAGATCGAAATTCAACAGTCAGATCTCGGTGACGGGCAG-3'(SEQ ID NO.6所示);BarZ:5'-CTTAA GCTTGCTGGTGATGTTGAATCCAACCCAGGTCCAATGAGCCCAGAACG ACGCCCG-3'(SEQ ID NO .7); BarF: 5'-CCGCTCGAGTCAGATCTCGG TGACGGGCAG-3' (shown in SEQ ID NO.8).

[0018] In a 50 µl reaction system, the HPT gene was amplified from pCAMBIA1301 using primers HPTZ1 and HPTF1; the Bar gene was amplified from pPZp-RCS2-Bar (Novagen, Madison, USA) using primers BarZ and BarF; the Bar gene was amplified from ...

Embodiment 2

[0019] Embodiment 2: Construction of plant expression vector

[0020] Firstly, the plant expression vector pCAMBIA1301 was double-digested with BglII and BstEII, and T4 DNA polymerase (Takara Baobiology) in the end smoothing kit was added, and then 5 U of T4 DNA ligase was added to identify the presence of the GUS gene in the vector by PCR, and screened to remove The vector of GUS gene is used as a novel marker gene expression vector. The pCAMBIA1301-GUS vector and the Hygfpbar fragment were digested with XhoI, and the recovered vector and fragment were ligated at a molar ratio of 1:10 or more. PCR was used to identify the direction of the inserted gene, and the clone of the hygromycin resistance gene on the promoter side was found for further identification, and a new marker gene plant expression vector pHygfpbar controlled by the CaMV35S promoter was constructed.

Embodiment 3

[0021] Example 3: Transformation of Rice and Arabidopsis

[0022] The strain used was Agrobacterium tumefaciens. The plasmid was introduced into Agrobacterium by electroporation. Pick a single bacterium into 25 ml YEB medium (50mg / l rifampicin) for overnight culture, transfer 5 ml of the bacterial liquid to 100 ml YEB medium (50mg / l rifampicin), and cultivate to OD 600 = 0.7-0.8, put the bacterial solution on ice for 10 minutes, centrifuge at 5000 rpm for 10 minutes, and collect the bacterial cells at 4°C, add 100ml sterile double distilled water to wash twice. Add 2 ml of 10% glycerol to suspend the bacteria and transfer to a 50 ml centrifuge tube. Centrifuge at 5500 rpm for 10 min at 4°C. Collect the cells, add 500 µl of 10% glycerol to suspend the cells, and transfer to a 1.5 ml centrifuge tube. Take 70µl competent cells and add 1µl recombinant plasmid pHygfpbar respectively. Mix well with a 200µl pipette tip removed, and transfer to a 0.1cm electric shock cup. Electri...

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Abstract

The invention discloses a novel fusion marker gene research for transgenic plants. The specific process of the research includes the steps that a 2A oligopeptide sequence (QLLNFDLLKLAGDVESNPGP) consisting of 20 amino acids of foot-and-mouth disease virus (FMDV) is introduced into the junction of two genes; in eukaryotic cells, oligopeptide is cleaved at C-termini; an FMDV2A connecting peptide coding sequence is in series connection with a green fluorescent protein GFP gene, a hygromycin resistance gene HPT and a herbicide resistance gene Bar to construct a constitutive expression unit of the three marker genes, and the constitutive expression unit is inserted into a plant expression vector so that the expression of the GFP gene, the HPT gene and the herbicide resistance gene Bar can be detected in the transgenic plants. By utilizing the research, transgenic positive plants can be screened through a plurality of methods, so that removal of the homozygous progeny and false positive materials of the transgenic plants is facilitated.

Description

technical field [0001] The invention belongs to the field of plant biotechnology, and specifically utilizes the method of FMDV 2A linking peptides to mark genes in tandem, and respectively verifies the tandem genes to determine that the plants are positive for transgenes. Background technique [0002] The transformation of exogenous genes into plants needs to be confirmed by screening markers at different stages. It is easy to obtain real transgenic plants with different screening markers, but plant gene expression requires a combination of promoters and terminators, so the construction of multiple marker genes is cumbersome. During the construction of plant gene expression vectors, the construction of target gene plant expression vectors is often limited due to the lack of vector expression components or the scarcity of restriction sites for multiple cloning sites. In order to use multiple marker genes in plant expression vectors and increase the reliability of transgenic p...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/82
CPCC12N15/8209
Inventor 彭日荷姚泉洪田永生高建杰许晶付晓燕李振军韩红娟王波王丽娟张福建黄悠楠张文慧
Owner SHANGHAI ACAD OF AGRI SCI
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