Bidirectional promoter bi-visual fluorescent protein report gene plant expression vector

A technology of bidirectional promoter and expression vector, applied in the field of bidirectional promoter dual visual fluorescent protein reporter gene plant expression vector, can solve the problems of promoter silencing, interaction, vector rearrangement or deletion, etc., and achieve the effect of easy screening

Inactive Publication Date: 2009-09-30
BEIJING FORESTRY UNIVERSITY
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Problems solved by technology

However, the use of multiple promoters may cause problems such as interaction bet

Method used

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  • Bidirectional promoter bi-visual fluorescent protein report gene plant expression vector
  • Bidirectional promoter bi-visual fluorescent protein report gene plant expression vector
  • Bidirectional promoter bi-visual fluorescent protein report gene plant expression vector

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Embodiment 1

[0018] Embodiment 1, construction general type plant bidirectional promoter expression vector

[0019] Construction of general-purpose plant bidirectional promoter expression vector

[0020] On the basis of the pBI121 vector, the p131 plasmid obtained by replacing the gus gene with the gfp gene, using the p131 plasmid as a template, and using D1 and D2 as primers, PCR amplifies a sequence from Pmini to the terminator:

[0021] D1: 5'-AAGCTTGGTACCCCCTTCGCAAGACCCTTC-3',

[0022] D2: 5'-GAAGCTTGTCACTGGATTTTGGTTTTAGG-3'.

[0023] PCR amplification conditions were: pre-denaturation at 94°C for 5mins; 94°C for 30s, 67°C for 30s, 72°C for 1min, 25 cycles; 72°C for 10mins; storage at 4°C.

[0024] The PCR product was recovered, connected with pMD18 T-Vector, and the cloning vector pT-MGN was constructed, and blue-white screening was carried out on LB medium. Pick a few white spots and culture them, extract the plasmids, digest with HindIII for preliminary verification, and send the...

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Abstract

The invention discloses a bidirectional promoter bi-visual fluorescent protein report gene plant expression vector. The promoter bi-visual fluorescent protein report gene plant expression vector is a recombinant expression vector of which the multiple cloning sites of an initial vector are inserted with the following DNA molecules: an EcoRI enzyme recognition sequence, an NoS terminator, an SacI enzyme recognition sequence, a kpnI enzyme recognition sequence, a red fluorescent protein gene, an SmalI enzyme recognition sequence, a BamHI enzyme recognition sequence, an XbalI enzyme recognition sequence, a bidirectional promoter, an XholI enzyme recognition sequence, an SpeI enzyme recognition sequence, a green fluorescent protein, an HindIII enzyme recognition sequence, an HpaI enzyme recognition sequence, an SalI enzyme recognition sequence, an NoS terminator and a Clal contained in turn from 5' end; and the nucleotide sequence of the bidirectional promoter is from the 5917th site to the 6834th site from the 5' end in a sequence 1 of a sequence list. The bidirectional promoter bi-visual fluorescent protein report gene plant expression vector can be applied to fusing functional genes by simple operation of direct enzyme cutting and connection.

Description

technical field [0001] The invention relates to a plant expression vector of a dual-visible fluorescent protein reporter gene with a bidirectional promoter. Background technique [0002] Transformation vectors containing two or more genes play an important role in gene therapy and the application of transgenic bodies for obtaining good traits. Common transgenic vectors expressing more than one gene include multiple promoters, internal ribosome entry site elements (IRES), splicing signals, and fusion proteins. However, using multiple promoters may cause problems such as interaction between two promoters, promoter silencing, vector rearrangement or deletion. [0003] Bidirectional promoters are of great interest for their ability to regulate two downstream genes. Usually, two genes arranged in a head-to-head manner have about less than 1000 bp of nucleotides between their transcription start sites, and the nucleotide region within this gene is less than 1 kb long, and both s...

Claims

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Application Information

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IPC IPC(8): C12N15/11C12N15/65C12N15/82C12N5/10C12N1/00C12P21/02
Inventor 盖颖王文棋张春晓陈雪梅蒋湘宁
Owner BEIJING FORESTRY UNIVERSITY
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