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Gateway cloning entry vector, construction method and use thereof

A carrier and pathway technology, applied in the field of molecular biology, can solve the problems of low success rate, high cost, and low success rate of PCR product fragments, etc.

Inactive Publication Date: 2008-11-12
KUNMING UNIV OF SCI & TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Gateway's BP reaction can be used to generate Gateway's entry clone, but in practice, the applicant found that the probability of success is not high when the PCR product fragments operated by this technology are large
Invitrogen has also developed two other methods for generating Gateway entry clones, one of which is TOPO cloning technology, but the cost of this technology is relatively high, and the probability of success is not high if the PCR product fragment is large

Method used

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  • Gateway cloning entry vector, construction method and use thereof
  • Gateway cloning entry vector, construction method and use thereof
  • Gateway cloning entry vector, construction method and use thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0078] Embodiment 1, construction of intermediate vector pUC118-T-PrbcS

[0079] Purify (operate according to kit instructions) pUC118-PrbcS-T-rbcS-3C (constructed and provided by Sugita et al., Sugita et al., 1987, MGG, 209: 247-256) with a plasmid extraction kit (Broadtech) , the rbcS-3C in pUC118-PrbcS-T-rbcS-3C was cut out with restriction endonuclease SphI (Fermentas), and the cut vectors pUC118-PrbcS-T and rbcS-3C were separated by agarose gel electrophoresis Fragment, recover the 4.6kb vector pUC118-PrbcS-T, and then use the ligase kit of TaKaRa to ligate (operate according to the kit instructions) the vector DNA fragment without rbcS-3C to generate an intermediate vector pUC118-PrbcS -T( figure 1 ), converting the high efficiency (10 8 ) Escherichia coli competent cells (DH5α, Tiangen Biochemical Technology), spread the transformed Escherichia coli on a plate added with ampicillin (Amp, 100 μg / ml), cultivate overnight at 37°C, and screen Amp-resistant recombinants T...

Embodiment 2

[0080] Example 2. Using point mutation technology to introduce NcoI site into the intermediate vector pUC118-T-PrbcS

[0081] Using the purified plasmid pUC118-Prbcs-T as a template, a pair of complementary primers (NcoI5 and NcoI3, NcoI3, figure 1 ), commissioned TaKaRa to synthesize. Add 25ng of purified plasmid pUC118-PrbcS-T to the point mutation reaction mixture as a template, and at the same time add 125ng of point mutation primers NcoI5 and NcoI3, 1μldNTP (2.5mM), 5μl of 10×KOD reaction buffer and 1μl of KOD polymerase (Toyobo Japan), add double distilled water to make the final reaction volume 50 μl. Heated at 95°C for 30 seconds on a PCR instrument, followed by 15 cycles of reaction at 95°C for 30 seconds, 55°C for 1 minute, 68°C for 10 minutes, and finally extended the reaction at 68°C for 10 minutes to synthesize The child chain of the mutation site. After the reaction was completed, the reaction mixture was cooled on ice for 2 minutes, 1 μl of restriction endonu...

Embodiment 3

[0082] Example 3. Using point mutation technology to change XmnI in Gateway's entry vector pENTR-2B multiple cloning site to HindIII

[0083] Using the purified plasmid pENTR-2B (purchased from Invitrogen) as a template, a pair of (HindIII5 and HindIII3) complementary primers (HindIII5 and HindIII3) for point mutations were designed according to the sequence near XmnI ( figure 2 ), entrusted Shanghai Shenergy Bocai to synthesize. Add 25ng of purified plasmid pENTR-2B as a template to the point mutation reaction mixture, add 125ng of point mutation primers HindIII5 and HindIII3, 1μldNTP (2.5mM), 5μl of 10×KOD reaction buffer and 1μl of KOD polymerase (Japan Toyobo), add double distilled water to make the final volume of the reaction 50 μl. Heated at 95°C for 30 seconds on a PCR instrument, followed by 15 cycles of reaction at 95°C for 30 seconds, 55°C for 1 minute, 68°C for 10 minutes, and finally extended the reaction at 68°C for 10 minutes to synthesize The child chain of ...

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Abstract

The invention discloses a gateway cloning technology entry plasmid vector. The vector contains a photoinducible promoter (Prbcs) of a small subunit gene of 1,5-bisphosphate carboxylase (Rubisco) and a green fluorescent protein (GFP) reporter gene. The invention also discloses a construction method and application of the vector. Through the vector, the gateway cloning technology can be utilized to quickly construct a photoinducible plant expression vector of a target gene to realize the high level expression of the target gene in plant leaves, the expression of the target gene is adjusted by light intensity, and expressed target protein can be positioned into chloroplast or cytoplasm.

Description

Technical field: [0001] The invention belongs to the field of molecular biology, and relates to an entry vector of pathway cloning (Gateway) technology, especially a Gateway entry vector containing a light-inducible promoter (PrbcS) and GFP, and the invention also relates to the construction and application of the entry vector . Background technique: [0002] Promoter is an important element in the regulation of gene expression, which regulates the expression of genes in quantity and quality. Promoters commonly used in plant genetic engineering can be divided into constitutive promoters and organ-specific promoters according to their mode of action and function. In plant transgenic operations, the most commonly used promoter is the 35S promoter from cauliflower mosaic virus (CaMV). This promoter directs the synthesis of 35S RNA during plant infection by CaMV, and is present in many tissues of plants. Can be highly expressed (Odell et al., 1985; Nature, 313:810-812), most s...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/63C12N15/65C12N15/82
Inventor 陈丽梅李昆志马莉宋中邦胡清泉
Owner KUNMING UNIV OF SCI & TECH
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