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Serine recombinases mediating stable integration into plant genomes

A plant genome and recombinase technology, applied in genetic engineering, recombinant DNA technology, and stably introducing foreign DNA into chromosomes, can solve problems such as no sequence or structural similarity

Pending Publication Date: 2021-11-05
格林维纳斯有限责任公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

These two families of recombinases operate under different recombination mechanisms and share no sequence or structural similarity

Method used

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  • Serine recombinases mediating stable integration into plant genomes
  • Serine recombinases mediating stable integration into plant genomes
  • Serine recombinases mediating stable integration into plant genomes

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0332] Example 1 Plasmid Construct

[0333] 1. Recombinase plasmid

[0334] Plasmids with recombinases are based on the pUC backbone. Each recombinase gene was modified to express a fusion protein with a C-terminal HA (influenza hemagglutinin) peptide and an SV40 nuclear localization signal (NLS). It is expressed under the control of cauliflower mosaic virus (CaMV) 35S promoter and nopaline synthase (NOS) terminator. figure 2 is a schematic diagram of a recombinase construct expressing a recombinase fusion protein.

[0335] Table 2 below describes the phage as the original source of the recombinase, the structure of the recombinase fusion protein used in this study, and the amino acid sequence identifier.

[0336] Table 2: Sources of Recombinase

[0337]

[0338] In experiments without effector plasmids, to balance the amount of DNA transfected into cells, a plasmid expressing the chloramphenicol acetyltransferase (CAT) gene was used as a control for transfection (Ulma...

Embodiment 2

[0349] Example 2. Genbank screening of lettuce and tobacco genomes did not find attP or attB sites

[0350] Screening of attB, attP and possible pseudo att sites was performed against public genome databases. Lettuce (Lactuca sativa) and Nicotiana benthamiana genomes in the Phytozome database (from the US Energy Joint Genome Institute, available at Phytozome.jgi.doe.gov / pz / portal.html) were examined. The Arabidopsis genome was searched at Arabidopsis.org, a database established by Arabidopsis Information Sources (TAIR).

[0351] These databases were searched for the attB and attP sequences listed in Table 3, allowing for 2 nucleotide mismatches or spacers to account for possible att "pseudo" sites. The att pseudosites identified in US9034650 were also screened against lettuce and tobacco genomes.

[0352] No attP or attB sites or canonical pseudosites were found in Arabidopsis, lettuce, and tobacco. Therefore, serine recombinase cannot incorporate reporter plasmids with att...

Embodiment 3

[0353] Example 3.SPβc2 recombinase integrates the 6.3kb plasmid containing attP but not attB into the lettuce genome

[0354] 1. Method

[0355] E. coli containing effector or reporter plasmids, respectively, were grown and DNA was extracted using the Qiagen Endo-Free Maxi kit (Cat# 12361, Qiagen, Inc., CA).

[0356] Mesophyll protoplasts were isolated from 3-5 week-old axenically grown lettuce plants as described by Tiwari et al., 2006 (Tiwari et al., "Transfection assay with protoplasts containing an integrated reporter gene", Methods Mol Biol, 2006 ; 323:237-44). Purified protoplasts were transfected with a plasmid expressing the recombinase using the standard polyethylene glycol method as described by Tiwari 2006 (supra).

[0357] The protocol for the subculture and regeneration of transfected protoplasts was as previously described (Jie et al., "Myo-inositol increases the plating efficiency of protoplasts of cabbage (Brassica oleracea var. capitata) cotyledon derived f...

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Abstract

Described is a serine recombinase that integrates large sections of foreign DNA into plant chromosomes through non-homologous recombination through attP or attB sites on the foreign DNA but without the engineering of corresponding attB or attP site on the plant chromosome. The serine recombinase-based method has several advantages over other systems, such as Agrobacterium. Also described are related methods, composition, and plants containing exogenous DNA.

Description

[0001] About the sequence listing [0002] This application incorporates by reference a Sequence Listing (shown below) concurrently filed as a text file through the US Patent Office's Electronic Filing System (EFS). The text file copy of the sequence listing submitted here is labeled "INX00466US-V4_ST25.txt", has a file size of 84947 bytes, and was created on October 14, 2019. This sequence listing is incorporated herein by reference in its entirety. Background technique [0003] Site-specific recombinases are common in prokaryotes and lower eukaryotes, but absent in higher multicellular eukaryotes such as plants. Site-specific recombinases catalyze recombination reactions between two DNA fragments containing short specific sequences. This process differs from homology-dependent recombinases, which catalyze recombination between two larger homologous sequences. [0004] Site-specific recombinases are broadly divided into two classes: tyrosine recombinases (also known as lam...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/22C12N15/82C12N15/90
CPCC12N15/90C12N9/22C12N15/8213C07K2319/00C12N15/8241
Inventor S·B·蒂瓦里A·特朗布莱J·卡森S·博杜帕利R·斯坦姆勒A·爱德华兹K·弗雷奇
Owner 格林维纳斯有限责任公司
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