Dual-elemental Ti plasmid with bidirectional promoter trapping and plasmid rescuing functions and construction method of dual-elemental Ti plasmid

Abstract

The invention relates to a dual-elemental Ti plasmid carrier for the transformation of T-DNA (Transferred-Deoxyribose Nucleic Acid) of plants and partial fungi and the construction of a T-DNA insertion mutant library and a construction method of the dual-elemental Ti plasmid carrier. The method comprises the following steps: firstly, performing module trapping on a promoter integrated with sGFP (Green Fluorescent Protein) reporter genes at two ends of a T-DNA area of a plasmid; optimizing and adjusting a nucleotide sequence before opening reading frames for the sGFP at two ends of the nucleotide sequence; secondly, integrating a functional module rescued by the plasmid into a target plasmid through an enzyme cutting site. Finally, the plasmid is integrated with a hygromycin resistance gene which is used for screening a transformant; the hygromycin resistance gene and the promoter thereof can be replaced freely through a Sac I enzyme cutting site so as to adapt to different screening purposes and species. The convenience in the efficiency, the gene cloning and the related gene function verification of transgene is seriously affected by the selection and the module configuration and optimization of the dual-elemental Ti plasmid carrier. The application range of the plasmid is enlarged and the screening and cloning efficiency of target functional genes is improved by organically integrating various plasmid functional modules into one plasmid. Thus, a good molecular tool for finding beneficial genes and verifying the functions of the beneficial genes is provided.

Description

technical field [0001] The invention relates to a binary Ti plasmid carrier used for T-DNA transformation of plants and some fungi and T-DNA insertion mutant library construction and a construction method thereof, belonging to the technical field of molecular variety improvement and transgene. Background technique [0002] Agrobacterium tumefaciens is a Gram-negative bacterium that can transfer and integrate a segment of DNA containing plant hormone and opine synthase genes on its Ti plasmid (Tumor inducing plasmamid) by infecting plant wounds into the genome of the recipient plant, eventually inducing the plant to develop tumors. After research, it was found that there are two independent regions in the Ti plasmid: one is the T-DNA region (transferred DNA region), which contains oncogenic genes; function during the integration process. Based on the structural characteristics of the Ti plasmid, the tumorigenic gene in the wild Ti plasmid was deleted, and appropriate select...

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Problems solved by technology

[0004] In the genome of eukaryotes, there are many regions that do not contain genes or do not affect gene expression, and the position of T-DNA integration

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