Universal unlabeled LIC vector pMF-LIC and construction method and application thereof

Abstract

The invention relates to a universal unlabeled LIC vector pMF-LIC and a construction method and an application thereof; a plasmid pJG100 is used as a template, primers having the sequence of SEQ ID NO.1 and SEQ ID NO.2 as a primer pair are used for PCR amplification, and a target fragment LIC sequence is obtained; the target fragment LIC sequence is subjected to double enzyme digestion by restriction endonucleases Xba I and Sac I and then is connected with a pMF-Npt II plasmid subjected to same double enzyme digestion, and the universal unlabeled LIC vector pMF-LIC is obtained. The pMF-LIC vector provided by the invention is safe in use and does not contain any vector skeleton sequence except an introduced target gene. The method is simple and convenient, and exogenous to-be-cloned PCR fragments do not need enzyme digestion and connection reaction; the method is efficient, does not depend on ligase, greatly simplifies the DNA recombination process and completely depends on paired annealing of viscous ends, and the vector has low self-connecting background; the vector can be processed in batch; and the cost is low.

Description

technical field [0001] The invention relates to the technical field of genetic engineering, in particular to a universal marker-free LIC carrier pMF-LIC and its construction method and application. Background technique [0002] When it comes to genetically modified technology, everyone will think of the safety of genetically modified organisms. Nowadays, the public is very concerned about the safety of GMOs, and the attention is mainly focused on some screening marker genes used in the transgenic process, most of these marker genes are genes encoding antibiotic resistance and herbicide resistance. Therefore, obtaining some genetically modified plants without marker genes may increase the public's acceptance of genetically modified products. So far, some transformed plants without marker genes have been obtained, but some methods still use marker genes during transformation, and then remove the screening marker genes by various means after positive transformants are obtained...

AI Technical Summary

Problems solved by technology

This method of cloning foreign genes into vectors is limited by the original restriction sites in the gene sequence, and is subject to certain limitations in application.

At the same time, if the laboratory wants to clone different genes into vectors, it is necessary to consider the restriction sites of each gene and vector, use different restriction sites, purchase different restriction enzymes, and pass multiple Restriction digestion and ligation to clone different genes is time-consuming and costly

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