Cloning method of kosteletzkya virginica KvNAC gene and construction method of expression vector of kosteletzkya virginica KvNAC gene

Abstract

The invention discloses a cloning method of a kosteletzkya virginica KvNAC gene and a construction method of an expression vector of the kosteletzkya virginica KvNAC gene. The cloning method comprisesthe steps of extracting total RNA of kosteletzkya virginica, performing cloning and sequence analyzing of the kosteletzkya virginica KvNAC gene, carrying out an RT-PCR reaction, and performing sequencing to obtain a full-length gene segment of the KvNAC gene; according to the obtained full length of the KvNAC gene, designing a primer for separation to obtain an ORF sequence of the gene; selectinghigh-fidelity Pfu DNA polymerase for PCR amplification to obtain a target segment; and taking 4 [mu]L of a PCR product and 1 [mu]L of a pEASY-Blunt Cloning Vector to carry out a ligation reaction soas to construct a recombinant cloning vector. The method is simple, convenient and effective; Isolation and cloning of the gene are realized in a new salt-resistant species, total RNA is successfullyextracted from the gene, and a spatial expression mode of the gene in a plant body is realized. The construction of the expression vector lays a foundation for the research of gene functions.

Description

technical field [0001] The invention relates to a method for cloning a gene and a method for constructing an expression vector thereof. Background technique [0002] The characteristics of plant sessile growth determine that plants can only passively suffer from the adverse effects of various abiotic stresses (high temperature, chilling injury, freezing injury, drought, high salt). In order to resist the influence of the adverse external environment, plants have gradually evolved a series of defense mechanisms at the physiological and molecular levels during the long-term evolution process. NAC (NAM, ATAF1 / 2, CUC2) transcription factors are a family of transcription factors unique to plants, which participate in the regulation of plant responses to various abiotic stresses. Seaside mallow is a new salt-tolerant species introduced from abroad. At present, there are few studies on the specific molecular mechanism of its salt-resistant ability, especially no related reports on...

AI Technical Summary

Problems solved by technology

Seaside mallow is a new salt-tolerant species introduced from abroad. At present, there are few studies on the specific molecular mechanism of its salt-resistant ability, especially no related reports on the KvNAC gene.

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