Method for preparation of plant suspension culture small cell line by interfering expansin gene Exp2

Abstract

Belonging to the field of biotechnologies, the invention relates to a method for preparation of a plant suspension culture small cell line by interfering an expansin gene Exp2. The method consists of: first, selecting forward fragments I and II from two conserved regions of gene Exp2 respectively, as well as corresponding reverse sequences I' and II'; constructing RNAi fragments of I-AC-II'-II-CT-I'; and screening out the agrobacterium tumefaciens engineering bacterium inserted in a pCAMBIA2300 plant expression vector; then taking licorice for example, performing agrobacterium-mediated transformation to obtain a transformed callus and its stably expressed suspension cell in order; and then, carrying out nursing culture, and making use of the suspension cell to screen a single cell line callus characterized by fast growth speed and good effect, and a suspension cell line formed thereby; and finally, conducting culture process optimization to obtain the optimum fermentation process parameters of suspension cell growth and product accumulation. The method provided in the invention can obtain the cell line with the advantages of large density, short culture cycle, low cost as well as high production efficiency, and can provide excellent provenances for large-scale development and production of medicinal plant effective components.

Description

technical field [0001] The invention patent belongs to the field of biotechnology. Background technique [0002] Cell suspension culture is one of the important means to produce plant secondary metabolites. However, the growth rate of plant suspension cells is slow and the culture period is long, which has become the key problem of many links and high cost of cell suspension culture. The invention utilizes the principle of RNAi to shorten the plant suspension cell culture period and increase the cell culture density by reducing the expression of the plant Exp2 gene. Contents of the invention [0003] Construction of plant cell expression vectors. [0004] By comparing the nucleotide sequences of the plant Exp2 genes registered in Genbank, the nucleotide sequences of 30-100 base pairs in each of the two conserved regions were selected as forward fragments I and II respectively, and based on them, respectively Set its corresponding reverse sequence (I' and II'); design ap...

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Problems solved by technology

However, the growth rate of plant suspension cells is slow, and the culture period has become a key problem such as many links and high cost of cell suspension culture.

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