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

A technology of suspension culture and swollenin, applied in the biological field, can solve the problems of high cost, long culture period and many links, and achieve the effects of low cost, short culture period and high production efficiency

Inactive Publication Date: 2012-12-26
TIANJIN UNIVERSITY OF SCIENCE AND TECHNOLOGY
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Problems solved by technology

However, the growth rate of plant suspension cells is slow, and the culture perio...

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

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Embodiment Construction

[0013] Taking Glycyrrhizae as an Example-Method of Interfering with Expansin Gene Exp2 to Prepare Glycyrrhiza Glycyrrhizae Suspension Culture Small Cell Line

[0014] Construction of plant cell expression vectors

[0015] Through the nucleotide sequences of all plant expansin genes registered in Genbank, the nucleotide sequences of 50 base pairs in each of its two conserved regions are selected as forward fragments I and II respectively, and their respective nucleotide sequences are set based on them. Corresponding reverse sequences (I' and II'); design appropriate primers based on two pairs of forward and reverse sequences, and during PCR amplification, the positive and negative fragments are introduced into restriction endonuclease sites (EcoR I and Hind III) and the corresponding terminal nucleotide sequences of GU-AG introns. The results are shown in Table 1. After digestion, re-ligation, transformation, cloning and identification, the four-fragment I-AC-II'-II-CT-I' seq...

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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...

Claims

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Application Information

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IPC IPC(8): C12N5/10C12N5/02C12N15/82C12N1/21C12R1/01
Inventor 李兴林肖天剑韩杨曹爱佳高洁张黎明赵俊满淑丽高文远
Owner TIANJIN UNIVERSITY OF SCIENCE AND TECHNOLOGY
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