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Artemisia apiacea4-(5'-cytidine diphosphate)-2-C-methyl-D-erythritol kinase coding sequence

A technology of cytidine diphosphate, -2-C-, applied in the field of coding sequence, Artemisia annua 4--2-C-methyl-D-erythritol kinase coding sequence, can solve problems that have not been reported yet, Achieve the effect of realizing large-scale production and increasing the content of artemisinin

Inactive Publication Date: 2012-04-25
SUZHOU TANGJI BIOTECH CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] After searching the literature of the prior art, it is found that there is no report related to the coding sequence of Artemisia annua 4-(5'-diphosphate cytidine)-2-C-methyl-D-erythritol kinase

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment

[0024] Step 1. Cloning of the coding sequence of Artemisia annua 4-(5'-cytidine diphosphate)-2-C-methyl-D-erythritol kinase (CMK)

[0025] (1) RNA extraction

[0026] Take the leaf tissue of Artemisia annua, grind it in liquid nitrogen, add it to a 1.5mL Eppendorf (EP) centrifuge tube containing the lysate, and after fully shaking, extract the total RNA according to the instructions of the TIANGEN kit. Use formaldehyde denaturing gel electrophoresis to identify the quality of total RNA, and then measure the RNA content on a spectrophotometer.

[0027] (2) Cloning of Full-length cDNA

[0028] According to the amino acid conservative sequence encoded by the related genes in Arabidopsis thaliana, using the principle of homologous gene cloning, the RACE (Rapid Amplification of cDNA Ends) method (Clontech SMART TM RACE kit) for full-length cDNA cloning in three stages:

[0029] ① Synthesis of first strand cDNA

[0030] Use SMART TM The 5'-CDS primer A and SMART II A oligo primers provided...

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PUM

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Abstract

The invention relates to an artemisia apiacea4-(5'-cytidine diphosphate)-2-C-methyl-D-erythritol kinase coding sequence in the technical field of gene engineering. The sequence is a sequence generated after one or a plurality of codons in nucleotide are replaced by degenerate codons of amino acid with same coding, as shown in 15-pisition to 1214-position of SEQ ID NO: 3 or shown in 15-position-1214-position of SEQ ID NO: 3. The method for improving the content of plant artemisinin by using the coding sequence includes the steps of: connecting the coding sequence to a plant expression regulatory sequence and constructing a plant expression carrier containing the artemisia apiacea4-(5'-cytidine diphosphate)-2-C-methyl-D-erythritol kinase coding sequence; transferring the expression carrier in step 1 to agrobacterium rhizogenes, then transferring agrobacterium rhizogenes to artemisia apiacea; through antibiotic screening, obtaining a transformation cell containing the artemisia apiacea4-(5'-cytidine diphosphate)-2-C-methyl-D-erythritol kinase coding sequence and regenerating a transgenosis plant. The coding sequence can improve the content of artemisinin in artemisia apiacea and the content of isoprene compound of other plants.

Description

Technical field [0001] The present invention relates to a coding sequence in the technical field of genetic engineering, in particular to a coding sequence of Artemisia annua 4-(5'-cytidine diphosphate)-2-C-methyl-D-erythritol kinase. Background technique [0002] Artemisia annua (Artemisia annua L.) is an annual herb of the genus Artemisia of the Compositae. Artemisinin is a sesquiterpene lactone compound containing a peroxy bridge structure isolated from its aerial part. It is currently recognized as the most effective treatment for malaria in the world, especially for cerebral malaria and anti-chloroquine malaria. effective. Currently, the most effective treatment for malaria recommended by the World Health Organization is artemisinin combination therapy (ACTs). In addition, with the gradual deepening of the pharmacological research of artemisinin, scientists have discovered that artemisinin and its derivatives also have anti-inflammatory, anti-schistosomiasis, anti-tumor an...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/54C12N9/12C12N15/82C12N15/84
Inventor 唐克轩王玥月翁纯纯杨可
Owner SUZHOU TANGJI BIOTECH CO LTD
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