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Sweet wormwood MYC2 transcription factor protein coding sequence and applications thereof

A kind of artemisia annua, base sequence technology, applied in the field of genetic engineering

Active Publication Date: 2014-02-26
SHANGHAI JIAO TONG UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Before the publication of the present invention, there has not been any publication or report on the protein coding sequence of the Artemisia annua MYC2 transcription factor mentioned in this patent application and its application

Method used

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  • Sweet wormwood MYC2 transcription factor protein coding sequence and applications thereof
  • Sweet wormwood MYC2 transcription factor protein coding sequence and applications thereof
  • Sweet wormwood MYC2 transcription factor protein coding sequence and applications thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0079] Cloning of MYC2 Transcription Factor Protein Gene of Artemisia annua

[0080] 1. RNA extraction (RNA extraction)

[0081] Take the leaf tissue of Artemisia annua, grind it in liquid nitrogen, add it to a 1.5mL Eppendorf (EP) centrifuge tube filled with lysate, shake it fully, and extract total RNA according to the instructions of the TIANGEN kit. The quality of total RNA was identified by formaldehyde denaturing gel electrophoresis, and then the RNA content was determined on a spectrophotometer.

[0082] 2. Cloning of Full-length cDNA

[0083] According to the conserved sequence of amino acids encoded by the MYC2 transcription factor protein gene in Arabidopsis, using the principle of homologous gene cloning, the Smart-RACE (Rapid Amplification of cDNA Ends) method (Clontech kit) was used to clone the full-length cDNA, divided into four proceed in stages:

[0084] (1) Synthesis of first-strand cDNA:

[0085] Using the 5'-CDS primer A and SMART2A oligo primers provid...

Embodiment 2

[0093] Sequence information and homology analysis of MYC2 transcription factor protein gene of Artemisia annua

[0094] The full-length cDNA of the novel Artemisia annua MYC2 transcription factor protein obtained in the present invention is 2357 bp in length, and the detailed sequence is shown in SEQ ID NO.3, wherein the open reading frame is located at 301-2175 nucleotides (1875 nucleotides). According to the obtained full-length cDNA sequence, the amino acid sequence of Artemisia annua MYC2 transcription factor protein was deduced, with a total of 625 amino acid residues, a molecular weight of 68.5kD, and an isoelectric point of 5.03. See SEQ ID NO.4 for the detailed sequence.

[0095] The protein amino acid sequence of the MYC2 transcription factor protein of Artemisia annua was searched for protein homology in the NCBI full database with the BLAST program, and it was found that it was similar to tobacco (Nicotianatabacum), grape (Vitis vinifera), vinca (Catharanthus roseus...

Embodiment 3

[0097] Amplification of Conserved Domain Sequence of MYC2 Transcription Factor of Artemisia annua

[0098] 1. According to the obtained full length of the Artemisia annua MYC2 transcription factor protein gene, the conserved domain comparison analysis was carried out in the NCBI database, and it was found that it contained 2 parts of the conserved domain, which were respectively located at the 65th amino acid sequence listed in SEQ ID NO.4 - the bHLH-MYC domain at position 245 and the HLH domain at position 442-492, the DNA sequence expressing the conserved domain fragment corresponds to the nucleotide sequence at position 493-1779 of SEQ ID NO.3, accounting for approximately 70% of the sequence of the open reading frame (nucleotides 301-2175 of SEQ ID NO.3) of the Artemisia MYC2 transcription factor protein gene. The conserved domain of the transcription factor protein is the main part of its regulatory role, so the MYC2 transcription factor fragment of Artemisia annua contai...

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Abstract

The invention discloses a sweet wormwood MYC2 transcription factor protein coding sequence in the field of bioengineering technology and applications of a modified plant of the coding sequence for improving the artemisinin content in sweet wormwood and for insect disease resistance. An isolated DNA molecular comprises a nucleotide sequence coding a polypeptide with sweet wormwood MYC2 protein activity. The nucleotide sequence has at least 70% of homology with a nucleotide sequence from the 301 nucleoside site to the 2175 site of the SEQ ID NO.3. The invention relates to a fusion gene construct containing the gene and a novel recombinant expression vector carrying the construct, and relates to a plant cell transformed by the expression vector, and a transgenic plant of the gene generated by the transformed cell and offspring of the transgenic plant, wherein the offspring comprises plant seeds and plant tissues. By expression of the gene in the plant, the artemisinin content in a transgenic sweet wormwood is significantly increased, and resistance of the transgenic sweet wormwood to botrytis cinerea is significantly enhanced.

Description

Technical field [0001] The present invention relates to the technical field of genetic engineering. Specifically, the present invention relates to a nucleotide sequence encoding a MYC2 transcription factor protein expressed in Artemisia annua. The invention also relates to the use of the nucleotide sequence encoding the protein. Background technique [0002] Artemisia annua L. is an annual herb of the genus Artemisia in the Asteraceae family. Artemisinin is a sesquiterpene lactone compound containing a peroxy bridge structure isolated from its aerial part. It is currently recognized as the most effective drug in the treatment of malaria in the world, especially for cerebral malaria and anti-malaria. Chloroquine malaria treatment has the characteristics of rapid action and low toxicity. Currently, the most effective treatment for malaria recommended by the World Health Organization is artemisinin combination therapies (ACTs). In addition, with the gradual deepening of pha...

Claims

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Application Information

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IPC IPC(8): C12N15/29C12N15/82C12N5/10C12N1/21A01H5/00C12R1/01
Inventor 唐克轩沈乾陆续付雪晴颜廷祥孙小芬王国丰
Owner SHANGHAI JIAO TONG UNIV
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