Southernwood 1-hydroxy-2-methyl-2-(E)- cyclobutenyl 4-diphosphoric acid reductase protein encoding sequence

A coding sequence, butenyl technology, applied in the field of protein coding sequence, can solve the problems of undiscovered and unseen HDR gene cloning

Inactive Publication Date: 2007-07-11
SOUTHWEST UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] In the analysis of existing documents, there is no cloning of the HDR gene on the pyruvate / phosphoglyceraldehyde biosynthetic pathway in Artemisia annua, and no reports of documents closely related to the subject of the present invention have been found so far

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0041] Cloning of 1-hydroxy-2-methyl-2-(E)-butenyl 4-pyrophosphate reductase gene from Artemisia annua

[0042] 1. Isolation

[0043] Artemisia annua was collected from Youyang, Chongqing, and the seeds were sown in the greenhouse. When the Artemisia annua seedlings grew to a height of 10 cm, DNA or RNA was prepared to be extracted.

[0044] 2. RNA isolation (RNA isolation)

[0045] Take part of the tissue, grind it with a mortar, add it to a 1.5mL EP tube filled with lysate, shake it fully, and then transfer it into a glass homogenizer. After homogenization, transfer to 1.5mL EP tube, and extract total RNA (TRIzol Reagents, GIBCO BRL, USA). The quality of total RNA was identified by formaldehyde denaturing gel electrophoresis, and then the RNA content was determined on a spectrophotometer.

[0046] 3. Cloning of Full-length cDNA

[0047] According to the nucleotide conservative sequence of the 1-hydroxyl-2-methyl-2-(E)-butenyl 4-pyrophosphate reductase gene of various pla...

Embodiment 2

[0059] Sequence information and homology analysis of 1-hydroxy-2-methyl-2-(E)-butenyl 4-pyrophosphate reductase gene from Artemisia annua

[0060] The full-length cDNA of the novel 1-hydroxyl-2-methyl-2-(E)-butenyl 4-pyrophosphate reductase gene sequence of the present invention is 1696bp in length, and the detailed sequence is shown in SEQ ID NO-1, wherein the open reading Boxes are located at nucleotides 36-1403 (1428 nucleotides). The amino acid sequence of Artemisia annua 1-hydroxy-2-methyl-2-(E)-butenyl 4-pyrophosphate reductase was deduced based on the full-length cDNA, with a total of 455 amino acid residues, a molecular weight of 51.44kDa, and an isoelectric point (pI) was 5.63. See SEQ ID NO.1 for the detailed sequence.

[0061] The full-length cDNA sequence of Artemisia annua 1-hydroxy-2-methyl-2-(E)-butenyl 4-pyrophosphate reductase gene and its encoded protein sequence were published in Non-redundant GenBank+EMBL+DDBJ using BLAST program Nucleotide and protein h...

Embodiment 3

[0063] Functional verification of 1-hydroxy-2-methyl-2-(E)-butenyl 4-pyrophosphate reductase protein or polypeptide from Artemisia annua in Escherichia coli

[0064] In this example, the full-length HDR coding sequence or fragment of Artemisia annua was constructed into a commercially available protein expression vector to express the protein, so that the host bacteria obtained the ability to grow on an auxotrophic medium.

[0065] According to the amino acid sequence of Artemisia annua HDR, design primers to amplify the protein coding region of HDR, and introduce restriction endonuclease sites on the forward and reverse primers (this depends on the pQE30 vector selected), so as to construct the expression vector . Using Artemisia annua RNA as a template, after RT-PCR amplification, the HDR gene of Artemisia annua was cloned into the pQE30 vector (Novagen) under the premise of ensuring the correct reading frame. The identified expression vector utilizes CaCl 2 Methods Escher...

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PUM

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Abstract

The invention discloses an arteannuin 1-hydroxy-2-methyl-2-(E)-cyclobutenyl 4-pyrophosphoric acid reductase protein coded sequence in the gene engineering domain, which is characterized by the following: the ribonucleotide sequence possesses 70% consanguinity with 36-1403th in the SEQ ID NO. 1, which an cross the 36-1403th in the SEQ ID NO. 1 under 45-55 deg. c.

Description

technical field [0001] The invention relates to a protein coding sequence, especially a protein coding sequence of Artemisia annua 1-hydroxyl-2-methyl-2-(E)-butenyl 4-pyrophosphate reductase, which belongs to the field of genetic engineering. Background technique [0002] Statistics in recent years show that more than 2 billion people in more than 100 countries and regions live in malaria-endemic areas. About 270 million people suffer from this disease every year, and nearly 3 million people die of malaria. Because of the serious harm of malaria, the World Health Organization began to implement a "Global Malaria Elimination Plan" in 1957, but the results have not been obvious so far. The reason is that since the 1960s, malaria parasites have become resistant to major antimalarial drugs such as chloroquine, making these drugs lose their antimalarial efficacy. For this reason, many countries in the world have invested a lot of manpower and material resources to research and d...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/53C12N9/02C12N15/79
Inventor 廖志华陈敏谌容
Owner SOUTHWEST UNIV
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