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Farnesyl pyrophosphoric acid synthetase fusion gene carrying plastid transport peptide and application thereof

A technology of farnesyl pyrophosphate and fusion gene, which is applied in the field of plant genetic engineering, can solve the problems that artemisinin has not been reported, and achieve the effect of increasing the content and activity of artemisinin

Inactive Publication Date: 2011-08-31
SOUTHWEST UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] At present, most of the relevant researches on Artemisia annua at home and abroad focus on the separation and extraction process of artemisinin, semi-synthesis or total synthesis, pharmacological effects, tissue culture, etc. It has not been reported that important related genes are transferred into Artemisia annua with high expression and stable inheritance, so as to obtain the content of artemisinin

Method used

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  • Farnesyl pyrophosphoric acid synthetase fusion gene carrying plastid transport peptide and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0029] Example 1: Cloning of the transit peptide lstp of Artemisia annua linalool synthase

[0030] Using the total RNA of Artemisia annua as a template, the 72bp lstp was amplified by RT-PCT method, and the amplified product was recovered and cloned by TA.

[0031] (1) RT-PCR amplification of the target fragment

[0032] Specific primers were designed according to the nucleotide sequence of the transit peptide of Artemisia annua linalool synthase published in GenBank, the Spe I restriction site was introduced into the upstream primer, and the Aat II restriction site was introduced into the downstream primer.

[0033] Upstream primer: 5'-cccactagtatggcatcaattagcttatttcc-3'

[0034] Downstream primer: 5'-cccgacgtctcttgctagaggacttg-3'

[0035]Extract the total RNA of Artemisia annua (the RNA extraction kit of Shanghai Huashun Biological Engineering Co., Ltd.), use RNA as a template, reverse transcribe into cDNA (TaKaRa RNA PCR 3.0Kit), use the above primers to carry out PCR am...

Embodiment 2

[0039] Embodiment 2: Artemisia annua farnesyl pyrophosphate synthase gene fps clone

[0040] Design specific primers according to GenBank published cDNA sequence information of Artemisia annua fps gene (GenBank accession number: AF112881), introduce Aat II restriction site at the 5' end, and remove the initiation codon ATG of the fps coding region, downstream The primers introduce the Bste II restriction site. The size of the PCR product should be 1042bp.

[0041] Upstream primer: 5'-cccgacgtcagtagtaccgatctgaaa-3'

[0042] Downstream primer: 5'-cccggtgaccctacttttgcctcttgtaaa-3'

[0043] Extract the total RNA of Artemisia annua (the RNA extraction kit of Shanghai Huashun Biological Engineering Co., Ltd.), use RNA as a template, reverse transcribe into cDNA (TaKaRa RNA PCR 3.0Kit), use the above primers to carry out PCR amplification with cDNA as a template, Prepare fps fragments. TA cloning was then performed. The PCR reaction conditions were 94°C for 3 minutes, followed b...

Embodiment 3

[0044] Embodiment 3: utilize pCAMBIA1304 vector to construct lstp-fps fusion gene

[0045] The vector plasmid pCAMBIA1304 was extracted from Escherichia coli, and the large vector fragment was recovered by double enzyme digestion with Spe I / Bste II.

[0046] The plasmid was extracted from the TA clone prepared in Example 1, digested with Aat II / Spe I, and the lstp fragment was recovered by agarose gel electrophoresis.

[0047] The plasmid was extracted from the TA clone prepared in Example 2, digested with Aat II / Bste II, and the fps fragment was recovered by agarose gel electrophoresis.

[0048] The above three fragments were ligated overnight at 16° C. under the catalysis of ligase to complete the construction of the lstp-fps fusion gene on the pCAMBIA1304 vector. The connection system is: T4 DNALigase 1ul, T4 DNALigase Buffer 1ul, pCAMBIA1304 vector recovery fragment 5ul, lstp recovery fragment 1.5ul, fps recovery fragment 1.5ul. The ligation mixture was transformed into ...

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Abstract

The construction and application of fusion gene of Farnesyl Pyrophosphate synthase carrying plastid transit peptide belongs to plant genetic engineering field. This invention uses the transit peptide lstp of artesunate linalool synthase and fps gene of Farnesyl Pyrophosphate synthase to construct the fusion gene lstp-fps, and transferred the fusion gene into artesunate, to make the transgenic artesunate increase artemisinin.

Description

technical field [0001] The invention belongs to the field of plant genetic engineering, and specifically uses the transit peptide lstp of artemisia annua linalool synthase and the gene fps of farnesyl pyrophosphate synthase; constructs lstp-fps fusion gene and its application in transgenic artemisia annua . 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...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/62C12N15/52C12N9/00C12N15/82
Inventor 廖志华陈敏谌容
Owner SOUTHWEST UNIV
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