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Artemisia annua L. PDR sub-family transport protein and functional verification method and application thereof

A technology for transporter and function verification, applied in the field of Artemisia annua PDR subfamily transporter and its function verification, can solve the problems such as the inability to effectively meet the market demand and the low content of artemisinin

Active Publication Date: 2016-05-18
SHANGHAI JIAO TONG UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] Aiming at the problem that the content of artemisinin in the plant Artemisia annua is low and cannot effectively meet the market demand, the inventor considers that if through the analysis of the secretory glandular hair transcriptome database of Artemisia annua, the relevant transporters involved in the artemisinin synthesis pathway can be screened out , and its function is verified, then genetic engineering can be used to improve the transport efficiency of the transporter, thereby increasing the content of artemisinin in Artemisia annua

Method used

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  • Artemisia annua L. PDR sub-family transport protein and functional verification method and application thereof
  • Artemisia annua L. PDR sub-family transport protein and functional verification method and application thereof
  • Artemisia annua L. PDR sub-family transport protein and functional verification method and application thereof

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Experimental program
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Effect test

Embodiment 1

[0050] Embodiment 1, cloning of Artemisia annua AaPDR1 gene

[0051] 1. Extraction of Total RNA from Artemisia annua Genome

[0052] 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 agarose gel electrophoresis, and then the RNA content was determined on a spectrophotometer.

[0053] 2. Cloning of the AaPDR1 gene of Artemisia annua

[0054] Using the extracted total RNA as a template, cDNA was synthesized under the action of PowerScript reverse transcriptase; gene-specific primers were designed according to the sequence of the AaPDR1 gene, and the AaPDR1 gene was amplified from the total cDNA by PCR and sequenced. The inventor has completed the whole genome sequencing and transcriptome sequencing of Artemisia annua, and the sequence of AaPDR1 gene comes fro...

Embodiment 2

[0060] Embodiment 2, the construction of the yeast expression vector containing AaPDR1 gene

[0061] The AaPDR1 gene was constructed on the yeast expression vector. In order to facilitate the construction of the expression vector, the restriction site of SpelI was introduced into the forward primer, and the restriction site of PstI was introduced into the reverse primer. The primers are shown in Table 3;

[0062] The PCR primers that table 3AaPDR1-PDR196 vector constructs

[0063]

Embodiment 3

[0064] Example 3, AaPDR1 transporter yeast mutant AD12345678 transport experiment

[0065] 1. Transformation of yeast AD12345678

[0066] 1.1 Competent state preparation

[0067] Take out the AD12345678 strain at -70°C and streak it on the YPD medium, culture it at 28°C for three days, pick the faster-growing clones and culture them overnight in 10 mL of YPD liquid medium (28°C, 220rpm), and test the next day After OD600, dilute the bacterial solution to a concentration of 0.4, continue to shake the bacteria for 2-4 hours, centrifuge at room temperature 2500rpm for 10min, remove the supernatant, then suspend with 40mL 1×TE (or sterilized water), centrifuge at room temperature 2500rpm for 10min, carefully remove the supernatant , suspended in 2 mL of 1×LiAC / 0.5×TE, and left at room temperature for 10 minutes, they are yeast competent cells.

[0068] 1.2 Yeast Transformation

[0069] Add 1.5 μL of plasmid into a sterile 1.5mL centrifuge tube, then add 10 μL of 10 mg / mL salm...

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Abstract

The invention discloses Artemisia annua L. PDR sub-family transport protein. Amino acid sequences of the protein comprise the amino acid sequence shown as SEQ ID NO:2 or the amino acid sequence of the protein is shown as SEQ ID NO:2 or nucleic acid codes crossed by the protein and complementary chains of nucleic acid of protein with the coded amino acid sequence shown in SEQ ID NO:2 under the highly-strict condition are achieved. Furthermore, the protein is named AaPDR1 by the inventor and is transport protein specifically expressed by Artemisia annua L. secreting type gladular trichomes, and it is proved through a yeast transport experiment and interference of Artemisia annua L. in the transport protein that the AaPDR1 participates in transporting an artemisinin synthetic route intermediate product dihydroartemisinic acid in Artemisia annua L.. The Artemisia annua L. PDR sub-family transport protein has great significance in providing high-yield and stable new drug resources for large-scale production of artemisinin.

Description

technical field [0001] The invention belongs to the technical field of genetic engineering, and in particular relates to a PDR subfamily transporter of Artemisia annua and its function verification method and application. Background technique [0002] Artemisia annua L. is an annual herb belonging to the family Asteraceae. Artemisinin, a sesquiterpene lactone oxide containing a peroxy bridge extracted from its aerial part, is currently the most widely used and effective antimalarial drug, especially for cerebral malaria and anti-chloroquine malaria. . Currently, artemisinin combination therapies (ACTs) are the most effective treatment for malaria recommended by the World Health Organization. However, the content of artemisinin in the plant Artemisia annua is low, which cannot fully meet the global market demand. Artemisia annua has glandular trichomes and nonglandular trichomes. There are a large number of secretory glandular hairs on the front and back of leaves, stems ...

Claims

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Application Information

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IPC IPC(8): C07K14/415C12N15/29C12Q1/68A01H5/00
CPCC07K14/415C12N15/8243C12Q1/6895
Inventor 唐克轩付雪晴石璞刘萌陈明慧马亚男郝小龙黎凌刘品孙小芬
Owner SHANGHAI JIAO TONG UNIV
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