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AaALDH1 gene promoter and use thereof

A promoter and gene technology, applied in genetic engineering, plant genetic improvement, DNA/RNA fragments, etc., can solve problems such as normal growth burden and harm of plants

Active Publication Date: 2016-04-20
SHANGHAI JIAO TONG UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

This promoter can drive the expression of foreign genes in all tissues and organs, which may cause certain burden and harm to the normal growth of plants

Method used

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  • AaALDH1 gene promoter and use thereof
  • AaALDH1 gene promoter and use thereof
  • AaALDH1 gene promoter and use thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0035] The acquisition of embodiment 1AaALDH1 gene promoter

[0036] 1. Cultivate aseptic seedlings of Artemisia annua

[0037] Artemisia annua seeds were soaked in 75% ethanol for 1 min, then soaked in 20% (w / v) NaClO for 20 min, rinsed with sterile water 3-4 times, blotted the surface moisture with sterile absorbent paper, and inoculated Cultivate on hormone-free MS solid medium at 25°C and 16h / 8h (sunlight / dark) light, after 14 days, you can get sterile seedlings of Artemisia annua.

[0038] 2. Genomic DNA extraction

[0039] Put a leaf of Artemisia annua (1cm 2 About the size, take it in an ice box), add 2 steel balls. Add 300μl TPSbuffer (operate in a fume hood, TPS contains mercaptoethanol), shake at 55-60Hz for 90s. Then add 300 μL TPSbuffer (fume hood). Water bath at 65°C for 1h (shake every 20min), and the time can be extended appropriately, up to 1.5h. Cool to room temperature and centrifuge at 10,000 rpm for 15 min at 4°C. Take 300 μL-400 μL of supernatant. ...

Embodiment 2

[0050] Example 2 Analysis of the cis-acting elements of the promoter proAaALDH1

[0051] The sequence length of the promoter proAaALDH1 obtained in Example 1 is 1620bp. In order to find cis-acting elements on the promoter, the promoter proAaALDH1 was analyzed with Plantcare (http: / / bioinformatics.psb.ugent.be / webtools / plantcare / html / ). The analysis found that the cis-acting elements on the promoter include: TATAbox, CAATbox, AE-box, ARE, GA-motif, GARE-motif and TCCC-motif, the details are shown in Table 4.

[0052] Table 4 Analysis of regulatory elements in the promoter proAaALDH1 sequence

[0053]

Embodiment 3

[0054] Example 3 The AaALDH1 gene promoter proAaALDH1 is connected into the pCAMBIA-1391z vector and transformed into Agrobacterium

[0055] The promoter proAaALDH1 of the AaALDH1 gene was fused with the GUS reporter gene, and the expression of the promoter driving the GUS gene in different tissue parts was studied. In order to facilitate the construction of the expression vector, a HindIII restriction site was introduced into the forward primer, and a BamHI restriction site was introduced into the reverse primer. The primers are shown in Table 5. The proAaALDH1 sequence added with the above two restriction sites was ligated into the pCAMBIA-1391z vector to obtain the pCAMBIA1391z-proAaALDH1 vector.

[0056] Subsequently, the pCAMBIA1391z-proAaALDH1 plant expression vector was transformed into Agrobacterium tumefaciens (EHA105).

[0057] Table 5 PCR primers used in the construction of pCAMBIA1391z-proAaALDH1 vector

[0058]

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Abstract

The invention discloses a promoter. The promoter can regulate and control the specific expression of a gene in secretion-type glandular hair, and a nucleotide sequence of the AaALDH1 gene promoter is shown as SEQ ID NO.1. The invention also provides an application of the promoter in the genetic engineering breeding by adopting plant secretion-type glandular hair tissues to express and produce metabolite. In addition, the invention also discloses a carrier and an expression cassette containing the promoter. By adopting the AaALDH1 gene promoter, a foundation is set for researching the expression regulation and control of the AaALDH1 gene and analyzing a cis acting element on the promoter, and the important significance on the genetic engineering breeding utilizing the plant secretion-type glandular hair tissues to express and produce the metabolite also can be realized.

Description

technical field [0001] The invention belongs to the field of bioengineering and relates to a promoter and its application, in particular to a transporter gene promoter (proAaALDH1) specifically expressed in plant secretory glandular hairs and its application in transgenic plants. Background technique [0002] Artemisia annua (Artemisia annua L.) is an annual herb of the genus Artemisia in the family Asteraceae. A sesquiterpene lactone peroxide, artemisinin, can be extracted from Artemisia annua. The World Health Organization calls it "the only effective malaria treatment in the world". Currently, the most effective treatment for malaria recommended by the World Health Organization is artemisinin-based combination therapies (ACTs). Plant Artemisia annua is currently the only natural resource to obtain artemisinin in the world, but its artemisinin synthesis is extremely low, only 0.1-0.8% of dry weight, which limits the large-scale commercial production of this drug. [0003...

Claims

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Application Information

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IPC IPC(8): C12N15/113C12N15/82C12N15/84A01H5/00
CPCC12N9/0008C12N15/8205C12N15/8243C12Y102/0101
Inventor 唐克轩刘萌石璞付雪晴沈乾何倩唐岳立孙小芬
Owner SHANGHAI JIAO TONG UNIV
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