Artemisia apiacea MYB transcription factor AaMYB15 and application thereof

A technology of transcription factor and Artemisia annua, applied in the field of genetic engineering, can solve the problems such as the undiscovered AaMYB15 gene sequence, and achieve the effect of increasing yield and content

Active Publication Date: 2021-04-16
SHANGHAI JIAO TONG UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

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  • Artemisia apiacea MYB transcription factor AaMYB15 and application thereof
  • Artemisia apiacea MYB transcription factor AaMYB15 and application thereof

Examples

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Embodiment 1

[0038] Embodiment 1, the cloning of Artemisia annua AaMYB15 gene

[0039] 1. Extraction of Total RNA from Artemisia annua Genome

[0040] 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 concentration was determined on a spectrophotometer.

[0041] 2. Cloning of Artemisia annua AaMYB15 gene

[0042] Using the extracted Artemisia annua total RNA as a template, calculate the dosage according to the RNA concentration, and obtain cDNA under the action of PowerScript reverse transcriptase; design gene-specific primers according to the nucleotide sequence (SEQ ID NO:1) of the AaMYB15 gene, The AaMYB15 gene was amplified from the total cDNA by PCR. After the PCR product was recovered and purified, it was connected to ...

Embodiment 2

[0044] Embodiment 2, the construction of the plant expression vector containing AaMYB15 gene

[0045] 1. Construction of overexpression vector pHB-AaMYB15-YFP

[0046] The AaMYB15 gene was amplified from the correctly sequenced blunt-end vector pLB and constructed on the plant expression vector pHB-YFP. In order to facilitate the construction of the expression vector, the forward primer introduced the restriction site of BamHI, and the reverse primer introduced the The restriction site of SpeI, the primer sequence is as follows:

[0047] Forward primer MYB15-YFP-F: SEQ ID NO.3

[0048] Reverse primer MYB15-YFP-R: SEQ ID NO.4

[0049] 2. Construction of antisense interference expression vector pHB-AaMYB15-antisense

[0050] After reverse-complementing the correct AaMYB15 nucleotide sequence, design primers to amplify the reverse-complementary sequence of the AaMYB15 gene from the blunt-ended vector pLB, and construct it on the plant expression vector pHB-YFP, in order to f...

Embodiment 3

[0053] Example 3. Dual fluorescence of the promoters of the key enzyme genes ADS, CYP71AV1, DBR2 and ALDH1 in the biosynthesis of artemisinin Construction of prime reporter vector

[0054] 1. PCR amplification of the promoters of key enzyme genes ADS, CYP71AV1, DBR2 and ALDH1 in artemisinin biosynthesis

[0055] According to the sequence information of the four key enzyme genes of artemisinin biosynthesis in the NCBI database, the promoter amplification specific primers of ADS, CYP71AV1, DBR2 and ALDH1 were respectively designed, and HindIII and PstI restriction sites were added in the upstream and downstream of the primers, respectively.

[0056] 2. Link the promoter fragment into the dual-fluorescein reporter vector

[0057] Using the method of homologous recombination, construct the pGreenII0800-LUC vector through the ClonExpress II One Step Cloning Kit (Novazyme, Nanjing) kit, and obtain the plant double luciferin detection reporter vectors pGreenII0800-ProADS, pGreenI...

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Abstract

The invention relates to an artemisia apiacea MYB transcription factor AaMYB15 and application thereof in the technical field of plant biology. The nucleotide sequence of the transcription factor AaMYB15 is shown as SEQ ID NO: 1, and the amino acid sequence of the transcription factor AaMYB15 is shown as SEQ ID NO: 2. The transcription factor AaMYB15 disclosed by the invention can be used for inhibiting the activity of key enzyme genes ADS, CYP71AV1, DBR2 and ALDH1 promoters in an artemisinin biosynthesis pathway and regulating and controlling the expression of corresponding genes in a negative direction, so that the biosynthesis of artemisinin is inhibited; and the artemisinin content of the transgene can be remarkably reduced, the artemisinin content of the transgene artemisia apiacea can be remarkably increased, and no harm is caused to plant growth and development. The AaMYB15 transcription factor can be applied to increase of artemisinin yield through an antisense interference expression method, and is of great significance to genetic engineering breeding of artemisia apiacea and large-scale production of artemisinin.

Description

technical field [0001] The invention belongs to the technical field of genetic engineering, and in particular relates to an Artemisia annua MYB transcription factor AaMYB15 and its application. Background technique [0002] Artemisia annua L., also known as Artemisia annua L., is an annual herbaceous plant belonging to Artemisia annua species in the genus Artemisia in the Asteraceae family. Artemisinin is a sesquiterpene lactone compound with antimalarial effect existing in Artemisia annua. In addition to the currently irreplaceable antimalarial effect, it is also effective in the treatment of lupus erythematosus-associated nephritis, cancer, diabetes and other diseases. It has shown potential, so artemisinin still has a broad research prospect and a very large market demand. In the case that other synthetic methods have not been put into use in large quantities at present, Artemisia annua is the only plant source of artemisinin, but the content of artemisinin in wild-type ...

Claims

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Application Information

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IPC IPC(8): C12N15/29C07K14/415C12N15/84A01H6/14A01H5/00
CPCY02A50/30
Inventor 唐克轩吴张宽玉严欣黎凌刘航
Owner SHANGHAI JIAO TONG UNIV
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