Artemisia apiacea MYB type transcription factor coding sequence AaMIXTA1 and application

Abstract

The invention discloses an artemisia apiacea MYB type transcription factor coding sequence AaMIXTA1. The nucleotide sequence of the coding sequence AaMIXTA1 is shown as SEQ ID NO.1, and the coding amino acid sequence of the coding sequence AaMIXTA1 is shown as SEQ ID NO.2. According to the artemisia apiacea MYB type transcription factor coding sequence AaMIXTA1, the density of secretory type glandular hairs of artemisia apiacea can be regulated and controlled; an artemisia apiacea AaMIXTA1 transcription factor overexpression vector is stably transformed into artemisia apiacea through a transgenic technology, therefore, the density of the secretory type glandular hairs on the artemisia apiacea epidermis can be effectively increased, and then the content of artemisinin in transgenic artemisia apiacea is increased; an artemisia apiacea AaMIXTA1 transcription factor interference vector is stably transformed into the artemisia apiacea through the transgenic technology, therefore, the density of the secretory type glandular hairs on the artemisia apiacea epidermis can be effectively decreased, and then the content of artemisinin in the transgenic artemisia apiacea is decreased. The artemisia apiacea MYB type transcription factor coding sequence AaMIXTA1 has the important significance on providing a novel high-yield stable medicine source for scale production of artemisinin.

Description

technical field

[0001] The invention relates to the technical field of genetic engineering, in particular to an Artemisia annua MYB transcription factor coding sequence AaMIXTA1 and its application. Background technique

[0002] Artemisia annua (Artemisia annua L.) is an annual herb belonging to the family Asteraceae. The sesquiterpene lactone oxide artemisinin, which is extracted from its aerial part and contains a peroxy bridge, is currently the most widely used anti-malarial drug with the best therapeutic effect, especially for cerebral malaria and anti-chloroquine malaria. Currently, artemisinin-based combination therapies (ACTs) are the most effective treatment for malaria recommended by the World Health Organization (WHO). However, the content of artemisinin in the plant Artemisia annua is low, and it cannot fully meet the annual demand of 200 tons in the global market. Artemisia annua has secretory glandular trichomes and non-glandular trichomes. There are a large ...

Examples

Embodiment 1

[0033] Embodiment 1, the cloning of Artemisia annua AaMYBL1 gene

[0034] 1. Extraction of Total RNA from Artemisia annua Genome

[0035] Take the leaf tissue of Artemisia annua, grind it in liquid nitrogen, add it to a 1.5mL Eppendorf 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.

[0036] 2. Cloning of Artemisia annua AaMIXTA1 gene

[0037] Using the extracted total RNA as a template, synthesize cDNA under the action of PowerScript reverse transcriptase; design gene-specific primers according to the sequence of the AaMIXTA1 gene, as shown in Table 1, amplify the AaMIXTA1 gene from the total cDNA by PCR, and sequencing.

[0038] Through the above steps, the full-length coding sequence (SEQ ID NO: 1) of the transcription factor in Artemisia annua was obtained and i...

Embodiment 2

[0043] Embodiment 2, the construction of the plant overexpression vector containing AaMIXTA1 gene

[0044] The AaMIXTA1 gene was constructed on the overexpression vector. In order to facilitate the construction of the expression vector, the restriction site of BamHI was introduced into the forward primer, and the restriction site of XbaI was introduced into the reverse primer. The construction map of the overexpression vector of AaMIXTA1 gene shown in Figure 1a , the primers are shown in Table 3;

[0045] Table 3 PCR primers constructed by AaPDR1-pHB vector

[0046]

Embodiment 3

[0047] Embodiment 3, the construction of the plant interference expression vector containing AaMIXTA1 gene

[0048] 1. Construction of intermediate vector pENTY-AaMIXTA1

[0049] Design upstream primers and downstream primers in the non-conserved region of AaMIXTA1 gene to construct interference vectors. Add CACC four bases before the ATG base of the upstream primer to construct the Gateway entry vector. According to Invitrogen pENTR TM / D- The operating steps of the Cloning Kit are to first amplify the AaMIXTA1 fragment with flat-end enzyme, recover and purify it, and connect it to the pENTR / D-TOPO vector through Gateway cloning technology.

[0050] 2. Construction of plant expression interference vector pHELLSGATE12-AaMIXTA1

[0051] According to Invitrogen LR The operation of II Enzyme kit, the interference fragment of AaMIXTA1 in the pENTR-AaMIXTA1 vector is recombined into two recombination sites that can form a hairpin structure in the RNA interference vector pH...

technical field

[0001] The invention relates to the technical field of genetic engineering, in particular to an Artemisia annua MYB transcription factor coding sequence AaMIXTA1 and its application. Background technique

[0002] Artemisia annua (Artemisia annua L.) is an annual herb belonging to the family Asteraceae. The sesquiterpene lactone oxide artemisinin, which is extracted from its aerial part and contains a peroxy bridge, is currently the most widely used anti-malarial drug with the best therapeutic effect, especially for cerebral malaria and anti-chloroquine malaria. Currently, artemisinin-based combination therapies (ACTs) are the most effective treatment for malaria recommended by the World Health Organization (WHO). However, the content of artemisinin in the plant Artemisia annua is low, and it cannot fully meet the annual demand of 200 tons in the global market. Artemisia annua has secretory glandular trichomes and non-glandular trichomes. There are a large ...

Examples

Embodiment 1

[0033] Embodiment 1, the cloning of Artemisia annua AaMYBL1 gene

[0034] 1. Extraction of Total RNA from Artemisia annua Genome

[0035] Take the leaf tissue of Artemisia annua, grind it in liquid nitrogen, add it to a 1.5mL Eppendorf 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.

[0036] 2. Cloning of Artemisia annua AaMIXTA1 gene

[0037] Using the extracted total RNA as a template, synthesize cDNA under the action of PowerScript reverse transcriptase; design gene-specific primers according to the sequence of the AaMIXTA1 gene, as shown in Table 1, amplify the AaMIXTA1 gene from the total cDNA by PCR, and sequencing.

[0038] Through the above steps, the full-length coding sequence (SEQ ID NO: 1) of the transcription factor in Artemisia annua was obtained and i...

Embodiment 2

[0043] Embodiment 2, the construction of the plant overexpression vector containing AaMIXTA1 gene

[0044] The AaMIXTA1 gene was constructed on the overexpression vector. In order to facilitate the construction of the expression vector, the restriction site of BamHI was introduced into the forward primer, and the restriction site of XbaI was introduced into the reverse primer. The construction map of the overexpression vector of AaMIXTA1 gene shown in Figure 1a , the primers are shown in Table 3;

[0045] Table 3 PCR primers constructed by AaPDR1-pHB vector

[0046]

Embodiment 3

[0047] Embodiment 3, the construction of the plant interference expression vector containing AaMIXTA1 gene

[0048] 1. Construction of intermediate vector pENTY-AaMIXTA1

[0049] Design upstream primers and downstream primers in the non-conserved region of AaMIXTA1 gene to construct interference vectors. Add CACC four bases before the ATG base of the upstream primer to construct the Gateway entry vector. According to Invitrogen pENTR TM / D- The operating steps of the Cloning Kit are to first amplify the AaMIXTA1 fragment with flat-end enzyme, recover and purify it, and connect it to the pENTR / D-TOPO vector through Gateway cloning technology.

[0050] 2. Construction of plant expression interference vector pHELLSGATE12-AaMIXTA1

[0051] According to Invitrogen LR The operation of II Enzyme kit, the interference fragment of AaMIXTA1 in the pENTR-AaMIXTA1 vector is recombined into two recombination sites that can form a hairpin structure in the RNA interference vector pH...

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