Coding sequence of AaMYBL1 protein of artemisia apiacea and application thereof

A coding sequence, Artemisia annua technology, applied in the field of genetic engineering, can solve the problem of not regulating glandular hair density and other problems

Active Publication Date: 2014-11-19
SHANGHAI JIAO TONG UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

In the field of secondary metabolism, there are relatively few reports on secretory glandular hairs
Especially in the study of the traditional Chinese herbal medicine Artemisia annua, the research mainly focuses on the transcriptional regulation of the synthesis key enzyme genes, and there is no report on the regulation of glandular hair density.

Method used

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  • Coding sequence of AaMYBL1 protein of artemisia apiacea and application thereof
  • Coding sequence of AaMYBL1 protein of artemisia apiacea and application thereof
  • Coding sequence of AaMYBL1 protein of artemisia apiacea and application thereof

Examples

Experimental program
Comparison scheme
Effect test

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 (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 formaldehyde denaturing gel electrophoresis, and then the RNA content was determined on a spectrophotometer.

[0036] 2. Cloning of Artemisia annua AaMYBL1 gene

[0037]Using the extracted total RNA as a template, cDNA was synthesized under the action of PowerScript reverse transcriptase; gene-specific primers (SEQ ID NO: 1 and SEQ ID NO: 2) were designed according to the sequence of the AaMYBL1 gene, and the total cDNA was obtained by PCR. The AaMYBL1 gene was amplified and sequenced.

[0038] Through the above steps, the full-length coding sequence (SEQ ID NO: 3) of the...

Embodiment 2

[0039] Embodiment 2, the construction of the plant binary interference expression vector containing AaMYBL1 gene

[0040] 1. Construction of intermediate vector pENTY-AaMYBL1

[0041] Design upstream primers and downstream primers in the non-conserved region of AaMYBL1 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 AaMYBL1 fragment with flat-end enzyme, recover and purify it, and connect it to the pENTR / D-TOPO vector through Gateway cloning technology.

[0042] 2. Construction of plant expression interference vector pHELLSGATE-AaMYBL1i

[0043] According to Invitrogen LR The operation of the II Enzyme kit is to recombine the interference fragment of AaMYBL1 in the pENTR-AaMYBL1 vector into two recombination sites that can form a hairpin structure in the RNA interference vecto...

Embodiment 3

[0045] Example 3. Agrobacterium tumefaciens-mediated AaMYBL1 interference vector genetically transforms Artemisia annua to obtain transgenic Artemisia annua plants

[0046] 1. Acquisition of Agrobacterium tumefaciens Engineering Bacteria Containing AaMYBL1 Interference Expression Vector

[0047] The plant binary interference expression vector containing AaMYBL1 in Example 2 was transformed into Agrobacterium tumefaciens by freeze-thaw method (such as EHA105, which is a publicly available biological material in the market, which can be purchased from CAMBIA Company in Australia, and the strain number is Gambar1) , and validated by PCR. The results showed that the plant binary interference expression vector containing AaMYBL1 had been successfully constructed into the Agrobacterium tumefaciens strain.

[0048] 2. Agrobacterium tumefaciens mediated AaMYBL1 gene transformation of Artemisia annua

[0049] 2.1. Preculture of explants

[0050] Artemisia annua seeds were soaked i...

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Abstract

The invention relates to a coding sequence of an AaMYBL1 protein of artemisia apiacea and an application thereof. The amino acid sequence coded by the coding sequence AaMYBL1 of an MYB-like type transcription factor of artemisia apiacea is shown as SEQ ID NO:4. The AaMYBL1 which codes a R3MYB type transcription factor takes part in regulation of density of glandular hairs of artemisia apiacea. An interference vector of the AaMYBL1 transcription factor of artemisia apiacea is transformed into artemisia apiacea by means of the transgenic technology, so that the density of the glandular hairs on the surface of artemisia apiacea can be effectively regulated, thus, the content of artemisinin is improved. The density of the glandular hairs on the surface of a blade of non-transgenic common artemisia apiacea is 24/square millimeter, and the density of the glandular hairs of a blade of transgenic artemisia apiacea inhibiting AaMYBL1 genetic expression is increased to 34/square millimeter; the quantity of total glandular hairs of each blade is increased from 61947 to 93683; correspondingly, the content of artemisinin is improved from 8mg/g DW of non-transgenic artemisia apiacea to 12mg/g DW. The coding sequence provided by the invention is of significance for providing a high-yield stable novel medicine source for scaled 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 AaMYBL1 protein coding sequence and application thereof. Background technique [0002] Plant metabolism is divided into primary metabolism and secondary metabolism. Primary metabolites (such as sugars, lipids and nucleic acids) exist in all plants and are necessary to maintain cell life activities, while plant secondary metabolites refer to a plant. A large class of small molecular organic compounds that are not necessary for plant growth and development, and their production and distribution are specific to species, tissues and organs, and growth and development. The surface of most plant leaves is not smooth, but has many types of hair-like structures. According to their function and shape, they are divided into two categories: one is cilia without secretory function; the other is glandular hair with secretory function. The main repr...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/29C07K14/415C12N15/84A01H5/00
Inventor 唐克轩张芳源
Owner SHANGHAI JIAO TONG UNIV
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