Preparation method of artemisinin AaORA protein, encoding gene and transgenic artemisinin plant

An artemisia annua and protein technology, applied in the protein field in the field of genetic engineering technology, can solve the problem that a single gene modification is difficult to work, and achieve the effect of reducing the content of artemisinin

Active Publication Date: 2012-07-11
SHANGHAI JIAO TONG UNIV SUBEI RES INST
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  • Abstract
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  • Claims
  • Application Information

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Problems solved by technology

Plant metabolic pathways are mostly multi-step reactions involving multiple enzymes. Due to the influ

Method used

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  • Preparation method of artemisinin AaORA protein, encoding gene and transgenic artemisinin plant
  • Preparation method of artemisinin AaORA protein, encoding gene and transgenic artemisinin plant
  • Preparation method of artemisinin AaORA protein, encoding gene and transgenic artemisinin plant

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0026] Embodiment 1, the cloning of Artemisia annua AaORA gene

[0027] 1.1. Extraction of Total RNA from Artemisia annua Genome

[0028] 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.

[0029] The quality of total RNA was identified by formaldehyde denaturing gel electrophoresis, and then the RNA content was determined on a spectrophotometer.

[0030] 1.2. RACE cloning of the AaORA gene of Artemisia annua

[0031] According to the conserved sequence of amino acids encoded by related genes in Arabidopsis, using the principle of homologous gene cloning, the RACE (Rapid Amplification of cDNA Ends) method (SMART of Clontech Company) was adopted. TM RACE kit) for full-length cDNA cloning in three stages:

[0032] 1.2.1. Synthesis of first-strand cDNA

[0033] Take advantage of SMART TM The 5'-CDS...

Embodiment 2

[0040] Embodiment 2, the construction of the plant binary interference expression vector containing AaORA gene

[0041] 2.1. Construction of intermediate vector pBlucescript::AaORA

[0042] The pBlucescriptSK+ and gus gene fragments were selected as basic components to construct the interference expression cassette pBlucescriptSK+::gus. Specifically, SmaI single-digested pBlucescriptSK+ and the plasmid containing the gus gene; recovered the gus fragment and the large pBlucescriptSK+ fragment; ligated the recovered product, transformed and screened, and verified by digesting the plasmid pBlucescriptSK+::gus.

[0043] The AaORA-specific fragments were inserted into the pBlucescriptSK+::gus vector in forward and reverse directions, respectively. Specifically, pGEM T-easy+AaORA and pBlucescriptSK+::gus were digested with XhoI / HindIII, the large fragments of AaORA and pBlucescriptSK+::gus were recovered, connected and transformed, single clones were picked, and the plasmid pBluc...

Embodiment 3

[0047] Example 3. Agrobacterium tumefaciens-mediated genetic transformation of Artemisia annua to obtain transgenic Artemisia annua plants

[0048] 3.1. Acquisition of Agrobacterium tumefaciens Engineering Bacteria Containing AaORA Interference Expression Vector

[0049] The plant binary interference expression vector containing AaORA in Example 2 was transformed into Agrobacterium tumefaciens (EHA105, which is a publicly available biological material in the market, which can be purchased from Australia CAMBIA company, and the strain number is Gambar 1) by using the freeze-thaw method , and validated by PCR. The results showed that the plant binary interference expression vector containing AaORA had been successfully constructed into the Agrobacterium tumefaciens strain.

[0050] 3.2 Agrobacterium tumefaciens mediated AaORA gene transformation of Artemisia annua

[0051] 3.2.1. Pre-cultivation of explants

[0052] Artemisia annua seeds were soaked in 75% ethanol for 1 min...

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Abstract

The invention relates to a preparation method of artemisinin AaORA protein, an encoding gene and a transgenic artemisinin plant. The amino acid sequence of the artemisinin AaORA protein is shown as SEQ ID NO: 6. The invention also relates to a preparation method of a nucleic acid sequence for encoding the artemisinin AaORA protein, a plant interference expression vector, an agrobacterium tumefacien strain and the transgenic artemisinin plant. An AaORA gene is cloned from artemisinin to construct the plant interference expression vector containing the AaORA gene, agrobacterium tumefacien EH105 is used as a medium to convert the AaORA genetic interference expression vector into the artemisinin; and the integration of an external target gene AaORA is detected by PCR (polymerase chain reaction), the artemisinin content in the artemisinin is determined by a high-efficiency liquid chromatography-evaporative light scattering detector, and the transgenic artemisinin plant with the artemisinin content reduced significantly is prepared via screening.

Description

technical field [0001] The invention relates to a protein in the technical field of genetic engineering, in particular to a method for obtaining an Artemisia annua AaORA protein, its encoding gene, and a transgenic Artemisia annua plant. 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. In recent years, with the gradual deepening of the research on the components of Chinese herbal medicines, it has been found that the active ingredients of many Chinese herbal medicines are secondary metabolites of plants, such as artemi...

Claims

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Application Information

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IPC IPC(8): C07K14/415C12N15/29C12N15/84C12N1/21A01H5/00C12R1/01
Inventor 唐克轩陆续江伟民张凌张利达张芳源沈乾吕宗友高尔娣
Owner SHANGHAI JIAO TONG UNIV SUBEI RES INST
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