Method for promoting terpene indole alkaloid content of Vinca rosea hairly root by transcription factor

A technology of indole alkaloids and transcription factors, applied in the biological field, can solve the problems that have not yet been discovered, and achieve the effects of increasing the content, meeting the needs of large-scale industrial production in the pharmaceutical industry, and solving the shortage of drug sources.

Inactive Publication Date: 2006-07-26
SHANGHAI JIAO TONG UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Because the g10h gene is not induced by ORCA3, the transcriptional regulator gene orca3 and the rate-limiting enzyme gene g10h are co-transformed into vinca, and the metabolic flow will continue to flow due to the global regulation of the transcriptional regulator ORCA3 and the role of G10H in breaking the rate-limiting bottleneck Flow toward TIAs biosynthetic direction, thereby obtain the periwinkle hairy root or plant of high yield of periwinkle TIAs, provide a new way for the large-scale production of periwinkle TIAs, but have not yet found the adoption transcription factor and key that mention with the theme of the present invention Enzyme gene co-transformation strategy to increase TIAs content in periwinkle hairy roots

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0019] Cloning of periwinkle orca3 and g10h genes

[0020] 1. Isolation

[0021] Soak the periwinkle seeds in 75% ethanol for 1 min, then soak in 2% NaClO for 10 min, rinse with sterile water 3-4 times, dry the surface with sterile absorbent paper, and inoculate them in hormone-free MS (Murashige and Skoog, 1962) Cultivate in solid medium at 25°C under 12h / 12h light to obtain sterile periwinkle seedlings. After the seedlings grow to about 5cm, cut the leaves and stems for RNA extraction.

[0022] 2. RNA isolation (RNA isolation)

[0023] Weigh 0.5 g of the periwinkle aseptic test-tube seedlings, quickly freeze them with liquid nitrogen, grind them quickly with a mortar, add them to a 1.5 mL Eppendorf tube filled with 1 mL TRIzol (TRIzol Reagents, GIBCO BRL, USA), shake them fully Afterwards, stand at room temperature for 5 minutes, add 200 μL of chloroform, shake vigorously for 15 sec, place at room temperature for 2-3 minutes, then centrifuge at 12,000 g for 15 minutes at 4...

Embodiment 2

[0030] Construction of plant binary expression vector containing orca3 and g10h genes

[0031] 1. Intermediate vector pCAMBIA1304 + build

[0032] Select pBI121 and pCAMBIA1304 as the basic elements to construct the binary plant expression vector pCAMBIA1304 + . Specifically, pBI121 and pCAMBIA1304 were digested with HindIII and EcoRI; the GUS expression cassette of pBI121 and the large fragment of pCAMBIA1304 were recovered; the recovered products were ligated, transformed and screened, and verified by plasmid digestion.

[0033] 2. Plant expression vector pCAMBIA1304 + + build of orca3

[0034] With the described pCAMBIA1304 + For the expression vector, replace the gus gene on it with orca3 in Example 1. Specifically, XbaI / SacI double enzyme digestion of pGEM T-easy+str and pCAMBIA1304 + , recovery of orca3 and pCAMBIA1304 + Large fragments, ligated transformation, picking single clones, extracting plasmids for PCR detection and enzyme digestion verification.

[003...

Embodiment 3

[0039] Agrobacterium rhizogenes mediated orca3 and g10h gene genetic transformation of periwinkle to obtain transgenic hairy roots

[0040] 1. Obtaining the engineering bacteria of Agrobacterium rhizogenes containing orca3 and g10h gene binary plant expression vector

[0041] The plant binary expression vector containing orca3 and g10h genes in Example 2 was transformed into Agrobacterium rhizogenes (such as C58C1), and PCR verification was performed. The results showed that the plant binary expression vector containing orca3 and g10h genes had been successfully constructed into the strain of Agrobacterium rhizogenes.

[0042] 2. Agrobacterium rhizogenes mediates orca3 and g10h gene transformation of periwinkle

[0043] 2.1. Preculture of explants

[0044] Cut periwinkle aseptic seedling blade (0.5cm * 0.5cm) and stem segment (0.5cm) explant in embodiment 1, inoculate on the pre-cultivation medium (MS+AS 100 μ mol / L), 25 ℃ of dark cultures 2d.

[0045] 2.2. Co-cultivation ...

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Abstract

The invention discloses a Vinca rosea trichoid root terpenes indole alkaloid content improving method of transcription factor in the biological domain, which is characterized by the following: cloning str and g10h clone form vinca rosea; gaining trans-gene Vinca rosea trihoid root by genetic conversing Vinca rosea; measuring the alkaloid content of Vinca rosea trichoid root by LC; measuring relative gene expression of biological synthesis of Vinca rosea trichoid root alkaloid by fluorescence quantitative PCRíú The important alkaloid content of trans-gene Vinca rosea trihoid root from the invention gets a distinctive increasing, which provides a new method of producing anti-cancer drugs VLB and LCR for clinic demand.

Description

technical field [0001] The invention relates to a method in the field of biotechnology, in particular to a method for increasing the content of terpene indole alkaloids (TIAs) in the hairy root of vinca by adopting a transcription factor metabolic engineering strategy. Background technique [0002] Vinca (Catharanthus roseus) is a plant of the genus Vinca in the family Apocynaceae. At present, more than 100 terpenoid indolealkaloids (Terpenoid indolealkaloids, TIAs) have been isolated from the whole plant of Vinca, such as Vinblastine (Vinblastine). ), Vincristine, Ajmalicine, Serpentine, Vindoline, Catharanthine, Lochneridine, Pivenli ( Perivine) and so on. Most TIAs are widely used in modern medicine due to their biological activity. For example, the famous antineoplastic drugs vinblastine and vincristine can be used to treat Hodgkin's disease, malignant lymphoma, acute lymphocytic leukemia, villous epithelial Cell carcinoma and other cancers, its sulfate has been widely...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/29C12N15/82C12Q1/68
Inventor 唐克轩龚一富廖志华苗志奇孙小芬
Owner SHANGHAI JIAO TONG UNIV
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