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Method for promoting terpene indole alkaloid content of Vinca rosea hairly root by transferring g10h gene

A technology of indole alkaloids and periwinkle, applied in biochemical equipment and methods, botanical equipment and methods, genetic engineering, etc., can solve undiscovered problems, increase content, and meet large-scale factory production in the pharmaceutical industry The effect of needing 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

Using genetic engineering means to transform the key enzyme gene g10h into periwinkle will break the speed-limiting bottleneck of TIAs biosynthesis, obtain periwinkle hairy roots or plants with high yield of periwinkle TIAs, and provide a new way for large-scale production of periwinkle TIAs approach, but have not yet found relevant reports on improving the content of TIAs in the hairy root of Changchun flower with the transformation g10h key enzyme gene mentioned in the subject of the present invention

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0019] Cloning of the g10h gene of periwinkle

[0020] 1. Isolation

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

Embodiment 2

[0030] Construction of Plant Binary Expression Vector Containing g10h Gene

[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 +g10h

[0034] With the described pCAMBIA1304 + For the expression vector, replace the gfp+gus gene on it with g10h in Example 1. Specifically, Bg1II / BstEII double enzyme cut pGEM T-easy+g10h and pCAMBIA1304 + , recovery of g10h and pCAMBIA1304 + Large fragments, ligated transformation, picking single clones, extracting plasmids for PCR detection and enzyme digestion verification. The results show that th...

Embodiment 3

[0037] Agrobacterium rhizogenes mediated g10h gene genetic transformation of periwinkle to obtain transgenic hairy roots

[0038] 1. Acquisition of plant expression vector Agrobacterium rhizogenes containing g10h gene

[0039] The plant expression vector containing the g10h gene in Example 2 was transformed into Agrobacterium rhizogenes (such as C58C1) and verified by PCR. The results showed that the plant expression vector containing g10h gene had been successfully constructed into the strain of Agrobacterium rhizogenes.

[0040] 2. Agrobacterium rhizogenes mediated transformation of periwinkle with g10h gene

[0041] 2.1. Preculture of explants

[0042] 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.

[0043] 2.2. Co-cultivation of Agrobacterium and explants

[0044] Put the described pre-cultivated periwinkle leaves and stem sec...

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Abstract

The invention discloses a Vinca rosea trichoid root terpenes indole alkaloid content improving method of trans-g10h gene in the biological domain, which is characterized by the following: cloning g10h gene form vinca rosea; constructing plant expression carrier of g10h gene; gaining trans-gene Vinca rosea trihoid root by genetic conversing Vinca rosea; measuring the alkaloid content of Vinca rosea trichoid root by HPLC; measuring relative gene expression of biological synthesis of Vinca rosea trichoid root alkaloid by fluorescence quantitative PCR. The important alkaloid content of trans-gene Vinca rosea trihoid root from the invention gets a distinctive increasing and 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 transforming a geraniol 10-decarboxylase (G10H) gene to improve the content of vinca hairy root terpene indole alkaloids (TIAs). 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 used clinically, and i...

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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