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Method for increasing content of vinca alkaloids in vinca by corotation of orca3/g10h genes

A periwinkle and alkaloid technology, which is applied in biochemical equipment and methods, botanical equipment and methods, angiosperms/flowering plants, etc., can solve the problems of high price, no commercial value, and inability to meet market demand, etc. The effect of reducing production costs

Active Publication Date: 2012-10-03
成都上交致远生物科技有限公司
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  • Abstract
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Problems solved by technology

[0003] However, since the content of these drugs in periwinkle is extremely small (only a few parts per million to a few parts per hundred thousand), the extraction of TIAs from natural plants is far from meeting the market demand, resulting in the shortage of medicinal components of periwinkle. The contradiction between supply and demand is quite serious (only 12kg of vinblastine and 1kg of vincristine can be produced each year), which makes the price extremely expensive (up to 6,000 US dollars / gram), and the use of chemical synthesis and semi-synthesis has no commercial value because the cost is too high

Method used

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  • Method for increasing content of vinca alkaloids in vinca by corotation of orca3/g10h genes
  • Method for increasing content of vinca alkaloids in vinca by corotation of orca3/g10h genes
  • Method for increasing content of vinca alkaloids in vinca by corotation of orca3/g10h genes

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0024] Example 1 Cloning of periwinkle orca3 and g10h genes

[0025] 1. Extraction of periwinkle genome total RNA

[0026] The total RNA of periwinkle genome is extracted by using the RNAprep pure plant total RNA extraction kit method provided by Tiangen Biochemical Technology Co., Ltd. The method steps are:

[0027] (1) Take 50-100 mg of periwinkle leaves and quickly grind them into powder in liquid nitrogen, add 450 μL RL (provided by the kit, add β-mercaptoethanol before use), and vortex vigorously to mix;

[0028] (2) Transfer all the solution to the filter column CS (provided by the kit, the filter column CS is placed in the collection tube), centrifuge at 12,000rmp for 2-5 minutes, carefully pipette the supernatant in the collection tube to RNase-free centrifugation In the tube, the tip should try to avoid contact with the cell debris in the collection tube;

[0029] (3) Slowly add 0.5 times the supernatant volume of absolute ethanol (usually 225 μL), mix well (precipi...

Embodiment 2

[0041] Example 2 Construction of plant binary expression vectors containing orca3 and g10h genes

[0042] 1. Construction of intermediate vectors pMDT18-g10h and pMDT18-orca3

[0043] The pMDT18 vector (Takara, Dalian) was selected as the basic element to construct the intermediate vectors pMDT18-g10h and pMDT18-orca3. Specifically, high-fidelity enzymes were used to amplify the full lengths of orca3 and g10h genes, and at the same time, SpeI and BstEII restriction sites were introduced before and after the orca3 gene, and NcoI and BstEII restriction sites were introduced at both ends of the g10h gene. The full length of the gene with restriction sites was ligated to the pMDT18 vector by ligase, and the correctness of the gene was confirmed by sequencing by Shanghai Yingjun Biotechnology Service Co., Ltd.

[0044] 2. Construction of single-gene plant expression cassettes CaMV 35S-orca3-nos terminator and CaMV 35S-g10h-nos terminator

[0045] Using pCAMBIA1304 as the expressi...

Embodiment 3

[0049] Example 3 Agrobacterium tumefaciens mediated orca3 and g10h gene transformation of periwinkle to obtain transgenic periwinkle plants

[0050] 1. Obtaining the dual plant expression vector Agrobacterium tumefaciens containing orca3 and g10h genes

[0051] The plant binary expression vector containing orca3 and g10h gene in embodiment 2 is transformed into Agrobacterium tumefaciens (such as EHA105, is that the market has publicly available biological material, can be purchased from Australian CAMBIA company, bacterial strain number is Gambar 1), And carry out PCR verification.

[0052] 2. Agrobacterium tumefaciens mediates orca3 and g10h gene transformation of periwinkle

[0053] (1) Pre-cultivation of explants

[0054] The periwinkle seeds used in the present invention are purchased from PanAmerican Seed Company, Pacifica Cherry Red (PCR). Periwinkle seeds were sterilized with 75% alcohol for 1 min, 20% NaClO for 5 min, rinsed with sterile water for 3 times, blotted t...

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Abstract

The invention relates to a method for increasing content of vinca alkaloids in vinca by corotation of orca3 / g10h genes; the method comprises the following steps: cloning orca3 gene and g10h gene; respectively constructing expression cassettes of orca3 gene and g10h gene, then orderly putting the expression cassettes in binary expression vector pCAMBIA 2300, constructing plant binary expression vector containing orca3 and g10h genes; transforming agrobacterium tumefaciens through a freeze-thawing method to obtain engineering bacteria; transforming vinca through an agrobacterium-mediated method and regenerating plants; detecting integration conditions of orca3 / g10h genes by PCR (polymerase chain reaction), measuring content of vinca alkaloids in transgenic vinca, and screening to obtain transgenic vinca plants in which content of alkaloids is increased. A high-yield and stable vinca alkaloid medicine source is provided by the invention, and is beneficial to producing vinca alkaloids in large scale at low cost.

Description

technical field [0001] The invention relates to a method for increasing the content of vinca alkaloids in the field of biotechnology, in particular to a method for co-transferring orca3 / g10h gene to increase the content of vinca alkaloids in periwinkle flowers. Background technique [0002] Vinca (Catharanthus roseus) belongs to the perennial herbaceous plant of Apocynaceae, which can produce more than 100 kinds of terpenoid indole alkaloids (Terpenoid Indole Alkaloids, TIAs) with anticancer activity. Among them, Vinblastine and Vincristine are important anti-tumor medicinal ingredients, and are also the most widely used natural anti-cancer medicinal ingredients. There are also anti-allergic medicinal ingredients Reserpine (Reserpine) and antihypertensive medicinal ingredients Ajmalicine (Ajmalicine). Wherein Vinorelbine is a relatively new type of vinca alkaloid drug developed by Pierre Fabre in the mid-1990s. Rosea) contains Vindoline (Vindoline) and vinblastine (Cathara...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/84A01H5/00
Inventor 唐克轩赵静雅王名雪潘琪芳徐菲
Owner 成都上交致远生物科技有限公司
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