Method for increasing content of camptothecin through co-transformation of double genes of transcription factor ORCA3 (Octadecanoie-responsive Cantharanthus AP2-doman protein 3) and key enzyme G10H (Geraniol 10-hydroxylase)

A transcription factor and key enzyme technology, applied in the biological field, can solve the problems of not significantly improving the yield of TIA metabolic end products, insufficient expression, etc.

Inactive Publication Date: 2011-10-12
SHANGHAI NORMAL UNIVERSITY
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Problems solved by technology

It must be pointed out that transcription factors are not omnipotent. Although a single transformation of an ORCA3 gene can regulate the coordinated expression of multiple key enzyme genes such as AS, TDC, DXS, CPR and STR, some genes such as the key enzyme gene G10H are not affected. Regulation of ORCA3
In the ORCA3 gene overexpressed cells, due to the insufficient expression of the key enzyme gene G10H, the production of TIA metabolic end products such as vinblastine was not significantly improved.

Method used

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  • Method for increasing content of camptothecin through co-transformation of double genes of transcription factor ORCA3 (Octadecanoie-responsive Cantharanthus AP2-doman protein 3) and key enzyme G10H (Geraniol 10-hydroxylase)
  • Method for increasing content of camptothecin through co-transformation of double genes of transcription factor ORCA3 (Octadecanoie-responsive Cantharanthus AP2-doman protein 3) and key enzyme G10H (Geraniol 10-hydroxylase)
  • Method for increasing content of camptothecin through co-transformation of double genes of transcription factor ORCA3 (Octadecanoie-responsive Cantharanthus AP2-doman protein 3) and key enzyme G10H (Geraniol 10-hydroxylase)

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0027] Embodiment 1 Obtaining of periwinkle CrORCA3 and CrG10H gene coding sequence

[0028] 1.1. Extraction of periwinkle total RNA and synthesis of cDNA first strand

[0029]Total RNA was extracted from periwinkle seedlings using the RNA prep pure plant kit provided by TIANGEN (see the instructions in the kit for the extraction steps). The fresh weight of periwinkle seedlings used to extract total RNA is about 0.1 g, and the DNA in the sample has been removed with DNase working solution during the extraction process. Measure the relative absorbance value of the extracted RNA on a spectrophotometer, and calculate the purity and concentration of the extracted RNA. After calculation based on the concentration of different RNA samples, the first-strand cDNA was synthesized with reverse transcriptase XL (AMV) using 0.5 μg RNA as the initial amount (for the operation steps, refer to the relevant instructions provided by Promega).

[0030] 1.2. Design of specific primers for CrOR...

Embodiment 2

[0032] Embodiment 2 Containing the construction of the plant expression vector of periwinkle CrORCA3 and CrG10H gene

[0033] 2.1. Intermediate vector pCAMBIA1304 + build

[0034] Using pBI121 and pCAMBIA1304 as materials, construct the plant expression vector pCAMBIA1304 + . Specifically, pBI121 and pCAMBIA1304 were digested with HindIII / EcoRI; the pBI121-GUS expression cassette and the large fragment of pCAMBIA1304 were recovered; ligation transformation was carried out, and single clone colonies were picked to extract plasmids for digestion verification. The results showed that the plant expression vector pCAMBIA1304 + The build was successful.

[0035] 2.2. Plant expression vector pCAMBIA1304 + - Construction of CrG10H

[0036] The successful pCAMBIA1304 constructed above + Basically, replace the GUS gene on it with the CrG10H gene cloned from periwinkle. Specifically, BamHI / SacI double enzyme cut pMD18T-CrG10H and pCAMBIA1304 + ; Recover CrG10H gene and pCAMBIA13...

Embodiment 3

[0040] Example 3 Agrobacterium rhizogenes mediated genetic transformation of CrORCA3 and CrG10H genes to obtain transgenic hairy roots

[0041] 3.1. Containing plant expression vector pCAMBIA1304 + -Acquisition of CrORCA3-CrG10H Agrobacterium rhizogenes Engineering Bacteria

[0042] The plant bivalent expression vector pCAMBIA1304 containing CrORCA3 and CrG10H gene in embodiment 2 + -CrORCA3-CrG10H was transferred into Agrobacterium rhizogenes C58C1, and a single clone colony was picked for PCR verification. The results showed that the plant expression vector containing CrORCA3 and CrG10H genes had been successfully constructed in Agrobacterium rhizogenes strain C58C1.

[0043] 3.2. Agrobacterium rhizogenes mediated CrORCA3, CrG10H gene genetic transformation of camptophylla

[0044] 3.2.1. Preculture of explants

[0045] Cut the sterile hypocotyls (1-3cm) of camptophylla and inoculate them into the pre-cultivation medium (B 5 ), cultured in the dark at 25°C for 2 days. ...

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Abstract

The invention discloses a method for increasing content of camptothecin in camptotheca hairy root in the technical field of biology. The method comprises the following steps of: cloning coding frame sequences of CrORCA3 and CrG10H genes from catharanthus roseus; constructing a plant bivalent high-efficiency expression vector including the genes; and genetically transforming the camptotheca into camptotheca hairy root genetically modified by CrORCA3 and CrG10H genes, by using the hair root agrobacterium-mediated technique. The camptothecin content in the obtained genetically-modified camptotheca hairy root is increased obviously. The content of the camptothecin is 5.4 times of the contrast. The content of hydroxycamptothecin is 2.3 times of the contract. The content of the hairy root camptothecin independently genetically-modified by CrORCA3 is increased by 1.5 times of the contract. The content of the hairy root camptothecin independently genetically-modified by CrG10H gene is not increased obviously. The invention provides a method for increasing content of the camptothecin in the camptotheca hairy root, thereby providing a novel method for producing the camptothecin and the hydroxycamptothecin as anti-cancer drugs of important clinical requirements.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to a method for increasing the content of camptothecin in camptothecin hairy roots through a dual-gene co-transformation metabolic engineering strategy of transcription factor ORCA3 and key enzyme G10H. Background technique [0002] Camptothecin (CPT) is the first alkaloid extracted from Camptotheca acuminata endemic to China and has significant antitumor activity. A variety of camptothecin drugs such as Irinotecan (Irinotecan) and Topotecan (Topotecan) have been certified by the US FDA (1992), and are widely used clinically to treat various malignant diseases such as ovarian cancer, rectal cancer, and leukemia. Tumor, the global market demand is very huge. At present, the production of camptothecin drugs in the world is mainly extracted from camptothecin and other plants. However, due to the limited amount of natural resources of camptothecin, the growth is very slow, and the content ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/82A01H5/06
Inventor 开国银李姗姗王伟付雪晴尤丽佳
Owner SHANGHAI NORMAL UNIVERSITY
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