Method for increasing content of camptothecin in camplotheca acuminata calluses by adopting camplotheca acuminata transgene aoc

A callus and transgenic technology, applied in the biological field, can solve problems such as undiscovered, low content and no commercial prospects, and achieve the effect of meeting the needs of large-scale factory production in the pharmaceutical industry and solving the shortage of drug sources

Inactive Publication Date: 2011-08-31
FUDAN UNIV
View PDF0 Cites 0 Cited by
  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] Although various methods have been tried to increase the content of camptothecin in camptothecin callus and cell culture, so far, the content of camptothecin produced by callus tissue and cell culture system is still too low to be commercially viable. prospect
Up to now, there is no report about the improvement of camptothecin content in camptothecin callus by transforming aoc gene

Method used

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
View more

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0024] Cloning of embodiment 1 camptophylla aoc gene

[0025] 1. Tissue Isolation

[0026] Soak the seeds of camptophylla with 75% (V / V) alcohol for 1 minute, and then soak them with 0.1% (V / V) HgCl 2 Soak for 5 minutes, rinse 3-4 times with sterile water, blot the surface moisture with sterile absorbent paper, inoculate in hormone-free MS (Murashige and Skoog, 1962) solid medium, and culture at 25°C under 12h / 12h light. The aseptic seedlings of Campylopa spp. can be obtained, and after the seedlings grow to about 5 cm, the leaves and stems are cut for RNA extraction.

[0027] 2. Isolation of RNA

[0028]Take by weighing 0.5g described camphor tree aseptic test-tube seedling, after quick-freezing with liquid nitrogen, grind with mortar rapidly, add and fill 1mL CTAB damping fluid [3%CTAB (W / V), 3%PVP (W / V) (Mw 40000), 25mM EDTA, 2.0M NaCl, 100mM Tris-HCl (pH 8.0), 0.5g / L spermidine and 0.1% DEPC (V / V)] in a 1.5mL Eppendorf tube, after full shaking, in Place at room temper...

Embodiment 2

[0032] Embodiment 2 constructs the plant expression vector pCAMBIA1304::p35S-aoc-nos containing aoc gene

[0033] Using pCAMBIA1304 as the expression vector, the aoc gene in Example 1 was used to replace the gfp+gus gene on it. Specifically, pMDT-simple+aoc and pCAMBIA1304 were digested with Spe I / BstE II, the large fragments of aoc and pCAMBIA1304 were recovered, ligated and transformed, single clones were picked, and plasmids were extracted for PCR detection and enzyme digestion verification. The results showed that the aoc gene had been successfully constructed into the plant expression vector pCAMBIA1304, thus obtaining the plant expression vector pCAMBIA1304::p35S-aoc-nos containing the aoc gene.

[0034] In this example, the key enzyme gene aoc of the camptojasmonic acid biosynthetic pathway is operably linked to the expression control sequence to form a plant expression vector containing the aoc gene. content of camptothecin.

Embodiment 3

[0035] Embodiment 3 Obtaining plant expression vector Agrobacterium tumefaciens engineering bacterium containing aoc gene

[0036] The plant expression vector containing the aoc gene in Example 2 is transformed into Agrobacterium tumefaciens (such as EHA105, which is a publicly available biological material in the market, which can be purchased from CAMBIA Company in Australia, and the strain number is Gambar 1), and PCR verification is carried out . Specifically, first prepare competent Agrobacterium tumefaciens (such as EHA105): cultivate Agrobacterium tumefaciens to 0.D 600 = 0.5, put the bacteria solution in ice bath for 30min, take the bacteria into a 1.5ml Eppendorf tube, centrifuge at 5000rpm at 4°C for 5min, remove the supernatant; use 0.1M CaCl sterilized by suction filtration 2 (Pre-cooling) 400 μl of suspended bacteria (mixed gently with a pipette gun), placed on ice for 30 minutes, centrifuged at 5000 rpm for 5 minutes, removed the supernatant, and washed with 100...

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to view more

PUM

No PUM Login to view more

Abstract

The invention relates to a method for increasing the content of camptothecin in camplotheca acuminata calluses by adopting a camplotheca acuminata transgene aoc, belonging to the technical field of biology. The method comprises the following steps of: cloning a gene aoc from camplotheca acuminata, constructing a plant expression vector containing the gene aoc and mediating with agrobacterium tumefaciens to introduce the gene aoc to camplotheca acuminata cells and regenerating the calluses; detecting the integration and expression conditions of the foreign target gene aoc by PCR (Polymerase Chain Reaction) and semiquantitative RT-PCR (Reverse Transcription-Polymerase Chain Reaction); measuring the camptothecin content in the camplotheca acuminata calluses by a high performance liquid chromatography detector; and scanning the transgenic camplotheca acuminata calluses, the camptothecin content of which is increased. The content of the camptothecin in the transgenic camplotheca acuminata calluses obtained by the invention is obviously increased. The method for increasing the content of the camptothecin in the culture of camplotheca acuminata cells is significant for solving the problem of deficient drug sources of the camptothecin and meeting the demands of large-scale industrialized production of pharmaceutical industry.

Description

technical field [0001] The invention belongs to the field of biological technology, and relates to a method for increasing the content of camptothecin in camptothecin callus, in particular to a method for improving the content of camptothecin in camptotheca callus by transforming the allene oxide cyclase gene (allene oxide cyclase, aoc). The method of camptothecin content in injured tissue. Background technique [0002] Camptotheca acuminata Decaisne (Camptotheca acuminata Decaisne), a perennial subtropical deciduous broad-leaved tree belonging to the family Nyssaceae, has aliases such as Eclipta acuminata and Ganzhang tree. This genus is only one species of Camptotheca acuminata, and it is a unique tree species in my country (Chinese Academy of Sciences China Flora Editorial Board, 1983). Since the 1990s, countries such as the United States, Japan, Canada, and the United Kingdom have actively invested a lot of manpower and material resources in research on the introduction ...

Claims

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to view more

Application Information

Patent Timeline
no application Login to view more
Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/82
Inventor 孙小芬皮妍蒋科技唐克轩
Owner FUDAN UNIV
Who we serve
  • R&D Engineer
  • R&D Manager
  • IP Professional
Why Eureka
  • Industry Leading Data Capabilities
  • Powerful AI technology
  • Patent DNA Extraction
Social media
Try Eureka
PatSnap group products

Browse by: Latest US Patents, China's latest patents, Technical Efficacy Thesaurus, Application Domain, Technology Topic.

© 2024 PatSnap. All rights reserved.Legal|Privacy policy|Modern Slavery Act Transparency Statement|Sitemap