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Papaver bracteatum with modified alkaloid content

A kind of alkaloid and poppy technology, applied in the plant field of the red poppy species, can solve the problems of changing the red poppy, not being informed, etc.

Inactive Publication Date: 2015-04-01
TPI ENTERPRISES LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0009] Furthermore, although mutagenesis has been used to alter the type of alkaloids produced by the annual poppy species, Papaver somniferum (see, for example, WO 98 / 02033), the inventors are not aware of any changes describing the alteration of Papaver somniferum (P. bracteatum) alkaloid types reported

Method used

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  • Papaver bracteatum with modified alkaloid content
  • Papaver bracteatum with modified alkaloid content
  • Papaver bracteatum with modified alkaloid content

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0178] Production of codeine / oripavine and / or morphine in the big red poppy

[0179] The nucleotide sequence listed under GenBank accession number GQ500139 (which encodes T6ODM) is cloned into an appropriate expression vector, such as pBI121 (Clontech), under the control of the cauliflower mosaic virus 35S promoter (CaMV 35S), This promoter directs constitutive expression in P. papyrus. The transgene was introduced into P. papyrus by Agrobacterium-mediated transformation using the method described by Solouki et al. (2009, supra). Transformants were selected using paromomycin and regenerated as described by Solouki et al. (2009, supra). Then, the transformants were screened for alkaloid content.

[0180] In said transformants, it is proposed that the strong constitutive expression of T6ODM together with the expression of COR leads to the accumulation of codeine in this transformant.

[0181] In order to increase the accumulation of morphine in the transformants, CODM activ...

Embodiment 2

[0192] Screening of Poppy Poppy against T6ODM, CODM and COR

[0193] A series of primers were used to amplify PCR products of different sizes at different positions in the T6ODM and CODM genes of P. somniferum. The same primers were also used to detect the existence of T6ODM and CODM genes or similar sequences in the genome of P. bracteatum. The PCR amplification conditions used were as follows: initial denaturation at 95°C for 120 s; 30 cycles of denaturation at 95°C for 30 s, primer annealing at 59°C for 30 s and extension at 72°C for 150 s; and a final extension step, 72 °C for 15 minutes. Reactions were performed using GoTaq Green Polymerase Mix (Promega) according to the supplier's instructions.

[0194] Table 2 below provides a summary of primer combinations used to amplify different regions of the T6ODM and CODM genes. The results of PCR amplification are shown in figure 2 , and the positions of the primers in the CODM and T6ODM genes are shown in image 3 middl...

Embodiment 3

[0202] Identification and isolation of T6ODM and CODM genes

[0203] The T6ODM and CODM genes were amplified from the opium poppy genome using the following primer sets:

[0204] About T6ODM:

[0205] 5'-ATGGAGAAAGCAAAACTTATGAAGCTAGG-3' (SEQ ID NO: 26) and

[0206] 5'-TTTCTGAAAGTAAAGGTACATTACATGTGG-3' (SEQ ID NO: 27); and

[0207] About CODM:

[0208] 5'-ATCTGACAAGAAAGTTCATCAAATATAGAGTTC-3' (SEQ ID NO: 28) and

[0209] 5'-CTTATTTCATCATATATTAAACACAATGTGGAG-3' (SEQ ID NO: 29).

[0210] By grinding tissue samples in liquid nitrogen and mixing with three times the sample volume of extraction buffer (1% sodium lauryl sarcosine, 100 mM Tris-Cl, 100 mM NaCl, 10 mM EDTA, pH 8.5) followed by phenol-chloroform extraction , precipitated with 0.1 volume of 3M sodium acetate (pH 4.8) and 1 volume of 100% isopropanol, washed with 1 mL of 70% ethanol, and resuspended in an appropriate volume of sterile distilled water with 40 μg / mL RNase A, whereas from Isolation of genomic DNA from ...

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Abstract

The present invention relates to genetically modified plants of the species Papaver bracteatum wherein the type or amount of one or more alkaloids produced by the plants has been modified. Specifically, the genetically modified plants have an increased expression of one or more of thebaine 6-O-demethylase, codeine O- demethylase and / or codeinone reductase relative to wild type P. bracteatum such that the genetically modified poppy plants produce an increased quantity of an alkaloid selected from codeine, oripavine and / or morphine relative to a wild type P. bracteatum. Also provided are progeny plants having the genetically modified poppy plants described above as a parent; mutant or derivative plants of the aforementioned plants; reproductive material derived from, straw produced from, straw concentrate produced from, latex derived from, or one or more isolated cells derived from, the aforementioned plants. Methods for producing an alkaloid from the aforementioned plants are also provided, together with nucleic acid and amino acid sequence variants of the 6-O-demethylase and codeine O-demethylase genes.

Description

field of invention [0001] The present invention relates to genetically modified plants of the species Papaver bracteatum, wherein the type or amount of one or more alkaloids produced by said plants has been altered. Background of the invention [0002] Opium poppies (Papaver somniferum) are cultivated commercially under regulated conditions in many countries. The latex obtained by cutting the characteristic capsule is called opiate and is a source of several pharmacologically important alkaloids. Morphine, codeine, thebaine, narcotine and papaverine are the most important alkaloids produced by this plant and are used in the pharmaceutical industry as analgesics, antitussives and antispasmodics. [0003] On a commercial scale, however, poppy straw and straw concentrates (rather than latex) are the most commonly used raw materials for the production of morphine and other poppy-derived alkaloids. For example, today, more than 50% of the world's morphine is produced from poppy...

Claims

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Application Information

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IPC IPC(8): C12N15/53A01H5/10C07D489/02A01H5/08C07D221/28
CPCC07D489/02C12N9/0006C12N9/0071C12N15/8243C12Y101/01247C12Y114/11031C12Y114/11032
Inventor 贾斯廷·泰勒·库姆斯贾罗德·戴维·里奇马克·阿尔弗雷德·特斯特达明·莱特富特迪帕·杰哈
Owner TPI ENTERPRISES LTD
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