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Method for increasing nicotine content in tobacco by utilizing camptotheca acuminate AOC (Allene Oxide Cyclase) gene transformation

A nicotine, gene technology, applied in the field of genetic engineering

Inactive Publication Date: 2011-09-14
FUDAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] At present, there is no application of genetic engineering technology mentioned in this patent application to transfer the enzyme gene of the jasmonic acid biosynthetic pathway of camptophylla, especially the allene oxide cyclase, into tobacco to increase the nicotine content of tobacco related reports, including patents and literature

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0026] Synthesis of the core fragment of camptothecadiene oxide cyclase gene, construction of expression vector and engineering bacteria.

[0027] 1. Obtaining the core fragment of camptotheca allene oxide cyclase gene

[0028] According to the full-length sequence of the camptothelen oxide cyclase gene, primers were designed between the 72nd base and the 812th base inside the gene, and the 741bp sequence was amplified as a gene fragment, and according to the plant expression vector pCAMBIA1304 The multiple cloning enzyme cutting sites, respectively designed two containing Speech I and Bst The PCR amplification primer of EII restriction endonuclease site and protection base, its primer sequence is:

[0029] P1: 5'-gg Actagt Atggctgcttcatcaact tc-3' (SEQ ID NO: 2), Speech The I restriction site is underlined;

[0030] P2: 5'-ga ggttacc Tcagtcagtgaagttaggaa-3' (SEQ ID NO: 3), Bst The EII restriction site is underlined.

[0031] Total RNA was extracted from the le...

Embodiment 2

[0040] Obtain genetically modified tobacco with increased nicotine content.

[0041] 1. Agrobacterium EHA105-CaAOC. Take it out from the refrigerator before use, inoculate in 50 ml YEB liquid (Rif + ,Str + , Kan + ), 28°C, 200rpm shaking culture twice.

[0042] 2. The second activation of OD 600 Add 100 μmol L when reaching 0.3 -1 Acetosyringone, continue to culture at 28°C, shake at 200rmp, OD 600 Centrifuge at 4000 rpm for 10 minutes at room temperature when reaching 0.6.

[0043] 3. Discard the supernatant, use MS (add 100 μmol L -1 Acetosyringone) liquid medium suspension, diluted to 5-20 times the original volume, so that the OD of the bacterial solution 600 =0.3 or so, called the conversion solution.

[0044] 4. Take young and strong leaves of sterile tobacco seedlings, remove the main veins, cut the leaves into leaf discs of about 0.8 cm×0.8 cm, and put them in MS (add 1 mg L -1 6-Benzylaminopurine, 1 mg L -1 Naphthalene acetic acid) medium to prevent deh...

Embodiment 3

[0052] Nicotine content assays in transgenic tobacco and non-transgenic tobacco controls.

[0053] 1. Nicotine extraction

[0054] 1) When the tobacco grows to about 20cm in the flower pot, take the middle leaves of the tobacco, remove the midrib, dry the leaves in an oven at 60°C, and grind them into powder. Among them, two wild-type and transgenic tobacco were respectively selected, one was treated with 100 μM methyl jasmonic acid, and the other was not treated.

[0055] 2) Place the powder in an oven at 60°C and dry to constant weight.

[0056] 3) Weigh 0.05g of the powder, extract with 1ml of 25mM sodium phosphate buffer (pH 7.8) at 30°C for 24h, centrifuge at 13.4krpm for 1min, and take the supernatant.

[0057] 4) The aqueous extract was filtered under reduced pressure, diluted 10 times with water, and stored at 4°C for testing.

[0058] 2. Determination of nicotine content

[0059] Use HPLC to optimize the conditions of the standard first, and then measure the sam...

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Abstract

The invention belongs to the technical field of genetic engineering, particularly relates to a method for increasing nicotine content in tobacco by utilizing camptotheca acuminate AOC (Allene Oxide Cyclase) gene transformation. In the method, a plant expression vector is constructed by using camptotheca acuminate allene oxide cyclase (AOC) genes; and the nicotine content in the tobacco is increased through over-expression of the camptotheca acuminate AOC genes in the tobacco by using a genetic engineering technology. The method comprises the process of: cloning the AOC genes from camptotheca acuminate; constructing a plant high-efficiency expression vector; introducing agrobacterium tumefaciens and performing genetic transformation on the tobacco; and measuring the nicotine content in transgenic tobacco leaves. By adopting the method, the camptotheca acuminate AOC genes can be over-expressed in the tobacco, and the nicotine content in the tobacco is increased.

Description

technical field [0001] The invention belongs to the technical field of genetic engineering, and in particular relates to a method for increasing the nicotine content of tobacco by utilizing transgenic technology. The invention also relates to tobacco cells and plants with high nicotine content obtained by genetic engineering and their cultured progeny, regenerated plants, plant tissues or seeds. Background technique [0002] Jasmonic acid is a ubiquitous hormone in the plant kingdom, which is involved in the signal transduction process of plant development and stress response under adversity stress. Endogenous and exogenous jasmonic acid compounds can significantly improve the tolerance of plants to mechanical damage, animal bites, pests, low temperature, high salinity and other adverse external environments. In plants, the synthesis pathway of jasmonic acid starts from the linolenic acid released from the cell membrane, and is processed by lipoxygenase, allene oxide syntha...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/52C12N15/82C12N5/10A01H5/00
Inventor 皮妍唐克轩蒋科技孙小芬
Owner FUDAN UNIV
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