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
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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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