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Cloning of cytochrome p450 genes from nicotiana

A technology of tobacco and tobacco plants, which can be used in genetic engineering, plant genetic improvement, enzymes, etc., and can solve the problems of low protein content and unstable purification.

Inactive Publication Date: 2012-01-18
U S SMOKELESS TOBACCO COMPANY LLC
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Furthermore, cloning of p450 enzymes has been hampered at least in part by the fact that these membrane-localized proteins are often present in low amounts and are often unstable during purification

Method used

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  • Cloning of cytochrome p450 genes from nicotiana
  • Cloning of cytochrome p450 genes from nicotiana
  • Cloning of cytochrome p450 genes from nicotiana

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0260] Development and ethylene treatment of embodiment 1 plant tissue

[0261] plant growth

[0262] Plants were grown in pots for 4 weeks in the greenhouse. The 4-week-old seedlings were moved to individual pots and grown in the greenhouse for 2 months. During the growth period, the plants were watered twice a day with 150 ppm of NPK fertilizer in the water. The expanded green leaves were separated from the plants for ethylene treatment as described below.

[0263] Cell line 78379

[0264] Tobacco line 78379 issued by the University of Kentucky, a burley tobacco line, was used as the source of plant material. 100 plants were grown, transplanted, and labeled with different numbers (1-100) using standard methods in the art for growing tobacco. Use recommended methods for fertilization and field management.

[0265] 3 / 4 of 100 plants converted 20-100% of nicotine to nornicotine. 1 / 4 of the 100 plants converted less than 5% of nicotine to nornicotine. Plant 87 had the lo...

Embodiment 2

[0274] Example 2: Isolation of RNA

[0275] For RNA extraction, medium-sized leaves of 2-month-old greenhouse plants were treated with ethylene as described above. Samples at 0 and 24-48 hours were used for RNA extraction. In some cases, leaf samples during the senescence process were taken from plants 10 days after flower-head removal. These samples are also used for extraction. Use the RNeasy Plant Mini (Qiagen, Inc., Valencia, California) Total RNA was isolated according to the manufacturer's protocol.

[0276] Tissue samples were ground to a fine powder in liquid nitrogen using a DEPC-treated mortar and pestle. Transfer approximately 100 mg of ground tissue to a sterile 1.5 mL eppendorf tube. Place sample tubes in liquid nitrogen until all samples are collected. Then, 450 μl of RLT buffer provided in the kit was added to each tube. Samples were shaken vigorously and then incubated at 56°C for 3 minutes. The lysate was applied to a QIAshredderTM spin column in a 2 ...

Embodiment 3

[0278] Embodiment 3: reverse transcription-PCR

[0279] First-strand cDNA was prepared using SuperScript Reverse Transcriptase (Invitrogen, Carlsbad, California) according to the manufacturer's protocol. The oligo dT primer mix enriched for poly A+ RNA consists of less than 5 μg of total RNA, 1 μl of 10 mM dNTP mix, 1 μl of Oligo d(T)12-18 (0.5 μg / μl) and up to 10 μl of DEPC-treated water composition. Each sample was incubated at 65°C for 5 minutes and then placed on ice for at least 1 minute. The following components were added in order to prepare a reaction mixture: 2 μl 10X RT buffer, 4 μl 25 mM MgCl 2 , 2 μl 0.1 M DTT and 1 μl RNase OUT Recombinant RNase Inhibitor. Pipette 9 μl of reaction mix into each RNA / primer mix and mix gently. Incubate at 42°C for 2 minutes, add 1 μl of Super Script IITM RT to each tube, and incubate at 42°C for 50 minutes. The reaction was terminated at 70°C for 15 minutes and cooled on ice. The samples were collected by centrifugation, 1 μl R...

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Abstract

The present invention relates to p450 enzymes and nucleic acid sequences encoding p450 enzymes in Nicotiana, and methods of using those enzymes and nucleic acid sequences to alter plant phenotypes.

Description

[0001] This application is a divisional application of the Chinese invention application (invention name: cloning cytochrome P450 gene from tobacco; application date: October 15, 2004; application number: 200480030385.0 (international application number: PCT / US2004 / 034065). technical field [0002] The present invention relates to the cloning of cytochrome P450 genes from tobacco. In particular, the present invention relates to nucleic acid sequences encoding cytochrome P450 enzymes (hereinafter referred to as P450 and P450 enzymes) in Nicotiana plants and methods of using those nucleic acid sequences to alter plant phenotypes. Background technique [0003] Cytochrome P450s catalyze enzymatic reactions of a variety of chemically distinct substrates, including oxidative, peroxidative, and reductive metabolism of endogenous and heterologous substrates. In plants, p450s are involved in biochemical pathways including the synthesis of plant products such as phenylpropanoids, alka...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/53C12N15/82C12Q1/68A01H5/00C12N9/02
CPCC12N9/0077C12N15/8243
Inventor D.徐
Owner U S SMOKELESS TOBACCO COMPANY LLC
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