Construction method for cold-resistant transgenic tobaccos based on fatty acid desaturase gene

A desaturase, construction method technology, applied in the fields of botany equipment and methods, biochemical equipment and methods, genetic engineering, etc., can solve problems such as plant damage

Inactive Publication Date: 2014-12-10
LANZHOU UNIVERSITY OF TECHNOLOGY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0005] The object of the present invention is to provide a method for constructing a cold-resistant transgenic tobacco base...

Method used

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  • Construction method for cold-resistant transgenic tobaccos based on fatty acid desaturase gene
  • Construction method for cold-resistant transgenic tobaccos based on fatty acid desaturase gene
  • Construction method for cold-resistant transgenic tobaccos based on fatty acid desaturase gene

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Experimental program
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Embodiment 1

[0020] The construction of embodiment 1 plant genetic transformation vector

[0021] Stearoyl carrier protein desaturase (SAD) is a key enzyme in the desaturation process of fatty acid in plants, and the degree of unsaturation of cell membrane lipids is positively correlated with the cold resistance of plants. The expression of SAD gene is low in plants, and SAD enzyme is the first step of stearoyl desaturation, and also the rate-limiting step of a series of subsequent desaturation reactions. Therefore, the present invention inserts the cloned spinach SAD gene into the downstream of the 35S promoter of CaMV to construct a plant genetic transformation vector pBI121-13 capable of highly expressing the SAD gene. At the same time, the antisense SAD gene was also constructed into pBI121 to obtain the recombinant plant genetic transformation vector pBI121-6 as a negative control.

[0022] First, the total RNA of spinach was extracted by phenol / chloroform extraction, and a pair of s...

Embodiment 2

[0027] Plant genetic transformation vectors such as embodiment 2pBI121, pBI121-13, pBI121-6 transform tobacco

[0028] Take out Agrobacterium tumefaciens LBA4404 stored in a -70°C refrigerator, streak it on a YEB plate, and culture it at 28°C until a single colony grows. Pick a single colony and culture it overnight in 5ml YEB medium at 28°C and 250rpm. Take 2ml and inoculate in 40ml YEB to continue culturing. After about 3 hours, pour into a pre-cooled centrifuge tube, ice bath for 30 minutes, centrifuge at 6000rpm for 10 minutes, and suspend the precipitate in 1ml of 0.1M CaCl 2 In the solution, draw 200μl and distribute it in a pre-cooled centrifuge tube, and add about 800ng pBI121, pBI121-13, pBI121-6 respectively. Ice bath for 5 minutes, put in liquid nitrogen for 8 minutes, take it out and immediately put it in a 37°C water bath for 5 minutes, add YEB to 1ml, incubate at 28°C and 250rpm for 4-5 hours, centrifuge, suspend the precipitate in 200μl YEB, and coat Culture ...

Embodiment 3

[0030] Embodiment 3 dot hybridization and Southern hybridization screening transgenic tobacco

[0031]Prepare the nylon membrane for hybridization, soak the nylon membrane with double-distilled water, soak in 2X SSC solution for 5 minutes, apply suction and filter, add 150 μl 2X SSC to wash, wait until the DNA is completely dry, place it on filter paper saturated with 0.4M NaOH for denaturation 5 minutes, then neutralize with 2XSSC saturated filter paper for 5 minutes, bake the film in a vacuum oven at 80°C for 1 hour, and store it in a dry place for later use.

[0032] Digest pBI121 plasmid with BamHI and EcoRI double enzymes, electrophoresis to check that the digestion is complete, and recover GUS+NOS by low melting point agarose gel electrophoresis Ter fragments as probe DNA. The radioactive labeling of the probe DNA was labeled by the Random Primer method.

[0033] Hybridization: Boil the radiolabeled DNA probe at 100°C for 5 minutes, cool in an ice bath, and add EDTA to...

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Abstract

The invention provides a construction method for cold-resistant transgenic tobaccos based on a fatty acid desaturase gene. The construction method comprises the following steps: firstly, amplifying out stearoyl carrier protein desaturase gene (SAD) of spinach by virtue of an RT-PCR (reverse transcription-polymerase chain reaction) process, and connecting the stearoyl carrier protein desaturase gene to a plant genetic transformation carrier pBI121; cloning and recombining a correct carrier by virtue of nucleotide sequence analysis and transforming the correct carrier into agrobacterium tumefaciens LBA4404; transforming tobacco aseptic seedling explants by the agrobacterium tumefaciens containing recombinant plasmids by virtue of a leaf disc method, screening out a kanamycin resistant plant and transplanting the kanamycin resistant plant into a flower pot, and collecting seeds after the kanamycin resistant plant is mature; sowing the seeds on an MS culture medium containing kanamycin to germinate and grow; extracting nuclear DNA (deoxyribonucleic acid) from leaves of the kanamycin resistant plant to carry out PCR (polymerase chain reaction) detection, dot blotting and Southern cross identification; and finally, by taking molecularly-identified positive transgenic tobaccos as experimental materials, carrying out conductivity measurement and chlorophyll content measurement respectively under low temperature stress, wherein the cold resistance of SAD transgenic tobaccos is obviously improved.

Description

technical field [0001] The invention belongs to the technical field of genetic engineering, and in particular relates to a method for constructing a cold-resistant transgenic tobacco based on a fatty acid desaturase gene. Background technique [0002] The cold resistance of plants, especially crops, has always been a research hotspot at home and abroad. If crops can grow normally at lower temperatures, it will have great economic significance for crops to resist low temperature and cold damage, especially for preventing agricultural disasters such as late spring cold and early frost in crop planting in our country. [0003] Existing research results have shown that the ability of plants to resist low temperature stress is positively correlated with the unsaturation of membrane lipids in plant cells, that is, the higher the unsaturation of membrane lipids, the stronger the resistance of plants to low temperature environments. Stearoyl carrier protein desaturase (SAD) is a ke...

Claims

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Application Information

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IPC IPC(8): C12N15/84A01H5/00
Inventor 马建忠刘丹肖成斌马燕林王永刚张伟杰
Owner LANZHOU UNIVERSITY OF TECHNOLOGY
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