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Plant linolenic acid synthetase gene CsFAD2-2 and application thereof
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A technology of linolenic acid and synthetase, applied in the field of genetic engineering, can solve problems such as inability to synthesize and lack
Inactive Publication Date: 2019-07-30
JILIN AGRICULTURAL UNIV
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Problems solved by technology
However, due to the lack of necessary dehydrogenase in the human body, basic fatty acids such as linoleic acid cannot be synthesized, so it can only be taken from daily diet
Method used
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Embodiment 1
[0014] Example 1 The extraction of flax mustard total RNA
[0015] 1) Frozen Arabidopsis thaliana material (50-100mg) is fully ground for half an hour under liquid nitrogen, and the ground fine powder is white without obvious graininess;
[0016] 2) After adding an appropriate amount of RNAiso, homogenate, let stand at room temperature for 5 minutes, and then centrifuge at 12,000g, 4°C for 5 minutes;
[0017] 3) Transfer the supernatant to a new 1.5ml centrifuge tube, add 200ml chloroform, shake and mix, let stand at room temperature for 5 minutes, then centrifuge at 12,000g, 4°C for 15 minutes;
[0018] 4) Transfer the supernatant to a new centrifuge tube, add isopropanol equal to the volume of the supernatant, let stand at room temperature for 10 minutes, centrifuge at 12,000g, 4°C for 10 minutes;
[0019] 5) Discard the supernatant, slowly add 1ml of 75% ethanol along the wall of the centrifuge tube, gently and slowly invert the centrifuge tube to mix, centrifuge at 12,000...
Embodiment 2
[0021] Example 2 Reverse transcription of RNA into the first strand of cDNA
[0022] 1. Take 4 μL of RNA sample as a template for reverse transcription;
[0023] 2. Add the following ingredients in turn:
[0024]
[0025] 65°C for 5 minutes, then quenched on ice and then added the second reaction solution, the composition is as follows:
[0026]
[0027] React at 30°C for 10 minutes, at 42°C for 45 minutes, and finally at 95°C for 5 minutes.
Embodiment 3
[0028] Example 3 Flax mustard CsFAD2-2 gene cloning
[0029] Primers for amplifying the coding region were designed according to the sequence of the CsFAD2 gene of linseed quince (GenBank accession number: JN831156), and introduced at the 5' ends of the primers Sma I and Sac I Restriction sites and protective bases (CsFAD2-F: 5'-CCCCGGGATGGGTGCAGGTGGAAGAAT-3', CsFAD2-R: 5'-CGAGCTCTCATAACTTATTGTTGTACCA-3'). Using the flax mustard cDNA as a template, PCR was carried out, and after the product was detected by agarosegel electrophoresis, the target gene fragment was recovered with an agarose gel recovery kit. Then, the recovered fragments were connected to the cloning vector, transformed into Escherichia coli competent cells, spread on LB solid medium containing 50 mg / L kanamycin, and cultured overnight at 37°C, single clones were selected for PCR detection, and the positive clones Sequencing analysis was carried out, and the sequencing results are shown in SEQ ID No.1. ...
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Abstract
The invention discloses a plantlinolenic acid synthetase gene CsFAD2-2 with the nucleotide sequence shown in a sequence table SEQ ID No.1, an expression vector which is inserted in the gene shown inthe sequence table SEQ ID No.1 and application of the plantlinolenic acid synthetase gene CsFAD2-2 in increasing the content of plantlinolenic acid. Camelina sativa (L.)Crantz cDNA serves as a template, and a reverse transcription PCR technology is utilized for cloning the CsFAD2-2 gene. By means of the gene, oleic acid and linoleic acid in plants can be catalyzed to generate linolenic acid, andthe content of the linolenic acid in the over-expressed transgenic plants is 2.56 times that of wild-type plants. High-quality candidate genes are provided for increasing the content of the linolenicacid of crops through genetic engineering breeding.
Description
technical field [0001] The invention belongs to the field of genetic engineering, in particular to plant linolenic acid synthase gene CsFAD2-2 and its application in transgenic plants. Background technique [0002] Polyunsaturated fatty acids (Polyunsaturated fatty acids, PUFAs) refer to straight-chain fatty acids with two or more double bonds and 16-22 carbon atoms. number related. PUFAs are divided into two categories, omega-6 and omega-3. Among them, omega-3 polyunsaturated fatty acids have important biological significance for human metabolism and development. Studies have shown that the intake of fish oil or omega-3 PUFAs in the human body can effectively inhibit the occurrence of inflammation and produce immune effects. Omega-3 polyunsaturated fatty acids are beneficial to increase the level of calcium in the human body and prevent bone loss. Most patients with osteoporosis are due to the lack of essential fatty acids in the body, among which γ-linolenic acid (Gamma...
Claims
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