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Separated diacylglycerol acylase gene, expression vector and application thereof

A technology of diacylglycerol acyl and expression vectors, which can be applied in application, genetic engineering, plant gene improvement, etc. It can solve the problems of unclear interaction and influence on content, and achieve the goal of improving edible value, simple and easy method, and remarkable effect Effect

Inactive Publication Date: 2013-08-28
SOUTHWEST UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

Previous studies (Shockey et al., Plant Cell 2006, 18: 2294-2313; Kroon et al., Phytochemistry 2006, 67: 1166-1176) found that in developing seeds, DGAT catalyzes the final stage of TGA synthesis of acyl CoA and diacylglycerides. One step, although the interaction between various acyltransferases is not clear, DGAT is a key rate-limiting step in the synthesis of oils and fats, and it has been clarified that it directly affects the content and composition of TAG in seeds

Method used

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  • Separated diacylglycerol acylase gene, expression vector and application thereof
  • Separated diacylglycerol acylase gene, expression vector and application thereof
  • Separated diacylglycerol acylase gene, expression vector and application thereof

Examples

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Effect test

Embodiment 1

[0046] 1. Extraction of DNA

[0047] Extraction method of fungal DNA

[0048] Firstly, the fungus was cultivated with liquid medium; after the fungus had been cultivated, the mycelia were suction-filtered and ground with liquid nitrogen quick-freezing; the ground powder was collected and added to the prepared fungal DNA extraction buffer 3ml (0.2mol / LTris HCl (pH7. 5), 0.5mol / L NaCl, 0.01mol / L EDTA, 1% (W / V) SDS) and 0.1ml of β-mercaptoethanol, shake well and centrifuge at 8000rpm for 5min at room temperature, take the supernatant after centrifugation; extract with chloroform 2 times (add 3ml of chloroform each time, centrifuge at 8000rpm for 5min at room temperature, and take the supernatant after centrifugation); add 2 / 3 volume of isopropanol and 1 / 10 of NaAc (pH5.2) to the supernatant and freeze overnight at -20°C Centrifuge at 10,000 rpm for 5 minutes at room temperature after overnight, remove the supernatant; dissolve the precipitate with 300 μL TE; mix and extract once...

Embodiment 2

[0062] 1. Cloning of lipolytic yeast DGAT gene

[0063] According to the sequence of lipolytic yeast DGAT gene (GenBank accession number: XM_504700), primers (sequences 3 and 4) were designed, and a fragment of about 1.5 kbp was obtained by PCR amplification from the genome of lipolytic yeast. The sequence analysis after cloning the amplified DNA fragment showed that it was the DGAT gene of Saccharomyces lipolytica. Then, specific primers (SEQ ID NO: 5) were designed to modify the obtained lipolytic yeast DGAT gene, and finally the Y1DGAT gene sequence shown in SEQ ID NO: 1 was obtained.

[0064] 2. Cloning of Aspergillus flavus DGAT gene

[0065] According to the Aspergillus flavus DGAT gene sequence (GenBank accession number: XM_002378082), primers (sequences 8, 9) were designed, and a fragment of about 1.4 kbp was amplified from the Aspergillus flavus cDNA in vitro. The amplified DNA fragment was cloned and then sequenced, and the sequence 12 was analyzed and showed to be...

Embodiment 3

[0073] 1. Mass induction of transgenic yeast

[0074] (1) Inoculate the monoclonal colonies containing pYES-DEST52 into 15 ml of SC-U yeast deficiency selection medium (0.67% YNB, 1× amino acid, adenine, 2% glucose), shake the bacteria overnight at 28°C and 200rpm;

[0075] (2) Measure OD600, and calculate the bacteria that are finally inoculated into 100ml of yeast galactose-induced expression medium (0.67% YNB, 1×amino acid, adenine, 2% galactose, 10% raffinose) according to the OD value liquid volume. The principle of inoculation is to make the initial OD value of yeast induction medium 0.4;

[0076] (3) Inoculate 100ml of yeast induction medium with the calculated amount of the original bacterial solution in the second step; inoculate 10 groups with a total volume of 1L.

[0077] (4) Shake the bacteria at 200 rpm at 28°C for 20 hours, then filter the bacteria liquid and collect the bacteria;

[0078] 2. Yeast cell protein extraction

[0079] (1) Resuspend fresh or froz...

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Abstract

The present invention relates to a separated diacylglycerol acylase gene, an expression vector and application thereof. The gene can be used to improve the oil content in the plant seeds.

Description

technical field [0001] The invention belongs to the field of plant genetic engineering, and specifically relates to a transferase gene, yeast expression vectors and plant expression vectors containing the gene, and transformants of these expression vectors. The present invention also relates to the application of the above-mentioned genes in improving plant traits. Background technique [0002] The shortage of traditional fossil energy will make biodiesel and fuel ethanol gradually replace petroleum and develop into a new basic industry and new energy industry to meet the large demand for liquid energy in the post-petroleum era. Biodiesel refers to a renewable diesel fuel that can replace petrochemical diesel oil, which is made from oil crops, wild oil plants, engineering microalgae and other aquatic vegetable oils, animal oils, and catering waste oils through transesterification process. Compared with diesel, it has high energy density, good lubrication performance, safe s...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/54C12N15/63C12N15/81C12N15/82C12N15/84C12N1/19A01H5/00A01H5/10
Inventor 侯磊裴炎梁爱敏周庆璋张丹丹李先碧宋水清李德谋张瑞芝肖月华罗明金丹罗小英
Owner SOUTHWEST UNIV
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