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Soybean oleosin gene GmOLEO1 as well as encoded proteins and application thereof

A soybean oil body and protein technology, applied in the field of genetic engineering, can solve the problem that the oil body protein gene is blank, and achieve a good application prospect

Inactive Publication Date: 2017-05-24
HENAN AGRICULTURAL UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the research on the effect of oleosin gene on soybean oil content is still blank

Method used

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  • Soybean oleosin gene GmOLEO1 as well as encoded proteins and application thereof
  • Soybean oleosin gene GmOLEO1 as well as encoded proteins and application thereof
  • Soybean oleosin gene GmOLEO1 as well as encoded proteins and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0038] Example 1: Cloning of soybean oleosin gene GmOLEO1 and construction of plant expression vector

[0039] (1) Design primers, extract RNA, reverse cDNA:

[0040] The total RNA of soybean Williams 82 leaves was extracted with a plant total RNA extraction kit (DP432, Tiangen), and the integrity of the RNA was detected by 1% agarose gel electrophoresis; cDNA was synthesized with reference to TaKaRa Primer Script TM The RT reagentkit with gDNA Eraser kit explains the operation. The primer sequence of design amplification gene GmOLEO1 is as follows:

[0041] Seq ID NO.3: GmOLEO1-F 5'-CCTACCACATTAATTACTCACTCTTCACTCA-3';

[0042]Seq ID NO.4: GmOLEO1-R 5'-TCAACTTTAACGCTCATTCCTGCATTCAT-3'.

[0043] (2) PCR amplification, the specific steps are as follows:

[0044] Step 1: Prepare PCR reaction solution (50μl system) according to the following components: 10×PCR Buffer (25μl), ddH 2 O (9 μl), dNTP (10 μl), GmOLEO1-F (1.5 μl), GmOLEO1-R (1.5 μl), cDNA (2 μl), KOD FX enzyme (1 μl...

Embodiment 2

[0053] Embodiment 2: Cultivation of GmOLEO1 gene overexpression transgenic soybean

[0054] (1) Disinfection and germination of seeds

[0055] The surface disinfection of soybean seeds was sterilized by dry chlorine gas. Pick clean seeds that are mature and plump, without disease spots, and without hardness, and arrange them in a single layer in a 90*15mm petri dish; open the lid of the petri dish and put it in a desiccator, place a 500ml glass beaker in the desiccator, and use a 100ml measuring cylinder Measure 75ml of commercial bleach into the beaker, measure 3ml of 12M HCl with a 10ml measuring cylinder, and slowly add along the wall of the beaker; cover the lid of the desiccator to ensure that the vessel is sealed, let stand overnight, 10-16 hours, and sterilize After completion, cover the culture dish and transfer it to a sterile ultra-clean bench, open the lid of the culture dish, and blow with strong wind for 25 to 40 minutes to remove residual chlorine; sow the steri...

Embodiment 3

[0062] Example 3: Verification of genetically modified materials

[0063] Because the vector used for the transgene encodes glufosinate acetyl CoA transferase (PAT), which can catalyze the free aminoacetylation of glufosinate, thereby inactivating the herbicide glufosinate. Basta, the herbicide used for identification, was diluted 1000 times (concentration: 200mg / L), and the transgenic seedlings were sprayed. The negative plants withered and died, and the positive plants showed obvious resistance and maintained good growth. DNA was extracted from the leaves of the positive plants that survived the herbicide detection (CTAB Plant Genomic DNA Rapid Extraction Kit: Zhongding Company, Cat. No. DN14-100T), and PCR was used to detect the marker bar gene to further screen positive materials. The primer sequence of bar gene is as follows:

[0064] Seq ID NO.7: Upstream primer 5'-ATGAGCCCAGAACGACGC-3';

[0065] Seq ID NO.8: Downstream primer 5'-ACGTCATGCCAGTTCCCGT-3'.

[0066] Final...

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Abstract

The invention belongs to the technical field of genetic engineering and specifically discloses a soybean oleosin gene GmOLEO1 as well as encoded proteins and application thereof. The gene has a nucleotide sequence shown as Seq ID NO.1. The soybean oleosin gene GmOLEO1 is obtained by cloning from soybean genome first, and the gene GmOLEO1 disclosed by the invention is introduced into plant cells by utilizing a plant expression vector, so that a transgenic plant of which the oil content is increased can be obtained. Compared with non-transgenic soybeans, soybeans in which the gene GmOLEO1 disclosed by the invention is overexpressed have the advantage that the oil content of the soybean seeds is obviously improved, and the gene GmOLEO1 plays an important role in the aspect of improving the oil content of plant seeds, particularly the soybean seeds. The achievement of the invention can be applied to regulating the soybean oleosin genes through biotechnologies, has significances for cultivating soybean varieties with high oil content and has excellent application prospects.

Description

technical field [0001] The invention relates to the technical field of genetic engineering, in particular to a soybean oil body protein gene GmOLEO1 and its encoded protein and application. Background technique [0002] Oils in plants play a vital role in plant cell division, growth and development. Vegetable oil is not only one of the important foods for human beings, but also a kind of renewable and environmentally friendly biomass energy, which is an important energy source for human production and life. [0003] The synthesis of lipids in plant seeds can be divided into three stages: the first stage is the synthesis of fatty acids in the plastid using sucrose as the main carbon source, the second stage is the synthesis of triacylglycerol in the endoplasmic reticulum, and the third stage is the formation of Triacylglycerols combine with oleosin to form oil bodies. Oil bodies are subcellular organelles that store triacylglycerols in plant seeds. They store liquid triacyl...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/29C07K14/415C12N15/82A01H5/00
CPCC07K14/415C12N15/8247
Inventor 张丹褚姗姗李红岩
Owner HENAN AGRICULTURAL UNIVERSITY
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