Medicago truncatula Gaertn MtWOX11 gene and application thereof in increasing fatty acid content of seeds

A technology of alfalfa, gene, applied in the field of genetic engineering

Active Publication Date: 2018-03-09
SHANDONG UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the search found that the nucleotide sequence and amino acid sequence of the WOX family gene Mt WUSCHEL relatedhomeobox 11 (MtWOX11) of Medicago truncatula has not been reported yet, and its application in improving the fatty acid content of legume seeds has not been reported either.

Method used

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  • Medicago truncatula Gaertn MtWOX11 gene and application thereof in increasing fatty acid content of seeds
  • Medicago truncatula Gaertn MtWOX11 gene and application thereof in increasing fatty acid content of seeds
  • Medicago truncatula Gaertn MtWOX11 gene and application thereof in increasing fatty acid content of seeds

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0022] Example 1 Cloning and expression analysis of Medicago truncatula MtWOX11 gene

[0023] 1.1 Obtaining and identification of the mtwox11 mutant of Medicago truncatula

[0024] Through the public website https: / / medicago-mutant.noble.org / mutant / database.php, the Tnt1-marked mutant library of Medicago truncatula was screened, and using Thermal asymmetric interlaced-PCR (TAIL-PCR) technology, 22,000 mutants Mutant lines in which Tnt1 was inserted into the MtWOX11 gene were screened out.

[0025] 1.1.1 Extract the total RNA of Medicago truncatula mtwox11

[0026] (1) Put 0.1g of tissue material into a liquid nitrogen precooled mortar, and fully grind it into powder in liquid nitrogen;

[0027] (2) After the liquid nitrogen evaporates to dryness, transfer it to a 2ml centrifuge tube immediately, add about 1ml of Invitrogen's TRIzol extract for every 100mg of material, after melting, repeatedly suck and blow with a sample gun, shake and mix the sample vigorously, and make Th...

Embodiment 2

[0086] Embodiment 2, the construction of plant expression vector

[0087] The full-length cDNA sequence of MtWOX11 was constructed into the pENTR / D-TOPO intermediate vector, and then the full-length CDS sequence of MtWOX11 was connected to the pEarleyGate103 overexpression vector by LR recombination. A plant expression vector that overexpresses the MtWOX11 gene driven by the CaMV35S promoter is obtained.

[0088] 2.1 The full-length CDS sequence of MtWOX11 was constructed into the pENTR / D-TOPO vector, and the method was the same as above.

[0089] 2.2 LR recombination was connected to pEarleyGate103 overexpression vector.

[0090] (1) Reaction system:

[0091] pENTR / D-TOPO vector: 1 μl

[0092] pEarleyGate103 overexpression vector: 1 μl

[0093] 2X Fuzyme Master Mix: 0.5μl

[0094] (2) Incubate the reaction mixture at 25°C for 2-3 hours.

[0095] (3) The above system was transformed into Escherichia coli DH5α.

[0096] 2.3 Identification of recombinants

[0097] (1) PC...

Embodiment 3

[0101] Embodiment 3, preparation and transformation of Agrobacterium competent

[0102] 3.1 Preparation of Competent Agrobacterium AGL1 / EHA105

[0103] 1. Pick a single colony of Agrobacterium tumefaciens from the YEP plate (containing 50 μg / ml rifampicin), inoculate it in YEP liquid medium (containing 50 μg / ml rifampicin), culture at 200 rpm / min at 28° C. overnight.

[0104] 2. Inoculate 2ml of the overnight culture solution into 50ml of YEP liquid medium containing the same antibiotic and cultivate to OD under the same conditions 600 up to 0.5.

[0105] 3. Bacteria solution was placed in an ice bath for 30 minutes, centrifuged at 5000 rpm for 10 minutes at 4°C, and the bacteria were collected.

[0106] 4. Resuspend the bacteria in 10 ml of 0.15 mol / L NaCl in an ice bath, and collect the bacteria by centrifugation.

[0107] 5. Resuspend in 1ml 20mmol / L ice-cooled CaCl 2 In the solution, divide the bacterial liquid into 1.5ml Eppendorf tubes at 200 μl / tube, freeze in liqui...

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Abstract

The invention discloses a Medicago truncatula Gaertn WOX family gene Mt WUSCHEL related homeobox11 (MtWOX11). The nucleotide sequence of the gene is shown in SEQ ID No. 1. The invention also disclosesapplication of the gene in increasing the fatty acid content of legume seeds. The analysis of transgenic lines obtained by a genetic transformation method proves that the overexpression of the gene can significantly increase the fatty acid content of seeds of legumes. The gene provided by the invention has wide application prospects, is used for improving the quality of legumes or creating novellegume economic crops, and is of great significance to the production of legume cash crops in China.

Description

technical field [0001] The invention relates to a WUSCHEL relatedhomeobox (WOX) family gene and application thereof, in particular to a Medicago truncatula WOX family gene Mt WUSCHEL relatedhomeobox 11 (MtWOX11) and its application in increasing the fatty acid content of leguminous plant seeds, belonging to the field of genetic engineering. Background technique [0002] In recent years, with the improvement of my country's economic level and people's quality of life, the demand for soybeans has also increased day by day. Soybean is one of the important economic leguminous crops, which can not only provide high protein, but more importantly, it can provide oil. However, at present, the quality of soybeans in my country is not high, lacking market competitiveness, and a large amount of soybeans needs to be imported. Therefore, cultivating new soybean varieties with high oil and high protein has become a very urgent task at present. [0003] At present, research work on soybe...

Claims

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Application Information

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IPC IPC(8): C12N15/29C12N15/82A01H5/10A01H6/54
CPCC07K14/415C12N15/8247
Inventor 韩璐周传恩蒋洪娇周欣
Owner SHANDONG UNIV
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