Fusion gene GAD-GLP-1 and culture method of diabetes diet therapy type cucumber

A GAD-GLP-1, -1GLP-1 technology, applied in the field of plant genetic engineering, can solve the problem of undiscovered diabetes diet type cucumber, etc., achieve the effect of lowering blood sugar level and improving symptoms of diabetes

Inactive Publication Date: 2010-09-08
NANKAI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

After searching the literature, no literature or patent reports have been found on the transformation and cultivation of diabetic dietary cucumbers using GAD and GLP-1 genes

Method used

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  • Fusion gene GAD-GLP-1 and culture method of diabetes diet therapy type cucumber
  • Fusion gene GAD-GLP-1 and culture method of diabetes diet therapy type cucumber
  • Fusion gene GAD-GLP-1 and culture method of diabetes diet therapy type cucumber

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0044] Construction of a fusion gene GAD-GLP-1. Including the following steps:

[0045] (1) Cloning the GAD gene into a cloning vector to construct pMD-GAD. The PCR primers used in the experiment were designed according to the sequence of the GAD gene:

[0046] P1: 5′- GGATCC ATGGCATCTCCGGGCTCTG-3′,

[0047] P2: 5′- GATATC CTTTAAATCTTGTCCAAG-3′,

[0048] BamH I was introduced upstream of p1, and EcoRV site was introduced downstream of p2, both of which are underlined.

[0049] (2) Cloning the GLP-1 gene into a cloning vector to construct pMD-GLP-1. The PCR primers used in the experiment were designed according to the sequence of the GLP-1 gene:

[0050] P3: 5′- GATATC AAGCATTCTGAGGGAAC-3′,

[0051] P4: 5′- GTC GAC TTAACGGCCATCTACGAC-3′

[0052] An EcoRV site was added to the 5' end of P3, and a Sal I site was added to the 3' end of p4. Both are underlined.

[0053] (3) The cloning vector pMD-GAD was digested with BamHI and EcoRV, and the GAD fragment was obtai...

Embodiment 2

[0055] Construction of a Ti expression vector containing GAD-GLP-1 fusion gene

[0056] The cloning vector pMD-GAD-GLP-1 was digested with BamHI and SalI, and the GAD-GLP-1 gene with cohesive ends was obtained after digestion. Plasmid pX6 was completely digested with Spe I, and the cohesive ends of the vector and gene fragments were blunted. The purified GAD-GLP-1 gene fragment was ligated with the dephosphorylated linear vector pX6. Such as image 3 As shown, the exogenous gene has been connected into the vector pX6, and the transformant selected for forward connection is the expression vector used in the present invention, the Ti expression vector pX6-GAD-GLP-1 containing the GAD-GLP-1 fusion gene .

[0057] Transforming the above-mentioned Ti expression vector pX6-GAD-GLP-1 into Agrobacterium LBA4404 can obtain an engineering strain containing the GAD-GLP-1 fusion gene.

Embodiment 3

[0059] Breeding of transgenic cucumber expressing GAD-GLP-1 fusion protein

[0060] Take the cotyledon node of cucumber as the explant, infect with the suspension of the engineering strain described in claim 4 for 15 minutes, transfer to the co-culture medium for co-cultivation; then transfer the cotyledon node to the screening medium, and wait for the resistant bud to elongate When the buds grow to about 1.5-2cm, transfer them to the bud elongation medium; when the buds grow to 3-4cm, transfer to the rooting medium; when the roots grow to more than 4-6cm, and some lateral roots grow on the adventitious roots, grow Robust plants can be transplanted; the preparation of the culture medium is shown in Table 1.

[0061] Table 1 Medium used in this experiment

[0062]

[0063] according to Figure 6 As shown, kanamycin-resistant plants were obtained through cotyledonary node organogenesis pathway and 30 mg / L kanamycin resistance selection. After the obtained resistant plants ...

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Abstract

The invention relates to a fusion gene GAD-GLP-1 and a culture method of diabetes diet therapy type cucumber. The fusion gene GAD-GLP-1 provided by the invention is shown as a sequence list, and comprises a section of glutamate decarboxylase gene GAD and a section of glucagon-like peptide-1 gene GLP-1, the glutamate decarboxylase gene GAD and the glucagon-like peptide-1 gene GLP-1 are connected through an EcoRV site, two lysines are respectively added at both sides of the EcoRV site, and the four amino acids simultaneously realize the effect of peptide connection. The invention is characterized in that the fusion gene in a cloning vector pMD-GAD-GLP-1 is connected into a Marker-free plant expression vector pX6 after the BamHI and SalI enzyme digestion recovery, a selective-marker-free plant expression vector pX6-GAD-GLP-1 is obtained through construction, and the selective-marker-free plant expression vector pX6-GAD-GLP-1 is converted into agrobacterium rhizogenes LBA4404 to be construction into engineered strains. The invention successfully converts the GAD-GLP-1 fusion gene into a cucumber self-bred line 2M1 by an agrobacterium rhizogenes mediate method, and obtains transgene plants of stably expressed GAD-GLP-1 fusion protein. Through the intragastric administration on diabetes model bandicoot of the transgene cucumber provided by the invention, the blood sugar level can be reduced, and the diabetes symptom can be relieved.

Description

【Technical field】: [0001] The invention belongs to the field of plant genetic engineering, and in particular relates to a transgenic cucumber expressing a fusion protein GAD-GLP-1 related to diabetes treatment and a cultivation method thereof, which can prevent and treat diabetes by directly eating the cucumber. 【Background technique】: [0002] Diabetes mellitus is a metabolic disorder caused by absolute or relative insufficient secretion of insulin and decreased sensitivity of target tissue cells to insulin. It is a systemic chronic metabolic clinical syndrome characterized by hyperglycemia, hyperlipidemia and hyperviscosity. . In recent years, diabetes itself and its complications have seriously endangered people's physical and mental health. The number of diabetic patients in the world is increasing year by year, and tends to be younger. With social progress and changes in people's lifestyles, diabetes has become an important chronic non-infectious epidemic disease that...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/62C12N15/82C12N1/21A61K36/42A61P3/10C12R1/01
Inventor 李明刚赵磊王翠艳丁东风王瑞菊陈苗
Owner NANKAI UNIV
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