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Sorghum 14-3-3 protein GF14c gene, recombined vector thereof and expression method of gene

A recombinant vector and gene technology, applied in the fields of botany equipment and methods, biochemical equipment and methods, genetic engineering, etc., can solve the problems that have not yet been reported on 14-3-3 protein gene research.

Pending Publication Date: 2019-06-21
GUIZHOU UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

But so far, there is no research report on 14-3-3 protein gene in sorghum at home and abroad

Method used

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  • Sorghum 14-3-3 protein GF14c gene, recombined vector thereof and expression method of gene
  • Sorghum 14-3-3 protein GF14c gene, recombined vector thereof and expression method of gene
  • Sorghum 14-3-3 protein GF14c gene, recombined vector thereof and expression method of gene

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0032] Example 1: Acquisition and Analysis of Sorghum 14-3-3 Protein GF14c Gene

[0033] According to the reported protein sequence of the maize GF14c gene, blastP searched the sorghum genome database (https: / / phytozome) to find the sorghum homologous gene GF14c (gene number Sb03g028430). Search the NCBI database with the GF14c protein, download the 14-3-3 genes in other species, and use the MEGA 7.0 software to construct an unrooted evolutionary tree with Neighbor-Joining (NJ) (bootstrap=1000). figure 1 It can be seen that GF14c and maize GF14c are clustered into one branch and have the closest relationship.

Embodiment 2

[0034] Example 2: Construction and Identification of Sorghum 14-3-3 Protein GF14c Gene Recombination Vector

[0035] 1. Extract sorghum RNA and reverse transcribe cDNA

[0036] The sorghum BTx623 material was taken, and the total RNA at the seedling stage was extracted with an RNA extraction kit (Tiangen Biochemical Technology (Beijing) Co., Ltd.), and cDNA was reverse-transcribed with a reverse transcription kit (Promega).

[0037] 2. Using cDNA as a template to amplify the GF14c gene;

[0038] Primers were designed to amplify GF14c gene using cDNA as template.

[0039] Primers are as follows:

[0040] Upstream primer: GF14c-F: GC GAATTC The underline of ATGGCATCAGCAGAGCTTT is the restriction site of BamHI;

[0041] Downstream primer: GF14c-R: CG CTCGAG TTACTGCCCCTCGCTCGA is underlined as the XhoI restriction site;

[0042] The upstream and downstream primers are shown in SEQ ID NO.3 and 4 respectively.

[0043] The PCR amplification system used for gene amplification...

Embodiment 3

[0048] Example 3: Induced expression of GF14c protein

[0049] 1. Obtain the recombinant prokaryotic expression strain of GF14c

[0050] The single clone successfully picked and sequenced was inoculated into 50ug / mL kanamycin liquid medium, cultured overnight at 37°C and 200rpm, and pET-28a-GF14c was recombinantly expressed according to the plasmid mini-extraction kit of Nanjing Novizan Biotechnology Co., Ltd. The vector was extracted, and the recombinant expression vector was transformed into Escherichia coli BL21(DE3), JM109(DE3), Rosetta(DE3), BL21(DE3)pLysS, Tuner(DE3), NovaBlue(DE3) respectively to detect the expression of GF14c protein .

[0051] 2. Cultivate the activated strain overnight

[0052] The above-mentioned recombinant prokaryotic expression strains were activated by culturing overnight. For example, transfer BL21(DE3), JM109(DE3), Tuner(DE3), and NovaBlue(DE3) strains to 50ug / mL kanamycin liquid medium, and transfer Rosetta(DE3), BL21(DE3)pLysS strains Tr...

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Abstract

The invention discloses a sorghum 14-3-3 protein GF14c gene. The nucleotide sequence of the gene is as shown in SEQ ID NO.1. The invention further discloses protein of a sorghum 14-3-3 protein GF14c gene code, and the amino acid sequence of the protein is as shown in SEQ ID NO.2. The invention further discloses a recombined vector and an expression method comprising the sorghum 14-3-3 protein GF14c gene. By the aid of a protein sequence of a corn GF14c gene, a sorghum homologous gene GF14c is acquired from a sorghum gene database, the overall length of the gene is 771bp, a prokaryotic expression vector is built according to the gene and purified by a protein purification system, and a foundation is laid for further study of the a protein crystal structure and biological characteristics bya purification result, so that bases are provided for improvement of sorghum stress resistance by a gene editing method.

Description

technical field [0001] The invention relates to a sorghum 14-3-3 protein GF14c gene, a recombinant vector and an expression method thereof, belonging to the field of biotechnology. Background technique [0002] Sorghum (Sorghum bicolor (L.) Moench), also known as water millet and chestnut, is an important cereal crop with strong resistance to adversity. It is an important raw material, and it is also a kind of animal husbandry crop that has been widely used in recent years. Sorghum has a long history of cultivation in my country, and it was not cultivated on a large scale before the founding of the People's Republic of China. It was not until the 1970s, with the development of economy and society, that my country gradually paid attention to and cultivated sorghum on a large scale. Sorghum is a short-day C4 plant. Compared with other energy crops, sorghum has more obvious photosynthetic efficiency and higher yield advantages, so it is known as a "high-energy crop". In recen...

Claims

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Application Information

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IPC IPC(8): C07K14/415C12N15/29C12N15/70
Inventor 谢鑫蒋君梅任明见李向阳孙涛
Owner GUIZHOU UNIV
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