Sorghum drought response gene SbJAZ1 and recombinant vector and expression method thereof

A recombinant vector and gene technology, which can be used in chemical instruments and methods, botanical equipment and methods, biochemical equipment and methods, etc., and can solve the problems that have not yet been reported on JAZ protein research.

Pending Publication Date: 2019-07-05
GUIZHOU UNIV +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, JAZ protein has been found in Arabidopsis thaliana, corn, grape, Hevea brasiliensis, etc., but so far, there has been no research report on JAZ protein in sorghum at home and abroad

Method used

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  • Sorghum drought response gene SbJAZ1 and recombinant vector and expression method thereof
  • Sorghum drought response gene SbJAZ1 and recombinant vector and expression method thereof
  • Sorghum drought response gene SbJAZ1 and recombinant vector and expression method thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0032] Example 1: Acquisition and Analysis of Sorghum Drought Response Gene SbJAZ1

[0033] According to the reported protein sequence of the rice JAZ1 gene, blastP searched the sorghum genome database (https: / / phytozome) to find the sorghum homologous gene SbJAZ1 (gene number is Sb08g005080, and its nucleotide sequence is shown in SEQ ID NO.1). Search the NCBI database with the SbJAZ1 protein (its amino acid sequence is shown in SEQ ID NO.2), download the JAZ1 gene in other species, and use MEGA 7.0 software to construct a no root phylogenetic tree, by figure 1 It can be seen that SbJAZ1 and maize JAZ1 are clustered together and have the closest relationship. It can be seen that the SbJAZ1 gene is a drought-responsive gene in sorghum.

Embodiment 2

[0034] Example 2: Construction and Identification of Sorghum Drought Response Gene SbJAZ1 Recombinant Vector

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

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

[0037] 2. Using cDNA as a template to amplify the SbJAZ1 gene;

[0038] Primers were designed to amplify the SbJAZ1 gene using cDNA as a template.

[0039] Primers are as follows:

[0040] Upstream primer: SbJAZ1-F: GC GAATTC ATGGCCGCCGAGCAG underlined is the EcoRI restriction site;

[0041] Downstream primer: SbJAZ1-R:CG AAGCTT The underline of TCAGCTGAGGCTGAGCGC is the HindIII 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 a...

Embodiment 3

[0048] Example 3: Induced expression of SbJAZ1 protein

[0049] 1. Obtain the recombinant prokaryotic expression strain of SbJAZ1

[0050] The single clone successfully sequenced in Example 2 was selected and inoculated into 50ug / mL kanamycin liquid medium, cultured overnight at 37°C and 200rpm, and the pET-28a - The SbJAZ1 recombinant expression vector was extracted, and the recombinant expression vector plasmid was transformed into Escherichia coli expression strains BL21(DE3), JM109(DE3), BL21(DE3)pLysS, Tuner(DE3), Rosetta(DE3), and the expression of SbJAZ1 protein was detected.

[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) strains to 50ug / mL kanamycin liquid medium, transfer Rosetta(DE3), BL21(DE3)pLysS strains to 50ug / mL In mL kanamycin+50ug / mL chloramphenicol liquid medium, cultivate the activate...

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Abstract

The invention discloses a sorghum drought response gene SbJAZ1. The nucleotide sequence of the sorghum drought response gene SbJAZ1 is shown as SEQ ID NO. 1. The invention also discloses a protein encoded by the sorghum drought response gene SbJAZ1, and the amino acid sequence of the protein is shown as SEQ ID NO. 2. The invention further discloses a recombinant vector containing the sorghum drought response gene SbJAZ1 and an expression method. According to the method, a sorghum homologous gene SbJAZ1 is obtained from a sorghum genome database through the protein sequence of a rice JAZ1 gene,the full length of the gene is 606bp, a prokaryotic expression vector is constructed according to the gene, the condition of soluble expression of the gene is optimized, meanwhile, the protein purification system is used for purifying the gene, the result lays a foundation for further researching the protein crystal structure and biological characteristics, and a basis for further provided for improving the drought resistance of sorghum through a gene editing method.

Description

technical field [0001] The invention relates to a sorghum drought response gene SbJAZ1, a recombinant vector and an expression method thereof, and belongs 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 ...

Claims

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Application Information

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IPC IPC(8): C12N15/29C07K14/415C12N15/70C12P21/02
CPCC07K14/415C12N15/70
Inventor 谢鑫蒋君梅任明见李向阳孙涛
Owner GUIZHOU UNIV
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