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Calcium ion binding protein derived from stem nodule as well as encoding gene and application thereof

A technology for binding proteins and calcium ions, applied in the field of calcium ion binding proteins and their coding genes and applications

Active Publication Date: 2013-08-14
CHONGQING UNIV OF POSTS & TELECOMM
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  • Summary
  • Abstract
  • Description
  • Claims
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AI Technical Summary

Problems solved by technology

[0006] For the stem mustard, there are no reports on the sequence of its calcium ion-binding protein and the gene function. The present invention has cloned the first calcium ion-binding protein of the stem mustard, and a large number of experimental studies show that the protein has the ability to improve plant resistance. The role of stress resistance provides new content and new basis for the research and application of plant stress resistance genes

Method used

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  • Calcium ion binding protein derived from stem nodule as well as encoding gene and application thereof
  • Calcium ion binding protein derived from stem nodule as well as encoding gene and application thereof
  • Calcium ion binding protein derived from stem nodule as well as encoding gene and application thereof

Examples

Experimental program
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experiment example 1

[0022] Experimental Example 1: Cloning of the BjCBP1 gene of BjCBP1

[0023] 1 Main reagents: column type small amount plant total RNA extraction kit (W7021) was purchased from Shanghai Huashun Biotechnology Co., Ltd.; DNA purification and recovery kit was purchased from Tiangen Biochemical Technology Co., Ltd.; 5'-Full RACE Kit kit , M-MLV Reverse Transcriptase, Premix Ex Taq, DL2000DNA Marker, and pMD19-T vectors were all purchased from Dalian Bao Biological Engineering Co., Ltd.

[0024] 2 Experimental methods and steps:

[0025] Acquisition of BjCBP1 gene sequence fragment SEQ ID NO.4: The total RNA of the tuber mustard stem was extracted by using the column type small amount plant total RNA extraction kit method, and a small part of the total RNA was detected by UV spectrophotometer for OD260 / OD280 The ratio of 1.92, the brightness of 28S by agarose gel electrophoresis is about 2 times that of 18S, indicating that the RNA has high purity and no degradation, which meets t...

experiment example 2

[0057] Experimental example 2: Prokaryotic expression and characteristic detection of BjCBP1 gene BjCBP1 in Escherichia coli BL21(DE3)

[0058] 1. Main reagents: His-Tag protein purification kit was purchased from Beijing Kangwei Reagent Biotechnology Co., Ltd.; T4DNA ligase, restriction endonuclease BamH I and HindШII were purchased from Dalian Bao Biological Engineering Co., Ltd.; the prokaryotic expression plasmid pET was included -32a (+) DH5α strains and Escherichia coli strains are preserved by our laboratory.

[0059] 2. Construction of recombinant prokaryotic expression vector pET32a-BjCBP1

[0060] 2.1 Primer design: Design upstream and downstream primers according to the coding region sequence of BjCBP1 gene SEQ ID NO.3, and introduce restriction site BamH I (GGATCC) and HindШ (AAGCTT) sequences respectively. The primers are:

[0061] Upstream primer pET-F: 5'-CGGATCCATGAAATTCGCAAAACTG-3'

[0062] Downstream primer pET-R: 5'-CAAGCTTTCATCGCTGGAGATCCA-3'

[0063] 2....

experiment example 3

[0069] Experimental Example 3: Construction of the recombinant plant expression vector pCAMBIA1302-BjCBP1 of the BjCBP1 gene of the mustard calcium ion binding protein

[0070] 1 Main reagents: Common plasmid extraction kit was purchased from Tiangen Biochemical Technology Co., Ltd.; T4DNA ligase, restriction endonuclease BamH I, Bgl II and BstE II were purchased from Dalian Bao Biological Engineering Co., Ltd.; strains containing plasmid pCAMBIA1302 maintained by the laboratory.

[0071] 2 Construction of recombinant plant expression vector pCAMBIA1302-BjCBP1

[0072] 2.1 Primer design: Design upstream and downstream primers according to the sequence of the coding region of BjCBP1 gene SEQ ID NO.3, and introduce restriction site BamH I (GGATCC) and BstE II (GGTCACC) sequences respectively. The primers are:

[0073] Upstream primer BjCBP1-Bam: 5'-GGATCCATGAAATTCGCAAAACTG-3'

[0074] Downstream primer BjCBP1-Bst: 5'-GGTCACCTCATCGCTGGAGATCCA-3'

[0075] 2.2 PCR amplification:...

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Abstract

The invention discloses a calcium ion binding protein derived from stem nodule as well as an encoding gene and application thereof. The calcium ion binding protein gene BjCBP1 has a base sequence as shown in SEQ NO.1; the constructed recombinant prokaryotic expression vector pET32a-BjCBP1 consists of a sequence SEQ ID NO.1 and a prokaryotic expression vector pET32a(+); the recombinant expression vector pCAMBIA1302-BjCBP1 consists of the sequence SEQ ID NO.1 and a plant expression vector pCAMBIA 1302; the plant expression vector is used for plant genetic transformation; the BjCBP1 genes are subjected to over-expression under the start of a CaMV35S promoter, and lots of BjCBP1 proteins are synthesized, so that the adverse situation resistance of plants is improved.

Description

technical field [0001] The invention relates to the field of genetic engineering, in particular to a calcium ion-binding protein derived from stem mustard and its coding gene and application. Background technique [0002] Calcium ion signaling plays an important role in many cellular activities, such as cell metabolism, gene expression, cytoskeleton dynamic changes, cell cycle, cell death, signal transduction and other processes. [0003] In plant cells, one of the functions of calcium ions is as a second messenger. Many external environmental signals, such as light, biotic stress, abiotic stress, plant hormones, etc., will stimulate the Ca in the cytosol. 2+ Concentration increases, thereby triggering calcium ion signaling. The first responder in the calcium ion signaling pathway is the calcium ion binding protein, which binds Ca 2+ A later conceptual change exposes the hydrophobic face, thereby activating or inhibiting the activity of other proteins, thereby initiating s...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/29C07K14/415C12N15/70C12N15/84A01H5/00
Inventor 向浏欣蔡应繁刘吉军王小艳付于银
Owner CHONGQING UNIV OF POSTS & TELECOMM
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