Soybean salt-tolerant gene GmCIPK2 and application thereof

A salt-tolerant gene, soybean technology, applied in application, genetic engineering, plant genetic improvement and other directions to achieve the effect of improving salt tolerance

Active Publication Date: 2015-06-24
SHANDONG CROP GERMPLASM CENT
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, there are few reports on the CBL gene in soybean and its effect on plant salt tolerance.

Method used

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  • Soybean salt-tolerant gene GmCIPK2 and application thereof
  • Soybean salt-tolerant gene GmCIPK2 and application thereof
  • Soybean salt-tolerant gene GmCIPK2 and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0018] Example 1: Yeast cDNA library construction of soybean 371005

[0019] Collection of salt-tolerant soybean germplasm: the inventor of the present invention collected and screened out a kind of salt-tolerant, low-temperature sensitive soybean germplasm (soil salt concentration (‰) in Qijia Village, Mingji Township, Lijin County, Dongying City in October 2009 ): 1.5, latitude / °: 37.59, longitude / °: 118.22, altitude / m: 10). According to the number starting with 37 in Shandong Province, the 1005th resource was named 371005 (Collection Number of Germplasm Resources in Coastal Areas of Shandong Province); its relative salt damage index: 18.00.

[0020] 1.1 Extraction of total RNA from soybean 371005 plants:

[0021] (1) Put the soybean 371005 material into a pre-cooled mortar, add liquid nitrogen and quickly grind it into a uniform powder;

[0022] (2) After the liquid nitrogen volatilizes, quickly transfer the material into a pre-cooled centrifuge tube, add 1ml TRIZOL solut...

Embodiment 2

[0135] Embodiment 2, Arabidopsis flower transformation of Agrobacterium-mediated GmCIPK2

[0136] 2.1 Overexpression vector construction

[0137] (1) Using the Gateway system of Invitrogen Company, the GmCIPK2 gene was connected to the pDONR221 vector by BP reaction (see image 3 ), the following primers SEQ ID No.9-10 are added to GmCIPK2 by the PCR reaction of high-fidelity DNA polymerase;

[0138] P3 (upstream primer): 5'-AAAAAAGCAGGCTTCATGGAGTACCCATACGACGT-3' (SEQ ID No.9)

[0139] P4 (downstream primer): 5'-AGAAAGCTGGGTCGGTACGTCGTATGGGTACTCCAT-3' (SEQ ID No.10);

[0140] (2) Take 25 μl of PCR product, use TE Buffer (10mM Tris-HCl pH 7.5, 1mM EDTA), 1 / 2 volume of 30% PEG 8000 / 30mM MgCl 2 The solution was diluted 4 times to a final concentration of 10% PEG and 10 mM MgCl 2 , vortexed, centrifuged at 13,000rpm for 15min;

[0141] (3) Gently move the supernatant and resuspend the pellet with TE to make the final concentration of DNA >10ng / μl;

[0142] (4) Add the follow...

Embodiment 3

[0172] Embodiment 3, Arabidopsis thaliana salt tolerance verification of transgenic GmCIPK2 gene

[0173] (1) extract the DNA sample of the plant, and use the following primers SEQ ID No.11-12 to pass through the PCR reaction of Taq DNA polymerase, verify and obtain the transgenic GmCIPK2 gene positive plant;

[0174] P5 (upstream primer): 5'-ATGGAGTACCCATACGACGTACCAG-3' (SEQ ID No.11)

[0175] P6 (downstream primer): 5'-CTGGTACGTCGTATGGGTACTCCAT-3' (SEQ ID No.12);

[0176] (2) Disinfecting two different types of Arabidopsis seeds respectively: Arabidopsis wild type (negative control) and Arabidopsis transgenic plants overexpressing soybean GmCIPK2 gene;

[0177] (3) On the 1 / 2MS medium with a NaCl concentration of 0.5%, cultivate two kinds of Arabidopsis seeds through disinfection;

[0178] (4) After 10 days of normal culture, it was found that the Arabidopsis transgenic plants overexpressing the GmCIPK2 gene could grow normally, while the Arabidopsis wild-type plants could...

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Abstract

The invention discloses a soybean salt-tolerant gene GmCIPK2 and application thereof. A nucleotide sequence of cDNA of the soybean salt-tolerant gene GmCIPK2 is shown in SEQ ID No.1. According the application, a yeast two-hybrid system of Clontech is firstly utilized for constructing a yeast cDNA library of soybean 371005, soybean salt-tolerant gene GmCBL3 is cloned, a bait protein is constructed, and an interacting protein GmCIPK2 of a protein GmCBL3 is obtained by virtue of a yeast two-hybrid method; then a gateway is utilized for carrying out BP reaction on the gene GmCIPK2 to link the gene GmCIPK2 to pDONR221, massive cloning is carried out in escherichia coli DH5alpha by virtue of a conversion method, and then LR reaction is carried out to link the gene GmCIPK2 to an expression vector pK2GW7; the expression vector pK2GW7 is transferred into Arabidopsis by virtue of agrobacterium mediation, so that the salt tolerance of transgenic plants is obviously improved.

Description

technical field [0001] The invention belongs to the technical field of biological genetic engineering, and in particular relates to a soybean salt-tolerant related gene GmCIPK2 and its application. Background technique [0002] my country is one of the countries where saline soil is widely distributed, and the salinization of land has become one of the main obstacles restricting the development of my country's agriculture, especially the soybean industry. Among the current soil improvement methods, chemical improvement and biological improvement are the focus of current research. For chemical improvement, although the effect is quick, it is easy to introduce new ions to cause secondary pollution, and the capital investment and technical requirements are high, so it is difficult to implement large-scale land restoration. Biological improvement can effectively reduce costs and is the most economical and effective measure for salinized land restoration. [0003] The ultimate ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/54C12N15/84A01H5/00
Inventor 王俊峰孔维国余华马玉敏谢坤白静
Owner SHANDONG CROP GERMPLASM CENT
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