Soybean salt-tolerant gene GmCBL3 and application thereof

A salt-tolerant gene and soybean technology, applied in the fields of application, genetic engineering, plant genetic improvement, etc., to achieve the effect of improving salt tolerance and salt/drought resistance

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 GmCBL3 and application thereof
  • Soybean salt-tolerant gene GmCBL3 and application thereof
  • Soybean salt-tolerant gene GmCBL3 and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0017] Cloning of embodiment 1, GmCBL3

[0018] 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.

[0019] 1.1 Extraction of total RNA from soybean 371005 plants:

[0020] (1) Put the soybean 371005 material into a pre-cooled mortar, add liquid nitrogen and quickly grind it into a uniform powder;

[0021] (2) After the liquid nitrogen volatilizes, quickly transfer the material to a pre-cooled centrifuge tube, add 1ml TRIZOL solution for every 50-100mg of tissu...

Embodiment 2

[0106] Example 2, Agrobacterium-mediated Arabidopsis transformation of soybean GmCBL3 and Arabidopsis AgCBL1 genes

[0107]2.1 Overexpression vector construction

[0108] (1) Referring to the instructions of the Gateway kit, use primers SEQ ID No.4-5 to add a linker to GmCBL3 through the PCR reaction of high-fidelity DNA polymerase, and amplify the full-length fragment of the target gene with the linker:

[0109] P2 (upstream primer): 5'-AAAAAAGCAGGCTTCATGGTGCACTGTTTAGATGG-3' (SEQ ID No.4);

[0110] P3 (downstream primer): 5'-AGAAAGCTGGGTCGCCAACACCAGAGAAACTGA-3'; (SEQ ID No. 5).

[0111] The primers SEQ ID No.6-7 are used to add adapters to AgCBL1 through the PCR reaction of high-fidelity DNA polymerase to amplify the full-length fragments of the target gene with adapters.

[0112] P4 (upstream primer): 5'-AAAAAAGCAGGCTTCATGGGCTGCTTCCACTCAAAGGCAG-3' (SEQ ID No.6);

[0113] P5 (downstream primer): 5'-AGAAAGCTGGGTCTGTGGCAAATCTCATCGACCTCCGAA-3' (SEQ ID No. 7).

[0114] (2) Ta...

Embodiment 3

[0147] Embodiment 3, Arabidopsis thaliana salt tolerance verification of transgenic GmCBL3 and AgCBL1 genes

[0148] (1) Extract the DNA sample of the plant, and utilize the following primers to pass through the PCR reaction of Taq DNA polymerase, verify and obtain the transgenic GmCBL3 gene positive plant,

[0149] P6 (upstream primer): 5'-ATGGTGCACTGTTTAGATGGATTAA-3' (SEQ ID No.8)

[0150] P7 (downstream primer): 5'-TCAGCCAACACCAGAGAAACTGACA-3' (SEQ ID No.9);

[0151] Utilize following primer similarly by the PCR reaction of Taq DNA polymerase, verify and obtain transfection AgCBL1 gene positive plant,

[0152] P8 (upstream primer): 5'-ATGGGCTGCTTCCACTCAAAGGCAG-3' (SEQ ID No.10)

[0153] P9 (downstream primer): 5'-TCATGTGGCAATCTCATCGACCTCC-3' (SEQ ID No. 11).

[0154] (2) Disinfect three different types of Arabidopsis seeds respectively: Arabidopsis wild type (negative control), Arabidopsis transgenic plants overexpressing Arabidopsis AgCBL1 gene (positive control) ) and...

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Abstract

The invention discloses a soybean salt-tolerant gene GmCBL3 and an application thereof. A cDNA sequence of the soybean salt-tolerant gene GmCBL3 is as shown in SEQ ID No.1. The method comprises the following steps: firstly, cloning the soybean salt-tolerant gene GmCBL3; carrying out BP reaction on the gene GmCBL3 by virtue of a Gateway system to connect to pDONR221; obtaining a lot of clones from escherichia coli DH5alpha through a transformation method; carrying out LR reaction to connect the gene GmCBL3 to an expression carrier pK2GW7, and expressing in the escherichia coli DH5alpha; screening transgenic escherichia coli, and extracting transform plasmids; and finally transferring the transform plasmids to agrobacterium, and transferring into arabidopsis thaliana, so that transgenic positive plants grow in an external salt stress condition. Comparative analysis proves that the salt resistance of the transgenic plants is obviously improved in comparison with that of non-transgenic plants and other transgenic plants of transforming arabidopsis CBL1 genes.

Description

technical field [0001] The invention belongs to the technical field of biogenetic engineering, and in particular relates to soybean salt-tolerant gene GmCBL3 and its application. Background technique [0002] my country is the origin and main cultivation country of soybean, and soil salinization has become one of the main obstacles restricting the development of soybean industry. Improving the drought and salt tolerance of plants through conventional breeding methods is time-consuming, laborious and difficult, and it has been difficult to breed truly resistant varieties. Molecular biology and reverse genetics have provided new ideas, new ways and methods for soybean breeding. The new opportunity provides the possibility to improve the salt tolerance of soybean. Understanding the salt tolerance of soybeans and further revealing its molecular mechanism will help to breed new salt-tolerant varieties of soybeans, expand the planting range of soybeans, reduce the impact of facto...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/55C12N15/84A01H5/00
Inventor 王俊峰余华马玉敏谢坤白静辛富刚
Owner SHANDONG CROP GERMPLASM CENT
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