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Arabidopsis AtPGK2 gene for enhancing salt tolerance of plants and application of arabidopsis AtPGK2 gene

A technology of Arabidopsis thaliana and salt tolerance, applied in the field of plant genetic engineering, can solve problems such as unclear physiological functions, achieve wide application value, and improve salt tolerance

Inactive Publication Date: 2014-10-29
JIANGXI AGRICULTURAL UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Entering this century, although recent studies have shown that the content of PGK protein in plants increases significantly under salt stress conditions (Kosova et al., 2011; Wang et al., 2013), but for its physiological functions, especially those involved in plant response to salt The function of coercion is unclear

Method used

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  • Arabidopsis AtPGK2 gene for enhancing salt tolerance of plants and application of arabidopsis AtPGK2 gene
  • Arabidopsis AtPGK2 gene for enhancing salt tolerance of plants and application of arabidopsis AtPGK2 gene
  • Arabidopsis AtPGK2 gene for enhancing salt tolerance of plants and application of arabidopsis AtPGK2 gene

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0028] Example 1: Arabidopsis Encoding 3-Phosphoglycerate Kinase Gene AtPGK2 clone

[0029] (1) The total RNA of Arabidopsis plants was extracted by the nitrile guanidine-phenol isosulfate method. For the use, preparation and specific operation steps of the drug, refer to the method of Huang Peitang et al. (2002).

[0030] (2) According to known Arabidopsis AtPGK2 Design specific primers for the CDS sequence of the gene and introduce them in the upstream primers Nco I restriction site, introduced in the downstream primer BstE II restriction site.

[0031] Upstream primer: 5'-CATG CCATGG CTTCCACCGCCGCAACTGCA-3' (introduced Nco I restriction site)

[0032] Downstream primer: 5'-G GGTAACC TTAAACAGTGACTGGCGTTGCT-3' (introduced BstE II restriction site)

[0033] (3) Take 1 mg RNA as the template for reverse transcription, with P 2853 For primers using Promega reverse transcriptase ImProm-II TM For reverse transcription, P 2853 The primer sequences, reverse trans...

Embodiment 2

[0049] Embodiment 2: Utilize pCAMBIA1301 vector construction " CaMV 35S - AtPGK2 "Fusion gene

[0050] (1) Extract vector pCAMBIA 1301 plasmid (purchased from CAMBIA Company) from Escherichia coli, use Nco I / BstE Recover large vector fragments after II double enzyme digestion.

[0051] (2) Reclaimed to embodiment 1 AtPGK2 The CDS fragment of the gene is used Nco I / BstE II double enzyme digestion, and the digested fragments were recovered by agarose gel electrophoresis (same as in Example 1).

[0052] (3) Ligate the above two fragments under the catalysis of ligase at 16°C overnight to complete the " CaMV 35S - AtPGK2 "Fusion gene construction.

[0053] Connection system:

[0054] Reagent Amount added (μl) AtPGK2 CDS fragment of the gene (50 ng μl -1 ) 2.0 Large fragment of pCAMBIA1301 vector (50 ng μl -1 ) 3.0 Solution I Ligase 5.0

[0055] (4) Transform Escherichia coli DH5α competent cells with the ligation mixture...

Embodiment 3

[0061] Embodiment 3: Preparation of transgenic Arabidopsis plants

[0062] (1) " CaMV 35S - AtPGK2 "The fusion gene was transformed into Arabidopsis thaliana. The specific transformation method used the Floral dip method mediated by Agrobacterium (Clough and Bent, 1998). The obtained seeds were subjected to 50 mg l -1 For hygromycin resistance screening, normal growing plants were transferred to soil for culture.

[0063] (2) Real-time quantitative RT-PCR detection of transgenic plants: the transgenic Arabidopsis thaliana identified by PCR in Example 2 was multiplied, and after 50 mg l -1 Hygromycin resistance screened for T 3 To generate homozygous transgenic lines, obtain wild-type and transgenic Arabidopsis seedlings grown for 1 week respectively, extract the total RNA of plant tissue according to the method of Example 1 and reverse transcribe it into mRNA, use the following primers, reaction procedures and reaction System for PCR reaction:

[0064] AtPGK2 Gene detec...

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Abstract

The invention clones an arabidopsis 3-phosphoglycerate kinase gene AtPGK2 for enhancing salt tolerance of plants, and discloses an application of the arabidopsis 3-phosphoglycerate kinase gene AtPGK2. The nucleotide sequence of the gene is shown in SEQIDNO: 1, and the gene also comprises genes which have homologies of 90-100% with the SEQIDNO: 1 nucleotide sequence. Meanwhile, the invention also provides a construction and transgenosis method of a recombinant vector to apply the gene. The method can be used for cultivating new varieties of transgenic plants with stronger salt tolerance, and has wide application values.

Description

technical field [0001] The invention belongs to the field of plant genetic engineering, in particular, it uses molecular biology technology to obtain a gene capable of enhancing the salt tolerance of plants. AtPGK2 and its application in transgenic plants. Background technique [0002] Soil salinization is very serious all over the world, and salt stress is one of the important abiotic stresses affecting global crop yields (Parvaiz and Satyawati, 2008). my country's saline soil has a large area, wide distribution, and many types, accounting for about 10% of the country's land area, and the salinization and secondary salinization are increasing every year, which threatens the sustainable development of agricultural production and seriously affects plants. The growth and development of economic crops and the yield and quality of commercial crops (Sun Jianchang et al., 2008). Therefore, it is a very urgent task to cultivate new varieties of transgenic crops with enhanced salt ...

Claims

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Application Information

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IPC IPC(8): C12N15/54C12N15/10C12N15/84A01H5/00
Inventor 刘栋李卫春马利霞程建峰
Owner JIANGXI AGRICULTURAL UNIVERSITY
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