Peanut aquaporin gene AhAQ1 and coded protein and gene cloning method thereof

A technology of aquaporin and cloning method, which is applied in the field of plant stress resistance gene research to achieve the effect of increasing expression

Inactive Publication Date: 2010-03-17
SHANDONG PEANUT RES INST
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, there is no relevant report in peanut. Therefore, the cloning of peanut aquaporin gene and the study of its expression will help to clarify the molecular me

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0023] Example 1 Molecular Cloning of Peanut Aquaporin Gene AhAQ1

[0024] Luhua 28 peanut seeds were selected as experimental materials. After being induced by salt, the total RNA of peanut tissues was extracted with Trizol reagent (Tiangen) at room temperature, and treated with DNase I (Takara) to remove genomic DNA contamination in the samples. cDNA was obtained by reverse transcription using Promega's RT-PCR system. The primers for the coding region of peanut AhAQ1 gene were designed according to the EST information in the NCBI database: forward primer: 5'-CACACTTACCCATCTCATCAC-3', reverse primer: 5'-CAATAATCAAG-CACTTGCATT-3'. Takara's Ex-Taq enzyme was used for PCR amplification, and the PCR product was recovered and ligated with MD18-T simple vector (Takara). The ligated product was transformed into E.coli Top 10 competent cells, and positive clones were screened by the blue-white method. The screened positive clones were further verified by PCR and then sequenced to ob...

Embodiment 2

[0025] Example 2 Sequence information and characteristic analysis of AhAQ1

[0026] The AhAQ1 gene of the present invention is 969bp long, and its open reading frame is 861bp, located at 58-918bp. Analysis by BioXM software showed that AhAQ1 encoded a total of 287 amino acids. The bioinformatics analysis of the sequence predicted that the molecular weight of the protein was 30.57Kda, and the isoelectric point was 9.04. The analysis results also showed that AhAQ1 is a highly hydrophobic protein containing six transmembrane domains. Sequence comparison analysis also shows that AhAQ1 has a very high similarity with aquaporins of maize, barley, tobacco, olive, Arabidopsis, etc. Phylogenetic analysis showed that AhAQ1 should belong to PIP2 type protein.

Embodiment 3

[0027] Example 3: Study on the expression of AhAQ1

[0028]The partial sequence of peanut AhAQ1 gene was used to design primers, and semi-quantitative RT-PCR was used to analyze the changes of the gene expression and the expression differences in different peanut tissues under salt stress. The results showed that under salt-induced conditions, the expression level of the gene in roots, stems, and leaves all increased, but the expression level began to increase after 6 hours of induction in stems and leaves, and continued to increase in leaves, while in stems The expression level of the decreased after 24 hours of salt stress. The expression level in roots began to increase steadily after 1 hour of salt stress. The results showed that the expression of AhAQ1 gene responded to salt stress, and it was inferred that it played a certain role in the process of salt resistance, but the degree of response was different in different tissues.

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Abstract

The invention relates to research field of plant stress resistance gene. The invention provides a peanut aquaporin gene AhAQ1 gene order and a coded protein order thereof. The invention also providesa method for cloning the aquaporin gene AhAQ1 in a peanut. The expression analysis of the aquaporin gene AhAQ1 shows that the expression of the gene is obviously increased in different tissues of thepeanut under the condition of salt stress and also shows that the gene is related to the plant salt resistance. The transgene test analysis also shows that the overexpression of the gene can enhance the salt resistance of the peanut to a certain extent.

Description

technical field [0001] The invention relates to the field of plant stress resistance gene research, in particular to an aquaporin gene AhAQ1 related to peanut salt tolerance, its encoded protein and a gene cloning method. Background technique [0002] Peanut is an important oil crop and economic crop in my country. From the perspective of the production and supply of domestic edible vegetable oil, peanut oil accounts for 25% of the total. It is the only bulk oil that does not need to be imported in my country, and it is the main oil crop that guarantees the supply of oil in my country. In recent years, with the increasing shortage of oil resources and energy resources in the world, more and more attention has been paid to the production of peanuts. Expanding the planting area of ​​peanuts has become an important way to solve the shortage of oil resources in my country. In line with the principle of "not competing for land with food crops", improve the stress resistance of p...

Claims

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Application Information

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IPC IPC(8): C12N15/29C12N15/10C07K14/415
Inventor 禹山林江燕杨庆利陈明娜潘丽娟朱凤任增凯
Owner SHANDONG PEANUT RES INST
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