Wheat salt-tolerant gene TaAAP3 and application thereof

A wheat salt-tolerant gene and genetic technology, applied in the fields of application, genetic engineering, DNA/RNA fragments, etc., to achieve the effect of improving milk salt capacity

Inactive Publication Date: 2021-02-09
CROP RES INST SHANDONG ACAD OF AGRI SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, whether the AAP gene is related to the salt tolerance of plants has not been reported so far

Method used

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  • Wheat salt-tolerant gene TaAAP3 and application thereof
  • Wheat salt-tolerant gene TaAAP3 and application thereof
  • Wheat salt-tolerant gene TaAAP3 and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0029] Example 1 Cloning TaAAP3 Gene from Wheat

[0030] 1. Primer Design

[0031] Search in NCBI with the mRNA sequence of Goat grass AAP3 (GeneBank: XM_020311037.1) to obtain the wheat mRNA sequence (GenBank: AK449433.1). According to this sequence, the primer sequences were designed as follows:

[0032] TAAPCF1: ATGGGGGAGAACGGCGTGGGCAAGAACTAC (SEQ ID No. 2),

[0033] TAAPCR1:

[0034] GGACTAGTCC TCAGTCAGTTGTGAAGAACGGCTTGTAG (SEQ ID No. 1);

[0035] Using the above primers (the base with a horizontal line at the bottom of the primer is the restriction site of the introduced Spe1 endonuclease, which is convenient for the construction of the subsequent expression vector) to perform PCR amplification on the genomic DNA of the new upland wheat variety Jimai 262.

[0036] 2. PCR reaction system and procedures

[0037] 50μl reaction system:

[0038]

[0039]

[0040] The PCR reaction program is: the PCR reaction program is: 94°C for 4min; 94°C for 30s, 60°C for 30s, 7...

Embodiment 2

[0048] Example 2 Expression pattern of TaAAP3 gene under salt stress

[0049] Design primers using the cDNA sequence of TaAAP3 that has been cloned

[0050] QAAP3F: CGCCTACTCCTATTCGCTCATCC (SEQ ID No. 4),

[0051] QAAP3R: ACCCTGACTTGGGCCACCTCT (SEQID No.5); select the plump seeds of Jimai 262, place them in a petri dish with a diameter of 9 cm, and cultivate them in a light incubator at a constant temperature of 25°C for 3 days, then select seedlings with the same growth state and transplant them to plastic In the culture box, when the 1 / 2 Hogland culture medium is cultivated to two leaves and one heart, it is used for salt stress treatment: the material to be treated is placed in an aqueous solution containing 250mmol / L NaCl for growth; the treatment time is 0h, 6h, 12h and 24h, 48h, 24 hours after rehydration, all the parts are roots, stems and leaves. RNA extraction and reverse transcription were performed on the collected materials. Wherein the RNA extraction method is: ...

Embodiment 3

[0073] Construction of embodiment 3 expression vector (PTCK303-TaAAP3) and wheat transgene

[0074] The PMD-18T plasmid containing the target gene TaAAP3 gene was double-digested with BamHI and SpeI endonucleases, and the small fragments after digestion were gel recovered, and at the same time, BamHI and SpeI endonucleases were used to treat PTCK303 ( Figure 4 ) for double enzyme digestion ( Figure 5 ), the large fragments were recovered from the gel, and the two fragments recovered from the gel were ligated using T4 ligase. The ligation product was transformed into Escherichia coli competent cells, and the positive colonies were verified by colony PCR. Then, the positive colonies were expanded and cultivated, the plasmid was extracted, and the plasmid was double-digested with BamHI and SpeI endonucleases to verify whether the size of the fragment after digestion was the same as the size of the target gene. Transformation of plasmids into Agrobacterium for wheat transgenes...

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Abstract

The invention relates to the technical fields of gene engineering, molecular biology and genetic breeding science, and particularly relates to a wheat salt-tolerant gene AAP3 and application thereof.The invention discloses a genome sequence of an amino acid permease gene TaAAP3 and an expression mode of the gene under the salt stress. An overexpression vector containing the gene TaAAP3 and a wheat transgenic plant for overexpression of the gene are obtained. Experiments prove that according to the invention, the salt tolerance of transgenic wheat is remarkably higher than that of a control (non-transgenic wheat), and the wheat salt-tolerant gene AAP3 can be used for salt-tolerant breeding of wheat and has huge application value.

Description

technical field [0001] The invention relates to the fields of genetic engineering, molecular biology, and genetic breeding science and technology, in particular to a wheat salt-tolerant gene AAP3 and its application. Background technique [0002] Soil salinization has become a growing problem in global agriculture. China's salinized and secondary salinized land accounts for about 10% of the country's cultivated land. Among them, the Bohai Rim region is the main distribution area of ​​saline-alkali land in my country, with more than 10 million mu of saline-alkali wasteland and more than 40 million mu of medium-low-yield fields affected by saline-alkali. Since salt stress affects plant growth, photosynthesis, protein synthesis, energy metabolism, and lipid metabolism, etc., under the situation of increasing population and decreasing cultivated land area, it is necessary to accelerate the cultivation, improvement, promotion and application of new varieties of salt-resistant cr...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/52C12N9/00C12N15/82A01H5/00A01H6/46
CPCC12N9/00C12N15/8273
Inventor 韩冉刘成李根英李玉莲汪晓璐宫文萍程敦公李豪圣刘建军刘爱峰曹新有宋健民郭军翟胜男李法计訾妍
Owner CROP RES INST SHANDONG ACAD OF AGRI SCI
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