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Method for Improving Aluminum Tolerance of Plants Using Peanut ahgsnor Gene

A gene and peanut technology, which is applied in the field of peanut AhGSNOR gene to improve the aluminum tolerance of plants, can solve the problems such as no aluminum tolerance has been found, and achieve the effect of improving tolerance

Active Publication Date: 2021-06-22
GUANGXI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the application of this gene to improve the aluminum tolerance of commercial crops such as peanuts and tobacco has not been found so far.

Method used

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  • Method for Improving Aluminum Tolerance of Plants Using Peanut ahgsnor Gene
  • Method for Improving Aluminum Tolerance of Plants Using Peanut ahgsnor Gene
  • Method for Improving Aluminum Tolerance of Plants Using Peanut ahgsnor Gene

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0043] Acquisition of the DNA fragment of the AhGSNOR gene. The specific steps are as follows: take 0.2 g of peanut root tips, and use Trizol reagent (Invitrogen) to extract total RNA. RNA was reverse transcribed into cDNA using the RevertAid(TM) first Strand cDNA Synthesis Kit (ThermoScientific). According to the peanut AhGSNOR gene sequence (SEQ ID NO: 1), an upstream primer (5'- CTCGAG ATGGCAACTCAAGGTCAAGT-3', where the underline is the Xho I site) and the downstream primer (5' GTC GAC TGCATCTGTTGAAAGAACAC-3', wherein the underline is the Sal I site), and the peanut cDNA was used as a template for PCR amplification. The PCR reaction conditions were: pre-denaturation at 95°C for 2 min; denaturation at 95°C for 30 s, annealing at 58°C for 30 s, extension at 72°C for 90 s, and 35 cycles. The PCR product was gel-recovered using a DNA purification and recovery kit (Tiangen), the recovered product was ligated to the pMD19-T vector, positive clones were screened and sequenced...

Embodiment 2

[0045]The overexpression vector of AhGSNOR gene was constructed. The specific steps are as follows: extract the pMD-AhGSNOR positive cloning plasmid and the pBI121-EGFP carrier plasmid in Example 1 (the carrier can be obtained through open and commercially available commercial channels), and use restriction enzymes XhoI and Sal I to carry out double enzymes respectively. Cut, and use the DNA purification and recovery kit (Tiangen) to recover the digested fragments. The digested fragments were ligated with a DNA ligation kit (Takara). The ligation reaction system was 4 μL of the product recovered from pMD-AhGSNOR gel and 1 μL of the product recovered from pBI121-EGFP gel. The reaction conditions were 16° C. for 3 h. The ligated product was transformed into DH5a competent cells, single colonies were screened for resistance using 100 mg / L kanamycin, and the obtained positive clones were sequenced and verified. The positive clone with correct sequencing was named as pBI121-AhGSNO...

Embodiment 3

[0047] The AhGSNOR gene was introduced into tobacco plants through the genetic transformation system mediated by Agrobacterium. Specific steps are as follows:

[0048] (1) Cultivation of sterile tobacco seedlings: Soak tobacco seeds in 75% ethanol for 1 min, then soak in 0.1% mercuric chloride for 5 min, and rinse with sterile water for 3-5 times. The sterilized seeds were inoculated onto MS basic medium with sterile tweezers, and placed in a 26°C incubator for cultivation.

[0049] (2) Acquisition of leaf discs: when the sterile seedlings induced by seeds grow to a leaf width of 1-1.5cm, the leaves are cut off and the back of the leaves are scratched, and inoculated with 1.0mg / L 6-BA and 0.2mg / L In the solid medium of L NAA, culture in the dark for 2 days.

[0050] (3) Infection: Draw 2 mL of the Agrobacterium EHA105 containing pBI121-AhGSNOR in Example 2 and inoculate it into 100 mL of YEP liquid culture containing 25 mg / L rifampicin, 25 mg / L streptomycin and 100 mg / L kana...

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Abstract

The invention discloses a method for improving the aluminum tolerance of plants by applying the peanut AhGSNOR gene. The polynucleotide sequence of the AhGSNOR gene comprises: (a) the nucleotide sequence shown in SEQ ID NO: 1; (b) It encodes the amino acid sequence shown in SEQ ID NO:2. The AhGSNOR gene is transformed into tobacco plants through transgenic technology, and its tolerance to aluminum stress is found to be significantly improved, indicating that the gene plays a positive role in the plant's resistance to aluminum stress. The invention has great significance for improving the aluminum-resistant ability of crops and creating the aluminum-resistant germplasm of plants by using the genetic engineering technology.

Description

technical field [0001] The invention relates to the technical field of plant genetic engineering, more specifically, the invention relates to a method for improving plant aluminum tolerance by using peanut AhGSNOR gene. Background technique [0002] Aluminum is the most abundant metal in the earth's crust, and its chemical form depends on pH. In acidic soil (pH<4.5), Al 3+ Is the main soluble and toxic form of aluminum. Therefore, the adverse effects of acidic soil on plant growth are related to Al 3+ are closely related to toxicity. Soluble Al 3+ It can inhibit root growth, affect the ability of plants to absorb water and nutrients, and limit plant growth and productivity. In recent years, a lot of research has been carried out on the mechanism of plant detoxification against aluminum and the internal and external adaptation mechanism of plants to aluminum toxicity. However, the limited gene function research limits the understanding of the mechanism of plant aluminu...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/29C12N15/82A01H5/00A01H6/54A01H6/82
CPCC07K14/415C12N15/8271
Inventor 何龙飞潘春柳詹洁肖冬王爱勤
Owner GUANGXI UNIV
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