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Application of the peanut annexin gene ahann6 in improving the resistance of plants and microorganisms to high temperature and oxidative stress

An annexin and microbial technology, applied in the biological field, can solve problems such as unreported research, achieve the effects of reducing adversity stress damage, important economic benefits and application prospects, and improving resistance

Active Publication Date: 2021-05-25
SUN YAT SEN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] Currently, the peanut annexin gene AhANN6 Studies on the resistance of plants and microorganisms to high temperature and / or oxidative stress have not been reported

Method used

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  • Application of the peanut annexin gene ahann6 in improving the resistance of plants and microorganisms to high temperature and oxidative stress
  • Application of the peanut annexin gene ahann6 in improving the resistance of plants and microorganisms to high temperature and oxidative stress
  • Application of the peanut annexin gene ahann6 in improving the resistance of plants and microorganisms to high temperature and oxidative stress

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0035]Example 1 peanutAhann6 Cloning of gene cDNA sequences

[0036](1) Preparation of peanut seed materials: peanut seeds, peeling sub-leaves;

[0037](2) Total RNA extract of peanut seeds: Extraction of total RNA extraction kit with polysaccharide polyphenol plants of Tiangen Endochem Technology (Beijing) Co., Ltd.;

[0038](3) Synthesis of cDNA first chain: use Takara's PrimescriptTM Rt Reagent Kit withgdna Eraser (Perfect Real Time) Kit Reverse Transcription Synthesis CDNA First Chain;

[0039](4)Ahann6 Cloning of genes: Template with peanut cDNA, clone by PCRAhann6 CDNA fragment, increased enzyme in primerSMA I andSAC I.

[0040]Positive primer: SEQ ID NO: 3:

[0041]5'-tcccCCGGG AtggcaActctTtatTGCTCCCAGCA-3 '

[0042]Reverse primers: SEQ ID NO: 4:

[0043]5'- cGAGCTC Tagtctgtttcccaacaatgtg-3 '

[0044]PCR reaction system: 1 μL cDNA template, 1 μL forward primer, 1 μL reverse primer, 1 μl DNTP (10 mm), 5 μL 10 × Ex-Taq PCR Buffer, 0.5 ml Ex-Taq enzyme (TAKARA products) Finally, the deionized water is f...

Embodiment 2

[0049]Example 2 Arabidopsis genetic transformation

[0050]First, Treatment of Arabidopsis Transformation

[0051]Pre-treatment of Arabidopsis transformation, flowering period Arabidopsis, main moss or side moss to 6-8 cm, remove the appearance of pods and have blossom buds, and the water is pouring enough to keep a high humidity environment.

[0052]Second, the preparation of Agrobacterium bacteria

[0053](1) Inoculation with Agrobacterium Eha105 to 5 ml LB liquid medium (including 30 mg / L of ptamycin 50 mg / l and rifumping 30 mg / L), 200 rpm, 28 Culture in ° C for 24-36 hours;

[0054](2) Add the bacterial solution to a new LB liquid medium (including 30 mg / L and Li Fuxing 30 mg / L), 28 ° C, 200 rpm shocking culture 16 -20 hours, OD600 is 1.2-2.0;

[0055](3) Room temperature collected bacteria, 5000 rpm, centrifugation for 10 minutes of precipitation bacteria;

[0056](4) Heavy suspension to OD600 with freshly prepared dipped medium (5% sucrose solution, Silwetl-77) hemacterium to OD600 is 0....

Embodiment 3

[0060]Example 3 Q-PCR detection of transgenic plants.

[0061](1) Extraction of total RNA: Seeding the screened homogenous germal plant and wild-type medullary seeds on 1 / 2 ms medium, after two weeks, according to RNAPRep Pure, Tiangen Biochemical Technology (Beijing) Co., Ltd. PLANT KIT (POLYSACCHARIDES & POLYPHENOLICS-RICH) polysaccharide polyphenol plant Total RNA extraction kit instructions for the step of extracting RNA;

[0062](2) Synthesis of the first chain CDNA: Using Takara's PrimescriptTM Rt Reagent Kit withgdna Eraser (Perfect Real Time), follow the instructions step;

[0063](3) Q-PCR reaction:

[0064]Ahann6 Gene specific primers:

[0065]Positive primer: SEQ ID NO: 5: 5'- TTGCGGAATCGCTTGTGTG -3 ';

[0066]Reverse primers: SEQ ID NO: 6: 5'- agcagccacatctttcca -3 '.

[0067]Reference geneActin2 Primers:

[0068]Positive primers: SEQ ID NO: 7: 5'- cactgcaccaagcagcatgaaga-3 ';

[0069]Reverse primers: SEQ ID NO: 8: 5'-aatggaacccgatccagacAct -3 '.

[0070]The reaction system of the fluorescent quantit...

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Abstract

The invention discloses peanut annexin gene AhANN6 Application in improving the resistance of plants and microorganisms to high temperature and oxidative stress. The present invention constructs AhANN6 The positive-sense overexpression vector of the gene was transformed into the model plant Arabidopsis. The results showed that the transgenic Arabidopsis had stronger tolerance to high temperature stress. By constructing AhANN6 The prokaryotic expression vector of the gene is transformed into Escherichia coli, and the results show that the gene can improve the high temperature resistance and anti-oxidation ability of the prokaryotic Escherichia coli. The above results indicated that the peanut annexin gene AhANN6 It has broad application prospects in the field of agricultural production and microbial industry such as the promotion of plant and microorganism resistance to high temperature and anti-oxidative stress through gene transformation, as well as the improvement of crop stress-resistant varieties.

Description

Technical field[0001]The present invention belongs to the field of biotechnology, and more particularly to a peanut membrane protein gene.Ahann6 Application in Plant and Microbial Anti - high Temperature and Oxidation Stress.Background technique[0002]High temperature is one of the important factors affecting plant growth development and crop yield. Plant is unable to change its own living environment as a solid-growing organism, however plants develop a series of complex defense mechanisms to respond to the outside world during long-term evolution . Plants to high temperature reaction processes and tolerance mechanisms are typically associated with signal molecules and proteins with protective functions, heat-resistant proteins protecting other proteins from other proteins from other proteins, repairing damaged proteins; antioxidant enzyme systems removing reactive oxygen (Reactive Oxygen Species, ROS) is capable of avoiding harm of cells and their membrane systems.[0003]peanut(Arac...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C07K14/415C12N15/29C12N15/82A01H5/00A01H6/20
CPCC07K14/415C12N15/8271
Inventor 黎茵冯兰兰
Owner SUN YAT SEN UNIV
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