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Peanut cysteine protease encoding gene AhRD21A as well as expression vector and application thereof

A technology of cysteine ​​protease and coding gene, applied in application, genetic engineering, plant genetic improvement, etc., can solve problems such as threats to food safety, life and health, economic losses in peanut production, and pesticide residues

Inactive Publication Date: 2021-04-16
FUJIAN AGRI & FORESTRY UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Bacterial wilt caused by R. solanacearum is an important disease in peanut production. When the disease is serious, it often causes a decline in peanut production and quality, and brings great economic losses to peanut production.
At present, in production, the occurrence and spread of the disease are mainly reduced through agricultural control and the use of chemical agents, but the efficiency of this control method is not very high and it will cause problems such as pesticide residues, which greatly threatens food safety and people's health. Life and health

Method used

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  • Peanut cysteine protease encoding gene AhRD21A as well as expression vector and application thereof
  • Peanut cysteine protease encoding gene AhRD21A as well as expression vector and application thereof
  • Peanut cysteine protease encoding gene AhRD21A as well as expression vector and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0016][Example 1] AHRD21A and AHRRS5 interaction verification

[0017]The cDNA of the peanut oil 92 is used as a template with anti-Blue blight cultivation species.Ahrd21a withAhrrs5 (CN104480117A) Full length CDS sequence is connectedpgad In the T7 (AD) and PGBKT7 (BD) vectors, the yeast strain Y2H GOLD was circulated, and PGADT7-T and PGBKT7-P53 were used as a positive control, PGADT7-T and PGBKT7-LAM as a negative control, in the second acknowlednerability SD- TRP / -LEU culture gains a transformed positive transformant, and a gradient dilution is used to grow a positive clonal sub-positive cloning point in four deficient medium SD-TRP / -LEU / -HIS / -ADE / X-α-Gal / ABA. 5 days later, transformed positive clones in 10-110-210-3Different dilutions can grow normally and blue, and the positive control is also blue clone, and the negative control is not growing, indicating that there is interaction between AHRD21A and AHRRS5.figure 1 A).

[0018]The Gateway system will be separatelyAhrd...

Embodiment 2

[0019][Example 2]Ahrd21a Excessive expression vector construction and verification

[0020]according toAhrd21a Full length gene CDS sequence, design specific primer

[0021]AHRD21A-ATTB1-F: Ggggacaagttgtacaaaaaaagcaggcttcatgcttcatccttccccc;

[0022]AHRD21A-ATTB2-R: GgggacccttgtacaagaAagctgggtcagcactgctcacccattgaag.

[0023]CDNA of peanut oil 92 in anti-blue bumper was carried out in PCR amplification as a template, using Takara's high-fidelity enzyme primestar®MAX amplified, PCR reaction system: 1 μL of cDNA as template, 10 μL 2 × primestar®Max Mix, the positive and actuator is 0.5 μL, hydrating to 20 μL. Reaction conditions: 95 ° C prevalence 5 min; 95 ° C 30 S, 55 ° C 30 S, 72 ° C 3 min, 25 cycles. PCR product agarose gel electrophoresis was subjected to BP reactions to the PDONR207 no-load connection, 1 μL of the PDONR207, 1 μL, BP enzyme 0.25 μL, 25 ° C, and Night, 2 ° C The product was converted to E. coli DH5α sensitudinal cells, screening positive clones were sequenced, correctly read cl...

Embodiment 3

[0024][Example 3]Ahrd21a Expression mode analysis after anti-and hyperbraese peanuts

[0025]For analysisAhrd21a Genes in anti-and hyperbrapasculent peanuts in response to the expression differences of cyprotype infestation, this study adopts real-time fluorescent quantitative PCR method confrontation, soft blight peanut materials Yue oil 92 (YY 92) and new particles (XHXL) After the shear leaf method is treated with cypros, the expression of the gene is analyzed at different time points. Design of the fluorescent quantitative PCR primers (AHRD21A-QPCR-F: CTaaggtgtgtgaacatgatga, AHRD21A-QPCR-R: CacatctTcctgtgtgaatca), using CTAB method to extract the total RNA of peanut leaves before and afterfeeding infection, according to PrimescriptTM Reverse Transcriptase Reverse enzyme (Purchased from Takara Company) The instructions were subjected to 1 μg of total RNA to synthesize single-stranded cDNA, dilute a single-stranded cDNA, 2 μL as a template, with aHactin as an intercephase gene (AHACT...

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Abstract

The invention discloses a peanut cysteine protease encoding gene AhRD21A, the construction of an overexpression vector of the peanut cysteine protease encoding gene AhRD21A and the application of the peanut cysteine protease encoding gene AhRD21A in tobacco bacterial wilt resistance genetic engineering. The gene contains a nucleotide sequence as shown in SEQ ID No. 1. The CaMV 35S promoter-driven overexpression vector agrobacterium-mediated transformation bacterial wilt susceptible variety Honghua Dajinyuan is constructed through a Gateway system, molecular detection and ralstonia solanacearum inoculation recognition are carried out on transgenic plants, and overexpression of the CaMV 35S promoter-driven overexpression vector agrobacterium-mediated transformation bacterial wilt susceptible variety Honghua Dajinyua in tobaccos can significantly improve bacterial wilt resistance of transgenic tobaccos. The result shows that the AhRD21A may participate in the resistance reaction of the plant to the bacterial wilt, which has an important gene resource for the bacterial wilt resistance genetic engineering breeding application of the plant.

Description

Technical field[0001]The present invention belongs to the field of plant gene engineering technology, which discloses a transduction gene encoding gene.Ahrd21a The Construction of Optimistic Expression Vectors and Its Application in Tobacco Anti-Blue Blood Sex.Background technique[0002]Peanuts is one of the important oil crops in my country, rich in nutritional value. Blood blight caused by cylatophytes is an important disease in peanut production. When the disease is seriously caused, it will cause a decline in production and quality, bringing great economic losses to peanut production. At present, it is mainly based on the use of agricultural prevention and chemicals to reduce the occurrence and spread of the disease, but this effect is not very high and will result in pesticide residues, and greatly threaten food safety and people's Life is healthy. Therefore, we need to find a green environmentally friendly, effective scientific prevention measures, and the cultivation of diseas...

Claims

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Application Information

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IPC IPC(8): C12N15/57C12N9/50C12N15/84A01H5/00A01H6/82
Inventor 张冲陈玉婷楚盼盼陈华蔡铁城杨强熊发前庄伟建
Owner FUJIAN AGRI & FORESTRY UNIV
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