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Genetic engineering application of wild soybean NADPH oxidase gene GsRbohA1

A wild soybean and genetic engineering technology, applied in the field of genetic engineering, can solve the problem of lack of Rboh gene, etc., and achieve the effect of improving the resistance of Spodoptera litura

Active Publication Date: 2022-07-22
NANJING AGRICULTURAL UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, there is no report that Rboh gene is involved in insect resistance in soybean

Method used

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  • Genetic engineering application of wild soybean NADPH oxidase gene GsRbohA1
  • Genetic engineering application of wild soybean NADPH oxidase gene GsRbohA1
  • Genetic engineering application of wild soybean NADPH oxidase gene GsRbohA1

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Experimental program
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Effect test

Embodiment 1

[0025] 1) Cloning of wild soybean NADPH oxidase gene GsRbohA1

[0026] With wild soybean variety W11 [1] Leaves were used as materials, and total RNA was extracted using the plant total RNA extraction kit DP432 (Tiangen, Beijing, China), according to the reverse transcription kit (TaKaRa Primer Script). TM RT reagent kit, Japan) for reverse transcription to obtain first-strand cDNA. Using the sequence of GsRbohA1 (Glysoja_004816, GenBank: KHN35050.1) in the NCBI database as a template to design specific primers, GsRbohA1 was cloned from wild soybean leaf cDNA by PCR reaction. The primer sequences are shown in SEQ ID NO.5 and SEQ ID NO.6. The PCR program is as follows: pre-denaturation at 95°C for 3 minutes, denaturation at 95°C for 15 seconds, annealing at 60°C for 15 seconds, extension at 72°C for 120 seconds, a total of 35 cycles, and finally 72 ℃ of extension for 7 minutes, the wild soybean GsRbohA1 gene sequence is obtained after sequencing, wherein the coding region se...

Embodiment 2

[0036] 1) Cloning of the promoter of wild soybean NADPH oxidase gene GsRbohA1

[0037] Take wild soybean W99 and W11 [1] The leaves were used as materials, and the total DNA was extracted using the plant genomic DNA extraction kit DP305 (Tiangen, Beijing, China). Using the DNA sequence 2000bp upstream of the ATG initiation codon of the GsRbohA1 gene as a template, specific primers were designed, and the promoter sequence of the GsRbohA1 gene was cloned from the genomes of wild soybean varieties W11 and W99 by PCR reaction. The primer sequences are shown in SEQ ID NO.15 and SEQ ID NO.15. ID NO.16, GsRbohA1 was obtained after gel purification of PCR product, ligation of T vector, transformation of Escherichia coli, selection of positive single clones for sequencing A and GsRbohA1 G Haplotype promoter sequence, the sequence is shown in SEQ ID NO.2 and SEQ ID NO.3 ( Image 6 ).

[0038] 2) GsRbohA1 A Increase the expression level of GsRbohA1

[0039] To identify the two hapl...

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Abstract

The invention discloses an application of a wild soybean NADPH (Nicotinamide Adenine Dinucleotide Phosphate) oxidase gene GsRbohA1. The invention relates to an application of a wild soybean NADPH oxidase gene GsRbohA1 as shown in SEQ ID NO.1 in gene engineering modification of the resistance of soybeans to prodenia litura. The active oxygen content of the soybean hairy roots over-expressed with the GsRbohA1 is increased, and the resistance to prodenia litura is increased. The soybean hairy roots for inhibiting the expression of the gene have no significant change in resistance to prodenia litura. The wild soybean resistance haplotype GsRbohA1A transcripts more GsRbohA1, accumulates more ROS (reactive oxygen species), and has stronger resistance to prodenia litura. According to the present invention, the wild soybean NADPH oxidase gene GsRbohA1 has the potential of introducing the wild soybean resistance haplotype GsRbohA1A into the cultivated soybean so as to improve the resistance of the soybean to the prodenia litura;

Description

technical field [0001] The invention relates to the application of wild soybean NADPH oxidase gene GsRbohA1, and belongs to the field of genetic engineering. Background technique [0002] Soybean is the main oil crop in the world and is widely used in food, feed, industrial production and other side industries. However, soybeans will be threatened by a variety of insect pests during the whole growth period, which will seriously affect the yield of soybeans. Wild soybean is a closely related wild species of cultivated soybean. Although the phenotypic difference between the two is relatively large, there is no reproductive isolation. Wild soybeans live in the wild for a long time and are subject to natural selection. They have strong stress resistance and richer genetic diversity. It is an effective strategy for soybean resistance breeding to mine resistance genes from wild soybean, and transfer the resistance genes of wild soybean into cultivated varieties through genetic e...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/53C12N9/02C12N15/82A01H5/00A01H6/54
CPCC12N9/0036C12N15/8286C12Y106/03001
Inventor 王慧喻德跃杜海平
Owner NANJING AGRICULTURAL UNIVERSITY
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