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Myosin-5 and application of myosin-5 to cyanoacrylate drug resistance treatment

A myosin, disease resistance technology, applied in the field of genetic engineering

Active Publication Date: 2019-12-06
NANJING AGRICULTURAL UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Although no phytopathogenic Fusarium strains resistant to cyclostrobin have been found in the field, the target pathogenic fungi are prone to develop resistance through ultraviolet mutagenesis and agent domestication under laboratory conditions, among which the moderately resistant and highly antibacterial The strains accounted for a large proportion and showed high fitness, indicating that myosin inhibitors have a high risk of resistance

Method used

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  • Myosin-5 and application of myosin-5 to cyanoacrylate drug resistance treatment
  • Myosin-5 and application of myosin-5 to cyanoacrylate drug resistance treatment
  • Myosin-5 and application of myosin-5 to cyanoacrylate drug resistance treatment

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 2

[0065] Example 2 Isolation and Cloning of Myo5 Gene cDNA

[0066] The cloning method of Fusarium myosin-5 cDNA, its specific steps are:

[0067] According to the myosin-5 gene sequence in Example 1, the intron sequence is removed, and the myosin-5 gene cDNA is artificially synthesized by chemical synthesis, and the sequence (the DNA sequence removes the intron) is as SEQ No.2 shown.

Embodiment 3

[0068] Example 3 Construction and Fungal Transformation of RNAi Vectors for Different Segments of Myo5 Gene cDNA

[0069] (1) Divide the cDNA of the Myo5 gene into 8 different segments, named respectively as Myo5-1 (cDNA start and stop sites: 1nt-473nt), Myo5-2 (cDNA start and stop sites: 456nt-938nt), Myo5-3 ( cDNA start-stop site: 939nt-1455nt), Myo5-4 (cDNA start-stop site: 1381nt-1915nt), Myo5-5 (cDNA start-stop site: 1828nt-2313nt), Myo5-6 (cDNA start-stop site: 2253nt-2739nt ), Myo5-7 (cDNA start-stop site: 2649nt-3206nt) and Myo5-8 (Fusarium graminearum and F.asiaticum cDNA start-stop site 3149nt-3645nt, F.Moniliform, F.oxysporum, Magnaporthe oryzae, Botrytis cinerea, Verticilliumdahliae and Sclerotinia sclerotiorum cDNA starting and ending sites 3149nt-final base), design specific PCR primers to amplify the forward sequence of each segment, taking Fusarium graminearum and F.asiaticum as examples, the specific primer sequences are as follows:

[0070] mRNAi-1F:

[007...

Embodiment 6

[0131] Embodiment 6 Myo5 dsRNA is to the determination of Fusarium live drug efficacy

[0132] (1) Myo5 dsRNA preparation preparation

[0133] 1) Using the full length of Myo5 cDNA as a template, synthesize 4 kinds of Myo5 gene dsRNA Myo5-9(1nt-1200nt), Myo5-10(901nt-2100nt), Myo5-11(1801nt-3000nt), Myo5-12( 2446nt-3645nt), dsRNA was synthesized using Invitrogen's RNAi Kit AM1626 kit (see the kit instruction manual for specific operation steps), the sequences of the four Myo5dsRNAs are the same as those of the corresponding template after U (uracil) replaces T (thymine);

[0134] 2) dsRNAs corresponding to Myo5-1, Myo5-2, Myo5-3, Myo5-4, Myo5-5, Myo5-6, Myo5-7 and Myo5-8 segments alone or in combination with different segments (Myo5-1+Myo5-3, Myo5-2+Myo5-4, Myo5-3+Myo5-5, Myo5-4+Myo5-6, Myo5-5+Myo5-7, Myo5-6+Myo5-8), As a template for the synthetic segment of interference dsRNA in vitro, 14 kinds of Myo5dsRNA were synthesized, and the dsRNA was synthesized using Invitrogen...

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Abstract

The invention provides application of a myosin-5 gene Myo5 segment to prevention and treatment of plant fungal diseases and / or enhancement of plant disease resistance. Myosin-5 gene is derived from fusarium graminearum, fusarium asiaticum, fussrium moniliforme, fusarium oxysporum, magnaporthe oryzae, botrytis cinerea, verticillium dahliae or sclerotinia sclerotiorum. The myosin-5 gene Myo5 segmentis siRNA of 15-30nt randomly generated after the combination of an Myo5dsRNA segment and a dsRNA segment or full-length or partial Myo5dsRNA is subjected to RNase digestion. The invention also provides an in vitro interference preparation containing the myosin-5 gene Myo5 segment. The myosin-5 gene Myo5 RNA interference technology has the green and safe outstanding advantages that the drug sensitivity of pathogenic fungi is improved, the drug resistance level is reduced, the pathogenicity is interfered, the disease resistance of plants is enhanced, and the specificity of plant diseases is prevented and treated.

Description

【Technical field】 [0001] The invention belongs to the technical field of genetic engineering, and relates to Fusarium myosin-5 gene Myo5, an RNAi carrier of the gene, in vitro preparation of the gene Myo5 dsRNA and the application of the gene in preventing and treating plant diseases. 【Background technique】 [0002] Cyclostrobin is a new type of cyanoacrylate myosin inhibitor independently developed by my country. It has strong bactericidal activity, high specificity, can reduce the pollution of Fusarium DON toxin, and has the effect of increasing plant production. At present, the market share of this fungicide continues to increase, and it has become an efficient and effective method for controlling important diseases of major crops such as wheat scab (Fusarium spp.), rice bakanae (F.fujikuroi) and vegetable root rot (Fusarium spp.). One of the selective fungicides. Although no phytopathogenic Fusarium strains resistant to cyclostrobin have been found in the field, the tar...

Claims

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Application Information

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IPC IPC(8): C12N15/31C12N15/82C07K14/37A01H5/00A01H6/20
CPCC07K14/37C12N15/8282
Inventor 周明国宋修仕谷凯鑫侯毅平段亚冰王建新
Owner NANJING AGRICULTURAL UNIVERSITY
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