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Novel method for inhibiting arabidopsis thaliana living nutritional type oomycete downy mildew infection based on small RNAs

A vegetative, Arabidopsis technology, applied in the biological field, can solve the problem of no reliable and efficient Hpa genetic transformation method

Active Publication Date: 2019-05-10
HANGZHOU NORMAL UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0007] To the best of our knowledge, there is currently no reliable and efficient method for genetic transformation of Hpa

Method used

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  • Novel method for inhibiting arabidopsis thaliana living nutritional type oomycete downy mildew infection based on small RNAs
  • Novel method for inhibiting arabidopsis thaliana living nutritional type oomycete downy mildew infection based on small RNAs
  • Novel method for inhibiting arabidopsis thaliana living nutritional type oomycete downy mildew infection based on small RNAs

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0028] Embodiment 1: experimental method

[0029] 1. Strains, isolation and propagation of pathogenic bacteria: On Ws-eds1, Emoy2 and Cala2 isolated from Arabidopsis biotrophic oomycetes were maintained. Sporulation assays were performed 7 days after inoculation, at the end of the Hpa life cycle. To quantify sporulation, 10 infected seedlings from each replicate were placed in a solution containing 250 μl H 2 0 in Eppendorf test tubes. Vortex samples and count the number of conidia with a hemocytometer.

[0030] 2. The synthesis of sRNA and DNA oligonucleotide: this method takes Hpa-CesA3 gene (HpaG810051) as the target gene, and the sense and antisense sRNAs of different lengths are designed:

[0031] Hpa_CesA3_RNA_AS_24 5'-GCCGCAUCGCACGUACCUCAGUAC-3' (as shown in SEQ ID NO.1);

[0032] Hpa_CesA3_RNA_AS_25 5'-GCCGCAUCGCACGUACCUCAGUACG-3' (as shown in SEQ ID NO.2);

[0033]Hpa_CesA3_RNA_S_25 5'-CGUACUGAGGUACGUGCGAUGCGGC-3' (as shown in SEQ ID NO.3);

[0034] Hpa_CesA3_RN...

Embodiment 2

[0046] Example 2: Screening of sRNA-mediated silencing target genes in HPA

[0047] The main components of the cell wall of oomycetes are β-glucan and cellulose. We targeted the cellulose synthase gene as a target for sRNA-mediated silencing. Using Pfam, we identified that M4BU64 in Hpa belongs to the cellulose synthase gene family. In computer simulation analysis, it was found that this gene corresponds to HpaG810051 in the Emoy2 genome and exists as a single-copy gene. EnsemblProtists gene annotation showed that HpaG810051 had no intron, and the open reading frame encoded a predicted protein of 1144 amino acids (molecular mass 127.028 kDa). BLASTX search of the database showed that HpaG810051 has high similarity with CesA3 protein of other oomycetes, so we named HpaG810051 as Hpa-CesA3. Then we obtained the nucleotide and amino acid sequences of the CesA3 genes of Albucia, Vitiligo, P. lettuce, Phytophthora capsici, P. infestans and Peronospora genus CesA3, and compared t...

Embodiment 3

[0049] Embodiment 3: Hpa-CesA3 antisense sRNA inhibits Hpa sporulation

[0050] Since Hpa is an obligate biotrophic pathogen growing on Arabidopsis, we examined whether HPA-CesA3 has any homology to the Arabidopsis CesA gene. A BLASTN search of the Arabidopsis database found no significant similarities. Subsequently, we designed 25-nt sense and antisense RNA oligonucleotides from the 5' region of the gene without any homology among other genes in the Hpa genome. Add positive and antisense sRNAs to HPA spores at 5, 10, and 20 μM concentrations, respectively, and inoculate 7-day-old Arabidopsis seedlings. On the 7th day (dpi) after inoculation, detect Hpa sporulation situation ( figure 1 ), against the control plants ( figure 1 a) There is no significant difference in the sporulation of plants inoculated with 5, 10, and 20 μM sRNA spore suspension. while the inoculations contained 5 and 10M antisense RNA ( figure 1 The spore suspensions of b and 1c) significantly reduced sp...

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Abstract

The invention provides a novel method for inhibiting arabidopsis thaliana living nutritional type oomycete downy mildew infection based on small RNAs, and belongs to the technical field of biology. Cellulose synthase A3 of arabidopsis thaliana living nutritional type oomycete is taken as a target gene to synthesize antisense sRNAs with the length >=25nt, and the germination of arabidopsis thalianaliving nutritional type oomycete spores is inhibited through the antisense sRNAs to inhibit the arabidopsis thaliana living nutritional type oomycete downy mildew infection. The method has the advantages of reliability and effectiveness, and it is indicated that a simple and effective sRNA method has potentials on deciphering genetic functions in specialized biotrophic pathogens and the controlling of plant diseases.

Description

technical field [0001] The invention belongs to the field of biotechnology, and in particular relates to a novel method for inhibiting Arabidopsis biotrophic oomycete downy mildew infection based on small RNA. Background technique [0002] Noncoding small RNAs (sRNAs) of 20 to 30 nucleotides (nt) long are known to be involved in the regulation of gene expression and defense in eukaryotes. Different types of RNAs, such as double-stranded RNA (dsRNA) and small interfering RNA (siRNA), can trigger homologous RNA degradation or inhibit mRNA translation. This process, known as RNA silencing, plays an important role in a variety of biological processes, including innate immunity and development. [0003] In plant-microbe interactions, plants and microbes can exchange RNA molecules that are then integrated into the RNA silencing machinery in the interacting recipient cells. Such cross-kingdom RNA transfer has been demonstrated for the first time between fungi and plants. Botryti...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/113A01N57/16A01P3/00
Inventor 马穆特·托尔洪益国奥斯曼·泰利章鹏程
Owner HANGZHOU NORMAL UNIVERSITY
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