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Specific gene sequence of colletotrichum fructicola and application of specific gene sequence

A gene sequence, anthracnose technology, applied in the application, genetic engineering, plant genetic improvement and other directions, can solve the problems of high professional background requirements for instruments, equipment and analysts, time-consuming and laborious, cumbersome steps, etc., and achieves simple and rapid identification steps. Reproducible, low-cost results

Active Publication Date: 2020-10-27
NORTHWEST A & F UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Although the results of this technical method are reliable, the steps are cumbersome, time-consuming and labor-intensive, and require high professional background of equipment and analysts

Method used

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  • Specific gene sequence of colletotrichum fructicola and application of specific gene sequence
  • Specific gene sequence of colletotrichum fructicola and application of specific gene sequence
  • Specific gene sequence of colletotrichum fructicola and application of specific gene sequence

Examples

Experimental program
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Embodiment 1

[0027] The species-specific DNA sequence of the anthracnose fruit of the present invention is 19878bp long, and its sequence is shown in SEQ ID No.1, and its acquisition and identification method is described as follows:

[0028] (1) Select more than 20 Ascomycete genomes in the GenBank public database, and perform OrthoMCL clustering on the encoded proteins of the entire genome, and screen out OrthoMCL clusters specific to the fruiting anthracnose species;

[0029] (2) Relying on the G. anthrax omics resources (17 genomes of fruiting anthracnose strains and 8 genomes of fruiting anthracnose relatives) obtained by the previous sequencing of the fungal research laboratory of Northwest Agriculture and Forestry University, OrthoMCL specific for the obtained fruiting anthrax Cluster clusters were analyzed by tBlastn and genome comparison analysis to verify the species specificity of the gene and the conservation among strains within the species of Anthrax fruit;

[0030] (3) On th...

Embodiment 2

[0041] This embodiment is an example of the development of the rapid detection kit for fruit-born anthracnose species. The kit of this embodiment includes conventional PCR reagents (commercialized kit AP111 of Beijing Quanshijin Biotechnology Co., Ltd.), and the sequence is SEQ ID No.2 Primer P1-F and primer P1-R whose sequence is SEQ ID No.3.

[0042] The specific steps of using the kit for rapid detection of fruit anthracnose are as follows:

[0043] (1) extracting the genomic DNA of the strain to be tested;

[0044] (2) DNA amplification: PCR amplification system: 10xEasy Taq Buffer 2.5μL, 2.5mM dNTPs 2μL, 10μM forward primer (SEQ ID NO.2) 0.5μL, 10μM reverse primer (SEQ ID NO.2) 0.5 μL, 1.0 μL of 100ng / μL DNA template, 0.2 μL of Easy Taq DNA polymerase, and make up to 25 μL with ddH2O. The PCR reaction program is: pre-denaturation at 94°C for 2 minutes; denaturation at 94°C for 30 s, annealing at 55°C for 30 s, extension at 72°C for 1 min, 40 cycles; extension at 72°C fo...

Embodiment 3

[0047] This embodiment shows that the rapid detection kit of fruit anthracnose bacteria (C. fructicola), secret anthracnose bacteria (C.aenigma), banana anthracnose bacteria (C.musae), Siamese anthracnose bacteria (C.siamense) ), the PCR amplification detection results of species such as C. gloeosporioides. details as follows:

[0048] Test strains: 17 test strains were used in this embodiment, including 6 fruit anthracnose bacteria, 3 cryptic anthracnose bacteria, 4 banana anthrax bacteria, 3 Siamese anthrax bacteria, and 1 glyospora anthracnose bacteria. All strains are stored in the China Agricultural Microorganism Culture Collection Management Center or the Fungal Laboratory of Northwest A&F University. The species classification information of the strains is confirmed based on the polygenetic phylogenetic analysis. The specific information of the strains is shown in Table 1:

[0049] Table 1 Example 3 test strain information table

[0050] serial number stra...

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Abstract

The invention discloses a specific gene sequence of colletotrichum fructicola and application of the specific gene sequence. According to the invention, the specific gene sequence of the colletotrichum fructicola is obtained based on whole genome comparison of the colletotrichum fructicola and closely related species thereof, and the specific gene sequence is further utilized to design a polymerase chain reaction (PCR) amplification primer to detect the colletotrichum fructicola, and a related kit is developed. The invention can specifically detect the colletotrichum fructicola (C. fructicola)and distinguishes the colletotrichum fructicola from the closely related species including colletotrichum aenigma (C. aenigma), colletotrichum siamense (C. siamense), colletotrichum musae (C. musae)and the like; and the invention has the advantages of being fast, simple, convenient and accurate.

Description

technical field [0001] The present invention relates to the detection technology of plant pathogenic organisms, in particular to a specific gene sequence of fruit anthracnose bacteria and its application. Background technique [0002] Colletotrichum fructicola is a pathogenic species belonging to the gloidospore anthracnose complex, which is widely distributed around the world and can infect and harm dozens of plants such as avocado, cocoa, mango, pear, apple, strawberry, tea, tobacco, etc. , is a very important class of plant pathogens. Anthracnose fruit can also be used as an endophyte to co-exist with plants without causing obvious disease symptoms. At present, the species identification of Anthrax fructose is strictly dependent on polygenic phylogeny, which needs to be amplified to obtain ribosomal DNA gene transcription spacer region (intergenic spacer region, ITS), actin (Actin), β-tubulin (β-tubulin) -Tubulin), Glyceraldehyde-3-phosphate dehydrogenase (GADPH), Calmo...

Claims

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Application Information

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IPC IPC(8): C12Q1/6895C12Q1/686C12N15/11C12N15/31
CPCC12Q1/6895C12Q1/686C12Q2565/125
Inventor 梁晓飞孟亚楠郭云忠朱明旗孙广宇
Owner NORTHWEST A & F UNIV
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