Ribosome RNA gene of erysiphe alphitoides and application thereof
A powdery mildew and pathogenic bacteria technology, applied in the direction of DNA/RNA fragments, application, genetic engineering, etc., can solve the problems of uncertain pathogenic fungal species, high variability of ITS segments, fungal species research, etc., and achieve good application prospects Effect
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Embodiment 1
[0056] Embodiment 1 detects the acquisition of the nucleotide sequence of the ribosomal RNA gene of the oak powdery mildew pathogen Erysiphe alphitoides
[0057] 1. Experimental method
[0058] (1) High-throughput sequencing
[0059] The leaves with typical powdery mildew lesions were randomly found in the affected oak garden, collected, cut off the lesions in the oak leaves, and the material of the lesions was cut and fully ground with liquid nitrogen. The total DNA was extracted using Dingguo fungus DNA extraction kit, according to its operating instructions, the extracted total DNA is stored at -20°C; according to the Illumina library construction process, the total DNA is constructed as a paired-end high-throughput sequencing library with a fragment size of 500bp; Illumina Hiseq2500 is used for sequencing The instrument performed high-throughput sequencing on the constructed DNA library, and a total of 11.17M pairs of sequencing fragments were measured. The sequencing rea...
Embodiment 2
[0069] Verification experiment of embodiment 2 ribosome assembly result
[0070] 1. Experimental method for verification of ribosome assembly results
[0071] (1) PCR amplification reaction
[0072] The leaves with typical white diseased spots on the back were randomly found in the diseased oak garden, collected, and the diseased area in the oak leaves was cut off, and the material of the diseased spots was cut and fully ground with liquid nitrogen to extract the total DNA of the diseased leaves of the oak tree. Store at -80°C;
[0073] Design 4 sets of primers for verification of ribosome assembly results (respectively primer set oak1, primer set oak2, primer set oak3 and primer set oak4), the nucleotide sequences of the 4 sets of primers are shown in Table 1 respectively; The total DNA was used as a template, and the above four sets of primers were used for PCR amplification. The PCR amplification reaction system is shown in Table 2.
[0074] Table 1 PCR amplification rea...
Embodiment 3
[0087] Embodiment 3 detects the method for oak tree powdery mildew pathogen Erysiphe alphitoides
[0088] 1. Detection method
[0089] (1) High-throughput sequencing
[0090] The leaves with typical powdery mildew lesions randomly found in the oak tree garden where the disease occurred were used as samples to be tested. The lesion area in the oak leaves was cut, and the lesion material was cut and fully ground with liquid nitrogen. The total DNA was extracted using Dingguo Fungal DNA Extraction Kit, according to its operating instructions, the total DNA after extraction is stored at -20°C; according to the Illumina library construction process, the total DNA is constructed into a paired-end high-throughput sequencing library with a fragment size of 500bp; use The Illumina Hiseq2500 sequencer performed high-throughput sequencing on the constructed DNA library, and the sequencing read length was 125 bp at both ends.
[0091] (2) Assembly of microbial genome sequences
[0092]...
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