Molecular markers of resistance to fenpropathrin in Neoseius pasteurii and its application
A technology of Neoseiid mite and fenothrin, which is applied in the field of molecular biology, can solve the problem that it is difficult to identify sensitive strains and resistant strains of Neoseiid mite, and achieves optimization of PCR system and amplification. Procedural, stable and specific effects
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Embodiment 1
[0024] Example 1 Acquisition of full-length sodium ion channel gene of Neoseiusius pastereii to fenpropathrin-resistant and sensitive strains
[0025] 1. Materials
[0026] 1.1 Source of tested mite
[0027]Sensitive strain: Neoseiius pasteruii was collected from the lemon leaves around the orchard of the Citrus Research Institute of the Chinese Academy of Agricultural Sciences. After collection, it was reared indoors for multiple generations without contact with pesticides. At low levels, there was no apparent resistance to fenpropathrin.
[0028] Resistant strains: Sensitive strains of Neoseius pastereius were used as raw materials for resistance screening, and they were reared indoors in contact with fenpropathrin. From 35.79mg / L to 22190.91mg / L, the resistant population increased by 619.96 times compared with the sensitive population. After that, it will be treated with chemicals at regular intervals depending on its population density to ensure that its resistance will...
Embodiment 2
[0055] Example 2 Obtaining of Molecular Markers of Neoseiius pasterei's Resistance to Fenpropathrin
[0056] Using the sequence comparison software BioXM2.6, the sequences of the sodium ion channel genes in the resistant and sensitive strains of Neoseius pasteruii were compared. The comparison results are as follows: figure 2 As shown, it is found that there are mutation sites on the three base segments of A, B, and C. There are a total of 5 SNP sites. At the 3228th and 3243rd positions, the G base mutation of the sensitive strain becomes the resistance strain. A base; at the 3844th, 4730th and 4874th positions, the A base of the sensitive strain was mutated into the G base of the resistant strain. That is to say, the molecular markers of the resistance to fenpropathrin of Neoseius pastereii from the 3228th position to the 4874th position are obtained, and the nucleotide sequence of the molecular marker is shown in SEQ ID No.1.
[0057] The mutation of the base eventually le...
Embodiment 3
[0058] Example 3: Acquisition of special detection primers for the resistance of Neoseiius pasterei to fenpropathrin
[0059] 1. Primer design: Select the nucleotide sequences near the upstream of the 3228 site and the downstream of the 4874 site of the target gene, and use the Primer Premier5.0 primer design software to design a specific 1754bp base that can quickly clone the entire mutation region. Sex positive and negative primers, the primer sequence is as follows:
[0060] F: 5'-CAACCTTTCACAGGCCAATC-3'; R: 5'-TTGCGTTGTAATACTTCTTCTG-3'.
[0061] 2. Primer verification: Collect samples of Neoseius pastereius to be tested in the citrus nursery of the Citrus Research Institute of the Chinese Academy of Agricultural Sciences, obtain cDNA according to the method in Example 1, and perform PCR amplification according to the following reaction system: PCR Master Mix 25 μL , ddH 2 O18 μL, 2 μL each of the above-mentioned specific positive and negative primers, and 3 μL of the cDN...
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