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A kind of locust β-d-glucoside hydrolase gene and its application
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A technology of glucoside and hydrolase, applied to locust β-D-glucoside hydrolase gene and its application field, can solve problems such as unfavorable agricultural environment, green, sustainable development and the like
Active Publication Date: 2022-05-31
LINYI UNIVERSITY
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At present, traditional chemical control methods are mostly used for the prevention and control of Asian locusts, which is not conducive to the green and sustainable development of the agricultural environment
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Embodiment 1
[0026] Example 1 Acquisition of the gene encoding β-D-glucoside hydrolase BG of Locust asiatica
[0028] use RNA isolation reagents were used to extract total RNA from Locust asiatica samples. Specific steps are as follows:
[0029] 1) Put the homogenizer on ice and add 1 mL RNA isolation reagent and 100-200 mg of locust asiaticus tissue, triturated.
[0030] 2) Transfer the homogenate to a 1.5 mL centrifuge tube, let stand for 5 min at room temperature, and centrifuge at 13000 r for 5 min at 4°C.
[0031] 3) Transfer the supernatant to a clean 1.5mL centrifuge tube, add 200μL of chloroform, and vortex for 15s. Place at room temperature for 5 min, and centrifuge at 13000 r for 10 min at 4 °C.
[0032] 4) Transfer 400 μL of supernatant to a new 1.5 mL centrifuge tube, add 200 μL of chloroform, vortex for 30 s, place at room temperature for 5 min, and centrifuge at 13000 r for 10 min at 4°C.
[0033] 5) Aspirate 3...
Embodiment 2
[0065] Example 2 Synthesis of dsRNA and its application in pest control
[0066] 1. Synthesis of dsRNA
[0067] Adopt T7 RiboMAX TM The Express RNAi System kit synthesizes dsRNA. Specific steps are as follows:
[0068] 1) Synthesis of dsRNA primers
[0069] Primers were designed according to the cloned gene fragments, and T7 promoter was introduced into the 5' end of the primers. The primer sequences are as follows:
[0070] BG-2F: 5'-GGATCCTAATACGACTCACTATAGGCAATCTCATTATCAGTATAAC-3' (SEQ ID NO: 3);
[0071] BG-2R: 5'-GGATCCTAATACGACTCACTATAGGCATCCCCATTCACCATGGTGACG3' (SEQ ID NO: 4).
[0073] The bacterial liquid plasmid of the positive cloned strain in Example 1 was extracted with a kit, and the plasmid was used as a template to carry out PCR amplification with BG-2F and BG-2R to obtain the target fragment containing the T7 promoter sequence, with a size of 645bp.
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Abstract
The invention discloses a locust β-D-glucosidehydrolasegene, the nucleotide sequence of which is shown in SEQ ID NO:1. According to this gene, primers are designed to synthesize dsRNA for interfering with the β-D-glucosidehydrolasegene, and the dsRNA is introduced into locusts to perform RNA interference on the β-D-glucosidehydrolase gene, which can inhibit the normal molting or laying eggs of locusts, and then It provides a new method for the green and sustainable prevention and control of pests, and also provides technical support for the creation of new biopesticides.
Description
technical field [0001] The invention relates to the field of biotechnology, in particular to a locust β-D-glucoside hydrolase gene and application thereof. Background technique [0002] Insect pests on crops can reduce productivity and compromise food security. Traditional pest control methods are mostly chemical control, but they rely too much on the use of chemical pesticides, which can easily lead to problems such as pest resistance, rampant, and serious residual poisoning, which seriously damages the ecological environment and the quality and safety of agricultural products. Therefore, it is particularly important to develop and create green, efficient and sustainable pest control technologies. [0003] The Asian locust Oedaleus asiaticus Bey-Bienko is one of the dominant species of locusts with the most serious damage in northern my country. It can feed on grasses and grain crops, and seriously damage the agricultural and animal husbandry production in the northern reg...
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