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Nucleotide sequence for controlling insect attack and method thereof
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A nucleotide and polynucleotide technology, applied in the field of control of the double-spotted firefly beetle, can solve the problems of very different effects and damages
Active Publication Date: 2019-12-10
BEIJING DABEINONG BIOTECHNOLOGY CO LTD
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Although the use of RNAi technology for pest control is known in the art, in view of the wide variety of insects, not only the effect of this technology in different insects varies greatly, but also the use of this technology as a measure to control insect infestation A critical factor is also the selection of the most appropriate target genes, those whose loss of function leads to severe disruption of essential biological processes and / or death of the organism
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no. 1 example
[0110] The first embodiment, the determination of the target sequence of the two-spot firefly beetle
[0111] 1. Extraction of total RNA from the two-spot firefly beetle
[0112] Using the newly hatched instars of the two-spotted firefly beetle as materials, the RNA was extracted by the conventional Trizol method, purified by conventional methods, and treated with DNase to obtain a concentration of ≥300ng / μL, a total amount of ≥6μg, and OD 260 / 280 1.8-2.2 for total RNA samples.
[0113] 2. Isolation of mRNA and synthesis of cDNA
[0114] The mRNA with polyA was isolated with oligo-dT magnetic beads, and then the first-strand cDNA was synthesized with random hexamers and Invitrogen's Superscript II reverse transcriptase kit.
[0116] From the analysis of the larval library, the expression level is moderate, and it may be involved in the screening of important metabolic pathways. A target gene c35112 of the two-spotted firefly beetle was s...
no. 2 example
[0121] The second embodiment, the acquisition of dsRNA
[0122] Referring to the instructions of MEGAscript RNAi Kit from thermoFisher Company, dsRNAs of the above 18 target sequences were synthesized respectively, i.e. r1-dsRNA to r18-dsRNA; and the product size was detected by agaroseelectrophoresis with a mass concentration of 1%. ) to measure the concentrations of the above-mentioned r1-dsRNA to r18-dsRNA respectively.
no. 3 example
[0123] The third embodiment, the ability of feeding dsRNA to identify the control ability of the two-spot firefly beetle
[0124] The separated and purified r1-dsRNA to r18-dsRNA were mixed and added to the feed at the ratio of 50 μg / g and 5 μg / g feed respectively (feed formula refers to Development of an artificial diet for the westerncorn rootworm, Entomologia Experimentalis et Applicata 105: 1–11,2002.), to obtain c35112-r1-50 to c35112-r18-50 feed and c35112-r1-5 to c35112-r18-5 feed respectively, wherein the control group feed CK added irrelevant dsRNA (SEQ ID NO :29), the other conditions are exactly the same. Use the prepared feed to feed the newly hatched larvae of the two-spotted firefly beetle. Put 30 newly hatched larvae with a hatching time of no more than 24 hours in each dish, and the feed mixed with dsRNA is changed every two days and fed until the 12th day , count insect mortality every two days from the beginning of feeding, and measure the expression of targ...
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Abstract
The invention relates to a nucleotide sequence for controlling insectattack and a method thereof. A separated polynucleotide sequence comprises (a) a polynucleotide sequence as shown in SEQ ID NO:1;or (b) a polynucleotide sequence of at least 15 or 17 or 19 or 21 continuous nucleotides as shown in the SEQ ID NO:1, wherein a double-stranded RNA containing at least one strand complementary to thepolynucleotide is taken by a coleoptera pest to inhibit growth of the coleoptera pest; or (c) an arbitrary polynucleotide sequence as shown in SEQ ID NO:3 to SEQ ID NO:20; or (d) a polynucleotide sequence which is hybridized with or complementary to the polynucleotide sequence limited in the (a), (b) or (c) under a strict condition. Multiple target sequences for controlling the target gene c35112of the coleoptera pest monolepta hieroglyphica are disclosed for the first time along with high efficiency, specificity, convenience and low cost.
Description
technical field [0001] The invention relates to a nucleotide sequence and a method for controlling insectinfestation, in particular to a method for controlling the two-spotted firefly beetle by using RNAi technology to reduce or close the expression of target sequences in the two-spotted firefly beetle. Background technique [0002] Field crops are often the target of insectattack. Over the past few decades, there have been some substantial advances in the development of more effective methods and compositions for insect infestation in crops. Chemical pesticides are relatively effective means to control pest infestation. However, the use of chemical pesticides also has many disadvantages. First of all, chemical insecticides are non-selective. People intend to use chemical insecticides to control insects that are harmful to various crops and other plants, but due to their lack of selectivity, chemical insecticides will also be harmful to non-target organisms. Cause damag...
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