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Issue:ISSN 2095-1353
           CN 11-6020/Q
Director:Chinese Academy of Sciences
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Your Position :Home->Past Journals Catalog->2017年54 No.5

Comparison of the efficacy of different dsRNA delivery methods to silence antenna-rich genes in Locusta migratoria
Author of the article:SHI Xue-Kai1, 2, 3** ZHANG Yi-Wei1, 3 ZHU Kun-Yan4 MA En-Bo1, 3 ZHANG Jian-Zhen1, 3 LIU Xiao-J
Author's Workplace:1. Institute of Applied Biology, Shanxi University, Taiyuan 030006, China; 2. College of Life Sciences Shanxi University, Taiyuan 030006, China; 3. Modern Research Center for Chinese Medicine, Shanxi University, Taiyuan 030006, China; 4. Kansas State University, Manhattan KS 66506–4004, America
Key Words:antennae, RNAi technology, injection method, soaking method, brushing method
Abstract:

[Objectives]  RNAi has been widely used in functional genomic studies of Locusta migratoria. The antennae of locusts have a large number of the odor binding proteins, transporter proteins and odor degradation enzymes, which play important roles in olfactory signal transduction. The optimal dsRNA delivery methods for the corresponding genes have not, however, been well established. We compared the effectiveness of different dsRNA delivery methods using RT-qPCR to measure the gene expression levels obtained by each method. The results provide a theoretical basis for the future functional study of antenna-rich genes in locusts and a foundation for developing a new method for controlling locust plagues based on the molecular targets in the olfactory mechanism. [Methods]  LmCYP3117C1 was chosen as the target gene. mRNA expression of this gene was measured in six tissues; antennae, maxillary palp, wing, tarsus, midgut, and Malpighian tubule, dissected from the fifth-instar L. migratoria. Double-stranded LmCYP3117C1 RNA was dissolved in different solvents (DEPC water, acetone and DEPC water (1︰1) mixed solvent, 0.1% Triton X-100). Three methods; injection (antennal socket and abdominal injection), soaking, and brushing, were tested and the expression levels of the LmCYP3117C1 gene measured with RT-qPCR after each method. [Results]  The LmCYP3117C1 gene was expressed in all six tissues. Expression was highest in the antennae, which was 3.78, 2.10, 10.84, 363.48, and 365.16-fold that in the maxillary palps, wings, tarsi, midgut, and Malpighian tubules, respectively. Expression levels of LmCYP3117C1 were significantly reduced 24 h after injecting dsCYP3117C1 into both antennal sockets. This technique reduced target gene expression in female and male nymphs by 76.84% and 87.51%, respectively, whereas expression in other tissues (maxillary palp, wing, tarsus, and other parts of the whole body) did not change significantly. Injecting dsCYP3117C1 into the abdomen of females and males reduced expression of the target gene by 68.60% and 61.10%, respectively. In addition, this technique significantly reduced expression of the target gene in the tarsi, whereas that in the maxillary palps, wings and other parts of the whole body did not obviously change in females. LmCYP3117C1 gene expression levels decreased in the maxillary palps and other parts of whole body, and did not undergo obvious change in the wings and tarsi of males. In addition, gene expression was not significantly different in males and females 24 h after soaking antennae in dsCYP3117C1 dissolved in different solvents (DEPC water, acetone and DEPC water (1︰1) mixed solvent, 0.1% Triton X-100) for 1, 3, 5 min, respectively. Gene expression levels did not significantly changes in male and female antennae 24 h after brushing them with dsCYP3117C1 dissolved in DEPC water and 0.1% Triton X-100, respectively. [Conclusion]  Injection of dsRNA into the antennal socket significantly reduced LmCYP3117C1 gene expression but soaking and brushing antennae with dsRNA did not.

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