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不同RNAi方法对飞蝗触角高表达 基因沉默效率的比较
Comparison of the efficacy of different dsRNA delivery methods to silence antenna-rich genes in Locusta migratoria
史学凯1, 2, 3** 张艺伟1, 3 朱坤炎4 马恩波1, 3 张建珍1, 3 刘晓
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DOI:10.7679/j.issn.2095-1353.2017.094
作者单位:1. 山西大学应用生物学研究所,太原 030006;2. 山西大学生命科学学院,太原 030006; 3. 农业有害生物综合治理山西省重点实验室,太原 030006;4. 美国堪萨斯州立大学,曼哈顿,KS 66506–4004
中文关键词: 触角,RNAi技术,注射法,浸泡法,涂抹法
英文关键词:antennae, RNAi technology, injection method, soaking method, brushing method
中文摘要:

【目的】 RNAi技术在研究飞蝗功能基因组学方面已经日趋成熟,飞蝗触角中富含有丰富的气味结合蛋白、转运蛋白以及气味降解酶等,这些蛋白在昆虫嗅觉信号传导中发挥着重要的作用,但是关于高效降解这些基因的相关RNAi方法还未知,因此本文通过分析不同RNAi方法对飞蝗触角高表达基因的沉默效率差异变化,以此探索一种高效降解飞蝗触角高表达基因的方法,为后续研究基因功能提供理论依据与技术指导,为从飞蝗嗅觉机制方向设计防控蝗灾的分子靶标奠定基础。【方法】 LmCYP3117C1基因为目标基因,飞蝗为实验材料,选取飞蝗5龄若虫解剖触角、下颚须、翅、跗足、中肠、马氏管6个组织部位,利用实时荧光定量PCR技术分析基因不同组织部位的表达特异性。用不同溶剂溶解dsRNADEPC水,丙酮和DEPC水(1︰1)混合溶剂0.1% Triton X-100),分别采用注射法(触角窝注射和腹部注射),浸泡法和涂抹法3种方法干扰靶标基因,利用实时荧光定量PCR技术分析基因表达抑制情况,研究不同RNAi方法对基因表达量变化的影响。【结果】 LmCYP3117C1基因在飞蝗若虫6个组织部位均有表达,在触角的表达量最高,分别是下颚须、翅、跗足、中肠、马氏管的3.78倍、2.10倍、10.84倍、363.48倍、365.16倍。通过在两边的触角窝注射dsCYP3117C124 h后飞蝗雌雄虫触角LmCYP3117C1表达量显著降低,雌虫和雄虫沉默效率分别为76.84%87.51%,但在其他组织部位(下颚须、翅、跗足、其余整虫)基因表达量没有发生显著变化;飞蝗腹部注射dsCYP3117C124 h后雌雄虫触角的基因表达水平均发生显著降低,其中雌虫触角沉默效率达68.60%,雄虫沉默效率达61.10%,另外,飞蝗雌虫跗足LmCYP3117C1基因表达量也发生显著降低,但下颚须、翅和其余整虫没有明显变化;飞蝗雄虫下颚须,其余整虫LmCYP3117C1基因表达有显著降低,翅和跗足没有发生明显变化;飞蝗触角浸泡于溶于不同溶剂的dsCYP3117C1溶液中DEPC水、丙酮和DEPC水(1∶1)混合溶剂、含0.1% Triton X-100DEPC水),分别浸泡135 min24 h检测雌雄虫触角LmCYP3117C1基因表达情况,结果显示没有发生明显变化;将dsCYP3117C1分别溶于DEPC水和含0.1% Triton X-100DEPC水中涂抹飞蝗触角,24 h后发现飞蝗雌雄虫触角LmCYP3117C1基因表达水平无显著变化。【结论】 飞蝗触角浸泡法和涂抹法干扰LmCYP3117C1没有显著效果,腹部注射法对飞蝗触角LmCYP3117C1的沉默效率较低,触角窝注射法可以高效降解LmCYP3117C1,可作为飞蝗触角高表达基因RNAi的主要干扰方法。

英文摘要:

[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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