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    脱氢表雄酮对美国白蛾幼虫G6PD活性及几丁质合成的影响

    Effects of dehydroepiandrosterone on G6PD activity and chitin synthesis in Hyphantria cunea larvae

      摘要
    • 摘要:
      目的 以农林业重要害虫美国白蛾为研究对象，明确脱氢表雄酮（DHEA）对幼虫体内葡萄糖-6-磷酸脱氢酶（G6PD）活性及磷酸戊糖途径（PPP）的抑制效果，进而解析PPP途径在幼虫生长发育及几丁质合成中的功能。
      方法 通过基因克隆获得美国白蛾HcG6PD基因，并利用生物信息学软件分析其氨基酸组成、理化特性及结构特征。采用实时荧光定量PCR技术分析HcG6PD基因在不同发育阶段的表达模式；测定DHEA处理后5龄幼虫体内G6PD活性、PPP关键基因表达水平及中间产物含量，以明确DHEA对G6PD及PPP的抑制作用；进一步检测几丁质合成通路关键基因的表达水平、表皮几丁质含量及染色情况，分析G6PD抑制对美国白蛾5龄幼虫几丁质合成的影响。
      结果 （1）DHEA处理5龄幼虫72 h后，G6PD活性显著降低，PPP关键基因（HcG6PD、Hc6PGD、Hc6PGL）的转录水平分别降至对照组的18%、17%和13%，PPP中间产物果糖-6-磷酸（F6P）的含量仅为对照组的18%。（2）DHEA处理导致幼虫生长及蜕皮受阻，处理72 h后5龄幼虫体重增长量仅为对照组的13%，且幼虫蜕皮时间较对照组延迟约4.8 d。（3）DHEA处理抑制了美国白蛾5龄幼虫几丁质合成通路关键基因（HcGNA、HcPAGM、HcUAP、HcCHSA）的转录水平，导致表皮几丁质结构变薄，且含量仅为对照组的27%。
      结论 DHEA通过抑制G6PD的活性、下调Hc6PGL和Hc6PGD的转录水平及F6P含量，显著抑制了美国白蛾幼虫体内PPP代谢途径；DHEA介导的G6PD的活性及PPP代谢途径抑制显著抑制了美国白蛾幼虫生长发育及表皮几丁质合成。

       

      Abstract:
      Objective This study investigated the inhibitory effect of dehydroepiandrosterone (DHEA) on glucose-6-phosphate dehydrogenase (G6PD) activity and the pentose phosphate pathway (PPP) in larvae of Hyphantria cunea, a major agricultural and forestry pest, further examining the role of the PPP in larval growth, development, and chitin biosynthesis.
      Method The HcG6PD gene was cloned from Hyphantria cunea, and the amino acid composition, physicochemical properties and structural characteristics of HcG6PD were analyzed by bioinformatics software. The expression pattern of HcG6PD at different developmental stages was analyzed by RT-qPCR. The G6PD activity, the expression levels of key genes and intermediate product content in PPP in the 5th instar larvae of H. cunea were measured after treating with DHEA. Meanwhile, the inhibitory effect of DHEA on G6PD and PPP was analyzed. To evaluate the effects of G6PD inhibition on chitin synthesis, the relative expression levels of key genes in the chitin synthesis pathway and the chitin content in epidermis of the 5th instar H. cunea larvae were determined. Meanwhile, chitin in the epidermis was stained after treating with DHEA.
      Result (1) After treating the 5th instar larvae with DHEA for 72 hours, the activity of G6PD significantly decreased, and the transcriptional levels of the key genes of PPP (HcG6PD, Hc6PGD, Hc6PGL) were only 18%, 17%, and 13% of control, respectively. The content of fructose-6-phosphoric acid (F6P) of key genes of PPP was only 0.18 times of control. (2) DHEA treatment led to the obstruction of larval growth and molting. After treating with DHEA for 72 hours, the mass gain of the 5th instar larvae was only 13% of control, and the molting time of larvae was delayed by approximately 4.8 days compared with control. (3) The transcription levels of key genes (HcGNA, HcPAGM, HcUAP, HcCHSA) in chitin synthesis pathway of the 5th instar H. cunea larvae were inhibited by DHEA. This inhibition led to a reduction in epidermal chitin content, which was only 27% of control, as well as a thinning of epidermal chitin structure.
      Conclusion DHEA significantly inhibites the PPP metabolic pathway in H. cunea larvae by suppressing G6PD activity, reducing the transcriptional levels of Hc6PGL and Hc6PGD, and the content of F6P. The inhibition of G6PD activity and PPP induced by DHEA significantly depresses the growth and development, and the epidermal chitin biosynthesis in H. cunea larvae.

       

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