生物防治研究与应用团队杨珊同学在《Pest Management Science》发表论文

近日，生物防治研究与应用团队博士生杨珊以第一作者在农林科学一区Top期刊《Pest Management Science》，在线发表了研究论文“Knockdown of hexokinase in Diaphorina citri Kuwayama(Hemiptera:Liviidae)by RNAi inhibits chitin synthesis and leads to abnormal phenotypes”(https://onlinelibrary.wiley.com/doi/abs/10.1002/ps.7049)，通过RNAi技术敲低柑橘木虱己糖激酶来抑制其几丁质合成及增加死亡率的最新报道，为柑橘木虱RNAi控害提供新途径。

柑橘是我国南方最重要的水果之一，柑橘黄龙病(Huanglongbing, HLB)又称柑橘“癌症”，严重危害柑橘产业的发展。柑橘木虱Diaphorina citri Kuwayama是已知唯一柑橘黄龙病传播的媒介，目前防治柑橘木虱主要依靠化学防治，长期、大量喷洒农药造成残留、抗性增加、杀死有益生物，带来严重的农业面源污染，因此，寻求控制柑橘木虱的新途径显得尤为迫切。昆虫几丁质合成和降解影响昆虫的蜕皮和变态，对昆虫的生长发育至关重要，操纵几丁质合成的调节正在成为研究开发新的害虫控制的有效途径；本研究以探寻非化学防治柑橘木虱特异性靶标为基准，发现柑橘木虱己糖激酶DcHK可以作为RNAi技术抑制其生长发育和几丁质合成的特异性靶标。研究表明DcHK在柑橘木虱三龄若虫、成虫和马氏管中的转录水平更高（图1）；针对柑橘木虱RNAi所需的dsRNA，呈现剂量依赖性，其中600ng/μLdsDcHK足以击倒内源性DcHK的表达（图2），36hmRNA表达水平最低，显著影响其几丁质合成途径中其他关键基因（DcTre、DcG6PI、DcGNAT、DcGFAT、DcPGM、DcUAP和DcCHS）的相对mRNA表达水平，且导致柑橘木虱死亡在给药36h时高达26.1%、体重减轻达0.168mg、畸形率在若虫期和成虫期分别为6.46%和21.4%远高于对照组（图3）。

                                                           A                                                                          B

图1：己糖激酶在柑橘木虱不同发育阶段及不同组织中的转录水平

Figure 1. Expression profiles of DcHK at different developmental stages (A) and various body segments and tissues (B) of D. citri. Relative mRNA levels of DcHK were analyzed using RT-qPCR. β-actin is used as an internal control. Each bar indicates a mean ± SEM, and different letters above each bar represent significant differences (P<0.05; ANOVA, Tukey’s multiple comparisons test) from three independent experiments.

图2：柑橘木虱RNAi的剂量测定

Figure 2. Effects of feeding dsDcHK on mortality rate in D. citri. Results for control (dsGFP) and treatment groups (dsDcHK of 200, 400, 600, and 800 ng/µL) 72 h post-RNAi are shown. β-Actin was used as the internal control for normalization. Values shown in the figure are means and standard deviations. Different letters indicate significant silencing relative to other treatments (P<0.05; ANOVA, Tukey’s multiple comparisons test).

图3：RNAi后对柑橘木虱表型的影响

Figure 3. Effects of RNAi on D. citri representative phenotypes after oral administration of dsRNA. A: Fifth instar nymph with oral dsGFP. B: The emerging adult worms 72 h after oral administration of dsGFP. C: Emerging mature adults 72 h after oral administration of dsGFP. D: The phenotype of adult wing after oral administration of dsGFP for 72 h. F: Fifth instar nymphs 72 h after oral administration of dsDcHK. The body color darkens, the abdomen shrinks, and no stratum corneum split is observed. F: Adults that have just emerged 72 h after oral administration of dsDcHK fail to spread their wings normally. G: Mature adults 72 h after of oral administration of dsDcHK fail to spread their wings normally, and their wings are deformed. H: Mature adults 72 h after oral administration of dsDcHK, whose hindwings have a folded-wing phenotype. I: Mature adults 72 h after oral administration of dsDcHK, with nicks in the forewings. J: Mature adults, 72 h after oral administration of dsDcHK, are smaller and show large individual differences, relative to dsGFP.

       该论文通讯作者为团队夏斌教授。近年来，团队注重柑橘主要害虫非化学防控的应用基础研究，同时开展其天敌的生物防治研究与应用，把实现农产品绿色、有机目标作为己任！