灰飞虱鞘氨醇激酶时空表达分析及其对杀虫剂胁迫的响应

【目的】明确灰飞虱(Laodelphax striatellus Fallén)鞘氨醇激酶(sphingosine kinase,SK)的分子特征,研究SK在携带水稻条纹病毒(Rice stripe virus,RSV)与健康灰飞虱两个种群的时空表达及其对杀虫剂胁迫的响应。【方法】利用PCR技术克隆灰飞虱SK(LsSK)基因序列;使用荧光定量PCR(qRT-PCR)技术分析LsSK在带毒种群与健康种群灰飞虱1—5龄若虫及成虫期的表达差异,并检测该基因在雌雄虫头、唾液腺、中肠、马氏管、卵巢和精巢等组织中相对表达量;采用手动微量点滴仪将3种杀虫剂(吡虫啉、噻嗪酮和溴氰菊酯)点滴于灰飞虱4龄若虫中胸背板,确定3种杀虫剂的半致死浓度(LC_50);根据半致死浓度的3种杀虫剂处理试虫后检测LsSK的表达动态;采用注射双链RNA(dsRNA)的方法沉默带毒种群和健康种群4龄若虫体内LsSK后,用半致死浓度的3种杀虫剂处理试虫并分析死亡率。【结果】克隆得到一段长度为1 282 bp的LsSK基因片段(Gen Bank登录号:KT989975)。氨基酸系统进化树显示LsSK与其他半翅目昆虫SK聚在同一支。LsSK氨基酸序列包含SK酶的4个保守区域C1—C4,其中SK激酶的活性位点——DAG活性区域位于C1—C3区域。q RT-PCR结果显示LsSK在带毒种群4龄若虫中表达量最高,3龄次之;健康种群5龄若虫表达量最高,1、4龄次之。且LsSK在带毒种群3、4龄表达量显著高于健康种群同时期若虫(P0.05);带毒成虫LsSK表达量显著高于健康成虫(P0.05)。LsSK在带毒雄成虫各个组织中的表达量均显著高于同种群雌成虫和健康雌雄成虫的各相应组织,且在唾液腺和马氏管的表达量最高,头部次之。用半致死浓度的3种杀虫剂(吡虫啉LC_50为6.5 ng·μL~(-1),噻嗪酮为500 ng·μL~(-1),溴氰菊酯为37.5 ng·μL~(-1))处理带毒和健康种群的4龄若虫后,试虫LsSK含量变化与响应速度均不同。噻嗪酮处理后LsSK水平升高且响应最为迅速,溴氰菊酯处理后LsSK表达水平无变化(P0.05)。用3种杀虫剂处理带毒和健康试虫,其死亡率分析结果表明,两种群注射dsSK组3种杀虫剂处理后死亡率均显著高于两种群注射dsGFP组和不注射组。【结论】克隆了LsSK基因片段且其在带毒灰飞虱4龄若虫中表达量最高。沉默LsSK后,带毒和健康种群灰飞虱在杀虫剂处理后的死亡率均显著上升,表明SK基因有利于灰飞虱抵抗杀虫剂胁迫。

Spatio-Temporal Expression of Sphingosine Kinase and Its Response to Insecticide Stress in Laodelphax striatellus Fallén

【Objective】The objective of this study is to understand the characteristics of sphingosine kinase (SK) and research on the spatio-temporal expression of SK and its response to insecticide stress in Rice stripe virus (RSV) -infected Laodelphax striatellus Fallén (small brown planthopper, SBPH) and RSV-free L. striatellus.【Method】A SK sequence (LsSK) from L. striatellus was cloned using PCR. qRT-PCR was employed to analyze the expression pattern of SK gene at different stages (1st-5th instar nymphs, female and male adult) and various organs (head, salivary, midgut, malpighian tubules, ovary and spermary) of the two above mentioned L. striatellus colonies. In order to calculate the sublethal concentration (LC₅₀) of three insecticides (deltamethrin, buprofezin and imidacloprid), those insecticides solutions were applied topically to the mesonotum of L. striatellus 4th-instar nymphs with a hand microapplicator. The SK mRNA level of L. striatellus was detected after exposure to LC₅₀ of three insecticides. After knockdown SK of two L. striatellus colonies by using dsRNA injection, L. striatellus was exposed to LC₅₀ of three insecticides and the mortality was recorded.【Result】a LsSK sequence from L. striatellus was cloned and its length is 1 282 bp(GenBank accession number KT989975). According to the phylogenetic analysis, SK of L. striatellus was clustered together with those from hemipteran insects. The amino-acid sequences of LsSK contained four conserved domains (C1-C4), and the catalytic domain–Diacylglycerol active site formed within C1-C3. qRT-PCR results showed that LsSK was most highly expressed in the 4th-instar nymphs of RSV-infected L. striatellus, followed by 3rd-instar nymphs. In RSV-free L. striatellus, LsSK was most highly expressed in 5th-instar nymphs, followed by 1st- and 4th-instar nymphs. Furthermore, LsSK levels were significantly higher in RSV-infected 3rd- and 4th-instar nymphs than that in RSV-free 3rd- and 4th-instar nymphs(P＜0.05). At the adult stage, LsSK levels were significantly higher in RSV-infected adults than in RSV-free adults (P＜0.05). Interestingly, LsSK showed extremely higher expression in the tissues of viruliferous males than that of viruliferous females and nonviruliferous adults. The highest expression of LsSK was found in salivary and malpighian tubules of viruliferous males, followed by heads. After exposure to sublethal concentrations (LC₅₀) of three insecticides (imidacloprid 6.5 ng·μL^-1, buprofezin 500 ng·μL^-1 and deltamethrin 37.5 ng·μL^-1), the mRNA levels of LsSK and response speed of two L. striatellus colonies were different. Exposure to buprofezin led to significantly increased expression of LsSK while no significant changes was observed after exposure to deltamethrin(P＞0.05). The response of LsSK expression to buprofezin was relatively faster than to the other insecticides. Injection of L. striatellus with dsRNA against SK reduced the mRNA level of LsSK greatly, and silencing of SK led to increased susceptibility in RSV-infected and RSV-free L. striatellus to these three insecticides.【Conclusion】The LsSK was identified and cloned, and it was found significantly high expression in the 3rd and 4th instar of RSV-infected L. striatellus. Silencing the SK resulted in increased susceptibility in L. striatellus to three insecticides, indicated that SK plays a role in facilitating L. striatellus to response to insecticidal stress.