柑橘全爪螨脂肪酸结合蛋白基因克隆及对饥饿胁迫的响应

Cloning the Panonychus citri fatty acid binding protein gene,and the response of this gene under food deprivation

【目的】脂肪酸在昆虫的生长发育和繁殖中起着重要的作用,而脂肪酸结合蛋白(Fattyacid bindingprotein,FABP)主要参与机体脂肪酸的特异性结合和转运等。本研究旨在克隆柑橘全爪螨Panonychuscitri脂肪酸结合蛋白基因,并分析其对饥饿胁迫的响应。【方法】运用RT-PCR结合cDNA末端快速扩增(RACE)技术克隆获得柑橘全爪螨脂肪酸结合蛋白基因全长并分析其序列特征,采用实时荧光定量PCR技术检测了该基因在柑橘全爪螨生长发育不同阶段的表达量差异;将柑橘全爪螨雌成螨在不同饥饿胁迫处理后,统计其存活率、产卵量、卵孵化率、每雌每日产卵量以及PcFABP基因的相对表达量。【结果】柑橘全爪螨PcFABP基因的cDNA全长为907 bp,包括119 bp的5′非编码区,402 bp的开放阅读框(ORF)以及387bp的3′非编码区,编码133个氨基酸。氨基酸序列分析表明,PcFABP基因与二斑叶螨Tetranychus urticae的同源性最高,与其他蛛形纲的相似性为50%左右。PcFABP在柑橘全爪螨不同的生长发育阶段(卵、幼螨、前若螨、后若螨、雌成螨、雄成螨)均表达,在卵、幼螨和后若螨中的表达量较高,前若螨和成螨中表达量较低。与饲喂24和48 h相比,柑橘全爪螨雌成螨饥饿处理24和48h、先饥饿24h后复饲24h、先饲喂24h后饥饿24h后,其存活率和产卵量均显著降低(P<0.05);饥饿胁迫处理后柑橘全爪螨雌成螨产卵后卵的孵化率与对照组相比并未达到显著差异(P>0.05)。此外,对PcFABP基因的表达分析显示先饥饿24h在复饲24h的相对表达量最高,饥饿48 h的相对表达量最低。【结论】PcFABP基因可能参与柑橘全爪螨的生长发育,并且柑橘全爪螨可能通过调控PcFABP基因的表达来对饥饿胁迫进行响应。

[Objectives]To clone the Panonychus citri fatty acid binding protein gene and analyze its response to food deprivation.[Methods]The full-length cDNA sequence of the PcFABP gene was,for the first time,cloned using RT-PCR in combination with RACE technology.The relative expression levels of the PcFABP gene in different developmental stages of P.citri were examined using RT-qPCR.In addition,the survival rate,oviposition,hatch rate,fecundity(eggs/female/day)and the expression of the gene,were measured after a period of food deprivation.[Results]The results demonstrate that the full-length cDNA sequence of PcFABP is 907 bp long,including 119 bp 5′-UTRs,402 bp ORFss,and 387 bp 3′-UTR,and encodes a deduced protein of 133 amino acids.Amino acid sequence alignment suggests that the P.citri FABP gene is most homologous to the Tetranychus urticae FABP gene,and has a similarity to the PcFABP genes of other arachnids of about 50%.The PcFABP gene was expressed in all developmental stages of P.citri,but expression in the egg,larva and deutonymph were significantly higher than in other developmental stages.The survival and oviposition rates of P.citri females were significantly lower than those of the control group after being deprived of food for 24 h and 48 h.Allowing females to feed for 24 h after 24 h of food deprivation,or depriving them of food for 24h after allowing them to feed for 24 h,also significantly reduced female survival and oviposition rates relative to the control group.The hatching rate of the eggs laid by females subject to food deprivation was not,however,significantly different from that of the control group.The relative expression of the PcFABP gene was highest in females that fed for 24 h after being deprived of food for 24 h,and lowest in those that were deprived of food for 48 h.[Conclusion]PcFABP may play an important role in the growth and development of P.citri,and may also be involved in the response of this species to food stress.