嗜虫书虱几丁质合成酶2基因的克隆与功能研究

Cloning and Functional Analysis of Chitin Synthase 2 Gene in Liposcelis entomophila

研究嗜虫书虱(Liposcelis entomophila)几丁质合成酶2(Liposcelis entomophila chitin synthase 2,LeCHS2)基因的表达模式及其在嗜虫书虱发育过程中的生物学功能,以期为嗜虫书虱的防治及特异性杀虫剂的新靶标筛选提供理论基础。本研究克隆获得了LeCHS2,其完整的编码区序列(coding sequence,CDS)为4524 bp,编码1507个氨基酸,运用生物信息学软件分析发现,其具有17个跨膜结构域,预测蛋白分子的质量为172.9 kDa,理论等电点为6.24,不具有信号肽。LeCHS2的氨基酸序列与其他8种昆虫几丁质合成酶2(chitin synthase 2,CHS2)的同源性为49.05%~53.67%。系统发育树显示,LeCHS2与人体虱(Pediculus humanus corporis)CHS2亲缘关系最近。实时定量PCR(real time quantitative PCR)检测LeCHS2基因在嗜虫书虱不同发育阶段和不同组织部位的表达模式,发现LeCHS2在嗜虫书虱的中肠和后肠中高表达,说明该基因主要在中肠、后肠中发挥作用;若虫期的表达量高于成虫期的,在若虫期第1天、第11天表达量最高。为研究LeCHS2的功能,本研究采用壳聚糖(chitosan,CS)与三聚磷酸钠(tripolyphosphate,TPP)交联产生纳米级递送载体,形成CS-TPP-dsLeCHS2的复合物进行RNA干扰(RNA interference,RNAi)。连续饲喂CS-TPP-dsLeCHS224、48、72、96 h后,LeCHS2基因表达量显著降低,实时定量PCR检测其干扰效率,LeCHS2基因表达量分别下降79.4%、47.0%、40.0%和25.0%,连续饲喂8 d后嗜虫书虱的存活率为56.6%。干扰LeCHS2后,对腹部外部形态观察发现,嗜虫书虱的体长缩短了17%,腹部厚度减少了18%,体重显著下降;组织切片观察发现,围食膜缺失导致肠道上皮细胞裸露,说明LeCHS2的功能与中肠围食膜的形成有关,对其生长发育至关重要。研究结果表明采用饲喂法干扰LeCHS2后对嗜虫书虱围食膜的形成有破坏作用,从而影响其进食和消化吸收,使其生长发育受阻并最终死亡。

The expression pattern of Liposcelis entomophila chitin synthase 2(LeCHS2)gene and its biological function in the develop-ment of L.entomophila were studied in order to provide a theoretical basis for the prevention and control of L.entomophila and to investi-gate new targets for pest management.LeCHS2 was cloned and the coding sequence(CDS)of the LeCHS2 was 4524 bp in length,en-coding 1507 amino acids.Bioinformatics software analysis found that it contained 17 transmembrane helices,the predicted molecular weight of the protein was 172.9 kDa,with a theoretical isoelectric point of 6.24 and no signal peptide.The amino acid sequence of LeCHS2 shared 49.05%-53.67%identity with CHS2 from the other 8 insects.The phylogenetic tree showed that LeCHS2 had the clo-sest relationship with Pediculus humanus corporis CHS2.The expression patterns of LeCHS2 in different developmental stages and dif-ferent tissues of Liposcelis entomophila were detected by real time quantitative PCR.It was found that LeCHS2 was primarily expressed in the midgut and hindgut,indicating that the gene mainly plays a role in the midgut and hindgut.The relative expression of LeCHS2 in numph was higher than in adults,and the highest expression was found at the 1st and 11th of the nymph stage.To study the function of LeCHS2,a RNA interference(RNAi)method was developed using dsRNA with a nano carrier formed by chitosan(CS)cross-linked with sodium tripolyphosphate(TPP).After continuous feeding of CS-TPP-dsLeCHS2 for 24,48,72,and 96 h,the expression of LeCHS2 gene decreased significantly.The interference efficiency was detected by real-time quantitative PCR.The interferential effi-ciency of LeCHS2 after continuous feeding on CS-TPP-dsLeCHS2 for 24,48,72,and 96 h was 79.4%,47.0%,40.0%and 25.0%,respectively,and after 8 d of continuous feeding,the survival rate of L.entomophila reached 56.6%.After interfering LeCHS2,obser-vation of the external morphology of the abdomen showed that the body length was shortened by 17%,and the abdominal thickness was reduced by 18%,and the body weight decreased significantly.Observation of tissue sections revealed that the absence of periesophage-al membrane led to the exposure of intestinal epithelial cells.It showed that the function of LeCHS2 was related to the formation of mid-gut peritrophic membrane,which was vital for the growth and development of L.entomophila.Interfering LeCHS2 gene affects its feeding,digestion,and absorption,and thus hinders the development and causes the mortality of L.entomophila.