【摘要】 通过研究Hc38基因对捻转血矛线虫L3期幼虫发育的影响,探索Hc38基因功能,为利用该基因防治捻转血矛线虫的进一步研究提供依据。针对Hc38基因序列设计并合成特异性dsRNA和siRNA,采用浸泡的方法对捻转血矛线虫L3期幼虫进行RNA干扰(RNAi)试验,利用实时荧光定量PCR分析Hc38基因在L3期幼虫中的转录抑制效果,同时将dsRNA和siRNA浸泡24h后的L3期幼虫感染绵羊,研究Hc38基因的沉默对捻转血矛线虫L3期幼虫在绵羊体内发育的影响,定期采集绵羊粪便检查虫卵数,30d后剖检绵羊皱胃,检查荷虫数的变化。实时荧光定量PCR检测表明,各试验组Hc38基因的相对含量显著低于对照组(P<0.01),浸泡72h后,Hc38-dsRNA组和Hc38-siRNA组Hc38基因的表达量分别仅为对照组的32.27%和14.93%。绵羊体内干扰沉默效果显示,试验组Hc38干扰组虫卵减少率最高,为50%,减虫率最高,为48.6%。通过浸泡法导入的dsRNA和siRNA均能有效抑制Hc38基因的转录,同时Hc38基因的沉默与幼虫的发育密切相关,为捻转血矛线虫及其他寄生线虫的基因功能研究提供一种新的方法。更多还原

【Abstract】 The aim of this research was to elucidate the function of Hc38 gene from Haemonchus contortus and provide scientific basis for the further research on Hc38 gene of animal-parasitic nematodes as well as the prevention and control of Haemonchus contortus.Specific double-stranded RNA(dsRNA) and small interference RNA(siRNA) was designed according to Hc38 gene sequence.Then RNA interference of L3 larvae of Haemonchus contortus was performed by soaking method.RT-PCR was utilized to detect the inhibition of Hc38 gene on the transcription of L3 larvae of Haemonchus contortus.After soaked with dsRNA and siRNA for 24 hours,L3 larvae of Haemonchus contortus was utilized to infect sheep in order to study the effect of Hc38 gene silencing on the development of L3 larvae of Haemonchus contortus.Feces were collected regularly to calculate the ovum of Haemonchus contortus.Worm burdens of Haemonchus contortus were also calculated through abomasum necropsy 30 days later.Indicated by RT-PCR,the content of Hc38 gene in treated group was significantly lower than that in the control group(P<0.01).After soaked for 72 hours,the expression of Hc38 gene in Hc38-dsRNA group and Hc38-siRNA group was 32.27% and 14.93% the amount of the control group.According to in vivo result of RNAi sheep,the amount of ovum in treated group was reduced by 50% and the amount of parasite was reduced by 48.6%.The transcription of Hc38 gene was inhibited in both dsRNAs group and siRNAs group after soaking.Silencing of Hc38 was closely related with the larvae development in Haemonchus contortus.This study provided a new method for the gene function research of Haemonchus contortus and other parasitic nematodes.更多还原