| The small brown planthopper(Laodelphax striatellus Fallen,SBPH)is one of the most economical important pest on rice,not only feeding directly but also transmitting rice stripe virus(Rice stripe virus,RSV)with a persistent,circulative-propagative manner,which has caused serious economic losses to rice production.Himetobi P virus(HiPV)was first isolated from the SBPH populations in 1992.HiPV is an ssRNA belonging to the genus Cripavirus in the Dicistroviridae family.Previous studies of interactions between RSV and SBPH,found that RSV ribonucleoprotein(RNP)interacted with capsid protein VP1 of HiPV,suggesting that HiPV facilitated potentially the accumulation of RSV in insect vector.For this aim,this paper focused on the relationship between HiPV VP1 and RSV in insect vector SBPH.1.Sequencing and analysis of HiPV genome of SBPH populations in JiangsuAfter sequencing the HiPV genome of SBPH populations in Jiangsu,the homology was analyzed through compared with the reported genome sequences of Japanese,Zhejiang,Jiangsu SBPH HiPV and the genome of Japanese BPH HiPV.It was found that SBPH HiPV genome of Jiangsu showed considerable high homology when compared with the other genomos which sequences homology of isolated objects from were 98.8%,99.5%,99.7%and 98.4%respectively.2.Investigation of the relationships between HiPV and RSV in SBPHTo investigate the relationships between HiPV-VP1 and RSV-RNP in SBPH,two viral titers in single insect were examined by real-time quantitative PCR(qRT-PCR),based on HiPV genome sequence of SBPH population in Jiangsu.The results showed that the expression levels of VP1 and RNP genes were different in single insect.In general respectively,the accumulation of RSV was higher in individual insects that held more VP1 expression,and when the level of VP1 was lower,RSV titers decreased accordingly in SBPH.What's more,quantitative analysis of the two viruses in SBPH groups indicated that when the level of VP1 was higher in SBPH,RSV also held a higher quantity,which suggested that HiPV-VP1 facilitateed the replication of RSV in insect vector.3.The effects of HiPV expression levels on RSV-replication and RSV-acquisition ability in SBPHRNAi was performed in SBPH by feeding dsRNA,which was synthesized using the HiPV-VP1 gene PCR product as a template,in order to investigate the effects of HiPV expression levels on RSV-replication and RSV-acquisition ability in SBPH.The results showed that the expression of HiPV-VP1 was reduced in SBPH after feeding dsVPl,and the processes of RSV replication and accumulation also slowed down,which suggested that HiPV-VP1 facilitated potentially the accumulation of RSV in insect vector.Meanwhile,after feeding dsVPl,RSV-free nymphs were transferred onto RSV-infected leaves for virus acquisition.The viruliferous rate of SBPH was detected after in 10-day.The results showed that the virus-acquisition rates of SBPH from different treatments were similar without a significant difference.4.Screening of HiPV-VP1 interaction protein factors in SBPHTo find the special interacting proteins of HiPV-VP1,HiPV-VP1 was used as a bait to screen the receptors in the yeast two-hybrid cDNA library of high viruliferous SBPH after construct pGBKT7-VP1 vector.As a result,ten interaction proteins were found,in which there were at least 3 genes with full ORF.Those possible interacting proteins not only provide new clues for further studies on the interaction between HiPV-VP1 and SBPH,but also are useful to find out the roles of HiPV-VP1 in RSV transmission. |
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