| The wheat yellow dwarf disease was caused by Barley yellow dwarf virus(BYDV).In China,it is a very serious wheat virus disease.There are four main strains of BYDV in China,among which GAV and PAV are the mainstream strains.In the field,BYDV was transmitted by vector aphids,Sitobion miscanthi(Takahashi)(which was mistakenly identified as Sitobion avenae(Fabricius)in China,and is still commonly used)as the dominant species of wheat aphid in the field,the two viruses could be transmitted simultaneously,which caused serious losses to wheat.BYDV was transported by the way of cyclic and non-propagative in aphids.However,there are midgut barrier and salivary gland barrier in aphid,and there are interacting proteins in aphid to help virus particles break through the transmission barrier,but the types and functions of interacting proteins are not clear at present.Other researchers used the yeast two-hybrid technique to screen out the protein of BYDV-GPV interacting with the protein CYP4G15 in the Rhopalosiphum padi.Our laboratory used yeast two-hybrid technology and Bi FC technology to screen and identify the proteins Lwr and Ntf that interact with the protein of BYDV-GAV in S.miscanthi.Under these circumstances,CYP4G15,Lwr,and Ntf genes of S.miscanthi were studied in this study,and used q RT-PCR technology to study the expression of target genes in various tissues of S.miscanthi.Meanwhile,the functions of CYP4G15,Lwr and Ntf involved in the acquisition,transmission,andretention of BYDV-GAV and BYDVPAV were verified by RNAi technology.The main results are as follows:1.The full length of the CYP4G15 cds sequence was amplified and we were found that CYP4G15 contained a transmembrane domain with no signal peptide on it and subcellular localization in the endoplasmic reticulum.It is closely related to Myzus persicae,but far related to Diptera,Lepidoptera,and Coleoptera.2.The cephalothorax,abdomen,gut,and hemolymph tissues of the uninfected and infected aphid were dissected and results showed that here were no significant differences in CYP4G15,Lwr and Ntf expression in the cephalopthorax and abdomen infected with GAV aphid.The expression of CYP4G15 in hemolymph tissue was significantly higher than that in gut,while there was no significant difference in Lwr and Ntf expression between hemolymph and gut.The expression levels of CYP4G15,Lwr,and Ntf in the gut of BYDV-GAV infected S.miscanthi were significantly decreased compared with those of non-infected aphid,and the expression levels of Ntf in the cephalopthorax were significantly increased,while the expression levels of CYP4G15,Lwr,and Ntf in other parts of aphid showed no significant difference.3.Primers for in vitro synthesis of ds RNA were designed according to gene sequences,and specific ds RNA was synthesized externally.ds RNA was delivered into the aphid by microinjection,and the silencing efficiency of target genes was detected by q RT-PCR.The three target genes all reached the highest interference efficiency at 48 h,which was 51.53%for CYP4G15,55.89% for Lwr,and 49.61% for Ntf.ds RNA of the three target genes did not affect aphid fitness parameters such as mortality and fecundity.4.RNAi experiments showed that(1)CYP4G15 can help aphid to acquire BYDV-PAV,promote the spread of BYDV-PAV,but reduce the amount of retained BYDV-PAV;However,it only promoted the acquisition of BYDV-GAV,had no significant effect on the transmission and retention of BYDV-GAV.(2)Lwr could assist in the acquisition of BYDV-PAV,but had no effect on the transmission and retention of BYDV-PAV.Lwr inhibited aphid acquisition of BYDV-GAV,but promoted the transmission of BYDV-GAV,and had no significant effect on the retention of BYDV-GAV.(3)Ntf could assist aphis communis to acquire BYDV-PAV and BYDV-GAV,but had no effect on the transmission and retention of BYDV-PAV and BYDVGAV.In conclusion,this study found that CYP4G15,Lwr and Ntf expression were different in tissues of infected and uninfected S.miscanthi.In addition,CYP4G15,Lwr and Ntf play important roles in the acquisition,transmission and retention of BYDV-PAV and BYDV-GAV by the aphis.This study will lay a foundation for further research on the transmission mechanism of the wheat yellow dwarf disease and new control methods based on the three genes. |
