Screen Of Important Genes From Sitobion Avenae And Effect Analysis Of RNA Interference

Wheat aphids,small sap-sucking insects in Homoptera Aphididae,is one of the most important wheat pests in the world and can cause yields losses by 10%-30%.Wheat aphids damage wheat by taking wheat sap,resulting in die-back of branches on plants or even the death of the entire plant.Furthermore,wheat aphids can be vectors of yellow dwarf virus and other viral diseases,and thus enhance the damage of the virus to wheat plants.There were currently two ways to control wheat aphids.One is by application of pesticides and the other is by planting varieties of wheat aphids resistance.The latter is recognized as the most environment-friendly,economical and effective way.Hybridization breeding is at present the main method for the development of new varieties of resistance to wheat aphids,by which insect resistant genes from common wheat or alien relatives.However,effectiveness of hybridization breeding is limited by the lack of useful gene resources of aphids resistance.So,screening and identification of resistance genes against aphids from wild species of wheat has great significance in the development of new resistant varieties of wheat.A novel RNA interference(RNAi)technique named by host-induced gene silencing(HIGS)opens a new way for breeding new wheat varieties of diseases and pets resistance.RNAi is phenomenon that the specific gene expression is silenced at transcriptional level by double-stranded RNA.HIGS is a RNAi technology to silence genes in plant pathogens by expressing a dsRNA construct against specific genes endogenous to the pathogen in the host plant.HIGS is achived by transfer a vector containing the antisense hairpin pathogen target gene into a host plant,which will produce dsRNA of pathogen specific genes and lead to the down-regulation of pathogens endogenous gene in the host plant.HIGS technology has been used in many both studies of gene function and disease control of fungal pathogens.For a succesful HIGS the target genes(the pathogen-derived genes)play a large role.Thus,screening and identification of the genes for aphid growth and reproduction is the foundation for the production of new whaet varieties against wheat aphids derived from HIGS technology.The research aims at providing new resistance aphid sources genes and new technologies for the development of aphid-resistance wheat by(1)the identification and screening of new genes of wheat aphids resistance,and(2)screening and identification of important genes in wheat aphid by analysis the effect of RNAi silencing.The research results are summarized as followings:1.Screening and identification of the aphid resistant genes in alien relatives of wheat and chromosome mapping of the resistant genes.Total of 78 of alien addition lines with genetic backgrounds of common wheat Chinese Spring(CS)have been scored by using home-made aphid identification devices at seedling stage following a evaluation criteria by aphid index.The results show that a CS-Agropyron elongatum addition line is highly resistance to aphid,and other seven alien addition lines derived from Ae.geniculata,Aegilops caudata,Ae.peregrina and Secale cereale are medium resistance to aphids.In the identified materials,8 alien addition lines show aphids resistance(including high resistance and medium resistance),accounting for 10.2%;59 lines are susceptible to aphids,accounting for 73.1%;the remaining 11 lines are high susceptible to aphids,accounting for 16.7%.In the study,Chinese Spring is susceptible,so the resistance can be located to alien chromosomes by the comparison of resistance with addition lines.Thus that high resistance gene(s)is located on chromosome 6E of Elytrigia elongata,and other 7 medium aphid resistance genes were respectively located on Elytrigia elongata 5E?Aegilops geniculata 1Mg Aegilops caudata D,Ae.peregrina 1Sv and 5Uv,Secale cereale 7R and Aegilops speltoides 6S chromosome.The research can provide valuable resistant genes for wheat breeding of aphids resistance,and for gene fine mapping?cloning and genetic mechanisms of resistance to wheat aphids.2.A dsRNA feeding system is established for screening important genes of aphid growth and reproduction.The wheat aphids are cultured in the cells of a sterile 24-well cell culture plates plusing a piece of filter paper at the bottom,and holding 3cm above water level.The celles are covered by double layers of UV sterilized paraffin film containing artificial diet quote from Chen.Wheat aphids then are cultured at 25 ? with photoperiod of 16h:8h(L:D).Proper feeding time(1-11 days)for RNAi screening is based on the standard curve of aphid survive.The best feeding results in the study is by using fourth instar aphids hungry five hours before adding artificial diet,and then replacing artificial diet every 48 hours,replacing new plates after seven days.3.Based on the gene sequences of aphididae such as Acyrthosiphon pisum,Myzus persicae,aphis gossypii,Rhopalosiphum padi collected from the NCBI database,56 pairs of specific primers are designed for gene cloning of wheat aphids.Total 37 candidate genes fragments are cloned,dsRNA of 29 candidate genes are successfully synthesized,DNA sequence comparsion of 19 candidate genes confirmed they are all conserved sequences in Aphididae,above 90%homology.4.Two important genes(aquaporin and ATP synthase gene)of wheat aphids are identified by RNAi silencing using dsRNA feeding system.Aquaporin gene silence by feeding dsRNA causes a aphid mortality rate of 40.6%,whereas dsRNA feeding of ATP synthase gene leads to a aphid mortality rate of 26.5%.In opposition,silence of the glucose dehydrogenase gene by dsRNA feeding enhances aphid growth and development,decreasing the mortality rate by 19.5%.5.Three candidate genes(water channel protein,chromosomal kinesin,ATP synthase)which significantly increase aphid mortality by feeding dsRNA,and glucose dehydrogenase encod gene which significantly decreased aphid mortality by feeding dsRNA are chosen to analyse target gene expression levels by RNAi by using qRT-PCR.The results showed that expression level of aquaporin gene,chromosome kinesin gene and ATP synthase gene are decreased by 74.8%,36.6%and 44.6%respectively,whereas the expression level of glucose dehydrogenase encoding gene increases by 209.0%.By comparison of aphid mortality and endogenous gene expression levels induced by dsRNA silencing,aphid mortality level of gene by dsRNA accords with the gene expression levels.Therefore,the mortality of wheat aphid by feeding dsRNA can be a reliable indicator for a gene silencing,it will be a good way to provide target genes for wheat breeding of aphid-resistance by HIGS technology.6.Five genes(aquaporin,ribosomal protein,ATP synthase,DNA damage and the acetylcholine receptor binding protein)are also used to design multiple silence target sites in for the same gene.The results show that the same gene silencing at different target sites can cause significantly different aphid mortality rate.Therefore,multiple silence target sites maybe should be choosed for a gene silencing,then the most effective silence target sequences are selected as target genes for HIGS.