| RNAi is produced in the process of resisting the invasion of foreign virus,and it can identify the double stranded RNA(ds RNA)by special enzymes and degrade the specific messenger RNA(m RNA)in eukaryotic organism.Virus induced gene silencing(VIGS)technology is the target gene integrated into the genome of the virus,resulting in a large number of ds RNA by virus infected host and proliferation process,which induced silencing of plant pathogen specific m RNA and reduced gene expression,to prevent pests and diseases.RNAi plays an important role in the study of fungal gene function,and the transformation method of filamentous fungi is the most widely used.This research through the ds RNA feeding method,it is found that the induction of wheat aphid aquaporin gene(Wsa)silencing can seriously affect the wheat aphid growth and reproduction;co-culture method of screening effect of gentisate dioxygenase gene greatly on the growth rate of wheat take-all using synthetic ds RNA and Gaeumannomyces graminis(Gdo).On this basis,this study carried out two parts:(1)The BSMV-VIGS mediated on wheat aphid aquaporin gene(Wsa)effect on aphid growth;(2)The hairpin RNAi vector of plant and fungi was constructed in our laboratory and the vector p BHt2-CHSA-Intron into Agrobacterium mediated wheat Agrobacterium transformation(Agrobacterium tumefaciens mediated transformation,ATMT),analyzes the Take-all gentisate dioxygenase gene(Gdo)fragments of RNAi on the growth of take-all.The main research contents and conclusions are as follows:1.Analysis on RNAi effect of aquaporin gene(Wsa)in wheat aphid:The success of the aquaporin gene of aphids(Wsa)connected to the BSMV vector and get the recombinant plasmid.Statistical analysis of spike Wang 126,Xinong 979,China spring 3 wheat varieties were inoculated with BSMV virus vector on wheat aphid reproduction number effect.The results showed that Chinese spring had the most significant inhibitory effect on wheat aphid.China spring on s.avenae growth rate has dropped68.60%,Xinong 979 on s.avenae proliferation rate has dropped 60.00%,suiwang126 on s.avenae proliferation rate has dropped 60.45% at twenty-fifth days.The growthrate of aphids in Chinese spring wheat was counted at thirtieth days,and the growth rate was reduced to about 30.10%.2.The take all of wheat gentisate dioxygenase gene(Gdo)RNAi effect analysis:Take all of wheat gentianic acid dioxygenase gene(Gdo)vector of RNA interference,a new carrier is conducive to connect the expression vector.Through the method of inverting connection,the same sequence of two foreign DNA fragments were successfully added to the new interference vector p BHt2-CHSA-Intron,use the method of ATMT,and the RNA interference could be produced.The carrier may be in fungi in vivo validation of gene interference effect,obvious effect by enzyme connection into the plant RNA interference vector p FGC5941,then transformation the plant can provides great convenience for the study of plant resistance to fungal transformation.Through t test comparison co-culture medium take all disease and non transformed take-all colony diameter growth has significant difference(t=0.037<0.5)in hygromycin resistant 100?g/m L B(Hyb).Compare the diameter of candidate transformants and non transformant that growth in non resistance PDA,through the t test,there is 1 candidate transformant reached a significant level.There are great differences between different candidate transformants,which may be related with the T-DNA insertion take-all genomic DNA sites.The diameter growth rate of the non transformants was faster than that of the transformants,and the growth of the candidate transformants was inhibited. |
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