The Application Of Artificial MicroRNAs Mediated Gene Silencing In Phytophthora Sojae

Phytophthora sojae belongs to oomycete which has a close genetic relationship with algae but distant with fungi.Disease caused by Phytophthora sojae bring about 10-20 billion dollars in economic losses to the global soybean production each year.Therefore,understanding the molecular mechanism of asexual and sexual production in Phytophthora sojae is particularly important for disease prevention and control.PEG-mediated protoplast transformation is an important method to study the molecular mechanism of Phytophthora sojae.The expression of exogenous gene in Phytophthora sojae result to gene silencing.However,this method is not mature because of the long transformation cycle,low silencing efficiency,meanwhile,integration of exogenous sequence into the genome is likely to cause insertional mutagenesis of other genes near the target gene.Therefore,the optimization of Phytophthora sojae transformation method is always a topic for researchers.In the past decades,RNA interference(RNAi)has been a broad and effective tool to down-regulated gene expression in eukaryotic(Hannon,2002).Micro RNA(miRNA)are a class of non-coding single-stranded RNA about 22 nucleotides encoded by endogenous genes,and it involved in down-regulation of genes after the transcription in animals and plants.Using the natural microRNA skeleton as a precursor,then the small artificial interfering RNA fragments merged to the precursor,that was a complete structure of artificial microRNA(amiRNA),it regulates the transcription level of target genes with high specificity and efficiency.The method has been successfully applied in Arabidopsis,rice and algae.In this study,we using PsGK5,PsCDC14,PsAVR3b,PsAVR1d,PsIMPA as the target genes and designed its amiRNA silencing vector,then we explored the effect of gene silence on Phytophthora sojae.Use the natural miRNA in Phytophthora sojae as precursor to construct amiRNA vectors.According to the design principle of artificial microRNA,we select the 5 ’end sequence of the target gene to avoid the formation of secondary structure.The sequence selected subject to ACG(N)18T feature that the 5 ’end of three bases must be ACG,3’ end base must be T,18 bases in the middle can be any sequence.Thus,the same target gene may have a number of different sequence selection.In theory,the worse amiRNA’s stability is,the more likely to form a complex with RISC,silencing efficiency is higher.We choose the sequence with low free energy as target sequence.Four primers were designed when we input the nucleotide sequence ACG(N)18T into the program amiRNA primer creator.Based on the design principles above,we constructed 8 amiRNA silencing vectors for verify its silencing efficiency,the 8 amiRNA silencing vectors are pTOR::GK5 ami-1,pTOR::GK5ami-2,pTOR::GK5ami1-2,pTOR::CDC14ami-1,pTOR::CDC14 ami-2,pTOR::CDC14 ami1-2,pTOR::AVR3b ami and pTOR::AVR1d ami.The pTOR::PsIMPA ami was used for comparing the efficiency of gene silencing mediated by stable silencing and amiRNA.Verification of gene silencing efficiency mediated by amiRNA We transformed amiRNA vectors into the protoplasts of Phytophthora sojae by PEG-CaCl2-mediated protoplast transformation,and we selected 28 transformants randomly for each vectors to verificate it’s silent efficiency and analysis the phenotype of silent mutants.We found that,the efficiency of gene silencing mediated by amiRNA is 40%to 70%;and,By the study of PsIMPA we found that the proportion of silent mutants in all transformants is higher than traditional stable transformation.Silence mutants accounted for about 25%.By analysis the phenotype of PsGK5,PsCDC14,PsAVR3b,PsAVR1d silent mutants,we found the comeuppance phenotype variation in mutants.