Analysis Of MiRNA By High-throughput Sequencing And Selecting Of Reference Genes For Two Populations Of Aphelenchoides Besseyi

Aphelenchoides besseyiis one of the most important pathogenic nematodes that endanger rice,mainly distributed in most of the world's rice growing areas.In C hina,it widely distributed amnong the 24 provinces,which occursin the Bohai Sea rice area currently.It not only harms rice,but also harms strawberries,chrysanthemum and other ornamental plants and cash crops.It caused major economical losses in our country.At present,the prevention and control of A.besseyi is still mainly usingnematocides,which has brought some negative impacts on the environment and human health.With the development of biotechnology and genetic engineering technology,molecular biology method has become an important and new method to study plant nematode control.In this paper,N10 populations of rice as the host,and the S24 population of the strawberry as host were analysis of micro RNA by high-throughput sequencing.The different expressed mi RN As of the two host populations were studied,and the four highexpression of mi RN As(mi R-1,mi R-34,mi R-71 and let-7-5p)in four life stages of the two populations was analyzed.Bioinformatics analysis was performed on the sequencing results,and the function of some mi RN As of A.besseyi was studied by q PCR,in situ hybridization and mi RN A mimics.The main research results are as follows:1.The mi RNA sequencing results and mi RN A differential expression profiles of A.besseyiwere obtained.810 and 733 known mi RNAs were obtained in N10 and S24 libraries,among which 413 were differentialmi RN As,254 mi RNAs wereup-regulated in N10 library,and 159 mi RN As were up-regulated in S24 library.2.The expression of 15 differentially expressed mi RNAs in N10 and S24 populations was verified by q PCR.mi R-1,mi R-34,mi R-71 and let-7-5p were analyzed in four life stages of N10 and S24 by using q PCR.The results showed that the expression of 15 different mi RN As was consistent with the sequencing results.The mi RNAs of mi R-1(mi R-1,mi R-34,mi R-71 and let-7-5p)were significantly different among the different life stages in the two populations,and the relative expression of mi R-34 in the eggs lower than other life stages,which was the largest multiple of the difference.3.The position expression of mi R-34 in A.besseyiwas studied by mi RN A in situ hybridization.It was found that it was mainly located in near the gonad of the male and female,and it was clearly visible.4.Using the transcriptome data of the A.besseyi,the four reference fragments(actin,GAPDH,UBC and ?-tubulin)were cloned.The stability of actin,GAPDH,UBC and ?-tubulinwere evaluated under three experimental conditions,and acquires the more stable reference gene UBC,which lays a foundation for the further study on the expression of certain m RNA genes of A.besseyi.The target genes of mi RNAs of Aegilops tauschii were analyzed by GO and KEGG annotation and enrichment5.The target genes of mi RNAs of A.besseyi were studied were analyzed by GO and KEGG annotation and enrichment.It was found that the target genes of differentialmi RN As were mainly expressed in growth,development,cell differentiation,catalytic transportetc..The target genes of mi R-1,mi R-34,mi R-71 and let-7-5pwere mainly enriched in the functions of signal transduction pathway,binding ligation and enzyme catalysis.6.Using of immersion method to detecte the effect of expression of target mi RN Aby mi RN A mimics treatment for plant parasitic nematode.The expression level of mi R-34 in treatment groups was increased to highest at 24 hourscompared with the control and the clear water group,while the difference between the co ntrol group and the clear water group was not significant,indicating that the mi RN A mimics played a role in increasing the expression of endogenous mi R-34 expression.In this paper,we obtained the mi RN A library and expression profile of A.besseyiand the optimal reference gene needed for the quantitative analysis of its m RN A genes.It is understood that some mi RNAs may have biological function,which laid a foundation for further studyon the mechanism and function of mi RN As in A.besseyi.It has great influnces to explorethe new method of controlling A.besseyi.