The Establishment And Application Of Over-expression System Of Small RNAs Induced By Tomato Yellow Leaf Curl China Virus

MicroRNAs(miRNAs)and trans-acting siRNAs(tasiRNAs)of small RNAs play important roles in plant growth and development,stress response and other physiological and biochemical process.Inrecent years,many small RNAs were known by high-throughput sequencing.However,studying the function of miRNAs through expressing artificial miRNAs(amiRNAs)using transgenic method is laborious and time consuming.In this study,the process of the construction of Tomato yellow leaf curl China virus(TYLCCNV)and the amplification of amiRNAs was simplified.Using athmiR319 backbone,the over-expression system of small RNAs was successfully established in Nicotiana benthamiana.Moreover,the over-expression system of small RNAs was optimized,and the athmiR390 backbone was chosen to be used in further research.The main results were as follows:1.pGEM-1.7A and pBinPLUS-2m?-Sub,separately containing 1.7A and 2m? of TYLCCNV,were integrated to one vector pBinPLUS-1.7A-2m?-Sub(named p1.7A+2m?).The target fragment can be constructed to the vector by one step of double enzyme digestion,which greatly simplified the process of constructing vector.Moreover,the high infection efficiency of the vector was proved by TYLCCNV-PDS and TYLCCNV-35S.2.Designing amiRPDS targeting report gene PDS and using athmiR319 backbone,TYLCCNV-amiRPDS(319L)was constructed.Nicotiana benthamiana plants were infected by Agrobacterium containing TYLCCNV-amiRPDS.Two to three weeks after infection,leaf vein in Nicotiana benthamiana showed discontinuous light bleaching phenomenon,verifying the TYLCCNV can be used to establish over-expression system of small RNAs.3.The original miRNA in miRNA backbone was replaced by amRNNA through designing primers,which changed the amplification process of amiRNA from three rounds PCR to one round PCR,facilitating the amplification process of amiRNA tremendously.4.Selecting athmiR319,slymiR159 and athmiR390 backbone,TYLCCNV-amiRPDS(319),TYLCCNV-amiRPDS(159)and TYLCCNV-amiRPDS(390)containing three different amiRPDS backbone were respectively constructed.Although amiRPDS could be overexpressed by all three vectors,which made the veins produce light bleaching phenomenon lasting more than one month,the analysis by RT-qPCR found miR390 backbone generated more amiRPDS,so miR390 backbone was choose to be used in further research.5.Designing amiRSu and amiRPCNA targeting the endogenous gene Su and PCNA respectively,TYLCCNV-amiRSu and TYLCCNV-amiPCNA were constructed.After Agrobacterium infection,Nicotiana benthamiana plants seperately showed yellow leaf veins and arrest growth apical leaves phenomenon,and the relative expression of Su and PCNA were decresed significantly,suggesting the TYLCCNV-based over-expression system of small RNAs can be used to silence endogenous gene effectively.6.Nicotiana benthamiana plants infected by Agrobacterium containing TYLCCNV-amiR156 vector showed curly leaves.Using RT-qPCR,miR156 was upregulated 30 times,and the two target genes TC17947 and TC24007 expression was significantly reduced.In addition,Nicotiana benthamiana plants infected by Agrobacterium containing TYLCCNV-amiR396 became short.By RT-qPCR analysis,miR396 was increased 2 times,and the target gene GRF3 was dramatically downregulated.These results demonstrated that TYLCCNV-based over-expression system of small RNAs can be used to effectively study the function of endogenous miRNAs.7.Selecting 5D8(+)of TAS3 in Arabidopsis,TYLCCNV-5'D8(+)was constructed.After Agrobacterium infection,the leaves of Nicotiana benthamiana showed curly phenomenon and deflected to stem.Using RT-qPCR,mature 5'D8(+)was detected,and the two target genes ARF3 and ARF4 was significantly decreased.These results implied that TYLCCNV-based over-expression system of small RNAs can be used to effectively explore the function of endogenous tasiRNAs.