Establishment Of The Random Overexpression System For Plant Endogenous Small RNA

Small RNA is a category of conserved growth and development regulators in eukaryotes,which plays important roles in controlling the organogenesis,polarity formation,cell proliferation,tissue differentiation and resistance to environmental stresses of plants.As the first species of flowering plant whose genome has been well sequenced,Arabidopsis.thaliana has become an ideal model for the developmental and molecular studies for dicot plant due to its compact genome,short growth period,easy cultivation and genetic manipulation.Arabidopsis contains large amount of conserved and specific small RNA species,and exploring their biological functions is important for the understanding of plant development and the pracitice of molecular breeding.Although it is possible to overexpress small RNAs with artificial microRNA or synthetic small RNA techniques,only one small RNA species could be studied by either approach,which largely restricts the functional study and breeding application of the huge quantity of small RNAs in plant.The goal of this thesis is to establish a new genomic expression system,through which plant endogenous small RNAs could be randomly overexpressed;by constructing small RNA library,the biological functions and molecular mechanism of will be explored.The main results of this thesis including:(1)On the basis of the original tasiRNA expression vector pRDE-0 in our laboratory,the 35 S promoter in the pRDE-0 was replaced with maize Ubi promoter to allow the general application in both dicot and monocot plants.c(2)Three reporter constructs(pRDE-Ch42,pRDE-Trich,pRDE-Ft)of this system were obtained by synthesizing the complementary oligoes for each reporter gene,followed by annealing and ligating with the vector pRDE.The constructs were transformed into Arabidopsis thaliana by agrobacteria mediated flower dipping.Positive transgenic lines were obtained by screening at T1 generation.The technical system was proofed to be successful by observing the phenotype quantifing the target gene expressions.(3)Total RNA was extracted from Arabidopsis,from which small RNAs were purified and the small RNA random overexpression library was constructed.Deep sequencing results showed that the library contain huge amount of data and cover most of the small RNA loci in Arabidopsis genome,indicating the quality of the library is high enough for plant transformation.(4)The plasmid library was transformed into Arabidopsis by agribacteria mediated flowing dipping method.The transgenic plants were obtained by sreening,and the inserted small RNAs were identified for some the transgenic lines.In this study,a new method for random overexpression of plant small RNAs was established by constructing the vector,validating of the system by reporters,and randomly overexpressed the native small RNAs in Arabidopsis.The system provided news ideas for functional study of plant small RNAs and molecular breeding practice.