Construction Of CircRNA Overexpression Vector And Establishment Of Function Analysis System Based On CircR5998

In eukaryotic cells,apart from messenger RNA(mRNA)which codes for proteins,there are a wide variety of other RNAs.Although these RNAs do not code for proteins,they play an important role.Circular RNA(circRNA)is one of them.The structure of circRNA is different from the traditional linear RNA,it presents a closed loop and lacks complete free radical terminal.So it does not have the 5' cap and the 3' ploy(A)tail.Thus it has become a research hotspot in the scientific community.However,no system has been established to accurately express plant circRNA.Now,most of the researches remain in overexpression of linear structure,which depends on the circularization of linear structure.The low expression efficiency and poor accuracy of this system limit our understanding of the function and mechanism of plant circRNA.Therefore,it is very important to study the system of efficient expression of circRNA.In this project,we took Arabidopsis thaliana circR5998 as the research object,and a circRNA expression system was constructed by screening and verifying the circular region and surrounding structure,which can accurately,efficiently express the target gene.Based on this system,circR5998 was expressed in Nicotiana benthamiana leaves and its disease related functions were studied.The specific research content is as follows:1.An accurate and efficient circRNA expression vector was constructed based on the exon circularization principle.In this project,we regarded circR5998 as the research object,adopt the reverse complementary introns sequence,flanking introns sequence and both the reverse complementary sequence of introns and flanking introns mediated methods to construct circRNA expression vector,after the agrobacterium tumefaciens-mediated transformation with circRNA divergent primers by RT-PCR detection circRNA of circularization in Nicotiana benthamiana.We found that although the reverse complementary intron sequence can effectively promote the circularization,it will affect the accuracy of circularization.At the same time,it was also found that the flanking method supplemented by the reverse complementary introns sequence was the most accurate and complete strategy to promote circularization,and the longer reverse complementary sequence was more effective.The establishment of this system can better promote the expression of circRNA in vivo accurately,which is of great help to the further study on the function of plant circRNA.2.A new system for function analysis of circRNA was established.This project broke away from the traditional construction of transgenic plants.A new expression system was used to express the circRNA so that the function of the circRNA could be detected in the agrobacterium tumefaciens-mediated N.benthamiana transient expression system.In the experiment,we took circR5998 as the research object,used N.benthamiana to express circR5998 instantaneously,then inoculated N.benthamiana leaves with DC3000,and further identified the biological function of circR5998 by detecting some immune response related indicators,such as the expression level of disease resistance genes,the accumulation of reactive oxygen species,callose deposition,Ion leakage and pathogenicity analysis.This method can be used as a new system to study the function of circRNA,and it is also very helpful for us to study the function of circRNA in transgenic plants.3.Preliminary identification of circR5998 inhibition of the defensive response of DC3000 to N.benthamiana and its molecular mechanism.In this study,the use of agrobacterium tumefaciens-mediated N.benthamiana transient expression system as a new detection method was found that under DC3000 inoculation,N.benthamiana leaves expressing circR5998 were more susceptible to disease than those in empty vector,the expression level of disease resistance genes were relatively lower,and the accumulation of ROS and callose was relatively lower.Further study on the internal molecular mechanism revealed that the transcriptional linear-P5CS1,like circR5998,negatively regulated of the immune response of DC3000 to N.benthamiana.In addition,the results of qRT-PCR showed that circR5998 promoted the expression of linear-P5CS1,while linear-P5CS1 in turn inhibited the expression of circR5998,indicating that linear-P5CS1 could participate in the negative regulation of immune response of DC3000 to N.benthamiana by circR5998.But the exact biological mechanism remains to be determined.