Study Of Artificial MicroRNA Silencing FMO_GS-OX Gene Family

Artificial microRNA（amiRNA） is a gene silencing technology based on the theory of synthesis, process and target gene silencing of endogenesis microRNA. The basic principle of amiRNA is using endogenous microRNA precursor as a basic skeleton, microRNA/microRNA*is then replaced by amiRNA/amiRNA* which have the same length with microRNA/microRNA*, then the amiRNA precursor is connected with a plant expression vector and transfered into plants to make the amiRNA express and silence the target genes in transgenic plant. Glucosinolates（GSLs） are nitrogen and sulphur-rich secondary metabolites present in the order Brassicales. The hydrolysis products of GSLs are famous for cancer-preventive activity,There are crucial glucosinolate side chain modification enzymes, Flavin-containing monooxygenase(FMO_GS-OX), which catalyse sulfur-oxidizing reaction of methylthioalkyl GSLs（MT GSLs）changing into methylsulfinylalkyl GSLs（MS GSLs）in biosynthesis process of glucosinolates.There are five members of FMO_GS-OXfamily encoding FMOs were identified in Arabidopsis thaliana. They are At1g65860(FMO_GS-OX1), At1g62540(FMO_GS-OX2), At1g62560（FMOGSOX3）, At1g62570(FMO_GS-OX4) and At1g12140(FMO_GS-OX5). FMO_GS-OX1-4catalyse all kinds of MT GSLs changing into MS GSLs, but FMO_GS-OX5 catalyses only long-chain MT GSLs. it is widely believed that MS GSLs have higher efficiency of anti-microbial pathogens and cancerpreventive activities. Therefore, the study of FMO_GS-OX family influencing profile and contents of MS GSLs is important to the study of plant insect resistant and anti-microbial pathogens.In the previous studies,we used amiRNA technology to silence the important gene in synthetic route of aliphatic glucosinolates and obtained single-gene mutants successfully. In this study, we aimed to explore the technology of amiRNA silencing multi-gene, then make FMOGSOX1-4as the target genes(Based on the narrow limitation of design parameter, we excluded FMO_GS-OX5 which catalyses only long-chain MT GSLs)to design a amiRNA aim to silence FMO_GS-OX1-4and obtain Arabidopsis mutants with significant low content of MS GSLs to provide reliable material basis for the future study and establish the technology of amiRNA silencing multi-gene efficiently.The results show that the expression of FMO_GS-OX1-4 are inhibited in different levels in35S::amiRNA Arabidopsis transgenic plants, the expression of FMO_GS-OX1 and FMO_GS-OX4 downregulated obviously, and the expression of FMO_GS-OX2 and FMO_GS-OX3 down-regulated unobvious.These illustrate that the amiRNA we designed can silence FMO_GS-OX1-4efficiently, but thesilencing efficiency vary with target genes. Through analyzing the vary of profile and content of glucosinolates, we found that there are obvious difference between transgenic and wild type plants: compare with wild type, the transgenic plants have obvious low ratio of 4MS/（4MS+4MT）, which mean relative contents of 4MS GSLs are reduced in transgenic plants, and absolute contents of 4MS GSLs are also reduced in transgenic plants.In total, our study verifies that amiRNA can simultaneously silence multi-genes efficiently.We made amiRNA overexpression, then silencing four target genes FMO_GS-OX1-4 on expression level successfully, and obtained transgenic plants with remarkable phenotype so that provided effective gene silencing technology for the study of biological process controled by multi-genes with functional redundancy.