The Potential Regulation Of Plant Small RNA On Botrytis Cinerea Gene Expression

Plant endogenous sRNAs(small RNAs)are important for regulating of gene expression and have a variety of biological functions by activating and inhibiting the expression of specific genes,especially in the regulatory mechanism of host-pathogen interactions in plants.ta-siRNA(Trans-acting RNA)is a class of sRNAs which are generally produced by 22 bp microRNAs through targeting homologous gene and then cut by DCL4(Dicer-like 4).Based on the difference of the miRNAs target sites,TAS genes can produce a range of different ta-siRNAs.Subsequently,one strand of the double stranded ta-siRNAs combines with the AGO(Argonaute Protein)and forms RISC(RNA-induced silencing complex)which degrades target genes finally.Recently,the researchers developed artificial sRNAs to suppress the expression of one or more genes in virus,fungus and even insect,based on the mechanism of natural sRNAs synthesis and function.Fungus can cause many seriously harmful and widely distributed plant diseases.Fungal diseases are the most harmful of the plant diseases and account for about 70%-80%.The published study showed that the sRNAs in Botrytis cinerea could enter the host plants and attack their immune pathways.In this study,we use Solanaceae plants and Botrytis cinerea interaction systerm as our research subject.We tried to investigate whether the plant endogenous sRNAs can also enter the Botrytis cinerea during the host-pathogen interaction and regulate the Botrytis cinereous gene expression.In addition,we also tried to obtain transgenic plants which can resist fungal diseases through the ata-siRNAs(artificial ta-siRNAs)expression.The main research results are as follow:1.We chose different types of sRNAs related to disease resistance in Solanaceae plants.Constructed into the vector pNAH-OGG,carrying GFP maker protein,use their reverse complementary sequences as the target anchor sites.Through the PEG-mediated protoplast transformation method,we inserted each reconstructed pNAH-OGG vectors into the B05.10(Botrytis cinerea 05.10)genome.We obtained the positive transformed Botrytis cinerea lines by detecting the green fluorescent and researching the characteristics on molecular and protein level.Made the foundation for the study of Solanaceae plants and Botrytis cinerea interaction.2.It showed that the cultivated tobacco SumsunNN could get the pathological characteristics after Botrytis cinerea infection.Otherwise,we explored the best stage of infection and made the foundation for the study of whether the transgenic tobacco can resist to the diseases.3.Obtained the sequences information of important virulence genes for Botrytis cinerea.Based on these sequences information,we constructed the vectors pH7LIC1.0-TAS and pAMIRgfp(control is pAMIR54)which can express ta-siRNAs targeting Botrytis cinerea genes.Conformed the expression of ata-siRNAs by transient expression;pH7LIC1.0-TAS and pAMIRgfp were transformed into the tobacco plant and we obtained the T0 generation of transgenic tobacco.T0 generation was hyg(hygromycin)ridized to produce F1 generation;We validated by Northern Blot experiment that F1 generation could express ata-siRNAs;Then infected F1 generation by Botrytis cinerea.According to the disease spot size we found that the F1 generation could reduce the degree of disease primarily.