Study On Separation And Clone Of The MicroRNA(miRNA) In Tomato Fruit And Their Expression Levels In Tissue

MicroRNAs(miRNAs) are short 20-25 nucleotides RNA molecules that have been shown to regulate gene expressions in a variety of eukaryoutic systems.Many studied work had done to prove that microRNA have abroad roles in the development of organism involving evry aspect of cell physical activity .microRNAs have been widely studied as a developmental regulator of the eukaryoutic organism recent years. With the development of clone method and the bioinformatinal method, more and more miRNAs have been identified, the cloning and characterization of microRNA is still a hot academic domain for the cause of the identified microRNA be far from saturation. Arabiodopsis thalina is a molecular model plant, while the tomato is a model plant of the fruit maturation, so we can discover the miRNA that regulate the maturation of the fruit, then we can improve the quality of the tomato fruit (nutrition & smell), increase the output, make it be long conserved by gene engineering method, it also can be used in normal breeding as a molecular tag.In this study we use tomatoes'fruit as the experimental material, we used phenol/chloroform to extracted the total RNA. then used the polyglycol to get the low molecular weight RNA fraction (≦200nt),then we get 20~40bp small RNA used polyacrylamide gel. The small RNAs(20~40bp) were polyadenylated by poly(A) polymerase. A 5'RNA adaptor (5'-CGACUGGAGCGACACUGACAU GGACUGAAGGAGUAGAA-3') was ligated to poly(A)-tailed RNA. After RT-PCR, the PCR product was cloned into T vector, then choose the positive clones to sequence. After that we acquired 50 small RNAs, we searched their homology sequence in the tomato gene bank , got 4 candidate miRNAs, then we tested their secondary structure and did northern blot and RT-PCR to see their expression in the tomato fruit .at last we confirmed one of them is a new miRNA(5'-C UUCCUUCUUCAUCUUUCUCUC-3').In this study, we discovered one new miRNAs in tomato. these results establish good foundation to study the functions of the new miRNAs in the growth of the fruit in the future.