Font Size: a A A

Cloning And Characterization Of Microrna And Other Related Small RNA From Rice

Posted on:2006-11-08Degree:DoctorType:Dissertation
Country:ChinaCandidate:Y Z LuFull Text:PDF
GTID:1100360182465759Subject:Developmental Biology
Abstract/Summary:PDF Full Text Request
microRNAs, as an important regulator in eukaryote organism, have been profoundly studied in recent years in plants and animals. Many study work had done to pove that microRNA have abroad roles in the development of organism involving evry aspect of cell physical activity. The cloning and characterization of microRNA is still a hot academic domain for the cause of the identified microRNA be far from saturation. Arabidopsis Thaliana as a model plant had been sequenced in 2000 (The Arabidopsis Genome Initiative 2000) . However, the Arabidopsis Thaliana, as a dicotyledonous plant, the sense of only its microRNA is limited in plant. The oryza sativa, as a monocotyledonous plant and an important food crop, had been completed to sequence the draft of genome in 2002 (yu et al. 2002). This is the foundation for us to clone and characterization of microRNA frome oryza sativa. The cloning and characterization of microRNA in oryza sativa must have a great academic and realistic meaning. In addition, there are other endogenetic small RNA such as siRNA in organism. siRNA had been proved to regulate their target by the way of cleavage.Therefore, using the traditional methods used by other groups for cloning microRNA, we have cloned 178 sequence of tiny RNAs in oryza sativa. Among these tiny RNA, we have identified 9 microRNAs including miR167 and miR171 which were also been found in Arabidopsis Thaliana. The following is the summarization of our results.(1).The loci of all 178 sequence of tiny RNAs in genome is lie in all 12 chromosomes of rice. Among these tiny RNAs, 129 sequences belong to single-copy in rice genome and the other 49 sequences belong to multi-copy. The number of total loci of all tiny RNAs add up to726. The number of sequences in intergenic region is 130 (70%), in transposon is 6 (3%), antisense to gene is 28 (15%), sense to gene is 22(12%). The length of all tiny RNAs had a various range from 8 to 30 nucleotides. Whereas the size of majority tiny RNAs centralize in range from 20 to 25 nucleotides (the number of these RNAs is about 74% of all tiny RNAs). We list all genes which have not more than 3 nucleotides to a given RNA as their targets in Table 1 and we gained 86 unique genes belong to 42 tiny RNAs.(2).we have analyzed whether all tiny RNA could form stem-loop when joined with flanking sequence in genome through m-fold program and the results showed that 12 tiny RNAs have this property (exception the tiny RNAs which was in transposon. partial of their loci in rice genome can form stem-loop when join with flanking genomic sequence). Further more, the 9 of the 12 tiny RNAs, including the miR167 and miR171 which were found in Arabidopsis Thaliana, could be detected by Northern Blots at least one tissue. The miR167 we cloned in rice is one nucleotide "G" longer than what had been found in Arabidopsi Tlialiana. In addition, all these 9 tiny RNA have a more higher frequency of been sequenced than other tiny RNAs (exception the 6 RNAs belong to transposon). We list the 9 tiny RNAs as microRNA in Table 2. Among these 9 microRNA, 7 microRNA as newly identified microRNA in rice and all loci of the 7microRNA are single copy in rice genome. As found in Arabidopsi Thaliana (Llave et al., 2002a; Reinhart et al., 2002) , the length of precursors of partial microRNAs in rice were longer than that of animals. The first nucleotide of 5 microRNAs of the 9 microRNAs is the nucleotide "U". This trend is similarly to what had been reported in animals (Lee and Ambros, 2001; Lau et al., 2001; Lagos-Quintana et al., 2001) .(3). All the identified 9 microRNAs could steadily express at least one tissue of rice. miRl, miR2, miR5, miR6 and miR171 can express in all tested tissue; whereas the expression of miR3, miR4, and miR167 shown a specific pattern. For example, miR167 only slightly expressed in seedling stage and miR171 expressed at a higher intensity in flower than other tissue. The similar expression pattern of miR171 between rice and A. thaliana is consistent with its roles that was been proved in A. thaliana (Llave et al., 2002a Llave et al., 2002b;Rhoades et al. 2002) . The same expression pattern of same microRNA indicated that miR171 may play important similar roles in flowering plant.(4).The identification of miR167 and miR171 in rice may suggest the conservation of partial microRNAs between dicotyledon and monocotyledon. In addition, we also found that a partial fragment of microRl which contained 16 nt could be searched in the genome of Metasequoia glyptosiroboides and m-fold program revealed the partial fragment could form a putative precursor when joined with flanking sequence. Further more, miR4 could also meet matches with only two nucleotide mismatch in Triticum aestivum and could form back-fold with flanking sequence and the sequence of precursor of microR4 in Triticum aestivum was in the joint point between intron and extron of gene MRP-2, of which function was associated with multidrug resistance protein. Further analysis revealed that the first the nucleotide of MRP-2 cDNA was exactly in the middle of the microRNA4 in Triticum aestivum, indicating that microRNA4 maybe participate in the cleavage of MRP-2 mRNA precursor. This is for the cause of the proved mechanism that the loci of microRNA or siRNA cleavage the target gene are exactly in the middle of microRNA or siRNA (Llave et al, 2002b;Tang et al, 2002) .miR4 is a conservational microRNA in monocotyledonous plant of oryza sativa and Triticum aestivum. In addition, miR5 and its precursor could be found matches in Nipponbare strain, as similar case, it has a different nucleotide. Nipponbare is a very intimate strain to 9311. One different nucleotide of same microRNA between two stain suggest that the conservation is relative. Aberrance is existing in conservation at same time. (5).We list all genes which have not more than 3 nucleotide mismatches to microRNA as their targets. Through consulting the genome, the results indicted that four known gene families and two unknown protein families which comprise 16 unique genes presumably be the potential targets to 6 microRNAs of the 9 microRNAs (Table2.2). By the method of our predication, the target gene of miR171 in rice is also scarerow-like family as its targets in A. arabidopsis. The member of scarerow-like family is 9 of which 7 is perfect complementarity to miR171 in oryza sativa. The conservation of the same one microRNA and its targets in different plants suggest the miR171 play an important role in the controlling flowering. Thepotential target of miR2 in oryza sativa is similarly to Arabidopsis thaliana trehalose-6-phosphate phosphatase and show a perfect complementarity to miR2. The potential targets of miR4 are three. The prediction function of two is not very close, one is predicted to be putative Vesicle-associated membrane protein and the other is predicted to be putative ABC transporter-like protein. The predicted target of miRl has 3 mismatches nucleotide to miRl and its function is supposed to belong to a putative Terpene synthase family. The function of target of miR6 and miR167 were unknown. It seemed that the function of known genes involved a various range.In short, the 9 identified RNAs and the other 169 related tiny RNAs we cloned is an important supplement for oryza sativa and is foundation for revealing the function of these microRNAs.
Keywords/Search Tags:microRNAs, m—fold, rice
PDF Full Text Request
Related items