Discovery Of MicroRNAs In Wheat (Triticum Aestivum L.) And Brachypodium Distachyon L. And Functional Analysis Of Small RNA Biogenesis-related Gene RDR1 In Tomato (Lycopersicum Esculentum)

Small RNAs are 20-30 nucleotides long single-strand non-coding RNAs. They represent a new mechanism of gene regulation and play important roles in plant growth and development, responses to different environmental stresses as well as genome stability. High-throughput sequencing technologies, expression pattern and function analysis are important approaches to discover key small RNAs in crops and model plants.In this study, to discover and analyze important small RNAs in wheat (Triticum aestivum L.), we sequenced the wheat and Brachypodium distachyon small RNAs. In addition, we characterized the RNAi transgenic plants of LeRDR1 gene and laid foundations for studying its functions in the biogenesis of small RNAs in tomato (Lycopersicum esculentum). The main results are as following:⑴To discover miRNAs related to important traits in wheat, we constructed a small RNA library using the mixture tissues of root, shoot, leaf and young spike of bread wheat cv. Chinese Spring. A total of 457,165 unique small RNAs were obtained. It is shown that there are 39 putative novel wheat miRNAs and 70 conserved wheat miRNAs, which belong to 33 and 25 miRNA families, respectively. The expression analysis of these miRNAs indicated that some miRNAs have the tissue-specific expression pattern, for example, the tae-miR2015 expressed in root tissue exclusively. The target prediction results showed that wheat miRNAs'targets include transcriptional factors and homologoues of disease-resistant genes. Interestingly, of the three members of tae-miR2009 family, the targets of tae-miR2009-a and tae-miR2009-c are homologic with disease-resistant genes in rice, and their cleaving abilities are confirmed by 5′RACE. It is shown that wheat small RNAs may play important roles in responses to different stresses.⑵To mine important small RNAs in Brachyopdium, the reproductive tissues and vegetative tissues were used respectively to construct small RNA libraries: BdR and BdV. We obtained a total of 283,876 and 362,038 non-redundant sequences, respectively. Using the available EST sequences, we discovered 12 novel Brachypodium miRNAs (9 miRNA families) and 94 conserved miRNAs (28 miRNA families). The expression pattern, target prediction and result validation by RLM-5′RACE of some of these miRNAs were conducted. Comparative analysis of small RNA copy numbers between the two libraries showed that preferred miRNA members were usually used in different developmental stages. In addition, certain miRNAs expressed different patterns in different species. Compare with 24 nt small RNAs, 21 nt small RNAs are more conserved among genomes of wheat, Buachypodium and rice (Oryza sativa).⑶To study functions of LeRDR1 gene in development and biogenesis of small RNA in tomato, we isolated the full-length cDNA and genomic DNA of LeRDR1 gene. Gene structure analysis showed that LeRDR1 gene includes four exons and three introns. Tissue-specific expression in tomato inflorescence indicated that LeRDR1 gene play an important role in tomato reproductive development. To further study its function, we developed RNAi transgenic plants, which showed very interesting phenotypes. The transgenic plants showed big sepals and aberrant ovules. Plants with severe phenotypes, showed immature ovule-like organs growing out of a fruit. Real-time PCR experiments indicated that some MADS-box genes were indeed up-regulated in inflorescence of transgenic plants. SLMBP3, TAGL11 and TAGL1 were up-regulated in the sepals of transgenic plants. SLMBP3, TAGL11, TAGL1 and SLMBP20 were up-regulated in petals, TM4 and TAGL1 were up-regulated in carpels, respectively. The expression changes of these genes in each whorl may be direct reasons for the phenotypes of transgenic plants. We will perform comparative analysis the small RNAs between transgenic and wild-type tomato to investigate the roles of LeRDR1 gene in tomato development and biogenesis of small RNAs in tomato.Our studies uncovered novel small RNAs in wheat and Brachypodium and preliminarily characterized the function of LeRDR1 gene, these work laid foundations for further studies of wheat and tomato small RNAs.