| micro RNAs are a kind of signle strand nucleic acid, length of which vary from 19 to 24 nt. micro RNAs have the ability to slice, methylate and/or bind to cap structure of mRNA to repress target gene expression by recognizing uncompletely complimentary site. In the pathway of miRNA regulation network, some important factors such as AGO family, DCL1 and HEN1 et al., play important roles in developmental regulation of plant development. AGO2, one of the core elements of RNA induced silencing complex(RISC), is regulated by miR403. However, the function of miR403 and AGO2 is still unkown in tomato. Therefore, sequence of miR403 was obtained on the basis of bio-informatics analysis and miR403-function gained transgenic plants were employed to observe the phenotype and analysis expression of its target gene in tomato. The main results obtained in this study are as follows:1. According to the mature sequence of miR403(MI0001072) in Arabidopsis, the potential sequence of mature SlmiR403(S14362631) were found. By blasting with genomic sequence data of tomato with lower E value, the potential loci of miR403 was found. The potential sequence of pre-miR403 was analyzed by RNA structure software and the precusor of Sly-miR403 was found.2. SlAGO2 was comfirmed to be the target of miR403. To verify the biological function of miR403, 5’ RACE mapping was used to detect the cleaving site of AGO2 which proved the function of miR403 in tomato via Sl AGO2; meanwhile, the expression pattern of Sly-miR403 were detected as well in tomato.3. According to the sequence of Sly-pre-miR403, gene specific primers were designed to amplify the precusor of Sly-pre-miR403, and the amplified fragments were then cloned to pBi121, named as pBi121-miR403. According to the mature sequence of Sly-miR403, miR403-sponge fragments were designed and synthesized by gene synthesis company, then the miR403-sponge were constructed to pBi121, so named as pBi121-miR403-sponge. Transgenic plant of pBi121-miR403 and pBi121-miR403-sponge were obtained via Agrobacterium based transgenic method.4. Transgenic plants were observed, and the defects were described as delay of floral timing, leaf abnormality and ABA resistance during germination. Moreover, shooting abnormality was also observed in miR403-function gained transgenic plants.5. Target of miR403(SlAGO2) was found to be down-regulated. At the same time, SlAGO1 A and SlAGO1 B were up-regulated. Furthermore, the downstream miR156, miR159 and miR394 were over-expressed as the consequences of SlAGO1A/SlAGO1 B accumulation, which eventually affected the phenotype of the transgenic plants.In this study, pre-miR403 and its target gene AGO2 were indentified and isolated, and transgenic plants were obtained to analyze the function of miR403 and AGO2 in tomato,the mRNA of SlAGO2 was downregulated while SlAGO1 s were up-regulated in the miR403 over-expressing transgenic plants. Futhermore, the down-stream miR156, miR319 and miR159 were influenced which alter the phenotype in miR403-OX transgenic plants. This study will contribute to the further study on the the regulating function of miR403 in tomato and comprehention of the complex mechanism of miRNA regulation in plants. |
