Identification And Exprestion Of Poncirus Trifoliata And Citrus Micrornas And Their Targets

Plant miRNA is widely distributed in the plant genome and is an endogenous non-coding regulatory RNA, which is a class of eukaryotic gene expression of negative regulatory factors at the post transcriptional level, acting mainly by repressing gene translation or degrading targeted mRNAs. miRNA plays an important role in controlling plant development, flowering timing, metabolism and stress response. At present, miRNA methods of separation and identification include experimental methods (genetic screening, direct cloning, high-throughput sequencing, northern blotting, qRT-PCR) and bioinformatic methods. Citrus is a perennial tree fruit. Trifoliate orange is even the most important plant in citurs molecular biology and genomics research areas. It is of significance to carry out identification of miRNAs and their target genes in economically important plants, so as to achieve effective control and use of important genes from epigenetics to explain the apparent phenomenon of life. More important is to achieve effective regulation of important genes with the purpose of using them.In this study, the experimental trifoliate orange,’Nules’,’Gongben’, and’Newhall’ were used as materials to predict miRNAs in citrus and its target genes by bioinformatics. The miRNA and the target gene expression in citrus were systematically and comprehensively studied by northern blotting, miR-RACE,5’-RACE, qRT-PCR, PPM-RACE and RLM-RACE, and high throughput sequencing. The major findings are as follows:1. We developed a comprehensive strategy for identifying new miRNA homologs by mining the repository of available citrus expressed sequence tags (ESTs). By adopting a range of filtering criteria, we identified a total of 38 potential miRNAs.9,5,9, and 15 miRNAs in Poncirus trifoliata (ptr-miRNAs), Citrus clementine (ccl-miRNAs), Citrus reticulata (crt-miRNAs), and Citrus sinensis (csi-miRNAs), respectively, were from more than 430,000 EST sequences in citrus. Using potential miRNA sequences, we conducted a further BLAST search of the mRNA database and found 10 potential target genes in these citrus species. Eight miRNAs were selected in order to verify their existence in citrus by northern blotting, and the functions of several miRNAs in miRNA-mediated gene regulation are experimentally suggested. It appears that all these miRNAs regulate the expression of their target genes by cleavage, which is the most common situation of gene regulation mediated by plant miRNAs.2. In this study, we report an efficient method to determine the precise sequences of computationally predicted microRNAs (miRNAs) that combines miRNA-enriched library preparation, two specific 5’and 3’miRNA RACE (miR-RACE) PCR reactions, and sequence-directed cloning, in which the most challenging step is the two precise gene specific primers designed for the two RACE reactions. Real-time PCR was used to analyze the expression of each miRNA. The results show that the efficiency of this newly developed method was validated using nine trifoliate orange miRNAs predicted computationally. miRNA-mediated mRNA cleavage by RLM-5’RACE and sequencing were carried out to validate the miRNAs detected. Quantitative analysis indicated these miRNAs have variable expression. miR-RACE can successfully be used to validate sequences of bioinformatically predicted miRNAs in grape and apple, with the same miR-RACE primers as trifoliate orange. The efficient and powerful approach developed herein can be successfully used to validate the sequences of miRNAs, especially the termini, which depict the complete miRNA sequence in the computationally predicted precursor.3. We used Solexa sequencing to discover new microRNAs in trifoliate orange (Poncirus trifoliata) which is an important rootstock of citrus. A total of 13,106,753 reads representing 4,876,395 distinct sequences were obtained from a short RNA library generated from small RNA extracted from P. trifoliata flower and fruit tissues. Based on sequence similarity and hairpin structure prediction, we found that 156,639 reads representing 63 sequences from 42 highly conserved miRNA families, have perfect matches to known miRNAs. We also identified 10 novel miRNA candidates whose precursors were all potentially generated from citrus ESTs. In addition, five miRNA* sequences were also sequenced. These sequences had not been earlier described in other plant species and accumulation of the 10 novel miRNAs were confirmed by qRT-PCR analysis. Potential target genes were predicted for most conserved and novel miRNAs. Moreover, four target genes including one encoding IRX12 copper ion binding/ oxidoreductase and three genes encoding NB-LRR disease resistance protein have been experimentally verified by detection of the miRNA-mediated mRNA cleavage in P. trifoliata.4. To study the expression of miRNAs and their targets during development and maturation of Poncirus trifoliata in relation to different tissue growth, we cloned 10 full-length target genes of 9 miRNAs from a cDNA library of different developmental stages trifoliate roots, stems, leaves and flowers by bioinformatics methods and RACE techniques. Sequence analyses reveal that these target genes all contained the recognition sites. Real time PCR and PPM-RACE and RLM-RACE were used to determine the tissue expression patterns of miRNA, their target genes and shear miRNAs target gene products (5’and 3’end) in the 15 different organs of from children trifoliate orange and adult leaves, respectively. The results show that the trifoliate orange miRNAs and its target genes existence relation to growth and decline in different developmental stages and different organs. High expression of miRNA and its target gene expression is often low, while the shear miRNA target gene expression is also a product of high; when the miRNA expression is often low, and its target gene expression is high, whereas the products of miRNA target gene expression of shear capacity is also low.5. In this study, precise sequences, especially terminal nucleotides of fifteen csi-miRNAs and nine crt-miRNAs were validated by miR-RACE. The expression of these fifteen miRNAs and nine crt-miRNAs could be detected in Citrus sinensis and Citrus reticulata young leaf, mature leaf, old leaf, young stem, mature stem, old stem, flower bud, half open flower, open flower, and different developing fruits (15,45,75,105 and 145 days after full bloom, DAFB) by quantitative RT-PCR (qRT-PCR) and some of them showed tissue-specific expression. In addition, potential target genes for csi-miRNAs and crt-miRNAs have been experimentally verified by PPM-RACE and RLM-RACE, a newly developed method for mapping miRNA-mediated cleavage products. The results from PPM-RACE and RLM-RACE could not only map the cleavage site of the target mRNA and also detect the expression pattern of cleaved fragments that could indicate the regulatory function of the miRNAs on their targets. Our results confirm the importance of csi-miRNAs and crt-miRNAs in regulating developmental transitions, flower development and fruit ripening of C. sinensis and C. reticulate further broadens the knowledge of small RNA-mediated regulation that all the csi-miRNAs regulated their target genes by degradation mode. 6. In this study,27 known miRNAs from Arabidopsis were searched against citrus EST databases for miRNA precursors, of which thirteen searched precursor sequences could form fold-back structures similar to those of Arabidopsis. The ubiquitous expression of 26 potential citrus miRNAs as could be detected in citrus leaf, young shoot, flower, fruit and root by northern blotting and some of them showed differential expression in different tissues. Based on the fact that miRNAs exhibit perfect or nearly perfect complementarity with their target sequences, a total of 41 potential targets were identified for 15 citrus miRNAs. Four target genes had been experimentally verified by detection of the miRNA-mediated mRNA cleavage in Poncirus trifoliata. Overall, this study in the identification and characterization of miRNAs in citrus can initiate further study on citrus miRNA regulation mechanisms, and it can help us to know more about the important roles of miRNAs in citrus.