Identification Of Granulation Related MiRNA And Function Analysis During Citrus Fruits Storage

Juice sac granulation is one of the key issues affecting citrus quality during storage. Almost all citrus granulate, and this has become a problem of citrus industry. Previous studies have found many relevant factors of citrus juice sac granulation, but its molecular regulation mechanism is unclear. Sweet orange(Citrus sinensis Osbeck) which is prone to granulation was selected for this study. In this research, bioinformatic and molecular biology tools were used to predict miRNA and the corresponding target genes that relate to granulation during storage of sweet orange. The results of this study lay a foundation for miRNA in regulating juice sac granulation at post-transcriptional level. Major conclusions were listed as follows:1. Candidate miRNAs were screened by bioinformatic analysis, and 12 potentially unreported miRNAs were obtained. Subsequently, we predicted the target genes of 87 citrus miRNA and analyzed their function by using psRNATarget and Blast2 go program, respectively. A total of 279 non-duplicate candidate target genes were predicted, of which 187 have their functional annotation.2. The physiological processes of lignification, nutrition stress and development influenced the citrus juice sac granulation. Therefore, the screening condition was whether the target of miRNA plays a regulatory role in the lignification and any other process which may affect granulation. Eventually, 8 potential miRNA families(miR172, miR159, miR160, miR397, miR169, miR828, miR827, and miR393) were presumed to be related to citrus granulation.3. The evaluation system of the granulated grade was established by comparing the results of low field nuclear magnetic resonance(LF-NMR) and sensory grading. The results showed that there was no significant difference(P = 0.4982 < t = 0.7143) between the mathematical model and the score of the sensory results. Therefore, the scoring mathematical model of artificial intelligence can be applied to identify the granulation degree of citrus fruits.4. Real-time quantitative reverse transcription PCR(qRT-PCR) revealed that the relative expression of N-miR159a-3p, csi-miR397, csi-miR828, csi-miR827 decreased gradually with the increasing granulation degree, and the difference was significant(P <0.05) between control group and experimental group. Simultaneously, the expressions of these four miRNAs were significantly associated to the granulation degree with correlation coefficients of 0.886, 0.888, 0.944, and 0.896, respectively, whereas N-miR159a3 p and N-miR828 were the potentially unreported miRNAs.5. The content of lignin and phosphorus(P) were measured at different levels of granulation. Results showed that the balance of lignin and P were significantly related to citrus granulation with correlation coefficients of 0.972 and 0.960, respectively. The relative expression of Cs6g06890.1, Cs1g17590.1, which is the target of csi-miR397 and N-miR828, respectively were significantly correlated to granulated degree and lignin content. Furthermore, Cs5g10180.1, target of csi-miR827 was significantly correlated to the degree of granulation and P content. Therefore, the results indicate the likely role of the csi-miR397/Cs6g06890.1 and N-miR828/Cs1g17590.1 modules in granulation through regulation of the internal lignin activity. Csi-miR827/Cs5g10180.1 modules were correlated to granulation through regulation of the internal P activity, while P with the characteristics of water retention of the cells, so it may regulate citrus granulation by controlling moisture transfer.