| Sorghum [Sorghum bicolor (L.) Moench] is the fifth most cereal crop in the world and is widely used as a food, wine, sugar and feed crop. Sweet sorghum is a natural variant of common grain sorghum and is becoming one of the potential source of bioenergy due to the high sugar content in its stems. Therefore, research on molecular mechanisms of sugar accumulation in the stems of sweet sorghum, it is of great significance to breeding high sugar content of sweet sorghum varieties, sugar transport in stem and molecular mechanism of sugar accumulation. But the results on QTL for sugar content in the stems of sweet sorghum are inconsistent. The molecular mechanism of sugar accumulation in the stems remains unclear. In order to explore characteristic of changes of genes and microRNAs in sugar accumulation stage in the stems of sweet sorghum, identify genes and microRNAs may involved in sugar accumulation in the stems. In this study, after getting the data from sugar accumulation in the stems of sweet sorghum (Rio) and grain sorghum (BTx623), we determine the sampling period and parts of the tissue. The total RNA was extracted from the fourth internode of the stems and the second leaves at the heading stage and the dough stage. We analyzed the genes and miRNAs expressed from stems and leaves of sweet sorghum in sugar accumulation stage using high-throughput sequence. We selected corresponding period of grain sorghum as a control and identify genes and microRNAs associated with sugar accumulation in sweet sorghum. This study analysed the molecular mechanisms of sugar accumulation in sweet sorghum at the expression profile level. It is provide a basis for sugar accumulation in the stems of sweet sorghum.1. The digital gene expressed profile results were as follows:(1) Clean reads were mapped to the sorghum gene and genome,25405 genes were detected from eight samples. We used FDR<0.001 and an estimated absolute value of | log2 Ratio |≥1 as the threshold to judge the significance of each gene expression difference.6285 (4224 up-regulated,2061 down-regulated) and 5216 (1744 up-regulated,3472 down-regulated) genes were significantly differentially expressed in the leaves and stems of BTx623.6245 (4239 up-regulated,2006 down-regulated) and 5424 (2338 up-regulated, 3086 down-regulated) genes were significantly differentially expressed in the leaves and stems of Rio.(2) KEGG pathways analysis showed that:the significantly enriched KEGG pathways were 25,13,17 and 15 for RS1-VS-RS2, RL1-VS-RL2, BS1-VS-BS2 and BL1-VS-BL2, respectively. Differentially expresssed genes of RS1-VS-RS2 were widely involved in starch and sucrose metabolism, biosynthesis of secondary metabolites, plant hormone etc. These pathways may play an important role in sugar accumulation in the stem of sorghum.(3) MapMan analysis showed that:genes and pathways related to processes for the synthesis and degradation of starch and sucrose were markedly difference during sugar accumulation in the stems of sorghum. In the metabolism overwiew, genes related to processes for the synthesis and degradation of starch, the synthesis of sucrose were markedly up-regulated in the stems of sweet sorghum (RS1-VS-RS2), at the same time, genes related to sucrose degradation were suppressed. Hydrolysis of starch to sucrose, finally, sucrose largely accumulated in the stems of sorghum. In the stems of BTx623, genes related to processes for the synthesis and degradation of starch were down-regulated, so reducing the hydrolysis of starch to sugar, and then hydrolysis of sucrose, so sucrose can’t large accumulated in the stem.(4) Sucrose transporter analysis showed that, SUT1 is highly expressed in mature leaf tissues and is induced in post-sugar accumulation in the leaves of sweet sorghum, accelerate transporting sucrose from leaves to stems. SUT2 is expressed most strongly in stems and is induced in post-sugar accumulation in the stems of sweet sorghum. SUT2 play a role in directing sucrose to stem storage parenchyma cell, so sucrose larget accumulation in the stems.2. The microRNA results were as follows:(1) A total of 185 known miRNAs, belonging to 56 families, was identified from eight miRNA libraries. Reads that were not annotated were used to predict 634 novel miRNAs using Mireap software.(2) The differentially expressed miRNAs were analyzed, miRNAs with changes in expression levels that were greater than 2-fold or less than 0.5 and those with p-values less than 0.05 were selected. The results showed that 61 miRNAs in the leaves and 42 miRNAs in the stems showed significantly differentially expressed in BTx623. In Rio,64 miRNAs in the leaves and 40 miRNAs in the stems showed significantly differentially expressed. Among the novel miRNAs,60 miRNAs in the leaves and 30 miRNAs in the stems were differentially expressed at a significant level in BTx623 and 58 miRNAs in the leaves and 33 miRNAs in the stems in Rio were significantly differentially expressed.(3) We found 442 targets for 169 known miRNAs and 3771 targets for 264 novel miRNAs.12 novel miRNAs and 9 known miRNAs in RS1-VS-RS2 were specifically differentially expressed. Predicted target genes for differentially expressed known miRNAs, including NAC transcription factors and homeodomain zipper proteins. In addition, other predicted target genes were involved in a number of physiological and metabolic processes. The target genes of the novel miRNAs included the transcription factor, glucosyltransferase, protein kinase, cytochrome P450, transporter genes, etc. Differentially expressed miRNAs and their target genes seem to form a complicated regulatory network that plays a critical role in sugar accumulation stage.(4) We screened that a stem-specifically induced expressed novel miRNA and a leaf-specifically induced expressed novel miRNA in Rio.We validated the expression of them using real-time quantitative PCR. Our results provide a basis for further investigation of the potential role of these individual miRNAs in sugar accumulation. |
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