| miRNAs, which are a class of-22 nt non-coding small RNA molecules, play vital roles by compounding to transcripts of target genes to inhibit translation or degrade mRNA of target genes in animals and plants. At present, the studies of miRNAs are mainly focusing on the intracellular miRNAs functions in their own species. For example, miRNAs regulate the growth and development of root, leaf, and flower in plants, and have important roles in the development of organs in animals. Recently, released miRNA was reported to be secreted from cells to body fluid and circulate in body fluid in a surprisingly stable-state in mammals. Those miRNAs could be carried by lipoprotein or other RNA-binding molecules, or packed by microvesicles to deal with the harsh condition (e.g., RNase, and extreme pH). Circulating miRNAs in body fluid were guided to target cells to regulate the expression of target genes.In 2011, Zhang et al discovered that rice-derived miRNAs cross the mammalian gastrointestinal tract to the mouse bloodstream, liver, and other tissues, where they regulate cholesterol levels by reducing the amount of low-density lipoprotein receptor-associated protein 1 (LDLRAP1). These findings provide new insights for genetic regulation by food ingestion and raise the prospect of engineering food or using oral small nucleic acid to prevent or cure diseases. However, in several other recent studies of mice, pigtailed macaques, or insects(Helicoverpa zea and Spodoptera jrugiperda), plant-derived miRNAs were not detected after ingestion of plant-derived food. In this context, we used the silkworm, Bombyx mori, an oligophagous insect that feeds only on mulberry leaves, to study whether mulberry miRNAs can transfer to silkworm upon ingestion.High-throughput sequencing technology was applied to sequence the small RNA libraries of wild mulberry (Morus notabilis), cultivated mulberry leaves (Morus multicaulis, and Morus atropurpurea), and silkworm larvae hemolymph. Combined the small RNA data and bioinformatics, we identified the species of mulberry miRNAs in silkworm hemolymph. TA clone, Sanger sequencing and droplet digital PCR assay were used to detect mulberry miRNAs in silkworm multiple tissues. RNA-seq, RT-PCR, and phenotypic investigation were then performed to explore the physiological progresses associated with one of mulberry miRNA, miR166b, in silkworm. The main results obtained in this research were as follows.1. High-throughput sequencing technology was applied to identify conserved and novel miRNAs of M. notabilisHigh-throughput sequencing technology was applied to sequence three small RNA libraries of M. notabilis three tissues containing leaf, bark, and male flower. Using bioinformatics methods, we identified 85 conserved miRNAs belonging to 31 families, 262 novel miRNA located in 371 loci in the M. natabilis. According to the target prediction of miRNAs,89 and 332 potential target genes were obtained for 20 conserved miRNA families and 113 novel miRNAs, respectively.Evolutionary analysis of conserved miRNAs in M. notabilis showed most of M. notabilis known miRNAs were conserved just like them in other dicotyledon and monocotyledon. In addition, parts of M. notabilis miRNAs were extremely conserved when compared with other seven plants. Conserved miRNA families were expressed across a vast range. Some miRNAs were expressed in a tissue-specific/biased manner. Among mulberry conserved miRNAs,5 leaf-biased,5 bark-biased, and 8 male flower-biased miRNAs were identified. RT-PCR results showed that the expression pattern of several conserved miRNAs were consistent with them in high-throughput sequencing results.The characters of novel miRNAs showed that the length range of novel miRNA precursors was consistent with other reported plants. The minimal folding energy of novel precursors were lower than it in tRNA and rRNA.90 of miRNA* were identified in novel miRNAs, which further confirmed the accuracy of novel miRNAs identification. The expression level analysis of novel miRNAs showed novel miRNAs were expressed across a vast range. The expression level of overall novel miRNAs were much lower than it in conserved miRNAs. Among the novel miRNAs,58 leaf-biased,84 bark-biased, and 72 male flower-biased miRNAs were identified. RT-PCR results showed that the expression pattern of several novel miRNAs were consistent with them in high-throughput sequencing.The annotation of miRNA target genes showed the potential target genes of conserved and novel miRNAs were transcriptional factor and functional protein, respectively. Compared the pair of miRNA-target, we found many pairs of miRNA-target in M. notabilis were conserved with other plants, which showed these miRNA-target played important roles in plant biological process, such as involving in the growth and development of organs. The RT-PCR results of several target genes and their corresponding miRNA expression in three tissues revealed the expression pattern of miRNAs and their potential target genes were contrary, which was accord with regulation mode of miRNA for targets.The identification of conserved and novel miRNAs in M. notabilis would provide a basic data for analyzing whether mulberry-derived miRNAs entering to silkworm.2. Identification of cultivated mulberry miRNAs and comparative analysis of wild and cultivated mulberry miRNAsSolexa sequencing was applied to sequence the small RNA libraries of two cultivated mulberry (M multicaulis and M. atropurpurea) leaves. Combined the 21 release version data from miRBase, conserved miRNAs were identified in M.notabilis(192), M. multicaulis(180), and M. atropurpurea (151). The novel miRNAs were 177 and 167 in M. multicaulis and M. atropurpurea, respectively. After comparing the expression level of wild and cultivated mulberry, we found 100 differentially expressed conserved miRNAs with 75 miRNAs up-regulated and 25 miRNA down-regulated in cultivated ones.266 novel miRNAs were differentially expressed in the two kinds of mulberry. Although the number of differentially expressed miRNAs (DE miRNAs) was very high, the number of DE miRNAs with highly-expression level was very low. There was only 6 DE miRNAs whose RPM value was more than 100. The above results showed that as a whole, the difference of miRNA expression between the wild and cultivated mulberry was very small. The KEGG analysis showed the target for differentially expressed miRNAs was enriched in carbohydrate and amino acid metabolism, which indicated the content of saccharide and protein might be different in the two kinds of mulberry.3. Identification and classification of silkworm hemolymph small RNATo understand whether mulberry-dervied miRNAs existed in silkworm, the small RNA libraries of silkworm hemolymph were constructed and sequenced by solexa method. The mulberry-derived miRNAs were identified in silkworm hemolymph. In addtion, several small RNA species, containing miRNA, tsRNA, rRNA, snRNA, snoRNA, and mulberry-derived miRNAs were also identified.The alignment of hemolymph small RNAs and mulberry miRNAs showed 8 mulberry-derived miRNAs (mno-miR166b, mno-miR166c, mno-miR167e, mno-miR396b, mno-miR156c, mno-miR398, mno-miR162, and mno-miR159a) existed in hemolymph with low reads. The results showed that mulberry-dervied miRNAs could cross the silkworm gut to hemolymph. However, these low abundant mulberry-derived miRNAs might be resulted from the contamination of sequencing or material preparation.After aligned silkworm hemolymph small RNAs with reported silkworm miRNAs, we identified 133 known and 6 novel miRNAs in hemolymph. The expression level of whole hemolymph miRNAs was much lower than them in silkworm tissues (e.g. fat body and silk gland). The reason was that most of hemolymph miRNAs came from hemocyte miRNAs and released miRNAs secreted from tissues.The length distribution of hemolymph small RNAs showed tsRNAs had the most content in small RNAs, which counted for 86.99%, major ranged from 25 nt to 28 nt. Classification of tsRNAs revealed the top two in content was tsRNA-Asp (GAC) and tsRNA-His (CAC). Location of tsRNA distribution in tRNAs showed 99% tsRNAs were matched to 5’terminal of tsRNA-Asp (GAC) and tsRNA-His (CAC). The distribution patters of tsRNAs were likely to miRNAs. In addition, the expression level of the two tsRNAs were highly in silkworm hemolymph, very little in whole silkworm body, fat body, and silk gland. The above results indicated tsRNA-Asp (GAC) and tsRNA-His (CAC) might play special roles in hemolymph.The above analysis of hemolymph small RNAs might provide clues of understanding the function of silkworm hemolymph miRNAs and tsRNAs, and support the opinion of plant-derived miRNAs cross-kingdom transferring.4. Nonfunctional ingestion of plant miRNA-miR166b in silkworm In the present study, we confirmed mulberry-derived miRNAs entering to silkworm, and played roles in silkworm physiological progress.Stem-loop PCR, T-A clone, and Sanger sequencing results revealed seven mulberry-derived miRNAs (mno-miR166b, mno-miR166c, mno-miR167e, mno-miR396b, mno-miR156c, mno-miR398, and mno-miR159a) entered to silkworm hemolymph, which further demonstrated the seven mulberry-derived miRNAs came from silkworm hemolymph, not resulted from contamination of Solexa sequencing or sample preparation. In addition, T-A clone and Sanger sequencing showed four mulberry-derived miRNAs (mno-miR166b, mno-miR166c, mno-miR167e, and mno-miR396b) could enter to silkworm nine tissues containing fat body, silk gland, brain, ovary, testis, gut, salivary gland, prothoracic gland, malpighian tube. Droplet digital PCR analysis showed synthetic miR166b could enter to silkworm hemolymph and fat body, and lasting about 3 hours.RT-PCR results of 11 potential target genes for miR166b showed no significant expression change was observed before and after silkworm ingested synthetic miR166b, which indicated miR166b had no function on silkworm, or the 11 genes were not the target genes of miR166b. RNA-seq was performed using whole body of silkworm larvae 3 hours post feeding. Compared with silkworm larvae fed on mulberry leaves, only 30 genes were differentially expressed, of which 17 were up-regulated and 13 were down-regulated in larvae feeding with synthetic miR166b. Classification of 30 DGEs showed genes involved in immunity and stress comprised about 40% of the differentially expressed genes. We speculated the ingestion of synthetic miR166b might only activate the host response against non-self-molecules. RT-PCR results of 6 DEGs revealed the expression level of 3 DEGs were up/down regulated after silkworm ingestion of other two synthetic miRNAs (miR166c and miR167e), which indicated the expression change of 30 DEGs might be caused by nucleic acid, not the sequence of mR166b. In addition, phenotypic investigation showed no significantly difference was observed in the larvae weight, developmental speed, lethality, weight of pupae and cocoon after silkworm fed on mulberry leaves with or without synthetic miR166b. In addition, there was no significant overlap in the predicted miR166b target sites for the 30 differentially expressed genes. These results suggest that mulberry miR166b has no significant effect on the expression silkworm genes.Considering the results of high-throughput sequencing, T-A clone, ddPCR, RNA-seq, RT-PCR experiments and phenotypic investigation, we believed mulberry-derived miRNAs could enter to silkworm hemolymph and 9 tissues after ingestion. The synthetic miR166b could also enter to hemolymph and fat body, but basically playing no roles in silkworm physiological progress. |
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