| Allelopathy is an interesting ecological phenomenon that has been found for two thousand years, and it was first reported on rice in 1990’s. Many studies had been conducted to examine the mechanism on the formation of rice allelopathy in the last 10 years. However, large scale of gene expression and regulation pattern on both donor and receptor plants, especially on target weed from rice allelopathy system, is still not been presented. Recently,microRNAs(miRNAs) were known as a kind of important small non-coding RNA which functions in transcriptional and post-transcriptional regulation of gene expression, and comprehensive study on the miRNAs expression pattern is feasible with the development and the maturity of high-throughput sequencing technology. In order to investigate the gene regulation network on allelopthic rice PI312777 and barnyardgrass (Echinochloa crus-galli L.,receptor plant) respectively, high throughput sequencing technology was used to detect the miRNAs expression pattern on the co-cultured system, of which contained same number of PI312777 and barnyardgrass. miRNAs relevant to rice allelopathic effect were obtained by using GO term and KEGG pathway enrichment analysis, and the dynamic expression of these miRNAs on barnyardgrass under different rice/weed co-culture days and different rice/weed ratios was detected respectively with quantitative PCR(qPCR). In order to evaluate the biological function of the miRNAs,comparison of the change expression on miRNA target genes from the barnyardgrass under various treatment was also conducted, following by the determination of physiological change on these gene-involved pathways. In addition, the study also detected the microbe population numbers in the culture system, as well as a particular species named myxobacteria. The change expression of miRNAs on barrnyardgrass that being cultured in the nutrient solution contained Myxococcus xanthus (a kind of myxobacteria being separated from allelopathic rhizosphere) was also investigated.The Myxococcus xanthus and phenolic acid was then added respectively or together in the pre-sterilize soil, with the aim to evaluate allelopathic potential of them on barnyardgrass. From all the studies above, the gene expression and regulation way during the process of rice allelopathy on barnyardgrass and its underlying mechanism was indicated, and the rhizospheric biological processes was also illuminated. The details of results was showed as follow,1. Annotation and the following KEGG pathway enrichment analysis on the miRNAs from sRNA presented that the miRNAs expressed on the barnyardgrass had higher abundant than that expressed on PI312777, and the enrich expressed miRNAs(17 sequences) from barnyardgrass were relevant to plant hormone signal transduction,nucleotide excision repair, phenylalanine, tyrosine and tryptophan biosynthesis,tropane, piperidine and pyridine alkaloid biosynthesis, PPAR signaling pathway and P53 signaling pathway. However, only miRNAs relevant to tropane, piperidine and pyridine alkaloid biosynthesis was found enrich expressed on PI312777. Change expression level of the enrich expressed miRNAs from barnyardgrass were then detected on the bamyardgrass respectively co-cultured PI312777 and non-allelopathic rice Lemont, the results showed that most of the miRNAs expression level was enhanced in the barnyardgrass accompanied with PI312777, and the up-regulated folds of these miRNAs were increased following the extension of co-cultured day and peak at the 7th day. In contrast to the result, the miRNAs expression level was mainly down-regulated on the barnyardgrass accompanied with Lemont. These findings agreed with the outcomes of high-throughput sequencing, and both of the results suggested that rice allelopathy particularly influence on several miRNAs expression in barnyardgrass.2. Moreover, the similar tendency of enriched miRNAs changing expression was also found in different densities of rice/weed co-cultured system, which showed that highest increased folds were found in high ratio of rice/weed system, and barnyardgrass accompanied with PI312777 had higher increased folds than that of barnyardgrass accompanied with Lemont under same rice/weed co-cultured ratio.Overexpression of PAL (PAL-OE) gene on PI312777 presented higher allelopathic inhibition on barnyardgrass and also resulted in increased miRNAs expression level on the accompanied barnyardgrass, when compared to that co-cultured with wild type v of PI312777, while inhibited PAL (PAL-RNAi) gene expression on PI312777 had reserve results. The change expression level of miRNAs target genes was also detected, and the results showed that the target genes presented reverse expression change in comparison to the relevant miRNAs,which further confirm the biological function of enrich miRNAs. Determination on the IAA content and DNA damage(presented as apurinic/apyrimidinic sites, AP sites) on different treatment on barnyardgrass showed that barnyardgrass accompanied with PAL-OE rice lines had lowest IAA concentration and most AP sites on nucleotide, increasing of weed density on PI312777/barnyardgrass system resulted in increased AP sites but decreased IAA content on barnyardgrass, however, no significant difference was found in different Lemont/barnyardgrass co-cultured system, and IAA content in the barnyardgrass accompanied with Lemont higher than that of BYG accompanied with PI312777 under the same ratio, the reverse was true in the AP sites on barnyardgrass nucleotide. The results further confirmed the rice allelopathic effect on particular miRNAs expression in barnyardgrass.3. Both expression level of the enrich miRNAs and their target genes were further detected on different concentrations of phenolic acid treated barnyardgrass,and it was found that phenolic acids treatment significantly increase the miRNAs expression level on barnyardgrass and expression level of the target genes were reduced. However, Barnyardgrass being treated by phenolic acids mixture showed down-regulated expression level of most miRNAs. In whilst, barnyardgrass treated by terpenoids were showed no significant difference or down-regulation on the miRNAs expression. The results indicated that bamyardgrass under single phenolic acid treatment presented similar tendency in the change of miRNAs expression level, in comparison with rice allelopathic effect.4. In addition, the microbe populations in the hydroponic solution from different co-cultured system were also detected. It was found that both total bacterial and total fungal population numbers were increased when the rice was co-cultured with barnyardgrass, however, no positive correlation was found between rice allelopathic potential and bacterial or fungal population in the hydroponic solution.Specially, the resulted also showed that the number of Myxococcus species population was increased following the increment of weed density,the system of PAL-OE co-cultured with barnyardgrass contained highest Myxococcus sp. number and mono-cultured of PAL-RNAi lines had lowest contain of Myxococcus sp., but no significant difference on Lemont/barnyardgrass system. Most of the phenolic acids significantly promote the increasing of Myxococcus sp in the hydroponic solution.However, terpenoids showed no significantly influence on Myxococcus sp. numbers,and some of which even inhibited the proliferation of Myxococcus sp. Barnyardgrass cultured in the hydroponic solution contained with Myxococcus xanthus showed increased miRNAs expression level than control, and the folds was increased in the condition of Myxococcus xanthus with its metabolites, and the miRNAs target genes were present lowest expression level, compared to the Myxococcus-treated BYG and the control ones. Adding both Myxococcus xanthus and phenolic acid in the pre-sterilize soil presented highest allelopathic inhibition on barnyardgrass, in comparison to the outcome of single phenolic or Myxococcus xanthus used, and the highest Myxococcus xanthus numbers was also detected on the pre-sterilize soil that being added with Myxococcus xanthus and phenolic acid.All the results above indicated that phenolic acids release from allelopathic rice accessions function in promoted the proliferation of Myxococcus sp. in rice rhizosphere, the Myxococcus sp. interacts with phenolic acid and then changed on plant growth and DNA repair relevant miRNAs and their target gene expression on target barnyardgrass, which in turn to reduce IAA contents and increase AP sites, and the influence resulted in inhibition on BYG growth and development. |
PDF Full Text Request