| Azorhizobium caulinodans ORS571（A.caulinodans）is a kind of plant growth promotingrhizobacteria（PGPR）, it can convert molecular nitrogen to a form which can be used byplants, promote the course of plant growth, enhance the capacity of plant resistance toenvironmental stress, improve plant immunity against pathogen infection. This study revealsthe molecular regulatory mechanism of wheat seedlings in response to the A.caulinodans viaSmall RNA Sequencing results, which based on Illumina’s HiSeq2000sequencingtechnology. The main results are as follows:1. The colonization of A.caulinodans in the wheat seedlings were detected by laserscanning confocal microscope. The results showed that A.caulinodans can colonize in the rootwheat seedlings via root hairs and root tip breakages and then ascend endophytic migrationinto the leaves, eventually colonize around stomas, which further improve the colonizationand distribution pattern of A.caulinodans in wheat seedling.2. Based on Small RNA Sequencing results,15shoot and root miRNAs of6h,12h,24h,48h,72h and96h post inoculation were confirmed by Real Time PCR. The results show thatthe miRNAs timing expression pattern changed, the expression of miR399wasdown-regulated and then up-regulated, while the expression of other14miRNAs wereup-regulated, then down-regulated. According to the expression level, we found threeexpression patterns:(1) miR168, miR403and miR444, these miRNA expressions of leaf androot were almost at the same level. The root and leaf expression level of miR168both peakedat12hs post-inoculation. miR403peaked at12h and24h, miR444peaked at6h and12hrespectively.(2) The peak expression level of root was higher than that of leaf. These eightmiRNAs were miR156, miR159, miR160, miR164, miR167, miR169, miR393and miR827;and their root expression level peaked at12h with the exception of miR827, which peaked at24h.(3) The peak expression level of leaf was higher than that of root. These four miRNAswere miR171, miR398, miR399and miR1507. miR171, miR399and miR1507peaked inexpressions of leaf at24h, whereas miR398peaked at48h.3. The target genes of15miRNAs were predicted, the results showed that these targetgenes were most transcription factor genes. Combined with the timing expression differences,the molecular mechanism of interaction between wheat and A.caulinodans was speculated. Of the15miRNAs, miR168and miR403regulate their own metabolism in the form of negativefeedback; miR164, miR167, miR169, miR171, miR398and miR399regulate nitrogen,phosphorus and trace elements absorption and utilization of plant by regulating the expressionof their target genes; miR393, miR444, miR827and miR1507may make wheat producecertain defense response, so the wheat seedling can adapt the interaction environment;miR156, miR159, miR160, miR164and miR167play roles in wheat seedlinggrowth-promotion by regulating hormones and lateral root formation.4. Target genes of miR167were validated by Real Time PCR. The results showed that:（1）In root, the expression of miR167was up-regulated then down-regulated, but theexpression of its target genes were down-regulated then up-regulated.（2）In leaf, theexpression of miR167was down-regulated then up-regulated, and finally down-regulated. Butthe expression levels of its target genes were up-regulated then down-regulated, andup-regulated in the end. The expression level of target genes in root and leaf maintained at arelative high level after48h inoculation. The expression patterns of miR167and target genesshowed that miR167can regulate its target genes in a negative way.This research showed the endogenous colonization and distribution of A.caulinodans.The molecular mechanism of wheat seedling in response to A.caulinodans was clarified bymiRNAs vertification and targets prediction. The results were expected to explore endophyticnitrogen fixation, nitrogen fixation, photosynthetic nitrogen fixation in non-legume crops andprovided a basis to study the interaction mechanism and application of A.caulinodans as abio-fertilizer. |
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