Identification and biological analysis of Circ032768
Circ032768 is located in Arabidopsis chr4:13026572-13027129, which is composed of exons and belongs to ecircRNA (ecircRNA). The total length of circ032768 is 558bp and the parent gene is AT4G25500.The main function of the parent gene is the arginine/serine splicing factor RS40, which is mainly found in the leaves, roots, stems, flowers and seeds of Arabidopsis thaliana (Figure 1a).
In order to verify the existence of circ032768 in a circular form in Arabidopsis thaliana, this study designed convergence and divergence primers to amplify +R treated cDNA, -R treated cDNA and genomic DNA, respectively. Theoretical studies showed that only cDNA could expand the dispersion product, while genomic DNA could not.Moreover, the cDNA samples treated with the R enzyme have aggregation effect on circRNA so as to rule out false positives caused by gene rearrangement or potential PCR products.The results showed that circRNA032768 could only amplify the head-tail junction in cDNA samples, and the bands of circ032768 treated with +R enzyme were brighter than those treated with -R enzyme, indicating that circ032768 was resistant to RNaseR enzyme (Figure 1b).At the same time, the cDNA amplified products were further detected by Sanger sequencing in this study, and the test results were shown in Figure 1c. The Sanger sequencing quality was good.
Then, circ032768 complete sequence 558bp and the mature sequence of the left arm in the precursor of miR472 (FIG. 2a) were selected to analyze the possible interaction sites of ceRNA in circ032768 sequence with miR472 through RNAhybrid.In this study, five ceRNA action sites of miR472 were identified in circ032768 sequence with length of 588bp.At these 5 binding sites, circ032768 and miR472 are base complementary, and at least 9 complementary bases exist at each binding site, which is consistent with the characteristics of the action site of ceRNA (Figure 2b).
Omidvar et al. (2014) showed that miR472 could identify and bind dozens of CC-NBS-LRR genes.In order to further understand the interaction between miR472 and target genes, we analyzed the homology of interaction sites in Arabidopsis Thaliana gene data from NCBI database.The results showed that there were dozens of CC-NBS-LRR genes with high matching with miR472 binding sites. The top 25 sequences with high matching gene sequences were downloaded and Clustalx analysis results were used to show the regions of interaction sites.Among them, RPS5 (AT1G12220) sequence and miR472 had the largest number of interaction sites, reaching 17, accounting for 81% of the mature sequences of miR472. Therefore, RPS5 was selected as the predicted target gene of miR472 in this study for subsequent studies.In addition, in this study, G, C and T with possibly high homology were found in the upper and downstream regions of the interaction sites, which may have a recognition effect when miR472 interacts with RPS5 (Figure 2c).The sequence homology analysis of the 25 CC-NBS-LRR genes was conducted based on amino acid sequences, and the phylogenetic evolutionary tree was established by NJ method, maximum reduction method and maximum likelihood method, respectively. The results showed that the 25 CC-NBS-LRR genes existed in four categories.The sequence AT1G12290 and AT1G12220 belong to the same branch (FIG. 2d, e, f).
The MDA and SOD levels of plants in 7-day drought and 14-day drought were measured in normal plants. It was determined that Arabidopsis plants were affected by different degrees of drought stress.Compared with the control, MDA content and SOD activity in the 7 and 14 days of drought showed an increasing trend to varying degrees. In particular, compared with the normal plants, the leaves of the 14 days of drought showed a tendency to wither, and the increase trend of MDA content and SOD activity was extremely significant (Figure 3c, d, e).
In order to further verify the interaction regulation mode of circ032768-MIR472-RPS5, U6 was used as the internal reference of miR472, and Actin was used as the internal reference of circ032768 and RPS5. The expression levels of circ032768, miR472 and RPS5 were measured in drought-treated plants.The results showed that the expression levels of circ032768 and RPS5 were significantly increased after drought stress treatment, while the expression levels of miR472 were significantly decreased, and the variation characteristics were consistent with the rule of ceRNA mechanism.It was further verified that circ032768, miR472 and RPS5 were responsive to drought stress and provided data support for subsequent interaction studies of Circ032768-MIR472-RPS5 (Figure 3f).
Acquisition of transgenic plants and their response to drought stress
Using Arabidopsis genomic DNA as template, circ032768 with flank intron, intron 1 with 223bp and intron 2 with reverse complement were cloned from Arabidopsis DNA.The cloned PCR products were separated by 1.0% agarose gel electrophoresis, and were recovered, sliced and connected to PCAMBIA1303 by enzyme digestion. The cloned strains were screened, and the positive strains were obtained by sequencing and sequence alignment. Finally, the vector was amplified and extracted (see 4a for the sketch of the vector, see 4b for the electrophoretic picture of the vector construction).The stem ring sequence and mature sequence of miR472 were introduced into PTO2 vector by primer design. The cloned PCR product 2882bp was separated by 1.0% agarose gel electrophoresis, then recovered, digested and linked into PFGC5941. After screening, positive clones were obtained by sequencing and sequence comparison.Finally, the carrier was strengthened and extracted (see 4c for the electrophoretic picture of carrier construction).
Plants that overexpressed circ032768 and STTM-miR472 were initially obtained by Kan resistance screening. The study showed that some plants of STTM-miR472 and overexpressed circ032768 showed resistance to Kan, and Arabidopsis seedlings that could grow normally on Kan resistance medium were retained.Soil transfer culture was performed (FIG. 4d).Subsequently, the gene expression levels of circ032768 and miR472 in transgenic plants were determined by qPCR.Wild type plants were selected as control, transgenic plants with significantly increased circ032768 and RPS5 expression while decreased miR472 expression were screened for subsequent planting and sowing.The results showed that the expression of circ032768 in T3 generation plants overexpressing circ032768 increased by more than 3 times, RPS5 increased by more than 6 times, and the expression of miR472 decreased by more than 2 times, which was consistent with the change trend of ceRNA, especially in plant 5.circ032768 increased by 8.388 times, miR472 decreased to 0.101 of wild type, and RPS5 increased by 30.726 times (FIG. 4e).3, 4, 6 and 9 of STTM-miR472 plants were in line with theoretical expectations, and the results of plants 4 and 9 were the best. circ032768 increased by 5.711 and 8.055 times, respectively, while miR472 decreased to 0.365 and 0.130 of the wild type.RPS5 increased by 15.478 and 21.060 times (Figure 4f).
In order to further verify the response of circ032768 to drought stress, 3-week-old circ032768 overexpressed plants, STTM-miR472 plants and wild-type plants (selected with the same growth) were subjected to drought treatment for 14 days without watering.It was obviously observed that wild-type Arabidopsis thaliana plants had yellows and atrophic leaves after 14 days of drought treatment, while circ032768 overexpressed plants and STTM-miR472 plants still maintained green and normal leaf morphology despite varying degrees of damage (FIG. 5a).Wild type plants were used as control, and circ032768 overexpressed plants and STTM-miR472 plants were stained with NBT.The staining results showed that under normal treatment, wild type plants produced a little blue substance, while circ032768 overexpressed plants and STTM-miR472 plants produced almost no blue substance.However, under drought treatment, blue substances in wild-type plants showed a significant increase trend compared with normal treatment. circ032768 overexpressed plants and STTM-miR472 plants also produced less blue substances, but the amount of blue substances produced was much less than that in wild-type plants (FIG. 5b).The DPPH and ABTS radical scavenging rates of different plants were measured at the same time, and the results showed that the circ032768 overexpressed plants had the highest scavenging rate, followed by STTM-miR472, and the wild type had the lowest (FIG. 5c, d).
Oxidation-sensitive fluorophore 2 '7' -dichlorofluorescein (H2DCF) is commonly used to directly measure changes in intracellular H2O2 levels.The nonpolar diacetate of H2DCF (H2DCFDA) enters the cell and hydrolyzes into the more polar nonfluorescent compound H2DCF, which can be captured.Subsequently, H2O2 oxidizes H2DCF, which is catalyzed by peroxidase to produce highly fluorescent DCF.Therefore, the higher the fluorescence intensity of stomata, the higher the level of H2O2 in cells, and the stronger the stress of plants.Fluorescence results showed that the fluorescence intensity of wild-type plants was much higher than that of circ032768 overexpressed plants and STTM-miR472 plants under both drought stress and normal treatment conditions (Figure 6a-g).
The germination rates of circ032768 overexpressed plants, STTM-miR472 plants and wild-type plants were measured at the manna concentration of 0mm, 50mm, 100mm and 150mm, and the green plants were considered to be the survival plants, so as to determine the germination rate of the plants.Wild type, STTM-miR472 and overexpressed circ032768 plants were cultured from left to right. Each group was treated parallel for 3 times for calculation.The results showed that the germination rate of STTM-miR472 and circ032768 overexpressed plants increased significantly with the increase of mannitol concentration compared with the wild type.Especially at 150mm, the germination rate of wild-type plants was only 50.07%, while that of STTM-miR472 plants was 68.17% and that of overexpressed circ032768 plants was 76.51% (FIG. 6h-l).Then, the root length of circ032768 overexpressed plants, STTM-miR472 plants and wild-type plants were measured at the manna concentration of 0mm, 50mm, 100mm and 150mm, and the root length was measured by IamgeJ.Wild type, STTM-miR472 and overexpressed circ032768 plants were cultured from left to right. Each group was treated parallel for 3 times for calculation.The results showed that the root length of STTM-miR472 and overexpressed circ032768 plants increased significantly compared with that of wild type, but the difference of root length between different plants decreased gradually with the increase of mannitol concentration (FIG. 6m-q).
Analysis of interaction mechanism of Circ032768-miR472-RPS5
Previous studies have shown that circ032768 and miR472-RPS5 do respond to drought stress and are predicted to be strongly correlated with each other. However, how do Circ032768 and MIR472-RPS5 respond to drought stress and whether there is any interaction between the two and their target RPS5?These questions need further study.In this chapter, we will verify the co-transfection of interacting vectors into tobacco, verify the interaction between circ032768, miR472, and RPS5 through the GFP marker carried by the transfection vector, and infer and predict the drought response pathway of RPS5.First, this study will construct PTF486-35S: RPS5-eGFP vector, and extract PTF486-35S with high concentration and purity by large plasmid extraction:RPS5-eGFP plasmid was transfected into Arabidopsis thaliana protoplast for incubation. The fluorescence of protoplast was observed under laser confocal microscope, and PTF486 was used as the control.Fluorescence results showed that PTF486 was expressed in the cytoplasm, while the PTF486-35s: RPS5-EGFP vector with RPS5 inserted was expressed on the cell membrane, which demonstrated RPS5 expression (FIG. 7a).After that, the good interaction vectors (Figure 7b, c) were extracted, Agrobacterium was transformed, and the bacterial liquid was evenly mixed 1:1, and tobacco was injected. The fluorescence intensity of GFP was observed by laser confocal microscope, and the fluorescence intensity was analyzed by ImageJ software.POT2+PTF486 was used as blank control to exclude fluorescence interference.The results showed that the fluorescence expression of the vector with RPS5 gene was concentrated on the cell membrane and the fluorescence intensity of POT2+RPS5 was stronger than that of miR472+RPS5.However, miR472+PTF486 and POT2+PTF486 were expressed in the cytoplasm, and the fluorescence intensity hardly changed (Figure 7d, e).To further verify the interaction between miR472 and RPS5, the relative gene expression levels of GFP in the above four transient leaves were determined.The results showed that, consistent with the observed fluorescence intensity, the expression of GFP gene in POT2+RPS5 was significantly higher than that in miR472+RPS5, and there was no significant difference in the expression of GFP gene between miR472+PTF486 and POT2+PTF486 (Figure 7f).This indicated that miR472 could reduce the gene expression of RPS5, which was consistent with the action mode of miRNA and its target, namely miR472 participated in and mediated the degradation process of RPS5.
Since both circ032768 and miR472 are non-coding Rnas, the interaction between circ032768 and miR472 must be verified by RPS5, the target of miR472.Therefore, in this study, Agrobacterium miR472+ RPS5+1303, POT2+RPS5+1303, miR472+PTF486+1303, POT2+PTF486+1303, mi were mixed at 1:1:1Eight systems, R472+RPS5+circ032768, POT2+RPS5+circ032768, miR472+PTF486+circ032768 and POT2+ PTF486+circ032768,Thus, the interaction between circ032768 and miR472 was verified.The results showed that there was no significant difference in fluorescence intensity and GFP expression in the system with PTF486 vector.In addition to the RPS5 target gene, the system with POT2 was not significantly different from the system with PTF486 vector.However, when miR472 and RPS5 were present at the same time, the fluorescence intensity and GFP expression decreased significantly.The fluorescence intensity of miR472 +RPS5+1303 system decreased to 37.9% of POT2+PTF486+1303, and the GFP expression decreased to 33.3% of the control group.When miR472+RPS5 was addedto circ032768, the fluorescence intensity of miR472+RPS5+1303 system was 1.56 times that of miR472+RPS5+1303 system, and the expression of GFP was 2.89 times that of mir472 +RPS5+1303 system.Previous studies have shown that miR472 and RPS5 interact, and miR472 can reduce the expression of RPS5, which is also supportedby the results of this experiment.However, the addition of circ032768 can increase the expression of GFP in miR472+RPS5 system. The results are consistent with the ceRNA mechanism of circRNA, indicating that circ032768 can be used as ceRN to competitively bind miR472 molecules.Thereby increasing the gene expression of RPS5 (Figure 8a-c).
In order to further clarify the role of circ032768-miR472-RPS5 mechanism in drought stress, the wild type was used as the control in this study. RNA extraction and qPCR were performed on transgenic Arabidopsis thaliana obtained in 3.4.3 to identify the expressions of DERB2A, RD29A and RD29B, the key influencing factors of drought stress.The identification results showed that circ032768 overexpression and T3 generation of STTM-Mir472,The gene expressions of DREB2A, RD29A and RD29B in circ032768 overexpressed plants 4, 5, 7 and 8 and in STTMmiR472 plants 3, 4, 6 and 9 were significantly increased (Figure 8d).In particular, circ032768 overexpressed plants 5 and STTM-miR472 plants 4 and 9 showed an extremely significant upward trend.This is consistent with the upward trend of circ032768 and RPS5 measured in 3.4.3.This suggests that the circ032768-miR472-RPS5 mechanism plays an important role in drought response mechanism in Arabidopsis thaliana through DREB2A and its downstream factors RD29A and RD29B.