Selection And Functional Analysis Of Mirnas In Soybean Nodulation

Soybean is one of the most important crops with high economic value, symbiotic association between soybean and rhizobia bacterial results in specialized nitrogen fixing structures called root nodules, which provide nutrients for the development of soybean.The nodulation development has been studied by molecular biology, and more and more microRNAs have been discovered in legumes, it is reported that microRNAs play important roles during legumes development. The microRNAs which have functions on nodulation has rarely been discoloured. It is important to study the novel microRNA roles in nodulation, their identification might help advanced our understanding of nodulation.Our lab had identified a set of 35 novel miRNA families from soybean. We selected 17 moderately abundant miRNA families, each of which at least has10 sequence reads in our high throughput sequencing libraries for detailed analysis.Williams82 roots were inoculated with B. japonicum USDA110 and were harvested at different time points post inoculation. Six microRNAs had Northern expression patterns, they are miR482,miR1507,miR1511,miR1512,miR1515 and miR1521. These six miRNAs have different expression patterns at different time points, suggesting that these miRNAs might play different regulatory roles at different stages of nodule development. We examined the soybean genome to identify their genome location, and found they were located at 1th, 2nd, 10th, 11th, 17th and 18th chromosome, which indicates that they may play different roles in different soybean organs. Northern blot analysis was applied to different organs of Williams 82: root, stem, mature leaf, fully opened flowers, young pods, and developing seeds at stage R5 and R6. miR1512 was only expressed in roots, while others had differential expressions in various organs. Different bioinformatics softwares were used to predict the target of these microRNAs, and RNA mFold was used to predict the secondary structure of the targets. We found that the pre-miRNA varies in structure and length, the length is between 95bp~145bp, which means the miRNA has different regulation roles; mature miRNA sites in pre-miRNA also different, 3 miRNAs locat at 3' end at their corresponding pre-miRNA, while 2 at 5' end. All the 10 predicted pre-miRNAs had low minimal free energies between -36.6~ -63.8, indicates the validation of our prediction.These 6 miRNAs were used for Northern blot analysis by soybean Bragg at different time points, that is 0h,6h,12h,2d,6d, These time points were the ones observed in wild-type roots W82. The time course of miRNAs expression in Bragg roots inoculated with B. japonicum was strikingly similar to what was observed in Williams 82. 2 mutants were used for expression patten analysis next. We found these miRNAs had different expression patterns in different mutants. NFR1 and GmNARK1 signaling might not govern B. japonicum-induced expression of miR482 and miR1521; while both these pathways influence miR1511 and miR1512. Uniquely, the expression of miR1507 in response to B. japonicum was influenced only by GmNARK1 and not by GmNFR1. It was the opposite for miR1515 whose expression was influenced by GmNFR1, but not by GmNARK1.In order to further study the function of these miRNAs, we predicted their targets. We searched the soybean genome database, and got 19 sequences as candidate targets from 1505 ones. The 19 putative targets were analyzed by qualitative Reverse Transcriptase-PCR, RNAs were from W82 different organs and different time points same as previous described. The expression pattern of miR1521 and its target Gm0022x00379 gene showed a very good inverse expression pattern. Experimental validation of miR482, 1512, and 1515 targets was conducted by 5'RACE analysis. Cleavage products for all examined targets at the expected site of miR- binding except for the target of miR1512 were detected, these results suggested that the target gene prediction was reliable. For miR1512, some other regulation mechanisms existed to control their gene expression levels.miR482,miR1507,miR1512 and miR1515 were used for over expression analysis from the 6 microRNAs, these were selected based on the expression of their targets during nodule development. Two sets of gateway vectors were constructed, they were driven by a strong constitutive promoter from CsVMV, and the rhizobium-responsive soybean promoter ENOD40, respectively. Overexpressed miRNAs were validated by Northern blot analysis, the RNAs were isolated from transgenic hairy roots. Three repeats were conducted to study the function of miRNAs in nodulation by composite hairy root system. In CsVMV drove over expressed system, miR482 exhibited a dramatic increase about 2 fold in the formation of nodules compared to control vector roots, miR1515 had 1.5-fold higher nodule count compared to vector controls, which is statistically significant (P<0.05). These results provided genetic evidence that miR482 and miR1515 might regulate nodulation in soybean roots. Over-expression of miR1512, miR1507 did not alter nodule numbers significantly. In soybean ENOD40 drove over expressed system, roots expressing miR482 in a rhizobium-inducible manner exhibited a dramatic increase (2-fold higher) in the number of mature nodules compared to control vector roots, miR1512 increased root nodule numbers too. However, miR1515 add miR1507 did not significantly alter the number of nodules. The results suggested that miR482, miR1512, and miR1515 might play specific and important functions during soybean nodulation.