Identification Of MicroRNAs In Grafted Cucumber Seedlings And Its Response To Abiotic Stresses

Pumpkins (Cucurbita moschata) are usually used as rootstocks for cucumber (Cucumis sativus)grafting to improve cucumber growth and tolerance to abiotic stresses. Plant microRNAs (miRNAs) areinvolved in regulating plant growth and development, and response to various abiotic stresses throughcleavage of target genes at post-transcriptional level. However, the miRNAs-mediated regulation ongrafting-induced enhancement of growth and stress tolerances in cucumber still needs further study.With the aim of probing the molecular mechanism of miRNAs-mediated growth and resistance toabiotic stresses of grafted cucumber seedlings, we constructed eight small RNA libraries from leavesand roots of seedlings that were grafted in the following four ways:(1) hetero-grafted cucumberseedling, using cucumber as scion and pumpkin as rootstock;(2) hetero-grafted pumpkin seedling, usingpumpkin as scion and cucumber as rootstock;(3) auto-grafted cucumber seedling, and (4) auto-graftedpumpkin seedling. By high-throughput sequencing and bioinformatics analysis, a group ofgrafting-responsive miRNAs were identified. And after treatments of drought, high salinity, nitrogen orphosphorus deficiencies, the expressions of miRNAs and target mRNAs in hetero-grafted cucumberseedlings and auto-grafted cucumber seedlings were detected by quantitative real-time PCR (qRT-PCR).The main results are as follows:1. Identification of miRNAs in cucumber and pumpkin. By Solexa high-throughput sequencing andbioinformatics analysis such as BLASTn and Mireap,112known miRNAs belonging to40miRNAfamilies, and48novel miRNAs were identified. The target genes of these miRNAs were predicted.2. Identification of grafting-responsive miRNAs in cucumber. According to the sequencing data,compared with auto-grafted seedlings, expressions of75%known miRNAs were significantly changed.The expressions of10known and16novel miRNA as well19predicted target genes were detected byqRT-PCR, and the expressions of miRNAs and target genes were changed dynamically in leaves androots of hetero-grafted cucumber seedlings. The expression-changed miRNAs and target genes wereconsidered as grafting-responsive miRNAs and targets.3. miRNAs of grafted cucumber seedlings are involved in response to drought stress. Underdrought stress, compared with auto-grafted cucumber seedlings, expressions of most miRNAs in leavesof hetero-grafted cucumber seedlings were dramatically reduced after3h, and then induced after8hand24h of drought stress; expressions of most miRNAs in roots of hetero-grafted cucumber seedlingswere reduced after1h, while induced after3h and8h, and then reduced significantly after24h ofdrought stress. On the other hand, compared with auto-grafted cucumber seedlings, expressions of mosttargets in leaves of hetero-grafted cucumber seedlings were remarkably induced after3h, and thenreduced after8h and24h of drought stress; expressions of most targets in roots of hetero-graftedcucumber seedlings were significantly induced under drought stress.4. miRNAs of grafted cucumber seedlings are involved in response to salt stress. Under salt stress,compared with auto-grafted cucumber seedlings, expressions of miRNAs in leaves of hetero-grafted cucumber seedlings were significantly reduced after2h, while induced after6h and24h of salt stress;expressions of most miRNAs in roots of hetero-grafted cucumber seedlings were induced under saltstress. On the other hand, compared with auto-grafted cucumber seedlings, expressions of most targetgenes in leaves of hetero-grafted cucumber seedlings were reduced after2h, and then dramaticallyreduced after24h of salt stress; most target genes showed induced expressions in roots of hetero-graftedcucumber seedlings under salt stress.5. miRNAs of grafted cucumber seedlings are involved in response to nitrogen (N) or phosphorus(P) deficiencies. Compared with auto-grafted cucumber seedlings, expressions of most miRNAs inleaves of hetero-grafted cucumber seedlings were significantly reduced after1h of N or P deficiencies,and induced after3h of N or P deficiencies, and then reduced after8h of N or P deficiencies, andinduced after24h and48h of N deficiency, while reduced after24h and48h of P deficiency;expressions of most miRNAs in roots of hetero-grafted cucumber seedlings were induced remarkablyafter24h of N deficiency, while induced after3h of P deficiency, and reduced after8h and24h, andthen induced after48h of P deficiency. On the other hand, compared with auto-grafted cucumberseedlings, expressions of most target genes in leaves of hetero-grafted cucumber seedlings were reducedafter1h,3h and8h, and then induced after24h and48h of N deficiency, while induced after1h and3h, and then reduced after8h,24h and48h of P deficiency; most target genes in roots ofhetero-grafted cucumber seedlings showed induced expressions under N or P deficiencies.Our results are helpful for clarifying mechanisms of the miRNAs-mediated roles in improvinggrowth and abiotic stresses tolerance of grafted cucumber seedlings.