Precursor Cloning And Initial Research On Function Of MiRNAs Related To Ericoid Mycorrhizal Symbiosis In Vaccinium

The fruit of Vaccinum is rich in carbohydrate and a variety of trace elements,which has a variety of physiological activities.Vaccinum roots generally form mycorrhizal through mycorrhizal fungi symbiosis to enhance the ability of Vaccinum to resist environmental stress and absorb nutrients and water from the soil.Such mycorrhizal symbiosis with mycorrhizal fungi is commonly referred as ericoid mycorrhizal.Ericoid mycorrhiza is an endophytic mycorrhiza in which mycelia colonize cells.miRNAs play an important role in mycorrhizal formation.To search miRNAs involving in mycorrhizal symbiosis and their function mechanism,this study analyzed differentially expressed miRNA target gene function and the role in miRNA regulating their target genes through significantly high expression miRNAs in the process of mvcorrhizal infection.After cloning of miRNA precursor,we analyze relative expression of overexpressed and silenced miRN As related to mycorrhizal symbiosis.Main results for this study are as follows:Nine miRNAs with significant changes in mycorrhizal formation were selected from a large number of miRNAs.Vaccinum culture seedlings was inoculated with mycorrhizal fungi ZE103.Transcriptome sequencing of non-inoculated and inoculated for 1-,2-,3-week roots was performed.Transcriptome after filtered were mined to screen out nine target gene Unigene of differential expressed miRNAs.Go enrichment analysis of 149 miRNA target genes showed that miRNA target genes were mainly enriched in five subclasses:Cell,Cell part.Organelle,Cellular process and Binding.KEGG pathway analysis showed that miRNA target genes were mainly involved in the processing of some proteins and the formation of spliceosomes.Functions of these miRNA target genes are mainly distributed in mycorrhizal formation,cold tolerance,lateral root development and SPL transcription factor regulation.In most cases,the expression of miRNA target genes showed a decreasing trend with the increase of matching miRNA frequency.Precursor sequences of nine differentially expressed miRNAs were predicted through expressed sequence tags.PCR and cloning sequencing verified that the predicted sequences were the precursors of nine miRNAs.Structure analysis showed that nine pre-miRNAs precursors had hairpin loops,and miRNA in one arm showed typical stem-loop structure of pre-miRNA.In addition,multi-sequence alignment showed that pre-miRNA was less conserved in different species,while miRNA was more conserved in different species.The results of evolutionary analysis showed that the genetic relationship of pre-miRNA was close to that of the precursor sequence of poplar and sweet orange,but which was far from herbs such as flax mustard and short-stalk grass.Real-time PCR results showed that the relative expression levels of some pre-miRNAs in Vaccinum roots,stems and leaves were different.Among 9 pre-miRNAs.3.4 and 2 pre-miRNAs had relatively high expression levels in roots,stems and leaves respectively.Pre-miR157?pre-miR7122a and pre-miR535d expressed highly in roots,pre-miR166a,pre-miR162a,pre-miR168a and pre-miR156 expressed highly in stem,while pre-miR396b and pre-miR159a had relatively high expression level in leaf.miR157a-5p and miR535d overexpressed and silenced vectors were constructed based on the template PYBA-1132 vector,and the vectors were transformed into Vaccinum cells through agrobacterium-mediated transformation.The growth states of miRNA overexpressed and silenced plants were compared after the transformed plants were cultured.The growth status of miR157a-5p overexpressed seedlings was better than that of control plants and miR157a-5p silenced seedling plants,and the growth status of Vaccinum roots in each week was roughly similar to that of seedlings..However,in miR535d,seedling growth in miR157a-5p overexpressed and silenced plants were worse than control plants,while root growth in miR157a-5p overexpressed plants was better.The relative expressions of miR535d and miR157a-5p with their target genes in each plant were detected by real-time fluorescence quantification PCR.The results showed that miRNAs in overexpressed plants had a significant trend of increase,while the relative concentration of target genes was reduced.The miRNA expression in silenced plants was significantly reduced.The growth and physiological indicators of miR157a-5p transformed plants showed that the plants had poor growth potential,the activity of anti-stress related physiological active substances decreased,and the mycorrhizal formation was seriously delayed.The content of stress-related physiological active substances in miRNA silencing plants increased and the mycorrhizal formation efficiency was improved.