Identification And Analysis Of MicroRNA Related To Adversity Stress And Anthocyanin Biosynthesis In Sweet Potato

Sweet potato[Impomoea batatas(L.)Lam],one of the major food and pharmaceutical crops,is a hexaploid(2n = 6x = 90)dicotyledonous plant in the Convolvulaceae family.Sweet potato is an ancient crop in China,with a cultivation history of more than 400 years.The total planting area of sweet potato in China is about 50%of the total area of the earth.The output is nearly 100 million tons,which accounts for more than 80%of the total output of the world.Since the complex of its genetic background,molecular study of sweet potato is laggard comparing with rice,maize etc.The genome sequencing has just been completed and released last year.There are different colors of sweet potatoes which are white,yellow,orange and deep purple.Purple-fleshed sweet potato has been widely favored in light of its high anthocyanin contents,which have strong antioxidant activity.Anthocyanins are naturally occuring pigments belonging to the group of flavonoids,a subclass of the polyphenol family.In plant,anthocyanins have important roles in adversity resistance and plant development.As a naturally antioxidant nutritional food pigments,anthocyanins with high nutritional and health functions has been widely favored.The biothethesis pathway of anthocyanin has widely been studied.The basic framework of the anthocyanins biosynthesis pathway and some related enzyme genes and regulating genes has been revealed.Recently,a number of genes related to adversity resistance and anthocyanin synthesis have been excavated in sweet potato.MicroRNA(miRNA),a class of 20-24 nt endogenous non-coding small RNAs.MiRNA-mediated gene regulation plays a minor role indiverse biological processes,such as plant growth and development,primary and secondary metabolism and adversity response at post-transcription level.Increasely studies showed that miRNA is an important regulatory factor in anthocyanin biosynthesis.However,there are a few studies about miRNA participate in regulating anthocyanin metabolism and adversity resistance of sweet potato.In the study,the transcriptome,small RNA and degradome library from the tuberous roots of white-fleshed(Xushu-18)and purple-fleshed(Xuzishu-3)sweet potato cultivars were constructed.Firstly,we used small RNA and degradome sequencing technology to excavate miRNAs and corresponding targets,which involved in anthocyanin biosynthesis and responded to adversity stress,and the sequencing accuracy was confirmed by qRT-PCR.MYB transcription factor played an important role in regulating anthocyanin synthesis and stress response.Then,we used bioinformatics to identify and analyze the MYB transcription factor family of sweet potato based on the whole genome sequencing data of 1pomoea triloba(l.Iriloba).Finally,the target gene JbMYB4-like of ib-miR858b was cloned and analyzed,which will lay the foundation for functional study.The results in the study are showing as follows:1)A total of 45.4 Gb clean and high quality reads were obtained by transcriptome analyses,and 111,017 unigenes were assembled,which was used as a reference sequence for subsequent experiments.2)From small RNA sequencing data,a total of 191 known and 33 novel miRNAs were identified,among which 121 were differently expressed.For identifying the target genes of miRNAs,degradome sequencing technology was used in our study.A total of 180 target genes from 120 miRNA were identified.QRT-PCR was used to confirm the data accuracy from Small RNA and degradome sequencing.3)Based on the genome information of the diploid wild species and six ploidy species of the sweetpotato,we integrated anthocyanin phenotype,small RNA,degradome sequencing data,GO and KEGG toreveals that 26 key miRNAs and 36 corresponding targets were potentially involved in the anthocyanin biosynthesis,27 miRNAs targeted 38 genes response to adversity stress.4)A total of 160 ItbMYB were identified,which were distributed unequally along its 15 chromosomes.The result of phylogenetic analysis revealed that the 160 ItbMYB genes were divided into 18 subfamalies.In the same ItbMYB subfamily,the motif composition and number were highly conserved,but the number of exons and introns varied highly.5)Based on the classification criteria of Arabidopsis R2R3-MYB,the 120 R2R3-MYB transcription factors were further divided into 30 subgroups.From qRT-PCR result of 10 R2R3-MYB genes under drought and salt stress,we found 2 ItbMYB were inhibited by drought stress,3 ItbMYB and 5 ItbMYB were induced by drought stress and salt stress,respectively.6)In the study,a target gene of ib-miR858b was cloned,named IbMYB4-like.The length of the open reading frame was 771 bp,encoding 241 amino acids with two conserved domains,is a R2R3-MYB transcription factor.In phylogenetic tree,IbMYB4-Iikebe\onged to the class of monocotyledon R2R3-MYB transcription factor and have the closest relationship with Ipomoea nil.By bioinformatics analysis,the protein formula of IbMB4-likewasC2852H4751N955O1177S198 with molecular weight of 77599.67 kDa,and the isoelectric point was 5.00.IbMYB4-like had more phosphorylation sites and was a hydrophilic protein.IbMYB4-like shaped helix-corner-helix in space and was located in the nucleus.The results lay the foundation for further exploring its mechanism of function.In summary,we detected miRNAs and targets,which involved in anthocyanin biosynthesis and responded to adversity stress.It will provide a reference for highly anthocyanin and resistance breeding of sweet potato.These findings will help in understanding the molecular mechanisms underlying regulation of anthocyanin biosynthesis pathway and studying on the function of genes in sweet potato.