Molecular Basic Analysis Of MiR156 Regulating Mulberry Vegetative Phase Transition And Response To Silkworm Herbivory

Mulberry is perennial woody flowering plant,which is widely cultivated in the tropical,subtropical and temperate regions of the world.Mulberry leaves are the main food source for silkworm,“mulberry planting and silkworm rearing”as a traditional culture in china which can be traced back to over one thousand years ago.Mulberry branches,fruits and leaves are rich in flavonoids,polysaccharides,and terpenes.Moreover,mulberry has good salt tolerance and drought resistance,can adapt to a variety of harsh growth conditions and improve the surrounding ecological environment.miRNAs are small non-coding RNAs with a length of 20-24nt,which are widely involved in plant phase development and metabolism.Studies showed that miR156determined the juvenile-adult phase transition,coordinated the allocation of energy between plant growth and stress response and involved in response to biotic stresses(salt,drought,and fungal)and abiotic stresses in Arabidopsis thaliana,Populus trichocarpa,and Malus Domestica.Perennial woody plants need to go through a sufficient long juvenile phase before grow into maturity.The juvenile stage of mulberry lasts for 1-3 years,which severely restrict the process of mulberry breeding.It is of great significance to uncover the molecular mechanism of juvenile to mature stage transformation of mulberry to guide the breeding of mulberry.In this study,mulberry mature miR156s were identified based on mulberry genome and small RNA high-throughput sequencing data,the molecular basis of miR156 regulating mulberry traits was analyzed by bioinformatics,degradation sequencing and a series of molecular biological methods,this work preliminarily elucidated the molecular mechanism of miR156 regulating mulberry development and responding to bite stress,provided molecular reference for the new mulberry varieties breeding.The main results were listed as follows:1.mno-miR166b can be used as a reference gene for mulberry small RNA RT-qPCR experiments.The reliability of the result of RT-qPCR can be ensured by the stable internal reference gene.To verified the stable internal reference for small RNA RT-qPCR in mulberry,the small RNA high-throughput sequencing data of three tissues(leaf,bark,and male flower)of Morus notabilis L.were analyzed and 7 miRNAs(mno-miR159a,mno-miR160b,mno-miR162,mno-miR166b,mno-miR168b,mno-miR393a,and mno-miR2111)with stable expression levels were identified,and standard deviation analysis showed that mno-miR166b had lowest standard deviation value.The result indicated that the expression level of mno-miR166b was the most stable among all miRNAs identified by sequencing,and it was most likely to become the reference gene in the mulberry small RNA RT-qPCR experiment.The expression stability of mno-miR166b and commonly used reference genes(5.8s r RNA and U6)in quantitative small RNA experiments were compared in mulberry stem apex,young leaves and mature leaves during mulberry juvenile and mature phase by four algorithms(ge Norm,Norm Finder,Best Keepe and?C_T).The result showed that mno-miR166b had the highest expression stability among three candidate reference genes.The expression level of miR156 in four mulberry tissues(root,bark,leaf,and male flower)was detected by RT-qPCR using mno-miR166b as a reference gene,which was consistent with the results of high-throughput sequencing of small RNA.These results indicated that mno-miR166b could be used as a stable reference gene to correct the experimental results of mulberry small RNA RT-qPCR.2.Identification of mulberry miR156 and its target geneThis study identified a total of 7 miR156 mature sequences respectively named mno-miR156a-g by analyzing the high-throughput sequencing results of small RNA in Morus notabilis L.three tissues(leaf,skin and male flower).The sequence length of miR156s were 21 or 22nt and the results of multi-sequence alignment showed that mature miR156 sequences were highly conserved.In mulberry miR156c significantly higher expressed than other miR156 sequences,and its base sequence was completely consistent with the miR156 mature sequence identified in Arabidopsis,soybean(Glycine max)and apple(Malus domestica).These results suggested that the role of miR156 in mulberry may be similar to that in Arabidopsis.Northern Blot and RT-qPCR were used to detect the expression levels of miR156 in three mulberry tissues(roots,bark and leaves)at different developmental stages.It was found that the expression level of miR156 in younger tissues was higher than that in mature tissues.which suggested that mno-miR156 is involved in the juvenile-adult phase transformation.Micro RNA degrades target genes at m RNA level or inhibits the translation process of target genes.A total of 115 miR156 target genes in mulberry were predicted using ps RNATarget software.GO analysis of predicted target genes showed that more than50%of the genes had"Binding"function.A total of 13 target gene sequences were obtained under strict prediction condition with the"expectation value"less than 2.5.and 7 target genes sequences belonged to SPL gene family,3 target genes sequences were WD40 repeat-like genes,1 sequence was fructose-1,6-diphosphatase gene and 2sequences could not found in mulberry database or NCBI(National Center for Biotechnology Information).Degradation sequencing data(P<0.05)revealed that 7sequences belong to SPL gene family(SPL2,SPL4,SPL6,SPL13,SPL15,SPL16A and SPL16B)were identified as miR156 target genes.RLM-5'RACE experiment confirmed the existence of a single degradation site in mulberry SPL2(11/12),SPL13(9/10)and SPL16A(11/12),and two degradation sites in SPL15(9/10 and 11/12)and SPL16B(11/12 and 12/13).Dual-luciferase reporter system verified that mno-miR156 directly inhibited the expression of SPL2,SPL7,SPL14,SPL15,SPL16A and SPL16B.Based on the experimental data of degradation library,RLM-5'RACE and dual-luciferase reporter system,9 SPL genes(SPL2,SPL4,SPL6,SPL7,SPL13,SPL14,SPL15,SPL16A and SPL16B)were identified as the target gene of miR156 in mulberry.Suppressed or over-expressed miR156 in mulberry,the expression level of SPL2,SPL4,SPL6,SPL7,SPL13,SPL14,SPL15,SPL16A and SPL16B were significantly increased or decreased,which verified the reliability of degradation sequencing data and molecular experiment results of mulberry.The result of RT-qPCR showed that the expression level of Morus002371?Morus011698,Morus017876,Morus024571,Morus014729,Morus027934,Morus021827 and Morus006755 changed significantly after suppressed or over-expressed miR156 in mulberry.These genes were from ps RNATarget prediction result(E?2.5)or degradation library data(P?0.5).Dual-luciferase assay verified that miR156could recognize the predicted miR156 cleavage site of Morus024571,Morus011698,Morus017876,Morus027934,Morus006755 and Morus021827.Based on the results of RT-qPCR and Dual-luciferase assay,we were of the opinion that these 6 genes(Morus024571,Morus011698,Morus017876,Morus027934,Morus006755 and Morus021827)were miR156target genes in mulberry.3.Identified the downstream regulatory network of SPL in mulberry15 sequences containing SBP domains were screened in mulberry database by bioinformatics methods,and named according to their evolutionary relationship with SPL gene in Arabidopsis thaliana.In this study,13 SPL genes were successfully cloned from Mulberry(except SPL3 and SPL5).,and one of the predicted sequences(SPL10)had two alternative splices(SPL10A and SPL10B).Tissue expression profile analysis revealed that the SPL genes were differentially expressed in 5 tissues(winter buds,male flowers,roots,bark and leaves).SPL6,SPL8,SPL10,SPL15 and SPL16A were highly expressed in winter buds.The expression levels of SPL2,SPL7 and SPL16B were the highest in male flowers.SPL1,SPL13 and SPL14 were mainly expressed in roots,while SPL4 and SPL12 were highly expressed in leaves and bark,respectively.This suggested that there were functional differences in SPL genes.The RT-qPCR results showed that other SPL genes except for SPL2 were highly expressed in mature leaves at the mature stage and lowly expressed in mature leaves at juvenile phase.The expression trends of SPL genes and mno-miR156 in mulberry leaves at different developmental stages were opposite,suggesting that SPL genes might be affected by age factors.miR172 had been identified in a variety of angiosperms and was a class of highly conserved plant-derived miRNA.High-throughput sequencing of small RNA showed that there were 6 miR172 genes named mno-miR172a-f in mulberry.Multiple sequence alignment analysis revealed that miR172 sequences in mulberry were very conserved.Small RNA RT-qPCR analysis showed that the expression of miR172 in mulberry leaves increased with age.When mno-miR156 was overexpressed in mulberry leaves,and the expression level of mno-miR172 was significantly decreased.Reduced the expression level of mno-miR156 in mulberry leaves by STTM technique,the expression level of mno-miR172 was significantly increased.Yeast one-hybrid experiment results revealed that 6 SPL genes(SPL2,SPL7,SPL10A,SPL10B,SPL15and SPL16A)could recognize a sequence containing two consecutive GTAC elements on the promoter of mno-miR172.Dual-luciferase assay confirmed that the fluorescence values of firefly fluorescent proteins were significantly increased after overexpression of 6 SPL genes(SPL2,SPL8,SPL10A,SPL10B,SPL15 and SPL16A),indicating that these 6 SPL genes could directly promote the expression of mno-miR172.Integrating the experimental results of the yeast single hybrid and dual-luciferase reporter system,6 SPL genes(SPL2,SPL8,SPL10A,SPL10B,SPL15 and SPL16A)were finally determined as the upstream target genes of mno-miR172,which directly promoted the expression of mno-miR172.Transcriptome data revealed that SPL genes in mulberry leaves respond to silkworm biting.The expression levels of SPL7 and SPL14 were significantly increased,SPL2and SPL16A were significantly decreased in Chuansang leaves bitten by silkworm,while the expression levels of almost all SPL genes were inhibited in Guishangyou 62leaves treated with silkworm biting.It was indicated that there was the difference between the two mulberry trees in response to silkworm biting treatment.RT-qPCR results showed that the expression levels of miR156 were significantly increased in Chuansang and Guishangyou 62 leaves treated with silkworm biting,indicating that miR156 in mulberry responded to the silkworm biting.The analysis of gene expression patterns in mulberry flavonoid synthesis pathway showed that the expression levels of TT2L2,GL3,TTG1,FNS,F3'H,DFR and LAR were significantly increased in Chuansang leaves bitten by silkworm,while the expression levels of FNS,F3'h,DFR,LAR and GL3 were significantly decreased in Guisangyou 62 leaves treated with silkworm biting.Dual-luciferase assay showed that SPL7 could recognize the gga CGTACa site on the Mn TT2L2 promoter and promoted the expression of Mn TT2L2.Therefore,after Chuansang leaves were bitten by the silkworm the expression of SPL7was increased,which promoted the expression of Mn TT2L2.Then,the activity of TT2L/GL3/TTG1 ternary complex was increased,which promoted the expression of genes(F3'H,DFR and LAR)involved in the proanthocyanidin synthesis pathway.In summary,we found that the expression level of miR156 in mulberry gradually decreased with the increase of age,and SPL gene and miR172 showed an opposite expression trend to miR156.There were 7 miR156s,15 SPL genes and 6 miR172s in mulberry.Nine SPL genes(SPL2,SPL4,SPL6,SPL7,SPL13,SPL14,SPL15,SPL16A and SPL16B)were the target genes of mno-miR156 and six SPL genes(SPL2,SPL8,SPL10A,SPL10B,SPL15 and SPL16A)can directly promote the expression of mno-miR172 in mulberry.The miR156/SPL/miR172 regulatory pathway was identified in mulberry and Arabidopsis thaliana,which indicated this pathway performed the same roles in mulberry.In mulberry,miR156 and SPL7 independently responded to silkworm baiting.This work uncovered the molecular mechanism of miR156/SPL/miR172regulation pathway,found miR156 and SPL7 independently responded to silkworm baiting,identified the molecular mechanism of Mn SPL/Mn TT2L2 in regulatory of catechin synthesis gene expression,our study provided basic molecular data for the mechanism study of mulberry development regulation and response to silkworm biting.