Screening Of Differentially Expressed RNA Between Rootstock And Scion Of Apple And The Function Of MdAGL24 Regulating Flower Buds

Grafting is widely used in apple industry.Grafting scions on dwarfing rootstocks（such as M9,T337 and M26）is beneficial to dwarf apple tree,accelerate flower,and improve fruit yield as well as quality.At present,‘Fuji’is the main cultivar of apple in China,and its total cultivated area accounts for about 75%.However,‘Fuji’shows difficultly flowering,poor quality of flower bud,and alternate bearing,which seriously restrict the development of higher-quality and higher-benefit in apple industry.The application of dwarfing rootstocks effectively solves the problems on flowering of Fuji.However,the molecular mechanism of dwarfing rootstocks regulating the flowering characters of Fuji scions is not clear.Therefore,we used‘Nagafu No.2’self-rooted seedling,M9 self-rooted seedling and‘Nagafu No.2’/M9grafted seedlings to analyze differentially expressed mRNAs,miRNAs and lncRNAs as well as flowering related RNAs which were caused by grafting in this study;this research will lay the foundation for elucidating the molecular regulation mechanism of dwarfing rootstocks promoting flowering of scion.Moreover,the key flowering gene MdAGL24 was selected as research object.We cloned MdAGL24,and analyzed its promoter and coding sequences,subcellular localization,and expression pattern.The function of MdAGL24 was studied in transgenic tomato.Yeast two-hybird and one-hybird assays were used to screen interacting proteins and transcriptional factors which regulated MdAGL24,and we verified interacting components of MdAGL24 through bimolecular fluorescence complementation（BIFC）and dual luciferase technologies.The main results were obtained as follows:1.Using phloem of the up and down of graft union of‘Nagafu No.2’/M9 at the thirtieth day after full blooming,RNA-seq was completed with three biological repetitions.There were about 36G sequencing data in 6 libraries.（1）there were 1,593 differentially expressed genes on up graft union/down graft union,of which 971 genes were up-regulated and 622 genes were down-regulated.These differentially expressed genes mainly involved in photosynthetic process,metabolic process,secondary metabolism,glycolysis,carbon fixation and fructose metabolism through GO and KEGG analyses.There were 45 transcription factors among them,and most of transcription factors belong to apple MADS family protein,such as MdAGL24,suggesting their important roles in dwarf rootstocks-mediated scion flowering.（2）In addition,lots of auxin Aux/IAA genes were significantly differentially expressed between on up and down graft union,indicating their important functions in flowering control.Therefore,we identified Aux/IAA（IAA）family gene in apple,analyzed their chromosomal location and synteny of between MdIAAs and Arabidopsis IAA genes;Real time fluorescence quantitative PCR（qRT-PCR）analysis showed that the expression levels of MdIAA22（MD09G1202300）和MdIAA51（MD17G1183500）were significantly lower in down graft union than up graft union,indicating that they may play important roles in dwarfing rootstocks inducing scion flowering.2.A total of 18 sRNA libraries with three biological repetitions were constructed using shoot tips of‘Nagafu No.2’（BS）,shoot tips of M9（RS）,and shoot tips（BG）,phloem of the up（UP）and down（DP）of graft union,as well as root tips（RG）of‘Nagafu No.2’/M9.（1）In BS,RS,BG,UP,DP and RG,11,686,623、11,590,477、11,775,098、11,874,809、11,663,829and 11,896,826 clean reads were respectively obtained.206 known miRNAs belonged to 42conserved miRNA families,and 976 new miRNAs were identified.（2）The results of cluster analysis indicated that all known and novel miRNAs had six significant response types to grafting of the six samples.Among the known miRNAs,30 were up-regulated in BG relative to BS,and 43 were up-regulated in RG relative to RS,115 were up-regulated in UP relative to DP;in the novel miRNAs,168,268 and 54 have the same expression pattern,respectively.（3）There were separately 1,051 and 7,473 target genes for known and novel miRNAs,such as MdmiR156-MdSPLs,MdmiR172-MdAP2,MdmiR171-MdAP2,MdmiR159-MdMYB and MdmiR393-MdARFs,respectively.These miRNAs and their target genes were predicted to involve in growth and development,and flowering of scion.3.Twelve RNA-Seq libraries with three biological repetitions were constructed with the young fruit（YT）,shoot tips（ST）,phloem（SP）and root tip（RT）from 4-year-old‘Nagafu No.2/T337/Malus Robusta’.（1）129,626、770,132、222,062、134,814、512 and 132,220,946 clean reads were identified in ST,SP,YF and RT,respectively.After filtering,1,726 hc-lncRNA(high confidence lncRNA were obtained.（2）There were respectively 522、551、553 and 631hc-lncRNAs in YF,RT,SP and ST;and 85、33、36 and 45 target genes of 850 hc-lncRNAs were characterized in above four tissues.（3）Further,the comparison of differentially expressed lncRNAs between rootstock-sicon revealed that 25 down-regulated and 16up-regulated lncRNAs in SP and 76 down-regulated and 43 up-regulated lncRNAs in ST compared with RT,including TCONS₀0118115,TCONS₀0090580 and TCONS₀0043576,with target genes of AP2 and AGL24,respectively.Our results indicated that lncRNA also participates in the molecular regulatory network of apple flowering.4.In order to reveal the function of MdAGL24,we cloned it,analyzed its promoter activity and expression pattern,constructed its transgenic tomato,and screened its interacting proteins and transcription factors.（1）Its length was 765 bp,encoding 254 amino acids;it contained highly conserved MADS and K-box domains.Subcellular localization analysis revealed that it was located in nucleus.qRT-PCR analysis showed that it mainly expressed in buds;moreover,it was obviously induced by GA₃ treatment,while did not respond to ABA,SA and MeJA treatment.（2）The core sequences of its promoter were identified by activity assay.Yeast one hybrid was used to screen upstream regulators of MdAGL24,and we identify that MdHY5 can target MdAGL24 through yeast one hybrid and double luciferase technologies.（3）A yeast two hybrid library was constructed with short shoot buds at different developmental stages;a total of 49 proteins which interact with MdAGL24 were obtained from the yeast two hybrid library,and 9 of them were transcription factors;Through BIFC,MdAGL24 has been found to interact with several proteins that directly regulate flowering,such as MdAGL19,MdAGL42,MdSOC1a/b and MdTIFY6b,indicating that MdAGL24 plays a direct role in flowering control.（4）The expression levels of flowering related genes MdFT and MdSPL3 was higher in overexpressing MdAGL24 tomato than wild type plants,and transgenic plants showed early flowering and early fruit.In summary,the expression patterns of differentially expressed mRNAs,miRNAs and lncRNAs in grafted apples were identified,which constructed the regulation network of dwarfing rootstocks-mediatied scion flowering induction.Moreover,the function of MdAGL24 and its interaction proteins were characterized.The findings in this study laid foundation for analyzing physiological and molecular mechanism of apple dwarf rootstock mediated scion flower formation in future.