Study The Role Of Several Key IAA Metabolism Genes In Dwarf Rootstock Induced Scion Dwarfing And The Role Of MiRNA In The Spur Type Apple Dwarfing

Apple dwarf and high density cultivation is the trend of the world’s apple production.Using dwarf rootstock and spur type varieties is the most important way.Although dwarf rootstock and spur type varieties are widely used in production,the dwarfing mechanism,especially the molecular mechanism,has not yet been fully revealed.In this paper,the expression level of key auxin synthesis,transport and metabolism genes were measured in leaves,phloem and roots of the Nagafu2/M9(dwarfing)and Nagafu2/MM106(semi vigorous).Some key different expression gene were also cloned and their function were also studied.We used high throughput mi RNA sequencing to identify the mi RNAs involving shoot development in shoot tips of ‘Nagafu2’(CF)and spur-type bud mutation ‘Yanfu6’(YF).From the perspective of auxin and miRNA,the mechanism of dwarf rootstock and spur type apple was revealed.The main findings are as follows:1.To identify auxin and auxin-related genes that may play roles in the composite tree’s auxin metabolism and dwarfing mechanism,the concentrations of IAA and the expression level of key auxin synthesis,transport and metabolism genes were measured in leaves,phloem and roots from the dwarfing Nagafu2/M9 and the vigorous Nagafu2/MM106.The results showed that the indole-3-acetic acid(IAA)content was lower in the dwarfing Nagafu2/M9 than in the vigorous Nagafu2/MM106.The IAA content in the Nagafu2/M9 rootstock’s phloem was higher than that of its scion phloem.The expression level of MdYUCCA8 andMdYUCCA10 a gene were significantly lower in the leaves and roots of Nagafu2/M9 than in that of the Nagafu2/MM106.The phloem and roots of the Nagafu2/M9 rootstock showed low expression levels of MdPIN1 b and MdPIN8 a expression.The auxin-conjugated gene MdGH3-5b and MdGH3-9a showed lower expression levels in the Nagafu2/M9 than in the Nagafu2/MM106.However,the Nagafu2/M9 showed higher levels of the auxin-conjugate hydrolase genes MdIAR3 c and MdILL6 c.2.The MdYUCCA8 gene was cloned in M9 and MM106.The gene has 1272 bp nucleotide sequence with 99.9% similarity in M9 and MM106,encoding 423 amino acids.The 1600 bp promoter sequence upstream of the MdYUCCA8 coding region was cloned in M9 and MM106 with 99.55% similarity.MdYUCCA8 promoter was induced by GA,MJ and Br.Spermidine and ethephon significantly inhibited the activity of MdYUCCA8 promoter.High temperature promoted the expression of MdYUCCA8.Transient expression assay in tobacco leaves showed that MdPIF4 a interacted with MdYUCCA8 promoter.Ectopic expression of MdYUCCA8 in Arabidopsis thaliana promoted stems growth and inhibited lateral branches growth.The MdYUCCA8 overexpression lines showed silique deformed.Overexpression of MdYUCCA8 promoted the root growth,the lateral root number and the lateral root length were significantly higher than the wild type.3.The MdYUCCA10 a gene was cloned in M9 and MM106.The gene has 1149 bp nucleotide sequence with 99.9% similarity in M9 and MM106,encoding 382 amino acids.The 1800 bp promoter sequence upstream of the MdYUCCA10 a coding region was cloned in M9 and MM106 with 99.55% similarity.MdYUCCA10 a promoter was induced by ABA and Spe.Ethephon significantly inhibitedMdYUCCA10 a promoter activity.Ectopic expression of MdYUCCA10 a in Arabidopsis thaliana promoted shootsand roots growth.The length of roots,the number of lateral roots and the length of lateral roots were significantly higher than those of wild type.4.Apple dwarfing rootstock M9 and vigorous rootstock MM106 were used as the material to clone MdABCB19.This gene has 3753 bp nucleotide sequence encoding a length of 1250 amino acids.MdABCB19 sequence in M9 and MM106 presented of a non-synonymous SNP,leading to the amino acid sequence different at the 885 th amino acids: M9 serine(Ser),MM106 alanine(Ala).Protein structure analysis found that the amino acid point mutation,leading to a shorter alpha helix in M9.Real time quantitative expression analysis showed that,MdABCB19 were differentially expressed in the rootstock M9 and MM106,Nagafu2/M9 and Nagafu2/MM106.Eighteen hundred bp upstream promoter sequences of M9 and MM106 MdABCB19 were also cloned and found 6 bp "CTCTGT" deletion at about the 170 bp upstream of the start codon of M9 MdABCB19 promoter,resulting in missing a 5’UTR Py-rich stretch motif.Promoter cis-acting element analysis shown MdABCB19 promoter region contained several light response motifs.The promoter activity was enhanced with increasing number of 5’UTR Py-rich stretch motif elements.5.A total of 700 mature miRNAs were identified,including 202 known apple miRNAs and 498 potential novel miRNA candidates.A comparison of mi RNA expression in CF and YF revealed 135 differentially expressed genes,most known miRNAs which were downregulated in YF.YF also had lower levels of GA,ZR,IAA,and ABA hormones,relative to CF.Exogenous applications of GA promoted YF shoot growth.In conclusion,auxin play an important role in the rootstock induced scion dwarf.The low expression level of auxin synthesis gene MdYUCCA8 and MdYUCCA10 a in Nagafu2/M9 probably induced the low auxin level.Missing a 5’UTR Py-rich stretch motif in the promoter of MdABCB19 decreased MdABCB19 expression in M9.The low expression of MdABCB19 decrease auxin transport and resulted in dwarf architecture.GA and miRNA play a key role in the spur development of YF.