| The cultivation area and yield of apple rank first in the world.Apple industry has important significance to the development of China’s economy and the adjustment of industrial structure.Dwarf rootstock planting cultivation is the direction and trend of apple development.Apple rootstock ’M26’ has become a widely used rootstock because of its dwarf tree architecture,high grafting survival rate,wide adaptability,strong resistance and early flowering and fruiting.However,’M26’ asexual propagation is more difficult to root.In this study,the effects of indole-3-butyric acid(IBA)and auxin polar transport inhibitor(NPA)on adventitious root formation,ethylene release,IAA,IBA and ABA content and auxin distribution in root tip of’M26’ were investigated.The differential genes related to auxin and ethylene during the formation of apple adventitious roots were screened by transcriptome analysis.At the same time,the NPA-related PIN family and the IBA-related ARF family were identified and their expression patterns during adventitious root formation were analyzed.The main results are as follows:1.Exogenous IBA promoted the formation of adventitious roots of ’M26’,which showed an increase in the number of adventitious roots,root volume,root surface area and root diameter.Compared with the control,NPA delayed the formation of basal callus and root primordium,thereby inhibiting the formation of adventitious roots.IBA+NPA did not find callus and root primordium formation in the first 20 days of treatment,and formed slender adventitious roots at 27 days.IBA induced the release of ethylene,NPA inhibited the release of ethylene,and the base of the plant was the main release site of ethylene.The auxin in the root tip of the four treatments was mainly concentrated in the meristem zone and elongation zone at 27 d,and the auxin under NPA treatment was the least.In summary,IBA promoted the formation of adventitious roots,and NPA inhibited the formation of adventitious roots.The qRT-PCR analysis showed that IBA induced the up-regulated expression of auxin-related genes MdIAA1like,MdIAA9,MdIAA12,MdIAA3,MdIAA6 and MdIAA4 and ethylene-related gene MdCRF2,but inhibited the regulated expression of MdRAP2-7,MdERF034-like and MdMLP-like while NPA treatment was the opposite,indicating that auxin and ethylene key genes were involved in the formation of adventitious roots.2.Bioinformatics was used to identify PIN gene families in the whole genome of apple,and their physical and chemical properties,evolutionary relationships,chromosome distribution and gene structure were analyzed.qRT-PCR was used to analyze the expression patterns of PIN gene family members during adventitic root.The results showed that there were 13 members of apple PIN gene family,which were distributed on 8 chromosomes.The amino acid length was between 357 and 660 aa,the protein molecular weight was between 38.84 and 71.61 kD,and the isoelectric point was between 6.93 and 9.35.Analysis of promoter elements showed that 13 PIN promoters contained cis-acting elements related to hormone,growth and stress response,of which 8 genes contained auxin response elements,suggesting that they may be involved in auxin regulation of adventitious root formation.Transcriptome analysis showed that 6 of the 13 PIN family members,MdPIN3,MdPIN4,MdPIN6,MdPIN7,MdPINII and MdPIN12,were up-regulated under IB A treatment.Further qRT-PCR analysis showed that MdPIN3,MdPIN4,MdPIN6 and MdPINI1 had a significant upward trend under IB A treatment,and the expression of MdPIN4 changed the most.It is speculated that MdPIN4 may play an important role in regulating the formation of adventitious roots of apple rootstocks.3.Bioinformatics was used to identify and analyze the ARF gene family in the whole genome of apple,and qRT-PCR was used to analyze the expression patterns of ARF gene family members in the adventitious root formation of apple.The results showed that a total of 29 MdARF genes were identified in apple,which were distributed on 16 chromosomes.The amino acid length was between 587-1151 aa,the protein molecular weight was 65.37-128.4 KD,and the isoelectric point was between 5.19-8.44,Subcellular localization prediction was located in the nucleus.The analysis of promoter action elements showed that auxin,methyl jasmonate and abscisic acid were more in hormone response elements,and low temperature and drought were more in abiotic stress response elements.Among the members of the ARF family,15 genes contain auxin and drought response elements,and 17 genes contain low temperature response elements.It is speculated that multiple MdARF genes may be involved in abiotic stress and growth and development.Seven genes(MdARF27,MdARF13,MdARF7,MdARF29,MdARF4,MdARF15 and MdARF23)with large differences were selected from the transcriptome results for qRT-PCR analysis.IBA treatment significantly induced the up-regulation of MdARF27,MdARF13 and MdARF4.MdARF7 and MdARF23 were down-regulated genes,but the overall change was small.Moreover,the expression levels of MdARF27 genes under IBA treatment at 15 d were 8 times higher than those under CK treatment,MdARF4 varied greatly.Suggesting that MdARF27 and MdARF4 genes play an important regulatory role in auxin involved in adventitious root formation. |
