| Adventitious roots(AR)play an important role in the vegetative propagation of apple rootstocks.At present,the studies on AR development mainly focus on anatomical observations of developmental processes,detection of endogenous hormones,cloning,expression,and functional analysis of related genes,as well as the development of adventitious root regulation techniques.The whole co-expression network in AR formation has not been well established in apple.In order to identify the molecular mechanisms underlying AR formation,the gene regulatory networks in the development of adventitious roots were analyzed and key candidate genes were discovered.In this study,apple rootstocks ‘T337’ were used as test material,transcriptomic changes that occur during key stages of AR formation were analyzed using high-throughput sequencing.The contents of endogenous hormones were determined.At the same time,the process of adventitious root formation was observed at the cellular level to understand the morphological changes of adventitious roots.The candidate gene family members were systematically identified.In addition,the expression profiles of genes in different tissues and stages were identified by RT-qPCR.These studies laid the foundation for further understanding on the molecular mechanism of adventitious root development in apple rootstocks.The results are as follows:1 The process of AR development in ‘T337’ apple rootstock can be divided into three periods: Cambium cell division,AR primordium formation and differentiation formation AR.Exogenous IBA-treated significantly induced the change of endogenous hormone content.The content of AUX and BR in S2 was significantly up-regulated;CTK and JA in S3 were also significantly up-regulated;but GA and ABA in S4 were significantly reduced.A total of 4294 difference expression genes during four stages AR development were identified by high-throughput sequencing.Genes were related to hormones,wounding,sugar signalling,root development,and cell cycle induction pathways.The expression profiles of 47 genes with knownfunction such as IAA17,PIN1,CKX,GA2OX2,EFE1,JAZ12,SUS4,CYCD1;1 and LRP1 were identified.Based on the above study,it is clear that IAA synthesis,signal transduction and transport are the key regulatory pathways in adventitious root formation.2 Through the transcriptome screening of different expression genes,it was found that the MdRR genes have significnat different during the adventitious root formation.Therefore,the MdRRs of apple rootstocks were screened.MdRRs proteins contained 218-777 amino acids and the isoelectric points are from 5.45 to 6.95.Results of exon-intron structures analysis showed that the MdRRs contained 5-10 exons and 4-9 intron.Conservative base sequence analysis showed that 3-10 conserved motifs existed in the Md RR proteins.In addition,the key numbers of MdRR10 and MdRR12 were cloned;the lengths of the sequences were 1914 bp and 744 bp,respectively,and 19 and 32 different nucleotides compared to Golden Delicious apple nucleic acid sequencec.Tissue-specific expressions indicated that 15 MdRR had significantly different expression patterns in different genotypes,as well as different tissues.In apple rootstock,the expression profiles of MdRRs in different tissues and stages were also identified by RT-qPCR.The expression levels of MdRRs were significantly changed by exogenous IBA-treated and MdRR1,MdRR10,MdRR12,Md RR12-1,MdRR18 and MdRR20 were highly expressed in root tissue.3 The candidate gene of CRFs were identified by bioinformatics analysis and named MdCRF1 ~ MdCRF11.Additionally,this study systematically analyzed its physical and chemical characteristics,gene structures,evolutionary relationships,promoter elements and Conservative base sequence.MdCRF proteins contained 137-435 amino acids and the isoelectric points are from 4.26 to 9.68.Results of exon-intron structures analysis showed that the MdCRF1 contained 2 exons and 1 intron;MdCRF11 contained 3 exons and 2 introns;other genes only have exons and withou t introns.Conservative base sequence analysis showed that 5-9 conserved motifs existed in the MdCRF proteins.Phylogenetic analysis showed that MdCRF family genes could be classified into three subgroups(G1,G2 and G3)according to its homology.In addition,the numbers of each subgroup were approximately equal.Tissue-specific expressions indicated that 11 MdCRF had significantly different expression patterns in different genotypes,as well as different tissues.The materials were ‘T337’ apple rootstock;RT-qPCR confirmed most of genes were highly expressed in root,stem and callus.At the same time the expression of these genes were down-regulated by rooting treatment.Additionally,we systematically analyzed the connection network of MdCRF proteins and predicted the potential link between genes.The results showed that MdCRF8,MdCRF10 and MdCRF11 might be involved in regulating adventitious root development in apple rootstock. |
