| In the practice of apple seedling breeding in China,the rooting ability of cloned dwarf rootstocks is generally poor,which has become an important bottleneck restricting the breeding of dwarf rootstocks and high-quality seedlings.Research on the molecular regulation mechanism of adventitious roots(AR),which is of great significance to develop apple clone rootstock breeding and molecular breeding.Studies found that mi RNAs play a wide role in the growth and development of plants.Our team found that mi R393 may be involved in the regulation of adventitious root development of apple rootstocks in the previous mi RNA sequencing.In this study,tissue culture seedlings of ’T337’ apple rootstock were used as test material.The functions of mi R393 and its target genes in AR development of apple rootstocks were in-depth analysis by bioinformatics analysis,RT-q PCR,and transgenic techniques.The main results are as follows:1.The evolutionary tree and multiple sequence alignment analysis of the mi R393 family revealed that mi R393 evolved conservatively among different species.We have cloned mdm-MIR393 a and mdm-MIR393 b from apple rootstock ’T337’,and found that the sequences of mdm-MIR393 a and mdm-MIR393 b were completely consistent with the genomic sequence of ’Golden Delicious’.In addition,different members of mi R393 precursor can form a stable stem-loop structure.The promoter of mi R393 precursor contains multiple cis-acting elements that respond to ABA,stress and light signals.It was predicted that there are 6 target genes of apple mi R393,of which 3 target genes of mdm-mi R393a/b/c belong to members of the auxin receptor protein Md TIR1/AFBs family.It was found that different members of the apple mi R393 family had similar expression patterns in different tissues of apple rootstock ’T337’ by RT-PCR;During the AR formation of apple rootstock ’T337’,mi R393 can change the expression level in response to the exogenous auxin,among which the expression level of mdm-mi R393a/b/c was significantly up-regulated at the critical period of AR formation.2.A total of 12 Md TIR1/AFBs family genes were obtained in apple by using the homologous genes of Arabidopsis thaliana to complete genome alignment.Phylogenetic trees of TIR1/AFBs family genes of Arabidopsis and apples were constructed,which could be classified into three subgroups(G1,G2 and G3).We have systematically analyzed the gene structures,conserved motifs,physicochemical properties,and promoter elements of the Md TIR1/AFBs family,and revealed that there are 1-14 exons and 0-13 introns existed in Md TIR1/AFBs;It was showed that 3-10 conserved motifs existed in the Md TIR1/AFBs proteins.The analysis of physical and chemical properties revealed that Md TIR1/AFBs proteins contained 222-1260 amino acids and the isoelectric point were from 5.40 to 9.22;The Md TIR1/AFBs promoter contained 18-44 cis-acting elements in response to hormones,stress,and light signals.The key member Md TIR1 A was cloned with a sequence length of 1764 bp.Tissue-specific expression analysis revealed significant differences in the expression levels of 12 genes in different tissues.RT-q PCR results showed that Md TIR1 A,Md TIR1 D,Md AFB1,Md AFB2,Md AFB3,Md AFB4 and Md AFB8 can respond to the induction of exogenous auxin in the critical period of AR development and the expression level was down-regulated.3.We identified Md TIR1 A was the target gene of mdm-MIR393 b through agrobacterium tumefaciens infiltration assay in Nicotiana benthamiana as well as degradome data analysis.The activity of mdm-MIR393 b promoter can be activated by exogenous IBA,but can be inhibited by NPA(auxin transport inhibitor).Overexpression of mdm-MIR393 b reduced the expression of the target gene Nt TIR1 in tobacco.Compared to wild type,the AR of transgenic tobacco were longer and decreased in number,and the transgenic tobacco showed a decreased sensitivity to auxin.The results indicating that mdm-MIR393 b can target Md TIR1 A and played a role in AR development in plants through auxin signal response. |
