Cloning And Expression Analysis Of BR-related Gene CBB1,miR492 And Its Target Gene BAK1 In T337

As the widely used dwarfing apple rootstocks, T337 has been applied in the production of apple. But itsgrowthmechanism has not been fully understood. Previous studies indicated that plant hormones play an important role on dwarfing rootstockstem elongation growth. This showed that plant hormones have an effect on dwarfing rootstock height.The study on the model plant Arabidopsis and rice showed that brassinosteroid（BR） plays an important role in regulating the internode elongation、 vascular differentiation、 leaf morphology、 breeding、 aging and disease resistance. This indicated thatthere is a close relationship between BR or its related gene and plant height. However,on the mechanism,no study wasreported in woody plants,especially in apple rootstocks. This study was designed to reveal thefunctionsof BR and its related gene on regulating the height of T337,to expand the hormonal research field on dwarfing rootstock.The T337 potted seedlings were used as plant materials. CBB1, the key synthesis gene of BR,were selected as the research object.At the same time, according to the results of degradation and miRNA sequencing, new apple miR492 and its target gene BAK1 which is involved with BR signal transduction were also choosen. And we explored their roles in growth and development of T337.The results are as follows:1 、 By using T337 cDNA as template,MdCBB1.1,MdCBB1.2 and MdBAK1.1,MdBAK1.2 were obtained,their length were 1781 bp, 1878 bp, 2090 bp and 2058 bp respectively. Their ORF（open reading frame, open read frame） were 1707, 1707, 1851 bp and 1848 bp, encoding 568, 568 and 617, 616 amino acid respectively;miR492 gene and its promoter were cloned by the T337 DNA,Their length is188 bp and235bp、708bp respectively.2、Bioinformatics analysis show that 2CBB1 genes were identified from the apple genome. With FAD_binding₄ domain, MdCBB1.1 and MdCBB1.2 are typical CBB1 protein;Phylogenetic tree analysis revealed that MdCBB1.1 and MdCBB1.2 existthe closest relationship with prediction of the apple CBB1 protein in the NCBI database.It was found that BL（synthetic analogue of BR） increased the expression level of MdCBB1.1 and MdCBB1.2 in xylem,significantly. MdBAK1.1 and MdBAK1.2contained the typical domains of BAK1 protein:LRRNT₂ 2, LRR₈, LRR₁ and Pkinase domain;MdBAK1.1 and MdBAK1.2 with the predicted apple BAK1 protein of NCBI database were lacted in the same clade and almost exactly the same,and the credibility of Phylogenetic treewas 100%. the expression level of MdCBB1.1,MdCBB1.2,MdBAK1.1 and MdBAK1.2in xylem was also increased by BL. It predicted that their expression level was induced by exogenous BR.And exogenous BL promoted the increase of T337 plant height.3、The structure of the mi R492 gene was found to meet the structural characteristics of mi RNA: Without an open reading,miR492 can’t encodea functional protein, its function depends on mature miR492 after cutting from a hairpin structure. And the length of miR492 is 21 nt, there are 3 mismatchbetween miR492 and the target genes. There are binding sites of BR transcription factor MdBZRs, non-E-box、 G-box and E-box elment, in the promoter region of miR492.In addition, the miR492 of T337 shared a high degree of homology with mi R172 in other species.The expression patterns of miR492 and MdBAK1.1 and MdBAK1.2 in different tissues and periods were just opposite. Firstly the expression level of MdBAK1 was decreased and then increased in xylem during the periods; however, Firstly the expression pattern of miR492 was reverse. The expression level of MdBAK1.1 and MdBAK1.2 was induced, but that of miR492 was suppressed. These indicated that there is a targeting relationship between miR492 and MdBAK1.4 、 The cleavage site of MdBAK1.1 and MdBAK1.2 was verified by 5 ’rapid amplification of cDNA ends（race）.The results show that mi R492 cut out MBAK1.1 and MBAK1.2at the 10 th and 11 th nucleotides, and the cleavage site is located in the protein kinase domain of MdBAK1 protein.Combined with degradation sequencing and qRT-PCR,MdBAK1.1 and MdBAK1.2 were confirmed as the target genes of mi R492.To sum up, it can be proved that BR can promote the increase of plant height of T337 through upregulating expression level of MdCBB1.1 、 MdCBB1.2 and MdBAK1.1 、MdBAK1.2,while miR492 suppress T337 stem elongation byinhibiting expression level ofMdBAK1.1and MdBAK1.2.