ITRAQ-based Proteomics Analysis Of Rooting In Mulberry Hardwood Cuttings And Expression Analysis The Genes MaTIR1 In Mulberry

Adventitious roots are formed from organs that typically do not develop roots, such as leaves and stems. They are essential for the successful clonal multiplication of elite genotypes of forest, horticultural, and agricultural plant species. In this paper, we investigated the proteome changes in Mulberry cuttings during AR formation for the first time, using iTRAQ based quantification. And the MaTIR1 gene, which significantly differential expressed during adventitious rooting, was analyzed by molecular cloning, bioinformatics analysis and expression analysis.The main results and conclusion were as follows:1 To identify the molecular processes that are involved in mulberry root primordium activation, formation, and outgrowth, iTRAQ-based proteome profiles of mulberry cuttings were collected. A total of 404,053 spectra were generated from the iTRAQ experiment. Mascot identified a total of 40,824 spectra matched to known spectra, 36,539 spectra matched to unique peptides, 14,944 peptides, 13,901 unique peptides, and 4,427 proteins(≥ 95% confidence). The expression of 595 proteins was altered significantly when root primordia were formed compared with the control cuttings(stage0 VS stage1), 274 of which were up-regulated and 321 down-regulated. Fully developed roots emerged in stage 2. In total, 231 proteins were differentially expressed compared with stage 1, where 100 proteins were up-regulated and 131 were down-regulated. GO and pathway enrichment analysis were performed to determine the functional subcategories and metabolic pathways that the differentially accumulated proteins were significantly enriched in. Results suggest proteins involved in hormone regulation, carbon metabolism, and flavonoids biosynthesis were significantly enriched, indicating potential contributions to adventitious roots formation.2 In this study, we cloned a gene from mulberry cuttings and denominated MaTIR1.(GeneBank accsssion NO.KJ787017). Sequence analysis showed that the open reading frame(ORF) of MaTIR1 is 1758 bp, and encodes 585 amino acids. The deduced protein is predicted to have a relative molecular weight of 65438.7, and its isoelectric point was 6.31. The bioinformatics analysis results also showed that the deduced protein have the F-Box domain and LRRs domain and have neither signal peptide nor transmembrance region. Quantitative real-time PCR analysis(qRT-PCR) showed that MaTIR1 gene expressed in all tissues, and that the highest level was in leaf following root. During the mulberry hardwood cutting and greenwood cutting rooting stage, the expression level of MaTIR1 was upregulated in initiation phase and expression phases. These results suggest that MaTIR1 may involves in the formation of adventitious root.