Role Of Multi-genes During Rooting Promoting By Rejuvenation In The Adventitious Rooting Of Walnut

Walnut (Juglans L.) is hard for cutting propagation. Rooting of softwood cuttings from mature walnut cultivars was successfully achieved in our research by the rejuvenation treatments such as buried stem etc., the rooting rate was more than 98%. This achievement has solved the problem of walnut vegetative reproduction and promoted progress on the whole plant cloning, as well as provided theoretical basis and research platforms for exploring the mechanism of organogenesis. Based on the former results, this research has studied the molecular mechanisms involved in the improvement of rooting by the rejuvenation treatment with a gene transcriptional regulatory network perspective, using RNA-seq technology and bioinformatics analysis. Results were as follows:1. High frequency and synchronous generation system of walnut softwood cutting was established.(1) Mature plants were successfully rejuvenated by repeating grafting and etiolation treatment. The rate of cutting rooting was higher than 98.0% after repeating grafted more than 10 times, while the initial rooting time could be controlled approximately at the ninth day;(2) The optimal cutting conditions was:stem section with half lignification,plant length at 14-18 cm,angles from 45°to 60°between the compound leaf petioles and stems.(3) Exogenous IBA+NAA (5 mg/m·1-1) has resulted better rooting effect than byIBA only;2. A precise sampling method was established for the transcriptome study of adventitious rooting in walnut using phenotype and histology investigation: ①Induction phase, 1～2d, there were no obvious phenotypic changes in the cutting-base. Histo logical observation found that the cambium cells began to change, nucleolus greaten, cytoplasm thicken, deeply stained; ②The cambium cell division vigorous phase: 3～5d, cuttings base thickened rapidly, irregular dot swelled on the surface. The cambium cells began to divide strongly, cell layers increased significantly; ③The root primordium formation phase: 6-8d, cutting base continued to enlarge, longitudinal gaps occured in the surface and widened with time. Some of the interfascicular cambium cells continued to divide. Periclinal and anticlinal division started, suborbicular root primordia formed gradualry;④Root primordia elongation phase:Adventitious roots which seemed like milky protuberances could be seen in the gap 9 days after cuttage. The lateral root primordia cells divided faster, the volume of the cell population increased and elongated, and vascular tissues formed gradually.3. Transcriptome sequencing of the critical-period-material accompanying the adventitious rooting between rejuvenated and mature cutting was carried out by RNA-seq technology. The results were as follows:(1) Quality control and filter was done on the original sequence and 290.0 Gb high-quality were obtained;(2) The clean reads was assembled using the Trinity software, resulting in 58920 Unigenes which corresponded to 99375 transcripts (N50=1485);(3) Through BLAST comparing with the Nr protein data, genomes and transcriptomes annotation information from 21 species were selected, eventually 14670 Unigenes were functionally annotated with blast aligning.4. The key factor related to the ability of adventitious roots generation by walnut rejuvenation was excavated by differential network analysis. First of all, during the adventitious root induction of cuttings in two kinds of state,962 DEGs were screened through the expression pattern of cluster analysis. According to the results of GO function enrichment and KEGG metabolic pathway, DEGs were mainly involved in botanic hormone signal transduction, biological rhythms, alpha-Linolenic acid metabolism and Linoleic acid metabolism etc. By comparing the differences of gene regulation networks among different cuttings states, significant differences in the patterns of gene expression and regulation during cuttage were caused by rejuvenation, while the variations in regulation relationships among members of the transcription factor family including ARF, MYB, MYB, NAC, WRKY, WOX and their target genes were mainly involved. Such variations could be the reasons for the improvement in the generation capacity of walnut adventitious roots with rejuvenation treatment.5. Validation of reference genes for qRT-PCR normalization during the adventitious rooting of walnut. With qRT-PCR technology, the expressions of 5 reference genes in 5 periods in the process of adventitious rooting were analyzed; Further, geNorm software was used to analyze the stability of reference genes, and the combination of GAPDH-ACT2 was confirmed to be the most optimum reference gene for analysis of gene expression during adventitious rooting; 10 Unigenes were randomly selected to validate the results of RNA-seq. Results showed that expression quantity calculated by RNA-seq was highly consistent with qRT-PCR, which proved the accuracy of RNA-seq sequencing result;6. The establishment of pipeline for genome-wide identification and function prediction of novel lncRNAs from RNA-seq data. Based on the public database (NCBI), the RNA-seq data from mulberry (Morus notabilis) bark, root, leaf, male flowers and buds were selected. Bioinformatics analysis software CNCI was used and 1133 new lncRNA were identified; Tissue specific expression analysis showed that 106 of them expressed specifically in five tissues; Combined with specific expression results and genome positioning information, the function prediction results showed that part of these new lncRNA could play an important regulative role in the development of mulberry root and flower.