| Cutting is the most widely used means in asexual reproduction of woody plants for easy operating and short breeding period. Mulberry(Morus alba L.) belongs to a hard-rooting tree species. Mulberry sapling bred through a regular cutting method has little roots, and the rooting parts are mainly located at both sides of a mulberry bud and a lower root primordial as well as a callus of incision. Mulberry sapling is not easy to survive by transplanting, thus, the cutting technique has not been in large-scale application on the mulberry sapling breeding. Inducting cortex rooting in mulberry cutting is an efficient seeding method researched by our research group in recent years. By using the technique, the rooting percentage of cutting may reach more than 90%, and the average rooting quantity of plants reaches more than 30. The rooting form performs a high-efficiency cortex rooting, of which the effect is obviously superior to that of the traditional cutting technique of callus rooting.In this thesis, based on the above technique, we studied on rooting mechanism of mulberry hard-wood cutting comprehensively from the physiology level, transcriptome level and proteome level, using relevant methods and technology thereof in order to provide a theory reference for the innovation of plant cutting seedling technique. The results of the study are mainly as follows.1. One-year hard branches of Yu 71-1 were used as cuttings to determine soluble total sugar content, soluble protein content, phenol content, indole acetic acid oxidase(IAAO) activity, peroxidase(POD) activity and polyphenol oxidase(PPO) activity in the processes of callus rooting and cortex rooting, which were compared and analyzed by setting the clear water treatment as control. The results showed that the respective physiological indexes presented different characteristics and change rules in different rooting types. Contents of soluble total sugar and soluble protein showed downward trends in treatments control and callus rooting, while in cortex rooting, they expressed rise after an initial decline. IAAO activity increased rapidly after a brief drop in cortex rooting, while in callus rooting it decreased after keeping a steady for a period of time. POD activity and PPO activity offered the same tendency of upgrade firstly then descending latter in both callus rooting and cortex rooting. Their activity in cortex rooting was higher than that in cortex rooting. Phenol content showed downward trend in cortex rooting, while in callus rooting, it expressed rise after a decline for a period of time.2. Cuttings base cortex in three development stages(normal stage-Stage1, expanding stage-Stage2, and rooting stage-Stage3) of mulberry hardwood cuttings rooting were used for transcriptome sequencing and sequence analying. 18729, 19044 and 19987 genes were identified in the respective development periods. A total of 8930, 9135, 2739 differential expression genes were identified in expanding stage vs normal stage(Stage 2 / Stage 1), rooting stage vs normal stage(Stage 3 / Stage 1) and rooting stage vs expanding stage(Stage 3 / Stage 2), respectively. By contrasting and annotating the differential expression genes on GO and KEGG databases, the differential expression genes in cuttings rooting mainly perform functions including metabolic process, establishment of localization, reproductive process, biological regulation, cell part, macromolecular complex, catalytic activity, etc. and participate in metabolic pathways, such as plant-pathogen interaction, plant hormone signal transduction, starch and sucrose metabolism, phenylpropanoid biosynthesis, flavonoid biosynthesis, etc.3. By comparing and analyzing three materials using for transcriptome sequencing on proteome, totally 4427 proteins were identified in the cuttings base cortex in a normal stage, an expanding stage and a rooting stage, wherein 2875 proteins were quantitatively analyzed. Differential proteins identified and quantified in the process of rooting were 595 in expanding stage vs normal stage(Stage2 / Stage 1), 660 in rooting stage vs normal stage(Stage3 / Stage1) and 231 in rooting stage vs expanding stage(Stage3 / Stage2), respectively. Proteins which may be closely related to rooting such as auxin IAA hydrolase, heat shock protein hsp70, lectin KM+, cytochrome b6, peroxidase, and the like, were selected from the proteins expressing obvious differences. By contrasting and annotating the differential proteins to GO and KEGG databases, the differential proteins in cuttings rooting, mainly perform functions including metabolic process, establishment of localization, biological regulation, response to stimulus, cell part, catalytic activity and binding, etc. and participate in metabolic pathways, such as biosynthesis of secondary metabolites, starch and sucrose metabolism, protein processing in endoplasmic reticulum, phenylpropanoid biosynthesis, glycolysis/gluconeogenesis, etc.4. Through performing a correlation analysis on the transcriptome sequencing result and the proteome analyzing result, nine genes, such as PPO1、PPO2、PPO3、ILL5、GH3.1、HSP83A、Cacybp、ANT1、SAUR2, etc., were selected from the differential genes and proteins having the same change trend, and expression changes of these genes in the rooting processes of callus rooting and cortex rooting were detected by q RT-PCR technique. The result indicated that the respective genes showed the corresponding expression characteristics in the cuttings rooting, and regulated the formation of adventitious roots through different functional mechanisms. Meanwhile, CDS sequences of the three PPO genes were cloned, and the result was uploaded in the Genebank database. The serial numbers were respectively KT371991, KT371992 and KT371993.The present research verified that cuttage rooting is a complicated and dynamic process co-regulated by multi factors such as an exogenous environment and an endogenous substance, etc. from variability of physiological indexes related to a rooting process for two different rooting forms of mulberry hard-wood cutting and nine important relevant gene expression quantities in conjunction with transcriptomics and proteomics analysis results of a base cortex during the cortex rooting process. Different processing methods, culturing conditions and the like may produce different rooting effects. In an entire adventitious root forming process, all from a physiological and biochemical substance to a regulatory gene, a protein, etc., including relevant enzymes and regulatory genes performing a defensible and protective function, genes and proteins relevant to hormone regulation, physiological substances relevant to cell proliferation and energy metabolism, etc., play significant roles. The respective regulatory genes and the physiological substances interact to together implement regulation to adventitious root formation and development. The results established good foundation to further develop the most efficient and practical mulberry cutting techniques.Although this thesis researched the mechanism of mulberry hard branch cuttings rooting from different aspects and made some achievements, it is only a tip of the iceberg for exploring the cuttings rooting mechanism, thus, further researches are required to perform. |
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