The Research On Different-expression Proteins And Function Analysis During Adventitious Roots Development Of Hybrid Larix Cuttings

Larix is an important fast-growing and ecological forestation species, but is difficult to root, which restricts the development of clonal forestry using cuttings, so how to improve the rooting rate and rooting quality become a research hotspot. Currently, the research on physiological and biochemical level of Larix rooting make some progress, but the molecular mechanism of cuttings rooting remains unclear.To make clear the molecular mechanism of cuttings rooting of hybrid Larix, anatomic development of adventitious roots was investigated, proteins expressed during adventitious roots development were extracted and identified using proteomics technique, and the expressions of some important proteins on transcription level were analyzed by gene cloning technology and qRT-PCR. The main results were as follows:1 The formation and the key development period of adventitious rootThe changes of morphology and anatomy were observed during adventitious root development, root primordia formed from cambium which opposite to pith rays, the formation of adventitious root was induce-to-root type. The period of adventitious roots development was about 55-60 days, the 14-18,25-30,35-40 days after cuttage were three key time-point for adventitious root induction, adventitious root primordia initiative cell formation and adventitious root primordia formation respectively.2 The relation between endogenous hormones and rooting traits, and the interaction between hormonesThe hormones content during adventitious roots development between different clones and treatments were determined. The hormones level of different donors, especially IAA and ABA, were important for cutting traits, the higher the IAA, the lower the ABA, the better the rooting traits. The effects of IBA on cuttings rooting were increasing IAA and decreasing ABA at the early stage of adventitious root development. The trend of (IAA+GA+ZR)/ABA between different clones or treatments were similar to rooting traits, so (IAA+GA+ZR)/ABA could be used as an index of rooting ability.The interaction between hormones was confirmed by measuring hormones content under different treatments. Compared with IBA using alone, IBA +6 BA and IBA + GA showed synergism, while the IBA + ABA showed antagonism. Therefore, the appropriate concentration of cytokinin or gibberellin could be used with auxin for promoting cuttings rooting.3 The regulation of LKCcO6b and LKMDH on adventitious root development The analysis on protein and transcription level of LKCcO6b and LKMDH which participated in the citric acid cycle and mitochondrial electron transport during adventitious root development showed these two proteins mainly played a regulation during adventitious roots induction, the protein and mRNA levels were up-regulated treated with IBA, which provided carbon skeletons and energy for cambium cells opposite to pith rays division and synthesis, promoted the formation of adventitious root initial cells.The expression analysis of LKCcO6b and LKMDH treated with combined hormones indicated that the appropriate concentration of cytokinin or gibberellic acid could increase the efficiency of auxin, while the abscisic acid could decrease the efficiency. These results were consistent with endogenous hormones changes treated with compound hormones. Therefore, LKCcO6b and LKMDH regulated adventitious roots formation and development.4 The molecular mechanism of adventitious root developmentThe function of differentially expressed proteins during key time-point of adventitious roots development between different clones (different genotype) or treatments (same genotype) were analyzed, the two-part research suggested the molecular mechanism of adventitious root development.1) The carbon and energy metabolism regulated adventitious root formation and development. Large amounts of carbon and energy metabolism related proteins were identified during adventitious root development of different clones or treatments, these proteins were significantly up-regulated in better-root clones or IBA treatments, especially at the adventitious root induction stage, which provided a stable source of carbohydrates and energy for cambium cells division and synthesis, promoted the formation of adventitious root initial cells.2) Signal transduction regulated adventitious root formation and development. Auxin signal transduction related proteins were identified during adventitious root development of different clones or treatments, these proteins were significantly up-regulated in better-root clones or IBA treatments. At the early stage of adventitious root induction, endogenous IAA appeared instantaneous peak higher than the threshold of IAA auxin signal transduction, while ubiquitin-related protein up-regulated for degradation the inhibitor of auxin signal transduction pathway, these changes jointly promoted the expression of genes down-stream of IAA signal transduction, regulated adventitious root morphogenesis.In summary, the formation and development of adventitious roots of Larix stem cuttings is a very complex and multi-gene regulation process, these results was a basis for study the mechanism of adventitious root development, while provided a reference for the other conifers.