| Racomitrium japonicum that grows on bare rock, thin soil or rock walls is a common drought tolerant mosses, and has strong vitality. And there must be a series of efficient and functional genes response to drought-stress in it. Compared to other higher plants, the growth model of R.japonicum is simple, and the haploid gametophyte is the dominant phase in the whole life. Taking R.aponicum as experimental material, it is very important to study drought-resistance molecular mechanism of mosses. This will have vital scientific value, theoretical and practical sinificance.Materials collected from field may not come from the same female parent. In the study, we successfully established in vitro regeneration system of R.japonicum, and can provide pure, homogenization material for subsequent test. Materials cultured under water deficit (GH) and normal condition (CK) were taken into analyzing the drought tolerance mechanism of R.japonicum by physiology and transcriptomics (RNA-Seq) technologies. This will lay the foundation and provide important information for molecular genetics mechanism of R.japonicum. The may results were as follows:(1) Concentration of 0.02% HgCl2 for 45～60 sencods was found to be successful in iniation cultures from fragments of gametophytes which were pre-immersed into the detergent for a period.(2) Protonema were successfully induced from fragments of gametophytes on the cut inoculated on MS medium with different concertrations of sucrose. The sutiable culture medium for protonem growth was MS1 medium with 1.0mg/L 2,4-D. The protonema could induce gametophytes, when they were transferred to the modified Beneck medium without any carbohydrate and phytohormone.(3) Under water deficit, the remarkable increased result of malondialdehyde (MDA) and electrolyte leakage further illustrated the increase of membrane lipid peroxidation and membrane permeability. Moreover, the accumulation of proline and soluble sugar enhance the ability of drought regulation are helpful to preserve the integrity of cell structure. In addition, dynamic changes of antioxidant system play important roles in the protection of plant/chloroplast response to water deficit. Furthermore, the changes of fluorescence parameters illustrated R.aponicum could reduce its photosynthesis in response to water deficit.(4) Taking GH and CK materials of R.japonicum as experimental materials, new generation of high-throughput sequencing technology was used to establish a transcriptome data library of R.japonicum. A total of 83 552 Unigene were generated. Matched against NR protein and NTdatabases,51 072 and 28 154 Unigene had annotations, and 31 027 Unigene had a COG classification and were divided into 25 COG categories. The Gene Ontology (GO) functional annotation with Nr annotation were obtained. Based on the sequence homology,21 049 Unigene were categorized into 41 functional groups.35 017 Unigene were assigned to 125 KEGG pathways, and involving in metabolic pathways and the biosynthesis of secondary metabolites and so on. Among differentially genes, eight kinds of genes involved in reactive oxygen seavengers, signal transduction, transcription factor, protection and repair of photosynthesis system, protein synthesis and degradation, sugar metabolism were ideniified. We examined some genes related to drought-resistance, such as transcription factor, signal transduction. The results showed that the expressions of these genes were all induced by dehydration or rehydration.This study provides insight into the effective physiology and transcriptome data of R.japonicum in response to dehydration under different conditions and establishes a platform for future researches for bryophytes. |
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