Physiological Response And Analyses Of Gene Expression Under Salt Stress In Guan Yin Bamboo

With the accelerated urbanization process in China, ormamental bamboo not only are used for urban residential area landscape but also widely used in the greening of river and the beach parks, Coastal areas are in serious water shorages due to global warming, Increased soil salinization and secondary salinization of soil is not conducive to the growth of oornamental bamboo, even cause death.causing major losses in the landscaping. Therefore, the screening salt-tolerant bamboo species and the breeding of salt-tolerant bamboo are particularly important. So far, the physiological, biochemical and molecular mechanisms of salt-tolerance in Bamboo is is rarely reported. In present study, One-year-old hedge bamboos (Bambusa multiplex var. riviereorum) grown in pots were watered with 1/4-strength modified Hoagland solution (MHS) respectively containing 0,0.1,0.3,0.5, and 0.8% of NaCl. Changes in physiological and biochemical indicators of leaves and plant biomass associated with salt tolerance were investigated at the 5th,9th,13th,17th,21st and 25th day. From the physiological and biochemical point of view, the mechanism of Bamboo response to salt stress is clarified. A normalized full-length cDNA library is established using the young leaf of Bambusa multiplex var. riviereorum treated with 0.5%NaCl and 1/4-strength modified Hoagland solution (MHS) and sequencing a large number of ESTs. The molecular mechanism of salt tolerance in Bambusa multiplex var. riviereorum is studied by real time PCR and transgenic technology. Prividing the value genes for the use of genetic engineering to improve salt tolerance of ormamental bamboo.The specific findings are as follows.1. One-year-old hedge bamboos (Bambusa multiplex var. riviereorum) grown in pots were subjected to five salt treatments as 0,0.1%,0.3%,0.5% and 0.8% NaCl for 25 days to investigate the effects of salt stress as characterized by growth, physiological and biochemical responses.Increasing salt stress significantly declined the Biomass of roots and shoots and root-to-shoot ratio. Salt stress significantly increased WSD but decreased RWC in leaves, Salt stress significantly affected Chlorophyll content in leaves, too. Chla, Ch1b and Ch1 (a+b) content critically decreased. increases in cell membrane permeability, malondialdehyde content and chla/b ratio. In this study, Superoxide dismutase, peroxidase and catalase activities and the protein content were increased, firstly, and decreased afterwards. As a result, Bambusa multiplex var.riviereorum plants can tolerate salt levels less than 0.5% NaCl without showing significant physio-morphological alterations.2. Based on SMART(switching mechanism at 5’end of RNA transcript) technology, a normalized full-length cDNA library was constructed for the tissue of young leaves of the Bamboo (Bambusa multiplex var. riviereorum) treated with 0.5%NaCl and 1/4-strength modified Hoagland solution (MHS) by DSN(duplex Specific nuclease) normalization method. The results showed:without amplication of the library, the cloning efficiency was 1.7×106 cfu·mL-1, the recombinant rate was 97 %, and the average size of inserted cDNA fragment was 1.5 kb. To generate EST information,600 clones were single-pass sequenced and 574 valid sequences from 403 bp to 1172 bp in length were generated with average size of 975 bp after vector trimming and discarding the sequences less than 400 bp, in which 30contigs and 513 singlets were obtained after initial assembly with Phrap program. The unigenes were subjected to Blast against the OryzasatiuaL, Zea, sorghum gene database which have complet sequencing and annotated..Based on the results of Blast analysis,324 unigenes (59.7%) were found to significantly match the proteins with known and putative function, and 94 unigenes (17.3%) matched significantly with those having unknown function in the non-redundant protein database. The remaining 125 unigenes ((23.0%) showed no significant homology to any protein in the OryzasatiuaL databases, indicated that they might be new genes. Homology blast results show that the ESTs encoded amino acid sequence of peroxidase, protein kinase, heat shock protein, zinc finger protein, dehydration response proteins, GTP-binding protein, transcription factor and other defence responses related proteins have a morehigh homology.The 324 unigenes of known and putative functions include 1442 GO terms classified into 3 categories. Among them,539 GO terms(37.4%) were grouped into the ’Biological Process’ category; 518 GO terms (35.9%) were related to the ’Cellular Component’ category and 385 GO terms (26.7%) were classified into the ’Molecular Function’ category. In addition,80 GO terms are involved in the response to stimuli, accounting for 24.7%.3. On based of ESTs analysising, the expression levels of 8 selected genes with potential salt-tolerance role in Bamboo(Bambusa multiplex var. riviereorum) of the control and treatment groups (treated with of 0.5% NaCl solution) at different treat time were analyzed using qRT-PCR with gene specific primers designed with primer premier software (version 5), and the results are as follows. The expression levels of Pti1, HSP, DRH3 and DRP were obviously induced in bamboo the treatment group at the first 8 days of NaCl treament, indicating that these unigenes might play a role in plant’s response to salt stress; the transcript levels of AATP and Cu/Zn-SOD were much higher in the treatment group than in the control group from the 4th day to the 12th day of salt stress, which is consistent with the increase mRNA in the treatment group at the same time. There was no obvious difference in expression abundance of NADPH-DO between the control and the treatment groups, indicating NADPH-DO might play different roles in salt stress. The transcript level of CAB is lower in the treatment group than in the control group, indicating that this unigene play different roles in salt stress.4. Protein kinase plays a very important role in the pathway of salt response. One full-length cDNA encoding a pti1 homologue was isolated from normalized full-length cDNA library of Bambusa multiplex var. riviereorum leaves and named as BmPti1. The gene sequence is 1 283 bp length, containing the complete open reading frame of 1092 bp, encoding a protein of 364 amino acids. PlantsP analysis showed that 65th-346th amino acids of the Bmpti1 protein is a ptotein kinase catalytic domain, which contains a tyrosine (Tyr) specific activity of protein kinase and an ATP binding site. It also shows the 68th -342th amino acid is a tyrosine kinase protein. BmPti1 amino acid sequence has high homology with that of the OryzasatiuaL, Zea, alfalfa, soybeans and other plants. It can be concluded that BmPtil may encode a functional Ser/Thr/Tyr protein kinase and involved in signal transduction in a similar mechanism with the other Ptil protein, exercising similar functions. Promoter element analysis indicated that BmPtil gene expression may be subject to the regulation of salicylic acid (SA), gibberellic acid, cold, drought and abscisic acid (ABA). QRT-PCR analysis showed that the BmPti1 protein expression was induced by salt. Futhermore, these results demonstrated that BmPtil represented a new Ptil-like gene and might play multiple roles in both plant biotic and abiotic resistant pathways. In order to analyzed the biological function of BmPtil. BmPtil gene was overexpressed on OryzasatiuaL. The followed analysises showed that the transgenic plants can enhance the salt resistance, the SOD activity and Ch1 content of transgenic plants can keep to a high level when treated with high salt.