Studies Of Molecular Mechanisms Of Stress Tolerance In Tamarix. Sp And Cloning Of Related Genes

Tamarix.spp has high ability in resisting stress enviroment, and can grow well in aridity and saline soils. The molecular mechanisms of stress tolerance in T.androssowii was studied by using differential display, suppression subtractive hybridization, analysis of EST and gene chip (cDNA microarray) methods, and some full-length sequence of stress resistance genes had been cloned from T.androssowii in this research..Two total RNA extraction methods were developed firstly. The main reagent of extraction buffer is borax, and the main extraction reagent is SDS and CTAB respectively in the two extraction methods. High quality RNA could be isolated using the two methods, and by using SDS method total RNA of fungi and insect could also be isolated. The method of LiCI precipitation RNA was also improved in this research, and RNA could be precipitated completely in 10 minutes by this improved method.Differential display technique was empolyed to study the gene expression of T.androssowii treated with NaHCO3 solution. Seventeen differentially expressed genes were obtained through Northern detection. BlastX analysis shows that two of these genes are homologous to the F-box protein family member; one gene is highly homologous to the translation initiation factor eIF. Additionally, two genes that are homologous to secretory peroxidase and peroxidases were also obteined, peroxidases are familiar proteins expressed in plant under stress. Five novel sequences closely related to salt and alkali resistance were obtained, which were expressed quite differentially when treated by NaHCO3 solution. These seventeen sequences were accepted by GenBank and their accession numbers were CD028574- CD028581 and CD028583-CD028591.The SSH method was carried out in analyzing the gene expression of T.androssowii under NaHCO3 stress. The cDNA of T.androssowii treated with NaHCO3 solution was used as tester and that of normally growing T.androssowii was employed as driver to study its gene expression under NaHCO3 stress. Total 36 genes related to NaHCO3 stress were obtained through Northern hybridization. BlastX analysis showed that these genes are homologous to the following genes: two antioxidant enzymes genes encoding Catalase and Peroxiredoxin respectively; one gene encoding trehalose phosphatase; a few genes encoding regulation proteins such as bZIP transcription factor, MADS-box protein, RNA-binding glycine-rich protein-1,CCCH-type zinc finger protein and F-box protein; one gene encoding early light-induced protein that can repair the photosynthetic apparatus damage induced by stress; The genes encoding cysteine proteinase and vacuolar processing enzyme which can defense the damage of stress, and the genes encoding lipid transfer protein precursor, polyubiquitin, chalcone synthase, NADP-dependent isocitrate dehydrogenase, and Oxygen-evolving enhancer protein 1 etc. Six new salt stress response genes were also obtained.Differential display technique was employed to study the gene expression of P.tenuiflora treated with NaHCO3 solution. Seven differentially expressed gene segments were obtained through Reverse Northern detection. Among them, six were induced by NaHCO3 , one was inhibited by NaHCO3 .The results of homologous analysis showed that one of the six gene segments induced by NaHCO3 solution was homologous to calcium-dependent protein kinases (CDPKs) , other five might be new sequences. The gene segment inhibited by NaHCO3 solution is homologous to putative adaptor protein. This research provides afoundation for further studying on the mechanism of salt-resistance of P.tenuiflora. The results indicated that under NaHCO3 stress, P.tenuiflora may positively regulate the stress signal transduction and its tolerance to stresses through the function of CDPK.s. These seven gene segments were accepted by GenBank, their accession numbers are CF198398-CF198404.The cDNA library of T.androssowii was constructed. The cDNA library was constructed from the tissue of T.androssowii treated with NaHCO3.This library primary titer was 5.7×105pfu, and the rate of recombination was 96%, the average size of inserts was 1.2kb, the titer of amplified library was 4.0×109pfu/mL.In an attempt to investigate its expression profile in response to NaHCO3 stress, expressed sequence tags (ESTs) analysis was carried out. A total of 2455 high-quality ESTs were generated, representing 1936 unique transcripts. Among which 1452(59.14%) ESTs showed similarity to the sequences in NCBI Nr database, and the remaining 1386 sequences are novel genes. The database-matched ESTs were further grouped into 12 function categories, i.e., metabolism-related category (18.53%), cell rescue, defense (18.80%), protein synthesis (6.75%), energy (4.61%), transport facilitation (6.75%), protein destination (7.09%), signal transduction (5.17%), cell growth, division (4.61%), transcription (6.75%), photosynthesis (8.82%), cell structure (1.79%), and function unknown (10.33%). Nearly 400 ESTs involved in salt tolerance were found; EST analysis revealed salt-resistance mechanism of T.androssowii is a complex system, involving the cooperation of many approaches, the salt-resistance mechanism involving the pathway such as signal tranduction and transcription regulating, scavenging reactive oxygen species, transporting of water and ions, osmolyte biosynthesis, protect dehydration damage and so on.Gene-chip method was used to study the differential expression of genes under drought and NaHCCH stress. The cDNAs from T.androssowii treated with drought and NaHCO3 stresses were labeled by fluorescence dyestuff Cy5 respectively, and cDNAs from control T.androssowii were labeled by fluorescence dyestuff Cy3. Gene expression profiles under NaHCO3 and drought stresses were obtained separately.Total of 89 genes differentially expressed were identified under NaHCO3, among them, 27 showed down regulated expression and 62 showed up regulated expression. BlastX analysis showed that the function of the differentially expressed genes could be grouped into some categorizations such as photosynthesis, reactive oxygen species eliminated, regulation of osmotic potential, regulation of gene expression and signal transduction, metabolism, development, ribosome assembly, protein breakdown and recycling, transporter, water channel proteins and so on. Based on this research, some function unknown or novel unreported genes that respond to salt stress were also identified, and these genes may have important functions in salt resistance of T.androssowii. Some important pathways of salt resistance in T.androssowii are revealed, and the gene expression profiling of T.androssowii under salt stress and without stress is obtained in this study.Total of 114 genes differentially expressed were identified under NaHCO3. Among them, 49 down-regulation expression and 65 up-regulation expression genes were obtained. Blast analysis showed these genes could be grouped into some categorizations such as dehydration protection, signal transduction and regulation, reactive oxygen species eliminated, photosynthesis, metabolism, ribosomal protein, protein breakdown and recycling, transport protein. Some function unknown and new genes related to drought stress were also found. That research revealed the diversity pathways of drought-resistance in T.androssowii.The comparing of the two gene expression profiles was conducted. The result showed that 14 genes weredown regulated and 31 genes were up-regulated both under NaHCO3 and drought stress, which showed there were high similarities in salt resistance and drought resistance of T.androssowii. One gene showed up-regulation under drought stress while down-regulation under NaHCO3 stress; Genes homologous to Lea protein and dehydration-induced protein RD22-like protein showed up regulation under drought stress and no significantly up regulation under salt stress. Genes homologous to S-adenosylmethionine decarboxylase proenzyme and water channel proteins were up regulated under salt stress but were no significantly up regulated under drought stress, that indicated there were obviously differences between salt resistance and drought resistance of T.androssowii. The above research denotes that the salt-tolerance capability of Tamarix.spp may be a complicated and interactive system involving multiple pathways and multiple salt-tolerant genes, but not only a single pathway of excreting salt through its salt glands.Bioinformatic methods and RACE method were employed in cloning the full-length genes. Totally, 22 full-length genes were obtained from T.androssowii. These genes included Superoxide dismutase, Oxygen-evolving enhancer protein 1, Cold acclimation protein, Metallothionein, Myb family transcription factor, Trehalose-6-phosphate synthase, Lipid transfer protein and Dehydration-induced protein RD22 protein, and sequence analysis of some genes were also performed.