Analizing Transcriptome Profiles And Studying Salt-resistant Genes Of Kosteletzkya Virginica Under Salt Stress

With the continuous expansion of saline soil and severity of soil salinization, salinity is becoming the most important limiting factor for food quantity and quality all over the world. Salt stress can restrain photosynthesis and lead to growth retardation and stagnation to plant. In addition, the excess ions in plant can bring about osmotic stress and ion toxicity, resulting in water imbalance and metabolic disorders. In face of the adverse effects, plant especially the salt-tolertant plant has developed various efficient regulation mechanisms and strategies. To reveal these mechanisms and strategies has great significance in theory and practice, which could help to take full advantage of plant resources, protect and restore ecology environment, increase and improve food nutrition as well as promote the sustainable development of modern agriculture.In this paper we selected Kosteletzkya virginica as study material to carry out our salt stress research. Our work focused on the molecular mechanisms of K. virginica under salt stress. On account of its resistance to high salinity, we studied the salt resistance mechanism and salt-risisting gene in K. virginica. We also studied the gene function futher at the same time.(1) High-throughput transcriptome sequencing was performed after the seedlings were stressed, sampled and extracted RNA. Large amount of data was abtained and there were as many as more than sixty million transcripts in every single library. In addition, the total length of the sequencing reached up to seventeen billion. The comparative analysis among different libraries revealed that the expressions of thousands of genes were altered in K. virginica after salt stress.Basing on the existing data from other species, the genes with changes in expression were given classifications, predictions of protein function and analysis of participated metabolic pathways. The results suggested that the genes involved in growth,development and photosynthesis were inhibited significantly, while the genes encoding ion channels and transporters were up-regulated. Expression analysis of the genes with KEGG database indicated that most of the genes involved in starch and sucrose decomposition reactions, amino acid and fatty acid metabolism as well as phytohormone metabolism and signal transduction were increased. Finally, qPCR was performed to verify the results of RNA-Seq and the result showed the outcome of RNA-Seq was credible.(2) The genes responding to salt stress were screened out by differential analysis among libraries. The full sequences of two genes were acquired by molecular cloning and they were named KvLEA and KvTIP3, respectively.The analysis on protein encoded by KvLEA discovered that this protein possessed many common properties belonging to LEA proteins. It showed high similarity to two LEA genes At2g36640 and At3g53040 in Arabidopsis. The tissue expression pattern displayed that this gene had a higher expression in root. This gene was induced by salt stress and in addition, with the increase of NaCl concentration, the expression of the gene increased synchronously. Meanwhile, research also showed that high temperature, chilling and ABA could induce the expression of this gene, too. Thus, this gene should have close relationship with abiotic stress resistance in K. virginica.The analysis of KvTIP3 revealed that it coded an aquaporin. The molecular weight, conserved functional motifs and transmembrane domains of this protein were the same with the typical features of aquaporins. The tissue expression profiles indicated that this protein should be a root-specific aquaporin. Moreover, this gene was not noly induced by salt stress, but it also responsed to chilling and ABA treatment. Thus, it was an important stress resistance gene of K. virginica.(3) KvHSP70 gene is another gene isolated from K. virginica. Bioinformatic analysis displayed that it encoded a HSP70 located in cytoplasm. The predicted protein of this gene had up to 96% similarity with other known HSP70 s. The transient transformation in onion epidermal cells proved that this protein situated in cytoplasm. The expression analysis discovered that it responsed to salt, heat as well as ABA. Gene engineering technology was applied to study its biological function. The transgenic tobacco expressing this gene was endowed with enhanced salt stress resistance. A number of physiological and biochemical parameters performed better than control under salt stress. Therefore, KvHSP70 was capable of advancing stress resistance of plant.