Cloning And Functional Analysis Of Salt Stress-related Genes In Halostachys Caspica

Soil salinization severely affects agricultural productivity. According toincomplete statistics, about20%of world’s agricultural land is affected by highsalinity. Identify salt tolerance related genes of halophytes and study on the functionof these gene, so as to understand the physiological and molecular mechanism of salttolerance in plants have become the most urgent tasks in agricultural research today.The development of salt tolerant crops by genetic engineering is one of thestraight-forward solutions.Halophytes has formed a unique salt tolerance mechanism in the process oflong-term adaptation to the salt stress environment to against the damage of salt.Salinity stress response involved multigenic and a number of processes, the maincause include the change of osmotic potential, produce toxicity ions, and oxidativestress, ultimately impacted the plants’ normal physiological and biochemicalprocesses, performance for the growth suppression or even death. Halostachyscaspica (Bieb.) C. A. Mey is a extremely salt-tolerant plants that grows on semi-desertsalt-alkaloid land, the leaves and stalks of H. caspica has evolved into assimilatingbranches, this unique morphological feature reflect one adaptation to salt stress, it isan ideal candidate for investigating the mechanisms of salt tolerance in plants. For thefully exploit and utilize of salt-tolerant plants resources improved crops has extensiveapplication foreground. Our research group has constructed a suppression subtractivehybridization forward library using H. caspica with salt treatment, It has identified86positive clones which involve in metabolism, energy, signal transduction, transport etal.39%of the unique EST has annotated as unknown function. This graduation thesishas chose three genes involved in water transport, hydrogen peroxide eliminates andunknown function, combining RACE and RT-PCR techniques to clone the full-lengthgene sequences, using qRT-PCR technology to detect gene expression profiling, andidentification their physiology function in salt tolerant by reverse genetics methods, and screening candidate gene for development tolerant crops. The main experimentalresults were as below:1. Cloning and function identification of a aquaporin gene from HalostachyscaspicaIn the majority of abiotic stress conditions such as salt, drought or cold willcause the dehydration of the cells, it is important to study influence of AQPs on thecellular consequences of abiotic stress stimuli. A novel aquaporin gene designatedHcPIP1was isolated from Halostachys caspica using RACE method. The ORF ofHcPIP1is858bp encoding285amino acids, the molecular weight is30.6kDa.HcPIP1shared high sequence identity with PIP1s of other plant species. qRT-PCRresults showed that the expression patterns of HcPIP1was obviously higher in rootthan assimilating branches, The HcPIP1transcripts was induced by salt treatment.Localization analysis showed that HcPIP1proteins tagged with green fluorescentprotein (GFP) were localized to the cell plasma membrane of the transgengicArabidopsis. Heterologous expression of HcPIP1in INVSc1enhanced the yeast cells’salt tolerance capacity. The growth of transgenic plants and WT was suppressionunder osmotic stress and ion stress conditions, but the restraining degree of transgenicplants is lower than WT, overexpression of HcPIP1in Arabidopsis has a betterphenotype and increased root elongation compared with WT under osmotic stress andsalt stress, but in the absence of stress control conditions, there was no significantdifference in growth between transgenic plants and WT. Our results suggest thatHcPIP1plays an important role in salt tolerance and can also enhance plants’tolerance to other abiotic stresses.2. Function identification of unkown polypeptide gene (HcUKPP) fromHalostachys caspicaFluorescence was obtained by confocal laser scanning microscopy from thehygromycin-resistant T1seedlings’ root. Fluorescence was found in cell nuclei, cytoplasm and plasma membrane in the root cells of GFP-expressing plants，butGFP-tagged HcUKPP protein was localized at the cell plasma membrane in the rootcells of HcUKPP-GFP-transgenic plants; Heterologous expression of HcPIP1inINVSc1enhanced the yeast cells’ salt tolerance capacity, this will lay the foundationfor the future study on the function of HcUKPP.3. Cloning and functional analysis of catalase gene from Halostachys caspicaA catalase gene from the Halostachys caspica (HcCAT1) was isolated byhomologous sequence cloning method. Sequence analysis showed that the ORF ofHcCAT1was1479bp, encodes492amino acids with a molecular mass of56.7kDaand a pI of6.84. HcCAT1shared high similarity with those from other plant species.A semi-quantitative PCR result showed that the expression of HcCAT1gene was upregulated by salt-stress.There combinant plasmid pET32a-HcCAT1was constructedand transformated into E.coli BL21(DE3), The fusion protein His-HcCAT1wasexpressed by IPTG induction. SDS-PAGE and Western blotting showed thatHis-HcCAT1was77.7kD, same size to the expected. Under low temperature,His-HcCAT1was expressed in soluble form, and had certain catalase activity. Saltstress experiments showed that E. coli carrying the recombinant plasmid pET32a-HcCAT1exhibited better growth phenotypes and higher growth rates than the controlbacteria in the LB medium supplemented with400mmol/L NaCl,400mmol/L KCland300mmol/L mannitol,demonstrating that HcCAT1could enhance the saltstress tolerance for E. coli cells. These resules will help to understand the molecularmechanism that Halostachys caspica for salt stress and oxidative stress.