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Salt Tolerance Analysis Of Transgenic Arabidopsis Thaliana Transformed By Vacuolar Proton Pump Genes From Halostachys Caspica

Posted on:2011-01-13Degree:MasterType:Thesis
Country:ChinaCandidate:Y Z HuFull Text:PDF
GTID:2120360305987208Subject:Biochemistry and Molecular Biology
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Abiotic stress especially salt stress cause extensive losses to agricultural production worldwide. The detrimental effects of salt on plants are impairment of water absorption and increasing toxicity of sodium in the cytosol. Vacuole is the biggest cell organ,and it plays a vital role in various physiological activities, such as adjustment of plant development and response to environmental stress. The research of salt tolerance mechanism has focused on membranous structure and enzyme activities since 1970s. More and more studies proved that the vacuolar-locate H+-translocating enzymes, H+-pyrophosphatase (H+-PPase) and H+-adenosine triphosphatase (H+-ATPase), can generate the proton electrochemical gradient to facilitate the Na+ compartmentalization, which will improve the plant growth under salt stress.Halostachys caspica (Bieb.) C. A. Mey, an extremely salt-tolerant short shrub, lives in the wilderness or semi-desert salt-alkaloid area, and it can survive even to 700 mM NaCl treatment. The morphological character of H. caspica is that its stem and leaves have assimilated as assimilating branches. Discovering the salt tolerance mechanism had significant interest in improving crop's salt tolerance. The present study mainly focuses on the function analysis of two tonoplast proton pumps from Halostachys caspica. The research comprises: isolation of vacuolar H+-PPase open reading frame from H. caspica; the construction of eukaryotic expression vectors pCAMBIA1301-HcVP1 and pCAMBIA1301-HcVHA-B harboring with HcVP1 and HcVHA-B; the transformation of Arabidopsis thaliana by Agrobacterium-mediated method; the salt tolerance analysis of two kinds of transgenic plants during different growing stages. The main results were as followed:The expression of HcVP1 and HcVHA-B was measured at different NaCl concentration with assimilating branches of H. caspica treated for 4d. As shown in Figure 5, HcVP1 and HcVHA-B transcript were induced by salt stress, and the expression of two genes enhanced substantially, with increasing salt concentration. The full length HcVP1 containing the open reading frame was cloned from the H. caspica cDNA by PCR using primers HcVP1-P1 and HcVP1-P2. To assess the biological function of HcVP1 and HcVHA-B in vivo, we generated transgenic Arabidopsis plants expressing HcVP1 and HcVHA-B driven by the cauliflower mosaic virus (CaMV) 35S promoter and with the plant selective marker hygromycin. After two generations of hygromycin-resistant selection, eight HcVP1 and three HcVHA-B transformants were identified by genome PCR and RT-PCR. The results showed that the HcVP1 and HcVHA-B genes had been integrated into the genome and transcribed normally in transgenic A. thaliana.Our study showed that overexpression of the H. caspica tonoplast H+-PPase and B subunit of H+-ATPase improved the salt tolerance of transgenic Arabidopsis. The germination rate of the overexpressing HcVP1 and HcVHA-B seeds were higher than the wild type seeds under salt stress, and the transgenic seedlings had a fresh weight greater than that of the wild type at the same NaCl concentration. Moreover, mature plants from HcVP1 and HcVHA-B transgenic lines grew well in the presence of 200 mM NaCl with relatively vigorous root system, whereas wild type plants exhibited chlorosis and died after 10 d of salt-stress treatment. The result of Na+ and K+ contents analysis reveal that the transgenic plants accumulate more Na+ and K+ in their leaf tissue no matter with and without salinity.In this work, although transgenic plants separately haboring HcVHA-B and HcVP1 both showed increased salt endurance, the overexpressing HcVHA-B Arabidopsis exhibited slight higher salt tolerance than HcVP1 transgenic plant in the seedling stage, while the transgenic Arabidopsis overexpressing HcVP1 was more salt tolerant than HcVHA-B transgenic plant in the mature stage. This phenomenon implies that the function of two proton pumps under salt stress may be different stages of plant development.
Keywords/Search Tags:H. caspica, HcVP1, HcVHA-B, transgenic A. thaliana, salt tolerance
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