Cloning And Salt Tolerance Analysis Of Lipid Phosphate Phosphatase Gene AtLPP1 From Arabidopsis Thaliana

In order to isolate lipid phosphate phosphatase gene from Arabidopsis,the amino acid sequence was get from Arabidopsis database.The cDNA fragment containing complete ORFs of 984bp was cloned from total RNA prepared from Arabidopsis leaves by the RT-PCR approach,and designated as AtLPP1.The predicted proteins of AtLPPl display high similarity to the other three LPPs,containing of 6 TM domains and a highly conserved acidPPc domain.The quantitative RT-PCR approach showed that the expression profiles of AtLPP1 was expressed in roots,leaves,siliques,flowers,stems.We also constructed GUS expression vector drived by AtLPP1 promoter and transformed to Arabidopsis by floral dip. The histochemical staining for GUS displayed the same results as the quantitative RT-PCR approach.In order to study the subcelluar localization of LPP1,we constructed the AtLPP1::YFP transiently expression vector and transformed to Arabidopsis protoplasts by PEG4000.The YFP fluorescence suggested that localized in plasma membrane and intracellular membrane.The previous research showed AtLPP1 was regulated by Gy,mastoparan,UV-B and harpin,while AtLPP2 did not affected by any stress,like a house-keeping gene(Pierrugues et al.2001).Katagiri et al(2005) found AtLPP2 is a negative regulator that participates early in the ABA signaling network during seed germination.We used AtLPP1-deficit mutant lppl to analyse the physiology of salt tolerance.We found lppl was sensitive to salt. One-month-old lppl and wide-type were watered with one-fourth strength Hoagland nutrient solution plus 100 mM NaCl.The ion analys indicated that lppl plants accumulate more Na~+ and retain less K~+ compared with wild-type plants.These suggest that AtLPP1 gene deficit affect ion homeostasis in plant and reduce the plant salt tolerance.We also found the content of Pi in lppl mutant was lower than wide-type after NaCl treatment. That's maybe because the AtLPP1 gene deficit result the lower of PA in plant.Based on this, we added PA to MS medium contained 100mM NaCl,and we found the lppl mutant can recovered from the salt stress more.These results suggest that PA contributes to the increase of salt tolerance in plant and the AtLPP1 gene deficient results the PA's lack.The contant of endogenesis PA assay confirm this conclusion more.Under salt stress,PA in lppl mutant was more decreasing than wide-type plant.These suggest that LPP1 in plant mainly reduced PA increase.Regarding germination,lppl mutant seeds were more sensitive than the wild-type seeds,also ABA.The germination rate was less than the wild type on MS plus 100mM NaCl.This may suggest that AtLPP1 is also involved in seed germination.