Cloning And Expression Analysis Of Phospholipase C Gene DsPLC From Dunaliella Salina

Dunaliella salina is an important model organism for the research of plant tolerance to saltstress, is also an ideal organism looking for plant salt-tolerant genes. Phospholipase C （PLC） is akey enzyme in inositol lphospholipids signal pathway, it can hydrolysis phosphatidylinositol-4,5-bisphosphate （PIP2） to produce inositol-1,4,5-triphosphate（IP3）and diacylglycerol（DAG）. IP3canlead to the release of intracellular Ca2+. From that we know PLC is in the upstream of the twoimportant second messenger Ca2+and DAG,it is very important to study its biologicalcharacteristics and function. In this article,we cloned the Phospholipase C gene from Dunaliellasalina, named DsPLC, the expression of DsPLC gene under salt stress was examined byReal-time fluorescence quantitative PCR （QRT-PCR）. These results of the current study laid afoundation for further illustrating the function and working mechanism of DsPLC.The mainexperiment methods and conclusions are as follows:1.Phospholipase C gene （GenBank Accession No. KF573428,named DsPLC）was successfullycloned by RT-PCR and RACE technology from Dunaliella salina. The full-length of cDNAsequence of DsPLC was2628bp, contained a48bp5′-untranslated region （UTR）, a798bp3′-UTR, and a1782bp complete ORF encoding a polypeptide of593amino acids.2. Bioinformatics analysis showed that DsPLC was a stable hydrophilic protein which had notransmembrane domains and signal peptide, located in the cytoplasmic matrix; there were twoconservative regions in DsPLC: a X structure domain in31-175and a Y structure domain in337-427, the His in46and93of X area is its active site.And there were multiple potentialphosphorylation sites; the main components of Secondary structure were random coil and alphahelix, the catalytic domain in the three-dimensional structure to form an active cavity; the aminoacid sequence of DsPLC has a very high similarity（74%）with the phospholipase C ofChlamydomonas reinhardtii （XP₀01696502.1）, further phylogenetic analysis showed thatDsPLC had the closest genetic relationship with the phospholipase C of Chlamydomonasreinhardtii and Volvox carteri.3.We investigated the expression changes of DsPLC under high salt stress （3mol/L NaCl） usingthe real-time quantitative PCR （QRT-PCR） method. The result of QRT-PCR showed that theexpression level of DsPLC was significantly up-regulated and reached the peak level at4h underhigh salt stress, which was9times more than that of the control group（1.5mol/L NaCl）,thedifference reached significant level （P<0.01）.As time progressed, the expression level decreasedbut still higher than normal.These results of the current study provide a new information to further explore themolecular mechanism of Dunaliella salina in response to the high salt stress, also laid a foundation for the further study of DsPLC in salt tolerance mechanism of Dunaliella salina atthe protein level.