Knockout And Functional Study Of SalA, VnxA And AkrA Related To Calcineurin In Aspergillus Nidulans

Calcium signaling system plays important roles in aspects of fungi's life,Aspergillus is no exception.Being the central regulator in the calcium signaling system(Ca2+/CaM/Calcineurin/CrzA),calcineurin is bound to interact with various proteins in living cells.To date,the transcription factors of calcineurin including Crz1 and NFAT have been reported.In the previous data,cnaA mutants show more remarkable defects than crzA mutants,which suggests that there must be another downstream proteins of calcineurin.So,to explore the new protein factors that may intetact with calcineurin in the signal transduction pathways can help us understand the molecular mechanisms involed in stress response of filamentous fungi calcium signaling pathways.We screen for two genes named salA.vnxA through searching an extensive literature,and study their function in Aspergilli.In Aspergillis,to our surprise,salA mutants and vnxA mutants haven't shown remarkable defects in hyphal growth or connidiation under low calcium conditions.The phenomenon also happened under metion ions stresses.salA mutants show weak resistance to azole drug ITZ.vnxA mutants display a little more sensitive to ITZ but a little bit resistance to cell wall disturbing agents such as Congo Red and Calcofluor white.These phenomenons make us think that the function of salA and vnxA played in yeast maybe not have introspecies conservatism between yeast and Aspergillli.On the other hand,we use the lower animals Culex quinquefasciatus NFAT gene as a query in a BLAST search and then we find the possible homologous protein AkrA,FapA,CdaA.Because of akrA mutants show more remarkable defects,it needs further investigation.Under low calcium conditions,the result shows that akrA mutants show remarkable defects in hyphe growth and less conidiation.These defects can be rescued by adding exogenous calcium.akrA mutants also play an important roles in maintain intracellular ion steady state,showing sensitive to Na+,K+,Mg2+,Li+.In addition to this,akrA mutants also show a little bit resistance to cell wall damage agents such as Calcofluor white and Congo Red;but show sensitive to azole drugs ITZ.With the help of GFP,we confirmed that protein AkrA located in Golgi.We decide to make a further investigation about it such as how akrA to contact with some key members of calcium signaling pathway.We construct ?akrA/?cnaA,? akrA/? midA,? akrA/? cchA and ?akrA/OE::cchA mutants.The phenotype of ?AakrA/?cnaA reacting to cell wall damage agents falls in between the phenotype of strain ?akrA and ?cnaA.Under low calcium condition,the phenotype of akrA mutants is similar to ?cchA and AmidA.Notably,the growth retardation of the AakrA?midA and AakrA?cchA double mutants in the low calcium conditions was reversed by the addition of external calcium(20 mM)in minimal medium.That makes us think that the function of akrA may have the overlapping with gene cchA and midA.However,the overexpression of gene cchA did not rescue AakrA defects under the low calcium conditions.That suggests that although AkrA likely has overlapping functions with CchA/MidA complex,it may also independently affect calcium signaling pathway.We also study the correlation between them from another perspective.We choose the recombinant aequorin expression method for this purpose.We also get the same conclusion in this way.In brief,gene salA and vnxA play different roles and function differently in yeast and Aspergilli.AkrA locats in Golgi,and plays important roles in calcium signaling pathway,ion homeostasis and the molecular mechanism of azole drug resistance.