Cloning And Stress Resistance Analysis Of Ribosomal Protein Genes (SpRPS3ae And SpRPL44) In Extreme Haiotolerant Aspergillus Sp.

Ribosomal proteins are the main components of ribosome，which arehighly conserved from yeast to mammals. Recent studies found thatgenes encoding ribosomal proteins are known to accomplish theimportant intracellular protein biosynthesis. Besides that, these genes alsoperform extra-ribosomal functions, such as DNA repair, regulation ofgene transcription, cell development and differentiation, and they are alsoinvolved in oncogenesis and congenital diseases. In this study, we usedan extreme halotolerant Aspergillus strain, named CCHA, as theexperiment material, which was isolated from air-dried wild vegetationsurface of solar salt field periphery. As a halophilic species, it is highlyadaptive to adverse environment. So we proposed that there might bediverse stress resistance genes in the extreme halotolerant Aspergillusstrain CCHA.In this study, we screened two genes,40s ribosomal protein gene SpRPS3ae and60s ribosomal protein gene SpRPL44from an extremehalotolerant Aspergillus strain CCHA yeast expression library constructedby our lab. We successfully cloned these two genes from Aspergillus. Thesequencing results showed that SpRPS3ae and SpRPL44contains a768bpand321bp open reading frame(ORF) respectively, which encoding apolypeptide of256and107amino acids, with an estimated molecularmass29.15kDa and12.09kDa. We transformed SpRPS3ae and SpRPL44genes into yeast, and found both of them could enhance the salt tolerancecapacity of yeast.In order to test the function of these two genes, we conducted astress analysis of Magnaporthe oryzae transformed with SpRPS3ae andSpRPL44genes. The result showed that both genes could promote the salttolerance capacity of M. oryzae. Additionally, they improved the abilityof M. oryzae to the resist hydrogen peroxide and affected thepermeability of cell membrance in M. oryzae.We further cloned ribosomal protein genes MoRPS3ae andMoRPL44from M.oryzae and transformed them into yeast respectively, thus getting four positive transformants (SpRPS3ae, SpRPL44,MoRPS3ae, MoRPL44). After the salt stress analysis, we found thatMoRPS3ae and MoRPL44had salt tolerance compared with the wild typestrain. Under the same salt stress level, yeast strains containsover-expressed SpRPS3ae and SpRPL44genes had higher viability thanyeast strains with MoRPS3ae and MoRPL44genes over-expressed.To further explore the function of SpRPS3ae and SpRPL44genes inplants, they were transformed into tobacco by leaf disc mediatedtransformation method, followed up by a salt stress analysis. Preliminaryresults demonstrated that the transgenic plants had higher salt tolerancethan the wild type. We successfully obtained the T2transgenicArabidopsis via Agrobacterium mediated transformation. It allows us tofurther explore function of SpRPS3ae and SpRPL44in halotolerantAspergillus sp.