Two Stress Tolerance Genes In Halophilic Aspergillus: Functional Analysis And Their Application

An extreme halophilic Aspergillus strain CCHA was isolated and characterized.To uncover the tolerance mechanism and/or isolate stress-tolerance genes, afull-length cDNA library in yeast was constructed through the Gateway technology.As a result,19candidate genes were screened using the cDNA library. Twocandidates (AgGlpF and AgRPL44), which appeared to be more resistant to highosmosis were further analyzed in Saccharomyces cerevisiae, Arabidopsis thaliana andNicotianata bacum. All the results suggeste that stress resistance genes in Aspergilluscontribute to the halophilic property, which might be valuable genes for tolerance-crops creation.The main results were summarized as follows:1. Identification and biologic analysis of the halophilic Aspergillus. A halophilicstrain CCHA was isolated from the surface of wild vegetation around saltern, and itwas identified as Aspergillus glaucus according to the morphological properties, ITSsequence comparison, and the multiple tolerance. The homology of ITS sequencebetween CCHA and each of other21reported Aspergillus strains was55.9%~100%.The strain was extremely tolerant to salt over31%.2. The cDNA library of halophilic Aspergillus was constructed using Gatewaytechnology. The cDNA library capacity was1.28×107colony forming unit; averageinserted fragment size was more than1000bp and the percentage of recombinationwas93%. The library storage capacity almost cover the entire genome of Aspergillus,and it meets the requirement for further research.3. The whole cDNA library plasmids were transformed into S. cerevisiae usingsmall-scale yeast transformation protocol downloaded from Invitrigen Web site. Yeasttransformants were screened by gradually increasing the concentration of NaCl（10%,12%, or15%NaCl）. We screened19salt tolerant transformants. Two candidates with higher resistant activities were selected for further analysis. The gene, termed asAgGlpF (GenBank Accession No. AHZ59733), contains typically six transmembranedomains and two conserved NPA motifs of the MIP family. In addition, it sharesamino acid homology up to94%with GlpF gene in Aspergillus ruber. Another genein this study is similar to60S ribosomal protein RPL44, therefore, we termed it asAgRPL44(Accession No. AHZ59732).4. Functional characterization and regulatory mechanism of AgGlpF.(1) Thefunction of AgGlpF were analyzed in yeast cells. Compared with the yeast cellsharboring empty vector (pYES2), the yeast cells harboring recombinant plasmid(pYES2-AgGlpF) displayed significant tolerance to salt, drought and metal ions,indicating its involvement in osmotic response.(2) A homologous genes NcAQP fromNeurospora crassa was searched by BlastP comparative analysis. Using mutant strainΔaqp, complementary expression vectors (pSur-AgGlpF and pSur-NcAQP) wereconstructed, and transformed into the mutant strain Δaqp. Compared with mutantstrain Δaqp, AgGlpF and NcAQP complementary strains were significantly resistantto salt. Intracellular glycerol content was examined in N. crassa under salt stressconditions, the results showed that glycerol content in AgGlpF complementary strainswere obviously higher than other three strains under the same osmotic stress. Thus,we conclude that the stress resistance are associated with intracellular glycerolaccumulation; In addition, this result was supported by the expression paterns ofseveral glycerol metabolism-related genes.(3) The plant expression vectorpGFPGUSPlus-AgGlpF was constructed for overexpression of AgGlpF inArabidopsis. The recombinant vectors were transformed into Arabidopsis with thefloral dip method. Germination and growth behaviours including survival rate, rootlength under salt and drought stress conditions were investigated, The results showedthat overexpressing AgGlpF enhanced plant stress tolerance to salt and droughtstresses.5. Moonlight function of AgRPL44. On the basis of preliminary analysis ofAgRPL44gene, its expression and stress resistance were characterized inMagnaporthe oryzae. As a result, the transformants displayed higher tolerance to salt and drought than the wide type, suggesting the potential value of AgRPL44for geneticapplications. Meanwhile AgRPL44gene can also improve the salt resistance intobacco, indicating its application in molecular breeding.