Regulation Of Sexual Sporulation By AcndtA Gene And Related Genes In Aspergillus Cristatu

Aspergillus cristatus is the dominant fungus during the fermentation of Fuzhuan brick tea.It reproduces sexually（Eurotium cristatum）during tea fermentation,producing golden yellow cleistothecia,so it is also known as the“golden flower fungus”.The quality of the tea increases with the number of cleistothecia that are produced by the fungus.Therefore,it is important to find the switch for A.cristatus to regulate development.However,the current knowledge of Aspergillus development is not fully understood.Previous studies have shown that the developmental regulation mode of this fungus was different from the model strain Aspergillus nidulans.Therefore,it is of great significance and value to study the sporulation mechanism of A.cristatus.Our previous study showed that Acndt A was an important regulatory factor for the sexual development of A.cristatus.Acndt A deletion mutants exhibited complete blockade of cleistothecium formation.Therefore,it is a good material for studying the regulatory mechanism of sexual and asexual sporulation of A.cristatus.In this study,we supplemented and overexpressed the AcrpnR gene for functional verification and completed transcriptome sequencing of Acndt A mutant strains and wild-type strains for analyzing the functional genes related to the mutant traits.Four important genes:GME9445（Acmrvl）,GME931（Acnrd A）,GME10066（Acbys1）and GME2916（AcrpnR）regulated by the Acndt A gene were screened and functionally verified using gene knockout,gene complementation and gene overexpression techniques.The main results were as follows:（1）Acndt A plays a key role in regulating sexual development,asexual sporulation and secondary metabolism.The Acndt A complementation and overexpression vectors were constructed.A total of 32complementary strains and 33 overexpression strains were obtained by Agrobacterium tumefaciens-mediated transformation.The results showed thatΔAcndt A strains had more aerial hyphae,and the cleistothecium formation was blocked.The OE::Acndt A strains had sparse hyphae and barren cleistothecia and the maturity of the cleistothecia were reduced.The conidial heads of the OE::Acndt A strains were significantly reduced and were smaller than that of the WT strain.The conidial yield of the OE::Acndt A strains was only approximately 1%of that of the WT strain.Moreover,theΔAcndt A strain was less sensitive to osmotic pressure and starvation,while the OE::Acndt A strain was the opposite.Acndt A also affects the production of pigments produced during sexual development of A.cristatus.TheΔAcndt A colonies were paler and produced fewer brown pigment and black droplets.The OE::Acndt A strains exhibited significantly reduced brown pigment and produced more orange-red pigment.All developmental defects,including cleistothecium formation,stress response and pigments production,were reversed by the re-introduction of the Acndt A gene in theΔAcndt A strain.（2）Transcriptome analysis of A.cristatus Acndt A mutant strains and wide type strains.RNA-seq was performed on the AC-592 and WT strains using the Illumina Hi Seq 4000 platform.A total of 1000 differentially expressed genes（DEGs）were identified,of which 633 up-regulated and 367 down-regulated.GO and KEGG pathway functional enrichment analysis revealed that ribosome-associated DEGs were generally down-regulated and significantly enriched.This suggest that a potential link exists between sexual sporulation and ribosome biogenesis.It was also found that 94 genes involved in biosynthesis of secondary metabolites,18 genes enriched in meiosis pathway,17 genes enriched in high-osmolarity MAPK pathway.A total of 17differentially expressed transcription factors were screened,and more than half were zinc finger transcription factors.A total of 98 sporulation-related genes were found,of which 25 genes have significantly differentially expressed.The transcription levels of most of these genes were not significantly different in Acndt A mutant and wild-type strains,indicating that there may be different sporulation-regulating pathways between A.cristatus and other Aspergillus.（3）A.cristatus GME9445（Acmrvl）and GME931（Acnrd A）genes are involved in sporulation and stress response,and Acnrd A also affects mycelial growth and branching.The GME9445 gene is 616 bp in length,includes two introns and encodes a predicted protein of165 amino acids that has a conserved domain of Marvel and 4 obvious transmembrane structures.GME9445 was named Acmrvl.A total of 6 Acmrvl gene deletion strains were obtained using gene knockout technology.The results showed that the number of ascospores and conidia ofΔAcmrvl strain increased.TheΔAcmrvl strains grow slightly faster under acidic environment.TheΔAcmrvl strain is less sensitive to osmotic pressure at 28℃.This is the first time that the function of such genes has been studied in Aspergillus.The GME931 gene is 1855 bp in length,includes one intron and encodes a predicted protein of579 amino acids that has two C₂H₂-type zinc finger domain.GME931 was named Acnrd A.A total of 2 deletion strains,93 complementary strains and 125 overexpression strains were obtained.The results showed that theΔAcnrd A strain grew very slowly with fewer aerial hyphae.The hyphae were short and dense with more branches.After the gene was supplemented,the growth rate of the colony and the morphology of the mycelium were restored to a large extent.The OE::Acnrd A strain grows slightly slower in the early stage,and grows faster than the wild-type strain in the later stage.The edge of the colony is fluffy.TheΔAcnrd A strains produced more ascospores and conidia.The conidia of the overexpression strain was reduced by 0.5 times that of the wild-type strain,and the number of ascospores was 1.5 times that of the wild-type strain.Moreover,theΔAcnrd A strains were less sensitive to SDS,and the OE::Acnrd A strain was more sensitive to SDS and less sensitive to menadione.（4）A.cristatus GME10066（Acbys1）gene is involved in sporulation and heat stress response,and GME2916（AcrpnR）gene balances sexual and asexual development.The GME10066 gene is 852 bp in length without intron and encodes a predicted protein of 283amino acids that has a Bys1 domain and a signal peptide at the N-terminus.A total of 5 Acbys1gene deletion strains were obtained using gene knockout technology.The results showed that the number of ascospores ofΔAcbys1 strains increased,while the number of conidia decreased.The growth of theΔAcbys1 strains was significantly slower than that of the wild-type strain under40℃.This is the first time that the function of such genes has been verified in filamentous fungi.The GME2916 gene is 2199 bp in length without intron and encodes a predicted protein of 732amino acids that has two C₂H₂-type zinc finger domain.GME2916 was named AcrpnR.Phylogenetic analysis showed that it has the closer relationship with Aspergillus ruber and Aspergillus glaucus.A total of 2 deletion strains,136 complementary strains and 56overexpression strains were obtained.The results showed that the deletion strain formed more aerial hyphae with fewer conidial heads,while the OE::AcrpnR strain had fewer aerial hyphae but more conidial heads than the WT strain.The number of conidia of OE::AcrpnR strain was 2.4times more than that of the WT strain.The complementation strain showed restoration of these phenotypic differences.TheΔAcrpnR strains produced more cleistothecium,while overexpression of AcrpnR resulted in delayed and inhibited sexual reproduction.With increasing sodium chloride concentration,the inhibitory effect of AcrpnR on sexual development became more obvious.Moreover,theΔAcrpnR strains were less sensitive to DTT and SDS,while the OE::AcrpnR strain was more sensitive to SDS.In addition,AcrpnR positively regulated the expression of genes of the central regulatory pathway of conidiation and negatively regulated the expression of sex-related genes.In conclusion,the function of Acndt A was verified.For the first time,gene deletion,gene complementation and gene overexpression techniques had been combined to analyze gene functions in A.cristatus.Some sporulation-related genes that were regulated by Acndt A had been screened and the function of several new sporulation genes（Acmrvl,Acbys1）were verified.These results provided insights into the developmental regulation mechanism of A.cristatus.