Alternative Splicing Analysis Of Lignocellulose Enzyme Genes In Aspergillus Niger And Enzymatic Properties Of Splicing Variants

Aspergillus niger plays an important role in the resource utilization of lignocellulosus because it can produce large amounts of lignocellulose degrading enzymes.Alternative splicing is an important gene expression regulation mechanism that is closely related to protein diversity.At present,it is unclear whether there are alternative splicing events in the lignocellulose degrading enzyme genes in Aspergillus niger genome.In this study,the alternative splicing events of Aspergillus niger lignocellulose degrading enzymes were identified by transcriptome analysis(RNA-seq),and the effect of alternative splicing on lignocellulose degrading enzymes were preliminarily revealed by the exogenous expression of splicing variants and the catalytic function of expression product enzymes.The main findings of this study are:(1)Transcriptome analysis identified the alternative splicing events of lignocellulose degrading enzyme genes in Aspergillus niger.Based on high quality of transcriptome data,we used both r MATS and ABLas analysis algorithms to analyze the alternative splicing events on a total of 56 lignocellulose degrading enzyme genes in A.niger strain CBS513.88 when cultured with glucose as the sole carbon source(Group G)and with wheat straw as the sole carbon source(Group WS)respectively.Based on ABLas analysis algorithm,a total of 21 lignocellulose degrading enzyme genes in both Group G and Group WS were found to have alternative splicing,13 genes were found to have alternative splicing in Group G while 14 genes in Group WS,with 6 genes being the same in both groups.The type of alternative splicing occurred was mainly intron retention(IR),which accounted for 82.8% of all alternative splicing events.(2)The alternative splicing events of xynF1,abn C,cbh C,bglM and eglD were successfully verified by RT-PCR and intron-specific PCR.The results showed that in addition to the normal spliced transcripts,the alternative 5’ end site of the 7th exon was detected in xynF1 transcripts,the 1st intron-preserved transcript was detected in abn C transcripts,IR events predicted by the ABLas algorithm were also detected in cbh C transcripts,the 2nd intron-preserved transcript was detected in bglM transcripts,and the 1st intron-preserved transcript was detected in eglD transcripts.All of them are consistent with the results of the ABLas analysis algorithm,which proves that the alternative splicing events analyzed by the ABLas algorithm are more accurate.(3)The catalytic functions and enzymatic properties of alternatively spliced isoforms and normal splicing products of xynF1 and eglD genes were analyzed.The results showed that XYNF1 had no corresponding activity of xylanase,while the splicing variant XYNF1-AS had xylanase activity.EGLD and splicing variant EGLD-AS did not have annotated activity of intracellular β-glucosidase,but had the function of lytic polysaccharide monooxygenase(LPMO).When acting on polysaccharides,the splicing variant EGLD-AS had greater preference for xylan substrates than EGLD.Enzymatic property studies showed that the optimal operating conditions for EGLD and EGLD-AS were both 40 ℃ and p H 7.0,and they had different types of metal ion sensitivity spectra,but they could tolerate most metal ions.In the synergy experiments with xylanases and cellulases,they showed different synergistic effects.In summary,this paper identified alternative splicing events in 21 lignocellulose enzyme genes and obtained catalytic active alternative splicing variants XYNF1-AS and EGLD-AS.This study provides a theoretical basis for elucidating the diversity mechanism of cellulase production in Aspergillus niger by alternative splicing,and the existence of numerous alternative splicing variants in A.niger also provides a basis for developing new resources of lignocellulose degrading enzymes.