Study On Promoter Activity Of SwnK Gene In Endophytic Fungi From Locoweed

Locoweed is a general term for toxic plants of Oxytropis and Astragalus in Leguminosae.Poisoning disease characterized by nervous system dysfunction occurs after long-term or excessive food intake by animals,which is called locoweed poisoning disease.Swainsonine(SW),an indolizidine alkaloid,is the main toxic ingredient of locoweed causing animal poisoning.A SW-producing fungus belonging to Alternaria alternata Alternaria Alternaria alternata is commonly coexisting in locoweed of different species and genera.This fungus is responsible for the generation of swainsonine in locoweed and is closely related to the content of swainsonine in locoweed.Studies have shown that the swainsonine biosynthesis gene cluster(swn)may be a key gene cluster involved in swainsonine biosynthesis in locoweed endophytic fungi,and it consists of multiple genes such as swnK,swnA,swnR,swnN,swnH1,and swnH2.Among them,swnK gene is the most important multifunctional protease-encoding gene in the synthesis of swainsonine,but its transcriptional regulation mechanism is still unclear.In this study,firstly,the non-coding region of swnK gene of the fungi UA003 and EA strains of Alternaria alternata was amplified and sequenced,and the bioinformatics prediction analysis on the structure and function of the 5’ non-coding region of swnK gene was performed.On this basis,the segment-by-segment deletion fragment of the promoter of UA003 swnK gene was cloned,and then the segment-by-segment deletion fragment of the promoter of UA003 swnK gene-PGL3-Basic luciferase reporter vector was constructed.The constructed luciferase reporter vector was transfected into the HEK-293T cell line,and the transcription initiation activities of each segment-by-segment deletion fragment of the swnK gene promoter were analyzed by luciferase reporting test to identify the core promoter region of the swnK gene,thereby laying a foundation for further research on the function of the swnK gene and its transcriptional regulation mechanism.The main research results are as follows:1.The 5’non-coding regions of UA003 and EA swnK genes were successfully amplified.The sequencing and comparison results showed that the sequence identity of the 5’ non-coding region of UA003 swnK gene with the 5’non-coding region of swnK gene of published KY365741.1 was 99.9%,and the sequence identity of the 5’ non-coding region of EA swnK gene with the 5’ non-coding region of swnK gene of published KY365741.1 was 90%.The results of bioinformatics analysis showed that the 5’non-coding region of UA003 swnK gene had CAATTOX and TATA box core promoter elements,as well as Box 4,G-box,I-box,MYBHvl binding site,MYB recognition site,TCA-element,GC-motif,AAGAA-motif,RY-element and other cis-acting elements.The region from-577 bp to-527 bp upstream of the swnK coding region may be the core promoter region,and the CpG islands are present at-855 bp to-754 bp and at-533 bp and-312 bp upstream of the swnK coding region.There were multiple transcription factor binding sites such as STB5,MSN4,YAP5,SOK2,and SPT23 in the non-coding region of UA003 swnK 5’.The 5’ non-coding region of eASNK gene has CAATTOX and TATA box core promoter elements,as well as Box 4,G-Box,GATA-motif,AACA-motif,ABRE,CGTCA-motif,TGACG-motif,GC-motif and other cis-acting elements.The region from-587bp to-537bp upstream of the swnK coding region may be the core promoter region,and CpG islands are present at-535 bp and-375 bp upstream of the swnK coding region.There were multiple transcription factor binding sites such as STB5,MSN4,wc-1,ABF2,and MOT3 in the non-coding region of EA swnK 5’.2.According to the bioinformatics analysis results of UA003 swnK gene promoter,five pairs of primers with 5’ end segment-by-segment deletion were designed respectively,and the promoter region of UA003 swnK gene was amplified and cloned with segment-by-segment deletion by PCR.The promoter segment-by-segment deletion fragments were double-cleaved and connected with the pGL3-Basic luciferase reporter vector,and five swnK gene promoter segment-by-segment deletion fragments-pGL3-Basic luciferase reporter vectors were successfully constructed.All the five fragments deleted segment by segment at the 5’ end had transcription initiation activity,and the transcription initiation activity of PS4 fragment with the deletion of-656 bp to-392 bp was decreased significantly(P<0.01),and the transcription initiation activity was decreased the most.Combined with the bioinformatics prediction results,it was determined that the core promoter region of UA003 swnK gene was located at-656 bp to-392 bp upstream of the non-coding 5’end of swnK gene.