Mining And Verification Of Herbicide Activity Related Genes In Talaromyces Purpureogenus CY-1

Microorganisms are an important source of active substances,and finding and discovering new active compounds from microorganisms is one of the important fields in the research and development of new pesticides.Talaromycos is a new fungal genus name proposed by Benjamin in 1955,widely present in environments worldwide.Talaromyces purpureogenus CY-1(hereinafter referred to as CY-1)is a fungus isolated from the soil of the Qinghai Tibet Plateau.Previous research results have shown that the metabolites of CY-1 have herbicide effects on various weeds such as amaranth,quinoa,solanum,and xanthium,and have potential development value as herbicides;However,there is a key bottleneck issue in directly utilizing fungi to develop drugs.The production of active ingredients in their metabolites is relatively low,making it difficult to achieve industrial levels.Exploring active related genes for microbial development is an effective approach.This paper takes CY-1 as the research object and obtains a strain with significantly improved herbicidal activity through mutation breeding.Genes related to herbicidal activity are screened out,and candidate genes and related functions are verified.This lays the foundation for genetic improvement of the strain and the construction of high-yield engineering strains.The main results obtained in this article are as follows:1.Using UV and diethyl sulfate(DES)mutagenesis to select CY-1 strains with significantly improved herbicide activity.A total of 3462 mutant strains were obtained through UV and DES mutagenesis,and one strain with significantly increased herbicide activity in the fermentation broth was screened.The CY-13-136 mutant strain,with a 20 fold dilution of the fermentation broth,had inhibitory rates of 52.61% and 56.56% on root and shoot growth of alfalfa,respectively.Compared with the wild strain CY-1,its activity increased by 40.01% and 40.95%,respectively.After 15 consecutive passages of the CY-13-136 mutant strain,it still showed good genetic stability.2.The transcriptome of mutant strain CY-13-136 was sequenced to study the changes of transcriptome.The transcriptome sequencing results showed that there were 965 differentially expressed genes between CY-1 and CY-13-136 strains,432 of which were upregulated and 533 down-regulated.Through GO and KEGG enrichment analysis,combined with gene function,a total of 8 candidate genes related to herbicidal activity,FUB1,RED2,CLM2,fsa1,TSTA,OXI1,gli A,and gli T,were identified.The q RT PCR technology was used to verify the up and down regulation of eight differentially expressed genes related to herbicidal activity.The verification results were consistent with the transcriptome sequencing results.The results showed that the expression level of genes related to herbicidal activity had changed between the screened mutant strains and wild strains.3.Verify through RNAi technology whether the toxin related genes gli T,gli A,and fsa1 are the target genes for the metabolism of herbicide active products by strain CY-1.Successfully constructed interference vectors p Silent-1 gli T,p Silent-1 gli A,and p Silent-1fsa1 for gli T,gli A,and fsa1 genes.The p Silent-1 gli T vector was genetically transformed using PEG4000 mediated method.Four interfering mutant strains were randomly selected,and real-time fluorescence quantitative PCR was used to determine the interference rate.The interference rates of the four interfering strains gli T 1,gli T 2,gli T 3,and gli T 4 were 52.17%,48.68%,50.35%,and 38.39%,respectively.After genetic transformation,the p Silent-1 gli A and p Silent-1 fsa1 vectors were randomly selected,and the interfering strains gli A 1 The interference rates of gli A 2 and gli A 3 are 69.10%,66.52%,and 64.47%,respectively.The interference rates of three interfering mutants fsa11,fsa12,and fsa13 were 76.41%,73.18%,and 71.58%,respectively.There was no significant difference in the inhibition rates of root length and bud length growth between the interfering strains and CY-1 in verifying the functions of these three genes,and there was no significant change in the weed control effect.Through mutation breeding of CY-1,a strain CY-13-136 with significantly improved herbicidal activity and good genetic stability was obtained.Eight candidate genes related to herbicidal activity were screened by transcriptome analysis of strains CY-1 and CY-13-136.And verify that genes gli T,gli A,and fsa1 are not key genes controlling the metabolism of herbicide active products in strain CY-1,among which the gli T gene may be closely related to pigment synthesis.