| The filamentous fungus Blakeslea trispora is an industrial strain that producesβ-carotene,whoseβ-carotene synthesis is regulated by light and mediated by the photoreceptor proteins Bt WC-1a,Bt WC-1b and Bt WC-1c,but the molecular mechanism of this process is not yet known.Research on the genetics and molecular biology of B.trispora,including the above is-sues,is constrained by the fact that efficient transformation systems and genetic manipulation techniques have not yet been established in B.trispora.In order to establish genetic manipula-tion tools applicable to B.trispora,the following aspects were investigated in this study.1)A method for the electrotransformation of B.trispora protoplasts was developed and optimized.Firstly,the optimal enzymatic digestion time applicable to the preparation of B.trispora protoplasts was determined,followed by single-factor experiments on different electric field intensities,nucleic acid concentrations,electrotransformation buffers and regeneration times to determine their optimal conditions,respectively.In addition,four horizontal orthogo-nal experiments were set up for the more significant four factors.The optimum conditions for the electroconversion of protoplasts by B.trispora were finally determined as follows:enzy-matic digestion for 3 h,voltage 0.4 kv,electric pulse 2 ms,nucleic acid mass concentration 2.5μg·m L-1 and regeneration time 3 h.2)A gene editing system based on CRISPR/Cas9 technology was explored for B.trispora.The predicted nuclear localization signal HTBNLS of histone H2B of B.trispora was selected as the nuclear localization signal of the Cas9 protein,and the endogenous Bt5S r RNA or the pre-dicted Bt U6 promoter was used as the promoter for sg RNA expression.The p UC57/p DHt/sk-Bt Cas9-Bt HTBNLS-Bt U6/Bt5S r RNA-sg RNA-hph（btwc-1c-target1/2/3）plasmids were con-structed and transformed into B.trispora respectively.The donor plasmid containing the neo-mycin gene neo and the homologous arm of btwc-1c was subsequently introduced into B.trispora that had ingested plasmid p DHt/sk-Bt Cas9-Bt HTBNLS-Bt U6/Bt5S r RNA-sg RNA-hph（btwc-1c-target1/2/3）by electroshock transformation of the protoplasts.However,this gene ed-iting system based on the construct failed to screen effectively for positive clones.In order to screen more effectively for positive transformants,the gene car B,which has a more pronounced phenotypic colour change,was selected as the target gene.The phosphorylated and thiophos-phorylated modified m Cherry red fluorescent protein gene was introduced into B.trispora with integrated cas9 and sg RNA expression frames based on electroshock transformation of proto-plasts.Despite extensive passaging,screening and sequencing analyses,no target gene knock-out strains were obtained,which may be related to the complex genetic background of the strains.3)To address the problem of genetic manipulation limitations,post-transcriptional level gene silencing technology,that is RNA interference and can be used to regulate the expression of target genes.RNA interference strains were constructed based on this technique targeting btwc-1a,btwc-1b and btwc-1c,and their regulation ofβ-carotene synthesis was analysed.The p Cambia1303-m U6-btwc-1a,p Cambia1303-m U6-btwc-1b and p Cambia1303-m U6-btwc-1c plasmids were constructed and transformed into B.trispora,and the transcript levels of these three target genes were analysed based on fluorescence quantitative PCR.The results showed that the relative transcript levels of btwc-1a,btwc-1b and btwc-1c were reduced by 72%,67%and 63%,respectively,compared to the control strain,and therefore the constructed RNA in-terference technology can be applied to the molecular biology of B.trispora.4)The photoreceptor Bt WC-1s were analysed based on RNA interference technology to influenceβ-carotene synthesis.RNA interference strains targeting btwc-1a,btwc-1b and btwc-1c did not differ significantly inβ-carotene synthesis compared to control strains when cultured under total darkness conditions.After blue light irradiation,there were significant differences in the synthesis ofβ-carotene in the btwc-1a and btwc-1c RNA-interfering strains compared to the control strains,with the highestβ-carotene content reduced by 1%and 36%,respectively,while there were no significant differences in the btwc-1b RNA-interfering strain,indicating that light-inducedβ-carotene synthesis was mainly associated with btwc-1a and btwc-1c but not btwc-1b.The relative transcript levels of theβ-carotene structural genes car B and car RA in RNA-interfering strains targeting btwc-1a and btwc-1c were then analysed.The relative tran-script levels of car B and car RA genes were significantly lower in the btwc-1a and btwc-1c RNA-interfering strains compared to the control strains,indicating that btwc-1a and btwc-1c affectβ-carotene synthesis and positively correlate with their regulation of the expression ofβ-carotene structural genes. |
