| Because of their poor mobility,fungi and plants are often exposed to extremes biological and non-biological stresses,such as temperature,drought,salinity,and heavy metals.Heat stress response(HSR)is one of the mechanisms evolved by eukaryotes such as fungi and plants to adapt to these environmental stresses.Heat shock transcription factors(HSFs)are the most important regulatory elements in the heat shock response,and play an important role in maintaining the normal function of organisms and the appropriateness to extreme stress environments.In the previous study,our laboratory got a strain of thermotolerant heterokaryoteyotic fungus called Trametes trogii S0301,which has the optimum growth temperature 37 ?,fast growth rate,and Laccase with high yield and good character.It has good prospects for the development and application of laccase resources.Previous studies showed that the heat shock transcription factor(HSFs)in this strain may be involved in the regulation of laccase expression.In order to further promote the establishment of functional genetic research methods such as the genetic transformation system in this strain and the mechanism of HSF in the heat tolerance and laccase expression regulation of the strain,this study was carried out as following:1.Identification of thermotolerant T.trogii S0301 homokaryotic strain and analysis of laccase production abilityProtoplast preparation and regeneration can release homokaryotic strain and heterokaryoteyotic strain from the mycelium of T.trogii S0301,which can form a mature single colony after regeneration.Based on the gene sequence of mating type B sites in the second generation genomic data of T.trogii S0301,the gene sequences of primers b1 and b2 were designed in the border regions of the corresponding genes,and the PCR amplification of this sequence can identify the homokaryotic strain and heterokaryoteyotic strain at the molecular level.The b1 type and b2 type genes were amplified by cloning in multiple single colonies,and T.trogii S0301 homokaryotic strains were obtained through protoplast preparation and regeneration.In this study,homocysteine regeneration rate reached 57.80-%.The b1 type homokaryon strain and the blb2 type heterokaryoteyotic strain were obtained by PCR verification,but the b2 type homokaryotic strain was not obtained.This establishes a molecular approach for the identification of homokaryons and heterokaryons.The clamp connection is an important basis for the identification of homokaryotic strain and heterokaryoteyotic strain.Observing the clamp connection of mycelium under the oil microscope is a commonly used method for rapid identification of homokaryotic strain.In this study,by inserting the regenerating plates and observing the mycelial structure of the regenerated single colonies under the oil microscope,we found that some regenerated single colonies do not have a clamp connection structure,and thus can be identified as homokaryotic strain,and this result is consistent with the PCR identifiction.There were significant differences in phenotype and enzyme production characteristics between the homokaryotic strain and heterokaryoteyotic strain of T.trogii S0301.The growth rate of the homokaryotic strain was faster than that of the heterokaryoteyotic strain and the mycelium was smooth.The mycelia of the heterokaryoteyotic strain grew densely and villous.In terms of laccase production,the rate of enzymatic production of heterokaryoteyotic in the non-copper-induced medium was higher than that of the homokaryotic in the early stage,but almost no enzymatic activity exsists in the later period and the laccase produced in the later period of the homokaryotic strain was higher.This suggests that there may be differences in the mechanism of laccase expression regulation between homokaryotic strain and heterokaryoteyotic strain.2.Cloning and sequence analysis of heat shock transcription factors in T.trogii S0301Studies have shown that there are three heat shock transcription factor genes in T.trogii S0301:HSF1,HSF2,and HSF3.Gene sequences of three heat-shocked transcription factors were obtained by designing partial sequence primers,the sequencing results confirmed these three HSF exists in T.trogii S0301.T.trogii S0301 was treated under different heat shock conditions and the total RNA was extracted.The full-length cDNA sequences of three heat-shocking transcription factors were obtained by reverse transcription.The sizes were:2289 bp,1824 bp,and 2103 bp.Compared the three mRNA sequences with their DNA sequences,the results showed that HSF1 contained 3 exons and 2 introns,whose intron length was 61-73 bp.HSF2 contained 4 exons and 3 introns.whose intron length was 51-59 bp;HSF3 contained 6 exons,5 introns,whose intron length was 51-643 bp.In the analysis of the sequencing results of the three heat shock transcription factors,it was found that HSF2 would undergo alternative splicing phenomenon under the heat shock.and different transcripts HSF2-1 and HSF2-2 of HSF2 were generated during the splicing process.For structural analysis of the gene,the full-length HSF2-1 gene contains 5 exons with a size of 36-972 bp,4 introns with a size of 51-68 bp.The full-length HSF2-2 gene contains 4 exons with a size of 64-972 bp,3 introns with a size of 59-68 bp.Compared with HSF2-2,HSF2-1 lacks a 68-bp sequence,and The sequence has obvious intron features This one intron,prevents HSF2-1 from forming a complete ORF like HSF2-2.3.Prokaryotic expression of different ORFs of HSF2 and preparation of polyclonal antibodiesDue to the high temperature resistance of T.trogii S0301 laccase,it is regulated by the heat shock transcription factor HSF.In order to understand its specific regulatory mechanisms,polyclonal antibodies against HSF2 were prepared,providing the basis for screening targets of HSF2.Through the analysis of HSF2-1 the alternative spliced transcripts of HSF2,two potential ORFs were found.ORF1 is located at the N-terminus with a size of about 246 bp.The size of ORF2 at the C-terminus is about 1077 bp.The two ORFs were designed with primers to construct a prokaryotic expression vector and induction results showed that the fusion protein could be expressed.ORF1 can express the fusion protein H2-S with a size of about 14 kDa,and ORF2 can express the fusion protein H2-L with a size of about 42 kDa.A prokaryotic expression vector for the complete ORF3 of HSF2 non-selective splicing transcript HSF2-2 was also constructed.ORF3 expressed the fusion protein H2-Q with a size of approximately 62 kDa.W H2-S and H2-L were purified under optimized conditions and got the corresponding antiserum by immunizing mice with H2-S and H2-L fusion protein.western-blot verification results show that H2-S antibody and H2-L antibody can specifically identify the corresponding immune-derived protein.Under different heat shock conditions,the total protein of T.trogii S0301 and its homokaryotic strain 19#,was extracted from the heat shocked strains.Total protein samples were detected by western-blot using H2-L and H2-S antibodies,respectively.The results showed that both H2-L and H2-S could recognize the full-length HSF2 protein in the total protein samples.The results of the two antibodies were similar,but there were significant differences in the recognition ability.The recognition ability of H2-S was stronger than that of H2-L.,and western-blot showed similar results in the heterokaryoteyotic strain T.trogii S0301 and the homokaryotic strain 19#,indicating that HSF2 plays the same role in homokaryotic strain and heterokaryoteyotic strain.The results of western-blot after quantification of total proteins showed that the expression of HSF2 in strain 19#of the same karyotype increased with the prolonged heat shock time,and was different from that in the heterokaryon strain T.trogii S0301.This suggests that the regulatory mechanisms of HSF2 in T.trogii S0301 and 19#may be different.In this study,Homokaryotic strain from T.trogii S0301 was isolated by protoplast isolation,and molecular biology methods for the identification of homokaryons were established.This can provide starting materials for the establishment of the genetic transformation system in this strain.Finding the different laccase expression regulation mechanisms between homokaryons and heterologous strains reminds further reserch.In addition,three HSFs were cloned from T.trogii S0301,and the alternative splicing of HSF2 was found.The preparation of HSF2 antibody was performed liad the foundation of screeing the HSF2 binding target genes and the mechanism of the laccase expression regulated by HSF2. |
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