| The transcriptional regular protein Hac1 from filamentous fungi is important for response of non-folding protein. Hac1 increased secretions of extracellular protein by increasing expressive quantity of genes such as molecular chaperone BIP1 of UPR, PDI1. The study on function of Hac1 could provide basic information for elucidating mechanism of filamentous fungi protein secreting regulation. This study used Penicillium decumbens 114-2 as original strain to construct expression cassette random integration of activated Hac1(Hac1i), expression cassette in-situ integration of non-activated Hac1(Hac1u) and expression cassette integration of r-pa by fusion PCR and nested PCR. Positive transformants successfully screened through protoplast trans-formation. Transcript profiles of Wild strains, Hac1 i strains and r-pa over- expressed strains were analyzed through sequence platform Illumina HiSeqTM 2000. In case to elucidating function of Hac1 in protein secreted regulation. It points out:1. Expression cassette Hac1 i randomly inserted by activated transcription regulation protein gene Hac1 was constructed,using enhanced green fluorescent protein as reporter gene and promoter of constitutive expression gpdA gene as cassette promoter. Hac1 i was transformed into wild strain 114-2 through protoplast transformation and positive transformants successfully screened and verified by PCR. Strong green fluorescence could be observed inside the hyphae. It Indicated that Hac1 i over-expressed strain Hac1-YGSJ was successfully constructed.2. Expression cassette randomly insert by exogenous protein r-pa was successfully constructed,using synthetic r-PA genes as template and induced cellulose CBHI promoters, signal peptides as direct sequence. The cassette was transformed into 114-2 through protoplast transformation. Positive transformants which integrated r-PA genes in genome was successfully screened by subculture and verified by PCR. Broth was collected from positive transformants with 2-3 days cultivation. And hydrolysis cycles were significantly observed on plate with active substrates and broth. It indicated that r-PA over-expressed strains we constructed could secrete active r-PA proteins to extracellular broth under directing of CBHI.3. Transcriptions of bipA, pdi A, xyn10 A,cel7A, cel7 B from 114-2 used bran and cellulose as carbon source, r-PA over-expressed strains and Hac1-YGSJ were analyzed by real-time fluorescence quantitative PCR,using act as reference gene. The RNA sampled after 48 h was suitable for transcriptome analysis.4. Transcriptomes were analyzed with sequence platform Illumina HiSeqTM 2000. Reads for following analysis were determined after sequence quality evaluation, clean reads filtration, SOAP2 comparative, reference sequence coverage analysis. Alternative splicing analysis, prediction and annotation of new novel transcripts, SNP analysis showed that: 10351 alternative splicing, 428 new novel transcripts, 1048 SNP were found in strain 114-2; 10776 alternative splicing, 423 new novel transcripts, 1244 SNP were found in strain Hac1-YGSJ; 11459 alternative splicing, 471 new novel transcripts, 174 SNP were found in strain r-PA. The gene expression variance analysis of 114-2 and Hac1-YGSJ showed that 2700 genes expressions were up and 1042 genes expressions were down in Hac1-YGSJ. Those variance expression genes are important. 2488 genes expressions were up and 695 genes expressions were down in r-PA compared with 114-2. Transcriptomes analysis result showed that response of non-folding protein could be triggered by over-expression of Hac1 active genes and exogenous protein r-PA, increasing genes expressions related to protein folding, protein transfer, glycosylation modification endoplasmic reticulum, vesicular transport on endoplasmic reticulum. |
PDF Full Text Request