Transcriptional regulation factor derived from trichoderma reesei and coding gene and application of transcriptional regulation factor
A transcriptional regulator, Trichoderma reesei technology, applied in applications, genetic engineering, plant genetic improvement, etc.
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Embodiment 1
[0046] Example 1: Cloning of the whole gene of transcription regulator TrPac1 in Trichoderma reesei
[0047] 1. Cloning of DNA sequence of TrPac1 gene
[0048] Genomic DNA was extracted from Trichoderma reesei strain QM9414 (preserved in the inventor's laboratory) as a template, and then primers were designed according to the information in the Trichoderma reesei genome database, and the target fragment was obtained by high-fidelity PCR.
[0049] (1) Extraction of Trichoderma reesei Genomic DNA
[0050] Wash the Trichoderma reesei spores grown on the PDA plate with sterilized water, filter with sterilized three-layer lens-cleaning paper after cleaning, and dilute the concentration to 10 6 Individuals / ml; Inoculate 1ml of spore suspension in a Erlenmeyer flask containing 80ml of CM medium, culture at 28°C, 180rpm for 24h. The cultivated mycelium was filtered with sterilized three-layer lens-cleaning paper, washed with distilled water for 2-3 times, collected, blotted dry with...
Embodiment 2
[0060] Embodiment 2: Cloning of transcription regulator TrPac1 gene cDNA sequence in Trichoderma reesei
[0061] 1. Extraction of Trichoderma reesei total RNA
[0062] Wash the Trichoderma reesei spores grown on the PDA plate with sterilized water, filter with sterilized three-layer lens-cleaning paper after cleaning, and dilute the concentration to 10 6 Individuals / ml; Inoculate 1ml of spore suspension in a Erlenmeyer flask containing 80ml of CM medium, culture at 28°C, 180rpm for 24h. The cultured mycelium was filtered with sterilized three-layer lens-cleaning paper, washed with distilled water for 2-3 times and then collected, blotted dry with absorbent paper, and the collected mycelium was immediately extracted with RNA extraction kit or firstly in Freeze in liquid nitrogen and store at -80°C for subsequent extraction. RNA extraction samples were immediately reverse-transcribed to obtain single-stranded cDNA.
[0063]The reverse transcription product was used as templat...
Embodiment 3
[0070] Embodiment 3: Construction of TrPac1 gene knockout vector
[0071] A gene knockout vector was constructed by a three-stage method, that is, the upstream and downstream 1000bp-2000bp sequences of the TrPac1 gene were respectively inserted into both ends of the hygromycin resistance gene.
[0072] First, the upstream and downstream fragments of the TrPac1 gene were respectively amplified from the Trichoderma reesei genome. Each fragment was about 1000bp in length, and they were connected to the pGEM-T vector respectively. HidIII and XbaI restriction endonuclease double enzyme cutting sites, after cut from the T vector, first connect the upstream fragment to pBS-Hyr with the same restriction endonuclease site, and then use HidIII and XbaI to digest the above vector, Connect the downstream fragment of the T vector to the restriction site to construct the gene knockout vector.
[0073] The primer sequences are as follows:
[0074] Pacup1: 5'-ATggtaccGGCCGTGTGACCGCTGGAGAGT-...
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