Phellinus baumii MADS-box transcription factor PbMADS1 as well as coding gene and application thereof

A technology of transcription factors and coding genes, applied in the fields of application, genetic engineering, plant genetic improvement, etc., can solve the problems of long growth cycle and lack of research on growth and development regulation mechanism, so as to improve the growth rate and shorten the artificial cultivation period of Phellinus linteus. Effect

Active Publication Date: 2021-09-24
HARBIN UNIV
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, Phellinus as a rare medicinal fungus has a long growth cycle, and the research on its growth and development regulation mechanism is still lacking.

Method used

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  • Phellinus baumii MADS-box transcription factor PbMADS1 as well as coding gene and application thereof

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Experimental program
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Effect test

Embodiment 1

[0020] Embodiment 1, the cloning of Phellinus Phellinus PbMADS1 gene

[0021] Phellinus strain DL101 (the Phellinus DL101, Phellinus baumii DL101, is preserved in the China Type Culture Collection Center, address: China Type Culture Collection Center, Bayi Road, Hongshan District, Wuhan City, Hubei Province, China Type Culture Collection Center, postal code: 430072 , the preservation date is April 25, 2011, and the preservation number is: CCTCC No: M 2011137) was activated with PDA medium, cultured at 25°C for 8-10 days, collected Phellinus mycelia, and used the fast-type plant genomic DNA respectively The extraction kit and the RNAprep Pure plant total RNA extraction kit were used to extract the Phellinus mycelium DNA and RNA, and the RNA samples that passed the test were reversed according to the instructions of the Reverse Transcriptase M-MLV (RNase H) kit. Transcribed into cDNA. The Phellinus Phellinus DNA extracted and the cDNA obtained by reverse transcription were stor...

Embodiment 2

[0026] Example 2, bioinformatics analysis of Phellinus PbMADS1 gene

[0027] Using ProtParam tool software on the ExPASy server to analyze the molecular weight, isoelectric point and other physical and chemical properties of the amino acid sequence encoded by the PbMADS1 gene, the results show that its molecular weight is 32.71kDa, and the isoelectric point is 6.97; the prediction of the conserved domain of the protein shows that PbMADS1 protein belongs to MADS superfamily( figure 2 ); using the ProtScale software on the ExPASy server to analyze the affinity / hydrophobicity of amino acids, the number of hydrophobic amino acids in PbMADS1 protein is more than that of hydrophilic amino acids, which belongs to hydrophobic proteins; using online tools TMHMM Server v.2.0 and SignalP 5.0server respectively The analysis of the transmembrane region and signal peptide of the protein shows that the PbMADS1 protein has no transmembrane structure and does not contain a signal peptide; the...

Embodiment 3

[0028] Example 3, Functional Identification of Phellinus Phellinus PbMADS1 Gene

[0029] The coding region of the PbMADS1 gene was cloned, using Phellinus cDNA as a template, primers were designed according to the coding region of PbMADS1, and BglⅡ restriction sites were introduced respectively,

[0030] FB: GGAagatctATGCAAGCAACTGACTCGCAAG

[0031] RB: GGAagatctTTACCCTCCTATACGTTGGCTGC,

[0032] The lowercase letters are the introduced BglⅡ restriction sites.

[0033] The pCAMBIA1301-gpd-gpd recombinant plasmid (the two CaMV35Spromoters on the pCAMBIA1301 vector had been replaced with the mushroom gpd promoter earlier) was digested with the restriction endonuclease BglⅡ and then gel-cut to recover, and the PbMADS1 The purified product of the gene coding region was ligated with T4 DNA ligase, transformed into Escherichia coli DH5α competent cells, positive clones were selected, and plasmids were extracted for sequencing.

[0034] The above-mentioned plasmids with correct sequen...

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Abstract

The invention discloses a phellinus baumii MADS-box transcription factor PbMADS1 as well as a coding gene and application thereof. The amino acid sequence of the transcription factor PbMADS1 is as shown in SEQ ID NO.3, and the sequence of the coding gene is as shown in SEQ ID NO.2. The invention further discloses application of the coding gene in regulation and control of the growth speed of phellinus baumii hyphae. Meanwhile, the invention also discloses an overexpression recombinant vector which comprises a transcription factor PbMADS1 coding gene sequence. Compared with a wild type (WT), the transgenic phellinus baumii strain (T) for expressing the PbMADS1 has the advantages that the mycelial growth speed can be increased by 17.5%, which indicates that the obtained transcription factor PbMADS1 participates in regulation and control of the growth and development of phellinus baumii mycelia, and the application of the transcription factor PbMADS1 in actual production is beneficial to shortening the artificial cultivation period of phellinus baumii.

Description

technical field [0001] The invention belongs to the field of genetic engineering of edible fungi, and in particular relates to a Phellinus MADS-box transcription factor PbMADS1 and its coding gene and application. Background technique [0002] Phellinus baumii is a very precious large medicinal fungus, which has a medicinal history of more than 2,000 years in my country. "Shen Nong's Materia Medica" describes it as "long-term use to make light of body and prolong life"; "Dictionary of Traditional Chinese Medicine" describes that it can cure many diseases in internal medicine. "Record Phellinus can control the diseases such as metrorrhagia, bloody stranguria, amenorrhea. In recent years, the academic circles have paid attention to the pharmacological activity of Phellinus Phellinus in 1968 when Japanese scholar Ikekawa found that Phellinus Phellinus extract had good tumor inhibitory activity and did not exhibit cytotoxicity. Subsequently, scientists have continued to study ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K14/37C12N15/31C12N15/80C12N1/15C12R1/645
CPCC07K14/37C12N15/80
Inventor 孙婷婷邹莉刘瑞鹏
Owner HARBIN UNIV
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