Ganoderma lucidum transcription factor GLHB-1 for regulating and controlling transcriptional activity of ganoderic acid synthetic genes LSS and SQS as well as coding gene and application thereof

Abstract

The invention provides a ganoderma lucidum transcription factor GLHB-1 for regulating and controlling the transcriptional activity of ganoderic acid synthetic genes LSS and SQS as well as a coding gene and application thereof, and belongs to the technical field of molecular biology and genetic engineering. According to the application, the transcription factor GLHB-1 is screened based on a key gene for regulating and controlling ganoderic acid synthesis of a transcriptome, the transcription factor can regulate and control the expression of key genes SQS and LSS in a ganoderic acid synthesis and accumulation pathway, and the transcription factor has important significance in the aspects of researching a ganoderic acid regulation and control mechanism, regulating and controlling ganoderic acid synthesis and accumulation, cultivating a new variety of ganoderic acid and the like.

Description

Technical Field

[0001] The invention belongs to the technical field of molecular biology and genetic engineering, and specifically relates to a ganoderma transcription factor GLHB-1 for regulating the transcriptional activity of ganoderic acid synthesis genes LSS and SQS, and a coding gene and application thereof. Background Art

[0002] Lingzhi (Ganoderma lucidum) It is a traditional large fungus with medicinal and edible properties in my country, with the effects of replenishing qi and calming the mind, relieving cough and relieving asthma. Ganoderma contains a variety of bioactive ingredients and has extremely high nutritional and health value. Triterpenes are the main bioactive secondary metabolites in Ganoderma, among which Ganoderma triterpenoid acids, referred to as Ganoderic acids (GAs), are the core components of Ganoderma's medicinal effects. Studies have shown that Ganoderic acids play an important role in tumor treatment, anti-oxidation, inhibition of cholesterol synthesis, inhibition of angiotensin converting enzyme activity, inhibition of histamine release, and anti-HIV. However, the main way to obtain Ganoderic acid is to extract it from Ganoderma fruiting bodies and spores, and the content of Ganoderic acid in Ganoderma is relatively low. Therefore, analyzing the biosynthetic pathway of Ganoderic acid and its regulatory mechanism is of great significance for cultivating Ganoderma varieties with high Ganoderic acid production.

[0003] Ganoderic acid is synthesized from acetyl-CoA through the mevalonate pathway (MVA). In the MVA pathway, there are two important branches for the synthesis of ganoderic acid, one branch is from farnesyl pyrophosphate to geranyl pyrophosphate, and the other branch is from farnesyl pyrophosphate to lanosterol. The key genes that have been cloned and reported in these two branches are FPS, MVD, AACT, LS, HMGR, HMGS, IDI, SQS and OSC genes, which directly regulate the synthesis of ganoderic acid and are the key to the synthesis and accumulation of ganoderic acid. Among them, squalene synthase (SQS) is the first key enzyme that catalyzes the transition of the MVA pathway to triterpene biosynthesis, and is also a key enzyme involved in the anabolism of ganoderic acid. It can catalyze the carbon flow in the isoprene pathway to triterpene biosynthesis. The activity of SQS is related to the production of triterpene compounds such as squalene, and is a key enzyme in the synthesis of triterpene compounds such as squalene. In recent years, studies have been conducted on cloning and constructing vectors of SQS genes in different plants such as ginseng, rice, and Paris polyphylla, which have enabled the exploration of the mechanism of triterpenoid synthesis in plants at the molecular level and the exploration of the optimal conditions for gene expression through gene transformation; lanosterol synthase (LSS) is a key enzyme in the cholesterol synthesis pathway, catalyzing the conversion of (S)-2,3-epoxysqualene into lanosterol. Patients with biallelic mutations in LSS will not only show diseases such as hypotrichosis, cataracts, alopecia with mental retardation (APMR) syndrome, and recessive neuroectodermal syndrome.

[0004] Based on the above background, this patent studies the interaction mechanism between SQS gene and LSS gene of Ganoderma lucidum and transcription factor HB-1, provides a certain basis for the regulatory mechanism of ganoderic acid, and is also of great significance for the cultivation of high-quality new varieties of Ganoderma lucidum. Summary of the invention

[0005] In order to solve the above problems, the present invention provides a Ganoderma transcription factor GLHB-1 for regulating the transcriptional activity of ganoderic acid synthesis genes LSS and SQS, as well as a coding gene and application thereof.

[0006] In one aspect, the present invention provides a Ganoderma transcription factor GLHB-1 that regulates the transcriptional activity of ganoderic acid biosynthesis genes LSS and SQS.

[0007] Specifically, the amino acid sequence of the transcription factor GLHB-1 is shown in SEQ ID NO.2.

[0008] SEQ ID NO.2: MSRSAADATFSAPSNPTDARRRQKSPRRSSSGSPSRHTLPTDPHTTQGASTSSNIVSQDHPATASSRVYTPSAPAPSSDPESSHRRRKRERSSPGSSSQGDMADTEMEGGPSKPSTSESAPEGAP PKKKRTRTLTTPHQAAVLHALLAQSRFPTTQMREEVGRSIGLSARKVQNQRQKARRPRGQATTTSAPLTRPPQFGPFTNAPPGTASDISPIAMIHSGSSVEGFFPRGPGGISEMQYSSAGPSSRGPPERFAP THRDAYLGEEYGRSGIPPTQLAGPGIPGSSRRGLLDDPRWRRPSQGFPAPPSTSAIELLEMYRATPRPGRPPGEEGPELTLSLPPVATRTRGPGSSPISPLSAGPYPTLASLSALEFSASAHRTRSSDEARR NLPPADVDFSTPRPPLNIPPPFTLQPQPLWDDPAFSPYNRRRSSPEQPHGAPPSFGESPTALHSPFGAEPLPSISTIVPMAPSGPSPVRSPLSPGHRTRFDPIRSTAGRAPPEHPVRPGTHDYTDEPPRNF.

[0009] In another aspect, the present invention provides a nucleic acid.

[0010] Specifically, the nucleic acid is a gene encoding the transcription factor GLHB-1.

[0011] More specifically, the nucleotide sequence of the nucleic acid is shown in SEQ ID NO.1.

[0012]

[0013] In yet another aspect, the present invention provides a recombinant vector.

[0014] Specifically, the recombinant vector comprises the above-mentioned encoding gene.

[0015] Specifically, the original vector of the recombinant vector includes but is not limited to: pGADT7-AD, pGreen II 62-SK, pET28a or pYES2.

[0016] Preferably, the recombinant vector is pGADT7-AD or pGreen II 62-SK.

[0017] In certain specific embodiments of the present invention, the recombinant vector is obtained by integrating SEQ ID NO.1 into pGADT7-AD or pGreen II 62-SK vector.

[0018] In certain specific embodiments of the present invention, SEQ ID NO.1 is integrated into the pGADT7-AD vector, and the homologous recombination primer sequences used for amplification are shown in SEQ ID NO.5-6; integrated into the pGreen II 62-SK vector, and the homologous recombination primer sequences of SEQ ID NO.1 used for amplification are shown in SEQ ID NO.7-8.

[0019] SEQ ID NO.5: CCATGGAGGCCAGTGAATTCATGTCACGCAGCGCC; SEQ ID NO.6: CGATTCATCTGCAGCTCGAGTGAAAATTACGGGGTGGTTCGTC; SEQ ID NO.7: CTAGAACTAGTGGATCCATGTCACGCAGCGCC; SEQ ID NO. 8: CGGTATCGATAAGCTTGAAAATTACGGGGTGGTTCGTC.

[0020] In yet another aspect, the present invention provides a genetically engineered cell.

[0021] Specifically, the genetically engineered cells contain the above-mentioned transcription factor GLHB-1, the nucleic acid encoding the transcription factor GLHB-1 and / or the above-mentioned recombinant vector.

[0022] More specifically, the genetically engineered cells include, but are not limited to, Escherichia coli, Bacillus subtilis, Streptomyces, Saccharomyces cerevisiae, Pichia pastoris, Kluyveromyces marxii or Ganoderma lucidum cells.

[0023] In another aspect, the present invention provides the use of transcription factor GLHB-1 in activating the expression of SQS and LSS genes.

[0024] Specifically, the SQS gene is the GLSQS gene; and the LSS gene is the GLLSS gene.

[0025] More specifically, the nucleotide sequence of the GLSQS gene is shown in SEQ ID NO.3; the nucleotide sequence of the GLLSS gene is shown in SEQ ID NO.4.

[0026] SEQ ID NO.4:.

[0027] In yet another aspect, the present invention provides the use of the transcription factor GLHB-1 in producing ganoderic acid and / or cultivating new varieties of ganoderma lucidum.

[0028] Compared with the prior art, the present invention has the following beneficial effects: The transcription factor GLHB-1 provided by the present application binds to the promoters of the GLSQS gene and the GLLSS gene and positively regulates the expression of GLSQS and GLLSS; The transcription factor GLHB-1 provided in the present application can regulate the key enzyme genes involved in the anabolism of ganoderic acid, thereby achieving the regulation of the ganoderic acid content in Ganoderma lucidum. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] Figure 1 This is a heat map of HB-1 gene expression provided in an embodiment of the present invention.

[0030] Figure 21 is a diagram of a recombinant vector provided according to an embodiment of the present invention, wherein A is pGADT7-HB-1 and B is p Green II62SK-HB-1.

[0031] Figure 3 This is the analysis result of the yeast single hybridization experiment provided in the embodiment of the present invention.

[0032] Figure 4 It is the analysis result of the dual luciferase reporter experiment provided by the embodiment of the present invention. Wherein, A is the analysis result of the dual luciferase reporter experiment; B is the fluorescence imaging result. DETAILED DESCRIPTION

[0033] The present invention will be further described in detail below in conjunction with specific examples. The following examples are not intended to limit the present invention, but are only intended to illustrate the present invention. The experimental methods used in the following examples are generally conventional, unless otherwise specified, and the materials, reagents, etc. used in the following examples are commercially available, unless otherwise specified.

[0034] Example 1 Screening of key genes regulating ganoderic acid synthesis based on transcriptome, the steps are as follows: 1. Mass spectrometry imaging was used to detect the content of ganoderic acid in the shell layer, skin layer and flesh layer of Ganoderma lucidum.

[0035] 2. Based on single-cell transcriptome data, identify the gene family members related to the synthesis of ganoderic acid in Ganoderma lucidum, and preliminarily screen the key enzyme protein sequences that catalyze the production of ganoderic acid from lanosterol in the ganoderic acid biosynthesis pathway.

[0036] 3. Based on the transcriptome data of different tissues of Ganoderma lucidum, the key genes that catalyze the production of ganoderic acid from lanosterol in Ganoderma lucidum were further screened through gene expression difference analysis.

[0037] 4. The second-generation transcriptome data was used to conduct correlation analysis with genes in the ganoderic acid biosynthesis pathway, and the gene GLHB-1 that promotes ganoderic acid synthesis was screened.

[0038] Gene expression heat map Figure 1 , it can be seen that the expression level of GLHB-1 in mycelium is the highest.

[0039] Example 2 To identify whether GLHB-1 can directly regulate the GLSQS and GLLSS genes, a yeast one-hybrid assay was performed. The experimental steps are as follows: 1. Construction of recombinant vector Using the gDNA of Ganoderma lucidum variety Xianzhi No. 2 as a template, the promoter region of 2000 bp before the start codon ATG of the GLSQS gene was cloned (the nucleotide sequence is shown in SEQ ID NO.3), the primer sequences for amplifying the GLSQS sequence are shown in SEQ ID NO.9-10, and the residues were inserted into the pAbAi vector (manufacturer: Shanghai Zeye Biological Co., Ltd.; product number: ZY8159; insertion sites: HindIII, XmaI) after restriction digestion and ligation to obtain the recombinant vector pAbAi-GLSQS.

[0040] SEQ ID NO.9:TAGGGTCTGCTCGATTTTCG; SEQ ID NO. 10: TATGACGAAAGAGTTGGTAG.

[0041] Using the gDNA of Ganoderma lucidum variety Xianzhi No. 2 as a template, the promoter region of 2000 bp before the start codon ATG of the GLLSS gene was cloned (the nucleotide sequence is shown in SEQ ID NO.4), the primer sequences for amplifying the GLLSS sequence are shown in SEQ ID NO.11-12, and the residues were inserted into the pAbAi vector (manufacturer: Shanghai Zeye Biological Co., Ltd.; product number: ZY8159; insertion site: HindIII, XmaI) by restriction digestion and ligation to obtain the recombinant vector pAbAi-GLLSS.

[0042] SEQ ID NO.11:CCCACGGAGCCGATGTC; SEQ ID NO. 12: TGTCTGAACGATGCG.

[0043] Using the cDNA of Ganoderma lucidum variety Xianzhi No. 2 as a template, the full-length coding region of the GLHB-1 gene was cloned (the CDS sequence of GLHB-1 is shown in SEQ ID NO.1), and the homologous recombination primer sequences used to amplify the GLHB-1 CDS region are shown in SEQ ID NO.5-6, respectively. The homologous recombination was integrated into the pGADT7-AD vector (manufacturer: Shanghai Zeye Biotechnology Co., Ltd.; product number: ZY6124; insertion site: HindIII, XmaI) to obtain the recombinant vector pGADT7-AD-GLHB-1. The plasmid map is shown in Figure 2 As shown in A.

[0044] 2. Yeast one-hybrid assay (1) Experimental groups The GLSQS group yeast one-hybrid experiment was divided into the following groups: Control group: The recombinant vector pAbAi-GLSQS and the pGADT7-AD empty vector were co-transformed into yeast competent cells Y2H (manufacturer: Shanghai Jingkang Bioengineering Co., Ltd.; catalog number: JK-G8044) as the control group.

[0045] Experimental group: The recombinant vector pAbAi-GLSQS and the pGADT7-AD-GLHB-1 recombinant vector were co-transformed into yeast competent cells Y2H.

[0046] The GLLSS group yeast one-hybrid experiment was divided into the following groups: Control group: The recombinant vector pAbAi-GLLSS and the empty vector pGADT7-AD were co-transformed into yeast competent cells Y2H.

[0047] Experimental group: The recombinant vector pAbAi-GLLSS and the pGADT7-AD-GLHB-1 recombinant vector were co-transfected into yeast competent cells Y2H.

[0048] (2) Experimental steps: The bacterial solution transferred with the corresponding vector was applied to the (SD / -ura / -leu) defective medium and cultured at 30°C for 3-4 days. More than 10 positive clones were selected and cultured in SD / -ura / -leu liquid medium at 30°C in a shaking incubator until the OD 600 >1.5, centrifuge at 1000rpm for 2min, discard the supernatant, resuspend with sterile water to OD600≈0.2, and spot the bacterial solution, diluted 1 / 10, 1 / 100, and 1 / 1000 times by volume, from left to right on the SD-Leu / -Ura solid culture medium containing Ab A (Aureo basidin A), and culture at 30℃ for 2~3d.

[0049] The results showed that the addition of 100 ng / mL Ab A to the SD / -ura / -leu defective medium inhibited the self-activation of the promoter, e.g. Figure 3 As shown, the growth ability of the combination of GLHB-1 co-transfected with GLSQS and GLLSS was significantly better than that of the control, indicating that GLHB-1 can directly bind to the GLSQS and GLLSS promoters in yeast cells.

[0050] Example 3 In this example, the dual luciferase system was used in tobacco to analyze the effect of GLHB-1 protein on the transcriptional activity of GLSQS gene and GLLSS gene.

[0051] 1. Vector construction pGreenII-62sk-GLHB-1 (abbreviated as 62SK-HB-1, map as shown in Figure 2B in the figure), pGreenII-0800-GLSQS and pGreenII-0800-GLLSS promoter recombinant vectors (original vector: pGreen II62-SK, manufacturer: Shanghai Zeye Biotechnology Co., Ltd., product number: ZY6124, insertion site: HindIII, XmaI). When constructing pGreenII-62sk-GLHB-1, the primer sequences used to amplify the GLHB-1 gene are shown in SEQ ID NOs.7-8; when constructing pGreenII-0800-GLSQS, the primer sequences used to amplify the GLSQS encoding gene are shown in SEQ ID NOs.13-14; the primer sequences used to construct pGreenII-0800-GLLSS are shown in SEQ ID NOs.15-16.

[0052] SEQ ID NO.13: CGATAAGCTTTAGGGTCTGCTCG SEQ ID NO.14:TCCCCCGGGTATGACG SEQ ID NO.15: CGATAAGCTTCGACTGGCTCC SEQ ID NO.16: TCCCCCGGGGTCCAAAATG 2. Vector transformation grouping: The corresponding vectors of each group were transformed into GV3101-pSoup-P19 Agrobacterium tumefaciens competent cells (manufacturer: Shanghai Weidi Biotechnology Co., Ltd.; catalog number: AC1003M) and stored at -80 °C.

[0053] (1) GLSQS group Control group: pGreen II 62-SK and pGreenII-0800-GLSQS; Experimental groups: pGreenII-62sk-GLHB-1 and pGreenII-0800-GLSQS.

[0054] (2) GLLSS group: Control group: pGreen II 62-SK and pGreenII-0800-GLLSS; Experimental groups: pGreenII-62sk-GLHB-1 and pGreenII-0800-GLLSS.

[0055] 3. Dual luciferase reporter assay Before use, Agrobacterium was streaked and activated on LB solid medium containing 50µg / mL kanamycin (Kan) and 25µg / mL rifampicin (Rif), and a single clone was picked and inoculated into 0.6mL liquid culture medium. After shaking for 16 hours, it was inoculated into 5mL and 20mL liquid culture medium in a ratio of 1:100 overnight until OD600>2.0 in the stable period. After centrifugation at 5000rpm for 10min at room temperature, the bacterial precipitate was collected, resuspended and mixed with 1×MMA solution until the OD600 of the final transcription factor and promoter was 0.5, and placed at room temperature away from light for 3h before injection into Nicotiana benthamiana.

[0056] Select a 4-5 week old healthy Nicotiana benthamiana plant and keep it away from light for 24 hours. Place it in the dark for 1-2 days after injection, then place it in a light cycle of 14 / 10 hours for 3-4 days, and then select a 0.5 cm 2 Add 500 μL of Glo lysis solution (manufacturer: Beijing Zhaosheng Lab Trading Co., Ltd.; catalog number: 0000566337) to the injection area and grind.

[0057] After full lysis, centrifuge at 12000rpm and 4℃ for 3min, take 50μL supernatant, add 50μL luciferin reaction substrate and react at room temperature for 10min, measure luciferase fluorescence A with a microplate reader, then add 50μL stop solution and react for 10min and measure Renilla fluorescence B, and calculate whether there is interaction between transcription factor and promoter by the ratio of A / B. The results of dual luciferase reporter experiment are shown in Figure 4 As shown, GLHB-1 can activate the expression of SQS and LSS.

[0058] It should be understood that the various forms of processes shown above can be used to reorder, add or delete steps. For example, the steps described in the disclosure of the present invention can be performed in parallel, sequentially or in different orders, as long as the desired results of the technical solution disclosed in the present invention can be achieved, and this document does not limit this.

[0059] Finally, it should be noted that the above content is only used to illustrate the technical solution of the present invention, rather than to limit the scope of protection of the present invention. Simple modifications or equivalent substitutions of the technical solution of the present invention by ordinary technicians in this field do not deviate from the essence and scope of the technical solution of the present invention.

Claims

1. A transcription factor GLHB-1, characterized in that The amino acid sequence of the transcription factor GLHB-1 is shown in SEQ ID NO.

2. The transcription factor GLHB-1 regulates the synthesis of ganoderic acid by regulating the expression of SQS and / or LSS.

2. A nucleic acid, characterized in that The nucleic acid encodes the transcription factor GLHB-1 according to claim 1.

3. The nucleic acid according to claim 2, characterized in that The nucleotide sequence of the nucleic acid is shown in SEQ ID NO.

1.

4. A recombinant vector, characterized in that: The recombinant vector comprises the nucleic acid according to any one of claims 2 to 3.

5. The recombinant vector according to claim 4, characterized in that The recombinant vector is obtained by integrating SEQ ID NO.1 into pGADT7-AD or pGreen II 62-SK vector.

6. The recombinant vector according to claim 5, characterized in that SEQ ID NO.1 was integrated into the pGADT7-AD vector, and the homologous recombination primer sequences used for amplification were shown in SEQ ID NO.5-6; it was integrated into the pGreen II 62-SK vector, and the homologous recombination primer sequences of SEQ ID NO.1 used for amplification were shown in SEQ ID NO.7-8.

7. A genetically engineered cell, characterized in that: The genetically engineered cell comprises the transcription factor GLHB-1 according to claim 1, the nucleic acid according to any one of claims 2 to 3 and / or the recombinant vector according to any one of claims 4 to 6.

8. Use of the transcription factor GLHB-1 according to claim 1 in activating the expression of SQS and LSS genes.

9. The use according to claim 8, characterized in that: The nucleotide sequence of the SQS gene is shown in SEQ ID NO.3; the nucleotide sequence of the LSS gene is shown in SEQ ID NO.

4.

10. Use of the transcription factor GLHB-1 according to claim 1, the nucleic acid according to any one of claims 2 to 3, and / or the recombinant vector according to any one of claims 4 to 6 in producing ganoderic acid and / or cultivating new varieties of ganoderma lucidum.

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