Efficient promoter for improving expression of green fluorescent protein in flammulina velutipes cells and application of efficient promoter

Abstract

The invention belongs to the field of genetic engineering and molecular biology, and particularly discloses an efficient promoter for improving expression of green fluorescent protein in flammulina velutipes cells and application of the efficient promoter. The promoter is selected from any one of the following nucleotide sequences: (a) a sequence as shown in SEQ ID NO.1-3; (b) a DNA molecular sequence hybridized with the sequence as shown in SEQ ID NO.1-3; (c) a DNA molecular sequence which has more than 90% similarity with the sequence as shown in SEQ ID NO.1-3 and can regulate and control the expression of the green fluorescent protein gene in flammulina velutipes; and (d) a sequence which is intercepted or assembled on the basis of the sequence as shown in SEQ ID NO.1-3, and the intercepted or assembled sequence can play a role of a promoter. The strong promoter disclosed by the invention can effectively improve the expression of green fluorescent protein, and has a good application prospect and important significance in the fields of flammulina velutipes genetic transformation and genetic engineering.

Description

Technical Field

[0001] The present invention belongs to the fields of genetic engineering and molecular biology, and particularly relates to a highly efficient promoter for enhancing the intracellular expression of green fluorescent protein in Flammulina velutipes and its application. Background Art

[0002] Flammulina velutipes ( Flammulina filiformis ) is deeply favored by consumers for its unique taste and rich nutrients. An efficient gene editing technology is of great significance for studying the growth and development of Flammulina velutipes and carrying out precise molecular breeding. In gene editing technology, enhanced Green fluorescent protein (abbreviated as eGFP) can often be used as a reporter gene to monitor gene expression and localization. eGFP can also be inserted under the control of the promoter of the target gene. By observing the fluorescence intensity of eGFP, the expression level and dynamic changes of specific genes can be monitored in real time. In addition, eGFP can be fused with other proteins or gene sequences. For example, by fusing eGFP with specific transcription factors, the localization and regulatory mechanisms of transcription factors can be studied. Therefore, how to enhance the fluorescence intensity of eGFP to improve gene expression and localization is extremely important. The present invention helps to promote the development of gene editing technology for Flammulina velutipes and has a guiding role in the molecular mechanism of the traits of Flammulina velutipes.

[0003] A promoter is a DNA sequence located upstream of the 5' end of a structural gene. As the key site where RNA polymerase and transcription factors bind, its core function is to drive the initial transcription of genes. As a cis-element in gene regulation, the promoter plays a crucial role in increasing the fluorescence of eGFP. They not only determine the expression intensity of the eGFP reporter gene but also regulate the temporal and spatial order of gene expression, thereby affecting the adaptability of organisms at different developmental stages and environmental conditions. Due to the direct influence of the promoter on transcription efficiency, they ultimately produce differential effects on the efficiency of gene expression. Therefore, developing a promoter that can improve the high expression and fluorescence intensity of the eGFP gene is of great significance for the study of gene function and genetic breeding improvement of Flammulina velutipes. Summary of the Invention

[0004] The purpose of the present invention is to provide a strong promoter for enhancing the highly efficient intracellular expression of the green fluorescent protein reporter gene in Flammulina velutipes, which can efficiently drive the expression of green fluorescent protein in Flammulina velutipes.

[0005] To achieve the above purpose, the present invention adopts the following technical solutions: A highly efficient promoter for enhancing the intracellular expression of green fluorescent protein in Flammulina velutipes, wherein the promoter is selected from any one of the following nucleotide sequences: (a)The nucleotide sequences shown in SEQ ID NO.1 - 3; (b)DNA molecular sequences that hybridize with the sequences shown in SEQ ID NO.1 - 3; (c)DNA molecular sequences that have more than 90% similarity with the sequences shown in SEQ ID NO.1 - 3 and can regulate the expression of the eGFP gene in Flammulina velutipes; (d)Sequences obtained by truncating or assembling based on the sequences shown in SEQ ID NO.1 - 3, and the truncated or assembled sequences can play the same role as the promoter.

[0006] Furthermore, the present invention provides a gene expression cassette, a recombinant plasmid or an engineered bacterium containing the above - mentioned strong promoter of Flammulina velutipes.

[0007] Preferably, the gene expression cassette is connected to the strong promoter of Flammulina velutipes in an expressible manner, and intracellular green fluorescent protein, a target gene and a terminator driven by the strong promoter of Flammulina velutipes are expressed.

[0008] The present invention also provides a transgenic strain cell line, a transgenic strain line or a transgenic strain variety containing the above - mentioned strong promoter of Flammulina velutipes.

[0009] Preferably, the method for obtaining the transgenic strain cell line, the transgenic strain line or the transgenic strain variety is the PEG - mediated method.

[0010] On the other hand, the present invention also provides the application of the above - mentioned high - efficiency promoter of Flammulina velutipes, the gene expression cassette containing the above - mentioned promoter of Flammulina velutipes, and the recombinant plasmid in the technical field of Flammulina velutipes gene editing or the technical field of plant molecular breeding.

[0011] Advantages of the present invention: Description of the drawings

[0012] Figure 1 Plasmid vector pBHg - eGFP map.

[0013] Figure 2 Fluorescence microscopy imaging of the control starting strain 6 - 3 and the transformants of the high - efficiency promoter. Specific implementation methods To better understand the present invention, the following further details the specific implementation manners of the present invention in conjunction with the drawings and embodiments. It should be noted that those skilled in the art should understand that these embodiments should not limit the protection scope of the present invention, and any improvements and changes based on the present invention should be regarded as falling within the protection scope of the present invention. The technical means used in the following embodiments are all conventional methods well - known to those skilled in the art. For the general equipment, materials, reagents, etc. mentioned, if not otherwise specified, they can be obtained through commercial channels.

[0015] Example 1: Transcriptome Sequencing Analysis and Screening of Strong Promoters in Flammulina velutipes 1. Transcriptome Sequencing and Analysis of Flammulina velutipes Mycelium stage, primordium stage, cap at the elongation stage, stipe at the elongation stage, cap at the mature stage, and stipe at the mature stage of Flammulina velutipes (strain L11) were selected for transcriptome sequencing. In all samples of the sequencing results, 3 commonly highly expressed genes were selected with an RPKM mean value ≥ 3500, which were respectively: gene7354 , gene4387 , gene6090 . The protein functions of each gene and the RPKM values in the 6 stages or parts are shown in Table 1 below: Table 1 RPKM Values of Highly Expressed Genes in Different Stages or Parts of Flammulina velutipes 2. Screening of Strong Promoters Based on Transcriptome Data According to the analysis of the transcriptome RPKM data, as shown in Table 1, the top 3 genes with an RPKM mean value ≥ 3500 were screened out: gene7354 , gene4387 and gene6090 . The first 2000 bp upstream of the gene was selected as the strong promoter region of the gene. The promoter nucleotide sequence of gene7354 is shown in SEQ ID NO.1, gene4387 The promoter nucleotide sequence of is shown in SEQ ID NO.2, gene6090 The nucleotide sequence of the promoter of is shown in SEQ ID NO.3, and the nucleotide sequence of eGFP is shown in SEQ ID NO.4.

[0016] Example 2: Construction of Flammulina velutipes Strong Promoter Vector 1. Extraction of Total DNA from Flammulina velutipes The mycelium sample of Flammulina velutipes (strain 6 - 3, preserved and provided by the Fujian Edible Fungi Germplasm Resource Conservation and Management Center) was scraped into a mortar and ground in liquid nitrogen. 0.1 g of the sample was taken into a 2 mL centrifuge tube, and total DNA was extracted by the CTAB method. The specific method was as follows: mercaptoethanol was added to lyse the cells, SDEB was used as a detergent, and extraction and elution were carried out with chloroform and DNA extraction phenol. Finally, DNA was precipitated with ethanol, and the obtained Flammulina velutipes DNA was stored at - 20°C.

[0017] 2. Construction of Flammulina velutipes Strong Promoter Vector Using the DNA of Flammulina velutipes (strain 6 - 3) as a template, 2×TransStart ® FastPfu PCR SuperMix high - fidelity enzyme was used for PCR amplification respectively of gene7354 , gene4387 andgene6090 The promoter fragments. The amplified promoter fragments were purified using a Universal DNA Purification Kit gel extraction kit. The promoter fragments of the three genes were ligated with the pBHg-eGFP vector at a ratio of 3:1 using a T4 DNA Ligase (5 U / μL) kit ( Figure 1 ) to obtain recombinant plasmids. The recombinant plasmids were transformed into DH5α competent cells by heat shock. After incubation, the bacterial solution was spread on the surface of LB solid medium containing Kanamycin and cultured at 37 °C for 16 h. Single colonies were picked for colony PCR verification, and further, the positive single colonies were sent to Tsingke Biotechnology Co., Ltd. for sequencing to verify the successful construction of the recombinant plasmids. The successfully constructed recombinant plasmid bacterial solution was amplified and cultured, and the recombinant plasmids were extracted using a TIANprep Mini Plasmid Kit. The successful recombinant plasmids were stored at -20 °C.

[0018] gene7354 、 gene4387 and gene6090 The names of the recombinant plasmids corresponding to the gene promoters are: pMYQ338, pMYQ324, and pMYQ346.

[0019] Table 2 gene7354 、 gene4387 and gene6090 Primers for amplifying the promoter sequences of the 3. Preparation and transformation of Flammulina velutipes protoplasts (1) Activate the Flammulina velutipes 6-3 strain using PDA medium. Take the agar block at the tip and inoculate it into liquid PDB medium at an inoculation amount of 1% and perform shake flask culture at 24 °C for 9 d; (2) Take 30 mL of the uncontaminated bacterial solution grown in the shake flask and transfer it to a 50 mL centrifuge tube, cover and seal it, centrifuge at 4500 rpm and 25 °C for 10 min, remove the supernatant, and take the precipitate.

[0020] (3) Wash the above precipitate with 40 mL of buffer (0.7M KCI and 10mM CaCI2·2H2O), centrifuge (4500 rpm, 25 °C, 10 min), and remove the supernatant; (4) Filter the 2% lysing enzyme (prepared with the buffer in step (3) and purchased from the Guangdong Institute of Microbiology) through a 0.22 μm pore size filter membrane to remove miscellaneous bacteria; (5) Add 10 mL of the filtered lysing enzyme to the mycelium and enzymatically digest it at 24 °C with shaking for 3 h; (6) Filter the enzyme solution through a 10 mL syringe plugged with absorbent cotton into a centrifuge tube to remove residues, and centrifuge the filtrate at 2500 g and 4 °C for 10 min; (7) Carefully discard the supernatant, add an appropriate amount of 1×STC (20% sucrose, 6.057% Tris·Cl (pH 8.0), 0.555% CaCl2) to the precipitate to suspend it, and centrifuge at 2000 g and 4 °C for 2 min; (8) Add an appropriate amount (about 300 μL) of 1×STC to suspend, count, and make the protoplast concentration 10 8 cells / mL; (9) Add 40 μg of recombinant plasmid DNA to 150 μL of protoplasts, gently mix, and let stand at room temperature for 25 min; (10) Add 1 mL of PTC (Protoplast Transformation Cocktail, a protoplast transformation mixed reagent prepared from 60% PEG-4000, 100 mmol / L Tris-HCl (pH 8.0), and 100 mmol / L CaCl2) in 2 - 3 portions, gently mix, and let stand at room temperature for 25 min; (11) Add the protoplasts obtained in step (10) to 10 - 15 mL of TB3 solid medium containing 50 μg / mL Kan and 100 μg / mL Hyg that has been melted and cooled to 45 - 55 °C (0.003% yeast extract, 0.003% casein amino acids, 20% sucrose, 1.3% agar powder), gently mix and pour into a petri dish, and incubate at 24 °C in the dark for 24 h; (12) Pour another 10 - 15 mL of TB3 solid medium containing 50 μg / mL Kan and 100 μg / mL Hyg into the petri dish; (13) After incubating in the dark at 24 °C for 5 d, pick out the transformants; (14) When the colony diameter of the transformant reaches 5 mm, pick the bacterial mass and transfer it to TB3 solid medium containing 300 μg / mL Hyg for screening the transformants, gene7354 、 gene4387 and gene6090 The transformants screened for the three promoter genes are named as shown in Table 3 below: Table 3 Transformant names Note: The last digit of the transformant name is the serial number when picking the transformants.

[0021] Example 3: Verification, Screening and Analysis of the Fluorescence Intensity of the eGFP Reporter Gene by Strong Promoters of Flammulina velutipes (1) Inoculate the starting strain 6-3 and the selected transformant strain into PDA medium with a coverslip, respectively, and culture them in the dark at 24°C. When the hyphae have climbed to 1 / 2 of the coverslip, place them under a fluorescence microscope that can excite a 488 nm light source, use a FITC filter set to observe and capture images, such as Figure 2 ; (2) Image J software was used to perform quantitative analysis on the starting strain 6-3 and the transformant images with a fixed threshold mode of Triangle. Three regions were intercepted on the image, and the maximum fluorescence value of each region was taken as the brightness value of the eGFP fluorescence point, so that the degree of green fluorescence change could be accurately and effectively evaluated. The data obtained from ImageJ software was input into Excel software. The results are shown in Table 4: Table 4 Fluorescence brightness values of starting strains and transformants Through the fluorescence intensity analysis and screening in Table 4, it is known that gene7354 The promoter of the target gene has the strongest fluorescence intensity for the eGFP reporter gene.

[0022] In summary, the present invention provides a powerful tool for the directional genetic improvement and improved variety selection of Flammulina velutipes by screening out a strong promoter that can significantly improve the green fluorescence intensity. This achievement is due to the application of modern biological methods such as transgenic technology or genome editing technology. In the field of edible fungi gene editing and molecular breeding, this technology shows broad application prospects.

[0023] Obviously, those skilled in the art can make various adjustments and expansions to the present invention based on the core spirit of the present invention without departing from its basic framework. If these changes and modifications are still within the scope of the claims of the present invention, or meet the standards of equivalent technologies, the present invention also expressly intends to include them in the scope of protection.

Claims

1. An efficient promoter for enhancing the intracellular expression of green fluorescent protein in Flammulina velutipes, characterized in that: The promoter is selected from the nucleotide sequences of any one of the following: (a) the nucleotide sequences shown in SEQ ID NO.1-3; (b) DNA molecular sequences hybridizing with the sequences shown in SEQ ID NO.1-3; (c) DNA molecular sequences having more than 90% similarity with the sequences shown in SEQ ID NO.1-3 and capable of regulating the expression of the eGFP gene in Flammulina velutipes; (d) sequences obtained by truncating or assembling on the basis of the sequences shown in SEQ ID NO.1-3, and the sequences after truncation or assembly can play the same role as the promoter.

2. A gene expression cassette for enhancing the intracellular expression of green fluorescent protein in Flammulina velutipes, characterized in that: The gene expression cassette comprises the promoter described in claim 1.

3. A recombinant plasmid comprising the promoter described in claim 1.

4. An engineered bacterium comprising the promoter described in claim 1.

5. Application of a promoter in enhancing the intracellular expression of green fluorescent protein in Flammulina velutipes, characterized in that: The nucleotide sequence of the promoter is as shown in SEQ ID NO.1-3.

6. Application of a promoter in gene editing of Flammulina velutipes, characterized in that: The nucleotide sequence of the promoter is as shown in SEQ ID NO.1-3.

7. Application of promoter in molecular breeding of Flammulina velutipes, characterized in that: The nucleotide sequence of the promoter is as shown in SEQ ID NO.1-3.

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