Agrobacterium tumefaciens-mediated pleurotus pulmonarius maize strain transformation method

Abstract

The invention discloses an agrobacterium tumefaciens-mediated pleurotus pulmonarius maize strain transformation method, and relates to the technical field of edible mushroom genetic transformation, the technical key points are as follows: maize (commonly known as foxtail millet in the north) is taken as a culture medium, pleurotus pulmonarius hyphae are successfully cultured, and agrobacterium tumefaciens-mycelium agrobacterium tumefaciens-mediated transformation is further realized; during transformation, hyphae are inoculated to a maize culture medium, and maize strains are triturated, so that the growth of pleurotus pulmonarius hyphae on maize is promoted, wounds are generated, and the infection efficiency of agrobacterium is improved; the method is simple and convenient to operate and high in conversion efficiency and has a very good application prospect.

Description

Technical Field

[0001] This invention relates to the field of genetic transformation technology of edible fungi, specifically to a method for Agrobacterium-mediated transformation of Pleurotus ostreatus strain. Background Technology

[0002] Pleurotus pulmonarius, commercially known as oyster mushroom, is an important edible fungus. Its fruiting body has a crisp and tender texture, a rich umami flavor, and is rich in nutrients such as protein, vitamins, total sugar, and crude fat. It also possesses certain anti-tumor effects and is known as the "MSG mushroom" due to its unique flavor.

[0003] The completion of genome and transcriptome sequencing of *Pleurotus pulmonaryus* in recent years has provided a valuable data platform for revealing and exploring genes related to matrix degradation, hyphal growth, primordium formation, and fruiting body growth and development. However, the functions of many of these genes remain unverified. Gene knockout, RNA interference, overexpression techniques, and CRISPR-Cas9 gene editing are important methods for studying gene function, with genetic transformation technology being a crucial component.

[0004] Therefore, the present invention aims to provide a method for Agrobacterium-mediated transformation of Pleurotus ostreatus strain, providing a stable and efficient genetic transformation method for edible fungi gene function research. Summary of the Invention

[0005] The purpose of this invention is to solve the above-mentioned problems and provide an Agrobacterium-mediated transformation method for Pleurotus pulmonarius in millet. Millet (foxtail millet) is used as a substrate to culture Pleurotus pulmonarius to obtain millet-mycelia, which are then transformed by Agrobacterium. During transformation, the mycelia are inoculated onto a millet culture medium. Crushing the millet mycelia creates wounds on the Pleurotus pulmonarius hyphae growing on the millet, which is conducive to Agrobacterium infection. This method is simple to operate, has high transformation efficiency, and has good application prospects.

[0006] To achieve the above objectives, the technical solution of the present invention is as follows:

[0007] This invention provides a method for Agrobacterium-mediated transformation of Pleurotus ostreatus, the method comprising the following steps:

[0008] S1. Preparation of *Pleurotus pulmonaryus* strain: The mycelium (with culture medium) of *Pleurotus pulmonaryus* strain 57-14 cultured on PDA was transferred to a culture bottle containing millet substrate and cultured at 20-24℃ for 10-15 days.

[0009] S2, Agrobacterium-mediated transformation of Pleurotus pulmonarius: Sterile medicine spoon is used to crush the millet spores of Pleurotus pulmonarius, and a spoonful of millet spores is put into the induced Agrobacterium suspension for about 3-4 hours of co-culture; then the millet spores are transferred to a culture dish, and the millet spores are transferred to a co-culture plate containing filter paper with tweezers, and cultured at 21℃ for 4 days.

[0010] S3, screening of Pleurotus pulmonarius pseudo-transformants: the millet spores of Pleurotus pulmonarius Tai Xiu single spore strain 57-14 are transferred to the primary screening medium MMP plate containing carboxin and cefotaxime with sterile tweezers, and cultured at 20-24℃ for about 7-10 days; the newly grown mycelial blocks are transferred to PDA medium containing carboxin and cefotaxime, and cultured at 20-24℃ for about 10 days for re-screening.

[0011] S4, verification of Pleurotus pulmonarius transformants: the DNA of the transformant strains obtained after screening is extracted, PCR verification is carried out, and 10 transformant PCR products are randomly taken for sequencing.

[0012] Compared with the prior art, the beneficial effects of the present application are:

[0013] In the present application, millet (millet) is used as the substrate to culture Pleurotus pulmonarius to obtain millet-mycelium, and Agrobacterium transformation is carried out, and the mycelium is inoculated into the millet medium during transformation, and the millet spores are crushed to cause wounds on the Pleurotus pulmonarius mycelium growing on the millet, which is conducive to the infection of Agrobacterium. Compared with the conventional method of using fruit body gills and potato dextrose (agar) cultured mycelium as transformation material, the transformation method of the present application has high transformation efficiency and convenient material acquisition. BRIEF DESCRIPTION OF DRAWINGS

[0014] Figure 1 is the method flow chart of Agrobacterium-mediated millet spore transformation of Pleurotus pulmonarius in the embodiment of the present application;

[0015] Figure 2 is the millet spore of Pleurotus pulmonarius 57-14 in the embodiment of the present application;

[0016] Figure 3 is the growth condition of Pleurotus pulmonarius transformants on the screening medium (containing carboxin and cefotaxime) in the embodiment of the present application;

[0017] Figure 4 is the electrophoresis map of PCR amplification of Pleurotus pulmonarius transformants in the embodiment of the present application, wherein M: DL5000 marker; +: plasmid as positive control; 0: blank control, -: untransformed 57-14 strain as negative control; 1-31: transformant strains. DETAILED DESCRIPTION

[0018] In order to make the person skilled in the art better understand the present application, the technical solutions of the present application will be further described in detail below in combination with the embodiments of the present application and the accompanying drawings. Obviously, the described embodiments are only a part of the embodiments of the present application, rather than all the embodiments. Based on the embodiments in the present application, all other embodiments obtained by those of ordinary skill in the art without creative work should belong to the protection scope of the present application.

[0019] It should be noted that the embodiments in the present application and the features in the embodiments can be combined with each other without conflict. The present application will be described in detail below in combination with the embodiments.

[0020] Embodiments:

[0021] 1. Strains

[0022] The Agrobacterium strain containing the Plesdi1 point mutation succinate dehydrogenase iron-sulfur protein subunit expression vector pPlesdi1 of the Pleurotus pulmonarius Tai Xiu single-spore strain 57-14 and the P. pulmonarius zizania strain were provided by the Edible Fungus Research Institute of Fujian Academy of Agricultural Sciences.

[0023] 2. Preparation of P. pulmonarius zizania strain

[0024] The mycelium (with culture medium) of the P. pulmonarius Tai Xiu single-spore strain 57-14 cultured on PDA was transferred to a cultivation bottle containing zizania substrate, and cultured at 20-24°C for 10-15 days. The zizania substrate formula: zizania (millet) 97%, gypsum 1.5%, light calcium carbonate 1.5%, water content after cooking controlled at 48%, loaded into the cultivation bottle, and autoclaved at 121°C for 3h.

[0025] 3. Agrobacterium-mediated transformation of P. pulmonarius

[0026] The Agrobacterium strain containing the binary expression vector pPlesdi1 was inoculated into the basic culture containing kanamycin and cultured overnight at 28°C; then, the bacterial solution was transferred into a 50mL sterile centrifuge tube and centrifuged at 9000g for 5min; the supernatant was discarded, and the cells were resuspended with the induction medium containing a final concentration of 200μmol / L acetyl-syringone (AS) and cultured at 28°C for 3-4h; the zizania strain of the P. pulmonarius Tai Xiu single-spore strain 57-14 was crushed with a sterile medicine spoon, and one spoonful of zizania strain was put into the induced Agrobacterium suspension; then, the zizania strain was transferred to a culture dish and transferred to a co-culture plate containing filter paper with sterile tweezers, and cultured at 21°C for 4 days. Figure 2

[0027] ​The basic culture medium (1L) formula is as follows: autoclave 941.5mL ddH2O, cool and add in the following order: 10mL K-buffer (pH7.0), 20mL MN, 1mL 1% CaCl2·2H2O, 10mL 20% glucose, 10mL 0.01% FeSO4, 5mL trace elements, and 2.5mL 20% NH4NO3.

[0028] Induction medium (1L): Autoclave 910.7mL ddH2O, cool, and then add in the following order: 10mL K-buffer (pH 7.0), 20mL MN, 1mL 1% CaCl2·2H2O, 5mL trace elements, 2.5mL 20% NH4NO3, 10mL 50% glycerol, 40mL 1M MES (pH 5.5), 10mL 20% glucose, and 0.0392g acetylsyleugenone.

[0029] Co-culture medium (1L): Add 5mL of 20% glucose and 1.5% agar, and the other components are the same as those of the induction medium.

[0030] The composition of trace elements (250mL) is as follows: 0.025g ZnSO4·7H2O; 0.025g CuSO4·5H2O; 0.025g H3BO4; 0.025g MnSO4·H2O; 0.025g Na2MoO4·2H2O.

[0031] 4. Screening of lung-shaped pleurotus transformants

[0032] Using sterile forceps, transfer the *Pleurotus erythrorhizon* strain 57-14 to MMP plates containing oxychloride (0.5 μg / mL) and cefotaxime (200 μg / mL) for initial screening. Incubate at 20–24°C for 7–10 days. Figure 3 The number of millet grains that grew mycelium on resistance plates was counted. The newly grown mycelial blocks were then transferred to PDA medium containing carbendazim (0.5 μg / mL) and cefotaxime (200 μg / mL) and cultured at 20–24°C for approximately 10 days for secondary screening. Figure 3 The MMP primary screening medium (1L) was formulated as follows: 0.5% peptone, 1% malt extract and 2.1% 3-(N-morpholino)propanesulfonic acid, with pH adjusted to 7.0.

[0033] 5. Validation of the pulmonary pleuropic transformant

[0034] DNA was extracted from the transformed strains obtained after screening and verified by PCR. The PCR primers were the plasmid loop sequence PlesdiHuanF:TGTACTGAATTAACGCCGA and the sdi1 gene sequence of Pleurotus pulmonale Plesdi1R:TCAAGTCAATAGTGTACGAC. The PCR system consisted of 12.5 μL PCR Mix, 1 μL each of PlesdiHuanF and Plesdi1R, and 10.5 μL sterile water. The reaction program was 94℃ denaturation for 4 min, 94℃ denaturation for 30 s, 58℃ annealing for 15 s, and 72℃ extension for 1 min, for 34 cycles. The PCR products from 10 transformants were randomly selected for sequencing.

[0035] 6. The positive conversion rate statistics show a conversion rate of 97.67%.

[0036] The above specific embodiments are merely explanations of the present invention and are not intended to limit the present invention. After reading this specification, those skilled in the art can make modifications to these embodiments without contributing any inventive step, but as long as they are within the scope of the claims of the present invention, they are protected by patent law.

Claims

1. A method for Agrobacterium-mediated transformation of Pleurotus ostreatus strain, characterized in that: The method includes the following steps: S1. Preparation of Pleurotus pulmonaryus corn spores: The mycelium of Pleurotus pulmonaryus Taixiu monosporus strain 57-14 cultured on PDA medium was transferred to a culture bottle containing corn substrate and cultured at 20-24℃ for 10-15 days to obtain Pleurotus pulmonaryus corn spores. S2. Agrobacterium-mediated transformation of Pleurotus pulmonale: The Pleurotus pulmonale strain was crushed and co-cultured in an induced Agrobacterium suspension for 3-4 hours; then the Pleurotus pulmonale strain was transferred to a petri dish, and the Pleurotus pulmonale strain was transferred to a co-culture plate containing filter paper with tweezers and incubated at 21°C for 4 days. S3. Screening of Pleurotus pulmonaryis transformants: Using sterile forceps, transfer the millet-like inoculum of Pleurotus pulmonaryis strain 57-14 to the primary screening medium MMP plate and incubate at 20-24℃ for 7-10 days; transfer the newly grown mycelial blocks to PDA medium and incubate at 20-24℃ for 10 days, and then perform secondary screening. S4. Validation of Pleurotus ostreatus transformants: DNA was extracted from the transformed strains obtained after screening, and PCR validation was performed. Ten PCR products of the transformants were randomly selected for sequencing.

2. The method for Agrobacterium-mediated transformation of Pleurotus ostreatus strain as described in claim 1, characterized in that: The corn substrate contains 97% corn, 1.5% gypsum and 1.5% light calcium carbonate; the moisture content of the corn substrate after cooking is 48%, and it is sterilized at 121°C for 3 hours in a cultivation bottle.

3. The method for Agrobacterium-mediated transformation of Pleurotus ostreatus strain as described in claim 1, characterized in that: Both the initial screening medium and the PDA medium in S3 contain carboxin and cefotaxime.

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