Application of CmHMG1 gene of Condylactis to enhance biocontrol ability in Condylactis

Abstract

The invention belongs to the field of microbial genetic engineering, and specifically relates to the application of the CmHMG1 gene of Shieldia spp. in enhancing the biocontrol ability of Shieldia spp. Overexpression of the CmHMG1 gene of Shieldia spp. in Shieldia spp. can significantly improve the ability of Shieldia spp. to parasitize Sclerotinia sclerotiorum, and is helpful to transform an efficient Shieldia spp. engineered strain.

Description

Technical Field

[0001] The invention belongs to the field of microbial genetic engineering, and specifically relates to the application of a CmHMG1 gene of Condylactis to enhance the biocontrol ability of Condylactis. Background Art

[0002] Sclerotinia sclerotiorum is an important plant pathogenic fungus with a worldwide distribution. It has a wide range of hosts and can successfully infect more than 450 species of plants in 75 families. Rapeseed sclerotinia caused by Sclerotinia sclerotiorum is common in rapeseed growing areas in my country, with an annual plant morbidity rate of 10% to 30%, and more than 80% in severe years, resulting in a 10% to 70% reduction in rapeseed production. According to a survey by the National Rapeseed Industry Technology System, the losses caused by rapeseed sclerotinia sclerotiorum in the Yangtze River Basin alone exceed 2 billion yuan per year. In the strategy of crop disease prevention and control, the application of disease-resistant varieties is the safest, most environmentally friendly and efficient measure for disease prevention and control, but there are currently no sclerotinia resistant germplasm resources available for production. Currently, chemical pesticide control is the mainstream control measure for rapeseed sclerotinia sclerotiorum, but the long-term use of chemical pesticides will lead to potential risks such as the production of drug-resistant strains and environmental pollution. To this end, developing environmentally friendly beneficial microbial control measures is currently the ideal way to solve the damage caused by rapeseed bacterial sclerotinia. It not only complies with the national policy of "reducing weight and medication", but also meets the national strategic needs of green development and ecological civilization construction.

[0003] Coniothyrium minitans is a parasitic fungus of Sclerotinia sclerotiorum, the pathogen of rapeseed sclerotinia disease. It can specifically parasitize and destroy the hyphae and sclerotia of Sclerotinia sclerotiorum, and has broad application prospects in the biological control of sclerotinia disease. The ability of sporulation and hyperparasitism is closely related to the biocontrol potential of Coniothyrium minitans. Exploring the genes related to sporulation and hyperparasitism of Coniothyrium minitans is conducive to the transformation of engineered Coniothyrium minitans strains with greater biocontrol potential.

[0004] HMGR, or 3-hydroxy-3-methylglutaryl-CoA reductase, catalyzes the synthesis of mevalonic acid (MVA) and is a key regulatory point in the isoprenoid biosynthesis pathway. Isoprenoids are widely distributed in nature and are a diverse class of compounds in natural substances. They are mainly related to the physiological regulation functions of cells, such as: gibberellins, abscisic acid and cytokinins are involved in cell differentiation; carotenoids, chlorophyll, etc. are related to photosynthesis; coenzyme Q is related to respiration, and sterols are related to the stability of cell membrane structure. In Saccharomyces cerevisiae, HMGR can affect yeast spore germination and growth. In filamentous fungi, it is not clear whether HMGR can regulate important growth and development processes such as filamentous fungal growth, sporulation and hyperparasitism.

[0005] The present invention utilizes the gene sequence encoding HMGR of Saccharomyces cerevisiae to compare and obtain the gene CmHMG1 encoding HMGR of the shield mold in the genome of the shield mold Cm2004 (deposit number: CGMCCN0.3852). In order to clarify the role of the gene CmHMG1 in the growth and development of the shield mold, especially in the sporulation and hyperparasitism process, the present invention analyzes the function of the gene CmHMG1 through gene knockout, interaction and overexpression technology, clarifies that CmHMG1 affects the growth, sporulation and hyperparasitism process of the shield mold, and overexpression of CmHMG1 can enhance the hyperparasitism ability of the shield mold, providing a biological control effect of the shield mold. Summary of the invention

[0006] The purpose of the present invention is to overcome the shortcomings of the prior art and provide an application of a CmHMG1 gene of Condylactis to enhance the biocontrol ability in Condylactis.

[0007] The objective of the present invention is achieved through the following technical solutions:

[0008] The application of the CmHMG1 gene of Condylactis to enhance the biocontrol ability of Condylactis, and the nucleotide sequence of the CmHMG1 gene of Condylactis is shown below.

[0009]

[0010] Furthermore, the amino acid sequence encoded by the CmHMG1 gene of the C.

[0011] MGAPTQRVKEDRSILGQIPRIQALKSDHEKVNIENFVGYAKVPLGLAGPLRIRGSENTDGAFVTPLATVEPTLVASCSRGSKALDRCGGVHFKVLGEAMSRAPVFAFADTAHAVAFAELLPSLHGRFAQEAESTSRHARLQKLTPHIIGSNVHVHFSYSCGDAAGQNMVTIATQRACEWLMESSKAQELRIRDI IIEGQMASDKKPSWGNVKELRGVQVVVWARLYDTVCKEVLGCTTERLFKVITLKEGGIHNGQFGCKINAANIIAAMFISCGQDAASVAEGCWSHLTPEYDWTTKELRLTMFFPSLPVAVVGGGTFYETQQEYLRLLRCDASGGKRRLAGLIGAFSLALDVTSSAAIANNTFAQSHANLARGKEQARGPVSLKEKL

[0012] Furthermore, the Coniothyrium minitans includes Coniothyrium minitans Cm2004, and the preservation number of Coniothyrium minitans Cm2004 is CGMCC NO.3852.

[0013] Furthermore, the biocontrol ability includes: one or more of the ability of the Shield mold to grow, the ability of the Shield enzyme to produce spores, and the ability of the Shield enzyme to heavily parasitize.

[0014] Furthermore, the ability of the shield enzyme to re-parasitize is the ability of the shield enzyme to re-parasitize Sclerotinia sclerotiorum.

[0015] Furthermore, the application method includes: overexpressing the Condylactis CmHMG1 gene in the Condylactis.

[0016] Furthermore, the method for overexpressing the CmHMG1 gene of the CmHMG1 fungus in the CmHMG1 fungus is as follows: connecting the CmHMG1 gene of the CmHMG1 fungus with a vector containing a Ptrp C promoter to form an overexpression vector, and introducing the overexpression vector into the CmHMG1 fungus using an Agrobacterium-mediated transformation system.

[0017] The beneficial effects of the present invention are:

[0018] The overexpression of the gene CmHMG1 of the present invention can significantly improve the ability of the Shieldia sclerotiorum to parasitize Sclerotinia sclerotiorum, and is helpful to transform a highly efficient Shieldia sclerotiorum engineering strain. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 Analysis of the conserved domains of CmHMG1 protein;

[0020] Figure 2 RT-PCR verification of CmHMG1 knockout and complementation transformants;

[0021] Figure 3 The colony morphology of the knockout and complementation transformants of C.

[0022] Figure 4 To test the ability of the shield mold to parasitize the sclerotia of Sclerotinia sclerotiorum;

[0023] Figure 5 RT-PCR verification (A), colony morphology (B), growth rate (C) and conidia production (D) of CmHMG1 overexpression transformants;

[0024] Figure 6 The ability of each transformant of the Shield Fungus to parasitize Sclerotinia sclerotiorum. DETAILED DESCRIPTION

[0025] The technical solution of the present invention is further described in detail below in conjunction with the accompanying drawings, but the protection scope of the present invention is not limited to the following.

[0026] The reagents and culture medium involved in the present invention are as follows:

[0027] Potato dextrose broth (PDB):

[0028] 200g peeled potato juice

[0029] Glucose 20g

[0030] ddH 2 0 to 1000ml. Add 20g agar to make PDA. Sterilize with moist heat at 121℃ for 30min.

[0031] Luria-Bertani (LB) medium:

[0032] Tryptone 10g

[0033] Yeast extract 5g

[0034] Sodium chloride (NaCl) 5g

[0035] ddH 20 to 1000 ml, adjust pH to 7.0 with NaOH, and add 20 g agar to obtain solid LB.

[0036] Agrobacterium induction medium (IM):

[0037]

[0038] ddH 2 0 to 1000 ml, add H 3 PO 4 Or adjust the pH to 6.0 with NaOH.

[0039] Agrobacterium co-cultivation medium (CO-IM):

[0040]

[0041] ddH 2 0 to 1000 ml, add H 3 PO 4 Or adjust the pH to 6.0 with NaOH.

[0042] K-Buffer:

[0043] K 2 HPO 4 200g

[0044] KH 2 PO 4 145g

[0045] ddH 2 0 to 1000 ml and adjust pH to 4.9.

[0046] MN Buffer:

[0047] MgSO 4 7H 2 O 30g

[0048] NaCl 15g

[0049] ddH 2 0 to 1000ml.

[0050] Regeneration medium (RM): 0.7 M sucrose, 0.5 g / L yeast powder, 20 g / L agar.

[0051] Protoplast preparation solution (protoplast buffer):

[0052] Snailase 0.01g

[0053] Lysing Enzymes 0.1g

[0054] 0.8M MgSO 4 10ml

[0055] The solution was shaken to promote dissolution and sterilized by filtration using a 0.22 μm filter membrane.

[0056] Protoplast suspension buffer STC: 1 M sorbitol, 50 mM Tris·HCl (pH 8.0), 50 mM CaCl 2 .

[0057] 60% PEG solution: weigh 60g PEG4000 and dissolve in 100ml ddH 2 In O.

[0058] KTC solution (KCl-Tris-CaCl 2 solution): 1.8M KCl, 150mM Tris·HCl (pH 8.0), 150mM CaCl 2 , prepared with deionized water, sterilized by high-temperature steam sterilization at 121℃ for 15min or sterilized by filtration through a 0.22μm filter membrane.

[0059] Protoplast transformation buffer (TB): Mix 60% PEG solution and KTC solution at a ratio of 2:1 (v:v) and prepare it for use immediately.

[0060] 0.2M acetosyringone (AS): 0.392 g of acetosyringone was dissolved in dimethyl sulfoxide (DMSO), then the volume was adjusted to 10 ml, divided into sterile 1.5 ml centrifuge tubes, and stored at -20°C for a long time.

[0061] Ampicillin (Amp): 50 mg / ml solution, stored at -20°C.

[0062] Hygromycin B (HYG): 50 mg / ml solution, stored at 4°C.

[0063] Cefaloxin (Cef): 10 mg / ml solution, stored at -20°C.

[0064] Kanamycin (Kana): 100 mg / ml solution, stored at -20°C.

[0065] Streptomycin (Str): 100 mg / ml solution, stored at -20°C.

[0066] Rifampicin (Rif): Prepare a 10 mg / ml solution in anhydrous methanol and store at -20°C.

[0067] Example 1 Obtaining the CmHMG1 gene of the C.

[0068] The Saccharomyces cerevisiae HMGR protein sequence (GenBank Acc. No. GHM90903) was downloaded from the NCBI website (https: / / www.ncbi.nlm.nih.gov / ), and a comparison search was performed in the genome of C. cerevisiae Cm2004 to obtain the HMGR homologous protein (SEQ ID NO: 2) and its encoding gene CmHMG1 (SEQ ID NO: 1) in C. cerevisiae. The CmHMG1 gene sequence includes an open reading frame of 1170 bp, without introns, and is presumed to encode a 389 aa protein. The conserved regions of the protein are as follows: Figure 1 .

[0069] Example 2 Functional study of CmHMG1 gene of C.

[0070] Gene knockout: CmHMG1 was knocked out using the split tagging method. The upstream and downstream sequences of the CmHMG1 gene were amplified and fused with the 3' and 5' ends of the hygromycin gene sequence. The two fusion fragments were transformed into Cm2004 protoplasts through PEG mediation, and the transformants that could grow on PDA plates containing 50μg / ml hygromycin B were screened and verified by RT-PCR. Figure 2 RT-PCR verification showed that transformants ΔCmHMG1-1 and ΔCmHMG1-2 were knockout transformants. The knockout transformants had white colonies, slow growth, and significantly reduced conidia production and ability to parasitize Sclerotinia sclerotiorum ( Figure 3 , Figure 4 , Figure 6 , Table 1).

[0071] Gene complementation: Amplify the full-length sequence of the gene CmHMG1 and connect it to the corresponding restriction site of the vector neop3300 to form the complementary vector pCHmgr. Use the Agrobacterium-mediated transformation system to transform the knockout transformant ΔCmHmgr-1, screen the transformants that can grow on PDA plates containing 30μg / ml neomycin, and perform RT-PCR verification. Figure 2 RT-PCR verification showed that transformants CmHMG1-C3 and CmHHMG1-C8 were gene complementation transformants. The colonies of complementation transformants were darker in color, grew faster, and formed a large number of mature conidiophores and conidia, and the ability to parasitize Sclerotinia was restored ( Figure 3 , Figure 4 , Figure 6 , Table 1).

[0072] Table 1 Growth and spore production ability of shield fungus

[0073]

[0074] Example 3 Effect of overexpression of CmHMG1 gene of C. cerana on biological characteristics of C. cerana

[0075] The CmHMG1 gene was connected to the pCETNS-4 vector with the Ptrp C promoter to construct an overexpression vector, and the overexpression vector was introduced into the C. cerevisiae Cm2004 using the Agrobacterium-mediated transformation system. After RNA was extracted, RT-PCR was performed to detect the expression level of CmHMG1. The results showed that the four screened transformants were CmHMG1 overexpression transformants ( Figure 5 ).

[0076] A: Growth and spore production of overexpressing transformants

[0077] After activating Cm2004 and the overexpression transformant, the young bacterial cake at the edge of the colony was inoculated in the center of the PDA plate. After culturing at 20°C for 10 days, the colony morphology was observed, the colony diameter was measured, and the growth rate was calculated. After culturing for 15 days, the conidia on the colony were washed off and the number of conidia was counted under a microscope. The results showed that overexpression of CmHMG1 did not affect the growth and sporulation of C. Figure 5 ).

[0078] B: Determination of the ability to parasitize Sclerotinia sclerotiorum

[0079] The Shield Fungus strain and the Sclerotinia sclerotiorum strain 1980 were inoculated on both sides of the PDA plate, 6 cm apart. After 20 days of confrontation culture, the 6 cm interval area was divided into 4 equidistant areas. Five hyphae were picked in each area and transplanted to the PDA plate for culture for 7-10 days. The cultured colonies were observed to determine whether they were Shield Fungus or Sclerotinia sclerotiorum, or mixed growth colonies of Shield Fungus and Sclerotinia sclerotiorum, so as to judge the ability of Shield Fungus to parasitize Sclerotinia sclerotiorum. The results showed that knocking out CMHMG1 weakened the parasitic ability of Shield Fungus, while overexpressing the gene could enhance the ability of Shield Fungus to parasitize Sclerotinia sclerotiorum ( Figure 6 ).

[0080] The above is only a preferred embodiment of the present invention. It should be understood that the present invention is not limited to the form disclosed herein, and should not be regarded as excluding other embodiments, but can be used in various other combinations, modifications and environments, and can be modified within the scope of the concept described herein through the above teachings or the technology or knowledge of the relevant field. The changes and modifications made by those skilled in the art shall not deviate from the spirit and scope of the present invention, and shall be within the scope of protection of the claims attached to the present invention.

Claims

1. The use of the CmHMG1 gene of the Shieldia spp. in enhancing the heavy parasitic ability of the Shieldia spp., characterized in that: The nucleotide sequence of the CmHMG1 gene of the Shieldia spp. is shown in SEQ ID NO: 1; Wherein, the application method comprises: overexpressing the CmHMG1 gene of the CmHMG1 fungus in the CmHMG1 fungus.

2. The use according to claim 1, characterized in that: The shield mold includes shield mold ( Coniothyrium minitans ) Cm2004, the shield mold ( Coniothyrium minitans )The deposit number of Cm2004 is CGMCCNO.3852.

3. The use according to claim 1, characterized in that: The heavy parasitism ability of the shield enzyme is the heavy parasitism ability of the shield enzyme on Sclerotinia sclerotiorum.

4. The use according to claim 1, characterized in that: The method for overexpressing the CmHMG1 gene of the Condylactis in the Condylactis is specifically as follows: connecting the CmHMG1 gene of the Condylactis with a vector containing a Ptrp C promoter to form an overexpression vector, and introducing the overexpression vector into the Condylactis using an Agrobacterium-mediated transformation system.

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