A recombinant vector for knocking out the Verticillium dahliae lncRNA28816 gene, its preparation method and application

Abstract

The present invention provides a lncRNA28816 gene knockout recombinant vector, a preparation method and an application thereof. The nucleotide sequence of the lncRNA28816 gene is shown as SEQ ID NO.1. By using the lncRNA28816 gene knockout recombinant vector, the pathogenicity of Verticillium dahliae to crops can be effectively reduced, and the resistance of crops to Verticillium dahliae can be improved.

Description

Technical Field

[0001] This application belongs to the field of microbial technology. Specifically, it relates to a recombinant vector for knocking out the lncRNA28816 gene and its preparation method, a Verticillium dahliae strain with the lncRNA28816 gene deleted and its preparation method, and a method for improving the resistance of crops to Verticillium dahliae. Background Art

[0002] Long non-coding RNA (lncRNA) is a class of nucleic acid molecules with a length greater than 200 nt, which usually widely participate in various physiological and pathological processes of organisms in the form of regulating gene expression, epigenetics, etc. Compared with plant and animal lncRNAs, fungal lncRNAs have only been studied in yeast, Trichoderma, etc. and participated in biological processes such as stress response, cell cycle regulation, gene silencing, etc., while there are few reports on the research of pathogenic fungi. As a major soil-borne pathogenic fungus in crop production, Verticillium dahliae has been widely studied on pathogenic-related genes (effectors, transcription factors, etc.) and its damage mechanism. It has been found that UvlncNAT-MFS in Ustilaginoidea virens participates in life processes such as the growth and development and stress response of Magnaporthe oryzae by forming an RNA duplex with UvMFS; GzmetE-AS in Fusarium graminearum regulates the expression of the GzmetE gene through the RNAi pathway and participates in the sexual and asexual reproduction of Fusarium graminearum. However, there is currently no relevant report on the pathogenic lncRNA of Verticillium dahliae. Summary of the Invention

[0003] The purpose of the present disclosure is to provide a recombinant vector for knocking out the lncRNA28816 gene and its preparation method, a Verticillium dahliae strain with the lncRNA28816 gene deleted and its preparation method, and a method for improving the resistance of crops to Verticillium dahliae in view of problems such as severe symptoms caused by Verticillium dahliae infecting plants in the prior art.

[0004] To achieve the above purpose, in the first aspect of the present disclosure, a recombinant vector for knocking out the lncRNA28816 gene is provided, and the nucleotide sequence of the lncRNA28816 gene is as shown in SEQ ID NO.1.

[0005] Optionally, in the recombinant vector for knocking out the lncRNA28816 gene, the lncRNA28816 gene is replaced by a hygromycin gene, and the nucleotide sequence of the hygromycin gene is as shown in SEQ ID NO.2.

[0006] Optionally, the nucleotide sequence of the recombinant vector for knocking out the lncRNA28816 gene is as shown in SEQ ID NO.3.

[0007] Optionally, the lncRNA28816 gene knockout recombinant vector is an lncRNA28816 gene knockout recombinant expression vector, and an expression cassette is inserted into the lncRNA28816 gene knockout recombinant expression vector, and the nucleotide sequence of the expression cassette is as shown in SEQ ID NO.4.

[0008] On the other hand, the present disclosure also provides a method for preparing the lncRNA28816 gene knockout recombinant vector, and the preparation method includes the following steps:

[0009] S1. Using the DNA of Verticillium dahliae as a template, performing PCR amplification with specific primers to obtain the upstream and downstream fragments of the lncRNA28816 gene;

[0010] S2. Connecting the upstream and downstream fragments of the lncRNA28816 gene to the 5' end and 3' end of the pCH expression vector and the hygromycin gene respectively to obtain the lncRNA28816 knockout vector.

[0011] Optionally, the sequences of the specific primers are as shown in SEQ ID NO.5-8.

[0012] On the other hand, the present disclosure also provides a Verticillium dahliae lncRNA28816 gene deletion strain.

[0013] On the other hand, the present disclosure also provides a method for preparing a Verticillium dahliae lncRNA28816 gene deletion strain, and the method includes the following steps: introducing the lncRNAC28816 gene knockout recombinant vector into a Verticillium dahliae strain.

[0014] On the other hand, the present disclosure also provides a method for improving the resistance of crops to Verticillium dahliae, and the method includes: attaching the lncRNA28816 gene deletion strain to the crop, and / or co-culturing the crop with the Verticillium dahliae lncRNA28816 gene deletion strain.

[0015] Optionally, the crop includes cotton.

[0016] Through the above technical solutions, the present disclosure provides a Verticillium dahliae lncRNA28816 gene knockout recombinant vector and its preparation method and application. By using the lncRNA28816 gene knockout recombinant vector to construct a Verticillium dahliae lncRNA28816 gene deletion strain, and attaching the Verticillium dahliae lncRNA28816 gene deletion strain to the crop or co-culturing with the crop, the pathogenicity of Verticillium dahliae to the crop can be effectively reduced, and the resistance of the crop to Verticillium dahliae can be improved.

[0017] Other features and advantages of the present disclosure will be described in detail in the following detailed description section. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 is the expression pattern of the lncRNA28816 gene during the infection process.

[0019] Figure 2 is the electrophoresis detection map of the lncRNA28816 gene deletion strain.

[0020] Figure 3 is the determination of the effect of the lncRNA28816 gene deletion strain on colony growth.

[0021] Figure 4 is the determination of the effect of the lncRNA28816 gene deletion strain on the pathogenicity of Verticillium dahliae.

[0022] Figure 5 is the determination of the disease index of cotton plants inoculated with the lncRNA28816 gene deletion strain.

[0023] Figure 6 is the determination of the effect of the lncRNA28816 gene deletion strain on the pathogenicity of Verticillium dahliae. DETAILED DESCRIPTION

[0024] The following detailed description of the present disclosure will be made with reference to the accompanying drawings. It should be understood that the specific embodiments described herein are for illustrative and explanatory purposes only and are not intended to limit the present disclosure.

[0025] In a first aspect of the present disclosure, a lncRNA28816 gene knockout recombinant vector is provided, and the nucleotide sequence of the lncRNA28816 gene is as shown in SEQ ID NO.1.

[0026] Optionally, in the lncRNA28816 gene knockout recombinant vector, the lncRNA28816 gene is replaced by a hygromycin gene, and the nucleotide sequence of the hygromycin gene is as shown in SEQ ID NO.2.

[0027] Optionally, the nucleotide sequence of the lncRNA28816 gene knockout recombinant vector is as shown in SEQ ID NO.3.

[0028] Optionally, the lncRNA28816 gene knockout recombinant vector is a lncRNA28816 gene knockout recombinant expression vector, and an expression cassette is inserted into the lncRNA28816 gene knockout recombinant expression vector, and the nucleotide sequence of the expression cassette is as shown in SEQ ID NO.4.

[0029] On the other hand, the present disclosure also provides a method for preparing the lncRNA28816 gene knockout recombinant vector, and the preparation method includes the following steps:

[0030] S1. Using the DNA of Verticillium dahliae as a template, performing PCR amplification with specific primers to obtain the upstream and downstream fragments of the lncRNA28816 gene;

[0031] S2. Connecting the upstream and downstream fragments of the lncRNA28816 gene to the 5' end and 3' end of the pCH expression vector and the hygromycin gene respectively to obtain the lncRNA28816 knockout vector.

[0032] Optionally, the specific primer sequences are as shown in SEQ ID NO.5-8.

[0033] On the other hand, the present disclosure also provides a Verticillium dahliae lncRNA28816 gene deletion strain.

[0034] On the other hand, the present disclosure also provides a method for preparing a Verticillium dahliae lncRNA28816 gene deletion strain, and the preparation method includes the following steps: introducing the lncRNAC28816 gene knockout recombinant vector into the Verticillium dahliae strain.

[0035] On the other hand, the present disclosure also provides a method for improving the resistance of crops to Verticillium dahliae, and the method includes: attaching the lncRNA28816 gene deletion strain to the crops, and / or co-culturing the crops with the Verticillium dahliae lncRNA28816 gene deletion strain.

[0036] Optionally, the crops include cotton.

[0037] The following further details the present disclosure through examples. The raw materials used in the examples can all be obtained through commercial channels.

[0038] Both lncRNA28816-1 and lncRNA28816-2 involved in the examples of the present disclosure are the Verticillium dahliae lncRNA28816 gene knockout recombinant vectors prepared by the present disclosure, and the nucleotide sequence of the lncRNA28816 gene is as shown in SEQ ID NO.1.

[0039] The culture medium used in the present disclosure is a culture medium commonly used in the art. Among them, Kan is kanamycin, Gen is gentamicin, Rif is rifampicin, Cef is cephalosporin, Hyg is hygromycin, and the nucleotide sequence of the hygromycin gene is as shown in SEQ IDNO.2.

[0040] Example 1

[0041] 1. Cloning of upstream and downstream fragments of lncRNA28816 gene

[0042] Using the sequences of SEQ ID NO.5 - 8 as specific primers and the genomic DNA of Verticillium dahliae Vd991 as a template, amplify the upstream sequence of lncRNA28816 with the EcoRⅠ restriction site of the pCH expression vector and the downstream sequence with the XbaⅠ restriction site. The PCR amplification system and reaction program are shown in Table 1 and Table 2 respectively; use 1% agarose gel electrophoresis to detect the PCR products, and recover the target fragments with a gel recovery kit (purchased from TransGen Biotech Co., Ltd., Beijing).

[0043] Table 1 PCR amplification system

[0044]

[0045] Table 2 Reaction program

[0046]

[0047] Ligation of the target fragment with the pCH expression vector: (1) Digest the pCH plasmid with EcoRⅠ to obtain a linearized vector, and use the single - fragment homologous recombination enzyme purchased from Novoprotein to ligate the upstream sequence of lncRNA28816 to the pCH expression vector; (2) Digest the pCH - lncRNA28816 upstream recombinant plasmid with XbaⅠ to obtain a linearized vector, and use the single - fragment homologous recombination enzyme purchased from Novoprotein to ligate the downstream sequence of lncRNA28816 to the pCH expression vector. Finally, obtain the recombinant vector of pCH - lncRNA28816 upstream - hygromycin - lncRNA28816 downstream. The nucleotide sequence of this recombinant vector is shown in SEQ ID NO.3, and the inserted expression cassette sequence in the recombinant vector is shown in SEQ ID NO.4.

[0048] The transformation method of the recombinant plasmid refers to the operation steps in the transformation instruction manual of Escherichia coli competent DH5α (purchased from TransGen Biotech Co., Ltd., Beijing): Add 5 μL of the ligation product to 50 μL of competent cells, gently mix; place on ice for 30 min, heat - shock at 42 °C for 45 s, and then place on ice for 2 min; then add 1 mL of LB liquid medium and culture at 37 °C and 220 rpm for 1 h; centrifuge to collect the bacteria, evenly coat on a solid LB medium plate with kanamycin resistance (100 μg / mL), and culture at 37 °C overnight (8 - 16 h).

[0049] Colony PCR identification: After overnight culture, pick a single colony with a sterilized pipette tip, first add 40 μL of ddH 2Pipette the suspension in the centrifuge tube containing O to form a bacterial solution, which is used as a template for colony PCR identification. Perform according to the reaction system in Table 1 and the reaction procedure in Table 2. Detect the PCR amplification products by 1% agarose gel electrophoresis, and select the colonies with appropriate amplified fragment sizes for shaking culture.

[0050] Extract the plasmid of Escherichia coli: Add the corresponding bacterial solution with correct colony PCR identification to 2 - 3 mL of LB liquid medium with kanamycin resistance, and culture at 37 °C and 220 rpm for 12 - 16 h. Extract the plasmid of Escherichia coli with reference to the operating steps of the plasmid extraction kit (purchased from TransGen Biotech Co., Ltd., Beijing).

[0051] Transformation of Agrobacterium tumefaciens AGL-1 competent cells: Take 100 μL of Agrobacterium tumefaciens AGL-1 competent cells, add 5 μL of recombinant plasmid DNA, incubate on ice for 30 min, freeze in liquid nitrogen for 2 min, incubate at 37 °C for 5 min, then add 1 mL of SOC liquid medium, culture at 28 °C and 220 rpm for 4 - 6 h, centrifuge at 5000 g for 1 min, leave about 100 μL of the bacterial solution, pipette it repeatedly and evenly coat it on the LB solid medium plate (Kan, Gen, Rif), and culture at 28 °C for about 48 h. After culture, pick a single colony for PCR amplification to detect whether it is a positive clone.

[0052] Agrobacterium-mediated genetic transformation: After 2 days, pick colonies for PCR verification. If the band size is correct, pipette 500 μL of the bacterial solution and mix it with glycerol at a volume ratio of 1:1 for preservation; Inoculate 500 μL of Agrobacterium tumefaciens into 20 mL of MM liquid medium (containing Kan and Rif), and culture it at 28 °C with shaking at 150 rpm for 24 - 48 h; Centrifuge to collect the cells, pour out the supernatant in the laminar flow bench and resuspend the cells twice with 10 mL of IM medium, then add acetosyringone (200 μg / mL) to the IM medium, dilute the mixture to OD600 = 0.15 - 0.20, and continue to culture with shaking until OD600 = 0.5 - 0.7; 3 days before co-culture, inoculate Verticillium dahliae into CM culture medium and culture it on a shaker at 25 °C and 150 rpm / min for 3 - 4 days. Filter the hyphae with a sterilized filter cloth during the increase of the bacterial solution OD600, and adjust the spore concentration to 5×10 6CFU / mL (20 spores per square in the middle grid), and a spore suspension was obtained; the sterilized microporous filter membrane (diameter 20 mm) was laid flat on the IM solid medium containing acetosyringone (200 μg / mL) with forceps, and the spore suspension and Agrobacterium were mixed in a 1.5 mL centrifuge tube at a volume ratio of 1:1. 100 μL was aspirated and evenly spread on the microporous filter membrane; sealed with parafilm and incubated upside down in an incubator at 25 °C for 2 d, then the microporous filter membrane was transferred to a PDA solid plate containing resistance (200 μg / mL Cef, 100 μg / mL Kan, 30 μg / mL Hyg), and continued to be cultured for more than 7 d until transformant colonies grew on the microporous filter membrane; the expression level of lncRNA28816 gene during the infection process was as Figure 1 shown. The expression level of lncRNA28816 gene increased during the infection process, reached the peak at the 5th day, about 25 times that of the uninfected expression level, and then the expression level gradually decreased.

[0053] Verification of knockout strains: Single spore isolation: Add 800 μL of sterile water to a 1.5 mL centrifuge tube, pick a single colony on the plate with a toothpick and add it to the sterile water, shake it, and pipette 10 μL and drop it on the PDA plate (containing the 4 antibiotics in step 6), roll it with the pre-sterilized glass beads and smear it evenly; new colonies will grow on the PDA plate after 2 d. Pick the new single colonies and repeat step 7 twice to obtain the third-generation single spores; due to the possible random insertion, pick single colonies into an appropriate amount of sterile water, activate them on the resistance plates of G418 and Hyg respectively, pick the strains with only Hyg resistance, culture and collect them, extract DNA, and perform PCR detection with the internal part of the target gene (test primers 1-F / R) and hyg fragment primers (Hyg-F / R), then design external primers (test primers 2-F / R and test primers 3-F / R) outside the upstream and downstream homologous arms for full-length (test primers 2F / 3R) amplification and sequencing, and compare the results. If the gene is replaced with hyg, it can be regarded as a successful knockout. The results are as Figure 2 shown. Re-detect and preserve after the third generation of single spores, and at least 2 strains of each gene should be preserved.

[0054] Example 2

[0055] This example is used to illustrate the application of the lncRNA28816 gene deletion strain of Verticillium dahliae in reducing the pathogenicity of Verticillium dahliae.

[0056] Inoculate the lncRNA28816 gene deletion strain into cotton: Use CM medium to shake culture and collect the spore suspensions of each strain and adjust the concentration to 5×10 6cells / mL, prepare approximately 300 mL; Take out the Junmian plants with 2 true leaves after growing for 3 weeks from the seedling pots, wash the soil from the roots, inoculate each plant with 2 lncRNA28816 gene deletion strains, inoculate 20 plants with each strain once, immerse their roots in the spore suspension for 30 min, then replant them in the culture substrate in groups of 5 (4 cups for each strain), quickly freeze the samples from the base of the cotton plant stems in liquid nitrogen, and store them at -80 °C for later use.

[0057] Set water and wild-type WT treatments as negative and positive controls respectively. All pathogenicity assays were repeated at least 3 times (20 plants for each mutant each time) until stable. The results are as Figure 3 shown. The lncRNA28816 gene deletion strain does not affect the growth of the colony.

[0058] Example 3

[0059] This example is used to determine the change in the disease index after inoculating the lncRNA28816 gene deletion strain into cotton.

[0060] After culturing in a humid and dark environment for 5 days, switch to an alternating light-dark culture. After about 3 weeks, collect the pathogenicity phenotypes, and count the incidence and disease index. Since the true leaves of the cotton plants were not included in the statistics, the disease index was divided into 4 grades: grade 0 is healthy; grade 1 is that one true leaf has Verticillium wilt symptoms; grade 2 is that two true leaves show Verticillium wilt symptoms; grade 3 is that two true leaves are completely wilted and fallen off, and even the plant dies. The results are as Figure 4 shown. The cotton plants inoculated with the lncRNA28816 gene deletion strain have leaves that are significantly more inclined to be healthy.

[0061] Observe the discoloration of the xylem at the base of the cotton plant stems. The results are as Figure 5 shown. The degree of brownization of the vascular bundles of the cotton plants inoculated with the lncRNA28816 gene deletion strain is significantly weakened.

[0062] Example 4

[0063] This example is used to determine the change in biomass after inoculating the lncRNA28816 gene deletion strain into cotton.

[0064] Extract the total DNA of the biomass samples according to the method provided in Example 1. Use fluorescence quantitative PCR (qPCR) to detect the biomass of fungi in the cotton root and stem parts after the strain infection. Use the 18S rDNA gene of cotton as an internal reference and the elongation factor VdEF-1α gene of Verticillium dahliae as the detection gene. Use 2 –ΔΔCtThe relative content of Verticillium dahliae DNA was detected by the method, and the qPCR reaction system was as follows (refer to Top Green qPCR SuperMix(+Dye II), TransGen). The QuantStudio3 Real-Time PCR system (Thermo Fisher Scientific) was used, and the program was pre-denaturation at 95°C for 3 min; 40 cycles: denaturation at 95°C for 15 s, annealing at 60°C for 20 s, extension at 72°C for 20 s (fluorescence intensity was captured here); complete extension at 72°C for 2 min, and the experiment was independently repeated 3 times.

[0065] The results are as Figure 6 shown. The use of the lncRNA28816 gene deletion strain can effectively reduce the fungal biomass in the cotton root and stem parts after Verticillium dahliae infection.

[0066] The preferred embodiments of the present disclosure have been described in detail above. However, the present disclosure is not limited to the specific details in the above embodiments. Within the technical concept of the present disclosure, various simple modifications can be made to the technical solutions of the present disclosure, and these simple modifications all fall within the protection scope of the present disclosure.

[0067] In addition, it should be noted that, in the above specific embodiments, the various specific technical features described can be combined in any suitable manner without conflict. To avoid unnecessary repetition, the present disclosure will not separately describe various possible combination methods.

[0068] Furthermore, any combination can be made between different embodiments of the present disclosure as long as it does not violate the idea of the present disclosure, and it should also be regarded as the content disclosed by the present disclosure.

Claims

1. A recombinant vector for knocking out the lncRNA28816 gene, characterized in that, the nucleotide sequence of the lncRNA28816 gene is as shown in SEQ ID NO.1; wherein, in the recombinant vector for knocking out the lncRNA28816 gene, the lncRNA28816 gene is replaced by a hygromycin gene, and the nucleotide sequence of the hygromycin gene is as shown in SEQ ID NO.2; the nucleotide sequence of the recombinant vector for knocking out the lncRNA28816 gene is as shown in SEQ ID NO.

3.

2. The recombinant vector for knocking out the lncRNA28816 gene according to claim 1, wherein, the recombinant vector for knocking out the lncRNA28816 gene is a recombinant expression vector for knocking out the lncRNA28816 gene, and an expression cassette is inserted into the recombinant expression vector for knocking out the lncRNA28816 gene, and the nucleotide sequence of the expression cassette is as shown in SEQ ID NO.

4.

3. A method for preparing the recombinant vector for knocking out the lncRNA28816 gene according to any one of claims 1-2, characterized in that, the preparation method comprises the following steps: S1. Using the DNA of Verticillium dahliae as a template, performing PCR amplification with specific primers to obtain the upstream and downstream fragments of the lncRNA28816 gene; S2. Connecting the upstream and downstream fragments of the lncRNA28816 gene to the 5' end and 3' end of the pCH expression vector and the hygromycin gene respectively to obtain the recombinant vector for knocking out the lncRNA28816 gene.

4. The preparation method according to claim 3, wherein, the sequences of the specific primers are as shown in SEQ ID NO.5-8.

5. A Verticillium dahliae strain with a deletion of the lncRNA28816 gene, characterized in that, the preparation method of the Verticillium dahliae strain with a deletion of the lncRNA28816 gene comprises: introducing the recombinant vector for knocking out the lncRNAC28816 gene according to any one of claims 1-2 into a Verticillium dahliae strain.

6. A method for preparing a Verticillium dahliae strain with a deletion of the lncRNAC28816 gene, characterized in that, the preparation method comprises: introducing the recombinant vector for knocking out the lncRNAC28816 gene according to any one of claims 1-2 into a Verticillium dahliae strain.

7. A method for improving the resistance of crops to Verticillium dahliae, characterized in that, the method comprises: attaching the Verticillium dahliae strain with a deletion of the lncRNA28816 gene according to claim 6 to the crop, and / or co-culturing the crop with the Verticillium dahliae strain with a deletion of the lncRNA28816 gene according to claim 6; wherein, the crop is cotton.

Images