CN113604477B - Lilium regale defensin antibacterial peptide gene LrDEF1 and application thereof - Google Patents

Abstract

The invention discloses a Minjiang lily defensin antimicrobial peptide gene LrDef1 , whose nucleotide sequence is as described in SEQ ID NO: 1, and encodes a protein with an amino acid sequence as shown in SEQ ID NO: 2. The present invention adopts functional genomics related technologies Studies have confirmed that the LrDef1 gene has the function of improving plant anti-fungal. The anti-fungal LrDef1 gene of the present invention is constructed on a plant expression vector and transferred to tobacco for overexpression. The transgenic tobacco plants have strong anti-fungal infection ability. The experimental results show that Transgenic tobacco overexpressing LrDef1 had a high level of resistance to infection by Fusarium oxysporum, Phomyces phloem, and Fusarium equisetia.

Description

A Minjiang lily defensin antimicrobial peptide gene LrDef1 and its application

technical field

The invention relates to the technical fields of molecular biology and genetic engineering, in particular to a defensin antimicrobial peptide gene LrDef1 of Minjiang lily with antifungal infection ability and its application.

Background technique

Pathogens are an important factor affecting plant yield and quality loss. Plant diseases caused by pathogens can reduce crop production by 20% to 40%. Among them, fungal diseases are the largest category of plant diseases, accounting for about 70% of the total number of diseases. ~80%. The traditional methods of controlling plant diseases are mainly the use of chemical pesticides, improvement of cultivation management measures and breeding of new resistant varieties. Although these methods have achieved certain results, the traditional breeding cycle is long, the effect of cultivation measures is poor, and pesticides are easy to cause problems such as environmental safety and food safety. With the rapid development of biotechnology, the use of genetic engineering to breed new disease-resistant varieties can not only overcome the many drawbacks of the above-mentioned control methods, but also minimize the damage to beneficial microorganisms in the soil and achieve sustainable agricultural development.

Plant antimicrobial peptides (antimicrobial protein, AMP) are a class of small, cysteine-rich, basic cationic peptides, which are part of the plant immune system and have inhibitory or killing effects on pathogenic microorganisms such as bacteria, fungi, and viruses. , plays a vital role in plant defense responses (Zasloff Michael. Antimicrobial peptides of multicellular organisms. Nature, 2002, 415 (6870): 389-395). Plant defensins are an important class of AMPs. When plants are subjected to biological stress, plant defensins can bind to fungal-specific plasma membrane components, causing the cell membrane of pathogenic bacteria to sag, embed in the cell membrane or be transported into the cell membrane, and then a series of biological processes occur. Chemical changes.

At present, a variety of defensin genes have been introduced into plants to enhance plant resistance. Tobacco ( Nicotiana megalosiphon ) defensin ( NmDef02 ) is a common plant defensin with antibacterial activity. Transgenic soybean ( Glycine max ) plants expressing NmDef02 exhibited antibacterial activity against soybean rust ( Phakopsora pachyrhizi ) and anthracnose pathogen ( Colletotrichum truncatum ) Higher resistance (Soto N , Y Hernández, Delgado C , et al. Field resistance to Phakopsora pachyrhizi and Colletotrichum truncatum of transgenic soybean expressing the NmDef02 plant defensesin gene. Frontiersin Plant Science, 2020, 11: 562). A NaD1 defensin was isolated from the flower buds of tobacco ( N. alata ). NaD1 can specifically bind to the fungal cell wall and enter the fungal cytoplasm, thereby triggering a rapid increase in the concentration of intracellular reactive oxygen species and permeabilizing the fungal cytoplasmic membrane. Ultimately leads to fungal cell death (Baxter AA , Poon IK , Hulett MD. The plant defensesin NaD1 induces tumor cell death via a non-apoptotic, membranenolytic process. Cell Death Discovery, 2017, 3(1): 402-410). In addition, NaD1 protein also has good antibacterial activity against various pathogenic fungi such as Fusarium oxysporum , Botrytis cinerea , and Candida albicans (Luo Xianlin, Zhao Degang, Zhao Yichen. Tobacco NaD1 Gene Prokaryotic expression and purification of . Molecular Plant Breeding, 2020, 18(11): 3502-3508).

Lily is the general term for plants of the genus Lilium in the family Liliaceae , and is a perennial bulbous flower. During bulb propagation and fresh-cut flower production, lilies are vulnerable to various pathogenic bacteria such as fungi, viruses, and bacteria. Dozens of lily diseases have been discovered so far, among which Fusarium wilt (also known as base rot and stem rot) caused by fungi of the genus Fusarium ( Fusarium spp.) is the most serious disease in lily production. Fusarium infestation of lily bulbs causes basal necrosis and scale rot and falls off, resulting in a decline in bulb quality; leaves turn yellow, wilt and droop after plants are infected with Fusarium, and plants wither and die early, seriously affecting the yield and quality of cut lily flowers; Fusarium oxysporum has the strongest pathogenicity and the highest isolation frequency, and is the main pathogen of lily wilt. Minjiang lily ( L. regale Wilson) is a unique species in China. It is only distributed in the valleys of the Minjiang River Basin at an altitude of 800-2700m to the rock crevices on the mountainside. It has strong resistance to fusarium wilt and is an important germplasm resource for modern lily breeding. Antimicrobial peptides are an important part of plant defense system, so the discovery and functional analysis of antimicrobial peptide genes in Minjiang lily have important research and application value.

Contents of the invention

The object of the present invention is to provide a Minjiang lily defensin antimicrobial peptide gene LrDef1 and its application, that is, in improving the resistance of tobacco to Fusarium oxysporum ( F. oxysporum ), Phoma herbarum ( Phoma herbarum ), Fusarium equisetum ( F. equiseti ) resistance applications.

The present invention clones the defensin antimicrobial peptide gene LrDef1 with antifungal activity obtained from Lily of Minjiang River. The nucleotide sequence of LrDef1 is shown in SEQ ID NO: 1. The full-length cDNA sequence of the gene is 501bp, including an open reading frame of 225bp , a 5' untranslated region of 37 bp, and a 3' untranslated region of 239 bp, encoding a protein with the amino acid sequence shown in SEQ ID NO:2.

The coding region of the LrDef1 gene in the present invention is the nucleotide sequence shown at positions 38-262 in the sequence table SEQ ID NO:1.

The present invention isolates and clones a complete cDNA fragment of an antifungal related gene of Lily Minjiang, uses Agrobacterium tumefaciens to transfer the target gene into recipient plants and overexpresses it, and further experiments verify whether the gene has antifungal properties. The activity of the fungus lays the foundation for the later use of the gene to improve the ability of tobacco and other plants to resist fungal diseases. The inventor named this gene LrDef1 .

The above-mentioned LrDef1 gene can be applied to improve the antifungal properties of tobacco, and the specific operation is as follows:

(1) Using the specific primers for amplifying LrDef1 , total RNA was extracted from the roots of Lily of the Minjiang River inoculated with Fusarium oxysporum, and amplified by reverse transcription-polymerase chain reaction (RT-PCR). The full-length coding region of LrDef1 is then connected to the pGEM-T vector, and the clone with the target gene is obtained by sequencing;

(2) Digest the pGEM-T- LrDef1 vector with restriction endonucleases Eco RI and Bam HI, recover the target gene fragment by gel recovery, digest the plant expression vector pCAMBIA2300s with the same endonuclease, and recover the desired vector by gel recovery large fragment, and then the obtained LrDef1 gene fragment was connected with the pCAMBIA2300s fragment to construct a plant overexpression vector, and then the constructed recombinant vector was mediated by Agrobacterium tumefaciens and expressed in tobacco;

(3) Screen the transformants with the resistance markers on the T-DNA of the recombinant vector, and obtain the real transgenic plants through PCR and RT-PCR detection, analyze the ability of the total protein of the leaves of the transgenic plants to inhibit the growth of pathogenic fungi, and finally screen out Transgenic plants with significantly enhanced resistance to fungi.

The present invention provides a new method for improving the resistance of plants to fungal diseases. Breeding disease-resistant plants by means of genetic engineering can overcome the shortcomings of traditional breeding, not only shortens the breeding cycle, but also is simple to operate and easy to obtain high-resistant materials. The LrDef1 gene from Lilium Minjiang in the present invention can enhance the resistance of plants to fungi, and when the gene is introduced into tobacco, new varieties and materials with fungal resistance can be produced; the use of genetic engineering technology to cultivate resistant plant varieties and materials has obvious advantages and irreplaceable importance; it can not only provide convenience for large-scale production of crops, medicinal materials, horticultural plants, etc., greatly reduce the use of chemical pesticides, but also save costs for agricultural production and reduce environmental pollution. Therefore, the present invention has Broad market application prospects.

Description of drawings

Fig. 1 is the PCR detection result figure of LrDef1 transgenic tobacco genome DNA of the present invention, among the figure: Marker is DL2000 DNA Marker (Dalian treasure biology); Positive control is the PCR product of plasmid pGEM-T- LrDef1 as template; WT is non-transgenic tobacco (wild type) total DNA is the product of template PCR;

Fig. 2 is the expression analysis result figure of LrDef1 transcript level in the positive LrDef1 transgenic tobacco of the present invention; Among the figure: Marker is DL2000 DNA Marker (Dalian treasure biological); WT is the PCR product of non-transgenic tobacco total RNA reverse transcription cDNA as template; Positive control It is the PCR product of plasmid pGEM-T- LrDef1 as a template;

Figure 3 is a diagram of the antibacterial effect of LrDef1 transgenic tobacco of the present invention on the growth of pathogenic fungi in vitro; the fungi shown in Figures a, b, and c are P. herbarum and F. equiseti respectively , Fusarium oxysporum ( F. oxysporum ); WT is the total protein of wild-type tobacco, and Buffer is the blank control, that is, no protein control (buffer used for protein extraction).

Detailed ways

The present invention will be described in further detail below by accompanying drawing and embodiment, but protection scope of the present invention is not limited to described content, method in the embodiment is conventional method unless otherwise specified, and the reagent that uses is conventional market unless otherwise specified. Reagents sold or prepared by conventional methods.

Example 1: LrDef1 Gene Cloning and Sequence Analysis

Lilium Minjiang was inoculated with Fusarium oxysporum, and total RNA was extracted from the roots 24 hours after inoculation. The inoculated roots of Lily Minjiang were ground into powder with liquid nitrogen, then transferred to a centrifuge tube, and the total RNA was extracted by guanidine isothiocyanate method. For RNA, reverse transcriptase M-MLV (promega) was used to synthesize the first strand of cDNA using total RNA as a template. The reaction system and operation process were as follows: take 5 μg total RNA, add 50 ng oligo (dT), 2 μL dNTP (2.5 mM), Add DEPC water to a reaction volume of 14.5 μL; after mixing, heat and denature at 70°C for 5 minutes, then quickly cool on ice for 5 minutes, then add 4 μL 5×First-stand buffer, 0.5 μL RNasin (200 U), 1 μL M-MLV (200 U ), mix well and centrifuge for a short time, incubate at 42°C for 1.5h, take it out and heat at 70°C for 10min to terminate the reaction; after the first strand of cDNA is synthesized, store it at -20°C for later use.

Using the synthesized first-strand cDNA as a template, the target gene LrDef1 was amplified, and the sequences of the upstream and downstream primers used were 5' TCGTCGTCCCCATCTCAG 3' and 5' AAATACAAGAGAACTTACTCCA 3' respectively. The target gene was amplified by Advantage ^TM 2 PCREnzyme (Clontech); PCR reaction conditions: 94°C 5min; 94°C 30s, 54°C 30s, 72°C 30s, 32 cycles; 72°C 7min; reaction system (20μL) 0.5μL cDNA, 2μL 10×Advantage 2 PCRBuffer, 0.4μL 50×dNTP Mix (10mM each), 0.4μL Forward Primer (10μM), 0.4μL Reverse Primer (10μM), 0.4μL Advantage 2 PCR Polymerase Mix, 15.9μL PCR- Grade water; After PCR, take 5 μL for agarose gel electrophoresis to detect the specificity and size of the amplified product.

For TA cloning of PCR products, the kit used was pGEM-T Vector System Ⅰ (Promega, USA). The reaction system and operation process were as follows: take 1.5 μL of PCR products, add 1 μL of pGEM-T Vector (50 ng/μL) and 2.5 μL 2×Ligation solutionⅠ, mix well and place at 16°C for overnight reaction. The ligation product was transformed into Escherichia coli DH5α by heat shock transformation method. Use LB solid medium containing ampicillin (Amp) to screen positive clones, select several single colonies, and after shaking the bacteria, use specific primers for amplifying LrDef1 to identify clones with multiple cloning sites inserted into LrDef1 . After sequencing, the final LrDef1 full-length cDNA was 501bp, which was found to contain a 225bp open reading frame (see Sequence Listing), LrDef1 encodes a protein containing 74 amino acids, its molecular weight is about 8.06kDa, and its isoelectric point is about 8.22. The protein sequence encoded by LrDef1 was analyzed with the help of bioinformatics software SignalP 5.0 to detect whether it had an N-terminal signal peptide. The results showed that there was a signal peptide at the N-terminus of LrDef1, so it was speculated that the protein was a secreted protein.

Embodiment 2: plant overexpression vector construction

The E. coli plasmid pGEM-T- LrDef1 inserted into LrDef1 and the plasmid of the plant expression vector pCAMBIA2300s were extracted using the SanPrep column plasmid DNA mini-extraction kit (Shanghai Sangong), and 1 μL was used for agarose gel electrophoresis to detect the extracted Integrity and concentration of the plasmid; use restriction endonucleases Eco RI (TaKaRa) and Bam HI (TaKaRa) to double digest the plasmids pGEM-T- LrDef1 and pCAMBIA2300s respectively (100 μL system), the reaction system and operation process are : Take 20 μL of pGEM-T- LrDef1 and pCAMBIA2300s plasmids, add 10 μL 10×K buffer, 4 μL Eco RI, 6 μL Bam HI, 60 μL ddH ₂ O in turn, mix well, centrifuge for a short time, and place at 37°C for overnight reaction; The excised product was electrophoresed on agarose gel, and then the LrDef1 fragment and the large fragment of the pCAMBIA2300s vector were gel-recovered, and the SanPrep column DNA gel recovery kit (Shanghai Sangong) was used for the whole process; 1 μL of the recovered product was passed through the agarose The size and concentration of the recovered fragments were detected by gel electrophoresis, and stored at -20°C for future use.

Use T4 DNA Ligase (TaKaRa) to connect the recovered LrDef1 DNA fragment and pCAMBIA2300s carrier fragment. The reaction system (20 μL) and the operation process are as follows: take 10 μL LrDef1 DNA fragment and add 2 μL pCAMBIA2300s carrier DNA, 2 μL 10×T4 DNA Ligase Buffer, 1 μL T4 DNA Ligase, 5 μL ddH ₂ O, mix well and centrifuge for a short time, then react overnight in a water bath at 16°C. Then, transfer the ligation product into Escherichia coli DH5α by heat shock transformation method, screen positive clones with solid medium containing 50 mg/L kanamycin (Kanamycin, Km), select a single colony and shake, and use the bacterial solution as a template Amplify the specific primers of LrDef1 for PCR, and select the clones that successfully connect LrDef1 and pCAMBIA2300s. If the detected strain is positive, add glycerol and store at -80°C for future use.

The pCAMBIA2300s- LrDef1 plasmid in the above-mentioned Escherichia coli was extracted and purified, and then the plant expression vector pCAMBIA2300s- LrDef1 constructed above was transformed into Agrobacterium tumefaciens LBA4404 competent cells by freezing and thawing with liquid nitrogen. The operation steps are as follows: take 2 μg pCAMBIA2300s- LrDef1 plasmid and add it to a centrifuge tube containing 200 μL competent cells, mix gently, and then put it in ice bath for 5 minutes, then transfer it to liquid nitrogen and freeze it for 1 minute, then quickly place it in a water bath at 37°C for 5 minutes, and then immediately Ice bath for 2 minutes, add 800 μL LB liquid culture and shake culture at 28°C for 4 hours; spread the activated Agrobacterium on the LB solid medium containing 50 mg/L Km, and culture at 28°C; select a single colony and shake it, and then expand LrDef1 -specific primers were added for PCR to detect whether pCAMBIA2300s- LrDef1 was transformed into Agrobacterium. For positive clones, add glycerol and store them at -80°C for later use.

Example 3: Plant genetic transformation mediated by Agrobacterium and screening of transgenic plants

The transgenic receptor in this experiment is tobacco. Soak the tobacco seeds in 75% alcohol for 30s, wash them with sterile water, soak them in 0.1% HgCl ₂ for 8 minutes, wash them several times with sterile water, and sow them in 1/2 On MS medium, culture in dark at 28°C for 6 days, transfer to light incubator after germination (25°C, 16 h/d light), and subculture once a month with 1/2 MS medium.

Take out the preserved Agrobacterium LBA4404 strain containing the pCAMBIA2300s- LrDef1 plasmid from the -80°C refrigerator, inoculate it in 5 mL of LB liquid medium containing 50 mg/L Km and 20 mg/L rifampicin, and culture it at 28°C until the medium is turbid . Pipette 1 mL of turbid bacterial solution onto LB solid medium containing 50 mg/L Km, and incubate at 28°C for 48 h; then scrape off an appropriate amount of Agrobacterium on the LB solid medium and inoculate it on the LB solid medium supplemented with 20 mg/L acetosyringone In the MGL liquid medium, shake culture at 28°C for 2-3h to activate Agrobacterium.

Take leaves of sterile tobacco seedlings and cut them into leaf discs of about 1 ^cm2 , soak them completely in the above-mentioned MGL liquid medium containing activated Agrobacterium for 15 minutes, use sterile filter paper to absorb the bacterial liquid on the surface of the leaves, and dry the leaves Place the plate on the co-culture medium for room temperature culture. The co-culture medium for tobacco transformation is MS + 0.02mg/L 6-BA + 2.1mg/L NAA + 30g/L sucrose + 6g/L agar, at 22°C under dark conditions Co-cultivate for 2 days.

Transfer the co-cultured leaf discs to the MS screening medium added with antibiotics to differentiate into seedlings, and screen transgenic plants at the same time; the tobacco screening medium is MS+0.5mg/L 6-BA+0.1mg/L NAA+30g/ L sucrose+6g/L agar+50mg/L Km+200 mg/L cephalosporin (cefotaxime sodium salt, Cef); transfer the culture bottle to a light incubator for cultivation during screening (25°C, 16 h/d light, 8 h/d dark), after the tobacco sprouted, it was subcultured with MS medium containing 50mg/L Km and 200mg/L Cef. Because the callus differentiation rate of tobacco is high, it is necessary to further screen the regenerated plants. The regenerated tobacco seedlings were moved to the MS medium containing 50mg/L Km to make them root, and finally the regenerated seedlings with better rooting were selected for further testing.

The genomic DNA of the leaves of transgenic tobacco plants was extracted by the CTAB method, and 1 μL of the extracted genomic DNA was detected by agarose gel electrophoresis for its integrity and concentration, and the genomic DNA of the transgenic plants was used as a template to perform PCR with specific primers for amplifying LrDef1 . After PCR, 8 μL of the product was used for agarose gel electrophoresis to detect positive transgenic plants. The amplification results of some transgenic tobacco plants were shown in Figure 1, and a total of 30 positive transgenic tobacco plants were screened.

Example 4: Expression Analysis of LrDef1 in Transgenic Tobacco and Functional Analysis of Anti-fungal Infection of Transgenic Plants

Extract total RNA from young leaves of positive transgenic plants and non-transgenic tobacco (wild type), reverse transcribe to generate the first strand of cDNA, and use this as a template to perform PCR with specific primers for amplifying LrDef1 , and analyze each transgenic single according to the PCR results. The expression of LrDef1 transcription level in the strain, the method of total RNA extraction and RT-PCR are the same as in Example 1. After the PCR is completed, 8 μL is used for agarose gel electrophoresis. The detection results of some individual strains are shown in Figure 2. A total of 20 transgenic individual plants were detected to express a large amount of LrDef1 at the transcriptional level, and these individual plants were numbered L1-L20.

Inoculate several pathogenic fungi stored in the laboratory on PDA solid medium (200g/L potato, 15g/L agar, 20g/L glucose), culture in dark at 28°C, and add plants when the colony grows to a diameter of about 2-3cm Protein, analysis of antifungal activity of transgenic plants in vitro. In order to prevent other bacteria from contaminating the extracted protein, the entire plant protein extraction process is performed aseptically. First, 1 g of transgenic tobacco plants (numbered L-8, L-9, L-16) and wild-type leaves were put into a mortar, and 1 mL of protein extract (1M NaCL, 0.1M sodium acetate, 1% PVP , pH6.0), fully ground. Transfer to a 1.5mL centrifuge tube, mix well and let stand overnight at 4°C. Centrifuge at 4°C for 30min (12,000g/min), take the supernatant into a new 1.5mL centrifuge tube, and measure the total protein concentration with an ultraviolet spectrophotometer. Adjust the total protein concentration of the transgenic and wild-type plants to 0.2 μg/μL, and then take 20 μL and drop them on the sterile filter paper of each fungal culture medium. In addition to adding the total protein of different transgenic tobacco plants on each fungal plate, the total protein of wild-type tobacco and the blank control (protein extract) were added in parallel. After culturing at 28°C for several days, the growth of the fungi was observed, and the in vitro antifungal activity of LrDef1 transgenic tobacco was evaluated accordingly. The results are shown in Figure 3. The growth of Fusarium thieves has obvious inhibitory effect.

sequence listing

<110> Kunming University of Science and Technology

<120> A Minjiang lily defensin antimicrobial peptide gene LrDef1 and its application

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<213> Lilium regale Wilson

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tcgtcgtccc catctcagtg gccgactatc tcttgcaatg gcgaagcttc ccaccatcct 60

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atgcctgtcg cagagccaca agttcaaggg gacctgtttg agggcggcca actgtgctag 180

tgtctgccag acggagggat tcaaaggagg ggtttgcgag ggcatccgcc gccgttgctt 240

ctgcgaagcc gactgtcact gatgcctgag ttcttggctt taataagtaa tgtcggacta 300

tccgagaaga ataagatgga cctggtgttg ttggttttac agtctcttct tcggtgtggg 360

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Met Ala Lys Leu Pro Thr Ile Leu Leu Leu Leu Phe Leu Val Met Ala

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Claims (2)

1. Lilium regale defensin antibacterial peptide geneLrDef1The nucleotide sequence is shown in SEQ ID NO. 1.

2. Lilium regale defensin antibacterial peptide gene as claimed in claim 1LrDef1In improving the resistance of tobacco to fusarium oxysporum: (Fusarium oxysporum) Phoma graminearum (A) and (B)Phoma herbarum) Fusarium equiseti (F.) hieron (F.)Fusarium equiseti) Use in resistance.

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