CN108251432A - Radix Notoginseng class PR gene PnPRlike and application - Google Patents

Abstract

The present invention discloses a notoginseng-type disease course-related protein gene PnPRlike and its application. The nucleotide sequence of the PnPRlike gene is shown in SEQ ID NO: 1, which encodes a disease-course-related protein; the invention is confirmed by functional genomics-related technical research The PnPRlike gene has the function of improving the resistance of plants to pathogenic fungi. The antifungal gene PnPRlike of the present invention is constructed on the plant expression vector and transferred to tobacco for overexpression. The transgenic tobacco plants have strong antifungal activity in vitro. Experimental results show : PnPRlike overexpressed transgenic tobacco has obvious inhibitory effect on the growth of Fusarium solani, Vitis vinifera and Nigeria oryzae.

Description

Panax notoginseng disease process-related protein gene PnPRlike and its application

technical field

The invention relates to related technologies in the fields of molecular biology and genetic engineering, in particular to a pathogen-related protein-like isoform (pathogen-related protein-like isoform) gene PnPRlike with antifungal activity and its application.

Background technique

During the growth and development of plants, they will be stressed by many biotic and abiotic factors, such as drought, cold, UV rays, trauma, infection by pathogens (fungi, bacteria, viruses), etc. Correspondingly, plants have evolved a series of defense mechanisms to resist adversity stress, and the activation and accumulation of pathogenesis-related proteins (PRs) is an important part of plant defense responses (Singh A, Kirubakaran SI, Sakthivel N. Heterologous expression of new antifungal chitinase from wheat. Protein Expr Purif, 2007, 56(1):100). When attacked by pathogens, plants respond to pathogenic bacteria and external stress by rapidly changing gene expression, inducing the re-synthesis of some special proteins, most of which are disease process-related proteins (Wen YJ, He HW, HuangQS, Liang S, Bin JH . Roles of pathogenesis-relative protein 10 in plant defense response. Plant Physiology Communications. 2008, 44: 585-592).

PR proteins are encoded by multiple genes. According to the similarity of amino acid sequences, serological relationships and biological activities, PR proteins are currently divided into 17 categories (PR1-PR17). PRs are expressed in plants as chitinases, sweet-like proteins , protease inhibitors, endopeptidases and peroxidases, etc. (van Loon LC, Rep M, Pieterse CM. Significance of inducible defense-related proteins in infected plants. Annu Rev Phytopathol.2006, 44: 135-162). PRs gene is a plant defense response gene, which is closely related to hypersensitive response (hypersensitive response, HR) and systemic acquire resistance (systematic acquire resistance, SAR). It is one of the hotspots of resistance research. The relative molecular weight of PRs is small, generally 10-40 kD, mostly acidic proteins, with a half-life of 40-70 h, so they can accumulate well in cells and cells; PRs have strong stability and low pH value remains soluble. Studies using cell separation techniques, immunofluorescence microscopy techniques, and immunogold labeling techniques have shown that PRs are mainly distributed in the intercellular spaces and vacuoles of plants, and their distribution is related to the isoelectric point and the affinity between the induced bacteria and plants.

Chitinases encoded by the PR3 gene family have attracted much attention in plant disease resistance defense responses. Chitinases are a class of glycosyl hydrolases with a size distribution of 20-90 kD that can hydrolyze chitin (Bhattacharya D, Nagpure A, Gupta RK. Bacterial chitinases: properties and potential. Critical Reviews in Biotechnology, 2007, 27(1 ): 21-28.). Among them, microbial chitinases are mostly 20-60 kD, plant chitinases are within 20-45 kD, and insect chitinases are 40-85 kD. Plant chitinases exist in plant stems, seeds, and flowers, and their expression is tissue-specific. Plant hormones ethylene (ETH), jasmonic acid (JA), salicylic acid (salicylic acid, SA) also induce the expression of chitinase. Ethephon induced the accumulation of chitinase in the intercellular spaces of cucumber leaves, and the induced effect was directly related to the perception of the mass concentration of ethephon in cucumber leaves. When ginseng ( Panax ginseng CA Meyer) was treated with methyl jasmonate (MeJA), the chitinase activity in the root system was significantly increased compared with the control. Exogenous salicylic acid treatment can induce the activity of chitinase and up-regulate the expression of chitinase gene in banana ( Musa paradisiacal ).

Thaumatin-like proteins (TLPs) in the PR5 family, as a member of the PRs family, are a class of defense proteins commonly found in higher plants. Effect (Datta K, Velazhahan R, Oliva N, et al. Over-expression of the cloned rice thaumatin-like protein (PR-5) gene in transgenic riceplants enhances environmental friendly resistance to Rhizoctonia solani causing sheath blight disease. Theoretical & Applied Genetics, 1999, 98(6-7):1138-1145.). At present, TLPs have been found in grape (Vitis vinifera ), tomato ( Lycopersicones culentum ), barley ( Hordeum vulgare ), wheat ( Triticuma etivum ), oat ( Avena sativa ) and some fungi. The amino acid sequence of thaumatin is highly homologous to the thaumatin of African plant Thaumatococcus daniellii , so it is called thaumatin. The homology of the two proteins is very high, but their functions are completely different. The sweet protein has a sweet taste and has no antifungal activity, while the sweet-like protein has no sweet taste and has an antifungal activity.

The protein encoded by PR10 has a molecular weight of 15-19 kD, an acidic isoelectric point, and no signal peptide sequence. It belongs to intracellular pathogenesis-related proteins (IPR). PR10 protein plays an important role in the process of plant defense against biotic stress such as fungi and abiotic stress (Liu JJ, Ekramoddoullah AKM. The family 10 of plant pathogenesis-related proteins: their structure, regulation, and function in response to biotic and abiotic stresses. Physiological and Molecular Plant Pathology, 2006, 68(1): 3-13.). PR10 protein belongs to ribonuclease-like, can degrade RNA, has in vitro nuclease activity and antibacterial activity (Edreva A. Pathogenesis-related proteins: research progress in the last 15years. General and Applied Plant Physiology, 2005, 31(1-2): 105-124.)

Panax notoginseng is a plant of the genus Panax in the Araliaceae family. It is a traditional precious Chinese medicinal material in China, mainly produced in the Wenshan area of Yunnan. Studies have shown that Panax notoginseng saponins have various biological activities such as anti-tumor, anti-virus, lowering cholesterol and improving immunity of the body, and are important drugs for preventing cardiovascular and cerebrovascular diseases. In recent years, the fungal diseases of Panax notoginseng have become more and more serious. The fungal diseases in the main producing areas of Sanqi in Wenshan, Yunnan are mainly root rot, black spot, round spot, phytophthora and gray mold. The perennial incidence rate of root rot is generally 5% to 20%, and the severe one can reach more than 70%, or even no harvest, and the longer the growth period, the more serious the disease (Yang Jianzhong, Cui Xiuming, Chen Yujun, etc. The effect of continuous cropping soil treatment on three The control effect of seven root rot. Specialty Research, 2010, 32(2):37.). The disease problem has become a serious obstacle restricting the development of Panax notoginseng planting industry. Isolate and clone the disease-resistant functional gene of Panax notoginseng, study the molecular interaction mechanism between Panax notoginseng and pathogens, and lay the foundation for the genetic breeding of Panax notoginseng for disease resistance.

Contents of the invention

The object of the present invention is to provide a kind of gene PnPRlike that encodes a class of disease course-related protein gene PnPRlike with antifungal activity obtained by cloning from Panax notoginseng. The nucleotide sequence of the class disease course-related protein gene PnPRlike is shown in SEQ ID NO: 1. The long sequence is 976bp, including an open reading frame of 717bp, 5' untranslated region of 96bp, and 3' untranslated region of 163bp, encoding the protein with the amino acid sequence shown in SEQ ID NO:2.

The coding region of the gene PnPRlike in the present invention is the nucleotide sequence shown in positions 97-813 in SEQ ID NO: 1 of the sequence table.

The present invention isolates and clones a complete cDNA fragment of an antifungal-related gene of Panax notoginseng, transfers the target gene into the recipient plant tobacco for overexpression through the mediation of Agrobacterium tumefaciens , and verifies whether the gene has The antifungal activity 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 PnPRlike .

The present invention relates to isolating a DNA fragment comprising PnPRlike and characterizing its function. Wherein the DNA fragment is shown in the sequence table, and the sequence analysis of the gene shows that the full-length cDNA of PnPRlike is 976bp, including a 717bp open reading frame, 96bp 5'-untranslated region (untranslated region, UTR) and 163bp 3'-UTR, where ORF encodes a protein with 238 amino acids. BLASTp analysis showed that the protein sequence encoded by PnPRlike was similar to that of carrot ( Daucus carota ) (XP_017223871.1), morning glory ( Ipomoea nil ) (XP_019190454.1), walnut ( Juglans regia ) The homology of the similar protein gene (XP_018849219.1) and the similar protein gene (XP_008237645.1) of plum blossom ( Prunus mume ) is high, respectively 76%, 68%, 69% and 67%, which indicates that It belongs to the process-related proteins in Panax notoginseng. The sequence shown in the overexpression sequence table SEQID NO: 1 can enhance the resistance of tobacco to Fusarium solani , Botryosphaeria dothidea and Nigrospora oryzae .

In the present invention, the notoginseng-type disease course-related protein gene PnPRlike is applied in improving the resistance of tobacco to pathogenic fungi, and the specific operations are as follows:

(1) Extract the total RNA from the roots of Panax notoginseng by the guanidine isothiocyanate method, use the extracted RNA as a template, and use oligo(dT) 18 as a reverse transcription primer, and perform reverse transcription-polymerase chain reaction (RT-PCR) polymerase chainreaction, RT-PCR) to amplify the PnPRlike coding region, then connect it to the pGEM-T vector, and obtain the clone with the target gene through sequencing;

(2) Digest pGEM-T- PnPR like with restriction endonucleases Bam HI and Eco RI, 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 PnPRlike 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 inhibitory activity of the transgenic plant protein on fungal growth, and finally screen out the anti-fungal Transgenic plants with significantly enhanced sex.

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 PnPRlike gene from Panax notoginseng in the invention can enhance the plant's resistance to several pathogenic fungi, and the gene can be introduced into tobacco to produce new varieties and materials with fungal resistance. 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, flowers, medicinal materials, etc., reduce the use of chemical pesticides, but also save costs for agricultural production and reduce environmental pollution. Therefore, the invention has broad market application prospects.

Description of drawings

Fig. 1 is the PCR detection result of partial PnPR like transgenic tobacco genomic DNA in the present invention, wherein Marker is DL2000 DNA Marker (Dalian Baobiology), consists of six DNAs of 2,000 bp, 1,000 bp, 750 bp, 500 bp, 250 bp and 100 bp Fragment composition; positive control is the PCR reaction of plasmid pGEM-T- PnPRlike as template; WT is the PCR of non-transgenic tobacco (wild type, wild type) total DNA as template;

Fig. 2 is the expression analysis result figure of PnPRlike transcript level in part positive PnPRlike transgenic tobacco in the present invention, wherein Marker is DL2000 DNA Marker (Dalian treasure biology); WT is the PCR product of non-transgenic tobacco total RNA reverse transcription cDNA as template; Positive Control: PCR product of plasmid pGEM-T- PnPRlike as template;

Fig. 3 is the effect diagram of PnPRlike transgenic tobacco in vitro inhibiting fungal growth in the present invention; wherein the fungi in a, b, and c illustrations are respectively Fusarium solani, Botrytis vine and Nigeria oryzae; WT is wild-type tobacco The total protein; Buffer is a blank control, that is, no protein control (buffer used for protein extraction).

Detailed ways

The present invention is further described below by accompanying drawing and embodiment, but protection scope of the present invention is not limited to described content, if the method in the present embodiment has no special instructions, all operate according to conventional methods, and the reagents used adopt conventional reagents if there are no special instructions Or reagents configured by conventional methods.

Example 1: PnPRlike full-length cDNA cloning and sequence analysis

The three-year-old Panax notoginseng leaves were used to extract total RNA, and the Panax notoginseng leaves were ground into powder with liquid nitrogen, then transferred to a centrifuge tube, and the total RNA was extracted by the guanidine isothiocyanate method, followed by reverse transcriptase M-MLV (promega) The first strand of cDNA was synthesized 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 each), and DEPC water to a reaction volume of 14.5 μL; After mixing, heat denaturation at 70°C for 5 min, then rapidly cool on ice for 5 min, then add 4 μL 5×First-stand buffer, 0.5 μL RNasin (200 U), 1 μL M-MLV (200 U), and mix Homogenize and centrifuge for a short time, incubate at 42°C for 1.5 h, take it out and heat at 70°C for 10 min to terminate the reaction. After the first strand of cDNA was synthesized, it was stored at -20°C for future use.

Using the synthesized first-strand cDNA as a template, the target gene PnPRlike was amplified, and the upstream and downstream primer sequences used were 5'CCAAATAACAATACTCTCCACAC3' and 5'TTAAAGTTTGAGTTTGTTTTACATT3', respectively. The target gene was amplified by Advantage ^TM 2PCR Enzyme (Clontech); PCR reaction conditions: 95°C for 1 min; 94°C for 30 s, 53°C for 30 s, 72°C for 1 min, 30 cycles; 72°C for 5 min; reaction system (10 μL) is 1 μL cDNA, 1 μL 10×Advantage 2 PCR Buffer, 0.5 μL 50×dNTP Mix (10 mM each), 0.2 μL forward primer (10 μM), 0.2 μL reverse primer (10 μM), 0.2 μL Advantage 2 PCR Polymerase Mix, 6.9 μL PCR-Grade water; after PCR, take 5 μL for agarose gel electrophoresis to detect the specificity and size of the amplified product.

The PCR product obtained has only one DNA band, and the PCR product is directly cloned by TA. The kit used is pGEM-T vector (Promega). The reaction system and operation process are as follows: take 1.5 μL of PCR product, add 1 μL of pGEM-T vector in turn (50 ng/μL) and 2.5 μL 2×Ligation solution, 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 use specific primers to amplify PnPRlike after shaking to identify clones with multiple cloning sites inserted into PnPRlike , and sequence the positives . The final obtained full-length cDNA of PnPRlike was 976bp, which was found to contain an open reading frame of 717bp through NCBI ORF finder (http://www.ncbi.nlm.nih.gov/gorf/gorf.html). ), PnPRlike encodes a protein containing 238 amino acids, the molecular weight of PnPRlike is about 26.64 kD, and the isoelectric point (pI) is about 5.69, indicating that the protein is an acidic protein. These include 32 acidic amino acids (D, E) and 32 basic amino acids (K, R, H).

Embodiment 2: plant overexpression vector construction

The Escherichia coli plasmid pGEM-T- PnPRlike inserted into PnPRlike 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 all Integrity and concentration of extracted plasmids; double digestion of plasmids pGEM-T- PnPRlike and pCAMBIA2300s with restriction endonucleases Bam HI (TaKaRa) and EcoR I (TaKaRa) respectively (100 μL system), reaction system and operation The process is: Take 20 μL of pGEM-T- PnPRlike and pCAMBIA2300s plasmids, add 10 μL 10×Kbuffer, 4 μL Bam HI, 6 μL EcoRI , 60 μL ddH ₂ O in sequence, mix well, centrifuge for a short time, and place at 37°C overnight Reaction; spot all digested products on agarose gel for electrophoresis, and then perform gel recovery on the PnPRlike fragment and the pCAMBIA2300s vector fragment respectively; take 1 μL of the recovered product to detect the size and concentration of the recovered fragment by agarose gel electrophoresis, Store at -20°C for later use.

Use T4 DNA Ligase (TaKaRa) to connect the recovered PnPRlike DNA fragment and pCAMBIA2300s carrier fragment. The reaction system (20 μL) and the operation process are as follows: take 10 μL of PnPRlike 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, the ligation product was transformed into Escherichia coli DH5α by heat shock transformation method, and positive clones were screened with solid medium containing 50 mg/L kanamycin (Km). Select a single colony and shake the bacteria, and use the bacterial liquid as a template to perform PCR with the specific primers for amplifying PnPRlike , and select the clone that successfully connects PnPRlike and pCAMBIA2300s. If the detected strain is positive, add glycerol and store it at -80°C for future use.

The pCAMBIA2300s- PnPRlike plasmid in the above Escherichia coli was extracted and purified. Subsequently, the plant expression vector pCAMBIA2300s- PnPRlike constructed above was transformed into Agrobacterium tumefaciens LBA4404 competent cells by freezing and thawing with liquid nitrogen. The operation steps are as follows: take 1 μg of pCAMBIA2300s- PnPRlike plasmid and add it to a centrifuge tube containing 200 μL of competent cells, mix gently and then place in ice bath for 5 minutes, then transfer to liquid nitrogen and freeze for 1 minute, and then quickly place in a 37°C water bath After 5 min, immediately ice-bath for 2 min, add 800 μL LB liquid culture and shake at 28°C for 4 h. The activated Agrobacterium was spread on LB solid medium containing 50 mg/L Km, and cultured statically at 28°C. Select a single colony and shake the bacteria, and then perform PCR with specific primers for amplifying PnPRlike to detect whether pCAMBIA2300s- PnPRlike is transferred 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 recipient in this experiment is tobacco. The tobacco seeds were soaked in 75% alcohol for 30 seconds, washed with sterile water, soaked in 0.1% HgCl ₂ for 8 minutes, washed several times with sterile water, and sowed in 1/2 2 MS medium, cultured in the dark at 28°C for 6 days, then transferred to the light incubator (25°C, 16h/d light) after germination, and subcultured once a month with 1/2MS medium.

Take out the preserved Agrobacterium LBA4404 strain containing pCAMBIA2300s- PnPRlike 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 cultivate it at 28°C until Medium is cloudy. 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 LB solid medium and inoculate it in acetosyringone supplemented with 20 mg/L MGL liquid medium, 28 ℃ shaking culture 2-3 h to activate Agrobacterium.

Take leaves of sterile tobacco seedlings and cut them into leaf disks of about 1 cm ² , soak them completely in the above-mentioned MGL liquid medium containing activated Agrobacterium for 15 min, blot the bacteria solution on the surface of the leaves with sterile filter paper, and put Leaf discs were placed on co-culture medium for room temperature culture. The co-culture medium for tobacco transformation was MS+0.02 mg/L 6-BA+2.1 mg/L NAA+30 g/L sucrose+6 g/L agar at 22°C without light conditions for 2 days.

The co-cultured leaf discs were transferred to the MS selection medium added with antibiotics to differentiate into seedlings, and the transgenic plants were screened at the same time. Tobacco selection medium is MS+0.5 mg/L 6-BA+0.1 mg/L NAA+30 g/L sucrose+6 g/L agar+50 mg/L Km+200 mg/L cefotaxime sodium salt , Cef); during screening culture, transfer the culture bottle to a light incubator for culture (25°C, 16h/d light, 8h/d dark), and after the tobacco sprouts, use 50 mg/L Km and 200 mg/L Cef Subculture on MS medium. Tobacco regenerated seedlings were transferred to MS medium containing 50 mg/L Km for rooting, and finally regenerated seedlings with better rooting were selected for further testing.

The genomic DNA of 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 PnPRlike . After PCR, 8 μL of the product was used for agarose gel electrophoresis to detect positive transgenic plants. The amplification results of some tobacco transgenic plants are shown in Figure 1. A total of 50 positive transgenic plants were screened from PnPRlike transgenic tobacco.

Example 4: Expression analysis of PnPRlike in transgenic tobacco and analysis of antifungal activity of transgenic plants

Extract total RNA from young leaves of positive transgenic individual 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 PnPRlike , and analyze each transgenic individual according to the PCR results. The expression of the PnPRlike transcript 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, take 5 μL for agarose gel electrophoresis. The detection results of some individual strains are shown in Figure 2 , A total of 42 transgenic individuals were detected to express a large amount of PnPRlike at the transcriptional level, and the numbers of these individuals were 1-41.

Inoculate several fungi preserved in the laboratory on PDA solid medium (200 g/L potato, 15 g/L agar, 20 g/L glucose) and culture in the dark at 28°C until the colony grows to a diameter of about 2-3 cm Protein was added to analyze the antifungal activity of transgenic plants in vitro. In order to prevent other bacteria from contaminating the extracted protein, the entire plant protein extraction process was performed aseptically. First, 1 g of transgenic tobacco plants (numbered 1, 2, and 4) and wild-type leaves were put into a mortar. Add 1mL protein extract (1M NaCl, 0.1M sodium acetate, 1% PVP, pH6), grind thoroughly; transfer to a 1.5 mL centrifuge tube, mix well, let stand at 4°C overnight, and centrifuge at 4°C for 30 min (12,000 g/ min), take the supernatant into a new 1.5 mL centrifuge tube, and take an appropriate amount to measure the total protein concentration with a UV spectrophotometer. Adjust the total protein concentration of the transgenic and wild-type plants to 1 μg/μL, and then take 20 μL and drop them on the sterile filter paper of each fungal culture medium, and add the total protein of different transgenic tobacco plants on each fungal plate At the same time, the total protein of wild-type tobacco and the blank control (buffer used for protein extraction) were added in parallel, and the fungal growth of each treatment was observed after culturing at 28°C for several days, and the in vitro antifungal activity of PnPRlike transgenic tobacco was evaluated accordingly. The results As shown in Figure 3, the PnPRlike transgenic tobacco protein has a strong inhibitory effect on the growth of Fusarium solani rot, Botrytis viticola, and Nigeria oryzae.

sequence listing

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Claims (2)

1. A gene PnPRlike related to the pathogenesis of Panax notoginseng, characterized in that: the nucleotide sequence is as shown in SEQ ID NO: 1, and the protein encoding the amino acid sequence is as shown in SEQ ID NO: 2. 2. The effect of the notoginseng class disease process-related protein gene PnPRlike described in claim 1 in improving the resistance of tobacco to Fusarium solani ( Fusarium solani ), Botryosphaeria dothidea , and Nigrospora oryzae application.