CN117247949A - Notoginseng disease course related protein 1 gene PnPR1-8 and its application - Google Patents

Abstract

The invention discloses a pseudo-ginseng disease course related protein 1 gene PnPR1-8 and application thereof, wherein the nucleotide sequence of the PnPR1-8 gene is shown as SEQ ID NO:1, coding a disease course related protein 1, and confirming that the PnPR1-8 gene has the capability of improving plant fungal infection resistance through functional genomics related technical research, constructing the PnPR1-8 gene into a plant expression vector and transferring the plant expression vector into tobacco for overexpression, wherein the resistance of the PnPR1-8 transgenic tobacco to Humicola insolens (Humicola fuscoatra) is obviously improved.

Description

Notoginseng disease course related protein 1 gene PnPR1-8 and its application

Technical Field

The invention belongs to the technical fields of molecular biology and genetic engineering, and particularly relates to a pseudo-ginseng disease course related protein 1 gene PnPR1-8 with antifungal infection capability and application thereof.

Background

During the whole growth of plants, they are subjected to stresses of various biotic and abiotic factors. Effector-triggered immunity (ETI) is an important component of natural plant immunity (Zhang J, zhou jm.plant immunity triggered by microbial molecular signature. Molecular plant.2010,3 (5): 783-793). This line of defense is activated when attacked by pathogens and a series of stress reactions such as ion flow exchange, production of reactive oxygen species and Salicylic Acid (SA) species, accumulation of secondary metabolites such as phytoalexins, tannins and phenolic compounds, synthesis of disease-associated Proteins (PRs) and the like (van Loon LC, rep M, pietessec M.Significa of inducibledefense-related proteins in infected plants. Annual review of bacteriology 2006, 44:135-162) occur.

PRs are a generic term for a class of proteins that plants are induced to produce and accumulate following biotic or abiotic stress, and were first discovered by van Loon et al in 1970 when tobacco (Nicotiana tabacum) leaves were infected with tobacco mosaic virus (van Loon LC, rep M, pieterse CM. Significa of inducibledefense-related proteins in infected plants. Annual review of phytodynamics.2006, 44:135-162). PRs are widely found in monocots and dicots, and some PR proteins exhibit potential antimicrobial activity in vitro and are associated with systemic acquired resistance (systemic acquired resistance, SAR) (Liu Y, liu Q, tang Y, et al, ntPR1a regulates resistance to Ralstonia solanacearumin Nicotiana tabacum via activating the defense-related genes, biochemical and biophysical research communications.2019,508 (3): 940-945). PR proteins are divided into 17 families according to characteristics of PR protein family in terms of structure, relationship and bioactivity (Kaur A, pati PK, pati AM, et al in-silico analysis of cis-acting regulatory elements of pathogenesis-related proteins of Arabidopsis thaliana and Oryza sativa. PLoS one.2017,12 (9): e 0184523). Plant PRs are of a wide variety of functions, such as transcription factors, protease inhibitors, antifungal agents, and the like. Some PRs also have hydrolase activities such as peroxidase, chitinase (Chitinase), and the like. PRs are distributed in relation to their isoelectric points and affinities, mainly in plant cell gaps and vacuoles (Jo BR, yu JM, jang S, et al cloning, expression, and purification of apathogenesis-related protein from Oenanthe javanica and its biological properties. Biological & pharmaceutical bulletin.2020,43 (1): 158-168).

Previous studies have demonstrated that PR proteins have inhibitory effects on the growth of many fungi. TdPR1.2 protein of PR1 family of Du Shixiao wheat (Triticum turgidum) has an inhibitory effect on the growth of the fungus Septoria tritici (septictrichl) (Ghorber M, zrtibi I, missaoui K, et al differential regulation of the durum wheat pathogenesis-related protein (PR 1) by cam module TdCaM1.3 protein. Molecular reports.2021,48 (1): 347-362). Constitutive high-efficiency expression of tobacco PR1-a enhances resistance to Phytophthora pathogens (Alexander D, goodman RM, gut-Rella M, et al, increased tolerance to two oomycete pathogens in transgenic tobacco expressing pathogenesis-modified protein 1a.Proceedings of the National Academy of Sciences of the United States of America.1993,90 (15): 7327-7331). Over-expression of the NPR1 gene into Arabidopsis thaliana (Arabidopsis thaliana) increased resistance to downy mildew (Peronospora parasitica) (Nandi A, kachroo P, fukushige H, et al, ethyl and jasmonic acid signaling affect the NPR1-independent expression of defense genes without impacting resistance to Pseudomonas syringae and Peronospora parasitica in the Arabidopsis ssi1 mutant. Molecular plant-micro interactions.2003,16 (7): 588-599).

Pseudo-ginseng [ Panax notoginseng (Burk) F.H.Chen ] has a long planting history and is mainly planted in southwest China. Notoginseng radix has extremely high medicinal value, has effects of promoting blood circulation, removing blood stasis, stopping bleeding, replenishing blood, and improving immunity of organism (Xie W, meng X, zhai Y, et al, panax notoginseng saponins: areview of its mechanisms of antidepressant or anxiolytic effects and network analysis on phytochemistry and pharmacology. Molecular.2018, 23 (4): 940), and in recent years, market demand of Notoginseng radix has been increasing. After the pseudo-ginseng is sown, the pseudo-ginseng is cultivated and grown for at least 3 years, and the root can be used as a medicine. In addition, pseudo-ginseng is a warm, moist and cool growing environment, but the unique planting environment causes pseudo-ginseng to suffer from various diseases and insect pests, and particularly fungus diseases typified by root rot often cause serious loss of yield and quality of pseudo-ginseng. In order to prevent and treat root rot, farmers kill soil in a large area or use a large amount of chemical pesticides, which not only increases the cost, but also causes environmental pollution easily due to the chemical agents (Wang Yong, ma Chengzhu, chenjun, etc. soil treatment has studied the control effect of root rot of pseudo-ginseng. Chinese journal of Chinese traditional medicine. 2008, (10): 1213-1214). One of the effective ways to solve the pseudo-ginseng disease is to cultivate resistant varieties, and resist fungal diseases through the defense reaction of plants.

Disclosure of Invention

The invention provides a disease course related protein 1 gene PnPR1-8 cloned from pseudo-ginseng, wherein the nucleotide sequence of the PnPR1-8 is shown as SEQ ID NO. 1, the ORF length of the gene is 702bp, and the protein with the amino acid sequence shown as SEQ ID NO. 2 is encoded.

The invention separates and clones the complete cDNA fragment of a antifungal related gene of pseudo-ginseng; transferring target genes into a receptor plant for overexpression through the mediation of agrobacterium tumefaciens (Agrobacterium tumefaciens); further experiments prove that whether the gene has antifungal activity or not, and lays a foundation for improving the capability of plants to resist fungal diseases by using the gene in the later period.

Another object of the present invention is to use the above-mentioned Notoginseng radix disease course related protein 1 gene PnPR1-8 in improving tobacco resistance to Humicola insolens (Humicola fuscoatra).

The operation of improving the antifungal property of tobacco by the pseudo-ginseng disease course related protein 1 gene PnPR1-8 is as follows:

(1) Extracting total RNA from pseudo-ginseng roots inoculated with PnPR1-8 by adopting a specific primer for amplifying PnPR1-8, amplifying ORF of PnPR1-8 by reverse transcription-polymerase chain reaction (reverse transcription-polymerase chain reaction, RT-PCR), connecting the ORF to pGEM-T vector, and sequencing to obtain clone with target gene;

(2) Cutting pGEM-T-PnPR1-8 vector and plant expression vector pCAMBIA2300s by using restriction enzymes EcoRI and BamHI, and recovering by gel to obtain target gene fragment and large vector fragment; then connecting the obtained PnPR1-8 gene fragment with a pCAMBIA2300s vector fragment to construct a plant super-expression vector; then transferring the constructed recombinant vector into tobacco for expression through the mediation of agrobacterium tumefaciens;

(3) And screening transformants by using a resistance marker on the recombinant vector T-DNA, obtaining positive transgenic plants by PCR and RT-PCR detection, analyzing the resistance of the transgenic plants to pathogenic fungi, and finally screening transgenic plants with obviously enhanced fungal resistance.

The invention provides a new method for improving the resistance of plants to fungal diseases, and the disease-resistant plants can be cultivated by a genetic engineering means to overcome the defects of traditional breeding, so that the breeding period is shortened, the operation is simple, and high-resistance materials are easy to obtain; the PnPR1-8 gene from pseudo-ginseng can enhance the resistance of plants to Humicola insolens, and the gene can be introduced into tobacco to generate new varieties and materials with fungal resistance. The cultivation of resistant plant varieties and materials by using genetic engineering technology has obvious advantages and irreplaceable importance; the invention not only can provide convenience for mass production of crops, flowers, medicinal materials and the like and reduce the use of chemical pesticides, but also can save cost and reduce environmental pollution for agricultural production, so that the invention has wide market application prospect.

Drawings

FIG. 1 is a PCR detection of a portion of the genomic DNA of PnPR1-8 transgenic tobacco according to the present invention, wherein Marker: DL2501 DNA Marker (Shanghai Jieli) is composed of five DNA fragments of 2,000bp, 1,000bp, 750bp, 500bp and 250 bp; positive control: PCR reaction with plasmid pGEM-T-PnPR1-8 as template; WT: PCR performed by taking non-transgenic tobacco (wild type) total DNA as a template;

FIG. 2 is a graph showing the results of expression analysis of PnPR1-8 transcription levels in partially positive PnPR1-8 transgenic tobacco according to the present invention, wherein Marker: DL2501 DNA Marker (Shanghai strapdown); WT: a PCR product taking non-transgenic tobacco total RNA reverse transcription cDNA as a template; positive control: PCR products with plasmid pGEM-T-PnPR1-8 as a template;

FIG. 3 is a graph showing the results of disease resistance identification of PnPR1-8 transgenic tobacco in the present invention; pnPR1-8 transgenic tobacco leaves inoculated with PnPR humicola insolens are shown; wherein WT is a wild type tobacco leaf, 8-6, 8-10, 8-11, 8-13 is a leaf of a PnPR1-8 transgenic tobacco strain.

Detailed Description

The present invention will be further illustrated by the following figures and examples, but the scope of the invention is not limited to the description, and the methods in this example are all performed according to the conventional methods unless otherwise specified, and the reagents used are the conventional reagents or the reagents configured according to the conventional methods unless otherwise specified.

Example 1: pnPR1-8 full-length cDNA clone and sequence analysis

Inoculating root tip of annual Notoginseng radix with mycelium of Pythium gracile (Humicola fuscoatra), collecting root 2 days after inoculation, and collecting rootThe Super total RNA extraction kit is used for extracting total RNA, measuring the concentration and performing agarose gel electrophoresis to analyze the quality of the total RNA.

Then Go ScriptTM Reverse Transcriptase System is adopted to synthesize a cDNA first strand by taking total RNA as a template, and the reaction system and the operation process are as follows: mu.g of total RNA was taken, 1. Mu.L of Oligo dT15 primer, 1. Mu.L of Random primer were added in this order, and the reaction volume was made up to 10. Mu.L with nucleic-Free Water; mixing, heating at 70deg.C for denaturation for 5min, cooling on ice for 5min, and sequentially adding 4 μL of 5×reaction Buffer and 4 μL of MgCl ₂ (25mM)、1μL PCR Nucleotide Mix、0.4μL RecombinantRibonuclease Inhibitor, 0.4 mu L Reverse Transcriptase and 1.2 mu LNuclease-Free Water, mixing, centrifuging briefly, standing at 25deg.C for 5min, cooling in 42 deg.C for 1.5h, taking out, heating at 70deg.C for 10min, and stopping the reaction; the cDNA first strand is synthesized and then stored at-20 ℃ for standby.

By synthesis of cDNA NoOne strand is used as a template to amplify target genes PnPR1-8, and the used upstream and downstream primer sequences are 5 'ATGGGGTTTCTTAGTATCTCTC3' and 5 'TCAGCACGAACGGATATGCC3'; by TAKARAExAmplifying a target gene; PCR reaction conditions: 95 ℃ for 5min;95℃30s,60℃30s,72℃1min,30 cycles; 72 ℃ for 5min; the reaction system (50. Mu.L) was 2. Mu.L of cDNA, 5. Mu.L of 10 XEx Taq Buffer (Mg-containing) ²⁺ 20 mM), 4. Mu.L dNTP Mix (2.5 mM each), 1. Mu.L upstream primer (5. Mu.M), 1. Mu.L downstream primer (5. Mu.M), 0.25. Mu.L TaKaRa Ex Taq (5U/. Mu.L), 36.75. Mu.L ddH ₂ O; after the PCR was completed, 50. Mu.L of the sample was subjected to 1% agarose gel electrophoresis to examine the specificity and size of the amplified product.

Detecting the PCR product band by electrophoresis, showing that the amplified product is a specific DNA band, and using SanPrep column type PCR product purification kit (Shanghai Ing) to cut gel and recycle to obtain the PCR amplified product; the recovered bands were then subjected to T-A cloning using pGEM-T Vector System I (TaKaRa), the reaction System and the procedure were: mu.L of PCR product was taken, and 0.7. Mu.L of pGEM-T vector, 0.9. Mu. L T4 DNALigase and 5. Mu.L of 2X Rapid Ligation Buffer were added in this order, and after mixing, they were allowed to react overnight at 16 ℃. The ligation product was transferred into E.coli DH 5. Alpha. Competence by heat shock transformation. Positive clones were screened with LB solid medium containing ampicillin (Amp). Selecting a plurality of single colonies in LB liquid medium containing Amp, and detecting cloning of the multiple cloning sites inserted into PnPR1-8 by using specific primers for amplifying PnPR1-8 after bacterial liquid is turbid. Sequencing the obtained positive clone, and analyzing that the final obtained PnPR1-8 full-length cDNA is 702bp through NCBIORF finder (http:// www.ncbi.nlm.nih.gov/gorf/gorf.html); pnPR1-8 encodes a protein of 233 amino acids having a molecular weight of about 25.55kDa and an isoelectric point of 8.48. Analysis of the protein sequence encoded by PnPR1-8 by means of bioinformatics software SignalP 4.1, detection of whether it has an N-terminal signal peptide, revealed the presence of a signal peptide at the N-terminal of PnPR1-8, and therefore the protein was presumed to be a secreted protein.

Example 2: construction of plant super-expression vector

The E.coli plasmid pGEM-T-PnPR1-8 inserted with the PnPR1-8 gene and the plant expression vector pCAMBIA2300s plasmid were extracted using SanPrep column type plasmid DNA miniprep kit (Shanghai) and 1. Mu.L was used for agarose gel electrophoresis to examine the integrity and concentration of the extracted plasmid. The recombinant plasmid pGEM-T-PnPR1-8 and the plant expression vector pCAMBIA2300s plasmid were simultaneously subjected to double digestion (50. Mu.L system) with restriction enzymes EcoRI (TaKaRa) and BamHI (TaKaRa) introduced in designing the primers; the reaction system and the operation process are as follows: respectively taking 15 mu LpGEM-T-PnPR1-8 and pCAMBIA2300s plasmids, and sequentially adding 7.5 mu L10 XK buffer, 2.5 mu L BamHI, 2.5 mu L LEcoI and 17.5 mu L ddH ₂ O, after being evenly mixed, the mixture is centrifuged for a short time and is put into a water bath kettle at 37 ℃ for enzyme digestion for 3 hours. And (3) carrying out agarose gel electrophoresis on all enzyme-digested products, then respectively carrying out gel recovery on large fragments of PnPR1-8 ORF and pCAMBIA2300s vectors by using a SanPrep column type DNA gel recovery kit (Shanghai, inc.) (the specific operation steps are the same), taking 1 mu L of recovered products, detecting the size and concentration of the recovered fragments by agarose gel electrophoresis, and storing at-20 ℃ for later use.

The recovered PnPR1-8 DNA fragment and pCAMBIA2300s vector fragment were ligated by using T4 DNALigase (TaKaRa), the reaction system (20. Mu.L) and the procedure were: mu.L of PnPR1-8 ORF was taken and added with 2. Mu.L of pCAMBIA2300s vector DNA, 2. Mu.L of 10 XT 4 DNALigase Buffer, 1. Mu. L T4 DNALigase, 5. Mu.L of ddH in this order ₂ O, after mixing evenly, centrifuging for a short time, and then carrying out a water bath at 16 ℃ for overnight reaction. The ligation product was then transferred into E.coli DH 5. Alpha. Using heat shock transformation, and positive clones were selected using solid medium containing 50mg/L kanamycin (kanamycin, kana). And (3) selecting a LB liquid medium containing 50mg/L Kana for single colony shake culture, using bacterial liquid as a template, carrying out PCR by using specific primers for amplifying PnPR1-8, selecting clones in which PnPR1-8 ORF and pCAMBIA2300s are successfully connected, adding glycerol into the detected positive strain, and storing at the temperature of-80 ℃ for later use.

Extracting and purifying pCAMBIA2300s-PnPR1-8 plasmid in the escherichia coli DH5 alpha, and transferring the recombinant plasmid pCAMBIA2300s-PnPR1-8 into the prepared competent cells of the agrobacterium tumefaciens LBA4404 by a liquid nitrogen freeze thawing method; the specific operation steps are as follows: adding 5 mu L of pCAMBIA2300s-PnPR1-8 plasmid into a centrifuge tube containing 50 mu L of competent cells, slightly mixing, ice-bathing for 30min, cooling in liquid nitrogen for 2min, rapidly placing in a water bath at 37 ℃ for 5min, ice-bathing for 2min, adding 500 mu L of LB liquid for shake culture at 28 ℃ for 4h; coating the activated agrobacterium on LB solid medium containing 50mg/LKana and 30mg/L Rif, and culturing at 28 ℃ in an inverted way; selecting a plurality of single colonies, shake culturing the single colonies in an LB liquid medium containing 50mg/LKana and 25mg/L Rif, performing PCR reaction by using a bacterial liquid as a template and then amplifying a specific primer of PnPR1-8, and detecting whether pCAMBIA2300s-PnPR1-8 is transferred into agrobacterium or not; for positive clones, glycerol was added and stored at-80℃until use.

Example 3: agrobacterium-mediated genetic transformation of tobacco and transgenic tobacco screening

The transgenic recipient for this experiment was tobacco (Nicotiana tabacum), and a batch of sterile tobacco seedlings was first cultured for genetic transformation experiments. Taking out the stored agrobacterium LBA4404 strain containing pCAMBIA2300s-PnPR1-8 plasmid from a refrigerator at the temperature of minus 80 ℃, inoculating 50 mu L of the strain into 50mL of LB liquid culture medium containing 50mg/L Kana and 30mg/L Rif, and culturing at the temperature of 28 ℃ until bacterial liquid is turbid; 1mL of the turbid bacterial solution was aspirated onto LB solid medium containing 50mg/L Kana and 30mg/L Rif, and cultured at 28℃for 48 hours. The agrobacteria on LB solid medium were then scraped off in appropriate amounts and inoculated into MGL liquid medium supplemented with 20mg/L Acetosyringone (AS) and cultured with shaking at 28℃for 4h to activate the agrobacteria.

Cutting young leaf of aseptic tobacco into pieces of about 1cm ² Is completely soaked in the MGL liquid culture medium containing the activated agrobacterium, and is subjected to dip dyeing at 25 ℃ for 15min. Sucking the bacterial liquid on the surface of the leaf disc by using sterile filter paper, placing the leaf disc on a co-culture medium, and co-culturing for 2 days under the dark condition at 22 ℃; the co-culture medium for tobacco transformation is MS+0.02 mg/L6-BA+2.1 mg/L NAA+30g/L sucrose+6 g/L agar.

Transferring the co-cultured leaf discs into MS screening culture medium with antibiotic to differentiate into seedling, and screening transgenic plant. Tobacco screening medium is MS+0.5 mg/L6-BA+0.1 mg/L NAA+30g/L sucrose+6 g/L agar+50 mg/LKana+200mg/L cephalosporin (cefotaxime sodium salt, cef); the flask was transferred to an illumination incubator for cultivation (25 ℃,16h/d illumination, 8h/d darkness) during the screening cultivation. After differentiation and sprouting of tobacco, subculturing is performed with MS medium containing 50mg/L Kana and 200 mg/LCef. And (3) transferring the regenerated tobacco seedlings to an MS culture medium containing 50mg/L Kana to root the regenerated tobacco seedlings, and finally selecting regenerated tobacco seedlings with better roots for PCR analysis.

Extracting genome DNA of transgenic tobacco plant leaves by adopting a CTAB method, and carrying out PCR reaction by using the genome DNA as a template and using specific primers of PnPR 1-8; after the PCR is finished, 8 mu L of the product is used for agarose gel electrophoresis to detect positive transgenic plants; the amplification result of part of tobacco transgenic plants is shown in figure 1, and 36 positive transgenic plants are screened from PnPR1-8 transgenic tobacco.

Example 4: analysis of expression of PnPR1-8 in transgenic tobacco and analysis of function of transgenic plant against fungal infection

Extracting total RNA from young leaves of positive transgenic plants and non-transgenic tobacco (wild type) respectively, performing reverse transcription to generate a first cDNA chain (the specific operation steps are the same as above), performing PCR with the first cDNA chain as a template by using specific primers for amplifying PnPR1-8, and analyzing the expression level of PnPR1-8 in each transgenic plant according to the PCR result; total RNA extraction and RT-PCR were performed in the same manner as in example 1; after completion of PCR, 8. Mu.L was used for agarose gel electrophoresis, and the detection results of a part of the individual plants are shown in FIG. 2.

Laboratory-preserved Humicola insolens were inoculated on PDA solid medium (200 g/L potato, 15g/L agar, 20g/L glucose) and dark-cultured at 28℃for 5 days. WT tobacco and PnPR1-8 transgenic tobacco leaves grown well and uniform in size in the incubator and fully stretched were taken and cut from the petioles with surgical scissors. Wounds with the same size are formed at the same position of the blades by using a sterile plastic gun head, and the equal-size humicola insoles are respectively inoculated. The inoculated leaves are placed on filter paper soaked by sterile water, cultured in an illumination incubator at 28 ℃, and water is added every day for moisturizing. After 7d of culture, collecting the leaves and observing the disease condition of the leaves of each strain; as shown in FIG. 3, after being inoculated with Humicola insolens, leaves of wild type tobacco form larger lesions, the leaves are yellow and rotten, and the symptoms of transgenic tobacco leaves are lighter, so that the area of the formed lesions is far smaller than that of the wild type tobacco. Obviously, pnPR1-8 transgenic tobacco has obvious resistance to PnPR humicola.

Claims (2)

1. A Notoginseng radix disease course related protein 1 gene PnPR1-8 has a nucleotide sequence shown in SEQ ID NO. 1.

2. Use of the pseudo-ginseng disease course related protein 1 gene PnPR1-8 according to claim 1 for increasing resistance of tobacco to humicola insolens (Humicola fuscoatra).