CN112159821B - Application of corn elicitor peptide gene ZmPep1 in improving verticillium wilt resistance of plants - Google Patents

Abstract

The invention discloses the application of the maize elicitor peptide gene ZmPep1 in improving the resistance of plants to Verticillium wilt. By cloning the elicitor peptide gene ZmPep1 in maize, a molecular biology method is used to construct a plant expression vector for constitutive expression of ZmPep1, Then, using genetic engineering method, ZmPep1 gene was introduced into plants, and transgenic Arabidopsis thaliana, tobacco and cotton lines with normal transcription and expression were obtained; The ZmPep1 gene can be used to improve the resistance of plants to Verticillium wilt, which is of great significance for plant disease resistance genetic engineering.

Description

Application of maize elicitor peptide gene ZmPep1 in improving plant resistance to Verticillium wilt

technical field

The invention relates to the field of genetic engineering, in particular to the application of the maize elicitor peptide gene ZmPep1 in improving the resistance of plants to Verticillium wilt, and also relates to the use of constitutively expressing the maize elicitor peptide ZmPep1 gene to improve the resistance of plants to Verticillium wilt. method.

Background technique

Plant diseases are one of the long-term natural disasters that endanger agricultural production, with an annual loss of more than 10% globally due to diseases (Feng Zhanshan et al., 2013). Plant diseases not only cause crop yield reduction, but also seriously threaten the quality and safety of agricultural products and international trade, and even cause food shortages and a series of social problems in severe cases (Punja, 2004). The harm of pathogenic bacteria not only causes direct economic losses, but also produces toxins, which brings serious food safety problems. Therefore, improving the disease resistance of crops is not only the key to solving the food problem, but also an important measure to improve food safety.

Verticillium wilt is a worldwide disease, and the incidence of Verticillium wilt has been reported in temperate, subtropical and tropical regions (PeggGF, 2002). Verticillium wilt is a soil-borne vascular pathogen with facultative nutritional properties (Steven J. Klosterman et al., 2009). Pathogens mutate rapidly, with many physiological races, can survive in soil for 20 years, and can infect more than 200 plant species, all plants except monocotyledonous plants, including various vegetables, fruit trees, crops, forest trees, flowers and plants, etc. All are hosts of Verticillium wilt, and the annual crop yield loss caused by Verticillium wilt reaches billions of dollars. Among them, the loss of potatoes infected with Verticillium wilt can reach more than 50%, lettuce can easily reach 100%, and cotton is in Years with severe incidence of Verticillium wilt are also likely to cause no harvest. Among all vascular diseases, the disease loss caused by Verticillium wilt is the most serious (Pegg GF, 2002; Steven J. Klosterman et al., 2009; Agrios G, 2005 ). At present, the only cloned gene for resistance to verticillium wilt is Ve1 from tomato. This gene also exists in lettuce, but the resistance is also lost after a few years. More importantly, most crops lack the antigen against verticillium wilt. , and it is difficult to obtain resistant antigens (Steven J. Klosterman et al., 2009).

Faced with the rapid mutation of pathogenic bacteria and the specificity of physiological races, the creation of materials with broad-spectrum resistance is the fundamental solution to the problem. Genetic engineering can overcome many of the deficiencies of traditional breeding, and more genes can be used and the sources are wider. In addition, genetic engineering can also achieve a broader spectrum of disease resistance against a wider range of pathogenic bacteria, with minimal impact on soil beneficial microorganisms (Owen Wally et al., 2010). However, transgenic plants with improved resistance to fungal and bacterial diseases have had very limited success compared to herbicide-resistant and insect-resistant transgenic plants that have been widely cultivated around the world for more than 10 years. There are also many successful reports of improving the resistance of transgenic crops to certain diseases. For example, overexpression of the chitinase gene CHIT36 from Trichoderma in carrots improved the resistance of transgenic plants to fungal diseases (Baranski R et al., 2008). Overexpression of defensin genes RsAFP2 and DmAMP of different origins in tomato and rice, respectively, improved their disease resistance (Jha S et al., 2009), and expression of the peak toxin gene in rice improved the resistance of rice to bacterial blight (Wei Shi, 2016) et al. Although these exogenous genes have achieved great success in improving plant disease resistance, there is no report on their application in production. The main problem is that the obtained resistance is only a certain pathogenic bacteria or a certain physiological race (or strain). Strong resistance, difficult to last and not broad-spectrum resistance (Yan-Jun Chen, 2012).

Improving plant self-defense is an important means to improve plant broad-spectrum and durable resistance. Plant hormones play an important role in plant defense responses, and the SA, JA and ET signaling pathways that regulate plant disease resistance have always been research hotspots. The defense signal peptides of insect resistance have attracted the attention of researchers, especially the role of defense signal peptides from plants in regulating plant immunity to insects and pathogens has been paid more and more attention (Yube Yamaguchi et al., 2011).

Plant defense signal peptides are a large class of peptide hormones, one of which is derived from precursor protein peptides without N-terminal secretion signals. Plant elicitor peptide Pep (Plant elicitor peptide) from maize is one of them. The Pep peptide family is widely present in plants and can regulate the resistance response of pathogenic bacteria (Huffaker A et al., 2006). However, it has been reported that the resistance responses of this pathogen are basically resistance to necrotrophic pathogens. Studies have shown that ZmPep1 of maize can regulate the resistance of maize to small leaf spot and stalk rot (Huffaker A et al., 2011). However, there is no relevant report on whether ZmPep1 can improve the disease resistance of other plants, especially to improve the resistance of facultative vegetative pathogens such as Verticillium wilt.

An important feature of plant defense signal peptides is that their precursor proteins or mature peptides accumulate in vascular tissues and amplify defense signals within vascular bundles, while also activating the JA signaling pathway (Yube Yamaguchi et al., 2013). Verticillium wilt is a typical vascular disease. After entering plants, it continues to infect and expand in vascular tissues. The JA signaling pathway plays an important role in the defense response of Verticillium wilt (Wei Gao et al., 2013). Combining the two characteristics of the accumulation of defense signal peptides and the spatial overlap of Verticillium wilt infection, the signal pathway activated by defense signal peptides and the overlapping of the defense signal pathways of Verticillium dahliae, the anti-Verticillium wilt bacteria were obtained. Defense signal peptides are of great significance for improving plant verticillium wilt resistance.

SUMMARY OF THE INVENTION

In view of this, one of the objects of the present invention is to provide an application of a maize elicitor peptide gene ZmPep1 in improving plant resistance to Verticillium wilt, combining the accumulation and signal transduction of ZmPep1 with the infection of Verticillium wilt in space. The two features of the overlapping of the signaling pathway activated by ZmPep1 and the defense of Verticillium dahliae in the signaling pathway, propose the strategy of the present invention.

To achieve the above object, the present invention provides the following technical solutions:

The application of the maize elicitor peptide gene ZmPep1 in improving plant resistance to Verticillium wilt, characterized in that: the nucleotide sequence of the maize elicitor peptide gene ZmPep1 is as shown in SEQ ID NO.1, or SEQ ID NO. The nucleotides shown in 1 are nucleotide sequences with the same function after substitution and/or deletion and/or addition of one or several bases.

In the present invention, the plant can be other plants that can be infected with Verticillium wilt, preferably, the plant is Arabidopsis, tobacco or cotton.

Another object of the present invention is to provide a method for improving plant resistance to Verticillium wilt by utilizing the maize elicitor peptide gene ZmPep1, by combining the maize elicitor peptide gene ZmPep1 (SEQ ID NO. 1) with a constitutive promoter. It is operatively linked to construct a plant expression vector constitutively expressing the maize elicitor peptide ZmPep1 gene, and then the maize elicitor peptide gene ZmPep1 is transformed into the plant through the transformant to obtain a transgenic plant.

To achieve the above object, the present invention provides the following technical solutions:

Using the maize elicitor peptide gene ZmPep1 to improve the resistance of plants to verticillium wilt, the maize elicitor peptide gene ZmPep1 is constitutively expressed in plants to obtain plants resistant to verticillium wilt;

The nucleotide sequence of the maize elicitor peptide gene ZmPep1 is shown in SEQ ID NO.1, or the nucleotide shown in SEQ ID NO.1 is substituted and/or deleted and/or added by one or several bases and nucleotide sequences with the same function.

In the present invention, the method for constitutively expressing the maize elicitor peptide gene ZmPep1 in plants is to construct a constitutive plant expression vector containing the maize elicitor peptide gene ZmPep1, and then obtain a transgenic plant mediated by Agrobacterium.

Preferably, the constitutive plant expression vector contains an expression cassette in which the expression of the maize elicitor peptide gene ZmPep1 is regulated by a constitutive promoter.

Preferably, the constitutive promoter is the CaMV35S promoter.

Preferably, the constitutive plant expression vector is obtained by passing the sequence shown in SEQ ID NO.1 into the BamHI and KpnI plant expression vector pLGN (CN105671076A) to obtain the plant expression vector pLGN-35S-ZmPep1.

In the present invention, the plant can be other plants that can be infected with Verticillium wilt, such as vegetables such as eggplant, tomato, pepper, lettuce, oil plants such as rapeseed and olive oil; the plant is Arabidopsis, tobacco or cotton.

In the present invention, the method for obtaining the gene ZmPep1 and the fusion of the promoter and the gene ZmPep1 to construct an expression vector for constitutively expressing the ZmPep1 gene are conventional methods in the art, and the vector used is a conventional vector used in the field of plant genetic engineering.

The method used for transforming the transformant into the plant is the Agrobacterium tumefaciens-mediated method, which is a commonly used method for plant transgenesis.

The third object of the present invention is to provide a plant expression vector with improved plant resistance to Verticillium wilt.

To achieve the above-mentioned purpose of the invention, the present invention provides the following technical solutions:

A plant expression vector capable of improving plant resistance to Verticillium wilt, the plant expression vector comprising an expression cassette for regulating the expression of a maize elicitor peptide gene ZmPep1 by a constitutive promoter, the nucleotides of the maize elicitor peptide gene ZmPep1 The sequence is shown in SEQ ID NO. 1, or the nucleotide sequence shown in SEQ ID NO. 1 is substituted and/or deleted and/or added by one or several bases and has the same function.

Preferably in the present invention, the gene ZmPep1 sequence (SEQ ID NO. 1) is operably linked with the constitutive promoter CaMV35S by using genetic engineering technology to construct a plant expression vector, and after the plants are transformed, maize is expressed under the action of the constitutive promoter The elicitor peptide gene ZmPep1. Preferred plant expression vectors have the vector structure shown in Figure 1 . Among them, the maize elicitor peptide gene ZmPep1 was positively connected to the CaMV35S promoter to form a plant expression vector pLGN-35S-ZmPep1 constitutively expressing the gene.

The fourth object of the present invention is to provide the application of plant expression vector.

To achieve the above-mentioned purpose of the invention, the present invention provides the following technical solutions:

The application of the plant expression vector in the preparation of transgenic plants with Verticillium wilt resistance.

The beneficial effects of the present invention are as follows: the present invention uses the method of molecular biology to construct a plant expression vector for constitutive expression of ZmPep1 by cloning the elicitor peptide ZmPep1 presuppressin gene ZmPep1 from maize, and then using the genetic engineering method to transform the ZmPep1 The genes were integrated into Arabidopsis, tobacco and cotton, and transgenic Arabidopsis, tobacco and cotton lines with normal transcriptional expression were obtained. When the disease index of wild-type Arabidopsis was 50.69, the disease index of transgenic Arabidopsis was only 15.40. When the disease index of wild-type tobacco was 57.93, the disease index of transgenic tobacco was only 15.39; when the disease index of wild-type cotton was 53.43, the disease index of transgenic cotton was only 7.92. It is shown that ZmPep1 can improve the resistance of plants to Verticillium wilt, and the invention is of great significance for promoting the application of ZmPep1 gene in plant disease resistance genetic engineering.

Description of drawings

In order to make the purpose, technical solutions and beneficial effects of the present invention clearer, the present invention provides the following drawings for description:

Figure 1 shows the map of the plant expression vector pLGN-35S-ZmPep1

Figure 2 shows the transcription and expression levels of ZmPep1 gene in transgenic Arabidopsis (ZmPep1-5, ZmPep1-8 and ZmPep1-10: independent transformants of transgenic Arabidopsis; WT: Colombia ecotype wild-type Arabidopsis. Values are for three techniques Mean of replicates, error bars are standard error (SD).

Figure 3 shows that ZmPep1 gene improves the resistance of Arabidopsis thaliana to Verticillium wilt (A: inoculated with Verticillium wilt for 14 days, the disease index of Arabidopsis strains; B: inoculated with Verticillium wilt for 14 days, the disease of Arabidopsis plants Disease resistance of transgenic Arabidopsis was evaluated by disease index. Data are the mean of three replicate experiments, and error bars are SD. ZmPep1-5, ZmPep1-8 and ZmPep1-10: ZmPep1 transgenic Arabidopsis independently transformed Sub.WT: Colombia ecotype wild type Arabidopsis thaliana.**: Compared with wild type Arabidopsis thaliana, the difference level of disease index reached extremely significant (p<0.01)).

Fig. 4 is the transcriptional expression level of ZmPep1 gene in transgenic tobacco (ZmPep1-1, ZmPep1-5 and ZmPep1-11: transgenic tobacco independent transformants. WT: wild-type tobacco. Values are the mean values of three technical replicates, and error bars are three standard error SD).

Fig. 5 is that ZmPep1 gene improves the resistance of tobacco to verticillium wilt (A: inoculated with verticillium wilt for 7 days, the disease index of tobacco strain; B: inoculated with verticillium wilt for 7 days, the disease of tobacco leaves. The disease resistance of transgenic tobacco Sex was assessed by disease index, values are the mean of three replicates, and error bars are SD. ZmPep1-1, ZmPep1-5 and ZmPep1-11: ZmPep1 transgenic tobacco independent transformants. WT: wild type tobacco.**: Compared with wild-type tobacco, the difference level of disease index reached extremely significant (p<0.01)).

Fig. 6 is the transcriptional expression level of ZmPep1 gene in transgenic cotton (ZmPep1-4, ZmPep1-5 and ZmPep1-7: transgenic cotton independent transformants. WT: wild-type cotton. Values are the mean values of three technical replicates, and error bars are standard error of three replicates).

Fig. 7 is that ZmPep1 transgenic cotton improves the resistance to Verticillium wilt (A: disease index of transgenic cotton leaves inoculated with Verticillium wilt for 5 days; B: inoculated with Verticillium wilt for 5 days, disease of cotton leaves. Disease resistance of transgenic cotton Disease index was used for evaluation. Values are the mean of three replicate experiments, and error bars are SD. ZmPep1-4, ZmPep1-5 and ZmPep1-7: ZmPep1 transgenic cotton independent transformants. WT: wild-type cotton.**: Compared with wild-type cotton, the difference level of disease index reached extremely significant (p<0.01)).

Detailed ways

The present invention is further described below with reference to the accompanying drawings and specific embodiments, so that those skilled in the art can better understand the present invention and implement it, but the embodiments are not intended to limit the present invention.

Example 1. Cloning of maize ZmPep1 gene

1. Extraction of total RNA from maize genome and synthesis of cDNA

The 14-day-old maize seedlings were taken, and the base of the petiole was lightly clipped with tweezers to induce the expression of ZmPep1 gene. After 6 hours, the damaged leaves were snap-frozen in liquid nitrogen immediately, and ground into powder, then the total RNA of the leaves was extracted with Promoga's plant rapid RNA extraction kit, and cDNA was synthesized by TaKaRa's one-strand cDNA synthesis kit. RNA extraction and cDNA synthesis were carried out according to the kit instructions. The detailed operation process is as follows:

Take fresh corn leaves and freeze them in liquid nitrogen and grind them into powder. Take about 100 mg of the powder and put it into a 1.5 mL centrifuge tube without nuclease. Immediately add 500 μL of RNA lysis solution. Tissue, then add 300 μL of diluent, invert the centrifuge tube 3-4 times to mix, and leave at room temperature for 3-5 min. 12,000 rpm, centrifuge for 5 min, take 500 μL of the supernatant into a new 1.5 mL nuclease-free centrifuge tube, add 250 μL of absolute ethanol, immediately mix with a pipette, then transfer the mixture into an RNA adsorption column, 10,000 rpm , centrifuge for 1 min, discard the filtrate, add 600 μL of rinsing solution to the adsorption column, repeat the rinsing for 2 times, transfer the adsorption column to the elution tube, add 100 μL of RNase-free and DNase-free water to the adsorption column, and leave it for about 3min Then, centrifuge at 10,000 rpm for 1 min, collect the eluted RNA solution and store it at -80°C.

Take 7 μL of the above extracted RNA solution, add 1 μL RNase-free DNase, 2 μL RNase-free DNase buffer, mix well, react at 42°C for 2 min in the PCR machine, then add 4 μL reverse transcriptase buffer, 1 μL reverse transcriptase buffer. Transcriptase, 1 μL reverse transcription primer mix, 4 μL double-distilled water without RNase and DNase, after mixing, react at 37 °C for 15 min to synthesize cDNA, and then react at 85 °C for 5 s to terminate the reaction. The synthesized cDNA was stored at -20°C.

2. Cloning of maize ZmPep1 gene

Find the ZmPep1 sequence (see SEQ ID NO.1) on the NCBI website, and design and synthesize the upstream and downstream primers of the complete coding frame according to the nucleotide sequence shown in SEQ ID NO.1, as follows:

Upstream primer F-ZmPep1: 5'-CGCGGATCCGCG ATGGATGAGCGCGGGGAGAA-3' (SEQ ID NO. 2);

Downstream primer R-ZmPep1: 5'-CGGGGTACCCCG CTAGTGGTGGTTCCCTCCAT-3' (SEQ ID NO. 3);

Using the cDNA synthesized above as a template and SEQ ID NO. 2 and SEQ ID NO. 3 as a primer pair, PCR amplification was performed using PrimesSTAR MAX DNA Polymerase. Amplification program: 98°C for 3 min; 30 cycles of amplification at 98°C for 10S, 57°C for 10S, and 72°C for 20S.

5 μL of amplified ZmPep1 fragment was added, 3 μL of pTOPO vector plasmid, 1 μL of DNA ligase, and 1 μL of buffer solution were added. After mixing, place at room temperature for 15 min. The reaction product was mixed with E. coli DH5α competent cells, and then transferred into 42 °C heat shock for 1 min 30 s. Escherichia coli DH5α, the transformed engineering bacteria were added to 500 μL of LB medium, cultured at 37°C at 200 rpm for 1 h, then spread on LB plates containing kanamycin, cultured at 37°C overnight, and a single colony was picked and inoculated into about 3 mL of LB medium. In the test tube, shake and culture at 200 rpm at 37 °C overnight, take an appropriate amount of bacterial liquid and send it to a sequencing company for sequencing, and store the positive cloned engineered bacteria containing the target fragment at -80 °C.

Example 2. Construction of plant expression vector constitutively expressing ZmPep1 gene and acquisition of engineered bacteria

The above-mentioned cloned vector engineering bacterial plasmids verified to be correct by sequencing were extracted, double-enzyme digested with BamHI and KpnI, and the digested product was subjected to agarose gel electrophoresis to recover the target fragment ZmPep1. At the same time, the plant expression vector pLGN was digested with BamHI and KpnI. After the digestion was completed, agarose gel electrophoresis was performed to recover large fragments. The recovered gene and vector fragment were ligated by T4 DNA ligase, and the ligated product was transferred into E. coli DH5α by heat shock method. The plasmid of the E. coli engineering bacteria was extracted, and then double-enzyme digestion was performed with BamHI and KpnI for verification. The transformed bacteria that obtained the ZmPep1 restriction fragment of the target gene was the plant expression vector pLGN-35S-ZmPep1 engineering bacteria, which was stored at -80 ℃. The vector map see picture 1.

The plasmids of E. coli engineering bacteria were extracted, and then Agrobacterium LBA4404 and GV3101 were transformed by electroporation. The transformed bacteria were added with Km (kanamycin) and Sm (streptomycin sulfate), Km and Rif (rifampicin). ) YEB plates were screened and cultured, and the resistant single colonies were added to the YEB liquid medium with Km and Sm, Km and Rif (rifampicin), and the cells were collected and the Agrobacterium plasmid was extracted, and then doubled with BamHI and KpnI. Enzyme digestion verified that the correct engineered bacteria were stored at -80°C.

Example 3. Genetic transformation of Arabidopsis, screening of transgenic Arabidopsis and analysis of transcriptional expression levels

1. Genetic transformation of Arabidopsis

Referring to Steven J.Clough and Andrew F.Bent's (1998) method of flower soaking transformation, the wild-type Arabidopsis thaliana was used as material for genetic transformation, and the seeds after Agrobacterium soaking were harvested after the seeds were mature.

2. Screening of transgenic Arabidopsis plants

The seeds harvested after genetic transformation by the flower dip transformation method were sterilized with 75% alcohol for 15 minutes, and then all inoculated on a screening plate with an additional 100 mg/L Km for germination. If the grown seedlings were green, they were transgenic plants. When the leaves are above, transplanted into the special soil for culturing Arabidopsis (grass carbon soil: vermiculite: perlite is 3: 1: 1), and the seedlings are harvested. Each plant is a transformant.

Arabidopsis screening medium: MS inorganic+MS organic+Km 100mg/L+2.5g/L Gelrite (factor), pH6.0

3. RNA extraction and cDNA synthesis from transgenic Arabidopsis plants

The transgenic Arabidopsis RNA was extracted and cDNA was synthesized according to the method of Example 1 using the young leaves of the transgenic plants as materials.

4. Analysis of ZmPep1 gene transcription and expression level in transgenic Arabidopsis

The transcriptional expression level of ZmPep1 gene in transgenic Arabidopsis was detected by Real-time PCR method.

A specific fragment of ZmPep1 gene was amplified using cDNA as template. The upstream and downstream primers of the ZmPep1 gene were ZmPep1UP: 5'-TTCTGCGGCTCCTGCTC-3' (SEQ ID NO. 4) and ZmPep1 DN: 5'-GTGGTTCCCTCCATTGC-3' (SEQ ID NO. 5). The Arabidopsis AtACT2 gene was used as the internal standard. The upstream and downstream primers of the AtACT2 gene were AtACT2 UP: 5'-TATCGCTGACCGTATGAG-3' (SEQ ID NO.6) and AtACT2DN: 5'-CTGAGGGAAGCAAGAATG-3' (SEQ ID NO.7).

The 20 μL Real-time PCR reaction system includes: 1 μL cDNA template, 1 μL upstream and downstream primers of the target gene, 10 μL 2×iQ SYBR Green Supermix, and 7 μL ddH ₂ O.

Real-time PCR amplification conditions: 95°C for 3 min; 94°C for 10s, 57°C for 30s, and 72°C for 30s, a total of 40 cycles of amplification. After amplification, the relative expression of ZmPep1 gene was analyzed by Gene Study software.

Real-time PCR results showed (Figure 2) that the ZmPep1 gene could be effectively transcribed and expressed in the transgenic Arabidopsis plants, and the obtained plants were ZmPep1 transgenic plants.

Example 4. Resistance of transgenic Arabidopsis to Verticillium wilt

1. Preparation of Verticillium wilt for inoculation of transgenic Arabidopsis

Pick a small amount of Verticillium decidua V991 strain preserved in solid PD medium (potato medium) and inoculate it into liquid PD medium, 180rpm, 26 ℃ shaking culture for 7d, and then press the ratio of 10% (bacteria liquid/PD medium) Inoculated into liquid PD medium, 180rpm, 26 ℃ shaking culture for 10d, filtered with four layers of sterile gauze to remove mycelium and impurities in the bacterial liquid, deionized water adjusted the spore concentration to 10 ⁸ /ml as the inoculated bacterial liquid.

2. Inoculation method for disease resistance identification of transgenic Arabidopsis

Arabidopsis thaliana seedlings that have been cultivated for one week are uprooted, then placed neatly in a 150mm petri dish with soil, poured into the mixed inoculum solution, the inoculation dose is 10mL/plant, soaked at room temperature for 24 hours, and then transplanted into In moist soil, 16 hours of light, 8 hours of dark culture, 20°C (dark culture)-24°C (light culture), and cultured in a light incubator with a humidity of 70%. Two weeks after inoculation, the disease grades of the plants were counted according to the standard of grade 0-4 and the disease index was calculated. Wild-type plants transformed with recipient material were used as controls.

Disease grade grading standard: grade 0: no disease in plant leaves; grade 1: disease in 0-25% of leaves; grade 2: disease in 25%-50% of leaves; grade 3: disease in 50%-75% of leaves; grade 4 : More than 75% of the leaves showed disease. The formula for calculating the disease index:

Disease index=(∑〖disease grade×number of plants〗)/(4×total number of inoculated plants)×100

3. The ZmPep1 gene improves the resistance of Arabidopsis to Verticillium wilt

Arabidopsis thaliana plants were inoculated with Verticillium wilt for 14 days, the disease index of wild-type plants reached 50.69, and the disease indexes of transgenic lines ZmPep1-5, ZmPep1-8 and ZmPep1-10 were 20.62, 15.40 and 21.36, respectively. The T test results showed that the disease index of the transgenic line was significantly decreased compared with the disease index of the wild-type control (A in Figure 3). Plant disease showed that after 14 days of inoculation with Verticillium wilt, the leaves of the wild-type plants basically developed disease, while the transgenic plants only developed disease at the base of individual leaves (B in Figure 3). The results showed that ZmPep1 gene could effectively improve the resistance of Arabidopsis to Verticillium wilt.

Embodiment 5, the genetic transformation of tobacco and the acquisition of transgenic tobacco

1. Tissue culture medium for tobacco genetic transformation

Seed germination medium: MSB (MS inorganic salt + B5 organic) + 1.0% agar powder, prepared with tap water, natural pH.

Genetic transformation co-culture medium: MSB (MS inorganic salt+B5 organic)+2mg/L NAA+0.5mg/L 6-BA+200μmol/L AS, pH5.6, solid medium with 1.0% agar powder for solidification.

Callus induction medium: MSB (MS inorganic salt+B5 organic)+2mg/L NAA+0.5mg/L 6-BA+1.0% agar powder, pH5.8.

Sprout induction medium: MSB (MS inorganic salt + B5 organic) + 2 mg/L 6-BA + 1.0% agar powder, pH 5.8.

Rooting medium: MSB (MS inorganic salt + B5 organic) + 1.0% agar powder, pH 6.0.

2. Genetic transformation of tobacco

The recombinant Agrobacterium containing the pLGN-35S-ZmPep1 plant expression vector was inoculated into liquid YEB medium, and cultured overnight at 28°C with shaking at 200 rpm to an OD600 of 1.0-1.2. After the bacterial solution was centrifuged, the bacterial cells were collected, and the bacterial cells were resuspended in an equal volume of MSB liquid medium, and the resuspended solution was the infusion solution for transformation.

The leaves of tobacco aseptic seedlings cultivated for 20 days were cut into leaf discs of 3-5 mm, infiltrated for 1 hr in the infusion solution, and the bacterial solution was removed. After the co-cultivation was completed, the explants were substituted into the callus induction medium supplemented with 100 mg/L kanamycin and 200 mg/L cephalosporin, and cultured in a photoperiod of 25 °C, 16 hr light/8 hr dark culture, and subcultured after 20 days. The seedling induction medium is subcultured once after 20 days, and the young shoots are produced at the edge of the leaf disc. analyze.

3. Acquisition and molecular identification of ZmPep1 transgenic tobacco

GUS histochemical staining of transgenic plants

GUS staining solution: 500mg/L X-Gluc, 0.1mol/LK ₃ Fe(CN) ₆ , 0.1mol/LK ₄ Fe(CN) ₆ , 1% Triton X-100(V/V), 0.01mol/L Na ₂ EDTA, 0.1 mol/L phosphate buffer (pH 7.0). The pLGN-35S-ZmPep1 plant expression vector contains the GUS gene controlled by the 35S promoter. Therefore, the transgenic plants can be rapidly identified by GUS histochemical staining. Referring to the method of Jefferson (1987), a little leaf tissue of Km-resistant seedlings was cut, added to GUS histochemical staining solution, stained at 37°C for 5h, and then decolorized with 95% ethanol until the green color was cleared. The last blue color is the transgenic plant, otherwise it is the non-transgenic plant.

4. Analysis of ZmPep1 transcriptional expression level

ZmPep1 transgenic tobacco plants used young leaves as materials, respectively extracted RNA from the leaves of GUS-positive and wild-type plants, and synthesized one-strand cDNA of each sample RNA according to the instructions of the cDNA one-strand synthesis kit, and then amplified the ZmPep1 gene using the cDNA as a template specific fragment. The upstream and downstream primers of the ZmPep1 gene were ZmPep1 UP: 5'-TTCTGCGGCTCCTGCTC-3' (SEQ ID NO. 4) and ZmPep1 DN: 5'-GTGGTTCCCTCCATTGC-3' (SEQ ID NO. 5). Using tobacco 18S gene as internal standard, the upstream and downstream primers of 18S gene were 18S UP: 5'-AGGAATTGACGGAAGGGCA-3' (SEQ ID NO. 8) and 18S DN: 5'-GTGCGGCCCAGAACATCTAAG-3' (SEQ ID NO. 9).

The 20 μL Real-time PCR reaction system includes: 1 μL cDNA template, 1 μL upstream and downstream primers of the target gene, 10 μL 2×iQ SYBR Green Supermix, and 7 μL ddH ₂ O.

Real-time PCR amplification conditions: 95°C for 3 min; 94°C for 10s, 57°C for 30s, and 72°C for 30s, a total of 40 cycles of amplification. After amplification, the relative expression of ZmPep1 gene was analyzed by Gene Study software.

The Real-time PCR results showed (Fig. 4) that the ZmPep1 gene could be effectively transcribed and expressed in the transgenic cotton plants, while the expression of this gene was not detected in the leaves of the wild-type plants.

Example 6. Resistance of transgenic tobacco to Verticillium wilt

1. Preparation of Verticillium wilt for inoculation of transgenic tobacco for disease resistance identification

The preparation method of Verticillium wilt for inoculation of transgenic tobacco for disease resistance identification is the same as that in Example 4, and the spore concentration is adjusted to 10 ¹⁰ spores/mL.

2. Identification and inoculation method of transgenic tobacco for disease resistance

Tobacco plants with 7-8 true leaves, cut the third to fifth leaves from the top to the bottom, wrap the petioles with moist absorbent paper, and place them in the inoculation box evenly and neatly, and gently squeeze with a pipette tip Press the junction of the main vein and the secondary vein of the leaf to achieve the purpose of artificial injury. Immediately after the injury, inoculate 10 μL of Verticillium wilt inoculum at the injury. Then cover the film to keep moisture, and place 16 hours light/8 hours dark culture, incubator at 20°C (dark culture)/26°C light, inoculate for 7 days, and count the disease grade of leaves according to the 5-level standard of 0-4. And calculate the disease index. Wild-type plants transformed with recipient material were used as controls.

Disease grade grading standard: grade 0: no disease in leaves; grade 1: disease in 0-25% of the leaf area; grade 2: disease in 25%-50% of the leaf area; grade 3: disease in 50%-75% of the leaf area; Grade 4: Disease on more than 75% of the leaf area. The formula for calculating the disease index:

Disease index=(∑〖disease grade×number of plants〗)/(4×total number of inoculated plants)×100.

3. ZmPep1 gene improves the resistance of tobacco to Verticillium wilt

Tobacco leaves were inoculated with Verticillium wilt for 7 days, the disease index of wild-type plants reached 57.93, and the disease index of transgenic lines ZmPep1-1, ZmPep1-5 and ZmPep1-11 were 16.29, 24.64 and 15.39, respectively. The T test results showed that the disease index of the transgenic line was significantly decreased compared with the disease index of the wild-type control (A in Figure 5). After 7 days of inoculation with Verticillium wilt, the diseased area of wild-type leaves exceeded 50%, while the diseased area of transgenic tobacco leaves was less than 10% (Fig. 5, B). The results showed that ZmPep1 gene could effectively improve the resistance of tobacco to Verticillium wilt.

Example 7. Genetic transformation of cotton

1. Common media for cotton genetic transformation

Basic medium: MSB (MS inorganic salt + B5 organic) (T.Murashige, 1962; O.L. Gamborg, 1968); Seed germination medium: 1/2MSB+20g/L sucrose+6g/L agar, prepared in tap water, natural pH ;

Co-culture medium: MSB+0.5mg/L IAA (indoleacetic acid)+0.1mg/L KT (6-furfuraminopurine)+30g/L glucose+100μmol/L acetosyringone+2.0g/L Gelrite (Sigma ), pH 5.4;

Screening sterilization medium: MSB+0.5mg/L IAA+0.1mg/L KT+75mg/L Km (Kanamycin)+500mg/Lcef (cephalosporin)+30g/L glucose+2.0g/L Gelrite , pH5.8;

Callus induction medium: MSB+0.5mg/L IAA+0.1mg/L KT+75mg/L Km+200mg/L cef+30g/L glucose+2.0g/L Gelrite, pH5.8;

Embryogenic callus induction medium: MSB+0.1mg/L KT+30g/L glucose+2.0g/L Gelrite, pH5.8;

Liquid suspension medium: MSB+0.1mg/L KT+30g/L glucose, pH5.8;

Somatic embryo maturation medium: MSB+15g/L sucrose+15g/L glucose+0.1mg/L KT+2.5g/L Gelrite, pH6.0;

Seedling medium: SH+0.4g/L activated carbon+20g/L sucrose, pH 6.0 (Schenk & Hildebrandt, 1972).

2. Methods of cotton genetic transformation

(1) Obtaining transformed explants

The seeds of the upland cotton cultivar Jimian No. 14 were shelled, the kernels were sterilized with 3% hydrogen peroxide for 60 min, rinsed with sterile tap water for 5-6 times, inoculated into the seed germination medium, and cultivated in the dark at 28°C for 5 days. Sterile hypocotyls were cut into 3-5 mm long segments and used as transformed explants.

(2) Preparation of Agrobacterium dipping solution for transformation

A single colony of Agrobacterium that integrates the plant expression vector pLGN-35S-ZmPep1 was obtained by the streaking method, and then a single colony was picked and inoculated into 10 mL liquid YEB (5 g/L sucrose) supplemented with 50 mg/L Km and 125 mg/L Sm (streptomycin) , 1 g/L bacterial yeast extract, 10 g/L bacterial tryptone, 0.5 g/L MgSO ₄ ·7H ₂ O, pH 7.0), cultured overnight at 28°C, 200 rpm, and then at a rate of 5% The bacterial liquid was inoculated into 20 mL of liquid YEB without antibiotics, and cultured at 28 °C and 200 rpm to an OD600 of about 0.5. Take 5 mL of the bacterial solution and centrifuge at 6000 rpm for 5 min to collect the bacterial cells, and then resuspend the bacterial cells in 5 mL of the non-Gelrite co-cultivation liquid medium.

(3) Genetic transformation of hypocotyl and induction of embryogenic callus

The explants were dipped with Agrobacterium dipping solution for 20 min, the bacterial solution was poured out, and the excess bacterial solution on the surface of the explants was removed with sterile filter paper. Cultured for 2 days, the hypocotyls were inoculated into the screening de-bacteria medium, and after 20 days were subcultured into the callus induction medium supplemented with kanamycin (Km) and cephalosporin (cef) for callus induction, and subcultured once every 20 days. , 60 days later, it was subcultured into embryogenic callus induction medium, and after obtaining embryogenic callus, liquid suspension culture was carried out to obtain a large number of embryogenic callus with consistent growth.

(4) Somatic embryo induction and seedling culture

The embryogenic callus cultured in liquid suspension was filtered through a 30-mesh stainless steel mesh, and the embryogenic callus under the sieve was inoculated into the somatic embryo maturation medium evenly and dispersedly, and a large number of somatic embryos were produced in about 15 days, which were substituted into the SH medium, Promote the further growth of somatic embryos. The regenerated seedlings with 3-4 leaves were transplanted into the greenhouse for propagation.

Example 8 Acquisition and molecular verification of ZmPep1 transgenic cotton

According to the cotton genetic transformation and regeneration method of Example 7, the ZmPep1 gene was transferred into the cotton genome. After induction of Km-resistant callus, embryogenic callus and somatic embryo, the somatic embryo became seedling, and then ZmPep1 transgenic cotton plants were obtained.

1. GUS histochemical staining of transgenic plants

The formula of GUS dyeing solution is the same as that in Example 5.2.

Cut a little petiole and leaf tissue of Km-resistant seedlings, add them to GUS histochemical staining solution respectively, stain at 37°C for 2 hours, and then decolorize with 95% ethanol until the green color is cleared. The last blue color is the transgenic plant, otherwise it is the non-transgenic plant.

2. Analysis of the transcriptional expression level of ZmPep1 gene

ZmPep1 transgenic cotton plants used young leaves as materials, respectively extracted RNA from the leaves of GUS-positive and wild-type plants, and synthesized one-strand cDNA of each sample RNA according to the instructions of the cDNA one-strand synthesis kit, and then amplified the ZmPep1 gene using the cDNA as a template specific fragment. The upstream and downstream primers of the ZmPep1 gene were ZmPep1 UP: 5'-TTCTGCGGCTCCTGCTC-3' (SEQ ID NO. 4) and ZmPep1 DN: 5'-GTGGTTCCCTCCATTGC-3' (SEQ ID NO. 5). The cotton histone HIS3 gene was used as the internal standard. The upstream and downstream primers of the HIS3 gene were GhHIS3 UP: 5'-GAAGCCTCATCGATACGTC-3' (SEQ ID NO. 10) and GhHIS3 DN: 5'-CTACCACTACCATCATGGC-3' (SEQ ID NO. 11) (Zhu YQ et al., 2003).

The 20 μL Real-time PCR reaction system includes: 1 μL cDNA template, 1 μL upstream and downstream primers of the target gene, 10 μL 2×iQ SYBR Green Supermix, and 7 μL ddH2O.

Real-time PCR amplification conditions: 95°C for 3 min; 94°C for 10s, 57°C for 30s, and 72°C for 30s, a total of 40 cycles of amplification. After amplification, the relative expression of ZmPep1 gene was analyzed by Gene Study software.

The Real-time PCR results showed (Fig. 6) that the ZmPep1 gene could be effectively transcribed and expressed in the transgenic cotton plants, while the expression of this gene was not detected in the wild-type plants.

Example 9 Resistance of ZmPep1 transgenic cotton to Verticillium wilt

1. Preparation of pathogenic bacteria for inoculation for disease resistance identification

The preparation method of transgenic cotton for identifying pathogenic bacteria for disease resistance is the same as that in Example 4.

2. In vitro leaf inoculation method

Cut the leaves of transgenic cotton young plants, arrange and align the petioles and insert them into the culture bottle containing the inoculated bacterial solution. The culture bottle containing the leaves was placed in a photoperiod of 16 hours of light, 8 hours of dark culture, a temperature change cycle of 20°C (dark culture) and 26°C (light), and continued moisturizing culture under 70% humidity conditions, and leaves were counted every two days. disease grade, and calculate the disease index. Wild-type plants transformed with recipient material were used as controls. Disease grade classification standard: grade 0: no disease in cotton leaves; grade 1: disease in less than 25% of the leaf area; grade 2: disease in 25%-50% of the leaf area; grade 3: disease in 50%-75% of the leaf area; Grade 4: Disease on more than 75% of the leaf area. The formula for calculating the disease index:

Disease index=(∑〖disease grade×number of plants〗)/(4×total number of inoculated plants)×100.

3. The ZmPep1 gene improves the resistance of cotton to Verticillium wilt

Verticillium wilt resistance identification was carried out on all transgenic plants according to the above method. The results showed that the disease index of wild-type cotton was 53.43 after inoculation for 5 days, and the disease index of transgenic lines ZmPep1-4, ZmPep1-5 and ZmPep1-7 were 23.61 respectively. , 7.92 and 14.96. The T-test results showed that the disease index of the transgenic cotton line was significantly lower than that of the wild-type cotton (A in Figure 7). 5 days after inoculation, the leaves of wild-type cotton showed severe disease, while the leaves of the transgenic cotton line showed only a few lesions (B in Figure 7). The results showed that ZmPep1 could significantly improve the resistance of cotton to Verticillium wilt.

The above-mentioned embodiments are only preferred embodiments for fully illustrating the present invention, and the protection scope of the present invention is not limited thereto. Equivalent substitutions or transformations made by those skilled in the art on the basis of the present invention are all within the protection scope of the present invention. The protection scope of the present invention is subject to the claims.

sequence listing

<110> Southwest University

<120> Application of maize elicitor peptide gene ZmPep1 in improving plant resistance to Verticillium wilt

<160> 11

<170> SIPOSequenceListing 1.0

<210> 1

<211> 417

<212> DNA

<213> Corn (Zea mays L.)

<400> 1

atggatgagc gcggggagaa ggaggaggag cacggagtag tggaggagga gacggcggcg 60

gttgtgctta aggaggtgga ggtggagatg gtcggcggct ctgaggaagc ctcggcggcg 120

ccgctcctcc tcgcgcaccc gtgctcgctt ctgcggctcc tgctccgcgc ctgcgccggc 180

tgcctggtgc gcctgctgca cggctactgc agcgacggcg acgacgacga cccaaaggct 240

gctgccgacg acgacgacga cgctgcgcct gaagctgctg cggcggcggc ggccgatggc 300

ggcgacaagg cagccaccta cttgtacatg caggaggtgt gggcagtgag gaggaggccg 360

acgacgcccg gccgtccgag agaaggttcc ggtggcaatg gagggaacca ccactag 417

<210> 2

<211> 32

<212> DNA

<213> Artificial Sequence

<400> 2

cgcggatccg cgatggatga gcgcggggag aa 32

<210> 3

<211> 32

<212> DNA

<213> Artificial Sequence

<400> 3

cggggtaccc cgctagtggt ggttccctcc at 32

<210> 4

<211> 17

<212> DNA

<213> Artificial Sequence

<400> 4

ttctgcggct cctgctc 17

<210> 5

<211> 17

<212> DNA

<213> Artificial Sequence

<400> 5

gtggttccct ccattgc 17

<210> 6

<211> 18

<212> DNA

<213> Artificial Sequence

<400> 6

tatcgctgac cgtatgag 18

<210> 7

<211> 18

<212> DNA

<213> Artificial Sequence

<400> 7

ctgagggaag caagaatg 18

<210> 8

<211> 19

<212> DNA

<213> Artificial Sequence

<400> 8

aggaattgac ggaagggca 19

<210> 9

<211> 21

<212> DNA

<213> Artificial Sequence

<400> 9

gtgcggccca gaacatctaa g 21

<210> 10

<211> 20

<212> DNA

<213> Artificial Sequence

<400> 10

gaagcctcat cgataccgtc 20

<210> 11

<211> 19

<212> DNA

<213> Artificial Sequence

<400> 11

ctaccactac catcatggc 19

Claims (8)

1. the application of maize elicitor peptide gene ZmPep1 in improving plant resistance to verticillium wilt, it is characterized in that: the nucleotide sequence of described maize elicitor peptide gene ZmPep1 is as shown in SEQ ID NO.1; For Arabidopsis, tobacco or cotton. 2. the method that utilizes the maize elicitor peptide gene ZmPep1 to improve plant resistance to verticillium wilt, characterized in that: the maize elicitor peptide gene ZmPep1 is constitutively expressed in the plant to obtain plants resistant to verticillium wilt; The nucleotide sequence of the maize elicitor peptide gene ZmPep1 is shown in SEQ ID NO.1. 3. method according to claim 2 is characterized in that: the method that described maize elicitor peptide gene ZmPep1 is constitutively expressed in plants is to construct the constitutive plant expression vector containing maize elicitor peptide gene ZmPep1 , then by agricultural Bacillus-mediated acquisition of transgenic plants. 4 . The method according to claim 3 , wherein the constitutive plant expression vector contains an expression cassette in which the expression of the maize elicitor peptide gene ZmPep1 is regulated by a constitutive promoter. 5 . 5. The method of claim 4, wherein the constitutive promoter is the CaMV35S promoter. 6. method according to claim 4 is characterized in that: described constitutive plant expression vector is to construct in the plant expression vector pLGN by the sequence shown in SEQ ID NO.1 by Bam HI and Kpn I double digestion, obtain plant Expression vector pLGN-35S- ZmPep1 . 7. The method according to any one of claims 3 to 6, wherein the plant is Arabidopsis thaliana, tobacco or cotton. 8. the application of a plant expression vector in the preparation of a transgenic plant with Verticillium wilt resistance, characterized in that: the plant expression vector contains an expression cassette that a constitutive promoter regulates the expression of the maize elicitor peptide gene ZmPep1 , and the The nucleotide sequence of the maize elicitor peptide gene ZmPep1 is shown in SEQ ID NO.1.

Images