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

Abstract

The invention discloses an application of a corn elicitor peptide gene ZmPep1 in improving the verticillium wilt resistance of plants, wherein an elicitor peptide gene ZmPep1 is cloned from corn, a plant expression vector of ZmPep1 constitutive expression is constructed by adopting a molecular biology method, and then a gene engineering method is utilized to introduce the ZmPep1 gene into the plants, so that transgenic arabidopsis thaliana, tobacco and cotton strains with normal transcriptional expression are obtained; the disease indexes of the obtained transgenic plants are obviously lower than those of wild type controls, the resistance to the verticillium wilt is obviously improved, and the ZmPep1 gene can be used for improving the resistance of the plants to the verticillium wilt and has important significance to plant disease resistance genetic engineering.

Description

Application of corn elicitor peptide gene ZmPep1 in improving verticillium wilt resistance of plants

Technical Field

The invention relates to the field of genetic engineering, in particular to application of a corn elicitor peptide gene ZmPep1 in improving verticillium wilt resistance of plants, and also relates to a method for improving verticillium wilt resistance of plants by using a constitutive expression corn elicitor peptide ZmPep1 gene.

Background

Plant diseases are one of natural disasters which endanger agricultural production for a long time, and more than 10% of the plant diseases are lost in the world due to the year of the diseases (Von Zhan et al, 2013). Plant diseases not only cause crop reduction, but also seriously threaten the quality safety and international trade of agricultural products, and when serious, also cause food shortage and a series of social problems (Punja, 2004). Pathogenic bacteria harm not only causes direct economic loss, but also can generate toxin, thereby bringing serious food safety problems. Therefore, improving the disease resistance of crops is not only a key to solving the food problem, but also an important measure for improving the food safety.

Verticillium wilt is a worldwide disease, and the onset of verticillium wilt is reported from temperate zone, subtropical zone to tropical zone (Pegg GF, 2002). Verticillium wilt is a pathogenic bacterium of the soil-borne vascular bundle, with facultative nutritional properties (Steven j. klosterman et al, 2009). The pathogenic bacteria have fast mutation frequency and multiple physiological species, can survive in soil for 20 years, and can infect more than 200 plant varieties, all plants except monocotyledons, including various vegetables, fruit trees, crops, forest trees, flowers and plants, and the like are hosts of verticillium wilt bacteria, and the crop yield loss caused by the verticillium wilt disease per year reaches billions of dollars, wherein the loss of the potato infected with the verticillium wilt bacteria can reach more than 50 percent, the lettuce is very easy to reach 100 percent, the cotton is easy to cause no grain harvest in the years with serious verticillium wilt disease, and the disease loss caused by the verticillium wilt bacteria is the most serious in all vascular bundle diseases (Pegg GF, 2002; Steven J. osteerman et al, 2009; Klrios G, 2005). At present, the only gene clonally obtained for resistance to verticillium wilt is Ve1 from tomato, which is also present in lettuce but resistance is lost after several years, and more importantly, most crops lack antigens for resistance to verticillium wilt and it is difficult to obtain resistant antigens (Steven j. klosterman et al, 2009).

In the face of rapid variation of pathogenic bacteria and specificity of physiological race, it is the fundamental to solve the problem to create a material with broad-spectrum resistance. The genetic engineering can overcome the defects of many traditional breeding, and the usable genes are more 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, the success of transgenic plants with increased resistance to fungal and bacterial diseases has been very limited compared to herbicide and insect resistant transgenic plants that have been grown extensively 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 carrot improves the resistance of transgenic plants to fungal diseases (Baranski R et al, 2008). Overexpression of defensin genes RsAFP2 and DmAMP from different sources in tomato and rice improves their disease resistance (Jha S et al, 2009), expression of peak toxin genes in rice improves resistance to bacterial blight in rice (Wei Shi, 2016), and so on. Although these exogenous genes have been successful in improving plant disease resistance, there is no report on their application to production, and the main problem is that the obtained resistance is only strong against a certain pathogen or a certain physiological race (or strain), and is difficult to persist and has no broad-spectrum resistance (Yan-Jun Chen, 2012).

Improving the self-defense ability of plants is an important means for improving the broad-spectrum and durable resistance of the plants. Plant hormones play an important role in plant defense response, SA, JA and ET signal pathways for regulating plant disease resistance are always hot points of research, and with the deepening and expanding of research, a new class of defense signal peptides capable of activating plant resistance to pathogenic bacteria and insects attracts attention of researchers in recent years, and particularly, the role of the plant-derived defense signal peptides in regulating plant immunity to insects and pathogenic bacteria is more important (Yube Yamaguchi ET al, 2011).

Plant defense signal peptides are a broad class of peptide hormones, one of which is derived from a precursor protein peptide without an N-terminal secretion signal. One of the maize-derived inducer peptides, Pep (plant organism peptide), is a Pep peptide family widely present in plants and capable of regulating resistance reaction against pathogenic bacteria (Huffaker A et al, 2006). However, it has been reported that the resistance response of this pathogen is essentially resistance to necrosomal disease trophoblasts. It has been shown that ZmPep1 from maize modulates maize disease resistance to northern leaf blight and stem rot (Huffaker A et al, 2011). However, it has not been reported that ZmPep1 can improve the disease resistance of other plants, particularly against facultative pathogenic fungi such as verticillium wilt.

Plant defense signal peptides have an important feature, their precursor proteins or mature peptides accumulate in vascular tissues and amplify defense signals within the vascular bundle, while activating the JA signaling pathway (Yube Yamaguchi et al, 2013). Verticillium dahliae is a typical vascular bundle disease, is continuously infected and expanded in vascular tissues after entering a plant body, and a JA signal path plays an important role in defense reaction of verticillium dahliae (Wei Gao et al, 2013). The two characteristics of the accumulation of the defense signal peptide and the spatial overlapping of the infection of the verticillium wilt bacteria, the signal path activated by the defense signal peptide and the overlapping of the defense of the verticillium wilt bacteria on the signal path are combined, and the obtained defense signal peptide capable of resisting the verticillium wilt bacteria has important significance for improving the verticillium wilt resistance of plants.

Disclosure of Invention

In view of the above, an object of the present invention is to provide an application of a corn elicitor peptide gene ZmPep1 in improving the resistance of plants to verticillium wilt, and provide a strategy of the present invention by combining two characteristics of spatial overlapping of accumulation and signal transmission of ZmPep1 and infection of verticillium wilt and overlapping of signal path of ZmPep 1-activated signal path and verticillium wilt defense.

In order to achieve the purpose, the invention provides the following technical scheme:

the application of corn elicitor peptide gene ZmPep1 in improving the resistance of plants to verticillium wilt is characterized in that: the nucleotide sequence of the corn inducer peptide gene ZmPep1 is shown in SEQ ID NO.1, or the nucleotide sequence shown in SEQ ID NO.1 is subjected to substitution and/or deletion and/or addition of one or more bases and has the same function.

In the present invention, the plant may be other plants which can be infected with verticillium wilt, and preferably, the plant is arabidopsis thaliana, tobacco or cotton.

The second purpose of the invention is to provide a method for improving verticillium wilt resistance of plants by using a corn elicitor peptide gene ZmPep1, which comprises the steps of operably connecting a corn elicitor peptide gene ZmPep1(SEQ ID NO.1) with a constitutive promoter to construct a plant expression vector for constitutively expressing a corn elicitor peptide ZmPep1 gene, and then transferring the corn elicitor peptide gene ZmPep1 into plants through transformants to obtain transgenic plants.

In order to achieve the purpose, the invention provides the following technical scheme:

the method for improving the verticillium wilt resistance of plants by using the corn elicitor peptide gene ZmPep1 comprises the steps of constitutively expressing the corn elicitor peptide gene ZmPep1 in the plants to obtain the verticillium wilt resistance plants;

the nucleotide sequence of the corn inducer peptide gene ZmPep1 is shown in SEQ ID NO.1, or the nucleotide sequence shown in SEQ ID NO.1 is subjected to substitution and/or deletion and/or addition of one or more bases and has the same function.

In the invention, the constitutive expression method of the corn inducer peptide gene ZmPep1 in the plant is to construct a constitutive plant expression vector containing the corn inducer peptide gene ZmPep1 and then obtain a transgenic plant through agrobacterium mediation.

Preferably, the constitutive plant expression vector contains an expression cassette for regulating expression of maize inducer peptide gene ZmPep1 from a constitutive promoter.

Preferably, the constitutive promoter is the CaMV35S promoter.

Preferably, the constitutive plant expression vector is a plant expression vector pLGN-35S-ZmPep1 obtained by passing the sequence shown in SEQ ID NO.1 through BamHI and KpnI plant expression vector pLGN (CN 105671076A).

In the invention, the plants can be other plants which can be infected with verticillium wilt, such as vegetables like eggplant, tomato, hot pepper, lettuce and the like, and oil plants like rape, olive and the like; the plant is Arabidopsis thaliana, tobacco or cotton.

In the invention, the method for obtaining the gene ZmPep1 and constructing the expression vector for constitutively expressing the gene ZmPep1 by fusing the promoter and the gene ZmPep1 is a conventional method in the field, and the used vector is a conventional vector used in the field of plant genetic engineering.

The method used for transferring the transformant into the plant is an agrobacterium tumefaciens mediated method, which is a commonly used plant transgenic method.

It is a further object of the present invention to provide a plant expression vector having improved resistance to verticillium wilt in plants.

In order to achieve the above purpose, the invention provides the following technical scheme:

a plant expression vector capable of improving the verticillium wilt resistance of a plant comprises an expression frame of a constitutive promoter for regulating and controlling the expression of a corn inducer peptide gene ZmPep1, wherein the nucleotide sequence of the corn inducer peptide gene ZmPep1 is shown as SEQ ID No.1, or the nucleotide sequence shown as SEQ ID No.1 is subjected to substitution and/or deletion and/or addition of one or more bases and has the same function.

Preferably, the sequence of the gene ZmPep1(SEQ ID NO.1) is operably connected with a constitutive promoter CaMV35S by applying a genetic engineering technology to construct a plant expression vector, and after a plant is transformed, the corn inducer peptide gene ZmPep1 is expressed under the action of the constitutive promoter. Preferred plant expression vectors have the vector structure shown in FIG. 1. Wherein, the maize inducer peptide gene ZmPep1 is positively connected to the CaMV35S promoter to form a plant expression vector pLGN-35S-ZmPep1 for constitutively expressing the gene.

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

In order to achieve the above purpose, the invention provides the following technical scheme:

the plant expression vector is applied to the preparation of transgenic plants with verticillium wilt resistance.

The invention has the beneficial effects that: according to the invention, an inducer peptide ZmPep1 prepro protein gene ZmPep1 is cloned from corn, a plant expression vector for constitutive expression of ZmPep1 is constructed by adopting a molecular biological method, and then a gene engineering method is utilized to integrate the ZmPep1 gene into arabidopsis thaliana, tobacco and cotton, so that transgenic arabidopsis thaliana, tobacco and cotton strains with normal transcription expression are obtained. The disease index of the wild type arabidopsis is 50.69, and the disease index of the transgenic arabidopsis is only 15.40. When the disease index of the wild tobacco is 57.93, the disease index of the transgenic tobacco is only 15.39; when the disease index of wild cotton is 53.43, the disease index of transgenic cotton is only 7.92. The invention shows that ZmPep1 can improve the resistance of plants to verticillium wilt, and has important significance for promoting the application of ZmPep1 genes in plant disease-resistant genetic engineering.

Drawings

In order to make the object, technical scheme and beneficial effect of the invention more clear, the invention provides the following drawings for explanation:

FIG. 1 is a map of plant expression vector pLGN-35S-ZmPep1

FIG. 2 shows the transcriptional expression level of ZmPep1 gene in transgenic Arabidopsis (ZmPep1-5, ZmPep1-8 and ZmPep 1-10: independent transformants of transgenic Arabidopsis; WT: Columbia ecotype wild type Arabidopsis; values are the average of three technical replicates and error bars are standard error SD).

FIG. 3 shows that ZmPep1 gene increases resistance of Arabidopsis thaliana to verticillium wilt (A: 14 days after inoculation of verticillium wilt bacteria, disease index of Arabidopsis lines; B: 14 days after inoculation of verticillium wilt bacteria, disease condition of Arabidopsis plants. evaluation of disease resistance of transgenic Arabidopsis thaliana is carried out by using disease index. data is average value of three times of repeated tests, error bars are standard errors SD. ZmPep1-5, ZmPep1-8 and ZmPep 1-10: ZmPep1 independent transformant of transgenic Arabidopsis thaliana. WT: ecological wild type Arabidopsis thaliana in Columbia. compared with wild type Arabidopsis thaliana, difference level of disease index reaches extreme significance (p < 0.01)).

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

FIG. 5 shows that ZmPep1 gene increases resistance of tobacco to verticillium wilt (A: index of disease of tobacco strain after inoculation of verticillium wilt for 7 days; B: disease of tobacco leaf after inoculation of verticillium wilt for 7 days; disease resistance of transgenic tobacco is evaluated by disease index, the value is average value of three repetitions, error bars are standard errors SD. ZmPep1-1, ZmPep1-5 and ZmPep 1-11: ZmPep1 independent transformant of transgenic tobacco. WT: wild type tobacco: difference level of disease index reaches very significant (p <0.01)) compared with wild type tobacco.

FIG. 6 shows the transcriptional expression levels of ZmPep1 gene in transgenic cotton (ZmPep1-4, ZmPep1-5 and ZmPep 1-7: independent transformants of transgenic cotton. WT: wild type cotton. values are the mean of three technical replicates and error bars are the standard error of three replicates).

FIG. 7 shows that ZmPep1 transgenic cotton has increased resistance to verticillium wilt (A: index of disease condition of 5 days after leaf inoculation of transgenic cotton with verticillium wilt pathogen; B: disease condition of 5 days after leaf inoculation of verticillium wilt pathogen; disease resistance of transgenic cotton is evaluated by disease condition index. the numerical value is the average value of three repeated tests, error bars are standard errors SD. ZmPep1-4, ZmPep1-5 and ZmPep 1-7: ZmPep1 transgenic cotton independent transformants. WT: wild type cotton. the difference level of disease condition index reaches a high significance (p <0.01)) compared with wild type cotton.

Detailed Description

The present invention is further described with reference to the following drawings and specific examples so that those skilled in the art can better understand the present invention and can practice the present invention, but the examples are not intended to limit the present invention.

Example 1 cloning of maize ZmPep1 Gene

1. Extraction of corn genome total RNA and cDNA synthesis

Corn seedlings grown for 14 days were taken, and the base of the petiole was lightly pinched with forceps to induce the expression of ZmPep1 gene. After 6 hours, the damaged leaves are cut and immediately quick-frozen in liquid nitrogen, ground into powder, then the total RNA of the leaves is extracted by using a plant rapid RNA extraction kit of Promoga, and then cDNA is synthesized by using a one-strand cDNA synthesis kit of TaKaRa. RNA extraction and cDNA synthesis were performed according to the kit instructions. The detailed operation flow is as follows:

taking fresh corn leaves, quickly freezing the fresh corn leaves by using liquid nitrogen, grinding the fresh corn leaves into powder, taking about 100mg of the powder, putting the powder into a 1.5mL centrifuge tube without nuclease, immediately adding 500 mu L of RNA lysate, repeatedly blowing the RNA lysate by using a pipette until no obvious blocky tissue exists in the lysate, then adding 300 mu L of diluent, reversing the centrifuge tube for 3-4 times, uniformly mixing, and standing at room temperature for 3-5 min. 12000rpm, centrifuging for 5min, taking 500 μ L of supernatant into a new centrifugal tube without nuclease of 1.5mL, adding 250 μ L of absolute ethyl alcohol, immediately blowing and uniformly mixing by a pipette, then transferring the mixed solution into an RNA adsorption column, 10000rpm, centrifuging for 1min, discarding filtrate, adding 600 μ L of rinsing solution into the adsorption column, repeatedly rinsing for 2 times, transferring the adsorption column onto an elution tube, adding 100 μ L of water without RNase and DNA enzyme into the adsorption column, standing for about 3min, 10000rpm, centrifuging for 1min, collecting eluted RNA solution and storing at-80 ℃.

And (3) adding 1 mu L of RNase-free DNase and 2 mu L of RNase-free DNase buffer solution into 7 mu L of the extracted RNA solution, uniformly mixing, reacting for 2min at 42 ℃ in a PCR instrument, then adding 4 mu L of reverse transcriptase buffer solution, 1 mu L of reverse transcriptase, 1 mu L of reverse transcription primer mixture and 4 mu L of double distilled water without RNase and DNase, uniformly mixing, reacting for 15min at 37 ℃ to synthesize cDNA, reacting for 5s at 85 ℃, and stopping the reaction. The synthesized cDNA was stored at-20 ℃.

2. Cloning of maize ZmPep1 Gene

The NCBI website searches a ZmPep1 sequence (shown in SEQ ID NO.1), and designs and synthesizes an upstream primer and a downstream primer of a complete coding frame according to a nucleotide sequence shown in SEQ ID NO.1, which comprises the following steps:

the upstream primer F-ZmPep 1: 5'-CGCGGATCCGCG ATGGATGAGCGCGGGGAGAA-3' (SEQ ID NO. 2);

the downstream primer R-ZmPep 1: 5'-CGGGGTACCCCG CTAGTGGTGGTTCCCTCCAT-3' (SEQ ID NO. 3);

PCR amplification was performed using PrimesSTAR MAX DNA Polymerase using the above synthesized cDNA as a template and SEQ ID NO.2 and SEQ ID NO.3 as a primer pair. And (3) amplification procedure: 3min at 98 ℃; amplification was performed for 30 cycles at 98 ℃ 10S,57 ℃ 10S,72 ℃ 20S.

5 mu L of ZmPep1 fragment obtained by amplification, adding 3 mu L of pTOPO vector plasmid, 1 mu L of DNA ligase and 1 mu L of buffer solution, placing at room temperature for 15min after uniform mixing, uniformly mixing a reaction product with escherichia coli DH5 alpha competent cells, then thermally shocking at 42 ℃ for 1min30s to transfer into escherichia coli DH5 alpha, adding 500 mu L of LB culture medium into the transformed engineering bacteria, culturing at 37 ℃ for 1h at 200rpm, then coating on an LB plate containing kanamycin, culturing overnight at 37 ℃, selecting a single colony, inoculating into a test tube containing about 3mL of LB culture medium, performing shaking culture at 37 ℃ for overnight at 200rpm, taking a proper amount of bacterial liquid, sending to a sequencing company for sequencing, and preserving the engineering bacteria containing the positive clone of the target fragment at-80 ℃.

Example 2 construction of plant expression vector for constitutive expression of ZmPep1 Gene and obtaining of engineering bacteria

Extracting the cloning vector engineering bacterium plasmid which is verified to be correct by sequencing, carrying out double enzyme digestion by using BamHI and KpnI, carrying out agarose gel electrophoresis on the product after enzyme digestion, and recovering a target fragment ZmPep 1. And carrying out double enzyme digestion on the plant expression vector pLGN by using BamHI and KpnI, carrying out agarose gel electrophoresis after enzyme digestion is finished, and recovering a large fragment. The recovered gene and vector fragment were ligated by T4 DNA ligase, and the ligation product was transformed into E.coli DH 5. alpha. by heat shock. Extracting the plasmid of the engineering bacterium of the escherichia coli, carrying out double enzyme digestion verification by using BamHI and KpnI, obtaining a transformation bacterium of the enzyme digestion fragment of the target gene ZmPep1, namely the plant expression vector pLGN-35S-ZmPep1 engineering bacterium, storing at-80 ℃, and showing a vector map in figure 1.

Extracting the plasmid of engineering bacteria of colibacillus, transforming Agrobacterium LBA4404 and GV3101 by electric transformation method, screening and culturing the transformed bacteria on YEB plates with additional Km (kanamycin) and Sm (streptomycin sulfate), Km and Rif (rifampicin), collecting the bacteria and extracting Agrobacterium plasmid from resistant single colony in YEB liquid culture medium with additional Km and Sm, Km and Rif, double enzyme digestion verification with BamHI and KpnI, and storing the correct engineering bacteria at-80 deg.C.

Example 3 genetic transformation of Arabidopsis, screening of transgenic Arabidopsis and analysis of transcription expression level

1. Genetic transformation of Arabidopsis thaliana

Genetic transformation was carried out using wild type Arabidopsis thaliana in Columbia as material, according to the floral dip transformation method of Steven J. Clough and Andrew F. bent (1998), and seeds after Agrobacterium maceration were harvested after seed maturation.

2. Screening of transgenic Arabidopsis plants

The seeds harvested after genetic transformation by a flower soaking transformation method are sterilized by 75% alcohol for 15min, then are inoculated on a screening plate with 100mg/L Km added for sprouting, if grown seedlings are green, the seedlings are transgenic plants, the plants are transplanted into special soil for culturing arabidopsis thaliana when the leaves of the plants are more than 2 (grass carbon soil: vermiculite: perlite: 3:1:1), and the seeds are harvested after seedling formation. Each plant is a transformant.

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

3. Extraction of transgenic arabidopsis plant RNA and synthesis of cDNA

The method of example 1 was followed to extract the RNA of transgenic Arabidopsis thaliana and synthesize cDNA using young leaves of transgenic plants as material.

4. Analysis of transcriptional expression level of ZmPep1 Gene in transgenic Arabidopsis

The transcription expression level of ZmPep1 gene in transgenic Arabidopsis is detected by using Real-time PCR method.

And amplifying a specific fragment of the ZmPep1 gene by using the cDNA as a template. The upstream and downstream primers of ZmPep1 gene are ZmPep1 UP: 5'-TTCTGCGGCTCCTGCTC-3' (SEQ ID NO.4) and ZmPep1 DN: 5'-GTGGTTCCCTCCATTGC-3' (SEQ ID NO. 5). The Arabidopsis AtACT2 gene was used as an internal standard. Upstream and downstream primers for the AtACT2 gene were AtACT2 UP: 5'-TATCGCTGACCGTATGAG-3' (SEQ ID NO.6) and AtACT2 DN: 5'-CTGAGGGAAGCAAGAATG-3' (SEQ ID NO. 7).

A20. mu.L Real-time PCR reaction system included: 1 mu L of cDNA template, 1 mu L of each of the upstream and downstream primers of the target gene, 10 mu L of 2 xiQ SYBR Green Supermix,ddH ₂ O 7μL。

real-time PCR amplification conditions: 3min at 95 ℃; amplifying at 94 ℃ for 10s,57 ℃ for 30s and 72 ℃ for 30s for 40 cycles. After amplification, Gene Study software was used to analyze the relative expression level of ZmPep1 Gene.

The Real-time PCR result shows (figure 2) that ZmPep1 gene in transgenic Arabidopsis plant can be effectively transcribed and expressed, and the obtained plant is ZmPep1 transgenic plant.

Example 4 resistance of transgenic Arabidopsis to Verticillium wilt

1. Preparation of verticillium wilt bacteria for disease-resistant identification and inoculation of transgenic arabidopsis thaliana

Selecting a small amount of verticillium dahliae V991 strain stored in a solid PD medium (potato medium), inoculating the strain into a liquid PD medium, carrying out shaking culture at 180rpm and 26 ℃ for 7d, inoculating the strain into the liquid PD medium according to the proportion of 10% (bacterial liquid/PD medium), carrying out shaking culture at 180rpm and 26 ℃ for 10d, filtering by using four layers of sterile gauze to remove hyphae and impurities in the bacterial liquid, and adjusting the spore concentration to 10 by using deionized water ⁸ One/ml was used as inoculum.

2. Transgenic arabidopsis disease-resistant identification inoculation method

The arabidopsis seedlings cultured for one week are uprooted, then placed in a 150mm culture dish with soil tidily, the evenly mixed inoculation bacterial liquid is poured, the inoculation dose is 10 mL/plant, after the seedlings are soaked and inoculated for 24 hours at room temperature, the seedlings are transplanted into moist soil, the seedlings are irradiated for 16 hours, dark culture is carried out for 8 hours, and the seedlings are cultured in an illumination incubator with the humidity of 70 percent (20 ℃ (dark culture) -24 ℃ (illumination culture). After 2 weeks of inoculation, the disease grade of the plants was counted according to the 0-4 grade standard and the disease index was calculated. Wild type plants transformed with the recipient material were used as controls.

Grading grade standard: level 0: the plant leaves have no diseases; level 1: 0-25% of leaf development; and 2, stage: 25% -50% of the leaves present with symptoms; and 3, level: 50% -75% of the leaves are diseased; 4, level: over 75% of leaves present with disease. The calculation formula of the disease index is as follows:

disease index (Σ [ (number of disease stages)/(number of inoculated plants)/(4 × total number of inoculated plants) × 100

3. ZmPep1 gene for improving resistance of arabidopsis thaliana to verticillium wilt

The disease index of wild plants reaches 50.69 when Arabidopsis plants are inoculated with verticillium dahliae for 14 days, and the disease indexes of transgenic lines ZmPep1-5, ZmPep1-8 and ZmPep1-10 are 20.62, 15.40 and 21.36 respectively. The T-test results showed a very significant decrease in disease index of the transgenic lines compared to that of the wild-type control (a in fig. 3). Plant disorders show that the leaves of wild plants basically have the disorders after 14 days of inoculation with verticillium wilt bacteria, while the leaves of transgenic plants only have the disorders in the individual leaves at the base (B in figure 3). The result shows that the ZmPep1 gene can effectively improve the resistance of arabidopsis to verticillium wilt.

Example 5 genetic transformation of tobacco and obtaining of transgenic tobacco

1. Tissue culture medium for tobacco genetic transformation

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

Genetic transformation co-culture medium: MSB (MS inorganic salt + B5 organic) +2mg/L NAA +0.5 mg/L6-BA + 200. mu. mol/L AS, pH5.6, solid medium added with 1.0% agar powder for solidification.

Callus induction medium: MSB (MS inorganic salt + B5 organic) +2mg/L NAA +0.5 mg/L6-BA + 1.0% agar powder, pH5.8.

Shoot induction medium: MSB (MS inorganic salt + B5 organic) +2 mg/L6-BA + 1.0% agar powder, pH5.8.

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

2. Genetic transformation of tobacco

The recombinant agrobacterium containing pLGN-35S-ZmPep1 plant expression vector is inoculated into liquid YEB culture medium, and is subjected to shaking culture at the temperature of 28 ℃ and the rpm of 200 overnight until OD 6001.0-1.2. And (3) centrifuging the bacterial liquid, collecting the bacteria, and suspending the bacteria by using an equal-volume MSB liquid culture medium, wherein the heavy suspension is a staining solution for transformation.

Culturing tobacco sterile seedling leaf for 20 days, cutting into 3-5mm leaf disc, dip-dyeing in the dip-dyeing solution for 1hr, removing bacterial liquid, inoculating the leaf disc into co-culture medium, and dark culturing at 24 deg.C for 2 days. After the co-culture is completed, the explants are subcultured into a callus induction culture medium added with 100mg/L kanamycin and 200mg/L cephamycin, the explants are subjected to photoperiod culture at 25 ℃ under 16hr illumination/8 hr dark culture, the explants are subcultured into a bud induction culture medium after 20 days, the explants are subcultured once after 20 days until buds are generated at the edge of a leaf disc, the buds are cut off and then substituted into a rooting culture medium to root and grow into seedlings, and the seedlings grow to 3-4 leaves and are transplanted into flowerpots for further analysis.

3. Acquisition and molecular characterization of ZmPep1 transgenic tobacco

GUS histochemical staining of transgenic plants

GUS staining solution: 500mg/L X-Gluc, 0.1mol/L K ₃ Fe(CN) ₆ ，0.1mol/L K ₄ Fe(CN) ₆ ，1％Triton X-100(V/V)，0.01mol/L Na ₂ EDTA, 0.1mol/L phosphate buffer (pH 7.0). The pLGN-35S-ZmPep1 plant expression vector contains a GUS gene under the control of a 35S promoter, so that transgenic plants can be quickly identified by GUS histochemical staining firstly. Leaf tissue from Km resistant seedlings was excised as described in Jefferson (1987), stained in GUS histochemical staining solution for 5h at 37 ℃ and destained to green by 95% ethanol. Finally, the blue plants are transgenic plants, otherwise, the plants are non-transgenic plants.

4. ZmPep1 transcript expression level analysis

The ZmPep1 transgenic tobacco plant takes young and tender leaves as materials, respectively extracts the RNA of GUS positive and wild plant leaves, synthesizes one-strand cDNA of each sample RNA according to the cDNA one-strand synthesis kit specification, and then takes the cDNA as a template to amplify the specific segment of ZmPep1 gene. The upstream and downstream primers of ZmPep1 gene are ZmPep1 UP: 5'-TTCTGCGGCTCCTGCTC-3' (SEQ ID NO.4) and ZmPep1 DN: 5'-GTGGTTCCCTCCATTGC-3' (SEQ ID NO. 5). The tobacco 18S gene is used as an internal standard, and the upstream and downstream primers of the 18S gene are respectively 18S UP: 5'-AGGAATTGACGGAAGGGCA-3' (SEQ ID NO.8) and 18S DN: 5'-GTGCGGCCCAGAACATCTAAG-3' (SEQ ID NO. 9).

A20. mu.L Real-time PCR reaction system included: 1 mu L of cDNA template, 1 mu L of each of upstream and downstream primers of target gene, 10 mu L of 2 xiQ SYBR Green Supermix, ddH ₂ O 7μL。

Real-time PCR amplification conditions: 3min at 95 ℃; amplifying at 94 ℃ for 10s,57 ℃ for 30s and 72 ℃ for 30s for 40 cycles. After amplification, Gene Study software was used to analyze the relative expression level of ZmPep1 Gene.

The Real-time PCR result shows (figure 4) that ZmPep1 gene in transgenic cotton plant can be effectively transcribed and expressed, and the expression of the gene is not detected in wild plant leaves.

Example 6 resistance of transgenic tobacco to Verticillium wilt

1. Preparation of verticillium wilt bacteria for transgenic tobacco disease-resistant identification and inoculation

The preparation method of verticillium wilt bacteria for identifying and inoculating transgenic tobacco disease resistance is consistent with the embodiment example 4, the concentration of spores is adjusted to 10 ¹⁰ spores/mL.

2. Transgenic tobacco disease-resistant identification inoculation method

Cutting the third to fifth leaves from the top to the bottom of 7-8 true-leaf tobacco plants, wrapping petioles with moist absorbent paper, uniformly and neatly placing in an inoculation box, slightly squeezing the junction of the main leaf vein and the secondary leaf vein of the leaves by using a pipette tip to achieve the purpose of artificial injury, and immediately inoculating 10 mu L of verticillium wilt germ inoculation liquid at the injured part after injury. Then covering with film for moisture preservation, culturing in incubator with 16hr illumination/8 hr dark culture, 20 deg.C (dark culture)/26 deg.C illumination, inoculating for 7 days, counting the disease grade of leaf according to 0-4 grade 5 standard and calculating disease index. Wild type plants transformed with the recipient material were used as controls.

Grading grade standard: grade 0, no disease of the leaf; grade 1, 0-25% of the leaf area presents diseases; grade 2, 25-50% of the leaf area is in disease; grade 3, disease symptoms appear in 50-75% of the leaf area; stage 4, more than 75% of the leaf area presents with symptoms. The calculation formula of the disease index is as follows:

disease index (Σ [ (number of disease stages × number of plants)/(4 × total number of inoculated plants) × 100.

3. ZmPep1 gene for improving resistance of tobacco to verticillium wilt

The verticillium dahliae is inoculated on tobacco leaves for 7 days, the disease index of a wild type plant reaches 57.93, and the disease indexes of transgenic lines ZmPep1-1, ZmPep1-5 and ZmPep1-11 are 16.29, 24.64 and 15.39 respectively. The T-test results showed a very significant decrease in disease index of the transgenic lines compared to that of the wild-type control (a in fig. 5). After 7 days of inoculation of verticillium wilt, the disease area of wild type leaves exceeds 50 percent, and the disease area of transgenic tobacco leaves is less than 10 percent (B in figure 5). The result shows that the ZmPep1 gene can effectively improve the resistance of tobacco to verticillium wilt.

Example 7 genetic transformation of Cotton

1. Common culture medium for cotton genetic transformation

Minimal medium MSB (MS inorganic salts + B5 organic) (T.Murashige, 1962; O.L.Gamborg, 1968); a seed germination culture medium is prepared from 1/2MSB, 20g/L of sucrose and 6g/L of agar and tap water, and the pH value is natural;

the co-culture medium comprises MSB +0.5mg/L IAA (indoleacetic acid) +0.1mg/L KT (6-furfurylaminopurine) +30g/L glucose +100 mu mol/L acetosyringone +2.0g/L Gelrite (Sigma), and the pH value is 5.4;

screening a degerming culture medium, namely MSB +0.5mg/L IAA +0.1mg/L KT +75mg/L Km (kanamycin) +500mg/L cef (cefamycin) +30g/L glucose +2.0g/L Gelrite, and pH is 5.8;

the callus induction culture medium comprises MSB, 0.5mg/L IAA, 0.1mg/L KT, 75mg/L Km, 200mg/L cef, 30g/L glucose and 2.0g/L Gelrite, and the pH value is 5.8;

the embryogenic callus induction culture medium comprises MSB +0.1mg/L KT +30g/L glucose +2.0g/L Gelrite, and pH5.8;

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

the somatic embryo maturation culture medium comprises MSB, 15g/L sucrose, 15g/L glucose, 0.1mg/L KT and 2.5g/L Gelrite, and the pH value is 6.0;

seedling medium SH +0.4g/L activated carbon +20g/L sucrose, pH6.0(Schenk & Hildebrandt, 1972).

2. Method for genetic transformation of cotton

(1) Obtaining of transformed explants

Removing shell of seeds of Gossypium hirsutum 14, sterilizing seed kernel with 3% hydrogen peroxide for 60min, rinsing with sterile tap water for 5-6 times, inoculating to seed germination culture medium, and dark culturing at 28 deg.C for 5 d. The sterile hypocotyls were cut into 3-5mm long sections as transformed explants.

(2) Preparation of Agrobacterium impregnation solution for transformation

Single colony of Agrobacterium incorporating plant expression vector pLGN-35S-ZmPep1 was obtained by streaking, and then single colony was picked and inoculated with 10mL of liquid YEB (5g/L sucrose, 1g/L yeast extract for bacteria, 10g/L tryptone for bacteria, 0.5g/L MgSO 1) supplemented with 50mg/L Km and 125mg/L Sm (streptomycin) ₄ ·7H ₂ O, pH7.0), cultured overnight at 28 ℃ and 200rpm, and then inoculated with 20mL of a liquid YEB containing no antibiotic at a ratio of 5%, cultured at 28 ℃ and 200rpm until the OD600 is about 0.5. And (3) centrifuging 5mL of bacterial liquid at 6000rpm for 5min to collect thalli, and suspending the thalli by using 5mL of co-culture liquid culture medium without adding Gelrite, wherein the suspended bacterial liquid is the agrobacterium tumefaciens staining solution for staining the explant.

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

The explant is impregnated with an agrobacterium impregnation liquid for 20min, a bacterial liquid is poured out, redundant bacterial liquid on the surface of the explant is absorbed by sterile filter paper, the impregnated hypocotyl is cut into sections and inoculated in a co-culture medium, dark culture is carried out at 26 ℃ for 2d, the hypocotyl is inoculated in a screening and degerming medium, 20d later is substituted into a callus induction medium with kanamycin (Km) and cef mycin (cef) added for callus induction, the subculture is carried out once at intervals of 20d, 60d later is substituted into an embryogenic callus induction medium, and liquid suspension culture is carried out after embryogenic callus is obtained, so that a large amount of embryogenic callus with consistent growth is obtained.

(4) Induction and seedling culture of somatic embryos

And (3) filtering the embryogenic callus cultured by liquid suspension, uniformly and dispersedly inoculating the screened embryogenic callus into a somatic embryo maturation culture medium, generating a large amount of somatic embryos in about 15 days, and subculturing the somatic embryos into an SH culture medium to promote further seedling formation of the somatic embryos. Transplanting the 3-4 leaf regenerated seedlings into a greenhouse for propagation.

EXAMPLE 8 acquisition and molecular validation of ZmPep1 transgenic Cotton

The ZmPep1 gene was transferred into the cotton genome according to the cotton genetic transformation and regeneration method of example 7. Following induction of Km resistant callus, embryogenic callus and somatic embryos, somatic embryos became plantlets, and ZmPep1 transgenic cotton plants were obtained.

1. GUS histochemical staining of transgenic plants

The GUS staining solution was formulated as in EXAMPLE 5.2.

Cutting a small amount of leaf stalk and leaf tissue of Km resistant seedlings, adding the cut leaves and leaf tissue into GUS histochemical staining solution respectively, staining for 2h at 37 ℃, and then decoloring by 95% ethanol until the leaves and leaf tissue are clean. Finally, the blue plants are transgenic plants, otherwise, the plants are non-transgenic plants.

2. Analysis of transcriptional expression level of ZmPep1 Gene

The ZmPep1 transgenic cotton plant takes young and tender leaves as materials, respectively extracts the RNA of GUS positive and wild plant leaves, synthesizes one-strand cDNA of each sample RNA according to the cDNA one-strand synthesis kit specification, and then takes the cDNA as a template to amplify the specific segment of ZmPep1 gene. The upstream and downstream primers of ZmPep1 gene are ZmPep1 UP: 5'-TTCTGCGGCTCCTGCTC-3' (SEQ ID NO.4) and ZmPep1 DN: 5'-GTGGTTCCCTCCATTGC-3' (SEQ ID NO.5), respectively. The cotton histone HIS3 gene is used as an internal standard. The upstream and downstream primers of HIS3 gene were GhHIS3 UP: 5'-GAAGCCTCATCGATACCGTC-3' (SEQ ID NO.10) and GhHIS3 DN: 5'-CTACCACTACCATCATGGC-3' (SEQ ID NO.11), respectively (Zhu YQ et al, 2003).

A20. mu.L Real-time PCR reaction system included: mu.L of cDNA template, 1. mu.L of each of the upstream and downstream primers of the target gene, 10. mu.L of 2 xiQ SYBR Green Supermix, and 7. mu.L of ddH2O 7.

Real-time PCR amplification conditions: 3min at 95 ℃; amplifying at 94 ℃ for 10s,57 ℃ for 30s and 72 ℃ for 30s for 40 cycles. After amplification, Gene Study software was used to analyze the relative expression level of ZmPep1 Gene.

The Real-time PCR results show (FIG. 6) that ZmPep1 gene in transgenic cotton plant can be transcribed effectively, while the expression of the gene is not detected in wild plant.

EXAMPLE 9 resistance of ZmPep1 transgenic Cotton to verticillium wilt

1. Preparation of pathogenic bacteria for disease-resistant identification and inoculation

The preparation method of the pathogenic bacteria for identifying the disease resistance of the transgenic cotton is the same as the embodiment example 4.

2. In vitro leaf inoculation method

Shearing tender plant leaves of transgenic cotton, arranging and aligning leaf stalks, inserting the leaves into a culture bottle containing inoculated bacterial liquid, culturing the leaves in the bacterial liquid for 24 hours (namely, inoculating for 24 hours), dumping the bacterial liquid, injecting a proper amount of sterile water into the culture bottle, placing the culture bottle containing the leaves into a 16-hour illumination system, carrying out 8-hour dark culture in a photoperiod, carrying out 20 ℃ (dark culture) and 26 ℃ (illumination) in a temperature change period, continuing to carry out moisture preservation culture under the condition of 70% humidity, counting the disease grade of the leaves once every two days, and calculating the disease index. Wild type plants transformed with the recipient material were used as controls. Grading grade standard: grade 0, no disease of cotton leaves; grade 1, disease occurs in leaf areas below 25%; grade 2, disease symptoms appear in 25-50% of leaf area; grade 3, 50-75% of leaf area shows diseases; grade 4, more than 75% of the leaf area presents with symptoms. The calculation formula of the disease index is as follows:

disease index (Σ [ (number of disease stages × number of plants)/(4 × total number of inoculated plants) × 100.

3. ZmPep1 gene for improving verticillium wilt resistance of cotton

The verticillium wilt resistance identification is carried out on all transgenic plants according to the method, and the result shows that: disease index of the wild type cotton at 5d of inoculation was 53.43, and disease index of the transgenic lines ZmPep1-4, ZmPep1-5 and ZmPep1-7 were 23.61, 7.92 and 14.96, respectively. The T-test results showed that the disease index of the transgenic cotton lines was very significantly lower than that of wild-type cotton (a in fig. 7). After 5 days of inoculation, the wild-type cotton leaves showed severe disease, while the leaves of the transgenic cotton lines showed only a few seed lesions (B in FIG. 7). The results show that: ZmPep1 can significantly improve the resistance of cotton to verticillium wilt.

The above-mentioned embodiments are merely preferred embodiments for fully illustrating the present invention, and the scope of the present invention is not limited thereto. The equivalent substitution or change made by the technical personnel in the technical field on the basis of the invention is all within the protection scope of the invention. The protection scope of the invention is subject to the claims.

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Claims (8)

1. Corn elicitor peptide geneZmPep1Use in increasing the resistance of plants to verticillium wilt, characterized in that: the maize inducer peptide geneZmPep1The nucleotide sequence of (A) is shown as SEQ ID NO. 1; the plant is Arabidopsis thaliana, tobacco or cotton.

2. Utilization of maize elicitor peptide genesZmPep1A method of increasing resistance of a plant to verticillium wilt, characterized by: the corn elicitor peptide geneZmPep1Constitutive expression in plants to obtain plants resistant to verticillium wilt;

the corn elicitor peptide geneZmPep1The nucleotide sequence of (A) is shown in SEQ ID NO. 1.

3. The method of claim 2, wherein: the corn elicitor peptide geneZmPep1The constitutive expression method in plant is to construct the gene containing corn elicitor peptideZmPep1And then obtaining the transgenic plant through agrobacterium mediation.

4. The method of claim 3, wherein: the constitutive plant expression vector contains corn inducer peptide gene regulated by constitutive promoterZmPep1Expression cassette for expression。

5. The method of claim 4, wherein: the constitutive promoter is CaMV35S promoter.

6. The method of claim 4, wherein: the constitutive plant expression vector is formed by passing a sequence shown as SEQ ID NO.1BamHI andKpni double restriction enzyme construction to plant TableObtaining a 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 preparing transgenic plants with verticillium wilt resistance is characterized in that: the plant expression vector contains a constitutive promoter to regulate and control corn inducer peptide geneZmPep1Expression cassette for expression of said maize inducer peptide geneZmPep1The nucleotide sequence of (A) is shown in SEQ ID NO. 1.

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