WO2022073260A1

WO2022073260A1 - Application of tomato hydroxyproline-rich systemin precursor protein gene slhypsys in improving resistance of plants to verticillium wilt

Info

Publication number: WO2022073260A1
Application number: PCT/CN2020/122401
Authority: WO
Inventors: 李先碧; 裴炎; 金丹; 范艳华; 于晓涵; 侯磊; 赵娟; 李美华; 郑雪丽; 唐梦
Original assignee: 西南大学
Priority date: 2020-10-09
Filing date: 2020-10-21
Publication date: 2022-04-14
Also published as: CN112159816B; CN112159816A

Abstract

Provided is an application of the tomato hydroxyproline-rich systemin precursor protein gene SlHypSys in improving the resistance of plants to Verticillium wilt. By means of cloning the tomato hydroxyproline-rich phylogenetic precursor protein gene, SlHypSys, and using molecular biology methods to construct a plant expression vector for constitutive expression of SlHypSys, then using genetic engineering methods to introduce the SlHypSys gene into a plant, transgenic Arabidopsis thaliana, tobacco, and cotton strains having normal transcriptional expression are obtained; the disease indices of all of the obtained transgenic plants are significantly lower than those of the wild-type control, resistance to Verticillium wilt is significantly higher, clearly indicating that the SlHypSys gene can be used for improving the resistance of plants to Verticillium wilt.

Description

Application of tomato hydroxyproline-rich systemin precursor protein gene SlHypSys in improving plant resistance to Verticillium wilt

technical field

The invention relates to the field of plant biotechnology, in particular to an application of tomato-rich hydroxyproline systemin precursor protein gene SlHypSys in improving plant resistance to Verticillium wilt.

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 (Pegg GF, 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 the defensin genes RsAFP2 and DmAMP from different sources 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 for insect resistance have attracted the attention of researchers, especially the role of plant defense signal peptides in regulating plant immunity to insects and pathogens (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 with N-terminal secretion signals. Such peptides include the hydroxyproline-rich systemin HypSys from plants such as tobacco, tomato, and petunia. Tomato hydroxyproline-rich systemin (SlHypSys) contains three hydroxyproline-rich glycopeptides, SlHypSys I, II, and III, with lengths of 20, 18, and 15 amino acids, respectively. These three mature peptides It comes from the same precursor protein, has the same function, and acts as a defense signal. SlproHypSys is synthesized in the vascular parenchyma cells of leaves and localized in the cell wall matrix when tomato plants are injured, induced by systemin and methyl jasmonate (Javier, 2005).

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, the object of the present invention is to provide a kind of tomato rich in hydroxyproline systemin precursor protein gene SlHypSys in improving the application of plants to Verticillium wilt resistance, utilize the accumulation of SlHypSys and the infection of Verticillium wilt bacteria In terms of spatial overlap, the signal pathway activated by SlHypSys and the signal pathway of Verticillium wilt defense overlap these two characteristics, the strategy of the present invention is proposed to use SlHypSys gene to improve the resistance of plants to Verticillium wilt.

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

Application of tomato-rich systemin precursor protein gene SlHypSys in improving plant resistance to Verticillium wilt, the nucleotide sequence of the tomato-rich systemin precursor protein gene SlHypSys as SEQ The nucleotide sequence shown in ID NO.1, or the nucleotide shown in SEQ ID NO.1, has the same function through the 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.

The second object of the present invention is to provide a method for improving plant resistance to Verticillium wilt by utilizing the tomato hydroxyproline-rich systemin precursor protein gene SlHypSys.

Using the method for improving the resistance of plants to verticillium wilt with the tomato systemin-rich systemin precursor protein gene SlHypSys, the tomato systemin-hydroxyproline-rich precursor protein gene SlHypSys was constitutively expressed in plants to obtain resistance to verticillium wilt. Verticillium wilt resistant plants;

The nucleotide sequence of the tomato-rich systemin precursor protein gene SlHypSys is as shown in SEQ ID NO.1, or the nucleotide shown in SEQ ID NO.1 is substituted by one or several bases and/or deleted and/or added nucleotide sequences with the same function.

Preferably, the method for constitutively expressing the tomato systemin-rich systemin precursor protein gene SlHypSys in plants is to construct a constitutive plant expression vector containing the tomato systemin-hydroxyproline-rich precursor protein gene SlHypSys , and then obtain transgenic plants through Agrobacterium-mediated transformation.

In the present invention, the constitutive plant expression vector is the expression of the tomato hydroxyproline-rich systemin precursor protein gene SlHypSys regulated by a constitutive promoter.

In the present invention, the constitutive promoter can be any constitutive promoter that can be expressed in plants, preferably, the constitutive promoter CaMV35S promoter.

In the present invention, 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-SlHypSys.

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, olive oil, and ornamental plants such as red leaves; preferably, the plant is a Arabidopsis, tobacco or cotton.

The specific operation is as follows: by obtaining the SlHypSys gene (SEQ ID NO. 1) from tomato, and operably linking it with a promoter, a plant constitutively expressing the tomato hydroxyproline-rich systemin precursor protein gene SlHypSys is constructed Expression vector, transform the vector into a host to obtain a transformant, and then transfer the transformant into a plant (such as Arabidopsis, tobacco and cotton, etc.), and obtain a transgenic plant after resistance screening and a series of molecular identifications.

In the present invention, the acquisition of the SlHypSys gene, and the fusion of the promoter and the SlHypSys gene to construct an expression vector for constitutively expressing the SlHypSys gene are conventional methods in the field, and the vector used is the 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.

A plant expression vector with improved plant resistance to Verticillium wilt, the plant expression vector contains an expression cassette for regulating the expression of a tomato-rich hydroxyproline systemin precursor protein gene SlHypSys by a constitutive promoter, and the tomato-rich The nucleotide sequence of the hydroxyproline-containing systemin precursor protein gene SlHypSys is shown in SEQ ID NO.1, or the nucleotide shown in SEQ ID NO.1 is substituted and/or deleted by one or several bases and/or added nucleotide sequences with the same function.

Preferably in the present invention, the SlHypSys gene 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, the tomato is expressed under the action of the constitutive promoter. Hydroxyproline-rich precursor protein gene SlHypSys. Preferred plant expression vectors have the vector structure shown in Figure 1 . Among them, the tomato hydroxyproline-rich precursor protein gene SlHypSys was positively connected to the CaMV35S promoter to form a plant expression vector pLGN-35S-SlHypSys constitutively expressing the gene.

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

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 first clones the hydroxyproline-rich systemin precursor protein gene SlHypSys from tomato, adopts the method of molecular biology to construct a plant expression vector constitutively expressed by SlHypSys, and then utilizes the gene Through engineering method, SlHypSys gene was introduced into Arabidopsis, tobacco and cotton, and transgenic Arabidopsis, tobacco and cotton lines with normal transcription and expression were obtained. When the disease index of wild-type Arabidopsis was 50.69, the disease index of transgenic Arabidopsis was only 13.52. When the disease index of wild-type tobacco was 50.85, the disease index of transgenic tobacco was only 9.11; when the disease index of wild-type cotton was 53.43, the disease index of transgenic cotton was only 17.22. The disease index of the transgenic plants was significantly lower than that of the wild-type control, and the resistance to Verticillium wilt was significantly improved. The invention is of great significance for promoting the application of SlHypSys 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 is the map of the plant expression vector pLGN-35S-SlHypSys.

Figure 2 shows the transcriptional expression levels of SlHypSys genes in transgenic Arabidopsis (SlHypSys-2, SlHypSys-3, SlHypSys-6, SlHypSys-12 and SlHypSys-15: independent transformants of transgenic Arabidopsis; WT: Columbia ecotype wild-type thaliana Arabidopsis; values are the mean of three technical replicates, error bars are standard error (SD).

Fig. 3 shows that SlHypSys gene improves the resistance of Arabidopsis to Verticillium wilt (A: 14 days of Verticillium thaliana, the disease index of Arabidopsis strains; B: 14 days of inoculation with Verticillium thaliana, 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. SlHypSys-3, SlHypSys-6 and SlHypSys-15: independent transformants of transgenic Arabidopsis 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 expression level of SlHypSys gene transcription in transgenic tobacco (SlHypSys-1, SlHypSys-2, SlHypSys-3, SlHypSys-4, SlHypSys-12 and SlHypSys-15: transgenic tobacco independent transformants; WT: wild-type tobacco. Values are Mean of three technical replicates, error bars are SD of three replicates).

Figure 5 shows that SlHypSys gene improves the resistance of tobacco to Verticillium wilt (A: inoculated with Verticillium dahlia for 7 days, the disease index of tobacco lines; B: inoculated with Verticillium dahlia for 7 days, the disease of tobacco leaves; disease resistance of transgenic tobacco Sex was evaluated by disease index, values are the mean of three replicates, and error bars are SD. SlHypSys-1 and SlHypSys-4: independent transformants of transgenic tobacco; WT: wild-type tobacco. **: compared with wild-type tobacco The difference level of disease index reached extremely significant (p<0.01)).

Figure 6 is the transcriptional expression level of SlHypSys gene in transgenic cotton (SlHypSys-1, SlHypSys-2 and SlHypSys-5: transgenic cotton independent transformants; WT: transgenic recipient wild-type cotton; values are the average of three technical replicates, Error bars are standard error of triplicate).

Fig. 7 SlHypSys transgenic cotton improves the resistance to Verticillium wilt (A: disease index of transgenic cotton leaves inoculated with Verticillium wilt for 5 days; B: disease of cotton leaves after inoculated with Verticillium wilt for 5 days; disease resistance of transgenic cotton Disease index was evaluated. Data are the mean of three replicate experiments, and error bars are SD. SlHypSys-1, SlHypSys-2 and SlHypSys-5: transgenic cotton independent transformants. WT: gene recipient 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 tomato SlHypSys gene

(1) Extraction of total RNA from tomato genome and synthesis of cDNA

A 4-week-old tomato seedling was taken, and the base of the petiole was lightly clipped with tweezers to induce the expression of SlHypsys gene. After 6 hours, cut the damaged leaves and immediately freeze them in liquid nitrogen and grind them into powder. Then use Promoga's Plant Rapid RNA Extraction Kit to extract the total RNA of the leaves, and then use TaKaRa's one-strand cDNA synthesis kit to synthesize cDNA, RNA Extraction and cDNA synthesis were carried out according to the kit instructions. The detailed operation process is as follows:

Quick-freeze fresh tomato leaves 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 RNase and DNase. Immediately add 500 μL of RNA lysis solution, and pipet repeatedly with a pipette until the lysate is in the lysate. If there is no obvious lumpy 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. 12000rpm, centrifuge for 5min, take 500μL of the supernatant into a new 1.5mL nuclease-free centrifuge tube, add 250μL of absolute ethanol, immediately mix with a pipette, then transfer the mixture into an RNA adsorption column, 10000rpm , 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 the RNA 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 tomato SlHypSys gene

Find the SlHypSys 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-SlHypSys: 5'-cgcggatccgcg atgatcagcttcttcagagc-3' (SEQ ID NO.2);

Downstream primer R-SlHypSys: 5'-cggggtaccccg ttaataggaagcttgaagaggc-3' (SEQ ID NO.3);

Using the above-mentioned synthetic cDNA as a template, and SEQ ID NO.2 and SEQ ID NO.3 as primer pairs, PCR amplification was performed using PrimesSTAR MAX DNA Polymerase. Amplification procedure: pre-denaturation at 98°C for 3 min; denaturation at 98°C for 10s, annealing at 57°C for 10s, extension at 72°C for 20s, and amplification for 30 cycles.

Add 5 μL of the amplified SlHypSys fragment, add 3 μL of pTOPO vector plasmid, 1 μL of DNA ligase, and 1 μL of buffer, mix well and place at room temperature for 15 min. The reaction product was mixed with Escherichia coli DH5α competent cells and mixed, and then heat-shocked at 42°C for 1 min 30s and transferred into Escherichia coli DH5α. The transformed engineered bacteria were added to 500 μL of LB medium, cultured at 37°C at 200 rpm for 1 h, and then spread on the Mycin LB plate, cultured at 37 °C overnight, pick a single colony and inoculate it into a test tube containing about 3 mL of LB medium, shake at 37 °C and 200 rpm overnight, take an appropriate amount of bacterial liquid and send it to a sequencing company for sequencing, positive cloning projects containing the target fragment The bacteria were stored at -80°C.

Embodiment 2, the construction of the plant expression vector that constitutively expresses SlHypSys gene and the acquisition of engineering bacteria

The above-mentioned cloned vector engineering bacterial plasmids verified to be correct by sequencing were extracted, and double-enzyme digested with BamHI and KpnI. The digested products were subjected to agarose gel electrophoresis, and the target fragment SlHypSys was recovered. 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 SlHypSyss digestion fragment of the target gene was the plant expression vector pLGN-35S-SlHypSys 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 screened and cultured on YEB plates with additional Km and Sm, Km and Rif (rifampicin), and single resistant colonies were obtained. Inoculate into YEB liquid medium with additional Km and Sm, Km and Rif (rifampicin), collect the cells and extract the Agrobacterium plasmid, and then double-enzyme digestion verification with BamHI and KpnI, the correct engineering bacteria are stored in - 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) dipping transformation method, the wild-type Arabidopsis thaliana was used as the material for genetic transformation, and the seeds after the Agrobacterium dipping were harvested after the seeds were mature.

(2) Screening of transgenic Arabidopsis plants

The seeds harvested after the genetic transformation by the flower dip transformation method were sterilized with 75% alcohol for 15 minutes, and then evenly inoculated on the screening plate with additional 100 mg/L Km (kanamycin) for germination. If the grown seedling is green, it is Transgenic plants were transplanted into the special soil for culturing Arabidopsis when the plants had more than 2 leaves (grassy carbon soil: vermiculite: perlite in a ratio of 3:1:1), and seeds were harvested after maturity. 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) Transcription and expression level analysis of SlHypSys gene in transgenic Arabidopsis

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

A specific fragment of SlHypSys gene was amplified using cDNA as template. The upstream and downstream primers of the SlHypSys gene are respectively SlHypSys UP: 5'-ttaccacctccttctccc-3' (SEQ ID NO.4) and SlHypSys DN: 5'-tacataatcgtgcctccc-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 AtACT2 DN: 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: pre-denaturation at 95°C for 3 min; denaturation at 94°C for 10s, annealing at 57°C for 30s, extension at 72°C for 30s, a total of 40 cycles of amplification. After amplification, the relative expression of SlHypSys gene was analyzed by Gene Study software.

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

Example 4. Resistance of transgenic Arabidopsis to Verticillium wilt

(1) Preparation of Verticillium wilt for inoculation of transgenic Arabidopsis

Pick a little of the Verticillium decidua V991 strain (Verticillium dahliae) preserved in solid PD medium (potato medium) and inoculate it into liquid PD medium, 180rpm, 26 ℃ of shaking culture for 7d, and then press 10% (bacteria liquid) The ratio of /PD medium) was 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 was adjusted to adjust the spore concentration to 10 ⁸ / ml as the inoculum.

(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) SlHypSys gene improves the resistance of Arabidopsis to Verticillium wilt

Arabidopsis plants were inoculated with Verticillium wilt for 14 days, the disease index of wild-type plants reached 50.69, and the disease index of transgenic lines SlHypSys-3, SlHypSys-6 and SlHypSys-15 were 13.52, 21.48 and 20.99, 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 the SlHypSys 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-cultivation medium: MSB (MS inorganic salt + B5 organic) + 2 mg/L NAA + 0.5 mg/L 6-BA + 200 μmol/L AS, pH 5.6, 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-SlHypSys plant expression vector was inoculated into the 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 sterile 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 SlHypSys transgenic tobacco

a. 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, 0.1 mol/L phosphate buffer (pH 7.0).

The pLGN-35S-SlHypSys plant expression vector contains the GUS gene controlled by the CaMV35S promoter. Therefore, the transgenic plants can be rapidly identified by GUS histochemical staining. According 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.

b. SlHypSys transcriptional expression level analysis

SlHypSys transgenic tobacco plants used young leaves as materials, respectively extracted RNA from GUS-positive and wild-type plant leaves, and synthesized one-strand cDNA of each sample RNA according to the instructions of the cDNA one-strand synthesis kit (methods and implementation of RNA extraction and cDNA synthesis). Example 1 is the same). Then a specific fragment of the SlHypSys gene was amplified using the cDNA as a template. The upstream and downstream primers of the SlHypSys gene are respectively SlHypSys UP: 5'-ttaccacctccttctccc-3' (SEQ ID NO.4) and SlHypSys DN: 5'-tacataatcgtgcctccc-3' (SEQ ID NO.5). The tobacco 18S gene was used as the internal standard. The upstream and downstream primers of the 18S gene are respectively Nt18S UP: 5'-aggaattgacggaagggca-3' (SEQ ID NO.8) and Nt18S 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 ddH2O.

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

Real-time PCR results showed (Fig. 4) that the SlHypSys gene could be effectively transcribed and expressed in transgenic tobacco plants, while the expression of this gene was not detected in leaves of 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) Inoculation method for disease resistance identification of transgenic tobacco

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 leaves according to the 5-level standard of 0-4. disease grade 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) SlHypSys 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 50.74, and the disease index of transgenic lines SlHypSys-1 and SlHypSys-4 were 9.11 and 15.43, 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 SlHypSys gene could effectively improve the resistance of tobacco to Verticillium wilt.

Example 7. Genetic transformation of cotton

(1) Common medium for cotton genetic transformation

Minimal 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 with 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/L cef (cephalosporin)+30g/L glucose+2.0g/L Gelrite, pH 5.8;

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) The method of cotton genetic transformation

Obtainment of transformed explants: the seeds of 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.

Preparation of Agrobacterium dipping solution for transformation: obtain a single colony of Agrobacterium that integrates the plant expression vector pLGN-35S-SlHypSys by streaking method, then pick a single colony and inoculate it with additional 50mg/L Km and 125mg/L Sm (streptomycin). ) 10mL liquid YEB (5g/L sucrose, 1g/L bacterial yeast extract, 10g/L bacterial tryptone, 0.5g/L MgSO ₄ ·7H ₂ O, pH7.0), 28 ℃, 200rpm culture Overnight, the bacterial solution was inoculated into 20 mL of liquid YEB without antibiotics at a ratio of 5%, 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. Culture for 2 days, inoculate the hypocotyls into the screening degerming medium, and substitute them into the callus induction medium supplemented with kanamycin (Km) and cephalosporin (cef) after 20 days to induce callus, and subculture once every 20 days. After 60 days, the cells were subcultured into the embryogenic callus induction medium, and after the embryogenic callus was obtained, 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 SlHypSys transgenic cotton

According to the cotton genetic transformation and regeneration method in Example 7, the SlHypSys gene was transferred into the cotton genome. After induction of Km-resistant callus, embryogenic callus and somatic embryos, the somatic embryos became seedlings, and then SlHypSys 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.

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) Transcriptional expression level analysis of SlHypSys gene

SlHypSys transgenic cotton plants used young leaves as materials, respectively, extracted RNA from GUS-positive and wild-type plant leaves, and synthesized one-strand cDNA of each sample RNA according to the instructions of the cDNA one-strand synthesis kit (RNA extraction and cDNA synthesis methods were the same as the embodiment). 1), and then use cDNA as a template to amplify a specific fragment of the SlHypSys gene. The upstream and downstream primers of the SlHypSys gene are respectively SlHypSys UP: 5'-TTACCACCTCCTTCTCCC-3' (SEQ ID NO.4) and SlHypSys DN: 5'-TACATAATCGTGCCTCCC-3' (SEQ ID NO.5). The cotton histone GhHIS3 gene was used as the internal standard. The upstream and downstream primers of the GhHIS3 gene were GhHIS3 UP: 5'-GAAGCCTCATCGATACCGTC-3' (SEQ ID NO. 10) and GhHIS3 DN: 5'-CTACCACTACCATCATGGC-3' (SEQ ID NO. 11) (Zhu YQ et al., 2003).

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 SlHypSys gene was analyzed by Gene Study software.

The Real-time PCR results showed (Figure 6) that the SlHypSys 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 SlHypSys transgenic cotton to Verticillium wilt

(1) Preparation of pathogenic bacteria for inoculation for disease resistance identification

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

(2) In vitro leaf inoculation method

Cut the young leaves of the transgenic cotton 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 grading 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:

(3) SlHypSys gene improves cotton resistance to Verticillium wilt

Verticillium wilt resistance identification of all transgenic plants was carried out according to the above method. The results showed that the disease index of wild-type cotton was 65.83 after inoculation for 5 days, and the disease index of transgenic lines SlHypSys-1, SlHypSys-2 and SlHypSys-5 were 23.61 respectively. , 18.65 and 16.56. 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). Five days after inoculation, the leaves of the 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 SlHypSys could significantly improve the resistance of cotton to Verticillium wilt.

The above experiments prove that the present invention introduces the tomato-rich systemin precursor protein gene SlHypSys into wild-type Arabidopsis thaliana, tobacco and cotton by transgenic means to obtain transgenic Arabidopsis thaliana, tobacco and cotton, and its transgenic plants The disease index after inoculation with Verticillium wilt was significantly lower than that of the wild-type control, thus proving that the gene can improve the resistance of Arabidopsis, tobacco and 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.

Claims

Application of tomato-rich systemin precursor protein gene SlHypSys in improving plant resistance to Verticillium wilt, characterized in that: the tomato-rich systemin precursor protein gene SlHypSys is rich in nucleosides The acid 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.
The application according to claim 1, wherein the plant is Arabidopsis, tobacco or cotton.
The method for improving the resistance of plants to Verticillium wilt by utilizing the tomato-rich systemin precursor protein gene SlHypSys, which is characterized in that: constituting the tomato systemin-rich systemin precursor protein gene SlHypSys in plants Expression to obtain plants resistant to Verticillium wilt;

The nucleotide sequence of the tomato-rich systemin precursor protein gene SlHypSys is as shown in SEQ ID NO.1, or the nucleotide shown in SEQ ID NO.1 is substituted by one or several bases and/or deleted and/or added nucleotide sequences with the same function.
The method according to claim 3, characterized in that: the method for constitutively expressing the tomato systolic-hydroxyproline-rich precursor protein gene SlHypSys in plants is to construct a tomato systolic-hydroxyproline-enriched precursor protein gene SlHypSys Constitutive plant expression vector for the somatic protein gene SlHypSys, and then mediated by Agrobacterium to obtain transgenic plants.
The method according to claim 4, wherein the constitutive plant expression vector is the expression of the tomato hydroxyproline-rich systemin precursor protein gene SlHypSys regulated by a constitutive promoter.
The method according to claim 5, characterized in that: the constitutive promoter CaMV35S promoter.
The method according to claim 5, wherein the constitutive plant expression vector is obtained by passing the sequence shown in SEQ ID NO.1 through the BamHI and KpnI plant expression vector pLGN to obtain the plant expression vector pLGN-35S-SlHypSys.
The method according to any one of claims 3 to 7, wherein the plant is Arabidopsis, tobacco or cotton.
A plant expression vector capable of improving the resistance of plants to Verticillium wilt, characterized in that: the plant expression vector contains an expression cassette for regulating the expression of a tomato-rich systemin precursor protein gene SlHypSys by a constitutive promoter, The nucleotide sequence of the tomato-rich systemin precursor protein gene SlHypSys is as shown in SEQ ID NO.1, or the nucleotide shown in SEQ ID NO.1 is substituted by one or several bases and/or deleted and/or added nucleotide sequences with the same function.
The application of the plant expression vector of claim 9 in the preparation of transgenic plants with Verticillium wilt resistance.