Application of tomato transcription factor LeMYB330 gene in improvement of plant broad-spectrum disease resistance

Abstract

The invention discloses application of a tomato transcription factor LeMYB330 gene in improvement of plant broad-spectrum disease resistance, and relates to the technical field of biology. The research finds that the expression quantity of the LeMYB330 gene in a LeMYB330 gene overexpression plant is increased, and the broad-spectrum disease resistance is enhanced; when the plant is infected by the elephant bean root-knot nematode and the meloidogyne javanica, the root knot number and the oocyst number of the plant are reduced, and the root knot index and the oocyst index are reduced; the disease index of bacterial wilt is reduced; the root-knot nematode resistance of plants is enhanced; and the method can be used for breeding tomato varieties capable of resisting the phagostimulant bean root-knot nematode, the meloidogyne javanica and the bacterial wilt.

Description

TECHNICAL FIELD

[0001] The application relates to the field of biotechnology, and particularly relates to application of a tomato transcription factor LeMYB330 gene in improving plant broad-spectrum disease resistance. BACKGROUND

[0002] In recent years, with the proposal of the strategy of high-quality economic development, China has paid extensive attention to the high-quality development of modern agriculture. Greenhouse facilities are currently widely used in agricultural development in China, and the planting area of various fruits and vegetables including tomatoes is gradually expanding. This approach can increase agricultural investment and production, but at the same time, continuous cropping, multiple cropping, and heavy cropping problems have gradually emerged. In addition, the suitable temperature and humidity environment in the protected field provides good breeding conditions for various root-knot nematodes (Meloidogyne spp.) and Ralstonia solanacearum, aggravating the occurrence and development of diseases (Sun et al., 2023).

[0003] Root-knot nematodes can parasitize more than 5,000 species of plants belonging to 114 families, including vegetables, fruit trees, cereals, and ornamental plants, among which the damage to Cucurbitaceae, Solanaceae, and Brassicaceae crops is the most serious (Ye et al., 2021). Root-knot nematodes not only destroy the root tissue and even the entire metabolic system of the host, but also exacerbate the composite infection of other pathogenic bacteria, inducing the occurrence of soil-borne diseases such as bacterial wilt. Both M. avanica and M. enterolobii have the characteristics of wide host range and serious damage, and are the main pathogens causing plant root-knot nematode diseases. Compared with other species of root-knot nematodes, M. enterolobii not only has a higher infection rate of host plant roots and induces more severe galls, but also overcomes the resistance of tomatoes carrying the Mi-1 gene and peppers carrying the N gene, and is one of the most serious root-knot nematodes recognized worldwide (Xu et al., 2023). Tomato bacterial wilt is a devastating soil-borne bacterial disease caused by Ralstonia solanacearum, which seriously restricts the development of the tomato industry (Hayward, 1991). Ralstonia solanacearum can infect the host through the gap or wound of the plant root tip, enter the tomato cells and tissues, and multiply and spread to the whole tomato plant in the vascular bundle, hindering water transport and causing the plant to wilt and die. The composite infection of root-knot nematodes and Ralstonia solanacearum aggravates the damage of the disease and makes it more difficult to control. Currently, there is no report on the creation of a tomato that is resistant to both root-knot nematodes and Ralstonia solanacearum.

[0004] Tomato (Solanum lycopersicum), also known as Lycopersicon esculentum, is a popular vegetable in China due to its delicious taste, high nutritional value, and good source of several important nutrients in human diet. According to statistics, about one-third of the world's tomato production comes from China. In addition, tomato has been used as a model plant for fruit ripening, disease response, genetics, and whole genome sequence studies for a long time. It is also an important basis for biological research and genetic improvement of all solanaceous crops such as potato, pepper, and eggplant. Therefore, tomato is one of the most widely grown vegetables in China, but various diseases and pests have a serious negative impact on its growth, quality, and yield (Shu et al., 2023).

[0005] Breeding of disease-resistant varieties can reduce the damage of root-knot nematode, Java root-knot nematode, and bacterial wilt from the source, which is one of the most economical and effective methods for disease control. Currently, only the Mi gene has been successfully used in tomato nematode resistance breeding, but the cowpea root-knot nematode can overcome the Mi resistance gene (reference). Therefore, it is urgent to explore new gene resources of host crops resistant to root-knot nematode to provide alternative strategies for the prevention and control of root-knot nematode disease. Tomato varieties with resistance to bacterial wilt such as "Huangshan 1" and "Huangshan 2" (Wu et al., 2009), tomato rootstock new variety "Chengta 1" (Su et al., 2023) can effectively reduce the occurrence of tomato bacterial wilt, but many resistant varieties are still in the laboratory screening stage (Phiri et al., 2024).

[0006] MYB transcription factors are one of the largest transcription factor families in the plant kingdom, and their members play important roles in regulating plant growth and development, secondary metabolite metabolism, biological and abiotic stress response, and other physiological processes. According to the PlantTFDB database, there are 127 MYB transcription factor members in tomato, which are divided into 18 subgroups according to domain similarity and phylogenetic topology. Currently, there are more and more reports on the expression characteristics of MYB transcription factors in tomato, but the role of MYB transcription factors in plant broad-spectrum disease resistance is still unclear (Wong et al., 2022).

[0007] Therefore, the present application is proposed. SUMMARY

[0008] In order to overcome the shortcomings and deficiencies of the prior art, the purpose of the present application is to provide an application of tomato transcription factor LeMYB330 gene in improving plant broad-spectrum disease resistance.

[0009] The application discloses a tomato transcription factor LeMYB330, overexpression of the gene can simultaneously improve the broad-spectrum resistance of tomato plants to C. cajani, C. javanica and P. solanacearum. Accordingly, the application provides application of a tomato transcription factor LeMYB330 gene in improving the broad-spectrum resistance of plants.

[0010] The application achieves the purpose by the following technical scheme:

[0011] Application of the tomato transcription factor LeMYB330 gene or related biological materials in improving the broad-spectrum resistance of plants.

[0012] Overexpression of the tomato transcription factor LeMYB330 gene improves the broad-spectrum resistance of plants.

[0013] Specifically, the method comprises the following steps:

[0014] (a) constructing Agrobacterium tumefaciens engineering bacteria containing a tomato LeMYB330 gene overexpression vector;

[0015] (b) transforming plant explants with the Agrobacterium tumefaciens engineering bacteria constructed in step (a) to obtain LeMYB330 gene overexpression plants;

[0016] (c) inoculating the LeMYB330 gene overexpression plants obtained in step (b) with C. cajani, C. javanica and / or P. solanacearum, and statistically analyzing the plant disease incidence.

[0017] Preferably, in step (c), the inoculation of C. cajani, C. javanica and / or P. solanacearum is specifically as follows: when the tomato LeMYB330 gene overexpression plants grow to four-leaf one-heart, C. cajani, C. javanica and / or P. solanacearum are inoculated.

[0018] The inoculated C. cajani and C. javanica are C. cajani and C. javanica in J2 stage and having an invasion activity.

[0019] The inoculation of C. cajani and C. javanica is performed for 5 weeks.

[0020] The inoculation of P. solanacearum is specifically performed by using a root injury inoculation method.

[0021] The inoculated P. solanacearum is a P. solanacearum bacterial suspension.

[0022] The inoculation of P. solanacearum is performed for 15 days.

[0023] Further, the application of the tomato transcription factor LeMYB330 gene or related biological materials in cultivating broad-spectrum resistance plants.

[0024] A method for cultivating a broad-spectrum resistant plant, comprising the following steps: overexpressing a tomato transcription factor LeMYB330 gene in a plant to obtain a broad-spectrum resistant plant.

[0025] Further, the plant includes a Solanaceae plant, but is not limited thereto.

[0026] Still further, the plant is a tomato.

[0027] Further, the broad-spectrum resistance includes at least one of Meloidogyne exigua resistance, Meloidogyne javanica resistance, and Pseudomonas syringae resistance.

[0028] The nucleotide sequence of the tomato transcription factor LeMYB330 gene is shown in SEQ ID NO: 1, and the encoded amino acid sequence is shown in SEQ ID NO: 2.

[0029] The related biological material is any one or a combination of the following biological materials:

[0030] (1) an expression cassette containing the tomato transcription factor LeMYB330 gene;

[0031] (2) a recombinant overexpression vector containing the tomato transcription factor LeMYB330 gene;

[0032] (3) a recombinant overexpression vector containing the expression cassette in (1).

[0033] (4) a recombinant microorganism containing the expression cassette in (1).

[0034] (5) a recombinant microorganism containing the recombinant overexpression vector in (2) or (3).

[0035] Further, the starting vector of the recombinant overexpression vector in (2) and (3) is a plant expression vector, including but not limited to plant binary expression vectors pCAMBIA series, pBI series, pFGC5941, etc.; still further, it is pCAMBIA1304.

[0036] Further, the host microorganism corresponding to the recombinant microorganism in (4) and (5) is selected from prokaryotes, etc.; the prokaryotes include bacteria of the genus Agrobacterium. More specifically, the prokaryote is Agrobacterium tumefaciens, and specifically can be Agrobacterium tumefaciens GV3101.

[0037] The expression amount of LeMYB330 gene in the LeMYB330 gene overexpression plant is increased, and the broad-spectrum disease resistance is enhanced. When the plants are infected by Meloidogyne exigua and Meloidogyne javanica, the number of root nodules and the number of egg capsules are reduced, the root nodule index and the egg capsule index are reduced, and the disease index of bacterial wilt is reduced; the plant resistance to root-knot nematode is enhanced.

[0038] The method for cultivating the transgenic tomato plant provided by the application can be specifically divided as follows:

[0039] The LeMYB330 transgenic tomato plant with the enhanced broad-spectrum resistance is cultivated, and the method comprises the following steps:

[0040] a) introducing the overexpression vector of LeMYB330 gene into the target plant to perform overexpression, so as to obtain the transgenic plant with overexpression of LeMYB330 gene;

[0041] b) inoculating the transgenic tomato plant with overexpression of LeMYB330 gene and the wild-type tomato plant respectively, so as to obtain the transgenic tomato plant with the enhanced resistance and the traits of the reduced number of root nodules and egg capsules, the reduced root nodule index and egg capsule index, and the reduced disease index of bacterial wilt.

[0042] In the above method, the LeMYB330 gene can be introduced into the target plant through a recombinant expression vector containing the gene, and the existing pFGC5941, pCAMBIA1300 and pBI121 or other derived plant expression vectors can be used.

[0043] The application has the following advantages and effects relative to the prior art:

[0044] The research of the application shows that the overexpression of LeMYB330 gene can simultaneously enhance the resistance of tomato plants to Meloidogyne exigua, Meloidogyne javanica and bacterial wilt, and can be used for the breeding of tomato varieties resistant to Meloidogyne exigua, Meloidogyne javanica and bacterial wilt. BRIEF DESCRIPTION OF DRAWINGS

[0045] Figure 1 A pCAMBIA1304-LeMYB330-HA overexpression vector is constructed; wherein A is a simple schematic diagram of the pCAMBIA1304-LeMYB330-HA overexpression vector; B is a linear gel electrophoresis diagram of LeMYB330-HA PCR amplification product and pCAMBIA1304 vector; lanes 1-2: LeMYB330-HA gene fragment amplification, lane 3: pCAMBIA1304 plasmid Spe I and Nco I double enzyme digestion, 4: uncut circular pCAMBIA1304 plasmid, M: D2000 DNA Marker.

[0046] Figure 2 Verification of LeMYB330 overexpression transgenic positive plants; wherein, A is PCR detection of LeMYB330 overexpression transgenic positive plants LeMYB330 gene, lane 1: pCAMBIA1304-LeMYB330-HA recombinant plasmid positive control; lanes 2-7: OE2, OE3, OE7, OE8, OE9, OE11 in turn; B is PCR detection of LeMYB330 overexpression transgenic positive plants HPT gene, lane 8: wild type tomato genomic DNA negative control; lanes 9-14: OE2, OE3, OE7, OE8, OE9, OE11 in turn; C is real-time fluorescent quantitative PCR detection of overexpression transgenic positive plants; CK is wild type tomato "Xinjinfeng No. 1" without transgene; OE is LeMYB330 overexpression plant; OE2, OE3, OE7, OE8, OE9, OE11 are six strains of LeMYB330 overexpression plants; compared with CK, *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001.

[0047] Figure 3 Root knot nematode phenotype observation results of tomato LeMYB330 overexpression plants inoculated with P. caricae 5 weeks; wherein, A is the representative disease characteristics of the roots of LeMYB330 overexpression plants and wild type; B is the statistical results of the disease condition of A figure. OE is LeMYB330 overexpression plant, CK + Wild type WT plant treated with nematode inoculation; CK - Wild type WT plant without nematode inoculation. Compared with CK + , *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001.

[0048] Figure 4 Root knot nematode phenotype observation results of tomato LeMYB330 overexpression plants inoculated with M. javanica 5 weeks, wherein, A is the representative disease characteristics of the roots of LeMYB330 overexpression plants and wild type; B is the statistical results of the disease condition of A figure. OE is LeMYB330 overexpression plant, CK + Wild type WT plant treated with nematode inoculation; CK - Wild type WT plant without nematode inoculation. Compared with CK + , *P<0.05.

[0049] Figure 5Phenotype observation results of Ralstonia solanacearum inoculation on tomato LeMYB330 overexpression plants; wherein, A is the disease characteristics of LeMYB330 overexpression plants and wild type; B is the disease statistics results of A figure. OE is LeMYB330 overexpression plants, CK + is the wild type WT plant inoculated with Ralstonia solanacearum. Compared with CK + Compared with CK, *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001. DETAILED DESCRIPTION

[0050] The application will be further described in conjunction with the examples and the accompanying drawings, but the embodiments of the application are not limited thereto. It should be understood that these examples are only used to illustrate the application and not used to limit the scope of the application. The operation methods not specified in the following examples are usually carried out according to the conventional conditions, or according to the conditions suggested by the manufacturers. The experimental materials, reagents and the like used in the following examples can be obtained from commercial channels unless otherwise specified.

[0051] In the examples, the observation method of the number of root nodules is as follows:

[0052] Acid fuchsin staining method is used for root nodule nematode infection root phenotype observation. The specific method is as follows:

[0053] (1) 3.5g acid fuchsin is dissolved in 250mL glacial acetic acid, and is fully dissolved to obtain acid fuchsin mother liquor;

[0054] (2) 2000mL ddH2O is added with 40mL acid fuchsin mother liquor;

[0055] (3) The root tissue is placed in a 100mL beaker, and 50mL of prepared acid fuchsin dyeing solution is added;

[0056] (4) The root tissue is heated using a microwave oven. During the heating process, attention should be paid to not let the root tissue pop out;

[0057] (5) The root tissue is taken out and observed under a stereoscope. The ddH2O is used for decolorization treatment, and the data is recorded. In the examples, the steps of culturing and inoculating C. cajan and M. javanica are as follows:

[0058] 1) C. cajan and M. javanica are preserved by the Nematode Research Room of South China Agricultural University. C. cajan and M. javanica infect and culture water spinach for about 2 months to form visible egg capsules in the root system, which can be used for experimental research.

[0059] 2) After washing the root of the water spinach with tap water, immerse the root in 0.5% sodium hypochlorite solution for 5 minutes, and then rinse with distilled water until the smell of sodium hypochlorite is not detected.

[0060] 3) Place the root tissue under a dissecting microscope, and gently pick up the egg mass with a pair of tweezers and place it in a small beaker containing ddH2O.

[0061] 4) Transfer the collected egg mass into a sterile petri dish lined with sterile absorbent paper using a pipette, and place the petri dish in a 28°C incubator. During the incubation period, make sure that the absorbent paper is kept moist and that the petri dish contains an appropriate amount of water to facilitate the hatching of the eggs.

[0062] 5) After 2-3 days, rinse the hatched Meloidogyne javanica and Pratylenchus vulnus J2 larvae from the top of the absorbent paper with distilled water and transfer them to the petri dish. The J2 stage nematodes obtained should be used as soon as possible within 24 hours to avoid affecting the activity of the worms.

[0063] 6) Inoculate the LeMYB330 gene overexpression plants when they have four leaves and one heart, with about 1000 J2 stage Meloidogyne javanica and Pratylenchus vulnus per plant. Water the plants lightly two days before inoculation, and then water them normally during the observation period.

[0064] Example 1

[0065] Cloning of the tomato LeMYB330 gene and construction of the vector

[0066] 1. Extraction of total RNA from tomato plants

[0067] Extract total RNA from the roots of tomato plants according to the instructions of the RNAprep pure Plant Kit (Cat. No. DP432) total RNA extraction kit for plants. The specific steps are as follows:

[0068] (1) Weigh 200 mg of root tissue from tomato seedlings at the 5-6 leaf stage, and quickly grind it into powder in liquid nitrogen. Add 450 μL of RL (1 mL of RL contains 20 μL of β-mercaptoethanol), and incubate at 56°C for 1-3 min.

[0069] (2) Transfer all the solutions to the filter column CS (which is placed in a collection tube), and centrifuge at 12000 rpm for 2-5 min. Carefully pipette the supernatant in the collection tube into an RNase-Free centrifuge tube, and make sure that the pipette tip does not touch the cell debris precipitate in the collection tube.

[0070] (3) Slowly add 0.5 times supernatant volume of absolute ethanol (usually 225 μL), mix well, and transfer the resulting solution and pellet into the adsorption column CR3, centrifuge at 12000 rpm for 30-60 sec, discard the waste in the collection tube, and place the adsorption column CR3 back into the collection tube;

[0071] (4) Add 350 μL of Deproteinization Solution RW1 to the adsorption column CR3, centrifuge at 12000 rpm for 30-60 sec, discard the waste in the collection tube, and place the adsorption column CR3 back into the collection tube;

[0072] (5) Preparation of DNase I Working Solution: Take 10 μL of DNase I Stock Solution and place it into a new RNase-Free centrifuge tube, add 70 μL of RDD Solution, and mix gently;

[0073] (6) Add 80 μL of DNase I Working Solution to the center of the adsorption column CR3, and let it stand at room temperature for 15 min;

[0074] (7) Add 350 μL of Deproteinization Solution RW1 to the adsorption column CR3, centrifuge at 12000 rpm for 30-60 sec, discard the waste in the collection tube, and place the adsorption column CR3 back into the collection tube;

[0075] (8) Add 500 μL of Rinse Solution RW to the adsorption column CR3, let it stand at room temperature for 2 min, centrifuge at 12000 rpm for 30-60 sec, discard the waste in the collection tube, and place the adsorption column CR3 back into the collection tube;

[0076] (9) Repeat step (8);

[0077] (10) Centrifuge at 12000 rpm for 2 min, discard the waste. Place the adsorption column CR3 at room temperature for several minutes to completely dry the residual rinse solution in the adsorption material;

[0078] (11) Place the adsorption column CR3 into a new RNase-Free centrifuge tube, and add 30-100 μL of RNase-Free ddH2O dropwise to the middle of the adsorption membrane, let it stand at room temperature for 2 min, centrifuge at 12000 rpm for 2 min, and obtain the RNA solution.

[0079] (12) Take 1 μL of the RNA solution for electrophoresis to detect integrity, and take another 1 μL for a nucleic acid concentration detector to measure the optical density at 260 nm and 280 nm wavelengths, and preliminarily estimate the purity and concentration of the RNA. The qualified RNA solution is immediately used or stored at -80°C for standby.

[0080] 2. Gene cloning and construction of Agrobacterium tumefaciens engineering bacteria

[0081] The total RNA of tomato was rapidly reverse transcribed into cDNA according to the Transcriptor First Strand cDNA Synthesis Kit (Cat. No. 04897030001) instruction manual of Roche. The LeMYB330 gene was amplified by PCR using the cDNA as a template and the primer pair MYB330-NcoI-F / MYB330-HA-SpeI-R, and the ORF sequence of the LeMYB330 gene of tomato carrying an HA tag sequence at the 3' end was obtained, which was denoted as LeMYB330-HA. The plant transformation vector pCAMBIA1304 was linearized by enzyme digestion with Nco I and Spe I restriction endonucleases, and then homologous recombination of the fragment and the vector was performed to construct the overexpression vector of the LeMYB330 gene of tomato, which was denoted as pCAMBIA1304-LeMYB330-HA. Figure 1 The overexpression vector of the LeMYB330 gene of tomato was constructed. Wherein, Figure 1 A is a simple schematic diagram of the pCAMBIA1304-LeMYB330-HA overexpression vector; Figure 1 B is a gel electrophoresis diagram of the LeMYB330-HA PCR amplification product and the linearized pCAMBIA1304 vector.

[0082] The sequencing was performed by Beijing Huada Genetech Co., Ltd., the sequencing result is shown as SEQ ID NO: 1, and the encoded protein sequence is shown as SEQ ID NO: 2. The results show that the cloned sequence is consistent with the sequence (XP_004241841.1) published in NCBI.

[0083] The obtained overexpression vector pCAMBIA1304-LeMYB330-HA was transformed into Agrobacterium tumefaciens GV3101 to obtain the Agrobacterium tumefaciens engineering bacteria A containing the overexpression vector of the LeMYB330 gene of tomato.

[0084] The primer sequences used are as follows:

[0085] MYB330-NcoI-F: 5'- CCATGGATGGGACGTTC ACCTTGTTGTG-3'; ACGGGGGACTCTTGACC MYB330-HA-SpeI-R: 5'- CTAGTTCAagcgtaatctggaa catcgtatgggtaGCAATATCTGTAAAATCCATTTG-3';

[0086] MYB330-HA-SpeI-R: 5'- CTAGTTCAagcgtaatctggaa catcgtatgggtaGCAATATCTGTAAAATCCATTTG-3'; AAGTTCTTCTCCTTTA MYB330-HA-SpeI-R: 5'- CTAGTTCAagcgtaatctggaa catcgtatgggtaGCAATATCTGTAAAATCCATTTG-3';

[0087] Note: The underlined sequence is the homologous sequence of the linearized vector pCAMBIA1304 plasmid, CCATGG and ACTAGT are Nco I and Spe I enzyme cutting sites, and the lower case sequence is the HA tag sequence.

[0088] Example 2

[0089] Construction of tomato LeMYB330 gene overexpression plants

[0090] Through the method of Agrobacterium-mediated genetic transformation, the tomato cotyledon was infected, the target vector pCAMBIA1304-LeMYB330-HA was transformed into the tomato cotyledon, and the transgenic plants obtained were screened by PCR amplification method using LeMYB330 primer pairs and hygromycin primer pairs.

[0091] The specific steps are as follows:

[0092] 1) Preparation of culture medium

[0093] Pre-culture medium: 4.43g / L MS powder + 30g / L sucrose + 8g / L agar powder + 0.2mg / L IAA + 2g / L 6-BA, pH 5.8.121℃, 0.11MPa sterilization for 20min.

[0094] Co-culture medium: 4.43g / L MS powder + 30g / L sucrose + 8g / L agar powder + 0.2mg / L IAA + 2g / L 6-BA + 200μmol / L + 200mg / L timentin, pH 5.8.121℃, 0.11MPa sterilization for 20min.

[0095] Induction and screening medium: 4.43g / L MS powder + 30g / L sucrose + 8g / L agar powder + 0.2mg / L IAA + 2g / L 6-BA + 200μmol / L + 200mg / L timentin + 5mg / L hygromycin, pH 5.8.121℃, 0.11MPa sterilization for 20min.

[0096] Rooting medium: 4.43g / L MS powder + 30g / L sucrose + 8g / L agar powder + 0.2mg / L IAA + 200mg / L timentin + 5mg / L hygromycin, pH 5.8.121℃, 0.11MPa sterilization for 20min.

[0097] 2) Culture of sterile seedlings

[0098] Tomato seed sterilization: Select healthy and vigorous tomato seeds, soak in ddH2O at room temperature for 5-10 min to fully wet the seeds; 75% alcohol sterilization for 30 s, with slight shaking during the period, then rinse with sterile ddH2O for 3-5 times; finally, immerse the tomato seeds in 10% sodium hypochlorite solution for 15 min, with constant shaking, and rinse with sterile ddH2O for 3-5 times. The sterilized tomato seeds are poured into a 50 ml sterile centrifuge tube, washed with sterile water for 3 times to wash off the residual sodium hypochlorite solution on the surface of the seeds.

[0099] Tomato seed germination: pour the surface sterilized seeds on sterile filter paper, dry the excess water on the surface of the seeds with a pipette, then transfer them to the prepared MS medium with tweezers, evenly sow about 30 seeds per bottle of medium. Incubate in a light incubator at 26°C in the dark for 3 days, then continue to cultivate in the light for 3-5 days (light 16 h, dark 8 h: light 26°C, dark 18°C, humidity 65%) after the radicle of the seeds grows out.

[0100] 3) Preparation of explants and Agrobacterium culture

[0101] Cotyledon explants are cut when the cotyledons are fully expanded and the true leaves have not yet emerged: use a sterile tweezers to remove the sterile seedlings from the medium and place them on sterile filter paper; use sterile surgical scissors to cut the cotyledon part and remove the ends of the cotyledon; cut the remaining cotyledon part in half and cut into explant fragments about 0.5 cm long; evenly place the cut cotyledon explant fragments on the pre-culture medium, with the back of the cotyledon facing down and tightly adhering to the medium; pre-culture in a light incubator at 25°C in the dark for 2-3 days.

[0102] On the same day of tomato cotyledon explant pre-culture, take the Agrobacterium tumefaciens engineering bacteria A containing the tomato LeMYB330 gene overexpression vector prepared in Example 1 to activate on the LB solid medium plate (50 μg / mL kanamycin + 50 μg / mL rifampicin), and incubate in a constant temperature incubator at 28°C for 2 days; pick a single colony into 1 mL of LB liquid medium (50 μg / mL kanamycin + 50 μg / mL rifampicin) and incubate at 28°C with 200 r / min shaking for 2 days to obtain seed liquid and perform PCR verification; take 1 mL of seed liquid into 100 mL of LB liquid medium containing the same hormones and incubate at 28°C with 200 r / min shaking for 12 h; centrifuge at 5000 r / min for 5 min to collect the recombinant Agrobacterium bacteria, dissolve the bacteria in MS liquid medium and adjust the OD 600 to 0.2, add AS (acetyl-syringone) at a final concentration of 200 μmol / L for transfection of tomato cotyledon explants.

[0103] 4) Transformation and regeneration

[0104] Take the pre-cultured tomato cotyledon explants in a sterile culture dish, add the prepared recombinant Agrobacterium bacterial suspension, the bacterial suspension submerges the explants for 15 min, and the sterile glass rod is constantly stirred gently during the process; after the infection is completed, the explants are taken out with sterile forceps, the surface residual bacterial solution is absorbed on sterile filter paper, and finally placed on co-culture medium flat plate at 26°C, dark culture for 2d. The tomato cotyledon explants after co-culture are transferred to the induction and screening medium, 5-8 explants are evenly placed on each flat plate, and the light culture is used to induce callus, and the callus rate is counted after 14d; the differentiation of the regenerated buds is observed and recorded after 35d, and the bud rate is counted. Subculture every 2 weeks, and the resistance screening hormone is hygromycin HPT (5mg / L).

[0105] 5) Rooting culture and transplanting

[0106] When the adventitious buds grow to 2-4cm, the adventitious buds with good growth are picked, the surrounding excess callus is cut off, and then transferred to the rooting culture medium, cultured in the light culture box (light for 16h, temperature: light 26°C, dark 18°C, humidity 65%), and induced to root. After the root system of the tissue culture seedlings grows well, the tissue culture bottle cap is removed and placed in the light culture box for hardening-off culture for 2d. After stable growth, the tissue culture seedlings are taken out from the tissue culture bottle using forceps, washed to remove the residual culture medium on the roots, and transplanted into a flowerpot containing sterilized substrate soil to obtain tomato LeMYB330 gene overexpression tomato plants.

[0107] Example 3

[0108] Molecular detection of transgenic plants

[0109] (1) PCR method for detecting transgenic plants at DNA level

[0110] Refer to the instruction manual of Tian Gen Plant Genomic DNA Kit (DP305-02) plant genomic DNA extraction kit, and quickly extract the DNA of the tomato transgenic plants, as follows:

[0111] 1. Take fresh plant tissue about 100mg or dry tissue about 30mg, and add liquid nitrogen to grind thoroughly.

[0112] 2. Quickly transfer the ground powder to a centrifuge tube preloaded with 700μL 65°C preheated buffer GP1 (add mercaptoethanol to the preheated GP1 before the experiment to make the final concentration 0.1%), mix well by inverting the centrifuge tube, and then place the centrifuge tube in a 65°C water bath for 20min, and invert the centrifuge tube several times during the water bath process to mix the sample.

[0113] 3. Add 700μL chloroform and mix thoroughly, and centrifuge at 12,000rpm (~13,400xg) for 5min.

[0114] 4. Carefully transfer the top aqueous layer from the previous step to a new centrifuge tube, add 700 μL Buffer GP2, and mix well.

[0115] 5. Transfer the mixed liquid to the adsorption column CB3, centrifuge at 12,000 rpm (~ 13,400 xg) for 30 sec, and discard the waste liquid.

[0116] 6. Add 500 μL Buffer GD (check if anhydrous ethanol has been added before use) to the adsorption column CB3, centrifuge at 12,000 rpm (~ 13,400 xg) for 30 sec, discard the waste liquid, and place the adsorption column CB3 into a collection tube.

[0117] 7. Add 600 μL Rinse PW (check if anhydrous ethanol has been added before use) to the adsorption column CB3, centrifuge at 12,000 rpm (~ 13,400 xg) for 30 sec, discard the waste liquid, and place the adsorption column CB3 into a collection tube.

[0118] 8. Repeat step 7.

[0119] 9. Place the adsorption column CB3 back into the collection tube, centrifuge at 12,000 rpm (~ 13,400 xg) for 2 min, discard the waste liquid. Place the adsorption column CB3 at room temperature for several minutes to dry the adsorption material completely.

[0120] 10. Transfer the adsorption column CB3 into a clean centrifuge tube, add 50-200 mL Elution Buffer TE to the middle of the adsorption membrane, and let stand at room temperature for 2-5 min, centrifuge at 12,000 rpm (~ 13,400 xg) for 2 min, and collect the solution into a centrifuge tube. Store at -20 °C.

[0121] 1) PCR detection of tomato LeMYB330 overexpression plants

[0122] The overexpression vector pCAMBIA1304-LeMYB330-HA contains a hygromycin resistance gene integrated into the plant genome. Therefore, we identified the overexpression positive transgenic plants by using LeMYB330 gene specific primers Pca MYB-F / Pca MYB-R and hygromycin screening gene primers HPT-F / HPT-R, respectively. The length of LeMYB330 gene detection fragment is 850 bp, and the length of hygromycin gene detection fragment is 750 bp. Figure 2 To verify the LeMYB330 overexpression transgenic positive plants. Among them, Figure 2 A is the PCR detection of LeMYB330 overexpression transgenic positive plants LeMYB330 gene; Figure 2 B is the PCR detection of LeMYB330 overexpression transgenic positive plants HPT gene;Figure 2 C is the relative expression of LeMYB330 gene in positive plants overexpressing transgene detected by real-time fluorescent quantitative PCR. CK is wild-type tomato "Xinjinfeng No. 1" without transgene; OE is LeMYB330 overexpression plant; OE2, OE3, OE7, OE8, OE9 and OE11 are six lines of LeMYB330 overexpression plants. The primer sequences are as follows:

[0123] LeMYB330 primer sequence:

[0124] Pca MYB-F: 5'-CATTTGGAGAGAACACGGG-3';

[0125] Pca MYB-R: 5'-GGGACAACTCCAGTGAAAAG-3';

[0126] Hygromycin primer sequence:

[0127] HPT-F: 5'-CTTGACATTGGGGAGTTTAGCGAGA-3';

[0128] HPT-R: 5'-CCCTTATCTGGGAACTACTCACACA-3';

[0129] (2) Detection of tomato LeMYB330 overexpression positive plants at transcriptional level by qRT-PCR

[0130] According to the instructions of Tian Gen RNA prep pure Plant Kit (DP432), the total RNA of tomato plants was extracted, and the specific steps were as follows:

[0131] 1) Homogenate treatment: 50-100 mg of plant leaves were rapidly ground into powder in liquid nitrogen, 450 μL of lysis solution RL (β-mercaptoethanol was added to a final concentration of 1% before use) was added, and it was mixed well by vortexing.

[0132] 2) Transfer all solutions to filter column CS (filter column CS was placed in a collection tube), centrifuge at 12000 r / min for 2-5 min, carefully aspirate the supernatant in the collection tube to the RNase-Free (RNAase-free) centrifuge tube, and the suction head should avoid contacting the cell debris precipitate in the collection tube as much as possible.

[0133] 3) Slowly add 0.5 times the volume of anhydrous ethanol (usually 225 μL, adjust according to the specific situation), mix well. At this time, if there is a precipitate, the resulting solution and precipitate should be transferred to the adsorption column CR3, and centrifuged at 12000 r / min for 30-60 s. Discard the waste liquid in the collection tube, and place the adsorption column CR3 back into the collection tube.

[0134] 4) Add 350 μL of the deproteinized solution RW1 to the adsorption column CR3, centrifuge at 12000 r / min for 30-60 s, discard the waste liquid in the collection tube, and place the adsorption column CR3 back into the collection tube.

[0135] 5) Preparation of DNase I working solution: take 10 μL of DNase I storage solution into a new RNase-Free centrifuge tube, add 70 μL of DNA digestion buffer (RDD), and mix gently.

[0136] 6) Add 80 ml of the DNase I working solution to the center of the adsorption column CR3, and place it at room temperature for 15 min.

[0137] 7) Add 350 μL of the deproteinized solution RW1 to the adsorption column CR3, centrifuge at 12000 r / min for 30-60 s, discard the waste liquid in the collection tube, and place the adsorption column CR3 back into the collection tube.

[0138] 8) Add 500 μL of the rinse solution RW (add an appropriate amount of anhydrous ethanol before use according to the bottle body requirements) to the adsorption column CR3, place it at room temperature for 2 min, centrifuge at 12000 rpm for 30-60 s, discard the waste liquid in the collection tube, and place the adsorption column CR3 back into the collection tube.

[0139] 9) Repeat step 8.

[0140] 10) Centrifuge at 12000 rpm for 2 min, discard the waste liquid. Place the adsorption column CR3 at room temperature for several minutes to completely dry the residual rinse solution in the adsorption material.

[0141] 11) Place the adsorption column CR3 into a new RNase-Free centrifuge tube, add 30-100 μL of RNase-Free ddH2O to the middle part of the adsorption membrane, place it at room temperature for 2 min, centrifuge at 12000 rpm for 2 min, and obtain the RNA solution.

[0142] According to the instructions of FastKing RT Kit (With gDNase) (TIANGEN, KR116), rapidly reverse transcribe the first strand of cDNA; using the first strand of cDNA obtained by reverse transcription as a template, perform qRT-PCR according to the Talent qPCR PreMix (SYBR Green) (TIANGEN, FP209) to quantitatively detect the relative expression amount of the target gene LeMYB330 in the LeMYB330 transgenic tomato plants Figure 2C). Primers are shown in Table 1. The 20 μL reaction system contains 10 μL 2×TalentqPCR PreMix, 0.6 μL forward primer, 0.6 μL reverse primer, 1 μL cDNA, and 7.8 μL ddH2O. The PCR three-step reaction program was used, and the reaction conditions were: 95°C pre-denaturation for 3 minutes; 95°C denaturation for 5 seconds, 55°C annealing for 10 seconds, 72°C extension for 15 seconds, and 40 cycles. Fluorescence data was collected at the end of the extension of each cycle. Tomato Actin gene was used as an internal reference. The specificity of the primers was determined based on the melting curve. The primer sequences are shown in Table 1. The relative gene expression was calculated according to the method of Livak and Schmittgen (Analysis of relative gene expression data using real-time quantitative PCR and the 22DDCT method, Methods, 2001, 25: 402-408). The experiments were repeated three times. According to Figure 2 C shows that the relative expression level of LeMYB330 gene in the LeMYB330 overexpressing plant OE8 is the highest, followed by the OE3 line.

[0143] Table 1 Real-time fluorescence quantitative PCR primers

[0144]

[0145] Example 4

[0146] The tomato plants OE8 overexpressing the LeMYB330 gene detected in Example 3 were inoculated with the root-knot nematode R. elegans and the root-knot nematode M. javanica. The specific method for inoculating the tomato plants OE8 overexpressing the LeMYB330 gene with the two root-knot nematodes is as follows:

[0147] Wild-type WT and overexpression plants, and wild-type WT plants were divided into two groups and inoculated separately. When the tomato plant grew to four leaves and one heart, it was inoculated with nematodes. Each plant was inoculated with about 1,000 J2 stage nematodes, and watered normally during the period. The tomato plants were cultivated in pots filled with high-temperature sterilized substrate soil. The nematode treatment time was 5 weeks. At 35dpi of inoculation, samples were taken and relevant indicators were measured. Three replicates were set for each treatment group. The results are shown in Figure 2. Figure 3 、 Figure 4 shown.

[0148] Figure 3 Observation of root-knot nematode phenotypes in tomato plants overexpressing LeMYB330 5 weeks after inoculation with R. Figure 3 A shows the representative pathogenesis characteristics of the roots of LeMYB330-overexpressing plants and wild type plants; Figure 3 B is the statistical result of the incidence of Figure A.

[0149] Figure 4 Root-knot nematode phenotype observation of tomato LeMYB330 overexpression plants after inoculation with Javanese root-knot nematode for 5 weeks. Figure 4 A is the representative disease characteristics of LeMYB330 overexpression plants and wild type roots; Figure 4 B is the statistical result of the disease in A.

[0150] As shown in Figure 3 , Figure 4 , we used LeMYB330 overexpression lines and wild type WT control plants as experimental materials, and inoculated each tomato plant with 1000 hatched J2 stage root-knot nematodes of Cajanus cajan and Javanese root-knot nematodes. After 5 weeks of culture, we found that the number of C. cajan root-knots and the number of egg capsules of LeMYB330 overexpression plants were significantly reduced compared with wild type, and the corresponding C. cajan root-knot index and egg capsule index were also significantly reduced. The number of Javanese root-knots of LeMYB330 overexpression plants was significantly reduced compared with wild type, and the corresponding Javanese root-knot index was also significantly reduced.

[0151] Example 5

[0152] Tomato LeMYB330 gene overexpression plant OE8 detected in Example 3 was inoculated with tomato bacterial wilt (Ralstonia solanacearum) for treatment, and the specific method was as follows:

[0153] Select 3 overexpression plants and control tomato plants each at the stage of transplanting to 4 leaves and 1 heart, and wash off the root soil with running water. Use the wounded root inoculation method: cut off the root tip (about 0.5 mm) with a sterile surgical scissors, then soak in the prepared bacterial suspension of Ralstonia solanacearum for 15 min. After the infection is completed, wash off the residual bacterial solution on the root surface with running water, and re-plant the tomato plants in the substrate soil (Ke Jingjing, 2022), continue to culture for about 2 weeks, take samples and measure the relevant indexes. The results are shown in Figure 5 .

[0154] Among them, the preparation steps of the bacterial suspension of Ralstonia solanacearum are as follows: take Ralstonia solanacearum strain GMI1000, streak culture on NA (chloramphenicol 15 mg / mL) medium, and incubate in a 28°C constant temperature incubator for about 48 h. Pick the dominant single colony and subculture, and the newly grown dominant single colony is cultured in 2 mL of NB medium at 28°C, 190 r / min on a shaker for 12 h. Take an appropriate amount of bacterial solution for expansion culture for 10-12 h. Pour the expanded culture bacterial solution into a 50 mL sterile centrifuge tube, centrifuge at 4000 r / min for 15 min, remove the supernatant, and resuspend the bacterial body with an equal volume of sterile water. Measure the OD 600 value and adjust it to 0.1 (about 10 8 CFU / mL) to prepare the bacterial suspension of Ralstonia solanacearum.

[0155] like Figure 5 As shown in the figure, this study inoculated overexpression plants and wild-type plants with Ralstonia solanacearum and found that LeMYB330 gene overexpression plants and CK + In comparison, CK + The number of diseased leaves and disease index of tomato plants in the group were significantly higher than those in the LeMYB330 gene overexpression plants at 3 to 15 dpi. At 15 dpi, the disease index of the LeMYB330 gene overexpression plants was 61.11 ± 1.39, which was significantly lower than that of the control by 36.11 ± 8.19. The results showed that LeMYB330 overexpression can significantly improve the resistance of tomato plants to bacterial wilt. Among them, wild-type plants not inoculated with R. solanacearum were used as the control (CK - ), the control plants grew normally and were not diseased.

[0156] In addition, it should be understood that after reading the above description of the present invention, those skilled in the art may make various changes or modifications to the present invention, and these equivalent forms also fall within the scope defined by the claims attached to this application.

[0157] References:

[0158] Ke Jingjing. Tomato immunity mediated by Pectin exoenzyme PehC of Ralstonia solanacearum and its application potential[D]. Wuhan: Doctoral Program of Huazhong Agricultural University

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[0167] Zhu L T. The Silencing Effect of Tomato Root Knot Giant Cell Apoptosis Related Genes MiPDCD6 and LeMYB330[D]. Guangzhou: South China Agricultural University, 2016.

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[0174] Shu P, Li Y, Xiang L, et al. SlNPR1 modulates chilling stress resistance in tomato plant by alleviating oxidative damage and affecting the synthesis of ferulic acid[J]. Scientia Horticulturae, 2023, 307: 111486.

[0176] Wong G R, Latif S N F B, Mazumdar P. Genome-wide investigation and comparative expression profiling reveal R2R3-MYB genes involved in Sclerotinia sclerotiorum defence in tomato[J].

[0177] Physiological and Molecular Plant Pathology, 2022, 121:101873.

[0178] Xun S, Ping W, Xin J, et al. Improvement of drought tolerance by overexpressing MdATG18a is mediated by modified antioxidant system and activated autophagy in transgenic apple[J]. Plant Biotechnology Journal, 2018, 16(2): 545-557.

[0179] Ye W M, Koenning S, Zeng Y S, et al. Molecular characterization of an emerging root-knot nematode Meloidogyne enterolobii in North Carolina, USA[J]. Plant Disease, 2021, 105(4): 819-831.

[0180] The above examples are the preferred embodiments of the present application, but the embodiments of the present application are not limited to the above examples, and any changes, modifications, substitutions, combinations, simplifications made without departing from the spirit and principles of the present application should be equivalent replacement methods, and are included in the protection scope of the present application.

Claims

1. Tomato transcription factor LeMYB330 gene or its related biological material in improving plant broad-spectrum resistance, characterized in that: Overexpression of tomato transcription factor LeMYB330 gene improves plant broad-spectrum resistance The amino acid sequence encoded by the tomato transcription factor LeMYB330 gene is shown as SEQ ID NO:

2. The biological material related to the tomato transcription factor LeMYB330 gene is any one or a combination of the following biological materials: (1) an expression cassette containing the tomato transcription factor LeMYB330 gene; (2) a recombinant overexpression vector containing the tomato transcription factor LeMYB330 gene; (3) a recombinant overexpression vector containing the expression cassette in (1); (4) a recombinant microorganism containing the expression cassette in (1); (5) a recombinant microorganism containing the recombinant overexpression vector in (2) or (3).

2. The tomato transcription factor LeMYB330 gene or the biological material related to the tomato transcription factor LeMYB330 gene for use in cultivating plants with broad-spectrum resistance.

3. The use according to claim 1 or 2, wherein: the plant is a Solanaceae plant, and further is a tomato; the broad-spectrum resistance includes at least one of Meloidogyne exigua resistance, Meloidogyne javanica resistance, and Pseudomonas solanacearum resistance.

4. Use according to claim 1 or 2, characterized in that: The nucleotide sequence of the tomato transcription factor LeMYB330 gene is shown as SEQ ID NO:

1.

5. The use according to claim 1 or 2, wherein: the starting vector of the recombinant overexpression vector in (2) and (3) is a plant expression vector, including plant binary expression vectors pCAMBIA series, pBI series, pFGC5941; the host microorganism corresponding to the recombinant microorganism in (4) and (5) is selected from prokaryotes; and the prokaryotes include bacteria of the genus Agrobacterium.

6. The use according to claim 5, wherein: the starting vector of the recombinant overexpression vector in (2) and (3) is pCAMBIA1304; the prokaryote is Agrobacterium tumefaciens.

7. The use according to claim 1, characterized in that: The method specifically comprises the following steps: (a) constructing Agrobacterium tumefaciens engineering bacteria containing the tomato LeMYB330 gene overexpression vector; (b) transforming plant explants with the Agrobacterium tumefaciens engineering bacteria constructed in step (a) to obtain LeMYB330 gene overexpression plants; (c) inoculating the LeMYB330 gene overexpression plants obtained in step (b) with Meloidogyne exigua, Meloidogyne javanica, and / or Pseudomonas solanacearum to perform stress treatment, and statistically analyzing the plant disease incidence.

8. The use according to claim 7, wherein: in step (c), the inoculation of Meloidogyne exigua, Meloidogyne javanica, and / or Pseudomonas solanacearum stress treatment is specifically as follows: when the tomato LeMYB330 gene overexpression plants grow to four-leaf one-heart, inoculate them with Meloidogyne exigua, Meloidogyne javanica, and / or Pseudomonas solanacearum.

9. The use according to claim 8, wherein: the inoculated Meloidogyne exigua and Meloidogyne javanica are J2-stage Meloidogyne exigua and Meloidogyne javanica with invasive activity; the Pseudomonas solanacearum stress treatment is specifically as follows: the root injury inoculation method is used to inoculate Pseudomonas solanacearum.

10. The use according to claim 8, wherein: The inoculation of Meloidogyne javanica stress treatment time is 5 weeks; The inoculation of P. solanacearum stress treatment time is 15 days.

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