Application of S1MLP1 gene in improvement of broad-spectrum disease resistance of tomatoes

Abstract

The invention provides an application of an S1MLP1 gene in promoting broad-spectrum disease resistance of tomatoes. According to the invention, through overexpression of the S1MLP1 gene, the capability of the tomato in resisting gray mold, late blight and tomato yellow leaf curl virus disease is greatly improved, the production cost is saved, the environment is protected, the postharvest quality of the tomato can be effectively improved, and the method is especially suitable for cultivation under facility environment conditions.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology, specifically relating to a... SlMLP1 Application of genes in improving broad-spectrum disease resistance in tomatoes. Background Technology

[0002] Tomatoes are one of the most widely cultivated horticultural products in my country, and their unique flavor and rich quality have always made them popular. my country has the largest tomato planting area and the highest tomato yield in the world, with an annual output of approximately 55 million tons, accounting for about 7% of the total vegetable production. In recent years, with the increasing tomato planting area in my country, tomato diseases have become increasingly serious, such as gray mold, late blight, and yellow leaf curl virus. Currently, the control of tomato diseases mainly relies on chemical pesticides to kill pathogens. While pesticides can control certain diseases to some extent, they also lead to problems such as toxic pesticide residues and environmental pollution. Furthermore, tomato production often faces threats from various types of pathogens, such as fungi, bacteria, oomycetes, and plant viruses. Therefore, cultivating broad-spectrum disease-resistant tomato germplasm is essential to fundamentally solve the problem of multi-pathogen invasion. However, currently, there are relatively few broad-spectrum disease-resistant genes that can be applied to tomato breeding. Summary of the Invention

[0003] In view of this, the present invention provides a SlMLP1 The application of genes in enhancing the broad-spectrum disease resistance of tomatoes enables tomatoes to resist gray mold, late blight, and yellow leaf curl virus.

[0004] To achieve the above objectives, the present invention adopts the following technical solution: In a first aspect, the present invention provides a SlMLP1 Application of genes in promoting broad-spectrum disease resistance in tomatoes.

[0005] In the above technical solution, the present invention SlMLP1 The gene encodes a major latex protein, a member of the MLP subfamily within the Bet v I supergene family, which plays a role in responses to biotic and abiotic stresses. For example, in cotton, GhMLP28 positively regulates resistance to Verticillium dahliae. Additionally, GhMLP423 confers resistance to herbivorous insects by activating SAR signaling and transcription of disease-related genes. SlMLP1 in this invention was screened by IP-MS using the broad-spectrum disease resistance negative regulator SlSRC2. This gene can be induced by Botrytis cinerea, indicating its involvement in plant-disease interactions. Overexpression... SlMLP1 It can increase its own alkaloid content, thereby improving its resistance to a variety of pathogens.

[0006] Preferably, broad-spectrum disease resistance includes resistance to gray mold, late blight, and tomato yellow leaf curl virus.

[0007] Preferably, the specific steps of the application are: isolating and cloning from tomatoes SlMLP1 Genes, utilizing the aforementioned SlMLP1 Gene overexpression vectors are constructed and introduced into the cells or tissues of the desired plant.

[0008] Preferably, the overexpression vector is introduced into the desired plant by introducing the overexpression vector into the cells of the desired plant through Agrobacterium-mediated genetic transformation.

[0009] Preferably, SlMLP1 The nucleotide sequence of the gene is any of the following: a. Has a nucleotide sequence as shown in SEQ ID NO:1; b. A complementary nucleotide sequence to the nucleotide sequence shown in SEQ ID NO:1; c. A sequence that has more than 90% homology with the nucleotide sequence shown in SEQ ID NO:1; d. A nucleotide sequence that encodes the same protein as a, b, and c, but is different from them due to the degeneracy of the genetic code.

[0010] Preferably, SlMLP1 The amino acid sequence encoded by the gene is shown in SEQ ID NO:2.

[0011] Secondly, the present invention provides a product containing the aforementioned SlMLP1 overexpression vectors of gene nucleotide sequences.

[0012] Thirdly, the present invention provides an engineered bacterium containing the aforementioned overexpression vector.

[0013] Preferably, the engineered bacteria is Agrobacterium GV3101.

[0014] Compared with the prior art, the beneficial effects of the present invention are as follows: (1) The present invention is achieved through SlMLP1 Overexpression of the gene greatly enhances the tomato's resistance to gray mold, late blight, and yellow leaf curl virus, saving production costs, protecting the environment, and effectively improving the post-harvest quality of tomatoes. It is particularly suitable for cultivation in facility environments.

[0015] (2) This invention uses transgenic methods to obtain broad-spectrum resistant plants. The method is economical, direct and effective, which solves the problem of scarce germplasm resources and greatly shortens the breeding cycle. Attached Figure Description

[0016] Figure 1 The invention described in Embodiment 1 SlMLP1 Diagram illustrating the construction process of a vehicle for expressing justice (excessive quantity); Figure 2 The invention described in Embodiment 4 SlMLP1 Image showing the expression level of positive (overexpression) plants; Figure 3 The invention described in Embodiment 5 SlMLP1 Graph showing the induction effect of gray mold; Figure 4 As provided in Embodiment 6.1 of the present invention SlMLP1 Statistical chart of disease incidence and lesion area on leaves inoculated with Botrytis cinerea using excessive amounts of detached material; Figure 5 The method described in Embodiment 6.2 of the present invention SlMLP1 Statistical chart of disease incidence and lesion area on leaves inoculated with late blight pathogens using excessive amounts of detached material; Figure 6 The method described in Embodiment 6.3 of the present invention SlMLP1 Image showing disease incidence and virus accumulation in tomato plants inoculated with excessive amounts of tomato yellow leaf curl virus; Figure 7 As provided in Embodiment 7 of the present invention SlMLP1 The graph shows the alkaloid content of materials after excessive inoculation with gray mold and tomato yellow leaf curl virus. Detailed Implementation

[0017] The present invention will now be described in further detail with reference to specific embodiments, so that those skilled in the art can more clearly understand the present invention.

[0018] The present invention will now be described in more detail with reference to specific embodiments. Based on the following description and these embodiments, those skilled in the art can determine the essential features of the invention, and various changes and modifications can be made to the invention to suit various uses and conditions without departing from its spirit and scope.

[0019] Example 1 SlMLP1 Cloning of genes SlMLP1 is an interacting protein identified by IP-MS screening of SlSRC2, a broad-spectrum disease resistance negative regulator in tomatoes. Furthermore... SlMLP1 It can be induced to express by Botrytis cinerea. Therefore, for SlMLP1 Cloning was performed. First, the genome was analyzed using the tomato genome database (https: / / solgenomics.net / ). SlMLP1The full-length open reading frame (ORF) was amplified, and the cDNA sequence is shown in SEQ ID NO: 1. Primers for amplifying the ORF were designed: the forward primer was (5' CATTTGGAGAGGACACGCTCGAGATGGGTTTAAAAGGCAAGTTG 3') and the reverse primer was (5' TCTCATTAAAGCAGGACTCTAGATTATTTCCCAACATGGTGAGTC 3'). The full-length ORF was amplified from the background material Ailsa Craig (hereinafter named A57) using PCR. SlMLP1 The ORF sequence was obtained. The amplification method involved first extracting RNA from tomato, and then using a reverse transcription kit (purchased from Nanjing Novizan Biotechnology Co., Ltd.) to synthesize the ORF sequence according to the kit instructions. SlMLP1 The cDNA of the gene was then amplified by PCR to obtain the required ORF fragment. The fragment was then recovered using a 1% agarose gel electrophoresis kit (OMEGA, see the instruction manual for specific procedures) after PCR detection.

[0020] The amplification reaction system is as follows: 2*Phanta Max Buffer 25μL dNTP Mix (10mM each) 1μL SlMLP1-OE-FW (10μM) 2μL SlMLP1-OE-RV (10μM) 2μL Phanta Max Super-Fidelity DNA Polymerase1μL 1 μL template cDNA ddH2O 18μL The reaction procedure is as follows: Pre-denaturation 95℃ for 3 min Denaturation at 95°C for 15 seconds Annealing at 55°C for 15 seconds Extend at 72℃ for 1 minute Extend completely at 72℃ for 5 minutes The denaturation, annealing, and extension processes are repeated 38 times.

[0021] Example 2 Construction of Overexpression Vector Using XhoI and XbaI (NEB) pHellsgate8 The plasmid (provided and preserved by our laboratory) was digested with enzymes (37℃, 3h). The digestion products were detected on a 1.0% agarose gel and recovered using a recovery kit. Then, the linearized plasmid and the amplified SlMLP1 gene fragment were ligated using a homologous recombination kit (Novizan, China). The specific reaction procedure is as follows: Exnase II 1μL 5X CE II Buffer 2μL Linearized cloning vector 25-100 ng Insert fragment amplification product 10-100 ng Add sterile water to a volume of 10 μL Connect at 37℃ for 30 minutes.

[0022] The ligation product was transformed into *E. coli* TransT1 (Weidi, China) using the heat shock method. Positive clones were screened on Spec resistant LB agar plates, and the bacterial culture was picked and shaken. PCR detection was then performed on the liquid bacterial culture. The first primer used for detection was CaMV 35S, and the second primer was... SlMLP1 Primers were used after amplification. Bacterial cultures with the correct banding pattern were sent to our sequencing facility (Tianyi Huayu). Sequence alignment was performed. After successful alignment, the correct bacterial culture was selected, incubated overnight with gentle shaking, and plasmids were extracted using a small-scale plasmid extraction kit (purchased from OMEGA, see instruction manual for details). The vector construction process is described below. Figure 1 .

[0023] The obtained recombinant clones were transformed into Agrobacterium GV3101 using an electroporator at 1800V. Positive clones were selected using LB agar plates containing 100 mg / L Rif and 50 mg / L Spec. The plates were then incubated overnight at 28°C with shaking at 200 rpm. 1 μL of the Agrobacterium culture was used as a template and electroporated at 35 s and... SlMLP1 The primers were then used for PCR detection.

[0024] Example 3 Genetic Transformation Tomato seeds (A57) were sterilized with sodium hypochlorite for 15 in (2% available chlorine) and sown on 1 / 2 MS medium (pH=5.8). They were cultured at 25±2℃ in darkness until germination, then transferred to a photoperiod of 1800 lx, 16 h light / 8 h dark. Cotyledons from 7-8 day old sterile seedlings were pre-cultured for 2 days (MS medium, pH=5.8). The seeds were then resuspended in MS medium to OD. 600Inoculate with ≈0.5% Agrobacterium solution for 3-5 min, blot off excess solution with sterile filter paper, and return to the pre-med medium. Co-culture in the dark for 2 days. Transfer to 1.0 ZR (MS + ZR (zeatin nucleoside) 1.0 mg / L + Cef (cephalosporin) 400 mg / L + Kan (kanamycin) 100 mg / L) for resistance selection. Subculture every two weeks. After resistant shoots appear, transfer explants to 0.2 ZR + Cef (cephalosporin) 200 mg / L + Kan (kanamycin) 100 mg / L. After 20-30 days, cut off the resistant shoots and insert them into rooting medium (RM) to induce rooting. Transplant plants with well-developed root systems into flower pots. See Table 1 for specific medium formulations.

[0025] Table 1. Formula for Tomato Genetic Transformation Culture Medium

[0026] Note: Except for MSO, all the above culture media contain 7.4 g / L of agar, and the pH of all culture media is 5.8.

[0027] Example 4: In the over-strain SlMLP1 Transcription level detection We performed positive detection on 8 T0 generation seedlings through genetic transformation. RNA was extracted from the positive plants and reverse transcribed into cDNA for expression level detection. The real-time PCR reaction system was: 5 μL SYBR Mix, 0.5 μL each of forward and reverse primers (10 nmol / L), and 4 μL of sample cDNA; the reaction program was: 95℃ for 30 s, 95℃ for 5 s, 55℃ for 10 s, 72℃ for 15 s, for a total of 45 cycles, followed by cooling at 40℃ for 10 s. Melting curves were then collected and analyzed. Each sample was tested in triplicate, using the tomato endogenous actin gene (GenBank accession no. BT013524) as an internal control. The obtained data were used... The Ct method was used for analysis. The results are shown below. Figure 2 Subsequent use of higher overdose ratios SlMLP1 -OE3, SlMLP1 -OE16 and SlMLP1 -OE17 will be used for subsequent experiments.

[0028] Example 5 Tomato SlMLP1 Induction by gray mold In order to investigate SlMLP1 To determine whether the fungus was involved in the infection of tomatoes by Botrytis cinerea, we inoculated AC plants with Botrytis cinerea and then sampled them at 0h, 1h, 3h, 6h, 12h, and 24h to extract RNA. This RNA was then reverse transcribed to form cDNA, which was detected using specific primers.SlMLP1 The expression status was analyzed using the tomato endogenous actin gene (GenBank accession no. BT013524) as an internal reference. The obtained data were used... The Ct method was used for analysis. The results showed that... SlMLP1 It can be induced by Botrytis cinerea, with high induction levels at 12h and 24h. This indicates... SlMLP1 It may be involved in the process of gray mold invading tomatoes. See details below. Figure 3 .

[0029] Example 6 SlMLP1 Functional identification 6.1 SlMLP1 Identification of resistance to gray mold in transgenic plants To verify SlMLP1 Regarding the function of tomatoes in resisting gray mold, the applicant... SlMLP1 Both the excess and control plants (A57) were inoculated with *Botrytis cinerea* spores from the underside of mature leaflets of the same compound leaf at the same location. The samples were kept at 22℃ for 72 hours under a constant temperature, 16 hours of light / 8 hours of darkness, and 75% relative humidity. The excess plants showed milder disease incidence and smaller lesion areas. We used ImageJ software to calculate the lesion area and performed statistical analysis on the data. The results showed that... SlMLP1 The lesion area of ​​the excess material was significantly smaller than that of the wild-type strain A57, indicating that... SlMLP1 It can positively regulate resistance to gray mold (see...) Figure 4 ).

[0030] 6.2 SlMLP1 Identification of resistance to late blight in transgenic plants To verify SlMLP1 Regarding the function of tomatoes in resisting late blight, the applicant... SlMLP1 For both excess and control plants (A57), mature leaflets from the same part of the compound leaves at different growth stages were inoculated and examined. 10 μL of a pre-treated late blight spore mixture was inoculated onto detached leaves. The inoculated leaves were placed in a humid, sealed plastic container and treated in darkness for 24 hours. After culturing for 5 days under a 16-hour light-8-hour dark cycle, the area of ​​lesions at the inoculation site was measured. Results showed that the excess strain exhibited less disease. We used ImageJ software to calculate the lesion area and performed statistical analysis on the data. The results indicated that… SlMLP1 The lesion area of ​​the excess material was significantly smaller than that of the wild-type strain A57, indicating that... SlMLP1 It can positively regulate resistance to late blight (see...) Figure 5 ).

[0031] 6.3 SlMLP1Identification of resistance of transgenic plants to yellow leaf curl virus disease To verify SlMLP1 Regarding the function of tomato resistance to yellow leaf curl virus disease, the applicant tested three-week-old... SlMLP1 Excess material was used for inoculation and identification of yellow leaf curl virus. The Agrobacterium concentration of TYLCSV was adjusted to OD. 600 =1.0, using the friction inoculation method, inoculated onto fully expanded leaves of the plants, with 3 leaves inoculated per plant. The inoculated material was placed in a 26°C incubator for 40 days. The inoculation results showed that, compared to the control material A57, the terminal leaves of the excess material were more flattened, and the yellowing of the leaf margins was lighter. RNA was further extracted from the virus-inoculated material and reverse transcribed into cDNA. The expression of TYLCSV was detected using specific primers, with the tomato endogenous actin gene (GenBank accession no. BT013524) used as an internal control. The obtained data were used... The Ct method was used for analysis. The results showed that... SlMLP1 The lower viral accumulation in the excess material indicates that SlMLP1 It has a relatively significant effect in resisting yellow leaf curl virus (e.g. Figure 6 (As shown).

[0032] In summary, the results of the inoculation experiments with the three pathogens indicate that tomatoes SlMLP1 It can negatively regulate resistance to gray mold, late blight, and tomato yellow leaf curl virus, making it a promising disease-resistant functional protein for application in tomatoes.

[0033] Example 7 SlMLP1 Detection of alkaloid content in transgenic plants after inoculation with pathogens In order to clarify SlMLP1 Whether there is a link between the function of regulating broad-spectrum resistance and disease-resistant alkaloid compounds was investigated. We measured the alkaloid content of plants before and after inoculation with *Botrytis cinerea* and yellow leaf curl virus. The results showed that before inoculation with the pathogens, SlMLP1 The alkaloid content in the excess material was not different from that in the control material AC. However, after inoculation with pathogens, SlMLP1 The alkaloid content of the excess material was significantly higher than that of the control material (e.g., Figure 7 (As shown). This indicates SlMLP1 The broad-spectrum resistance conferred on plants has a certain causal relationship with their alkaloid content.

[0034] gene sequence SEQ ID NO: 1 ( SlMLP1 (cDNA sequence) ATGGGGTTTAAAAGGCAAGTTGATCTCTCAAATAGAAACGAAGTGTTCTGGACATTTGCTTCATGAGCACTTCAAATCAAATCCACACAAAACATCCGCCATGGCTCCTGATAAAATAACAAATTTTACACTTCATGAAGGTCAATTAGGAAAAACTAACTGTTGTTAGCTGGAATTATATTCTTGGAGGAAAAGAGAGGCATGCCAAACAAGTCCTACACA TAGACGATGAGGAAAAATCAATCACTTTCAACTTTAAAGAAGGATATATGAATGAACTATACAAGTCAATGACACTGACACTTACATTGATTGCAGAGAAAAACTTTATCACTTGGACTCTTGTGTATGAGAAATTGAATGAAATACTCCAGAGCCCTTGGATTTTATAGAGTTTCTTATCTATCTCATCAAGGACCTTGAGACTCACCATGTTGGGAAATAA SEQ ID NO: 2 ( SlMLP1 (amino acid sequence) MGLKGKLISQIETKCSGHLLHEHFKSNPHKTSAMAPDKITNFTLHEGQLGKTNSVVSWNYILGGKERHAKQVLHIDDEEKSITFNFKEGYMNELYKSMTLTLTLIAEKNFITWTLVYEKLNENTPEPLDFIEFLIYLIKDLETHHVGK Unless otherwise specified, all raw materials used in this invention are existing substances that can be purchased directly from the market.

[0035] The above are merely preferred embodiments of the present invention and are not intended to limit the scope of protection of the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. A kind SlMLP1 Application of genes in promoting broad-spectrum disease resistance in tomatoes.

2. Use according to claim 1, characterized in that, Broad-spectrum disease resistance includes resistance to gray mold, late blight, and tomato yellow leaf curl virus.

3. The application according to claim 1, characterized in that, The specific steps of the application are: to isolate and clone from tomatoes SlMLP1 Genes, utilizing the aforementioned SlMLP1 Gene overexpression vectors are constructed and introduced into the cells or tissues of the desired plant.

4. Use according to claim 3, characterized in that, The overexpression vector is introduced into the desired plant cells through Agrobacterium-mediated genetic transformation.

5. The application according to claim 1, characterized in that, SlMLP1 The nucleotide sequence of the gene is any one of the following: a. Has a nucleotide sequence as shown in SEQ ID NO:1; b. A complementary nucleotide sequence to the nucleotide sequence shown in SEQ ID NO:1; c. A sequence that has more than 90% homology with the nucleotide sequence shown in SEQ ID NO:1; d. A nucleotide sequence that encodes the same protein as a, b, and c, but is different from them due to the degeneracy of the genetic code.

6. The application according to claim 1, characterized in that, SlMLP1 The amino acid sequence encoded by the gene is shown in SEQ ID NO:

2.

7. A composition comprising the nucleotide sequence of claim 5 SlMLP1 overexpressing the gene.

8. Engineered bacteria containing the overexpression vector of claim 7.

9. The engineered bacteria according to claim 8, characterized in that, The engineered bacteria is Agrobacterium GV3101.

Images