A tomato whitefly-resistant gene S1PR3 and its application

Abstract

The present invention discloses a tomato whitefly-resistant gene S1PR3 and its application, belonging to the field of plant genetic engineering technology. The nucleotide sequence of the gene is shown in SEQ ID NO.1; the CDS of the tomato whitefly-resistant gene S1PR3, whose CDS sequence is shown in SEQ ID NO.2; and the amino acid sequence of the expressed protein of the tomato whitefly-resistant gene S1PR3, whose amino acid sequence is shown in SEQ ID NO.3. The present invention demonstrates through real-time fluorescence quantitative technology that the expression level of the S1PR3 gene changes significantly after being attacked by whiteflies, indicating its role in tomato whitefly resistance. The present invention also constructs tomato plants overexpressing the tomato whitefly-resistant gene S1PR3 and detects the resistance phenotype of the S1PR3 gene when the plants are attacked by whiteflies, demonstrating its resistance to whiteflies. The present invention has important application value in the breeding of insect-resistant crop varieties.

Description

Technical Field

[0001] The invention belongs to the technical field of plant genetic engineering and relates to a tomato whitefly resistance gene S1PR3 and an application thereof. Background Art

[0002] Tomato (Solanum lycopersicum), a plant of the Solanaceae family native to Central and South America, serves as a key model organism for the study of flowering plants in the Solanaceae family. Due to its rich vitamins and lycopene, and its immune-boosting properties, the tomato plant is now widely cultivated worldwide, becoming one of the world's most popular and widely used fruits. However, tomato cultivation is often subject to a variety of pests and diseases, including the broad-spectrum plant pest Bemisia tabaci. Bemisia tabaci is one of the most destructive and difficult-to-manage omnivorous pests worldwide, causing significant damage to agricultural production. Studies have reported that common tomato cultivars have little resistance to whitefly. Whiteflies can directly feed on the phloem sap of tomatoes, causing stunted plant growth and development; they also secrete honeydew on tomato fruit and leaves, causing premature ripening and mildew. Whiteflies can also transmit various plant viruses, such as Tomato Yellow Leaf Curl Virus, causing significant losses to tomato production. Spraying insecticides with yellow plasters is the most common method for repelling whiteflies. However, long-term and excessive use of insecticides not only leads to insecticide resistance in whiteflies but also creates environmental and food safety issues. Genetic engineering, a core technology of modern biotechnology, boasts the advantage of increasing crop yields, improving nutrition, and reducing pesticide use by regulating the function of genes within an organism. Therefore, identifying insect-resistant genes in tomatoes and developing whitefly-resistant varieties are crucial for reducing whitefly damage. Summary of the Invention

[0003] The purpose of the present invention is to provide a tomato whitefly resistance gene S1PR3 and its application.

[0004] In a first aspect, the present invention provides a tomato chitinase gene S1PR3, which has significant resistance to whiteflies. The nucleotide sequence of the tomato chitinase gene S1PR3 is shown in SEQ ID NO.1.

[0005] SEQ ID NO.1 is:

[0006]

[0007] The CDS of the tomato whitefly-resistant gene S1PR3 has a nucleotide sequence as shown in SEQ ID NO.2;

[0008] SEQ ID NO.2 is:

[0009] ;

[0010] The amino acid sequence of the expressed protein of the tomato whitefly-resistant gene S1PR3 is shown in SEQ ID NO.3;

[0011] SEQ ID NO.3 is:

[0012] MKFNIVSPVALSCLFFLFLTGTLAQNAGSIVTRELFEQMLSFRNNDACPAKGFYTYDAFIAAAANSFPGFGTAGDDTARKKEIAAFFGQTSHETNGGSAGTFTGGYCFVKQIEQSDRYYGRGPIQLTH QSNYERAGQGIGVGQELVNNPDLVATDPIISFKTAIWFWMTEQDNKPSCHNVIIGQWTPSPKDTAANRVPGYGVITNIINGQFECGMGPNTAAESRIGFYRRYCGMLNVPTGENLDCNNQKNFAQG.

[0013] In a second aspect, the present invention provides a plant overexpression vector containing the tomato whitefly resistance gene S1PR3, the specific preparation method of which is as follows:

[0014] The SlPR3 gene was assembled using pBI121 as a vector. Through gene recombination technology, the SlPR3 gene was inserted into the pBI121 vector. The pBI121 vector was equipped with a 35S promoter element and a Kana gene expression cassette to ensure that the SlPR3 gene was overexpressed in transgenic plants in subsequent experiments. The Kana gene expression cassette was a screening marker for transgenic plant materials, and transgenic materials were screened using kanamycin.

[0015] In a third aspect, the present invention provides a tomato whitefly-resistant gene S1PR3 and a plant overexpression vector containing the tomato whitefly-resistant gene S1PR3 for enhancing the resistance of tomatoes to whiteflies.

[0016] The tomato whitefly-resistant gene SlPR3 and the plant overexpression vector containing the tomato whitefly-resistant gene SlPR3 can be used for tomato whitefly-resistant seedling cultivation. By transforming tomato plants with the plant overexpression vector containing the whitefly-resistant gene SlPR3, SlPR3 gene-overexpressing tomato plants are obtained.

[0017] Beneficial Effects: This study, using wild-type M14 tomato strains from the Ailsa Craig (AC) background, used fluorescence quantitative detection to detect changes in SlPR3 gene expression in tomato plant leaves during whitefly infestation. The SlPR3 gene was obtained through homologous cloning, and an overexpression vector was constructed and transformed into M14 to generate a SlPR3-overexpressing tomato line. The resistance phenotype of the SlPR3 gene in plants infested with whiteflies was tested, demonstrating its ability to resist whiteflies. This study has important application value in the development of insect-resistant crop varieties. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 The expression level of SlPR3 in the leaves of wild-type tomatoes was changed when they were attacked by whiteflies.

[0019] Figure 2 This is the plasmid map of the plant overexpression vector pBI121-35S:SlPR3.

[0020] Figure 3 This is the expression level detection map of the SlPR3 gene in the tomato SlPR3 overexpression strain.

[0021] Figure 4 The resistance of the SlPR3-overexpressing strain to whiteflies is shown; (a) shows the number of whiteflies on the tomato SlPR3-overexpressing strain in a 7-day selective experiment and the number of eggs S on the leaves on the 7th day; (b) shows the change in the number of whiteflies on the leaves of the tomato SlPR3-overexpressing plants in a non-selective experiment and the phenotype on the 7th day. DETAILED DESCRIPTION

[0022] The present invention will be further described below with reference to specific embodiments.

[0023] Example 1

[0024] In this example, quantitative PCR was used to investigate changes in SlPR3 gene expression after whitefly infestation. A forward primer (SEQ ID NO. 6) 5'-CAATGGACGCCATCCCCTAA-3' and a reverse primer (SEQ ID NO. 7) 5'-CGGACCCATCCCACATTCAA-3' were designed based on the exon region of the SlPR3 gene. Internal reference primers (forward primer (SEQ ID NO. 8) 5'-GGAACTTGAGAAGGAGCCTAAG-3' and reverse primer (SEQ ID NO. 9) 5'-CAACACCAACAGCAACAGTCT-3') were designed based on the Actin gene. Using a CFX connect quantitative PCR instrument, the number of cycles required to reach the fluorescence threshold was determined according to the manufacturer's instructions for the PCR system and protocol. The relative expression level of the SlPR3 gene in tomato plants after whitefly infestation was calculated.

[0025] like Figure 1 As shown in the figure, the expression level of SlPR3 increased to varying degrees after being attacked by whiteflies.

[0026] Example 2

[0027] The S1PR3 gene was identified and cloned through sequence comparison.

[0028] Based on the S1PR3 gene ID (Solyc02g082920), the corresponding CDS sequence was found in the Sol Genomics Network database. Full-length primers were designed using Primer 5.0. Restriction sites and homology arms were added to the 5' ends of the upstream and downstream primers, respectively. Primer specificity was confirmed by comparison with the NCBI database. PCR amplification of the target fragment was performed using wild-type M14 tomato cDNA as a template. The amplified fragment was positively screened and then sequenced. Sequencing results confirmed the complete S1PR3 gene.

[0029] I. Primer Design

[0030] The SlPR3 OE primer pair is SlPR3-OE-F and SlPR3-OE-R:

[0031] S1PR3-OE-F (SEQ ID NO. 4):

[0032] 5'ggatcttccagagatggatccATGAAGTTCAATATTGTATCACCCGT 3';

[0033] S1PR3-OE-R (SEQ ID NO. 5):

[0034] 5'ctgccgttcgacgatgagctcTTAGCCCTGGGCGAAGTTC 3';

[0035] II. Sequence Cloning

[0036] Step 1, PCR;

[0037] The PCR reaction system is: 2×HieffCanace AdavanceFast PCR Master Mix 25ul, SlPR3-Fprimer 2μl, SlPR3-Rprimer 2μl, cDNA 2μl, ddH2O to 50μl; the reaction program is: 98℃30sec; 98℃10sec, 60℃5sec, 72℃10sec, 32 cycles; 72℃1min to obtain PCR product.

[0038] Step 2, ligating the cloning vector;

[0039] Ligate with the pBI121 cloning vector. The ligation reaction system (10 μl) consists of 3 μl of enzyme-digested, linearized pBI121, 2 μl of recovered PCR product, and 1 μl of 2× HieffClone Enzyme Premix. Reaction conditions: Incubate at 50°C for 20 minutes to obtain the ligation product.

[0040] Step 3, E. coli transformation;

[0041] Take 50 μl of E. coli competent cells, add the target DNA (cloning vector ligation product in step 2), mix gently, ice bath for about 30 minutes, 42°C water bath for 60 seconds, and quickly place on ice for 2 minutes; add 700 μl LB liquid culture medium, 37°C, 200 rpm for recovery for 1 hour; centrifuge at 5000 rpm for 5 minutes, aspirate the upper 650 μl culture medium, mix the remaining bacterial liquid, evenly spread it on the LB screening culture plate containing Kana, and invert and culture at 37°C overnight.

[0042] Step 4: Positive clone screening and sequencing;

[0043] Single colonies were selected from the overnight culture plates and inoculated into LB liquid medium. Culture was incubated overnight at 37°C and 250 rpm to obtain bacterial suspension. PCR detection of recombinant transformants was performed using the bacterial suspension as a template. The PCR reaction system consisted of 10 μl of 2× RapidTaq MasterMix, 0.5 μl of 35S 3'F primer, 0.5 μl of SlPR3 R primer, 1 μl of bacterial suspension, and ddH2O to a total of 20 μl. The reaction procedure was: 95°C for 3 min; 95°C for 15 sec, 60°C for 10 sec, 72°C for 25 s, 32 cycles; and 72°C for 5 min. Positive results were detected by electrophoresis. Positive bacterial suspensions were sent to Qingke Biotechnology for sequencing.

[0044] Analysis of the sequencing results showed that the DNA of SlPR3 was 1158 bp long, as shown in SEQ ID NO.1, the CDS of SlPR3 was 762 bp long, as shown in SEQ ID NO.2, and the corresponding SlPR3 protein sequence was 253 amino acid residues, as shown in SEQ ID NO.3.

[0045] Example 3

[0046] In this example, an S1PR3 overexpression vector was constructed and genetically transformed into tomato.

[0047] Construction of the SlPR3 overexpression vector: Sequencing confirmed that the target gene had been successfully linked into the pBI121 cloning vector. The 5' end of the SlPR3 gene contains a 35S promoter element, ensuring overexpression of the SlPR3 gene in transgenic plants. The pBI121 vector also contains a Kana gene expression cassette, which serves as a selection marker for transgenic plant material. Transgenic material was screened using kanamycin.

[0048] To obtain a positive strain: Add the target plasmid (SlPR3 overexpression vector) to competent Agrobacterium GV3101 and place on ice for 30 minutes, incubate in liquid nitrogen for 1 minute, in a 37°C water bath for 5 minutes, and then rest on ice for 2 minutes. Add 900 μl of LB medium and incubate with shaking at 28°C for 1.5 hours. Centrifuge at 12,000 rpm for 5 minutes, discard the supernatant (approximately 800 μl), resuspend the cells, and evenly spread them on an LB plate supplemented with antibiotics (Rif 50 μg / ml, Kana 50 μg / ml). Incubate at 28°C for 2–3 days to identify a positive strain.

[0049] I. Tomato Seed Germination

[0050] Take an appropriate amount of wild-type M14 tomato seeds, disinfect them with 75% alcohol for 30s to 1min, wash them once with sterile water, disinfect them with 15% NaClO for 10min, shake them up and down every 1 to 2 minutes to ensure sufficient disinfection, wash them with sterile water 3 to 5 times, place the disinfected seeds on sterile filter paper to dry, and then inoculate them on M2 culture medium. The sown sterile tomato seeds are placed in an incubator at 25±3℃, 50% humidity, 60% light, and a photoperiod of 16h light and 8h dark.

[0051] II. Preparation before infection

[0052] A single colony of the positive Agrobacterium strain (obtained in Example 2) was selected and suspended in 100 ml of LB liquid medium. The culture was shaken at 250 rpm at 28°C for 14-18 hours. When the OD600 reached approximately 0.6, the culture was aliquoted into two 50 ml sterilized centrifuge tubes. The tubes were centrifuged at 5000 rpm for 10 minutes. The supernatant was discarded, and 5 ml of MS liquid medium was added to each tube. The pellet was resuspended by pipetting, and then 45 ml of MS liquid medium was added to each tube. After equal amounts of the resuspended cells were added, 100 μl of acetosyringone (As) solution, which promotes Agrobacterium infection, was added and mixed thoroughly.

[0053] III. Leaf Co-cultivation

[0054] In a clean bench, place the tomato seedlings in a sterile culture dish, pour in a shallow layer of MS liquid culture medium to keep it moist, cut off the cotyledons of the tomato with a knife, remove the top and base of the cotyledons, and cut the center into 2 to 3 small slices depending on the growth of the cotyledons.

[0055] Pour the resuspended Agrobacterium solution into a sterile culture dish containing tomato cotyledon slices, gently shake to mix, and infect for about 10 to 15 minutes; remove the cotyledon slices, place them on filter paper for about 10 minutes to allow them to fully dry, and inoculate them into the co-cultivation medium.

[0056] IV. Differentiation Screening and Culture

[0057] Remove tomato leaves that have been incubated at 28°C for 2-3 days and transfer them to selective medium. Change the medium every one to two weeks, and continuously monitor callus formation and adventitious bud differentiation. Once adventitious buds begin to differentiate into seedlings, remove seedlings with normal plant shape and transfer them to culture bottles for further culture.

[0058] V. Rooting, screening, culture and hardening and transplanting

[0059] When the seedlings grow to 2-4 cm in height, cut off the callus tissue blocks at the base of the seedlings and transfer the seedlings to the rooting medium. Change the medium every 10-15 days. When the plant height grows to 5-6 cm, select the seedlings with good rooting condition, open the bottle and harden the seedlings for 1-2 days, then take them out and gently wash off the excess culture medium attached to the roots of the seedlings. Move them to the nutrient soil, keep it highly moist, and gradually reduce the humidity in the culture environment according to the growth status of the seedlings until the new leaves of the seedlings grow.

[0060] The expression of SlPR3 was analyzed by RT-PCR in wild-type tomato plants (WT) with the same growth status and seedlings identified as positive. The relative expression of SlPR3 was determined based on the brightness of the bands presented by the overexpressing tomato plants and the wild-type materials. The results are shown in Figure 2. Figure 3 As shown, S1PR3 was not only overexpressed but also expressed at the highest levels in tomato overexpression lines 16, 18, and 19. Phenotypic experiments were performed on T2 homozygous lines 16-3 and 19-6 of overexpression lines 16 and 19.

[0061] Example 4

[0062] Phenotypic experiments were performed on the T2 generation homozygous strains 16-3 and 19-6 of the overexpression strains 16 and 19 in Example 3. The insect resistance of the SlPR3 overexpression strains was detected using M14 wild-type tomatoes as a control. The following experiments were repeated three times, with three biological replicates set up for each experiment.

[0063] (1) Select healthy six-leaf tomato seedlings with the same growth status, one wild type and one transgenic strain, and place them in a breathable experimental device. Blow 75 female and 75 male whiteflies into the device through the small hole at the top. Close the small hole and place it in an incubator at 25±3℃ and 16h / 8h light / dark cycle. On the 7th day, record the number of whiteflies surviving on different strains and calculate the survival rate of whiteflies. Take the leaves of tomato with four leaves from different strains, punch a hole in the leaf disc at the same position, and count the number of whiteflies laying eggs on the leaf disc under a microscope. The results are as follows: Figure 4 As shown in (a).

[0064] (2) Six-leaf seedlings of different tomato overexpression lines with consistent growth status and health were selected. A breathable insect cage was fixed on the fourth leaf position. 15 male and female whiteflies were sucked and blown into the small cage. The plants were placed in an incubator at 25±3℃ and a 16h / 8h light / dark cycle. The number of whiteflies on each plant was counted at the same time every day after the whitefly infestation. The number of whiteflies was counted for 7 days. Figure 4 As shown in (b) in .

[0065] In the overexpressing plants, the number of eggs laid and the number of meals taken by whiteflies were significantly lower than those in the wild-type plants, indicating that the SlPR3 gene has a whitefly resistance function. The SlPR3 gene in tomato has important application value in enhancing the resistance of tomatoes to whiteflies.

[0066] As described above, although the present invention has been shown and described with reference to specific preferred embodiments, it should not be construed as limiting the present invention itself. Various changes may be made to the form and details without departing from the spirit and scope of the present invention.

Claims

1. Overexpression of Tomato Whitefly Resistance Gene SlPR3 The application of the method for enhancing the resistance of tomatoes to whiteflies is characterized in that: The tomato whitefly resistance gene SlPR3 The CDS sequence is shown in SEQ ID NO.

2.

2. The use according to claim 1, characterized in that The tomato whitefly resistance gene SlPR3 The amino acid sequence of the expressed protein is shown in SEQ ID NO.

3.

3. The use according to claim 1, characterized in that Overexpression was performed using a plant overexpression vector.

4. The use according to claim 3, wherein the plant overexpression vector is constructed by: Assembly using pBI121 as vector SlPR3 Gene, through gene recombination technology, SlPR3 The gene was inserted into the pBI121 vector, which was equipped with a 35S promoter element and a Kana gene expression cassette.

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