Tomato bemisia tabaci resistance gene SlPR3 and application thereof

Abstract

The invention discloses a tomato bemisia tabaci-resistant gene SlPR3 and application thereof, and belongs to the technical field of plant genetic engineering, and the nucleotide sequence of the tomato bemisia tabaci-resistant gene SlPR3 is as shown in SEQ ID NO.1; the sequence of the CDS of the tomato bemisia tabaci-resistant gene SlPR3 is as shown in SEQ ID NO.2, and the amino acid sequence of the expression protein of the tomato bemisia tabaci-resistant gene SlPR3 is as shown in SEQ ID NO.3. The invention further discloses a preparation method of the tomato bemisia tabaci-resistant gene SlPR3. A real-time fluorescence quantification technology proves that the expression quantity of the SlPR3 gene is obviously changed after the SlPR3 gene is damaged by bemisia tabaci, so that the effect of the SlPR3 gene in tomato bemisia tabaci resistance is indicated. The invention also constructs an overexpression tomato plant of the tomato bemisia tabaci resistance gene SlPR3, detects the resistance phenotype of the SlPR3 gene when the plant is invaded by bemisia tabaci, proves the resistance effect of the SlPR3 gene on bemisia tabaci, and has important application value in the field of cultivation of insect-resistant crop varieties.

Description

Technical Field

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

[0002] Tomato (Solanum lycopersicum) is a Solanaceae plant native to Central and South America. It is an important model organism for the study of Solanaceae flowering plants. It is now widely planted around the world and has become one of the most popular and widely used fruits in the world because it is rich in vitamins and lycopene and can enhance human immunity. However, tomatoes are often attacked by a variety of pests and diseases during cultivation, including the broad-spectrum plant pest Bemisia tabaci. Bemisia tabaci is the most destructive and difficult to manage omnivorous pest in the world, causing great harm to agricultural production. Studies have reported that common cultivated varieties of tomatoes have low resistance to Bemisia tabaci. Bemisia tabaci can directly feed on the juice of tomato phloem, causing poor growth and development of plants; secrete honeydew on tomato fruits and leaves, causing abnormal ripening and mildew of tomatoes; Bemisia tabaci can also spread a variety of plant viruses such as Tomato Yellow Leaf Curl Virus, causing huge losses to tomato production. Spraying insecticides and treating with yellow boards is the most common way to repel whiteflies on the market. However, long-term and large-scale use of insecticides will not only make whiteflies resistant to pesticides, but also cause environmental and food safety problems. Genetic modification technology is the core of modern biotechnology. Its advantage is that it can increase crop yields, improve nutrition, and reduce pesticides by regulating the function of the organism's own genes. Therefore, exploring insect-resistant genes in tomatoes and cultivating whitefly-resistant varieties are of great significance to reducing the harm caused by whiteflies. Summary of the invention

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

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

[0005] SEQ ID NO.1 is:

[0006]

[0007] The CDS of the tomato whitefly resistance 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 resistance 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, and the specific preparation method 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 the 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 resistance gene SlPR3 and a plant overexpression vector containing the tomato whitefly resistance gene SlPR3 for enhancing the resistance of tomatoes to whiteflies.

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

[0017] Beneficial effects: The present invention uses the wild type M14 of tomato Ailsa Craig (AC) background as material, and detects the change of SlPR3 gene expression in tomato plant leaves when attacked by whiteflies through fluorescence quantitative detection technology. The SlPR3 gene is obtained by homologous cloning, an overexpression vector is constructed and M14 is transformed to obtain a tomato strain with overexpression of the SlPR3 gene, and the resistance phenotype of the SlPR3 gene when the plant is attacked by whiteflies is detected, proving that it has resistance to whiteflies and has important application value in the field of breeding 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 It is the expression level detection map of SlPR3 gene in tomato SlPR3 overexpression strain.

[0021] Figure 4 The resistance of the SlPR3 overexpressing strain to whiteflies is shown; (a) is 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) is 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 in conjunction with specific embodiments.

[0023] Example 1

[0024] In this example, the expression level of the SlPR3 gene after being attacked by whiteflies was investigated by fluorescent quantitative PCR technology. Based on the exon region of the SlPR3 gene, the fluorescent quantitative PCR forward primer (SEQ ID NO.6) 5'-CAATGGACGCCATCCCCTAA-3' and the reverse primer (SEQ ID NO.7) 5'-CGGACCCATCCCACATTCAA-3' were designed, and based on the Actin gene, the internal reference primers forward primer (SEQ ID NO.8) 5'-GGAACTTGAGAAGGAGCCTAAG-3' and the reverse primer (SEQ ID NO.9) 5'-CAACACCAACAGCAACAGTCT-3' were designed. Using the fluorescent quantitative PCR instrument CFX connect, the PCR system and program in the manufacturer's manual were referred to, and the number of cycles to reach the fluorescence threshold of the fluorescent quantitative PCR was obtained, and the relative expression level of the SlPR3 gene after the tomato was attacked by whiteflies was calculated.

[0025] like Figure 1 As shown, 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 by sequence comparison.

[0028] According to the ID of SlPR3 gene (Solyc02g082920), the corresponding CDS sequence information was found in the Solanaceae database (Sol Genomics Network), and the full-length sequence primers were designed using Primer 5.0, and restriction sites and homology arms were added to the 5' ends of the upstream and downstream primers, respectively. The specificity of the primers was determined by comparison in the NCBI database. The cDNA of wild-type M14 tomato was used as a template to PCR amplify the target fragment. The amplified fragment was sequenced after positive screening, and the sequencing results were compared to confirm that it was a complete SlPR3 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 was: 2×HieffCanace AdavanceFast PCR Master Mix 25ul, SlPR3-Fprimer 2μl, SlPR3-Rprimer 2μl, cDNA2μl, ddH2O to 50μl; the reaction program was: 98℃30sec; 98℃10sec, 60℃5sec, 72℃10sec, 32cycles; 72℃1min to obtain the PCR product.

[0038] Step 2, connecting the cloning vector;

[0039] The ligation reaction system (10 μl) was 3 μl of linearized pBI121 that had been cut with enzymes, 2 μl of recovered PCR products, and 1 μl of 2×HieffClone Enzyme Premix. The reaction conditions were: incubation 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 put 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 apply 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] A single colony was selected from the overnight culture plate and inoculated into LB liquid medium. The culture was cultured overnight at 37°C and 250 rpm to obtain bacterial solution. The bacterial solution was used as a template for PCR detection of recombinant transformants. The PCR reaction system was: 2×RapidTaq MasterMix 10μl, 35S 3'F primer 0.5μl, SlPR3 R primer 0.5μl, bacterial solution 1μl, ddH2O supplemented to 20μl; the reaction procedure was: 95°C 3min; 95°C 15sec, 60°C 10sec, 72°C 25s, 32cycles; 72°C 5min, electrophoresis detection was positive. The bacterial solution with positive detection was sent to Qingke Biotechnology Company for sequencing.

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

[0045] Example 3

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

[0047] Construction of SlPR3 overexpression vector: Sequencing confirmed that the target gene has been successfully connected to the pBI121 cloning vector. The 5' end of the SlPR3 gene contains a 35S promoter element to ensure that the SlPR3 gene is overexpressed in transgenic plants. The pBI121 vector is equipped with a Kana gene expression cassette, which is a screening marker for transgenic plant materials. Transgenic materials are screened by kanamycin.

[0048] Obtain positive strains: Add the target plasmid (SlPR3 overexpression vector) to Agrobacterium competent GV3101, place on ice for 30 minutes, liquid nitrogen for 1 minute, 37℃ water bath for 5 minutes, and stand on ice for 2 minutes. Add 900μl LB medium, shake culture at 28℃ for 1.5h; centrifuge at 12000rpm for 5 minutes, discard the supernatant (about 800μl), resuspend the bacteria and evenly spread on the LB plate with antibiotics (Rif 50μg·ml-1, Kana 50μg·ml-1), culture at 28℃ for 2-3 days, and identify the positive strains.

[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. Culture the sown sterile tomato seeds 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 picked and suspended in 100 ml LB liquid medium, and the culture was shaken at 28°C and 250 rpm for 14-18 h. When OD600 was about 0.6, it was divided into two 50 ml sterilized centrifuge tubes, centrifuged at 5000 rpm for 10 min, the supernatant was discarded, 5 ml MS liquid medium was added to each tube, and the bacterial liquid precipitate was resuspended by blowing with a pipette, and then 45 ml MS liquid medium was added to each tube. After the equal amount of resuspended bacterial liquid, 100 μl of acetosyringone (As) solution that promotes Agrobacterium infection was added and mixed thoroughly.

[0053] III. Leaf Co-cultivation

[0054] In the clean bench, place the tomato seedlings in a sterile culture dish, pour 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; take out 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 Culture

[0057] Take out the tomato leaves that have been co-cultured at 28℃ for 2-3 days and transfer them to the selection medium. Change the medium every one to two weeks, and continue to observe the callus formation and adventitious bud differentiation. When the adventitious buds begin to differentiate into seedlings, cut the seedlings with normal plant shape and transfer them to the culture bottle for further cultivation.

[0058] V. Rooting, screening, cultivation and seedling 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 rooting medium. Replace 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, gently wash off the excess medium attached to the roots of the seedlings, move them to nutrient soil, keep it highly moist, and gradually reduce the humidity in the culture environment according to the growth status of the seedlings until new leaves grow out.

[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. Figure 3 As shown, S1PR3 was not only overexpressed but also expressed at the highest level 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, and 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, and three biological replicates were set for each experiment.

[0063] (1) Select healthy six-leaf tomato seedlings with consistent growth status, one wild type and one transgenic strain, and place them in a breathable experimental device. Blow 75 female and male whiteflies into the device from the top hole. Close the 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 different strains with four leaves, punch holes in the same position on the leaf discs, and count the number of whiteflies laying eggs on the leaf discs 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 inverted fourth leaf position. Fifteen 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. After the whiteflies were infested, the number of whiteflies on each plant was counted at the same time every day for a total of 7 days. Figure 4 As shown in (b) in .

[0065] The number of eggs laid and the number of feedings of whiteflies in the overexpression plants were significantly less than those in the wild type, indicating that the SlPR3 gene has the function of whitefly resistance. The SlPR3 gene of tomato has important application value in enhancing the resistance of tomato 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. A tomato whitefly resistance gene S1PR3, characterized in that: The nucleotide sequence is shown in SEQ ID NO.1, the CDS sequence of the tomato whitefly resistance gene S1PR3 is shown in SEQ ID NO.2, and the amino acid sequence of the expression protein of the tomato whitefly resistance gene S1PR3 is shown in SEQ ID NO.

3.

2. A plant overexpression vector containing the tomato whitefly resistance gene S1PR3 according to claim 1, characterized in that: The method for constructing the plant overexpression vector is as follows: The SlPR3 gene was assembled using pBI121 as a vector. The SlPR3 gene was inserted into the pBI121 vector by gene recombination technology. The pBI121 vector was equipped with a 35S promoter element and a Kana gene expression cassette.

3. The use of the plant overexpression vector according to claim 2, characterized in that: Used to construct transgenic plants overexpressing the tomato whitefly resistance gene SlPR3.

4. Use of the tomato whitefly resistance gene S1PR3 according to claim 1 in enhancing the resistance of tomato to whitefly.

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