Tobacco bacterial wilt resistance gene NtPVS
Tobacco NtPVS knockout mutant created through CRISPR/Cas9 technology, it was found that the deletion of the NtPVS gene will cause the tobacco strain to be sensitive to blue wilt, providing the tobacco blue wilt resistance gene, solving the difficulty of effectively removing tobacco blue wilt in the existing technology. The problem of disease has achieved the resistance of tobacco to bacterium wilt.
Patent Information
- Application Number
- CN202411731460.1
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2024-11-29
- Publication Date
- 2025-02-25
AI Technical Summary
Tobacco green wilt is caused by green wilt bacteria. The prior art is difficult to effectively remove the disease, affecting the quality and yield of tobacco leaves.
Tobacco NtPVS knockout mutants were created through CRISPR/Cas9 gene editing technology, and it was found that the deletion of the NtPVS gene would make the tobacco strain more sensitive to infectious diseases, providing a tobacco blue wilt resistance gene NtPVS and its vector.
Tobacco's resistance to cyanobacteria has been achieved, new genetic resources have been provided, and support for the mining of genes related to cyanobacteria and the cultivation of new varieties of cyanobacteria.
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Abstract
Description
Technical Field
[0001] The invention belongs to the field of agricultural biotechnology, and in particular relates to a tobacco bacterial wilt resistance gene NtPVS. Background Art
[0002] Tobacco leaves are the main raw material for cigarettes, and their quality directly determines the quality of tobacco products. Tobacco bacterial wilt is a soil-borne bacterial disease caused by Ralstonia solanacearum (abbreviated as Ralstonia solanacearum). It is a typical vascular disease. When it occurs, it will cause the xylem vascular bundles of tobacco roots, stems, and leaves to be unable to transport water normally, causing tobacco plants to wilt or even die, which seriously affects and restricts the quality and yield of tobacco leaves.
[0003] Studies have shown that Ralstonia solanacearum is mainly spread by running water, fertilizers, diseased seedlings, humans and livestock, and production tools, and its transmission methods are diverse and complex. In addition, Ralstonia solanacearum can survive on host diseased remains for 7 months, in soil or compost for 2-3 years, and in soil for up to 8-25 years. As a stubborn pathogen, its eradication has always been the focus of research. Existing technologies mainly rely on the survival characteristics of Ralstonia solanacearum to eliminate it by rotating water and dryland crops, but this method not only has low sterilization efficiency, but also seriously affects the efficiency of tobacco planting.
[0004] Therefore, it is of vital importance to discover tobacco bacterial wilt resistance genes to enhance tobacco's resistance to bacterial wilt. Summary of the invention
[0005] The present invention utilizes CRISPR / Cas9 gene editing technology to create a tobacco NtPVS gene knockout mutant, and obtains a genetically homozygous NtPVS mutant through self-pollination.
[0006] By simultaneously infecting wild-type tobacco plants and NtPVS mutant tobacco plants with Ralstonia solanacearum and analyzing the disease index of the two, it was found that the deletion of the NtPVS gene would make the tobacco plants more sensitive to bacterial wilt infection, that is, the tobacco NtPVS gene has the ability to resist tobacco bacterial wilt.
[0007] In order to achieve the above object, the specific scheme adopted by the present invention is:
[0008] In a first aspect, the present invention provides a tobacco bacterial wilt resistance gene NtPVS, whose nucleotide sequence is shown in SEQ ID NO.1.
[0009] The second aspect of the present invention provides a vector comprising the tobacco bacterial wilt resistance gene NtPVS described in the first aspect, wherein the vector is pRGEB32-GhU6.7-NPTⅡ-NtPVS.
[0010] The tobacco bacterial wilt resistance gene NtPVS screened by the present invention can provide a new gene resource for the mining of tobacco bacterial wilt resistance-related genes and the breeding of new tobacco bacterial wilt resistance varieties. BRIEF DESCRIPTION OF THE DRAWINGS
[0011] Figure 1 This is a diagram of the induced expression pattern of the control material Yanyan 97 after inoculation with Ralstonia solanacearum in the RNA-seq data analysis of a specific implementation method.
[0012] Figure 2 This is a vector map containing the NtPVS gene in a specific embodiment;
[0013] Figure 3 This is a diagram showing the positive detection results of T0 generation transgenic tobacco materials in a specific embodiment;
[0014] Figure 4 This is a graph showing the growth of the NtPVS gene knockout strain and the control strain 15 days after inoculation with Ralstonia solanacearum in a specific embodiment;
[0015] Figure 5 This is a graph showing changes in disease index of NtPVS gene knockout strain and control strain in a specific implementation manner; DETAILED DESCRIPTION
[0016] The present invention is further described below by way of examples, but is not limited to these examples.
[0017] The experimental methods without specific conditions in the examples are usually carried out under conventional conditions and conditions described in the manual, or under conditions recommended by the manufacturer. The general equipment, materials, reagents, etc. used are all commercially available unless otherwise specified.
[0018] Example 1
[0019] This example briefly introduces the process of obtaining the tobacco NtPVS gene as follows.
[0020] Yanyan 97 material was sown in the greenhouse, and inoculated with Ralstonia solanacearum and water when it grew to the 6-leaf stage. Root samples were taken at 0h, 12h, 24h, 48h, and 72h. The roots treated with water were used as the control group. The transcriptome data were measured for inoculation mode induced expression analysis. The results showed that after inoculation with Ralstonia solanacearum, the NtPVS gene in Yanyan 97 had a significant up-regulated expression trend, while the NtPVS gene in the materials treated with water did not change in expression (such as Figure 1 As shown in the figure, CK is the blank control, R is the treatment with inoculation of Ralstonia solanacearum, and W is the clean water treatment).
[0021] Using the cultivated tobacco cultivar Yanyan 97 as a sample, Vazyme Plant DNA IsolationMini Kit was used to extract genomic DNA from tobacco leaves; Total RNA was extracted from tobacco leaves using the Super total RNA extraction kit and then Ⅲlst Strand cDNA Synthesis Kit (+gDNAwiper) reverse transcription kit reverse transcribes RNA into cDNA for later use.
[0022] By homology comparison, referring to the Arabidopsis gene sequence and the known tobacco partial gene sequence, the amplification primer sequences were designed as follows:
[0023] F: 5'-ATGGCCCCAGCTGCTGCAGTAC-3' (as shown in SEQ ID No. 2)
[0024] R: 5'-TTAAATTACAATAGAGTCCACCAGT-3' (as shown in SEQ ID No. 3)
[0025] Using the above prepared cDNA as template, PCR amplification was performed using the above primers:
[0026] Amplification system (50 μL):
[0027]
[0028] After mixing and centrifuging, PCR amplification was performed. The PCR reaction conditions were: 95°C for 1 min, 95°C for 15 sec, 56°C for 15 sec, 72°C for 3 min / 1 min, for a total of 35 cycles; 72°C for 10 min; 12°C Hold.
[0029] The amplified product was purified and sequenced to obtain the tobacco bacterial wilt resistance gene NtPVS, whose nucleotide sequence is shown in SEQ ID No. 1, including 1661 bp bases.
[0030] Example 2
[0031] Using the NtPVS gene sequence obtained in Example 1, this example further constructed a gene editing vector.
[0032] Step (1), using the CRISPR-P 2.0 website, select two specific sgRNA sequences in the NtPVS gene, namely Target1 sequence (5'-TTCATTCTCCCTCGCCAATCAGG-3', as shown in SEQ ID No. 4) and Target2 sequence (5'-ATGTTAATGTCCGCGGCTTCTGG-3', as shown in SEQ ID No. 5), then add the adapter sequence GTTTTAGAGCTAGAAATA downstream of the Target1 sequence to synthesize primer 1, after reverse complementation of Target1 and Target2, respectively, add the adapter sequence TGCACCAGCCGGGAAT downstream of the reverse complemented sequences of Target1 and Target2, respectively, to obtain primer 2 and primer 3, and further add the adapter sequence TTCTAGCTCTAAAAC upstream of the reverse complemented sequence of Target2 to synthesize primer 4, and then amplify them separately using the pGRT4 vector (researched and preserved by Huazhong Agricultural University) as a template, and then concatenate the two target sequences by overlap extension PCR to obtain the guide sequence of CRISPR / Cas9.
[0033] Step (2), PCR amplification of the guide sequence synthesized in step (1), the specific conditions are as follows:
[0034] Prepare the PCR reaction system: 2 μl Buffer, 0.3 μl 10 mM dNTPs, 0.5 μl Primer-S / A, 0.2 μl easyTaq (5U), 1 μl template, and add 15.5 μl ddH2O to make the total system 20 μl.
[0035] PCR amplification was carried out, and the PCR amplification conditions were as follows: pre-denaturation: 95°C, 4 min; denaturation: 95°C, 30 s; annealing: 55°C, 30 s; extension: 72°C, 20 s; cycle: 3 cycles; denaturation: 95°C, 30 s; annealing: 60°C, 30 s; extension: 72°C, 20 s; cycle: 28 cycles; final extension: 72°C, 5 min.
[0036] The amplified products were detected by 0.8% 1×TBE agarose gel electrophoresis.
[0037] Step (3), the PCR product synthesized in step (2) was connected to the pRGEB32-GhU6.7-NPTⅡ vector after restriction digestion by In-fusion, wherein the In-fusion connection system is: 1μl PCR product, 1μl linearized pRGEB32-NPTII-U6.7 vector (researched and preserved by Huazhong Agricultural University), 0.5μl Exnase, 1μl 5×CE Buffer, supplemented with 1.5μl ddH2O. The connection conditions are: 37°C water bath for 30min, placed on ice for 5min, and after the reaction, the reaction solution was transferred and stored at -20°C for future use.
[0038] Step (4): Take 5 μl of the reaction solution obtained in step (3) and transform it into 50 μl of DH5α competent cells. Gently flick to mix and place on ice for 30 min (do not shake); gently take out, heat shock at 42°C for 30 s, and immediately place on ice for 2 min; add 100 μl of LB liquid culture medium and culture at 37°C with shaking for 1 h; take 60 μl of the bacterial solution and spread it on LB solid culture medium (LB+50 mg / L Kan), and culture it upside down at 37°C overnight.
[0039] Step (5), pick the monoclonal clone prepared in step (4) and inoculate it into LB liquid culture medium (LB + 50 mg / L Kan), expand the culture until it becomes turbid, and follow the method of Beijing Quanshijin Company. Plasmid extraction was performed using the Plasmid MiniPrep Kit, and the resulting plasmid vector was pRGEB32-GhU6.7-NPTⅡ-NtPVS (see vector diagram). Figure 2 shown);
[0040] Step (6), transforming the successfully sequenced plasmid into EHA105 competent cells, preserving the strain for use in genetic transformation of tobacco.
[0041] Example 3
[0042] In this example, genetic transformation and tissue culture were performed to obtain plants in which the tobacco bacterial wilt resistance gene NtPVS was knocked out and edited. The relevant experimental process is briefly described as follows.
[0043] (1) Preparation of sterile seedlings
[0044] Disinfection: First, put the recipient material Yanyan 97 seeds into a 2mL centrifuge tube, add 700μL 75% ethanol solution and soak for 30sec in a sterile clean bench, aspirate the ethanol solution, and wash the seeds repeatedly with sterilized ddH2O for 3 times; add 700μL 15% H2O2 solution and soak for 8min; aspirate the H2O2 solution, and wash them repeatedly with sterilized ddH2O for 3 times.
[0045] Sowing: In a clean bench, use a high-temperature sterilized toothpick to evenly sow the disinfected seeds on the MS0 culture medium and place it in a 25℃ artificial climate chamber for cultivation.
[0046] Cultivation: When the seedlings grow to the cross stage, transfer the seedlings to MS0 solid culture bottles and continue culturing.
[0047] (2) Pre-culture of leaves / culture of Agrobacterium
[0048] Agrobacterium recovery: Streak the Agrobacterium EHA105 containing the target gene vector on a plate containing the corresponding antibiotics and culture in the dark at 28°C for 2 days until a single colony appears;
[0049] Pre-culture: After about 4 weeks, select robust and tender plants, cut the sterile seedling leaves into 1 cm2 leaf discs of about 0.5 cm*0.5 cm in a clean bench (discard the leaf veins), spread them on the TC culture medium for pre-culture, and culture them in a 25℃ artificial climate chamber under light for 2 days;
[0050] Small shaking: Pick a single clone and inoculate it into 1.5mL LB (LB+50mg / L Hyg+50mg / L Rif) liquid medium, 28℃, 200rpm, and culture for 24h;
[0051] Shake vigorously: Pipette 1 mL of the turbid bacterial solution and inoculate it into 50 mL of LB (LB + 50 mg / L Hyg + 50 mg / L Rif) liquid culture medium at 28°C, 200 rpm, and culture for 12 h;
[0052] Measurement: Use a UV spectrophotometer, set its wavelength to 600nm, set a blank group to calibrate the instrument and measure the absorbance of the sample. Measure the absorbance of the bacterial solution so that the absorbance OD value is in the range of 0.8 to 1.0, which can be used as the Agrobacterium infection solution.
[0053] (3) Infection and transformation of Agrobacterium
[0054] In the clean bench, place the pre-cultured leaves in a 9 cm sterile glass dish, add an appropriate amount of Agrobacterium liquid (make sure there is enough liquid to contact the material), leave it at room temperature for infection for 8 to 10 minutes (depending on the state of the leaves and liquid), and shake it from time to time.
[0055] After infection, take out the leaf disc and place it on sterilized filter paper to absorb the surface bacterial liquid, then place it on TC medium with the back of the leaf facing up, and culture it in the dark in a 25℃ artificial climate chamber for 3 days;
[0056] The leaves after co-cultivation were washed repeatedly 3-4 times with sterile ddH2O, and finally the excess water on the leaf surface was absorbed with sterile filter paper. After drying, the leaves were transferred to TD medium with the back of the leaves facing up and cultured in an artificial climate room at 25℃ under light for 14 days.
[0057] After 14 days, the leaves with positive callus on the edge were transferred to TS medium for further culture. Generally, small buds could be differentiated in about 15 days, and seedlings could be differentiated in 30 days.
[0058] When the differentiated buds grow to about 2 to 3 cm, cut them off and transfer them to TR medium first. Let the seedlings grow. After sampling and identification, transfer the positive plants to rooting bottles and continue to culture until they are ready for transplanting.
[0059] (4) PCR identification of transgenic tobacco
[0060] Using NtPVS-1F / NtPVS-1R primers, the plants were sent to a sequencing company for molecular testing to determine whether the NtPVS gene homozygous edited plants were obtained.
[0061] NtPVS-1F: 5'-CCCGTTTCGGACTTCTCTC-3' (SEQ ID No. 6)
[0062] NtPVS-1R: 5'-AGGCGTTCAACAATGTCTATC-3' (SEQ ID No. 7)
[0063] (5) Obtaining the homozygous editing mutant of the target gene NtPVS
[0064] The T0 generation of positive seedlings identified as successfully edited in step (4) were self-pollinated and harvested, and the seeds were planted to obtain the T1 generation. When the plants grew to 5-6 leaves, the leaves of individual plants were sampled and molecular detection was performed using Cas9F / Cas9R primers (the detection results were as follows Figure 3 The editing rate of T1 transgenic tobacco materials is shown in the following table.
[0065] Table 1 Editing rate of transgenic tobacco materials in T1 generations
[0066] Material Name Strain number Target-editing rate Target di-editing rate Edit Type NtPVS gene editing material 1 96％ 20％ Indel NtPVS gene editing material 2 100％ 36％ Indel NtPVS gene editing material 3 100％ 18％ Indel
[0067] Cas9F: 5'-GATCACCGACGAGTACAAGG-3' (SEQ ID No. 8);
[0068] Cas9R: 5'-CTCTTCCACCAGGAAGGACTC-3' (SEQ ID No. 9);
[0069] The leaves of the transformed plants obtained by gene editing were sampled and sent to BGI for molecular testing, which confirmed that the plants were gene-edited with the tobacco bacterial wilt resistance gene NtPVS.
[0070] Example 4
[0071] This example is mainly to observe the phenotype of the homozygous mutant of NtPVS gene editing. The following operations were performed:
[0072] The preserved Ralstonia solanacearum (LC3 strain) was streaked and cultured in a medium containing 2,3,5-triphenyltetrazolium chloride (TTC) for 3 days. When a single colony grew, a single colony was picked up and purified 2-3 times. The colony was grown in NA medium at 28°C for about 15 hours. The bacterial solution concentration was adjusted to OD600 = 0.1 using a spectrophotometer, and the bacterial suspension was placed at 4°C for use.
[0073] Prepare T1 transgenic lines with high editing rates and the control material Yanyan 97 for sowing and standby. When they grow to about 5-6 true leaves (about 35-45 days), randomly select 6 tobacco seedlings with similar plant height and growth from each of the three lines and the control group material, gently pull them out, wash the roots with clean water, soak them in a solution of Ralstonia solanacearum with OD600=0.1 for 5 minutes, take them out, and replant them in sterile small pots.
[0074] After inoculation of Ralstonia solanacearum, observe for disease occurrence about 7 days later, and investigate the disease occurrence of different transformed offspring according to the standard of "GB / T23222-2008 Grading and Investigation Methods of Tobacco Diseases and Insect Pests", record the date of the first disease occurrence of each transformed offspring after treatment, and record the occurrence of bacterial wilt in the number of diseased leaves. A total of 4 investigations were conducted to count the disease occurrence of different strains. The disease level is divided into 5 levels according to the disease level of greenhouse tobacco seedlings, specifically:
[0075] Level 0, the whole plant is disease-free
[0076] Level 1, occasional chlorotic spots on the stem
[0077] Level 2, black streaks on the stem
[0078] Level 3: Black streaks on the neck reach the top or more than 2 / 3 of the leaves on the diseased side wilt
[0079] Level 4: diseased plants are basically dead
[0080] The incidence rate and disease index of the offspring transformed with different genes were calculated in each survey, and the disease index was calculated based on the disease level.
[0081] Fifteen days after the inoculation of Ralstonia solanacearum, the second disease statistics were conducted and it was found that the disease conditions of the gene-edited materials of the three different strains of NTPVS were significantly aggravated compared with the control materials (e.g. Figure 4The NtPVS gene-edited plants showed severe wilting, with the disease level reaching level 2 or 3. However, the control materials occasionally had lesions on the leaves and stems, which were still generally at level 1. After completing the disease statistics for four times, the disease index of each strain after inoculation with Ralstonia solanacearum was calculated according to the disease index calculation formula (as shown in Figure 2). Figure 5 As shown, ntpvs is the NtPVS gene editing material, and WT is Yanyan 97).
[0082] According to the calculation results, compared with the control material, the disease index of the gene-edited material increased faster and more significantly over time, showing a weakened resistance to Ralstonia solanacearum. This shows that the functional loss of the NtPVS gene has a significant impact on the resistance response of tobacco to Ralstonia solanacearum infection.
[0083] It should be understood that after reading the above content 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.
Claims
1. A tobacco bacterial wilt resistance gene NtPVS, characterized in that: Its nucleotide sequence is shown in SEQ ID NO.
1.
2. A vector comprising the tobacco bacterial wilt resistance gene NtPVS according to claim 1.