CN115976046A - SlCAS gene and application of protein coded by same in regulation and control of tomato gray mold resistance - Google Patents

SlCAS gene and application of protein coded by same in regulation and control of tomato gray mold resistance Download PDF

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CN115976046A
CN115976046A CN202211279246.8A CN202211279246A CN115976046A CN 115976046 A CN115976046 A CN 115976046A CN 202211279246 A CN202211279246 A CN 202211279246A CN 115976046 A CN115976046 A CN 115976046A
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slcas
gene
tomato
gray mold
mold resistance
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石武良
贾承国
王喆
李斌
张明哲
王晓丽
张玉斌
秦建春
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Jilin University
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Jilin University
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Abstract

The invention is applicable to the technical field of microbial genetic engineering, and provides an application of an SlCAS gene and a protein coded by the SlCAS gene in regulation and control of tomato gray mold resistance, wherein a nucleotide sequence of the SlCAS gene is shown as SEQ ID NO.1, and an amino acid sequence of the SlCAS protein coded by the SlCAS gene is shown as SEQ ID NO. 2. The SlCAS gene is transferred into a tomato explant in an agrobacterium tumefaciens mediated mode, after a transgenic plant is inoculated with botrytis cinerea, phenotype observation and physiological index determination are carried out on the transgenic plant and a wild plant of a control group, the physiological mechanism of resisting gray mold of the transgenic plant is researched, the molecular mechanism of the related gene level is explored, the three overexpression tomato strains SlCAS-2, slCAS-4 and SlCAS-6 are verified to have obvious gray mold resistance, and the tomato gene SlCAS has obvious gray mold resistance.

Description

SlCAS gene and application of protein coded by same in regulation and control of tomato gray mold resistance
Technical Field
The invention belongs to the technical field of microbial genetic engineering, and particularly relates to an application of SlCAS gene and protein coded by the SlCAS gene in regulation and control of tomato gray mold resistance.
Background
Plant gray mold caused by Botrytis cinerea (Botrytis cinerea) belongs to a worldwide disease, and is a common fungal disease which is difficult to control in open and protected plants. Botrytis cinerea is a typical necrotrophic pathogenic fungus that can infect over 1400 different host plants, including almost all vegetables and fruit trees. The host plants can be attacked from the seedling stage to the fruiting stage, the transportation stage and the storage stage, so that huge economic loss is caused, particularly, in the rapid development stage of modern facility agriculture, a proper environmental condition is provided for the occurrence of gray mold, and the hazard of the gray mold tends to be improved year by year. Just because of its wide host and great harm, the famous botrytis cinerea is the second most important fungal pathogen in molecular plant pathology. At present, no effective method for preventing and treating the botrytis cinerea exists, and the traditional solution is to use a large amount of pesticide, so that the method not only relates to food safety, but also causes environmental pollution and seriously harms the health of people. Therefore, there is a strong need to deeply study the pathogenic mechanism of Botrytis cinerea to find an effective and environmentally friendly method for controlling Botrytis cinerea.
The tomato (tomato lycopersicum) is rich in nutrient components such as vitamins, lycopene, protein and the like, has a wide planting area in the world and is popular with consumers. But the disease seriously restricts the production and the preservation after the picking, and reduces the economic benefit of the production. With the popularization of greenhouse cultivation, the abuse of a large amount of pesticides and the increase of the resistance of the tomato to gray mold, the prevention and the treatment of the gray mold are increasingly difficult. Therefore, the method has important value in cultivating tomato gray mold resistant varieties.
Disclosure of Invention
The embodiment of the invention aims to provide an application of an SlCAS gene and a protein coded by the SlCAS gene in regulation and control of tomato gray mold resistance, and aims to solve the problems in the background art.
The embodiment of the invention is realized by applying the SlCAS gene and the protein coded by the SlCAS gene in regulation and control of tomato gray mold resistance, wherein the nucleotide sequence of the SlCAS gene is shown as SEQ ID No.1, and the amino acid sequence of the SlCAS protein coded by the SlCAS gene is shown as SEQ ID No. 2.
In a further technical scheme, the regulation and control of the tomato gray mold resistance are realized by cultivating a transgenic plant with an SLCAS gene overexpression.
In a further technical scheme, the method for acquiring the transgenic plant comprises the following steps:
step 1: the construction of an over-expression vector,
extracting total RNA of leaves of wild tomato plants, reverse transcribing to obtain a first cDNA chain, taking the obtained cDNA as a template, and taking a primer pair as follows:
SlCAS1-L:TACGAACGATACTCGACCCCATGGCACTTAGAGCTTCAGCSlCAS1-R:TAGAGTCGACGGATCCCCATCTGAAAGCAATTTGACAGTGCC；
PCR amplification is carried out, and DNA fragments of about 1100bp are recovered from the gel. The pCambia1300-YFP vector is digested by restriction enzyme SmaI to be linearized, and the linearized vector is recovered by gel electrophoresis. And connecting the target gene fragment to a pCambia1300-YFP vector by using a seamless cloning technology to construct an overexpression vector.
Step 2: agrobacterium tumefaciens mediated genetic transformation to obtain over-expressed plants,
and (3) transforming the recombinant plasmid 35S-SlCAS-YFP to agrobacterium tumefaciens GV3101 by a freeze-thaw method to obtain the recombinant agrobacterium tumefaciens. Selecting positive bacteria to carry out bacteria selection and bacteria preservation, storing a bacterial liquid in a refrigerator at minus 80 ℃ to carry out subsequent experiments, pre-culturing the tomato explants subjected to aseptic treatment for 2d, soaking the expanded cotyledon explants in the prepared agrobacterium liquid for 10-15 min, absorbing redundant bacterial liquid on sterilized filter paper, and then inoculating the sterilized filter paper on a pre-culture medium on which the sterilized filter paper is laid. Then carrying out subculture and rooting culture to obtain a T0 generation tomato plant, and selfing to obtain a T1 generation transgenic seed;
step 3, identifying the gray mold resistance function of the transgenic plant,
step 3.1: sowing and cultivating plant materials;
step 3.2: identification of transgenic Positive seedlings
DNA was extracted from fresh leaf samples by a method using 2% CTAB (cetyltrimethylammonium bromide) extraction buffer. Primer:
35SF：GACGCACAATCCCACTATCC，
SlCAS1RT2: TAGATGGAAGACGAGGGA; carrying out PCR amplification, and after the PCR is finished, carrying out PCR amplification according to the following steps of 1g: agarose and TAE buffer solutions were prepared at a ratio of 100mL, and the transgenic vector was used as a positive control, while a negative control was set. The plant with the corresponding strip with the overexpression vector is a positive plant, and the positive plant is selected for subsequent seed reservation and other experiments.
In a further technical scheme, the step 2 of the freeze-thaw transformation comprises the following specific steps:
step 2.1: the agrobacterium tumefaciens stored at the temperature of minus 80 ℃ is taken to be in a sensitive state at room temperature or palm for a moment until part of the agrobacterium tumefaciens is melted, and the agrobacterium tumefaciens is inserted into ice when the agrobacterium tumefaciens is in an ice-water mixed state.
Step 2.2: add 5. Mu.L (or less) plasmid DNA per 50. Mu.L competence, dial the tube bottom by hand and mix well, stand on ice for 5min, liquid nitrogen for 5min, water bath for 5min at 37 ℃ and ice bath for 5min.
Step 2.3: adding 500 mul LB liquid culture medium (without antibiotic), shaking culturing at 28 deg.C for 3-5 h.
Step 2.4: centrifuging at 6000rpm for 1min to collect bacteria, taking about 100 microliter of supernatant, lightly blowing and beating the heavy suspension bacteria block, coating the heavy suspension bacteria block on an LB (lysogeny broth) plate containing rifampicin and hygromycin, and inversely placing the heavy suspension bacteria block in a 28 ℃ incubator for culturing for 2-3 days.
According to a further technical scheme, the preparation of the agrobacterium liquid in the step 2 specifically comprises the following steps:
5 mu L of the preserved bacteria liquid is sucked and inoculated in 5mL LB liquid culture medium containing rifampicin and hygromycin (the specific dosage can be increased according to the proportion), and the liquid culture medium is subjected to shaking culture at the temperature of 28 ℃ and at the speed of 200rpm/min until the logarithmic phase. Determination of its 0D 600 When the value is about 0.8, centrifuging for 10min at 8000rpm in a centrifuge, discarding the supernatant, washing the precipitate twice with MS liquid culture medium, and diluting with MS liquid culture medium to a value of 0.3-0.4 to obtain the agrobacterium liquid for infection.
In a further technical scheme, the step 3.1 comprises the following specific steps:
the tomato is required to be subjected to germination accelerating treatment before planting, seeds are placed into a culture dish padded with two pieces of absorbent filter paper, and a proper amount of distilled water is added to perform germination accelerating in a culture room for 2-3 days in the dark. After germination, the seedlings are sowed in a nutrition pot which takes peat soil and vermiculite (the volume ratio is 3:1) as mixed substrates, and the tops of the seedlings are covered with the substrates with the same thickness. Placing in a culture chamber, wherein the growth environment is as follows: temperature is 25/20 ℃ (day/night), photoperiod is 16/8h, light-dark cycle, and illumination intensity is 600mol · m -2 ·s -1 1g of nutrient fertilizer and 1L of distilled water are added to prepare nutrient solution for culture.
In a further technical scheme, the PCR reaction system (10 μ L) in the step 3.2 is as follows:
Figure BDA0003897403110000041
the reaction conditions are as follows:
Figure BDA0003897403110000042
the SlCAS gene and the application of the protein coded by the SlCAS gene in regulation and control of the tomato gray mold resistance are realized by transferring the SlCAS gene into a tomato explant in an agrobacterium tumefaciens mediated mode, inoculating botrytis cinerea to a transgenic plant, performing phenotype observation and physiological index measurement on the transgenic plant and a wild plant of a control group, researching the gray mold resistance physiological mechanism of the transgenic plant, exploring a molecular mechanism of a gene level involved in the phenotype observation and physiological index measurement, verifying that overexpression tomatoes SlCAS-2, slCAS-4 and SlCAS-6 have obvious gray mold resistance, and ensuring that the tomato gene SlCAS has obvious gray mold resistance.
Drawings
FIG. 1 shows the predicted expression levels of tomato SlCAS gene in different tissues;
FIG. 2 shows the expression analysis of SlCAS gene under salt stress;
FIG. 3 shows the expression analysis of SlCAS gene under low temperature stress;
FIG. 4 shows the expression analysis of SlCAS gene under high temperature stress;
FIG. 5 is an expression analysis of SlCAS gene under drought stress;
FIG. 6 shows the expression analysis of SlCAS gene under abscisic acid (ABA) treatment;
FIG. 7 shows the expression analysis of SlCAS gene under Brassinolide (BR) treatment;
FIG. 8 shows the susceptible conditions of tomato leaves of different strains after in vitro inoculation of Botrytis cinerea;
FIG. 9 shows the leaf lesion spot area after MT and three CAS transgenic strains are inoculated with Botrytis cinerea in vitro for 3d treatment;
FIG. 10 is the disease index of different strains after spraying of Botrytis cinerea;
FIG. 11 shows the POD content after spraying with Botrytis cinerea.
Detailed Description
In order to make the objects, technical solutions and advantages of the present invention more apparent, the present invention is described in further detail below with reference to the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are merely illustrative of the invention and are not intended to limit the invention.
Specific implementations of the present invention are described in detail below with reference to specific embodiments.
The first embodiment is as follows:
1. the expression condition of SlCAS at different tissue parts of a wild tomato plant is shown in the specification, wherein the nucleotide sequence of the SlCAS gene is shown in SEQ ID No.1, and the amino acid sequence of the SlCAS protein coded by the SlCAS gene is shown in SEQ ID No. 2.
The used plant material is common experimental variety Micro Tom of tomato, and is bred and stored in a laboratory.
And detecting the expression conditions of the SlCAS in different tissue parts of wild tomato plants by using a real-time fluorescence quantitative technology, wherein the tissue parts comprise roots, stems, leaves, flowers, fruits (14 d after pollination) and mature red fruits. Total RNA extraction was performed using a kit manufactured by Beijing Quan Shi gold Biotech Ltd, a kit PrimeScript manufactured by TAKARA TM First strand cDNA synthesis was performed using RT Master Mix and fluorescent quantitative PCR assay was performed using CFX from Bio-Rad with 3 biological replicates per group. Analyzing the gene expression condition of the target gene in different parts or different plants according to the expression result of the target gene, and adopting 2 for data analysis -△△CT The method of (1). The reaction system and reaction conditions were as follows:
Figure BDA0003897403110000061
loading samples according to the table, taking the Actin of tomato as an internal reference gene, and the program is as follows: pre-denaturation 95 ℃ 30s, PCR reaction 95 ℃ 5s,60 ℃ 30s, for a total of 40 cycles. The melting curve procedure was 95 ℃ 5s,60 ℃ 1min,95 ℃ 15s,50 ℃ 30s. Each sample was repeated 3 times and the fluorescence change curve was analyzed after the reaction was completed.
Wherein, the primers for detecting the SlCAS are as follows:
SlCAS1RT1:CGTGGTTACAAGGGTGAA，
SlCAS1RT2:TAGATGGAAGACGAGGGA；
the primer of the Actin is as follows:
SlActin-F:TGTCCCTATCTACGAGGGTTATGC；
SlActin-R:AGTTAAATCACGACCAGCAAGAT。
2. SlCAS expression condition of wild type tomato plant after different periods of salt treatment
Salt treatment (200 mmol/L NaCl solution) is carried out on a batch of wild tomatoes, tomato plants are sampled at 0h, 1h, 3h, 6h, 12h and 24h of the beginning treatment respectively, 3 plants are taken each time, and the next time of sampling is not taken. The expression of SlCAS in different periods of salt treatment, the extraction of total RNA, the synthesis of cDNA first strand and the determination of expression level are the same as above.
3. Obtaining transgenic plants
1. Construction of overexpression vectors
Extracting total RNA of leaves of wild tomato plants, reverse transcribing to obtain a first cDNA chain, taking the obtained cDNA as a template, and taking a primer pair as follows:
SlCAS1-L:TACGAACGATACTCGACCCCATGGCACTTAGAGCTTCAGCSlCAS1-R:TAGAGTCGACGGATCCCCATCTGAAAGCAATTTGACAGTG CC；
PCR amplification is carried out, and DNA fragments of about 1100bp are recovered from the gel. The pCambia1300-YFP vector is digested by restriction enzyme SmaI to be linearized, and the linearized vector is recovered by gel electrophoresis. And connecting the target gene fragment to a pCambia1300-YFP vector by using a seamless cloning technology to construct an overexpression vector.
2. Obtaining over-expression plant by agrobacterium tumefaciens mediated genetic transformation
And (3) transforming the recombinant plasmid 35S-SlCAS-YFP to agrobacterium tumefaciens GV3101 by a freeze-thaw method to obtain the recombinant agrobacterium tumefaciens. The steps of the freeze-thaw method conversion are as follows:
(1) The agrobacterium tumefaciens stored at the temperature of minus 80 ℃ is taken to be in a sensitive state at room temperature or palm for a moment until part of the agrobacterium tumefaciens is melted, and the agrobacterium tumefaciens is inserted into ice when the agrobacterium tumefaciens is in an ice-water mixed state.
(2) Add 5. Mu.L (or less) plasmid DNA per 50. Mu.L competence, dial the tube bottom by hand and mix well, stand on ice for 5min, liquid nitrogen for 5min, water bath for 5min at 37 ℃, ice bath for 5min.
(3) Adding 500 mu L LB liquid culture medium (without antibiotic), shaking culturing at 28 deg.C for 3-5 h.
(4) Centrifuging at 6000rpm for 1min to collect bacteria, leaving about 100 microliter of supernatant, lightly blowing and beating the heavy suspension bacteria block, coating the heavy suspension bacteria block on an LB (lysogeny broth) plate containing rifampicin and hygromycin, and inversely placing the heavy suspension bacteria block in a 28 ℃ incubator for culturing for 2-3 days.
Selecting positive bacteria, selecting and preserving the positive bacteria, and storing the bacteria liquid in a refrigerator at the temperature of minus 80 ℃ for subsequent experiments.
Preparation of an agrobacterium liquid: sucking 5 μ L of the well-preserved bacterial liquid, inoculating to a culture medium containingRifampicin and hygromycin in 5mL LB liquid medium (the specific dosage can be increased proportionally), at 28 ℃, 200rpm/min shaking culture to logarithmic phase. Determination of its 0D 600 When the value is about 0.8, centrifuging for 10min at 8000rpm in a centrifuge, discarding supernatant, washing the precipitate twice by using an MS liquid culture medium, and then diluting to a value of 0.3-0.4 by using the MS liquid culture medium to obtain agrobacterium liquid for infection. After the tomato explants which are subjected to aseptic treatment are pre-cultured for 2d, the expanded cotyledon explants are soaked in the prepared agrobacterium liquid for 10-15 min, and the excess liquid is absorbed on the sterilized filter paper and then inoculated on a pre-culture medium on which the sterilized filter paper is laid. Then carrying out subculture and rooting culture to obtain T0 generation tomato plants, and carrying out selfing to obtain T1 generation transgenic seeds.
4. Identification of gray mold resistance function of SICAS overexpression tomato material
1. Seeding and cultivation conditions of plant material
The tomato is required to be subjected to germination accelerating treatment before planting, seeds are placed into a culture dish padded with two pieces of water-absorbing filter paper, and a proper amount of distilled water is added to be cultured in the dark of a culture room for 2-3 days for accelerating germination. After germination, the seedlings are sowed in a nutrition pot which takes peat soil and vermiculite (the volume ratio is 3:1) as mixed substrates, and the tops of the seedlings are covered with the substrates with the same thickness. Placed in a culture room, and the growth environment is as follows: temperature 25/20 deg.C (day/night), photoperiod 16/8h light-dark cycle, and illumination intensity 600mol · m -2 ·s -1 1g of nutrient fertilizer and 1L of distilled water are added to prepare nutrient solution for culture.
2. Identification of transgenic Positive seedlings
DNA was extracted from fresh leaf samples by a method using 2% CTAB (cetyltrimethylammonium bromide) extraction buffer. Primer:
35SF：GACGCACAATCCCACTATCC，
SlCAS1RT2: TAGATGGAAGACGAGGGA; PCR amplification was performed in the following PCR reaction system (10. Mu.L):
Figure BDA0003897403110000091
the reaction conditions are as follows:
Figure BDA0003897403110000092
after the PCR was completed, the reaction was carried out in a volume of 1g: agarose and TAE buffer solutions were prepared at a ratio of 100mL, and the transgenic vector was used as a positive control, while a negative control was set. And selecting the positive plants to carry out subsequent seed reservation and other experiments.
3. Phenotype observation of overexpression SlCAS (plant transformation and integration System) plant and wild-type plant
In the plant growth process, the wild type Micro Tom and SlCAS transgenic T2 generation plants in the same growth period have larger difference in plant height, and wild type plants are obviously higher than SlCAS overexpression plants. After two groups of plants sowed in the same batch grow for two and a half months, the height and the stem thickness of the plants do not change any more, phenotype observation is carried out on the two groups of plants, and the height and the stem thickness of the plants are measured. Selecting tomato plants with the same growth vigor in the same growth environment in each group, and measuring the plant height by taking the position of a cotyledon node to the apical meristem as a range, wherein the unit is cm; the thickness of the stem is measured in mm by a vernier caliper based on the diameter of the first internode.
4. Preparation of a conidia suspension of Botrytis cinerea
Botrytis cinerea (Botrytis cinerea) standard strain B05.10. The botrytis cinerea standard strain B05.10 is reactivated by using a PDA culture medium, a large number of conidia are generated on the surface of the culture medium when the culture medium is cultured to the 10 th day, the conidia on the surface of a culture dish are collected by using 1/2PDB culture solution, and hyphae are removed by filtration. Adjusting the concentration of spore liquid to 1 × 10 6 The spore and hyphae are collected when the spore germination rate is 80-90%, and the low-oxygen environment simulating the process of infiltrating Botrytis cinerea into the host is used as the control of the wild type strain. After liquid nitrogen treatment, the RNA is stored in an ultra-low temperature refrigerator at-80 ℃ for RNA extraction.
5. Cultivation of tomato
Tomato (Solanum lycopersicum cv Moneymaker) was cultured under conditions of 25 ℃, relative humidity 80%, photoperiod 10h light/dark for 7 weeks for the interaction experiment with botrytis cinerea.
6. Tomato and botrytis cinerea inoculation treatment and pathogenicity determination
Tomato and botrytis cinerea interaction group: the concentration of the spore suspension of the wild type strain B05.10 was adjusted to 1X 10 using the PDB medium, respectively 5 Perml, inoculated on mature tomato detached leaves. Wild type strains tomato leaves were inoculated with the cake and the cake was removed by punching with a sterile 200. Mu.l yellow pipette at the edge of a fresh colony. The concentration is 1 x 10 6 Spraying and inoculating a/ml spore solution on tomato leaves, then placing the tomato leaves in a sealed incubator with saturated humidity for culturing for 24 hours, and carrying out three independent treatments, wherein each treatment is sprayed with 20ml spore suspension; tomato control group: the treatment was carried out by spraying 1/2PDB medium and three independent treatments were carried out under the same conditions, each treatment being carried out by spraying 2 ml of 1/2PDB medium. Leaf lesion size was determined using ImageJ software and statistical analysis was performed. All groups of samples are mixed and treated by liquid nitrogen, and stored in an ultra-low temperature refrigerator at minus 80 ℃ to serve as a biological experiment.
And (3) data analysis:
1.SlCAS expression conditions of different tissue parts of wild type tomato plant
The expression conditions of SlCAS in different organs of wild tomato plants are detected by utilizing a qPCR technology, and the result shows that the expression level of SlCAS in leaves is the highest, and then flower buds and flowers are arranged, the expression level is higher in the early fruit development time, the expression level is reduced along with the maturation of fruits, and the expression level in roots is very low (as shown in figure 1).
2. Expression analysis of tomato SlCAS gene under different stresses
The expression condition of SlCAS in tomatoes under salt stress is researched, and real-time fluorescence quantification results show that after the tomatoes are subjected to the salt stress, obvious increased expression can be seen in the first 1h, but the expression level begins to gradually decrease along with the lapse of time, the expression level is lowest in 6h, but the expression level begins to gradually increase in the subsequent 18h, and the maximum value is reached in 24 h. This indicates that salt stress has a significant effect on the expression level of SlCAS in tomato (as shown in figure 2).
The expression condition of the SlCAS in the tomato under low-temperature stress is researched, and a real-time fluorescence quantification result shows that the expression quantity of the SlCAS gene is remarkably reduced in the first 1h under the low-temperature stress, and is increased in 3h-6h, but the whole SlCAS gene still shows a very obvious reduction trend compared with a control group. It shows that the low temperature stress has certain influence on the expression of the tomato SlCAS gene (shown in figure 3).
The expression condition of the SlCAS in the tomato under high-temperature stress is explored, and a real-time fluorescence quantitative result shows that the SlCAS gene is integrally up-regulated to different degrees in the whole time period under the high-temperature stress. Indicating that high temperature stress has a significant effect on the expression of the tomato SlCAS gene (as shown in fig. 4).
The expression condition of the SlCAS in the tomato under drought stress is explored, and real-time fluorescence quantitative results show that the gene expression level is reduced remarkably all the time after the drought stress is carried out at different time. Indicating that drought stress has a significant effect on expression of the tomato SlCAS gene (as shown in fig. 5).
Therefore, it can be speculated that the tomato SlCAS gene has certain functions and effects in the salt stress and temperature stress tolerance of plants and participates in the abiotic stress regulation of tomatoes.
3. Expression analysis of tomato SlCAS gene under different hormone treatment
Tomato plants were treated with abscisic acid (ABA) and Brassinolide (BR), and the effect of treatment with different hormones on the expression level of the SlCAS gene was analyzed. The results are shown in fig. 6 and 7: the gene expression level gradually decreases in the first 3h under the ABA treatment, but the expression level tends to increase extremely remarkably from 3h and tends to the original level after 1 d; under BR treatment, the SlCAS gene shows a remarkable rising trend in a short time, especially within 1h after treatment. The longer the treatment time, the slower the expression amount decreased. We speculate that the solanum lycopersicum gene is involved in the regulation process of these two hormones, where the response to BR treatment is more sensitive.
4. Phenotypic observation and statistical analysis of physiological data after inoculation of Botrytis cinerea
Selecting leaves of tomato lines SlCAS-2, slCAS-4, slCAS-6 and wild type MT with consistent growth vigor respectively, placing on a wet filter paper, inoculating botrytis cinerea stipe cultured at the same time, and observing phenotypic change after inoculating for 3d. The results are shown in FIG. 8. The leaves of the wild MT are withered and yellow in color and are seriously dehydrated, and the leaves inoculated with the botrytis cinerea cakes are already withered around the inoculation points and seriously damaged. While SlCAS-2, slCAS-4 and SlCAS-6 have significantly less MT damage than MT damage. The inoculated leaves of the SlCAS-6 are more serious than those of the SlCAS-2 and SlCAS-4, the whole leaves have yellowing signs, small areas around bacterial plaques are yellow and dry, the SlCAS-2 only has normal water loss, and the leaves of the SlCAS-4 hardly have any change.
For the experimental results of in vitro inoculation of botrytis cinerea, imageJ was used to perform statistical analysis on the lesion area of the leaves, and the statistical analysis results are shown in fig. 9: compared with wild MT, slCAS-2, slCAS-4 and SlCAS-6 all have different degrees of gray mold resistance, wherein the gray mold resistance of SlCAS-2 and SlCAS-4 is stronger.
Therefore, a treatment mode of spraying botrytis cinerea spore suspension is further performed on the strains SlCAS-2 and SlCAS-4, the plant infection conditions and POD (peroxidase) indexes are subjected to statistical analysis, and the results are shown in figures 10 and 11, and as can be seen in figures 10 and 11, the over-expressed tomatoes SlCAS-2, slCAS-4 and SlCAS-6 have remarkable gray mold resistance and the tomato gene SlCAS has remarkable gray mold resistance.
The above description is intended to be illustrative of the preferred embodiment of the present invention and should not be taken as limiting the invention, but rather, the intention is to cover all modifications, equivalents, and alternatives falling within the spirit and scope of the invention.

Claims (6)

  1. The application of the SlCAS gene and the protein coded by the SlCAS gene in regulation and control of tomato gray mold resistance is characterized in that the nucleotide sequence of the SlCAS gene is shown in SEQ ID No.1, and the amino acid sequence of the SlCAS protein coded by the SlCAS gene is shown in SEQ ID No. 2.
  2. 2. The SlCAS gene and the application of the protein encoded by the SlCAS gene in regulation of tomato gray mold resistance of tomato, as claimed in claim 1, wherein the regulation of tomato gray mold resistance is realized by cultivating a transgenic plant of SlCAS gene overexpression.
  3. 3. The SlCAS gene and the application of the protein coded by the same in regulation and control of tomato gray mold resistance, as claimed in claim 2, wherein the method for obtaining the transgenic plant comprises the following steps:
    step 1: the construction of an over-expression vector,
    extracting total RNA of leaves of wild tomato plants, reversely transcribing to obtain a first cDNA chain, taking the obtained cDNA as a template, and taking a primer pair as follows:
    SlCAS1-L:TACGAACGATACTCGACCCCATGGCACTTAGAGCTTCAGC，
    SlCAS1-R:TAGAGTCGACGGATCCCCATCTGAAAGCAATTTGACAGTGCC；
    performing PCR amplification, carrying out gel recovery on a 1100bp DNA fragment, carrying out restriction enzyme SmaI enzyme digestion on the pCambia1300-YFP vector to linearize the vector, and carrying out gel electrophoresis to recover a linearized vector; connecting a target gene fragment to a pCambia1300-YFP vector by utilizing a seamless cloning technology to construct an overexpression vector;
    and 2, step: agrobacterium tumefaciens mediated genetic transformation to obtain over-expressed plants,
    transforming the recombinant plasmid 35S-SlCAS-YFP to agrobacterium tumefaciens GV3101 by a freeze-thaw method to obtain recombinant agrobacterium, selecting positive bacteria to carry out bacteria selection and preservation, storing a bacterial solution in a refrigerator at minus 80 ℃ to carry out subsequent experiments, pre-culturing an aseptically processed tomato explant for 2 days, soaking the expanded cotyledon explant in the prepared agrobacterium bacterial solution for 10-15 min, absorbing redundant bacterial solution on sterilized filter paper, inoculating the sterilized filter paper onto a pre-culture medium paved with the sterilized filter paper, then carrying out subculture and rooting culture to obtain a T0 generation tomato plant, and selfing to obtain a T1 generation transgenic seed;
    step 3, identifying the gray mold resistance function of the transgenic plant,
    step 3.1: sowing and cultivating plant materials;
    step 3.2: identification of transgenic Positive seedlings
    Fresh leaf samples are taken to extract DNA, a method of extracting a buffer solution by adopting 2% hexadecyl trimethyl ammonium bromide for extracting genome DNA is adopted, and primers are as follows:
    35SF：GACGCACAATCCCACTATCC，
    SlCAS1RT2: TAGATGGAAGACGAGGGA; carrying out PCR amplification, and after the PCR is finished, carrying out PCR amplification according to the following ratio of 1g: agarose and TAE buffer solution are prepared according to the proportion of 100mL, the transgenic vector is used as a positive control, meanwhile, a negative control is set, the positive plant is obtained when the transgenic vector has a corresponding strip with the overexpression vector, and the positive plant is selected for subsequent seed reservation and experiments.
  4. 4. The SlCAS gene and the protein encoded by the same as claimed in claim 3, wherein the specific steps of the freeze-thaw transformation in step 2 are as follows:
    step 2.1: taking agrobacterium tumefaciens strain preserved at the temperature of minus 80 ℃, keeping the strain in a state of being infected by the agrobacterium tumefaciens at room temperature or palm for a moment until part of the strain is melted, and inserting the strain into ice when the strain is in a state of being mixed with ice water;
    step 2.2: adding 5 μ L plasmid DNA into every 50 μ L competence, dialing tube bottom with hand, mixing, standing on ice for 5min, liquid nitrogen for 5min, water bath at 37 deg.C for 5min, and ice bath for 5min;
    step 2.3: adding 500 mu L of LB liquid culture solution without antibiotics, and carrying out shaking culture at 28 ℃ for 3-5 h;
    step 2.4: centrifuging at 6000rpm for 1min to collect bacteria, collecting 100 microliter supernatant, lightly blowing out heavy suspended bacteria block, spreading on LB plate containing rifampicin and hygromycin, and culturing in 28 deg.c incubator for 2-3 days.
  5. 5. The SlCAS gene and the application of the protein encoded by the SlCAS gene in regulation of tomato gray mold resistance, as claimed in claim 3, wherein the preparation of the Agrobacterium liquid in step 2 specifically comprises the following steps:
    sucking 5 μ L of the preserved bacteria liquid, inoculating in 5mL LB liquid culture medium containing rifampicin and hygromycin, performing shake culture at 28 deg.C and 200rpm/min to logarithmic phase, and measuringTo its 0D 600 When the value is 0.8, centrifuging for 10min at 8000rpm in a centrifuge, discarding the supernatant, washing the precipitate twice with MS liquid culture medium, and diluting with MS liquid culture medium to a value of 0.3-0.4 to obtain the agrobacterium liquid for infection.
  6. 6. The SlCAS gene and the protein encoded by the same as claimed in claim 3, wherein the step 3.1 comprises the following specific steps:
    the tomato is required to be subjected to germination accelerating treatment before planting, seeds are placed into a culture dish padded with two pieces of water-absorbing filter paper, a proper amount of distilled water is added to perform germination accelerating in a culture room for 2-3 days in dark, and the tomato seeds are sowed after germination in a culture room with a volume ratio of peat soil to vermiculite of 3:1 mixing the materials in a nutrition pot, covering the top with the same thickness of the materials, placing the materials in a culture room, and growing in the environment: the day temperature is 25 ℃, the night temperature is 20 ℃, the photoperiod is 16 and 8h, the light and dark cycle is carried out, and the illumination intensity is 600 mol.m -2 ·s -1 1g of nutrient fertilizer and 1L of distilled water are added to prepare nutrient solution for culture.
CN202211279246.8A 2022-10-19 2022-10-19 SlCAS gene and application of protein coded by same in regulation and control of tomato gray mold resistance Pending CN115976046A (en)

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CN104131049A (en) * 2014-08-06 2014-11-05 华中农业大学 Phytophthora polysaccharide exciton Gep1 and application thereof to improvement of plant disease resistance
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CN104131049A (en) * 2014-08-06 2014-11-05 华中农业大学 Phytophthora polysaccharide exciton Gep1 and application thereof to improvement of plant disease resistance
CN107114369A (en) * 2017-03-24 2017-09-01 浙江大学 Applications of the phytosulfokine-α α in plant botrytis resistance is improved
WO2022073260A1 (en) * 2020-10-09 2022-04-14 西南大学 Application of tomato hydroxyproline-rich systemin precursor protein gene slhypsys in improving resistance of plants to verticillium wilt

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