Application of CaCRY2 gene in regulating plant resistance to Phytophthora capsici

Abstract

The present invention provides an application of the CaCRY2 gene in regulating plant resistance to pepper phytophthora, belonging to the technical field of plant molecular biology and plant genetic engineering. The gDNA sequence of the CaCRY2 gene is shown in SEQ ID NO: 1, and the cDNA sequence thereof is shown in SEQ ID NO: 2; inhibiting the expression of the CaCRY2 gene enhances the resistance of the plant to pepper phytophthora; and the plant is a Solanaceae plant. The present invention provides a pepper blight-related gene CaCRY2 and an application thereof, and silencing the CaCRY2 gene through a virus-induced gene silencing technique can enhance the resistance of pepper plants to blight; the present invention provides a favorable basis for improving plant disease resistance and cultivating disease-resistant varieties.

Description

Technical Field

[0001] The invention belongs to the technical field of plant molecular biology and plant genetic engineering, and particularly relates to the application of the CaCRY2 gene in regulating plant resistance to pepper phytophthora. Background Art

[0002] Cryptochrome CRYs are blue / UV-A light receptors of flavoproteins similar to photolyases, which are commonly found in prokaryotes and eukaryotes. Plant CRYs can be divided into three subfamilies: CRY1, CRY2 and CRY3, which mainly mediate the regulation of blue light on plant growth and development, such as photomorphogenesis, flowering process, biological rhythms and stomatal opening and closing. Among them, CRY2 is a protein that is localized in both the nucleus and the cytoplasm. It contains two domains: one is the photolyase homologous region (PHR) domain at the N-terminus, and the other is the variable C-terminal extension (CRY C-terminal extension, CCE) domain. In recent years, CRY2 has been reported to play a role in plant response to abiotic and biotic stresses. For example, CRY2 regulates blue light-dependent cold acclimation in Arabidopsis through the CRY2-COP-HY5-BBX7 / 8 module, and CRY2 regulates resistance protein-mediated virus defense by negatively regulating E3 ubiquitin ligases.

[0003] Pepper blight is a soil-borne disease caused by infection with pepper phytophthora. It has the characteristics of fast infection and rapid onset. It can break out in a large area in a short period of time, causing pepper production reduction or even complete crop failure. Pepper blight has a wide host range. In addition to pepper, it can also infect many plants such as tomatoes and tobacco in the Solanaceae family, cucumbers, watermelons and pumpkins in the Cucurbitaceae family, common beans, oil beans and yellow beans in the Leguminosae family, and cause serious losses. In addition, the disease caused by pepper phytophthora is a typical multi-cycle disease, which infects and spreads by producing spores of various forms such as oospores, sporangia, zoospores, and thick-walled spores. Once pepper blight breaks out, chemical control is feasible in the short term, but it is easy to cause environmental pollution and long-term use will cause plants to develop drug resistance. Among the prevention and control measures for pepper blight caused by pepper phytophthora, one of the ways is to select and cultivate disease-resistant varieties, and exploring disease-resistant genes is the key to disease-resistant breeding. Summary of the invention

[0004] In view of this, the present invention aims to provide an application of the CaCRY2 gene in regulating plant resistance to Phytophthora capsici, aiming to solve at least one technical problem in the background technology.

[0005] The present invention is achieved in that:

[0006] Application of the CaCRY2 gene in regulating plant resistance to Phytophthora capsici, wherein the gDNA sequence of the CaCRY2 gene is shown in SEQ ID NO: 1; and the cDNA sequence of the CaCRY2 gene is shown in SEQ ID NO: 2;

[0007] Inhibiting the expression of the CaCRY2 gene and enhancing the resistance of the plant to Phytophthora capsici;

[0008] The plant is a plant of the Solanaceae family.

[0009] Furthermore, the CaCRY2 gene is used as a target gene, and the expression of the CaCRY2 gene is inhibited through genetic engineering methods to enhance the plant's resistance to blight; the blight is a disease caused by pepper Phytophthora.

[0010] Furthermore, the genetic engineering method is a virus-induced gene silencing method.

[0011] Furthermore, the gene silencing method uses a silencing vector to induce silencing of the CaCRY2 gene in the plant, and the steps of constructing the silencing vector include:

[0012] Designing a primer pair using the cDNA sequence of the CaCRY2 gene as shown in SEQ ID NO: 2 as a template, amplifying the template, and recovering the amplified fragment by gel cutting to obtain a DNA fragment for silencing the CaCRY2 gene;

[0013] The tobacco rattle virus vector pTRV2 was double-digested with restriction endonucleases Xba I and Sma I, and the vector backbone was recovered by gel cutting;

[0014] The DNA fragment was connected to the vector backbone of the pTRV2 vector through homologous recombination and transferred into Escherichia coli DH5α to obtain a recombinant CaCRY2 gene silencing vector.

[0015] Furthermore, the plant is a Solanaceae plant that can be infected by Capsicum frutescens.

[0016] Compared with the prior art, the present invention provides a pepper blight-related gene CaCRY2 and its application. Silencing the CaCRY2 gene through virus-induced gene silencing technology can enhance the resistance of pepper plants to blight, providing a favorable basis for improving plant disease resistance and cultivating disease-resistant varieties. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 The CaCRY2 gene expression diagram of TRV:00 and TRV:CaCRY2 in Example 1 of the present invention;

[0018] Figure 2The phenotypic diagram of pepper seedlings before and after inoculation of pepper phytophthora by root irrigation in Example 1 of the present invention;

[0019] Figure 3 This is a comparison chart of the disease index of pepper seedlings 7 days after being inoculated with pepper phytophthora by the root irrigation method in Example 1 of the present invention;

[0020] Figure 4 The bright field and trypan blue staining images of the detached leaves in Example 1 of the present invention 3 days after inoculation of pepper phytophthora;

[0021] Figure 5 This is a comparison diagram of the lesion area of ​​the detached leaves in Example 1 of the present invention 3 days after being inoculated with pepper phytophthora;

[0022] Figure 6 The CaCRY2 gene expression diagram of WT, OE-1 and OE-2 in Example 2 of the present invention;

[0023] Figure 7 The phenotype diagram of tobacco seedlings before and after inoculation of pepper phytophthora by root irrigation in Example 2 of the present invention;

[0024] Figure 8 This is a comparison chart of the disease index of tobacco seedlings 5 ​​days after being inoculated with pepper phytophthora by the root irrigation method in Example 2 of the present invention. DETAILED DESCRIPTION

[0025] In order to make the purpose, technical solution and advantages of the present invention more clearly understood, the present invention is further described in detail below in conjunction with the embodiments. It should be understood that the specific implementation cases described herein are only used to explain the present invention and are not used to limit the present invention.

[0026] The gDNA sequence of the CaCRY2 gene involved in the present invention is shown as SEQ ID NO: 1, and its sequence is retrieved from the Solanaceae genome database SGN (https: / / solgenomics.net / ), with ID No. CA03g05280, and its cDNA sequence is shown as SEQ ID NO: 2; the present invention finds that inhibiting the expression of the CaCRY2 gene can enhance the resistance of plants to pepper phytophthora.

[0027] In a specific implementation, inhibiting the expression of the CaCRY2 gene can be achieved by a variety of ways, such as completely or partially limiting the expression level of the CaCRY2 gene, completely or partially limiting the protein activity expressed by the CaCRY2 gene, and performing gene mutation on the CaCRY2 gene. Specifically, it mainly includes methods such as gene knockout, RNA interference, antisense RNA technology, viral vectors, and protein level regulation. In the following embodiments, the gene silencing technology (Virus-Induced Gene Silencing, VIGS) using viral vectors is used as an example to partially limit the expression of the CaCRY2 gene.

[0028] In a specific implementation, the plant of the present invention refers to any plant that can be infected by Phytophthora capsici and cause blight, especially Solanaceae plants. In the following embodiments, pepper and tobacco belonging to Solanaceae plants are used as examples.

[0029] Example 1

[0030] This Example 1 is a pepper disease resistance experiment. The CaCRY2 gene is silenced in pepper seedlings by VIGS technology, thereby inhibiting CaCRY2 gene expression and studying how it regulates pepper plant resistance to Phytophthora capsici.

[0031] 1.1 Genetic material

[0032] Using pepper variety 'Hangjiao No. 12' as material, RNA was extracted from its leaf tissue and reversely transcribed into cDNA, the sequence of which is shown in SEQ ID NO: 2, and used as a template for the subsequent construction of CaCRY2 gene silencing vector.

[0033] 1.2. Construction of CaCRY2 gene silencing vector

[0034] The primer pair was designed using the sequence of the CaCRY2 gene in the Solanaceae genome database SGN (https: / / solgenomics.net / ) (its cDNA sequence is shown in SEQ ID NO: 2) as a template. The sequence of the primer pair is as follows:

[0035] Forward primer: 5'-TGAGTAAGGTTACCGAATTCTCTAGAGGTGGAACTCGGTAT-3', as shown in SEQ ID NO: 3;

[0036] Reverse primer: 5'-TTTAATGTCTTCGGGACATGCCCGGGCGAACTGCTCGGACAA-3', as shown in SEQ ID NO: 4;

[0037] The above primer pair was used to amplify the cDNA of the pepper variety 'Hangjiao No. 12' as a template to obtain a 232 bp specific fragment, and the amplified fragment was recovered by gel cutting; the tobacco rattle virus (TRV) vector pTRV2 was double-digested with restriction endonucleases Xba I and Sma I, and the vector backbone was recovered by gel cutting; the recovered amplified fragment and the vector backbone were connected by homologous recombination and transferred into Escherichia coli DH5α. After sequencing and comparison, the correct plasmid vector was the CaCRY2 gene silencing vector pTRV2:CaCRY2.

[0038] 1.3. Silencing the CaCRY2 gene in pepper seedlings

[0039] The constructed silencing vector pTRV2:CaCRY2 was transferred into Agrobacterium competent cells GV3101 to form pTRV2:CaCRY2 Agrobacterium culture solution; the TRV-expressing vector pTRV1 was transferred into Agrobacterium competent cells GV3101 to form pTRV1 Agrobacterium culture solution; the vector pTRV2 was transferred into Agrobacterium competent cells GV3101 to form pTRV2:00 empty Agrobacterium culture solution.

[0040] Taking pepper seedlings grown to the cotyledon stage as the object, the pTRV1 Agrobacterium solution and the pTRV2:CaCRY2 Agrobacterium solution were mixed in a volume ratio of 1:1, and the mixture was injected to infect the cotyledons of 'Hangjiao No. 12' pepper seedlings to obtain the CaCRY2 gene silenced pepper seedling material, which was named TRV:CaCRY2; the pTRV1 Agrobacterium solution and the pTRV2:00 empty Agrobacterium solution were mixed in a volume ratio of 1:1, and the cotyledons of 'Hangjiao No. 12' pepper seedlings were used as the control pepper seedling material, which was named TRV:00.

[0041] The relative expression of CaCRY2 gene in leaves of TRV:00 and TRV:CaCRY2 was detected. Figure 1 As shown by Figure 1 It can be seen that CaCRY2 gene silencing was successfully achieved in TRV:CaCRY2. Figure 1 The * indicates that the P value is less than 0.5, which means that there is a significant difference.

[0042] 1.4、Cultivation of Phytophthora capsici

[0043] Phytophthora capsici was inoculated on PDA medium (200 g / L potato + 20 g / L glucose + 17 g / L agar) and incubated in the dark for 4 to 7 days for activation. The medium with mycelium of Phytophthora capsici was punched into fungus blocks with a sterilized puncher and placed in a 10% V8 medium (100 mL V8 vegetable juice + 1 g CaCO 3+17g agar to 1000mL) in a culture dish and incubate in the dark at 28℃ for 7 days to induce sporangia. Add 10mL of sterilized water to the culture dish full of mycelium, refrigerate at 40℃ for 30min, and then transfer to room temperature (25℃~28℃) for 20min to stimulate sporangia to release spores. Use a sterile spreader to gently scrape off the mycelium on the surface of the culture medium, and collect the filtered liquid as the spore suspension. Use a hemocytometer to count the number of spores and dilute the spore suspension to the required concentration (1×10 5 zoospores·mL -1 ).

[0044] 1.5. Root irrigation method for inoculation of pepper with Phytophthora spp. and result analysis

[0045] Pepper seedlings with TRV:CaCRY2 and TRV:00 were used as inoculation objects. When the pepper seedlings grew to the stage of five leaves and one heart, they were inoculated with Phytophthora. 5 mL of diluted spore suspension of Phytophthora capsici was injected into the soil 1 cm away from the rhizomes of pepper seedlings.

[0046] The pepper seedlings were planted and cultured in a greenhouse. The specific growth conditions were set as follows: light intensity of 200 μmol·m -2 ·s -1 , temperature 19℃~25℃, photoperiod 12h.

[0047] The disease index of pepper seedlings is divided into 6 levels according to the disease situation, and the grading standards are shown in Table 1.

[0048] Table 1

[0049]

[0050] Seven days after inoculation with pepper phytophthora, three pepper seedlings were selected from each group for photographing. Figure 2 shown by Figure 2 It can be seen that before inoculation with pepper phytophthora, the pepper seedlings of TRV:00 and TRV:CaCRY2 did not wilt; after inoculation with pepper phytophthora, both groups of pepper seedlings wilted; and the degree of wilting of pepper seedlings in which the CaCRY2 gene was not silenced was more serious than that of pepper seedlings in which the CaCRY2 gene was silenced.

[0051] Seven days after inoculation with pepper phytophthora, the disease grades of pepper seedlings TRV:00 and TRV:CaCRY2 were counted and the disease index was calculated according to the grading standard in Table 1. The results are shown in Table 2. Figure 3 As shown, Figure 3 The * indicates that the P value is less than 0.5, which means that there is a significant difference.

[0052] Table 2

[0053]

[0054] Disease index = ∑ (disease level * number of plants with the disease level) / total number of investigated plants. The total number of investigated plants for TRV:00 and TRV:CaCRY2 was 10 plants, and the disease level before inoculation with pepper phytophthora was 0. Figure 3 It can be seen that among the pepper seedlings inoculated with pepper Phytophthora capsici, the pepper seedlings whose CaCRY2 gene was not silenced had a higher disease index than the pepper seedlings whose CaCRY2 gene was silenced.

[0055] 1.6. Inoculation of detached leaves with Phytophthora capsici and analysis of results

[0056] Based on the root irrigation inoculation test, further resistance identification was carried out by inoculating pepper phytophthora with detached leaves.

[0057] In vitro leaf inoculation: Leaves of pepper seedlings of TRV:00 and TRV:CaCRY2 were placed on 0.8% agar medium (8 g agar powder was diluted to 1000 mL) to keep them moist, and 20 μL of 1×10 5 zoospores·mL -1 of spore suspension.

[0058] The lesion area was measured after 3 days. The measurement method was as follows: preheat the trypan blue staining solution (according to the volume ratio, 95% alcohol: lactophenol = 2:1) for 1 minute, immerse the leaves, boil for 2 minutes, take out the leaves and place them in a culture dish containing trypan blue staining solution to continue soaking for about 12 hours, pour out the staining solution, add a decolorant (900g chloral hydrate dissolved in 600mL deionized water), replace the decolorant every 12 hours until the leaves are transparent, and finally place them in 95% alcohol to wash off the decolorant, measure and record the area of ​​dead cells in the leaves. Among them, lactophenol is 10mL of lactic acid, 9.3mL of phenol, 10mL of glycerol, 10mL of trypan blue, and ddH 2 O 10mL and mix well.

[0059] After 3 days, the detached leaves were photographed in bright field and after trypan blue staining. Figure 4 As shown, the lesion area is Figure 5 shown. Figure 4 and Figure 5 The results showed that the leaf lesions of pepper seedlings without CaCRY2 gene silencing were significantly larger than those of pepper seedlings with CaCRY2 gene silencing. Figure 5 The * indicates that the P value is less than 0.5, which means that there is a significant difference.

[0060] In this Example 1, the resistance of pepper seedlings with CaCRY2 gene silenced to blight was identified by counting the disease index of pepper seedlings and the lesion area of ​​detached leaves, and it was clarified that silencing the CaCRY2 gene can enhance the resistance of pepper seedlings to blight caused by pepper Phytophthora.

[0061] Example 2

[0062] This Example 2 is a tobacco disease resistance experiment, in which the CaCRY2 gene is overexpressed in tobacco plants through transgenic technology, and then the tobacco plants overexpressing the CaCRY2 gene are inoculated with pepper phytophthora to study how it regulates the pepper phytophthora resistance of tobacco plants.

[0063] 2.1 Genetic material

[0064] Using pepper variety 'Hangjiao No. 12' as material, RNA was extracted from its leaf tissue and reversely transcribed into cDNA, the sequence of which is shown in SEQ ID NO: 2, and used as a template for the subsequent construction of a CaCRY2 gene overexpression vector.

[0065] 2.2. Construction of CaCRY2 gene overexpression vector

[0066] The primer pair was designed using the sequence of the CaCRY2 gene in the Solanaceae genome database SGN (https: / / solgenomics.net / ) (its cDNA sequence is shown in SEQ ID NO: 2) as a template. The sequence of the primer pair is as follows:

[0067] Forward primer: 5'-TTACAATTACCATGGGGCGCGCCATGGAGAGTAATTCCAAGACAA-3', as shown in SEQ ID NO: 5;

[0068] Reverse primer: 5'-AACATCGTATGGGTAGGTACCTAGCTCTGTCTTACCAGTTTTGCAC-3', as shown in SEQ ID NO: 6;

[0069] Using the primer pair, the cDNA of the pepper variety 'Hang Jiao No. 12' was used as a template for amplification to obtain a 1929 bp full-length fragment of the CaCRY2 gene. The amplified fragment was recovered by gel cutting, and the vector PAC containing the CaMV 35S promoter was double-digested with restriction endonucleases Asc I and Kpn I. The vector backbone was recovered by gel cutting, and the recovered amplified fragment and the vector backbone were connected by homologous recombination and transferred into Escherichia coli DH5α. After sequencing and comparison, the correct plasmid vector was the CaCRY2-PAC vector, that is, the CaCRY2 gene overexpression vector.

[0070] 2.3 Tobacco genetic transformation

[0071] The CaCRY2-PAC vector was transformed into competent Agrobacterium GV3101 and then transferred into Nicotiana benthamiana ( Nicotiana benthamiana ) and obtained tobacco materials with overexpression of CaCRY2 gene. Two strains were selected and named OE-1 and OE-2 respectively; and a group of wild-type Nicotiana benthamiana was used as a control, and the tobacco material was named WT (wild type).

[0072] The relative expression levels of CaCRY2 gene in leaves of tobacco corresponding to WT, OE-1 and OE-2 were detected. Figure 6 As shown by Figure 6 It can be seen that overexpression of the CaCRY2 gene was successfully achieved in OE-1 and OE-2.

[0073] 2.4、Cultivation of Phytophthora capsici

[0074] The same as 1.4 in Example 1, the concentration was 1×10 5 zoospores·mL -1 of a spore suspension of Phytophthora capsici.

[0075] 2.5. Root irrigation inoculation of pepper with Phytophthora capsici and result analysis

[0076] OE-1, OE-2 and WT tobacco seedlings were used as inoculation objects. When the tobacco seedlings grew to 4 to 6 true leaves, they were inoculated with Capsici Phytophthora. 5 mL of diluted Capsici Phytophthora spore suspension was injected into the soil 1 cm away from the rhizomes of the tobacco seedlings.

[0077] The above tobacco seedlings were planted and cultured in a greenhouse, and the specific growth conditions were set as follows: light intensity 200 μmol·m -2 ·s -1 , temperature 19℃~25℃, photoperiod 12h.

[0078] Three tobacco seedlings were selected from WT, OE-1, and OE-2, and photographed before and 5 days after inoculation with Phytophthora capsici. Figure 7 As shown;

[0079] Five days after inoculation with pepper phytophthora, the disease grades of tobacco seedlings of WT, OE-1 and OE-2 were counted and the disease index was calculated according to the grading standard in Table 1. The results are shown in Table 3. The comparison of disease index is shown in Table 3. Figure 8 As shown, the grading standard of disease level is the same as Table 1 in Example 1; Figure 8 The * indicates that the P value is less than 0.5, which means that there is a significant difference.

[0080] Table 3

[0081]

[0082] Disease index = ∑ (disease level * number of plants with the disease level) / total number of investigated plants. The total number of investigated plants for WT, OE-1 and OE-2 were all 20, and the disease level was 0 before inoculation with pepper phytophthora.

[0083] Depend on Figure 7 It can be seen that the tobacco seedlings in the OE-1, OE-2 and WT groups did not wilt before inoculation with pepper phytophthora; after inoculation with pepper phytophthora, the tobacco seedlings all wilted and had varying degrees of leaf drop; the tobacco seedlings (OE-1 and OE-2) with overexpression of the CaCRY2 gene were more severely wilted than those in the WT group, and the leaf drop was more obvious.

[0084] Depend on Figure 8 It can be seen that after inoculation with pepper phytophthora, the disease index of tobacco seedlings overexpressing the CaCRY2 gene (OE-1 and OE-2) was higher than that of tobacco seedlings in the WT group.

[0085] This Example 2 identified that tobacco plants overexpressing the CaCRY2 gene had reduced resistance to blight by statistically analyzing the disease index, further clarifying that overexpressing CaCRY2 weakened tobacco plants' resistance to blight.

[0086] As demonstrated by Examples 1 and 2, the CaCRY2 gene, as a negative regulatory factor, can confer disease resistance to different plants against Phytophthora capsici and the blight caused by it. As a target gene, the CaCRY2 gene can be applied to: (1) detecting the expression level of the CaCRY2 gene or preparing a molecular marker for use in pepper blight-resistant breeding; (2) preparing products resistant to pepper blight, such as reagents and drugs that inhibit the expression of the CaCRY2 gene or the activity of the protein; (3) cultivating transgenic plants resistant to pepper blight, such as introducing nucleic acid molecules that inhibit the expression of the CaCRY2 gene into the target plant.

[0087] The above-mentioned embodiments only express several implementation methods of the present invention, and the description thereof is relatively specific and detailed, but it cannot be understood as limiting the scope of the patent of the present invention. It should be pointed out that, for ordinary technicians in this field, several variations and improvements can be made without departing from the concept of the present invention, which all belong to the protection scope of the present invention. Therefore, the protection scope of the patent of the present invention shall be subject to the attached claims.

Claims

1. Application of the CaCRY2 gene in regulating plant resistance to Phytophthora capsici, characterized in that: The gDNA sequence of the CaCRY2 gene is shown in SEQ ID NO: 1; The cDNA sequence of the CaCRY2 gene is shown in SEQ ID NO: 2; The regulation is to inhibit the expression of the CaCRY2 gene and enhance the resistance of the plant to Phytophthora capsici; The plant is a plant of the Solanaceae family.

2. The use of the CaCRY2 gene according to claim 1 in regulating plant resistance to Phytophthora capsici, characterized in that: The CaCRY2 gene is used as a target gene, and the expression of the CaCRY2 gene is inhibited through a genetic engineering method to enhance the plant's resistance to blight; the blight is a disease caused by pepper phytophthora.

3. The use of the CaCRY2 gene according to claim 2 in regulating plant resistance to Phytophthora capsici, characterized in that: The genetic engineering method is a virus-induced gene silencing method.

4. The use of the CaCRY2 gene according to claim 3 in regulating plant resistance to Phytophthora capsici, characterized in that: The gene silencing method uses a silencing vector to induce silencing of the CaCRY2 gene in plants, and the steps of constructing the silencing vector include: Designing a primer pair using the cDNA sequence of the CaCRY2 gene as shown in SEQ ID NO: 2 as a template, amplifying the template, and recovering the amplified fragment by gel cutting to obtain a DNA fragment for silencing the CaCRY2 gene; The tobacco rattle virus vector pTRV2 was double-digested with restriction endonucleases Xba I and Sma I, and the vector backbone was recovered by gel cutting; The DNA fragment was connected to the vector backbone of the pTRV2 vector through homologous recombination and transferred into Escherichia coli DH5α to obtain a recombinant CaCRY2 gene silencing vector.

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