Application of RcWRKY31 gene in enhancing resistance of Chinese rose to gray mold

Abstract

The invention discloses an RcWRKY31 gene and application of the RcWRKY31 gene in enhancing the resistance of Chinese roses to gray mold. The nucleotide sequence of the RcWRKY31 gene is shown as SEQ ID NO. 1. The RcWRKY31 gene is silenced and overexpressed in petals which are good in Chinese rose growth state and free of botrytis cinerea infection, and research results show that the petals treated by the silent RcWRKY31 gene are larger in scab area and lower in resistance to botrytis cinerea; the resistance of petals treated by overexpression of the RcWRKY31 gene to gray mold is enhanced.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of molecular breeding, and particularly relates to a RcWRKY31 application of a gene in enhancing resistance of Rosa hybrida to botrytis blight. BACKGROUND

[0002] Rosa hybrida (Rosa hybrida) is a perennial evergreen or semi-evergreen woody ornamental plant of Rosaceae Rosa. (Rosa) Rosa hybrida has high ornamental, economic and cultural values due to its elegant flower type, bright flower color and rich fragrance, and is one of the four major cut flowers and is widely used in landscaping, courtyard decoration, pot flower, perfume and cosmetic industries.

[0003] Rosa hybrida is one of the four major cut flowers, and botrytis blight is the most harmful fungal disease in the postharvest transportation process of Rosa hybrida cut flowers, which seriously damages the ornamental value and economic benefits of Rosa hybrida cut flowers. Rosa hybrida cut flowers are extremely vulnerable to botrytis blight in long-distance logistics transportation, and about 15-40% of Rosa hybrida cut flowers completely lose economic value due to postharvest rot. Chemical control, physical control, biological control and agricultural control all have certain drawbacks. Therefore, the botrytis blight of Rosa hybrida should be fundamentally controlled from the genetic breeding of Rosa hybrida. By studying the interaction between Rosa hybrida and botrytis blight, a new high-resistance Rosa hybrida variety is bred to reduce economic losses of the Rosa hybrida cut flower industry due to botrytis blight. At present, the specific mechanism of the RcWRKY31 gene in the resistance of Rosa hybrida to botrytis blight is not clear, and therefore it is necessary to study the function of the gene through gene editing or transgenic technology.

[0004] Therefore, a kind of RcWRKY31 application of a gene in enhancing resistance of Rosa hybrida to botrytis blight is developed. SUMMARY

[0005] To solve the problems in the prior art, the purpose of the present application is to provide a kind of RcWRKY31 application of a gene in enhancing resistance of Rosa hybrida to botrytis blight. The resistance of Rosa hybrida to botrytis blight is enhanced by overexpression of the gene, and a new high-resistance Rosa hybrida variety is bred.

[0006] To achieve the above purpose, the present application provides the following technical solutions: The present application provides a kind of RcWRKY31 application of a gene or its transcription and translation protein expression product in regulating botrytis blight of Rosa hybrida.

[0007] Beneficial effects: a disease resistance regulation gene RcWRKY31 is identified from the genome of Rosa hybrida variety 'Yueyue powder' RcWRKY31 , and it is found that the gene is highly expressed in petals of Rosa hybrida tissues, and the expression amount in roots and leaves is low. In the process of botrytis blight infection of Rosa hybrida flowersRcWRKY31 In the analysis of the expression of the gene, it is found that RcWRKY31 The expression of the gene is obviously increased in a certain period after the infection of the botrytis.Therefore, it is speculated by the present application that RcWRKY31 The gene may be involved in the regulation of the resistance of the Chinese rose to the botrytis.

[0008] Compared with the prior art, the present application has the following advantages: (1) According to the expression results of the gene in the petals of the Chinese rose, the gene in the petals is silenced and overexpressed, and it is found that RcWRKY31 The abnormal expression of the gene affects the resistance of the petals of the Chinese rose to the botrytis, and the overexpression of the gene can promote the resistance of the petals to the botrytis, and vice versa, the silencing can inhibit the resistance of the petals to the botrytis. RcWRKY31 RcWRKY31 (2) The gene in the petals of the Chinese rose in a good growth state without infection of the botrytis is silenced and overexpressed, and the research results show that the petals treated by silencing the gene have a larger lesion area and a lower resistance to the botrytis, and the petals treated by overexpressing the gene have a higher resistance to the botrytis, that is, RcWRKY31 The gene is of great significance to the regulation of the resistance of the Chinese rose to the botrytis.

[0009] RcWRKY31 RcWRKY31 RcWRKY31 RcWRKY31 BRIEF DESCRIPTION OF DRAWINGS

[0010] In order to more clearly illustrate the technical solutions in the embodiments of the present application, the drawings needed to be used in the embodiments or the prior art description will be briefly introduced as follows. Obviously, the drawings in the following description are only some embodiments of the present application, and other drawings can be obtained by those skilled in the art without any creative effort on the basis of these drawings.

[0011] Figure 1 is an expression analysis diagram of the present application. RcWRKY31 Rt: root; St: stem segment; Lf: leaf; Fl: petal.

[0012] Figure 2 is a subcellular localization diagram of the present application. RcWRKY31 Note: the subcellular localization in the leaf of Nicotiana benthamiana, RcWRKY31-GFP and DAPI (nuclear marker) are co-infiltrated into the tobacco leaf, and the fluorescence signal is observed by a confocal microscope.

[0013] Figure 3 is the change of the expression amount of RcWRKY31 with time after the infection of the botrytis.

[0014] Figure 4 ​​​​​Figure 3 is a diagram of the expression level of TRV and RcWRKY31-silenced genes on the third day after transiently silencing RcWRKY31 according to the present application Figure 4 Figure 3 is a diagram of the expression level of TRV and RcWRKY31-silenced genes on the third day after transiently silencing RcWRKY31 according to the present application Figure 4 Figure 3 is a diagram of the expression level of TRV and RcWRKY31-silenced genes on the third day after transiently silencing RcWRKY31 according to the present application Figure 4 Figure 3 is a diagram of the expression level of TRV and RcWRKY31-silenced genes on the third day after transiently silencing RcWRKY31 according to the present application Figure 4 Figure 3 is a diagram of the expression level of TRV and RcWRKY31-silenced genes on the third day after transiently silencing RcWRKY31 according to the present application

[0015] Figure 5 Figure 3 is a diagram of the expression level of TRV and RcWRKY31-silenced genes on the third day after transiently silencing RcWRKY31 according to the present application Figure 5 Figure 3 is a diagram of the expression level of TRV and RcWRKY31-silenced genes on the third day after transiently silencing RcWRKY31 according to the present application Figure 5 Figure 3 is a diagram of the expression level of TRV and RcWRKY31-silenced genes on the third day after transiently silencing RcWRKY31 according to the present application Figure 5 Figure 3 is a diagram of the expression level of TRV and RcWRKY31-silenced genes on the third day after transiently silencing RcWRKY31 according to the present application Figure 5 DETAILED DESCRIPTION DETAILED DESCRIPTION DETAILED DESCRIPTION

[0016] The technical solutions in the embodiments of the present application will be clearly and completely described below with reference to the drawings in the embodiments of the present application. Obviously, the described embodiments are only some of the embodiments of the present application, rather than all the embodiments. Based on the embodiments in the present application, all other embodiments obtained by those skilled in the art without creative work fall within the scope of the present application.

[0017] In the present application, the term "and / or" describes the association relationship of associated objects, which means that there can be three kinds of relationships, for example, A and / or B, which can mean that A exists alone, A and B exist together, and B exists alone. Wherein A and B can be singular or plural. The character " / " generally represents an "or" relationship between the front and rear associated objects.

[0018] In the present application, "one or more" means one or more, and "multiple" means two or more. "One or more of the following" or the like means any combination of these items, including any combination of single item or multiple items. For example, "one or more of a, b, or c", or "one or more of a, b, and c", can mean a, b, c, a-b (i.e. a and b), a-c, b-c, or a-b-c, wherein a, b, and c can be single or multiple.

[0019] It should be understood that the size of the sequence number of the above-mentioned processes in various embodiments of the present application does not mean the order of execution, and part or all of the steps can be executed in parallel or in sequence, and the execution order of the processes should be determined according to their functions and internal logic, and should not constitute any limitation on the implementation process of the embodiments of the present application.

[0020] The terms used in the embodiments of the present application are only for the purpose of describing specific embodiments, and are not intended to limit the present application. The singular forms "a", "said" and "the" used in the embodiments of the present application and the appended claims are also intended to include the plural forms, unless the context clearly indicates otherwise.

[0021] The weight of the related components mentioned in the specification of the embodiments of the present application can not only refer to the specific content of each component, but also represent the proportional relationship between the weights of each component. Therefore, as long as the content of the related components in the specification of the embodiments of the present application is enlarged or reduced in proportion, it is within the scope disclosed in the specification of the embodiments of the present application. Specifically, the mass mentioned in the specification of the embodiments of the present application can be µg, mg, g, kg, etc. Mass units commonly known in the chemical field.

[0022] The terms "first", "second" are only for the purpose of description, used to distinguish objects such as substances from each other, and cannot be understood as indicating or implying relative importance or implicitly indicating the number of technical features indicated. For example, without departing from the scope of the embodiments of the present application, the first XX can also be referred to as the second XX, and similarly, the second XX can also be referred to as the first XX. Therefore, the features limited by "first", "second" can explicitly or implicitly include one or more of the features.

[0023] The following will be described in detail in conjunction with the embodiments and the accompanying drawings. Based on the genomic sequence of the rose variety 'Yueyuefen', the present application mined potential disease resistance regulation genes RcWRKY31 by identifying and analyzing WRKY family members in the early stage RcWRKY31 The expression of the gene was significantly increased at a certain period of time after the infection of gray mold. After transient silencing or overexpression RcWRKY31 of the gene, it was found that the resistance of rose petals to gray mold was significantly changed. Therefore, the present application discloses RcWRKY31 the biological function of the gene in the disease resistance of rose to gray mold, thereby providing technical support for enhancing the resistance of rose to gray mold, promoting the research of rose functional genomics, gene editing breeding and transgenic breeding, mining and cultivating new varieties of rose with high resistance, and providing certain theoretical basis for the comprehensive prevention and control of gray mold and the cultivation of new varieties of rose.

[0024] The abbreviations appearing in the present application and their corresponding names are shown in Table 1.

[0025] Table 1

[0026] Example 1 The present application provides a kind of RcWRKY31 The application of gene or its transcription translation protein expression product in the regulation of Chinese rose gray mold. RcWRKY31 The nucleotide sequence of the gene is shown as SEQ ID NO: 1. RcWRKY31 The coding protein of the gene, the amino acid sequence of the coding protein is shown as SEQ ID NO.2. Example 2

[0027] 1. Plant material 1.1‘Yueyufen’ ‘Yueyufen’ is taken from the Jinning Baofeng base of the Flower and Ornamental Plant Institute of Yunnan Academy of Agricultural Sciences, and different tissue parts are used for tissue-specific analysis. ‘Carola’ is a modern cut flower rose variety, taken from the Baofeng base of the Flower and Ornamental Plant Institute of Yunnan Academy of Agricultural Sciences, and S3 stage petals are used for silencing and expression verification experiments.

[0028] 1.2 Tobacco The material for subcellular localization and transcription activation experiment is Nicotiana benthamiana. The seeds are sown in moist nutrient medium and covered with film, and cultured in the culture room.

[0029] Culture conditions: temperature 24±1℃, relative humidity 60-65%, photoperiod 16h / 8h 1.3 Strains and vectors Escherichia coli: DH5α, Agrobacterium tumefaciens strain EHA105, pSuper-1300 (Kan resistance) are purchased from Beijing Genki Biotechnology Co., Ltd. VIGS vectors are pTRV1 and pTRV2 purchased from HonorGene.

[0030] 1.4 Medium formula involved in the case (1) LB medium and YEB medium (Table 2) Table 2

[0031] (2) Experimental related medium (Table 3) Table 3

[0032] 2. Research methods 2.1 Extraction of total RNA RNAprep Pure polysaccharide polyphenol plant total RNA extraction kit (centrifugal column type) is used to extract total RNA from the roots, stems, leaves and petals of ‘Yueyufen’.

[0033] 2.2 cDNA synthesis (1) Preparation of genomic DNA removal reaction system (Table 4) Table 4

[0034] Mix well, 42 ℃, 2 min.

[0035] (2) Preparation of reverse transcription reaction system (Table 5) Table 5

[0036] Mix well by blowing, 37 ℃, 15 min; 85 ℃, 5 s. The product is stored at -20 ℃.

[0037] 2.3 Real-time fluorescent quantitative PCR Design the RT-qPCR specific primers of the gene with Primer premier5 (Table 6). Dilute the cDNA four times with ddH2O, and use the cDNA obtained by reverse transcription as the template for Real-Time PCR amplification. Set 3 biological replicates with UBI2 as the internal reference.

[0038] Table 6

[0039] The RT-qPCR reaction system is shown in Table 7: Table 7

[0040] The RT-qPCR reaction program is shown in Table 8: Table 8

[0041] 2.4 Vector construction 2.4.1 PCR amplification of target gene fragment PCR amplification of target gene uses high-fidelity enzyme (PhusionTM Plus PCR Master Mix). The PCR amplification reaction system is shown in Table 9: Table 9

[0042] The PCR amplification reaction program is shown in Table 10: Table 10

[0043] After amplification, 1% gel electrophoresis is performed, and the target band is selected for subsequent gel recovery.

[0044] 2.4.2 Glue Recycling The PCR products were recovered and purified using the DNA gel extraction kit (TaKaRaMiniBEST Agarose Gel DNA Extraction Kit Ver. 4.0).

[0045] 2.4.3 Vector double enzyme digestion Based on the restriction enzyme sites inserted according to the target fragment sequence, the vector is double-digested with the corresponding enzymes. The double digestion system is shown in Table 11: Table 11

[0046] After adding the system reagents on ice, perform double digestion of the vector according to the specific enzyme denaturation temperature.

[0047] 2.4.4 Homologous recombination The double-digested vector was then subjected to homologous recombination with the cloned target gene fragment to construct the vector. The homologous recombination system is shown in Table 12: Table 12

[0048] Procedure: Run the PCR machine at 50 ℃ for 15 min.

[0049] 2.4.5 Escherichia coli transformation (1) Remove the competent states from -80 ℃ and melt them in ice; (2) Take a 1.5 mL centrifuge tube, add 10 μL of recombinant product and 50 μL of DH5α competent cells, mix by pipetting, and let stand on ice for 30 min; (3) Heat shock in a 42 ℃ water bath for 90 s, then quickly transfer to ice and let stand for 2 min; (4) Add 500 μL LB to the centrifuge tube, incubate at 37 ℃, 200 rpm, for 1 h; (5) Centrifuge the cultured bacterial solution at 5000 rpm for 5 min; (6) Discard 400 μL of supernatant in a clean bench, mix well by pipetting, spread on LB solid medium containing antibiotics, and incubate overnight at 37 °C with the medium inverted.

[0050] 2.4.6 Microbial detection and sequencing (1) Shaking culture: Pick a single colony that has grown overnight and shake it in 500 μL of LB medium containing antibiotics for 3-4 h (37 ℃, 200 rpm).

[0051] (2) Bacterial culture PCR: The cultured bacterial culture was used for PCR amplification to detect whether the target band of the constructed vector met expectations. The bacterial culture PCR amplification system is shown in Table 13: Table 13

[0052] The bacterial culture PCR amplification reaction procedure is shown in Table 14: Table 14

[0053] The amplified products were used for 1% gel electrophoresis imaging, and those with band sizes that met expectations were sent to the company for sequencing.

[0054] 2.5 Plasmid Extraction After the test results are returned, sequence alignment is performed, and samples that meet the expectations are selected for inoculation and plasmid extraction is carried out using the plasmid extraction kit (TaKaRaMiniBEST Plasmid Purification Kit Ver.4.0).

[0055] 2.6 Agrobacterium-mediated transformation (1) When the competent state of the straw is taken out of the -80 ℃ freezer and melted into an ice-water mixture, it is inserted into ice; (2) Add 0.01-1 μg plasmid DNA to each 100 μL competent cells, mix well and then incubate on ice for 5 min, in liquid nitrogen for 5 min, in a 37 ℃ water bath for 5 min, and in an ice bath for 5 min. (3) Add 500 μL of antibiotic-free YEB liquid medium and incubate at 28 °C with shaking for 5 h; (4) Centrifuge at 5000 rpm for 2 min, discard 400 μL of supernatant in a clean bench, spread it onto YEB solid medium containing antibiotics, and incubate upside down at 28 ℃ for 5 days.

[0056] 2.7 Instantaneous infection (1) Vector primer design and vector construction Using EcoRI and KpnI as restriction sites, RcWRKY31 The silent fragment sequence (SEQ ID NO: 3) was inserted into the TRV2 empty vector, and primers were designed using homologous recombination to construct TRV- RcWRKY31 Carrier.

[0057] SmaI and KpnI were selected as restriction enzyme sites, and the enzyme was digested. RcWRKY31 The CDS sequence was inserted into the pSuper-1300 vector, and primers were designed using homologous recombination to construct... RcWRKY31 After obtaining Agrobacterium-positive strains using the -GFP vector, the following experiments were conducted.

[0058] (2) Bacterial culture Streak the bacterial culture on agar plates (containing 50 mg / L Kan / Rif) and incubate upside down at 28 ℃ for 5 days. Pick a single colony and gently shake it into 500 μL of YEB containing antibiotics for bacterial testing; if the bands are correct, shake it medium and then vigorously (28 ℃, 200 rpm).

[0059] (3) Collection and resuspension of bacteria Centrifuge at 5000 rpm for 8 min to collect bacteria, discard the supernatant, resuspend the bacteria in the infection solution, mix well by pipetting, and adjust to OD600 = 1.0. For transient silencing experiments, TRV1 and TRV2, TRV2- RcWRKY31 The bacterial cultures were mixed at a 1:1 volume ratio and incubated in the dark for 4-6 hours. During transient overexpression, Super-1300 and Super- RcWRKY31 Each bacteria can be collected and left to stand.

[0060] (4) Vacuum suction Vacuum pumps were used to inoculate 1.5 cm diameter petal discs made from the outermost petals of 'Carola' S3 grade flowers. The pressure was 0.082 MPa, aspirated for 10 min, held for 10 min, and then degassed for 10 min, ensuring the entire petal disc was fully immersed in the bacterial solution. This process was repeated three times. After inoculation, the discs were rinsed with sterile water and placed in 1% Agr culture dishes. They were incubated in the dark at 8 ℃ for 3 days, then transferred to a tissue culture room at (22 ± 1) ℃ with a light exposure of 16 h and a dark exposure of 8 h. The color changes of all petal discs were observed until complete, and the petal phenotype was recorded by photograph. ImageJ 15.1 software was used to measure and statistically analyze the petal color changes.

[0061] 2.8 Data Statistical Analysis Data used for statistical analysis were obtained from three biological replicates and three technical replicates. Data were statistically analyzed using GraphPad Prism 9.5 software, and the comparison between the two groups was performed using the student-t test (* P < 0.05, ** P < 0.01, *** P < 0.001).

[0062] 2.10 RcWRKY31 Primers used in sequence experiments, RcWRKY31 The gene sequences and primer lists are shown in Table 15: Table 15

[0063] Example 3 RcWRKY31 Validation and subcellular localization in different tissue sites Example 2: Total RNA was extracted from rose petals, leaves, roots, and stems and its quality was assessed. Genes with the most cis-regulatory elements related to meristem and plant growth and development were selected, and quantitative fluorescence expression analysis revealed differences in the expression of different genes in different tissues. Figure 1 The expression levels in different tissue sites, from highest to lowest, were: sepals, petals, leaves, and stem segments. RcWRKY31 ).in, RcWRKY31 The expression was more specific and at higher levels in the petals and sepals, indicating that... RcWRKY31 Genes play a role in the disease resistance of rose petals.

[0064] Simultaneously cloned RcWRKY31 CDS sequence of the gene, construction RcWRKY31 -GFP vector. Using an empty GFP vector as a control, EHA105 was transformed into tobacco leaves and injected. Observations were made under a laser confocal microscope 3 days later. Results showed... Figure 2 -GFP is located in the cell nucleus ( RcWRKY31 ). Example 4

[0065] silence RcWRKY31 The resistance to gray mold on the petals is weakened. First, detect botrytis cinerea infection. Figure 3 Changes in gene expression levels over time ( RcWRKY31 ),Discover RcWRKY31 The expression level gradually increased from 0 to 72 hours after Botrytis cinerea infection, indicating that... RcWRKY31 Related to the resistance of rose petals to gray mold.

[0066] To confirm RcWRKY31 The function of the gene in rose petals' resistance to gray mold was silenced using VIGS technology in 'Carola' rose plants. RcWRKY31 The petals were injected with gray mold. Observations were performed 3 days post-infection, revealing gene silencing (TRV2-). Figure 4 The petal lesion area was larger than that of the control (TRV). Figure 4 A, RcWRKY31 B), Detecting relative particle leakage rate to demonstrate gene silencing (TRV2- Figure 4 The degree of petal damage compared to the control (TRV) was also increased after gene silencing, as was the relative particle leakage rate of the petals. RcWRKY31 D). Simultaneously, the silenced gene (TRV2-) was detected. RcWRKY31 ) and control gene (TRV) petals Figure 4 Gene expression level ( RcWRKY31 C) The results showed that, compared with the control (TRV), RcWRKY31 The expression level was significantly reduced in silent plants, which further illustrates...RcWRKY31 Genes are associated with resistance to gray mold in rose petals. Example 5

[0067] overexpression RcWRKY31 Increased resistance to gray mold on hind petals To confirm RcWRKY31 The function of the gene in rose petal resistance to gray mold was investigated by overexpressing the gene in the 'Carola' rose plant through transient infection. RcWRKY31 The petals were injected with gray mold. Observations were performed 3 days post-infection, revealing overexpression of the gene (pSuper1300-). Figure 5 The area of ​​petal lesions was smaller than that of the control (pSuper1300). Figure 5 A, RcWRKY31 B), Detecting relative particle leakage rate to demonstrate gene overexpression (pSuper1300- Figure 5 The degree of petal damage compared to the control (pSuper1300) was compared to the relative particle leakage rate of the petals after gene overexpression. RcWRKY31 D). The overexpressed gene (pSuper1300-) was also detected. RcWRKY31 ) and control gene (pSuper1300) in the petals Figure 5 Expression level ( RcWRKY31 C) The results showed that, compared with the control (pSuper1300), RcWRKY31 The expression level was significantly increased in overexpressing plants, which further illustrates... ​ Related to the resistance of rose petals to gray mold.

[0068] The foregoing has shown and described the basic principles, main features, and advantages of the present invention. Those skilled in the art should understand that the present invention is not limited to the above embodiments. The embodiments and descriptions in the specification are merely illustrative of the principles of the invention. Various changes and modifications can be made to the invention without departing from its spirit and scope. The scope of protection of the present invention is defined by the appended claims, specification, and their equivalents.

Claims

1. A kind RcWRKY31 The application of a product expressing a gene or its transcribed and translated protein in regulating gray mold in roses, characterized by: The RcWRKY31 The nucleotide sequence of the gene is shown in SEQ ID NO.1.

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