CN106591323B

CN106591323B - Wild downy grape disease-resistant gene and application thereof

Info

Publication number: CN106591323B
Application number: CN201611202935.3A
Authority: CN
Inventors: 王西平; 王现行; 涂明星
Original assignee: Northwest A&F University
Current assignee: Northwest A&F University
Priority date: 2016-12-23
Filing date: 2016-12-23
Publication date: 2020-01-07
Anticipated expiration: 2036-12-23
Also published as: CN106591323A

Abstract

The invention discloses a wild downy grape disease-resistant gene and application thereof, wherein the full length of a complete open reading frame sequence is 1095bp, and 364 amino acids are coded. And a pCAMBIA2300-35S-VqWRKY52 overexpression vector is constructed, and is introduced into a model plant Arabidopsis thaliana by a catkin-dip dyeing method, so that the overexpression in the Arabidopsis thaliana obviously improves the resistance of the Arabidopsis thaliana to living parasitic powdery mildew and pseudomonas syringae, and reduces the resistance to botrytis cinerea. The wild vitis pubescens merchant-24 disease-resistant gene VqWRKY52 participates in an SA-mediated disease-resistant signal path, can enhance anaphylactic reaction, can be used for improving the capability of resisting living parasitic fungi powdery mildew and pseudomonas syringae of plants and regulating and controlling plant anaphylactic reaction by regulating and controlling the expression of VqWRKY52 gene, thereby regulating and controlling the reaction of the plants to different pathogenic bacteria.

Description

Wild downy grape disease-resistant gene and application thereof

Technical Field

The invention relates to the technical field of plant disease-resistant gene identification and genetic engineering, in particular to a wild downy grape disease-resistant gene and application thereof.

Background

Unlike animals which have mobile guard cells and an acquired immune system, the plant immune system can only rely on signals released by each plant cell and from the site of infection. During the long-term evolution of plant pathogenic bacteria and plants, various life strategies are evolved to help the plant to infect itself, so that a set of unique immune system is formed, wherein transcription factors play a very important role in regulation and control.

The WRKY transcription factor is an important transcription factor, and plays a role in positive regulation or negative regulation in the whole immune system network of a plant. The WRKY transcription factor is a special transcription regulation factor found in plants, the N end of the protein of the WRKY transcription factor contains a Zinc finger (Zinc-finger type) structure of highly conserved amino acid sequences WRKYGQK and CX4-5CXNHXH/C (X is any amino acid), and the downstream of the protein specifically binds to a nucleotide sequence of a target gene promoter region W box (T) TGACC (A/T), thereby regulating the expression of a corresponding gene and playing the molecular biological function of the gene. The classification of WRKY transcription factors is based on the number of domains contained in the transcription factors and zinc finger structures, and generally the WRKY transcription factors can be divided into three main classes: the 1 st class contains 2 WRKY conserved domains, and the zinc finger structure type is C2H2 type; class 2 contains only 1 WRKY conserved domain, and the zinc finger structure is C2H2 type; class 3 is the same as class 2, and contains only 1 WRKY conserved domain, but its zinc finger structure is C2HC type.

A large number of researches in arabidopsis thaliana and rice show that the WRKY transcription factors play a role in disease resistance, and most of the WRKY transcription factors are in inducible expression. Generally, the upstream of the WRKY transcription factor is activated by protein kinase MAPK phosphorylation in a MAPK signal pathway, and the downstream can be specifically combined with W-box, so that the expression of related disease-resistant genes is stimulated, and a disease resistance reaction is generated; one can be induced to produce protein directly by pathogenic bacteria, is mostly effector factor produced by pathogenic bacteria, makes WRKY protein expression quantity improve, WRKY gene regulates and controls the relevant disease-resistant gene again, thus improve the disease resistance of the plant, for example AtWRKY70 and AtWRKY18 improve the disease resistance of Arabidopsis by this regulation mode; multiple WRKY transcription factors can also regulate the resistance of plants to pathogenic bacteria through interaction, and compared with a single mutant, the triple mutants of AtWRKY18, AtWRKY40 and AtWRKY60 have the advantages that the resistance to pseudomonas syringae is increased, but the resistance to tomato gray mold is weakened. Over-expression of the rice OsWRKY23 gene in Arabidopsis can improve the expression level of disease course related protein and improve the resistance to pseudomonas syringae.

Resistance of plants to pathogen infection is mainly dependent on two major classes of phytohormone signalling pathways, of which the SA signalling pathway mainly mediates resistance of plants to living parasitic fungi, such as powdery mildew and the like; the JA/ET signaling pathway is primarily involved in regulating plant resistance to saprophytic bacteria, such as Botrytis cinerea and the like. Plant hormones often work with other response elements in regulating plant immunity, such as allergic reactions and reactive oxygen species bursts. The anaphylactic reaction is closely related to plant disease resistance, when living parasitic fungi infect plants, the plants can form large-area cell death at infection sites to prevent the fungi from obtaining nutrition from the plants, and the growth of the fungi is favorably limited, so that the disease resistance of the plants is improved. The SA signaling pathway can often stimulate anaphylactic reaction at an infection site or a far-end tissue, and simultaneously activate the expression of disease-resistant related protein and activate systemic acquired resistance. Plants respond to pathogenic bacterial reactions, often accompanied by bursts of reactive oxygen species, which play an important role in allergic reactions.

Research on disease-resistant related WRKY transcription factors in a model plant Arabidopsis is more, and research on grape disease-resistant functions of WRKY green-turning factors is not much. China has abundant wild grape resources, and some species and strains have strong disease resistance, such as the quotient-24 (S-24) but the quality of the grape resources is inferior to that of European grape species. The disease resistance of wild grapes is combined with the excellent quality of European grapes by making up for deficiencies, and the method has important significance for researching and breeding grape disease resistance by using the resources. Therefore, the disease-resistant related genes are discovered from wild grapes in China, the disease-resistant function of the wild grapes is researched, the regulation and control mechanism of the disease-resistant response of the wild grapes is known, the disease-resistant response key genes are found, and susceptible varieties are improved through genetic transformation, so that ideal varieties with excellent properties are obtained.

Disclosure of Invention

The invention aims to provide a wild downy grape disease-resistant gene, and verifies the function and possible action mechanism of the wild downy grape disease-resistant gene in disease resistance.

In order to realize the task, the invention adopts the following technical solution:

the wild downy grape disease-resistant gene is characterized in that the wild downy grape disease-resistant gene is a wild downy grape quotient-24 disease-resistant gene VqWRKY52, and the sequence of a coding region of the wild downy grape disease-resistant gene is as follows:

further research by the applicant shows that the wild downy grape disease-resistant gene can be used for improving the capability of resisting living parasitic fungi powdery mildew and pseudomonas syringae of plants. And the plant allergy can be regulated and controlled by regulating and controlling the expression of VqWRKY52 gene, so that the plant reaction to different pathogenic bacteria can be regulated and controlled.

The wild downy grape disease-resistant gene provided by the invention is a wild downy grape quotient-24 disease-resistant gene VqWRKY52 cloned from wild downy grape quotient-24 for the first time, and further researches the disease-resistant function and possible action mechanism of the wild downy grape quotient-24 disease-resistant gene VqWRKY52, so that a theoretical basis is provided for disease-resistant breeding of grapes and other crops.

The applicant constructs a pCAMBIA2300-35S-VqWRKY52 overexpression vector for the first time and introduces the vector into a model plant Arabidopsis thaliana by a catkin dip-dyeing method. The disease-resistant reactions of various strains of a transgenic strain and a wild control, which are over-expressed by a wild vitis heyneana-24 disease-resistant gene VqWRKY52, after powdery mildew, pseudomonas syringae and botrytis cinerea are inoculated are researched.

According to further research of the applicant, the wild downy grape commercial-24 disease-resistant gene VqWRKY52 can obviously improve the resistance of Arabidopsis to powdery mildew and pseudomonas syringae and increase the susceptibility to botrytis cinerea.

Under the infection condition of three pathogenic bacteria, the accumulation of Reactive Oxygen Species (ROS) in the transgenic line is obviously higher than that of a wild control, and the transgenic line has strong anaphylactic reaction, so that the strong anaphylactic reaction limits the growth of the pathogenic bacteria for living body erysiphe necator and semi-living body pseudomonas syringae, thereby enhancing the disease resistance to the pathogenic bacteria, but the strong anaphylactic reaction is beneficial to the infection of the saprophytic bacteria botrytis cinerea, so that the resistance of the transgenic line to the pathogenic bacteria is reduced. The results all show that the over-expression of wild downy grape merchant-24 stress-resistant gene VqWRKY52 in Arabidopsis enhances the plant allergic reaction, improves the living parasitic fungus resistance of the plant, and reduces the saprophytic fungus resistance.

Drawings

FIG. 1 is the response of wild downy grape quotient-24 disease-resistant gene VqWRKY52 to SA and JA. Wherein, a is relative expression quantity of wild downy grape quotient-24 disease-resistant gene VqWRKY52 observed by using a qRT-PCR method, b is activity analysis of wild downy grape quotient-24 disease-resistant gene VqWRKY52 promoter, which shows significant difference compared with a control, and which shows very significant difference compared with the control (0.01 < P <0.05, P < 0.01).

FIG. 2 is the expression pattern of wild downy grape quotient-24 disease-resistant gene VqWRKY52 promoter in different growth stages of Arabidopsis T3 generation plants; wherein, A is mature embryo which grows in MS culture medium for 24 hours, and the scale is 200 μm; b, 5-day-old seedlings with a scale of 500 μm; c, seedlings of 2 weeks old are shown, and the ruler is 2 mm; d, showing 3-week-old seedlings; e, showing the catkin, and the scale is 2 mm; f is flower with 1mm scale; g is the flower column and anther of the flower with the scale of 200 μm; graph H is silique and scale is 1 mm.

FIG. 3 is the response of the inoculation of white powdery mildew into potted seedlings of wild control, pad4 mutant, transgenic lines VqWRKY52(#28), VqWRKY52(#30) and VqWRKY52(# 33). Wherein, A is a morphological photo 7 days after each strain is inoculated with white powder; panel B shows trypan blue staining for powdery mildew-induced plant cell death, with the upper scale representing 200 μm.

FIG. 4 shows colony growth and active oxygen accumulation after inoculation of white powdery mildew on potted seedlings of wild control, pad4 mutant, transgenic lines VqWRKY52(#28), VqWRKY52(#30) and VqWRKY52(# 33). Wherein, A picture is inoculated with trypan blue for seven days to observe the growth of powdery mildew and the death of plant cells, arrows indicate the death of the plant cells induced by the powdery mildew, and a ruler on the picture represents 100 mu m; b is a graph of NBT vs O₂ ^-Staining was seen at 0 hours after inoculationAccumulation after 24 hours, 48 hours, and 72 hours, with 2cm on the scale; c is the statistics of the number of conidia seven days after inoculation, indicating a significant difference from the wild control, indicating a very significant difference from the wild control (0.01)<P<0.05，**P<0.01)。

FIG. 5 shows the expression of disease resistance related genes in wild control, transgenic lines VqWRKY52(#28), VqWRKY52(#30) and VqWRKY52(#33) after treatment with Erysiphe cichoracearum. The reference gene is Actin 2. Indicates significant differences compared to the wild control and indicates very significant differences compared to the wild control (. + -. 0.01< P <0.05,. + -. P < 0.01).

FIG. 6 is the response of pot seedlings of wild control, pad4 mutant, transgenic lines VqWRKY52(#28), VqWRKY52(#30) and VqWRKY52(#33) inoculated with Pseudomonas syringae. Wherein, A is a morphological photo 5 days after each strain is inoculated with pathogenic bacteria; b is the statistics of bacterial growth three days after inoculation; FIG. C shows the bacterial-induced death of the plant cells observed at 0 hour, 24 hours, 48 hours, and 72 hours after inoculation with the pathogenic bacteria, with trypan blue staining, and the scale on the graph represents 200 μm; d is graph H₂O₂And O₂ ^-Accumulation at 0 and 48 hours after inoculation with pathogenic bacteria, H₂O₂And O₂ ^-Staining with NBT and DAB, respectively. Each experiment was repeated three times, at least 6 leaves were treated in each independent experiment, and the scale on the graph represents 200 μm. Indicates a significant difference from the wild control (. about.0.01)<P<0.05)。

FIG. 7 shows the expression of disease resistance related genes in wild control, transgenic lines VqWRKY52(#28), VqWRKY52(#30) and VqWRKY52(#33) after inoculation with Pseudomonas syringae. The reference gene is Actin 2. Indicates significant differences compared to the wild control and indicates very significant differences compared to the wild control (. + -. 0.01< P <0.05,. + -. P < 0.01).

FIG. 8 shows the Botrytis cinerea inoculation reaction of potted seedlings of wild control, transgenic lines VqWRKY52(#28), VqWRKY52(#30) and VqWRKY52(# 33). Wherein, A is a morphological photo of each strain in vitro rosette leaf 3 days after being inoculated with pathogenic bacteria; b is a table used at 0 hour, 24 hours, 48 hours and 72 hours after the inoculation of pathogenic bacteriaTrypan blue staining is used for observing the plant cell death induced by bacteria, and the scale on the graph represents 2 cm; panel C is lesion diameter statistics three days after inoculation; d is a drawing H₂O₂And O₂ ^-Accumulation at 0 and 48 hours after inoculation with pathogenic bacteria, H₂O₂And O₂ ^-Staining with NBT and DAB, respectively. Each experiment was repeated three times, at least 6 leaves were treated in each individual experiment, and the scale on the graph represents 2 cm. Indicates a significant difference from the wild control (. about.0.01)<P<0.05)。

FIG. 9 shows the expression of disease resistance-associated genes in wild control, transgenic lines VqWRKY52(#28), VqWRKY52(#30) and VqWRKY52(#33) after inoculation with Botrytis cinerea. The reference gene is Actin 2. Indicates significant differences compared to the wild control and indicates very significant differences compared to the wild control (. + -. 0.01< P <0.05,. + -. P < 0.01).

The invention is described in further detail below with reference to the figures and examples.

Detailed Description

The application inventor utilizes Reverse Transcription-Polymerase Chain Reaction (RT-PCR), the first strand of cDNA synthesized by Reverse Transcription of wild downy grape business-24 leaf total RNA is taken as a template, homologous cloning technology is utilized, and a designed primer is adopted according to a European grape Henbinuo genome sequence to amplify to obtain the wild downy grape business-24 disease-resistant gene VqWRKY52, wherein the wild downy grape disease-resistant gene is the wild downy grape business-24 disease-resistant gene VqWRKY52, the full length of a complete open reading frame sequence is 1095bp, and 364 amino acids are coded.

In order to research the specific function of the wild vitis amurensis merchant-24 disease-resistant gene VqWRKY52 in the plant resistance to biotic stress in one step, the inventor of the applicant constructs a pCAMBIA2300-35S-VqWRKY52 overexpression vector and overexpresses the vector in a wild Arabidopsis plant. The over-expression strain with the wild vitis heyneana-24 disease-resistant gene VqWRKY52 is found to be capable of obviously improving the resistance of arabidopsis to powdery mildew and pseudomonas syringae and increasing the susceptibility to botrytis cinerea. The overexpression of VqWRKY52 in Arabidopsis enhances the plant anaphylactic reaction, improves the capability of resisting living parasitic fungi of plants, and reduces the capability of resisting saprophytic bacteria.

In the research, the applicant carries out stress treatment on three different types of pathogenic bacteria on seedling transgenic plants and wild controls, and determines the cell death condition, the accumulation of active oxygen and the expression condition of disease-resistant related genes, and the result shows that the wild downy grape quotient-24 disease-resistant gene VqWRKY52 is expressed by SA induction and plays an important role in the cell death process induced by the pathogenic bacteria.

The following steps are specific steps of the coding region sequence of wild downy grape merchant-24 disease-resistant gene VqWRKY52 and the experimental verification of the anti-biotic stress function.

A. In the early research and analysis, on the basis of the expression of grape WRKY family genes after pathogenic bacteria treatment and hormone treatment, a first cDNA chain synthesized by reverse transcription of wild downy grape Shang-24 leaf total RNA is used as a template by utilizing a homologous cloning technology, and a wild downy grape Shang-24 disease-resistant gene VqWRKY52 sequence is obtained through amplification, wherein the coding region sequence of the wild downy grape Shang-24 disease-resistant gene VqWRKY52 is as follows:

B. the complete open reading frame of the wild vitis mauritiana merchant-24 stress-resistant gene VqWRKY52 sequence is inserted into the downstream of a CaMV35S promoter, a plant over-expression vector is constructed, and the plant over-expression vector is introduced into wild type Arabidopsis thaliana Columbia C0 by an agrobacterium-mediated catkin dip-dyeing method. The screening yielded VqWRKY52 transgenic lines (#28, #30, #33) with good phenotype.

C. Referring to FIGS. 1-9, the VqWRKY52 transgenic strain (#28, #30, #33) was identified, which significantly improved the resistance of Arabidopsis to powdery mildew and Pseudomonas syringae, and increased susceptibility to Botrytis cinerea. In addition, the accumulation of Reactive Oxygen Species (ROS) in the transgenic lines after inoculation with pathogenic bacteria was also significantly greater than in the wild control, and had a stronger allergic response than in the control. The results all show that the wild downy grape quotient-24 disease-resistant gene VqWRKY52 plays an important role in a plant disease-resistant signal pathway.

The following are specific examples given by the inventors, and all the methods in the following are conventional ones unless otherwise specified. The wild downy grape quotient-24 disease-resistant gene WRKY52 is abbreviated as VqWRKY52 in the following examples.

Example 1: VqWRKY52 response to hormones and tissue-and spatio-temporal specific expression in Arabidopsis

In the previous research, the applicant found that VqWRKY52 is induced to be expressed by erysiphe necator, and in order to determine the response of VqWRKY52 to SA and JA, the expression of VqWRKY52 under the treatment of the two hormones was analyzed, and as a result, VqWRKY52 can be strongly induced to be expressed by SA but cannot be induced by MeJA when the pathogenic bacteria are treated for 1 hour and 12 hours (FIG. 1A).

To further verify this result, a 2107bp promoter region of VqWRKY52 was cloned and fused in front of GUS reporter gene to form Pro_VqWRKY52GUS reporter construct, transient transformation of tobacco to analyze promoter activity in response to hormones, Pro_35SGUS was used as a control. The results indicate that the VqWRKY52 promoter can be expressed by SA induction and not by JA induction (fig. 1B), which is consistent with the results of fig. 1A.

To see tissue-and spatio-temporal specific expression, Pro was used_VqWRKY52GUS stably transforms Arabidopsis thaliana to obtain T3 generation plants, and GUS staining analysis is carried out by using plants in different developmental stages. No significant GUS activity was detected during early germination (fig. 2A), and very low activity was found in the tip and root system of the cotyledons as the plants grew (fig. 2B). Plants grown in MS medium for two weeks had strong GUS activity in all tissues, especially in leaves (FIG. 2C). In the adult seedlings, the old leaves had stronger GUS activity than the young leaves (FIG. 2D). Strong GUS activity was found in the tissues other than the seeds in the anthers and pods throughout the Arabidopsis thaliana floral battings and pods (FIGS. 2E-H).

Example 2: response of VqWRKY52 transgenic Arabidopsis T3 seedlings to powdery mildew

In order to further determine the effect of VqWRKY52 in plant defense, 3T 3 transgenic lines (#28, #30, #33), an Arabidopsis Wild Type (WT) and a pad4 mutant are inoculated with Arabidopsis powdery mildew, the response of the Arabidopsis powdery mildew to pathogenic bacteria is observed, the inventor inoculates powdery mildew to adult seedlings growing for 3-4 weeks, observes the active oxygen, dead cell accumulation and pathogenic bacteria growth conditions of different lines, counts the conidia number of a single colony of the powdery mildew, and quantitatively analyzes the relative expression condition of disease-resistant related genes. The results showed that the three transgenic lines (#28, #30, #33) had less disease than the wild control (WT) after Erysiphe cichoracearum inoculation and the pad4 mutant had the most severe disease (FIG. 3A). Three transgenic lines (#28, #30, #33) developed strong erysiphe necator-induced cell death, whereas wild control (WT) cells died less, and the pad4 mutant, although the most severe, had little apparent cell death observed (fig. 3B). To further confirm the above results, we observed the growth of powdery mildew in the leaves of each disease-causing strain. The growth of powdery mildew was found to be significantly inhibited in 3 transgenic lines and there was significant cell death at the site of infection (FIG. 4A). The statistical number of conidia in the transgenic lines was significantly less than that of the WT plants (FIG. 4C).

O₂ ^-The accumulation of (D) is often associated with plant cell death, so it was further determined that the different genotypes were O at 0 hours, 24 hours, 48 hours, and 72 hours of powdery mildew inoculation₂ ^-Accumulation of (2). In all genotypes examined, no significant difference was observed between 0 and 24 hours, and a large burst of reactive oxygen species occurred. At 48 and 72 hours, the transgenic lines showed a greater amount of O compared to WT and pad4₂ ^-Aggregation, especially high at 72 hours, and the accumulation of reactive oxygen species appears speckled. (FIG. 4B) previous experiments have demonstrated that VqWRKY52 is expressed by SA but not by JA. To further understand the response of VqWRKY52 gene in plants responding to powdery mildew stress, the inventors examined the expression levels of disease-resistant related genes in the SA signaling pathway in wild control (WT) and transgenic lines (#28, #30, #33) by real-time quantitative PCR (fig. 5). AtICS1 gene is involved in SA biosynthesis and affects SA accumulation, and expression level is increased after 24 hours of pathogenic bacteria treatment, 4The peak was reached at 8 hours and the decrease started at 72 hours, and the expression level in all 3 transgenic lines was higher than that of the wild type at all 3 time points. AtEDS1 is also involved in SA-related signaling pathways, playing an important role upstream of SA biosynthesis. Its expression was similar to AtICS1 at 24 and 48 hours, but at 72 hours, the expression level was lower in the over-expressed line than in the wild type. In addition, the AtPR1 and AtPR5 genes were up-regulated in expression at 24, 48 and 72 hours after treatment, and the expression level of the over-expressed strain was higher than that of the wild type. The above experiments demonstrate that VqWRKY52 plays a role in SA-mediated signaling pathways, with enhanced expression of SA-related genes in over-expressed strains.

These results all indicate that the overexpression of wild vitis amurensis merchant-24 disease-resistant gene VqWRKY52 in Arabidopsis can obviously improve the resistance of Arabidopsis to powdery mildew.

Example 3: VqWRKY52 response of transgenic Arabidopsis T3 seedlings to Pseudomonas syringae

In order to determine the effect of VqWRKY52 in plant anti-pseudomonas syringae, 3T 3 transgenic strains (#28, #30, #33) and Arabidopsis Wild Type (WT) were inoculated with pseudomonas syringae, response to pathogenic bacteria was observed, adult seedlings growing for 3-4 weeks were inoculated with pathogenic bacteria, active oxygen, dead cell accumulation and pathogenic bacteria growth were observed for different strains, the number of pathogenic bacteria in leaves after 3 days of inoculation was counted, and the relative expression of genes related to disease resistance was quantitatively analyzed (FIGS. 6, 7). The results showed that the three transgenic lines (#28, #30, #33) had less disease after inoculation with pathogenic bacteria than the wild control (WT) (FIG. 6A). Meanwhile, the growth amount of bacteria in different genotypes was counted 3 days after inoculation, and it was found that the bacterial growth was inhibited in the overexpressed plants (fig. 6B). At 0h, no cell death was found for all genotypes, cell death began to appear in the transgenic lines at 24h, whereas WT did not appear, and massive cell death appeared in the transgenic lines at 48h and continued to increase at 72 h. WT cell death occurred at 72h comparable to the transgenic line at 24h (FIG. 6C). Massive cell death at 72h was likely associated with a reactive oxygen burst. Thus measuring H₂O₂And O₂ ^-Accumulation condition at 72 h. As a result, it was found that,at 72h, the over-expressing line accumulated more reactive oxygen species than the wild type (FIG. 6D).

To further understand the response of VqWRKY52 gene in plant anti-Pseudomonas syringae, the expression changes of disease-resistant related genes 0h, 6h, 12h and 24h after inoculation of pathogenic bacteria in wild control (WT) and transgenic lines (#28, #30, #33) were detected by real-time quantitative PCR (FIG. 7). The results show that AtPR1, AtPR5 and AtEDS1 induced expression in the wild type but were suppressed in the transgenic lines, in contrast to the response of the over-expressed lines to powdery mildew. This is also true for the JA pathway-associated gene AtPDF1.2. The above experiments demonstrate that the bacterially induced enhancement of cell death by overexpression of VqWRKY52 in arabidopsis is not matched to the expression of SA-associated genes.

These results indicate that the overexpression of wild vitis pubescens merchant-24 disease-resistant gene VqWRKY52 in Arabidopsis obviously improves the resistance of adult plants to pseudomonas syringae, and is related to cell death induced by bacteria, but the expression relationship with SA-related genes is not large.

Example 4: VqWRKY52 response of transgenic Arabidopsis T3 seedlings to Botrytis cinerea

Bacteria-induced cell death plays an important role in plants against live-parasitic pathogens, and applicants wanted to look at the response of over-expressed lines to saprophytic bacteria. 3T 3 transgenic lines (#28, #30, #33) and Arabidopsis Wild Type (WT) were inoculated with Botrytis cinerea to see their response to pathogenic bacteria; adult seedlings grown for 3-4 weeks were inoculated with pathogenic bacteria, different lines of active oxygen, dead cell accumulation and lesion diameter were observed, and relative expression of disease-resistance-associated genes was quantitatively analyzed (FIG. 8, FIG. 9). The results showed that the three transgenic lines (#28, #30, #33) had a more severe disease status after inoculation with pathogenic bacteria than the wild control (WT) (FIGS. 8A, C). Meanwhile, cell death was observed at 0h, 24h, 48h and 72h of the inoculation with Botrytis cinerea, and accumulation of active oxygen was observed at 0h and 48h (FIGS. 8B, D). The results show that the allergic reaction of the over-expression strain is stronger than that of the wild type strain after the over-expression strain is inoculated with the botrytis cinerea, and more active oxygen is accumulated. However, overexpression of VqWRKY52 enhanced susceptibility to botrytis cinerea.

To further understand the role of VqWRKY52 gene in regulation of Botrytis cinerea resistance, changes in expression of disease-resistance-related genes in wild control (WT) and transgenic lines (#28, #30, #33) at 0h, 12h, 24h and 48h after inoculation with pathogenic bacteria were examined by real-time quantitative PCR (FIG. 9). The results showed that the JA pathway-associated gene AtPDF1.2 was induced to be expressed 12h after inoculation in all genotypes, and that the expression level was higher in 3 transgenic lines than in the wild type. 24h after inoculation, the expression level in the transgenic lines began to decrease, while the wild type continued to increase, when the wild type was higher than the transgenic lines. In addition, the expression levels of AtPR2 and AtPR5 were suppressed at 12h, 24h and 48h after inoculation with Botrytis cinerea, relative to control 0h, but the expression levels in the transgenic lines were significantly higher than those in the wild type, suggesting that the expression of these genes was suppressed less in the transgenic lines than in the wild type. These results suggest that the SA signaling pathway is significantly enhanced in the transgenic lines, and that the SA signaling pathway is antagonistic to the JA signaling pathway, and that the enhancement of the SA signaling pathway may weaken the JA pathway, which is unfavorable for the resistance of plants to saprophytic bacteria, and may increase susceptibility.

The results show that the excessive expression of the wild downy grape quotient-24 disease-resistant gene VqWRKY52 in Arabidopsis obviously improves the susceptibility of adult plants to botrytis cinerea.

Nucleotide or amino acid sequence listing

<110> northwest agriculture and forestry science and technology university

<120> wild downy grape disease-resistant gene and application thereof

<160>

<210> 1

<211>1095

<212> coding region sequence of wild downy grape quotient-24 disease-resistant gene VqWRKY52

<213>

<220>

<400> 1

1 ATGGAGAACA TGGGAAGTTG GGAACAAAAG AATCTGATAA ATGAGCTTAC GAATGGGAGA

61 GAGCTAGCCA AACAGCTACA AATCCATCTC AGCGTGTCGT CTTCTTCACA TGACGCCCGT

121 GAATCCTTGG TCCAAAAGAT CCTAACTTCA TACGAGAAGG CCCTTTCATT GC TGAGGTGT

181 AGCGGCCCAG TGGGCGAGCC ACATGCCGCC GCAGCAGGCG GAGGAGGAGC AGTTGGAATA

241 GGGATGTCTG AGTCTCCACG CTCGCTCAGT GGGAGTCCTA GGAGCGAAGA TTCTGATAAG

301 GAGCAGGAGC ACAAAGATGG GTCTAGGAAG AGAAAGACAT TACCAAGGTG GACAAAGCAA

361 GTGCAGATGT CTCTAGGGCC AGGGCCAGAA GGCCCTCTTG ATGATGGGTT TAGTTGGAGG

421 AAGTATGGGC AGAAGGACAT TCTCGGAGCC AAATATCCAA GGAGCTATTA CAAATGCACT

481 CATAGAAATG CCCAAGGCTG TCTGGCTACA AAGCAAGTTC AAAGATCAGA CGATGACCCA

541 ACCATCTTTG AAATCACCTA CCGTGGAAGA CACACTTGCA CCCAAGTCTC CCAACAAATT

601 CCACCGCCGG CAGCACTGCC GGGAAACCAA GGACTGATGG ACAGCAAAGA TCATCAGCAA

661 TTCAACCTCC AACCCCAACA AAACCAGCAA CAAGCACAAG ACATGCTCTT GAGCTTCCAA

721 AGTGGCCTCA GAGTCGTAAC AGAAGGCTTG GACACACCAA GCGACCAAGC ATTTCCCCCC

781 TTTTGCTTCA CTTCAACGTC TAATATTAAG GTCGAAGAAC CCGGTTTCTC GCCTTCCATG

841 ATGGACAACA ATATCGTTGG TAATTTTCCT ACCTCATTTG TATCTCCTGC AGCATCTGGA

901 TCAAACTATT TTTCCATGTC GTCTGATGAA ATGAACAGCC TTGGAGGAAA CCAAAACCTG

961 CAGGCCCCCG AAGCCAATCT CAATGAGATA ATCTCAGCTG CTGCTTCAAC CACAAACCCT

1021 CAGTTTGAGC AGTTCCCGTT TGGTTCCATG GAATTCGATC CGAACTTTAA CTTCGACCAC

1081 CTGGGATTCT TTTAA

Claims

1. The wild vitis amurensis merchant-24 disease-resistant gene VqWRKY52 is used for improving the resistance of plants to powdery mildew, which is a living parasitic fungus, and the resistance of plants to pseudomonas putida, and plays a role in regulating and controlling plant allergy by regulating and controlling the expression of VqWRKY52 gene, so that the regulation and control of the reaction of plants to different pathogenic bacteria are achieved;

the coding region sequence of the wild downy grape merchant-24 disease-resistant gene VqWRKY52 is as follows: