A mango MYB transcription factor, interacting protein and its application in enhancing disease resistance

Abstract

The present invention provides a mango MYB transcription factor, an interacting protein and an application thereof in enhancing disease resistance. The mango MYB transcription factor is named MiMYB44 transcription factor, the nucleotide sequence of the encoding gene is shown in SEQ ID NO.1, the interacting protein is 1-aminocyclopropane-1-carboxylic acid oxidase ACO, and the MiMYB44 transcription factor can activate the plant disease resistance immune response, by increasing the content of malondialdehyde, superoxide anion, hydrogen peroxide, anthocyanin and ethylene, activating the transcription activity of disease resistance-related genes such as PR, thereby enhancing the plant disease resistance. Experimental results show that MiMYB44 is a key gene in the defense response of mango against bacterial diseases, can be used as a key gene resource for disease-resistant breeding, and helps in the selection of disease-resistant germplasm resources, the creation of resistant parents, and the breeding of disease-resistant varieties.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and in particular to a mango MYB transcription factor, an interacting protein and an application thereof in enhancing disease resistance. Background Art

[0002] Transcription factors are those that can interact with cis-acting elements in the promoter region to activate or inhibit the expression of downstream target genes, and play a key role in plant growth, emergency response, disease resistance, insect resistance, and stress resistance. The differences in transcription factor sequences and structures determine the differences in their functions, and differences in nuclear localization and oligomerization also affect their own activity. Plant transcription factors activate or inhibit the expression of induced genes by interacting with DNA functional domains, proteins, and other transcription factors, and play an important role in regulating plant growth. Transcription factors can also interact with transcription factors and other proteins to exert their functions.

[0003] MYB family transcription factors contain a highly conserved MYB DNA domain at their N-terminus, which is about 50-53 amino acid residues in length and usually contains 1 to 4 incomplete MYB repeat sequences (SONT domain). MYB family transcription factors are involved in various physiological activities of plants, such as growth and development, secondary metabolism, hormone signal transduction, disease resistance, and tolerance to biotic and abiotic stresses. For example, the R2R3-MYB genes AtMYB83 and AtMYB46 are direct targets of the Arabidopsis secondary cell wall-related protein AtSND1 (NAM-ATAF-CUC [NAC] domain protein 1), and their induction triggers the expression of AtMYB58, AtMYB63, and AtMYB85, thereby regulating secondary cell wall biosynthesis genes.

[0004] Mango is one of the important economic fruits in tropical regions and is widely welcomed by consumers around the world. Mango is often infected by pathogens and subjected to biological and abiotic stresses during its growth and development, causing huge economic losses. Mango bacterial black spot (Xanthomonas citri pv. mangiferaeindicae, Xcm) is a disease caused by Xanthomonas bacteria and is the most serious bacterial disease in mango production. In production, disease prevention and control is one of the necessary links in production, and the breeding of disease-resistant varieties is one of the important measures to solve disease prevention and control. High-quality mango reference genome sequencing data can promote molecular breeding and design breeding research of mango, while the high heterozygosity of the mango genome poses a huge challenge to the assembly of the mango genome.

[0005] In recent years, the research on mango transcription factors has mainly focused on bioinformatics fields such as bHLH and NAC. Analyzing the disease resistance mechanism related to transcription factors and providing genetic resources for disease-resistant breeding can accelerate the selection of disease-resistant varieties. However, there are currently no reports on the research on mango MYB transcription factors in plant disease resistance. Summary of the invention

[0006] In view of this, the present invention proposes a mango MYB transcription factor, an interacting protein and their application in enhancing disease resistance.

[0007] The technical solution of the present invention is achieved in this way:

[0008] A mango MYB transcription factor, wherein the mango MYB transcription factor is named MiMYB44 transcription factor.

[0009] It is further described that the nucleotide sequence of the gene encoding the MiMYB44 transcription factor is shown in SEQ ID NO.1.

[0010] A mango MYB transcription factor interacting protein, wherein the interacting protein is 1-aminocyclopropane-1-carboxylic acid oxidase ACO.

[0011] Application of a mango MiMYB44 transcription factor in enhancing plant disease resistance.

[0012] Application of a mango MiMYB44 transcription factor in enhancing the defense response of mango against bacterial diseases.

[0013] Application of a mango MiMYB44 transcription factor in positively regulating anthocyanin and ethylene content.

[0014] Application of a mango MiMYB44 transcription factor in positively regulating malondialdehyde content.

[0015] Application of a mango MiMYB44 transcription factor in positively regulating hydrogen peroxide content and superoxide anions.

[0016] A mango MiMYB44 transcription factor is used in positively regulating AtICS1, AtPR1, AtPR2, AtPR5, AtPDF1.2 and AtACS disease resistance genes.

[0017] The invention discloses an application of a mango MiMYB44 transcription factor in enhancing the disease resistance of mangoes. The MiMYB44 transcription factor forms a protein complex with ACO to activate the synthesis of endogenous ethylene, enhance the transcription of disease resistance-related genes, increase the synthesis and accumulation of disease resistance products, and improve the disease resistance of mangoes.

[0018] Compared with the prior art, the beneficial effects of the present invention are as follows: the present invention identifies the coding gene of the MiMYB transcription factor in mango, screens out 4 candidate genes from the aspects of structural domain, evolutionary tree, gene transcription level, etc. to explain the action mechanism of disease-resistant related genes, and establishes 4 MiMYBs transient expression systems in tobacco and mango, including virus-induced gene silencing (VIGS method) and transient overexpression based on the 35S promoter of cauliflower mosaic virus (CaMV), detects its transcription level, the growth rate of pathogens, and physiological and biochemical indicators, and finds that the plant disease resistance immune response activated by MiMYB44 has the best effect.

[0019] The present invention heterologously overexpresses the MiMYB44 gene in Arabidopsis thaliana. During the infection of Xcm and the plant pathogenic bacterium Pseudomonas syringae (Pst DC3000), the transgenic strains are found to enhance the disease resistance of Arabidopsis thaliana by increasing the content of malondialdehyde, superoxide anion, hydrogen peroxide, anthocyanin and ethylene, activating the transcriptional activity of disease resistance-related genes such as PR, and the results show that MiMYB44 can also be widely involved in disease resistance in Arabidopsis thaliana. The present invention screened and obtained the interacting protein 1-aminocyclopropane-1-carboxylateoxidase (ACO) of MiMYB44. MiMYB44 forms a protein complex with ACO, activates the synthesis of endogenous ethylene, enhances the transcription of disease resistance-related genes, increases the synthesis and accumulation of disease resistance products such as anthocyanin, improves the disease resistance of plants, hinders the growth rate of pathogens, and makes plants show disease resistance.

[0020] Experimental results show that MiMYB44 is a key gene in the defense response of mango against bacterial diseases. It can be used as a key gene resource for disease-resistant breeding, helping to identify disease-resistant germplasm resources, create resistant parents, and breed disease-resistant varieties. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 The phylogenetic tree of the mango and Arabidopsis MYB gene family members of the present invention is

[0022] Figure 2 The conserved domain and conserved motif of the mango MYB gene family members of the present invention

[0023] Figure 3 This is a diagram for identifying the T0 generation of the MiMYB44-overexpressing Arabidopsis transgenic strain of the present invention;

[0024] Figure 4 This is a graph showing the expression level of the transgenic Arabidopsis MiMYB44 gene of the present invention;

[0025] Figure 5This is a diagram for identifying the T3 generation of the transgenic Arabidopsis thaliana transgenic strain of the present invention;

[0026] Figure 6 This is a phenotype diagram of the MiMYB44 transgenic Arabidopsis thaliana of the present invention;

[0027] Figure 7 This is a graph showing the malondialdehyde content of MiMYB44 transgenic Arabidopsis thaliana of the present invention;

[0028] Figure 8 This is a graph showing the superoxide anion content in MiMYB44 transgenic Arabidopsis thaliana of the present invention;

[0029] Fig. 9 This is a graph showing the hydrogen peroxide content of MiMYB44 transgenic Arabidopsis thaliana of the present invention;

[0030] Fig.10 This is a graph showing the anthocyanin content of MiMYB44 transgenic Arabidopsis thaliana of the present invention;

[0031] Fig.11 This is a graph showing the ethylene content of MiMYB44 transgenic Arabidopsis thaliana of the present invention;

[0032] Fig.12 The transcription map of the disease resistance-related genes regulated by the overexpression of MiMYB44 gene in Arabidopsis thaliana according to the present invention;

[0033] Fig.13 The PCR product of the yeast two-hybrid positive clone strain of the present invention is shown in FIG.

[0034] Fig.14 This is a diagram showing the effect of the protein control experiment of the yeast two-hybrid interaction of the present invention. DETAILED DESCRIPTION

[0035] In order to better understand the technical content of the present invention, specific embodiments are provided below to further illustrate the present invention.

[0036] Unless otherwise specified, the experimental methods used in the embodiments of the present invention are all conventional methods.

[0037] Unless otherwise specified, the materials, reagents, etc. used in the embodiments of the present invention can be obtained from commercial sources.

[0038] Example 1 - Identification and analysis of mango MiMYB transcription factors

[0039] A total of 54 candidate members of the MiMYB family were detected and named in the mango genome. The size of MiMYB proteins in mango varies significantly, with the length of the encoded protein ranging from 215 to 1121 amino acids, the protein molecular weight ranging from 24.92 to 125.84 kDa, and the predicted isoelectric point ranging from 4.87 to 9.67. Figure 1 , Figure 2 , phylogenetic analysis between mango and Arabidopsis thaliana showed that MYBs in mango and MYBs in Arabidopsis thaliana were divided into 13 different groups, indicating that the MYB family was relatively conservative in the evolution of different plants. The present invention analyzes the expression profile of MiMYBs genes in mango in response to biotic and abiotic stresses. MiMYBs show different expression patterns, and some MiMYBs show higher transcription levels in response to specific periods during pathogen infection, resistance signal molecules, etc. Therefore, the present invention screens out 4 candidate genes from the aspects of structural domain, evolutionary tree, gene transcription level, etc. for explaining the mechanism of action of disease-related genes, namely MiMYB10, MiMYB14, MiMYB40, and MiMYB44. The nucleotide sequences of MiMYB10, MiMYB14, and MiMYB40 encoding genes are shown in SEQ ID NO.3-5.

[0040] Example 2- Functional analysis of MiMYBs genes

[0041] The MiMYBs of the present invention were silenced by VIGS method in tobacco and found that under the infection of Xcm and DC3000, the anthocyanin content of MiMYB10, -40 and -44 all showed a downward trend; in order to further verify its function, MiMYBs were transiently overexpressed and found that the anthocyanin content of MiMYB10, -40 and -44 all showed overexpression. It shows that MiMYB10, -40 and -44 have a positive regulatory function on the anthocyanin content during the growth and development of mango fruit. The growth of pathogens when silencing and overexpressing MiMYBs was detected, and it was found that its change trend was generally consistent with anthocyanins, which further verified that MiMYBs increased resistance to pathogen infection by changing the anthocyanin content, and also regulated physiological and biochemical reactions such as malondialdehyde and hydrogen peroxide to varying degrees.

[0042] The present invention performs VIGS silencing and overexpression of MiMYBs on tobacco and mango leaves respectively. After silencing MiMYB10, -40 and -44 genes, the ethylene content can be reduced under pathogen infection, while after overexpressing these three genes, the ethylene content is significantly increased, and the growth of the pathogen corresponds to it. This shows that the transcription levels of MiMYB10, -40 and -44 in tobacco leaves and mango leaves are positively correlated with the growth rate and ethylene content of the pathogen, and at the same time regulate the transcription of plant disease resistance-related genes and participate in the plant disease resistance defense process, and the plant disease resistance immune response activated by MiMYB44 is the best. In addition, the gene function displayed by MiMYB14 is different from that of MiMYB10, -40 and -44 in regulating plant defense.

[0043] The present invention also shows that, through the results of silencing and overexpressing MiMYBs in tobacco and mango leaves, except MiMYB14, the silenced tobacco and mango become more sensitive to the infection of Xcm and DC3000. The overexpression of tobacco and mango significantly improves the resistance to the infection of Xcm and DC3000, and also induces the expression of disease-resistant related genes, indicating that mango relies on the regulation of MYB genes when resisting infection by pathogens.

[0044] Example 3 - Genetic transformation of mango MiMYB44 gene into Arabidopsis thaliana

[0045] The invention adopts the wild-type material Arabidopsis thaliana of Columbia (Col-0) as the genetic transformation material. The strain and the vector are: Agrobacterium GV3101, 35S::GFP-MiMYB44 vector, containing 35S strong promoter and herbicide resistance gene, and the resistant Arabidopsis seedlings can be screened by spraying herbicide.

[0046] Arabidopsis thaliana was genetically transformed by Arabidopsis pollen tube introduction. The bacterial suspension of Agrobacterium GV3101 containing the 35S::GFP-MiMYB44 vector was used for flower soaking. The infected Arabidopsis thaliana was shaded with black plastic film for 24 hours, and then the black plastic film was removed. The seeds were cultured under normal conditions until they matured and harvested. The seeds were placed in an oven to dry, and after being taken out, color-changing silica gel was placed to prevent the seeds from becoming damp, and they were stored and used for subsequent experiments.

[0047] The harvested suspected transgenic seeds were planted in nutrient pots. When they grew to 2 true leaves, herbicides were sprayed for preliminary screening. The total DNA of the leaf genome of the suspected positive Arabidopsis plants was extracted by CTAB method, and PCR was used for positive identification. Specific primers were used to detect whether the target gene was inserted into the Arabidopsis genome. The transgenic Arabidopsis plants that were positive in PCR detection were selected, and the total RNA was extracted and the first chain of cDNA was reverse transcribed. The expression level of the MiMYB44 gene was detected by fluorescence quantitative detection. The internal reference gene was the Arabidopsis housekeeping gene Actin-2. The strains with relatively high gene expression were selected, harvested, sown, and identified again until the T3 generation homozygous Arabidopsis MiMYB44 transgenic seedlings were obtained for subsequent phenotypic observation experiments.

[0048] The identification results of transgenic Arabidopsis thaliana are as follows Figure 3 , Figure 4 shown. Figure 3 In the figure, M is DNA marker DL2000; 1 is 35S::GFP-MiMYB44 plasmid DNA as a positive control; 2 and 3 are two strains of Arabidopsis transgenic T0 generation OE-MiMYB44#1 and OE-MiMYB44#2, respectively. Figure 4The relative expression levels are different. After continuous reproduction, the positive seedlings are transplanted when they grow well and cultured under normal culture conditions. When the T3 generation positive plants grow to 8-10 leaves, samples are taken, and DNA of each transgenic line is extracted. PCR detection is performed on the positive plants using specific amplification primers to identify whether the gene has been successfully transferred into the Arabidopsis genome. The PCR product is detected by electrophoresis. Figure 5 As shown in the figure, M is DNA marker DL2000; 1 is 35S::GFP-MiMYB44 plasmid DNA as a positive control; 2, 3 and 4 represent the experimental seedling detection of Arabidopsis transgenic T3 generation. The results showed that the electrophoresis bands consistent with the size of the target fragment were obtained, indicating that the MiMYB44 gene has been successfully transformed and homozygous transgenic plants have been obtained, which can be identified as positive strains and then used in subsequent related experiments.

[0049] Phenotypic observation of MiMYB44 transgenic Arabidopsis infected by pathogens. Transgenic lines OE-MiMYB44#1 and OE-MiMYB44#2 were planted and the leaf phenotypes of Arabidopsis were observed when they grew to 8-10 true leaves ( Figure 6 , A), it can be seen that the leaves of wild-type Arabidopsis are flat and grow normally, while the two MiMYB44 overexpression lines show a phenotype of thick plants and larger leaf area compared with the wild type. In order to identify the disease resistance of transgenic Arabidopsis, Xcm and DC3000 were used to infect the two lines OE-MiMYB44#1 and OE-MiMYB44#2 with pathogens, and it was found that ( Figure 6 , B) Before treatment, WT and OE-MiMYB44#1, OE-MiMYB44#2 showed normal growth with green leaves; 2 days after infection, the front sides of the leaves of OE-MiMYB44#1, OE-MiMYB44#2 and WT were still green, while the back sides of OE-MiMYB44#1 and OE-MiMYB44#2 began to show partial purple; 4 days after infection, WT showed no obvious changes, while OE-MiMYB44#1 and OE-MiMYB44#2 also began to show small areas of purple on the front sides of the leaves, while the back sides turned purple over a large area.

[0050] Example 4- Determination of physiological and biochemical indicators of transgenic Arabidopsis thaliana under pathogen infection stress

[0051] Transgenic strains of MiMYB44 (OE-MiMYB44#1, OE-MiMYB44#2) and wild-type strains with consistent growth were selected for pathogen infection, and photos and samples were taken at 0d, 2d, 4d, and 6d of treatment, and then quickly frozen in liquid nitrogen and stored at -80℃ for later use. They were used for the determination of malondialdehyde, superoxide anion, hydrogen peroxide, anthocyanin, ethylene, etc., as well as the differential expression levels of disease resistance-related genes.

[0052] (1) Determination of malondialdehyde content in leaves of transgenic lines

[0053] In order to verify the function of MiMYB44 gene in Arabidopsis, the malondialdehyde content of two transgenic lines infected with Xcm and DC3000 was detected. Figure 7 A. Figure 7 B shows that the malondialdehyde content of the two transgenic lines was significantly upregulated when infected by Xcm and DC3000. At 0d of infection, there was no significant difference between OE-MiMYB44#1, OE-MiMYB44#2 and WT; at 2d, the malondialdehyde content of the two transgenic lines increased significantly, and the upward trend of OE-MiMYB44#1 was higher than that of OE-MiMYB44#2; the malondialdehyde content continued to increase at 4d and 6d of infection and reached the maximum value at 6d. This shows that overexpression of MiMYB44 in Arabidopsis can significantly increase the malondialdehyde content, indicating that MiMYB44 increases resistance to pathogen infection by regulating the malondialdehyde content.

[0054] (2) Determination of superoxide anion content in leaves of transgenic lines

[0055] In Xcm( Figure 8 A) DC3000( Figure 8 B) In the infection of these two pathogens, the superoxide anion content of the two transgenic strains showed an overall upward trend compared with WT. In Xcm infection, OE-MiMYB44#1 showed a continuous upward trend from 0 to 4 days, and the content was the highest. The superoxide anion content decreased at 6 days. In DC3000 infection, the superoxide anion content increased significantly by 1.26 times at 2 days, and then the increase in superoxide anion content slowed down and reached the highest value at 6 days. The superoxide anion content of OE-MiMYB44#2 in Xcm infection was higher than that in DC3000 infection, and showed a continuous upward trend in Xcm infection. In DC3000 infection, the content remained stable at 2 days and 4 days without significant changes, and increased at 6 days and reached the highest value.

[0056] (3) Determination of hydrogen peroxide content in leaves of transgenic lines

[0057] Depend on Fig. 9 It can be seen that in Xcm( Fig. 9 A) and DC3000( Fig. 9B) During infection, the two transgenic lines showed different trends. In Xcm infection, the hydrogen peroxide content of OE-MiMYB44#1 was not significantly different from that of WT at 0-2d infection, but increased significantly at 4d infection and reached a maximum value, which was 3.23 times that of WT. At 6d infection, the content decreased to no significant difference from 2d. The increase of hydrogen peroxide content in OE-MiMYB44#2 was roughly the same as that in OE-MiMYB44#1, and also increased significantly at 4d, which was 2.57 times that of WT. In DC3000 infection, although OE-MiMYB44#1 showed a continuous upward trend, the increase was small, and the hydrogen peroxide content did not change significantly. The hydrogen peroxide content of OE-MiMYB44#2 remained basically the same at 0-2d infection, and increased significantly and remained stable at 4-6d infection, which was 1.98 times and 1.81 times that of WT, respectively. These results indicate that MiMYB44 has a certain regulatory effect on the hydrogen peroxide content in transgenic Arabidopsis.

[0058] (4) Determination of anthocyanin content in leaves of transgenic lines

[0059] Depend on Fig.10 It can be seen that in Xcm infection ( Fig.10 A), the anthocyanin content of OE-MiMYB44#1 increased significantly during the infection process, and was 3.66 times that of WT at 6 days; the anthocyanin content of OE-MiMYB44#2 had no significant difference from WT at 2 days after infection, but increased significantly at 4 days and reached the maximum value, which was 7.57 times that of WT. The anthocyanin content decreased at 6 days, but there was no significant change overall. Under DC3000 infection ( Fig.10 B), OE-MiMYB44#1 showed a significant upward trend throughout the infection process, reaching a maximum value at 6 days of infection, which was 3.23 times that of WT; the overall increase in anthocyanin content in OE-MiMYB44#2 was lower than that in OE-MiMYB44#1, and there was no significant difference with WT at 2 days of infection, and the anthocyanin content was significantly higher than that of WT at 4 days to 6 days. These results show that MiMYB44 can also regulate anthocyanin content in transgenic Arabidopsis, thereby increasing resistance.

[0060] (5) Determination of endogenous ethylene content in leaves of transgenic lines

[0061] Under Xcm infection ( Fig.11 A), the ethylene content of OE-MiMYB44#1 increased significantly during the infection period of 0-2 days, and remained relatively stable as the infection time continued; the ethylene content of OE-MiMYB44#2 continued to increase during the infection period of 0-4 days and reached a maximum value, and the ethylene content tended to be stable at 6 days. Under DC3000 infection ( Fig.11B), OE-MiMYB44#1 showed significant differences compared with WT throughout the infection process, but the ethylene content remained relatively stable; OE-MiMYB44#2 also showed significant differences compared with WT throughout the infection process. The ethylene content showed an upward trend from 0 to 4 days of infection, but the increase was not large. The ethylene content increased significantly at 6 days and reached a peak value, which was 4 times that of WT. These results indicate that the overexpression of MiMYB44 gene can promote the ethylene content to a certain extent to maintain a high level.

[0062] (6) Determination of the transcription level of disease resistance-related genes in leaves of transgenic lines

[0063] The transcription level of disease resistance-related genes in MiMYB44 transgenic Arabidopsis was detected by qRT-PCR. Fig.12 It can be seen that at 2 days of Xcm infection, the expression levels of AtICS1, AtPR1, AtPR2, AtPR5, AtPDF1.2, and AtACS genes in OE-MiMYB44#1 and OE-MiMYB44#2 were significantly higher than those in WT. At the same time, except for AtPDF1.2, the expression level of OE-MiMYB44#2 was significantly higher than that in OE-MiMYB44#1. At 4 days of infection, the transcription level of AtPDF1.2 was low in WT and low in OE-MiMYB44#2. The expression levels of the remaining genes were significantly higher than those in WT, and the expression levels increased with the increase of infection time. These results indicate that the overexpressed MiMYB44 gene in Arabidopsis can improve its own resistance by regulating the transcription of disease resistance-related genes.

[0064] In summary, in order to verify and analyze whether the mango MiMYB44 gene has the ability to improve the disease resistance of mango, the present invention heterologously expresses the MiMYB44 gene in Arabidopsis to analyze the phenotype and physiological and biochemical reactions after pathogen infection. The results show that overexpression of MiMYB44 in Arabidopsis can increase the expression of malondialdehyde, superoxide anion, hydrogen peroxide, anthocyanin, ethylene content and disease resistance-related genes to a certain extent. MiMYB44 increases resistance to pathogens by stimulating a series of biological reactions and enhances the disease resistance of Arabidopsis. The present invention found that endogenous ethylene actively participated in the process of disease resistance regulated by MiMYB44. In the MiMYB44 Arabidopsis transgenic strains OE-MiMYB44#1 and OE-MiMYB44#2, the leaves and plant height were significantly larger and stronger than the wild type phenotype, further verifying that MiMYB44 may have the function of regulating leaf size.

[0065] Example 5-Screening and analysis of MiMYB44 interacting proteins

[0066] The present invention adopts YPDA culture medium, and defective culture medium: SD / –Trp, SD / –Leu, SD / –Ade / –His / –Leu / –Trp is mainly used to screen yeast positive clones. If the plasmid carrying pGBKT7 is successfully transferred into the yeast strain, it can grow on the SD / –Trp culture medium. If the plasmid carrying pGADT7 is successfully transferred into the yeast strain, it can grow on the SD / –Leu culture medium. If both enter the yeast cell, it can grow on the SD / –Leu / –Trp (DDO) culture medium. If both enter the yeast cell and interact with each other, it can grow on the SD / –Ade / –His / –Leu / –Trp culture medium.

[0067] The primers used are shown in Table 1 below:

[0068] Table 1 Primer sequences

[0069]

[0070] (1) Construction of bait vector and detection

[0071] Bait vector construction: Using the full-length cloning vector of MiMYB44 gene as a template, the MiMYB44 fragment containing restriction endonuclease cuts at both ends was ligated to the bait vector pGBKT7 with the corresponding endonuclease cuts by T4 DNA ligase, and then connected in a 16℃ water bath for more than 8 hours, and then transformed into Escherichia coli E coli DH5α, spread on LB solid culture medium with matching resistance, and inverted culture at 37℃ overnight; single colonies were selected for PCR detection, and the positive transformants with correct bands were sequenced; the successfully constructed plasmid was named pGBKT7-MiMYB44.

[0072] (2) Preparation and transformation of competent yeast

[0073] PEG / LiAc method for transformation of yeast competent cells: First, prepare yeast cell competent cells, take out and activate the AH109 yeast strain stored at -80℃, take out the plate after 3-4 days, inoculate it in 5mL YPDA culture medium, and culture it at 30℃ and 200r / min overnight. Add an appropriate amount of cells cultured overnight to 30mL YPDA, mix well, take out a small amount to measure the OD600 value, which is between 0.2-0.3, and culture it at 30℃ with shaking for 4h. Measure it every 3h until the OD600 value is between 0.4-0.6, collect the cells in a 50mL centrifuge tube, centrifuge it at 4℃ and 3000r / min for 5min, dry the culture medium with a pipette, and add 20mL Resuspend the cells in ddH2O, centrifuge at 3000r / min for 5min, prepare a 1×TE / LiAc mixed solution (10×TE:10×CH3COOLi:H2O=1:1:8) in advance, and resuspend the cells with 350μL of the prepared 1×TE / LiAc mixed solution, which are now AH109 yeast competent cells; after the yeast competent cells are prepared, genetic transformation is immediately carried out, and the salmon sperm DNA sample stored at -20℃ is taken out, boiled in water bath for 15min, and immediately placed in ice for 3-5min to completely denature it. Add 10 μL salmon sperm DNA + 0.1 μg plasmid + 100 μL AH109 yeast competent cells to each tube, mix well, prepare 1×TE:CH3COOLi:PEG mixed solution (10×TE:10×CH3COOLi:50%PEG=1:1:8), add 600 μL of the above mixed solution to each tube, mix well, shake at 30°C for 45 min, add 70 μL dimethyl sulfoxide (DMSO) and mix well (pipette blowing), place at 42°C for 20 min, immediately place on ice for 2 min, then centrifuge at 3000 r / min for 5 min, discard the supernatant, add 100 μL 1×TE or 0.9% NaCI and mix well, spread on the corresponding resistance plate, place the plate upside down in the incubator, and culture at 30°C for 3-4 days.

[0074] Single colonies grown on SD / -Trp-Leu plates were picked and streaked onto 4-deficient SD / -Trp-Leu-His-Ade medium. After culturing at 30°C for 3-4 days, strains growing well on 4-deficient medium were picked and transferred to 2-deficient yeast medium, cultured at 30°C and 180-200rpm for 2-3 days. The plasmids were extracted using a yeast plasmid extraction kit, and PCR amplification was performed using the universal primers of pGADT7 with the extracted plasmid as a template to obtain a single fragment, which was then sequenced. The sequencing results were then subjected to online Blastn comparison analysis by NCBI to obtain suspected interacting proteins.

[0075] (3) Isolation and identification of candidate interacting proteins and vector construction

[0076] The sequences obtained by sequencing were compared with NCBI to obtain 10 candidate interacting proteins. According to the experimental methods in the article, the full-length gene sequences of the candidate interacting proteins were isolated by gene cloning, sequencing, and alignment, and then constructed into the pGADT7 vector by enzyme digestion. Similarly, the recombinant plasmid successfully constructed into the pGADT7 vector was obtained by PCR specific fragment amplification, enzyme digestion verification, sequencing, etc., and was named pGADT7-X (X is the name of the candidate gene) for the verification of MiMYB44 and the candidate interacting proteins.

[0077] (4) Results and analysis

[0078] A. Screening of candidate interacting proteins: construct a recombinant plasmid pGBKT7-MiMYB44, co-transform the correctly sequenced recombinant plasmid and the pGADT7 library into yeast strains, use a yeast plasmid extraction kit to extract the plasmid of the yeast suspension grown on the medium lacking SD / -Trp-Leu-His-Ade, and use the universal primers of the yeast vector sequence to perform PCR amplification on the plasmid, such as Fig.13 , M: DNA marker DL2000; 1-10: represent 10 candidate interacting proteins, lane 3 is MiACO protein, the amplified single fragment was cut and recovered from the gel and then sequenced.

[0079] The sequence alignment results showed that multiple candidate interacting proteins were obtained (Table 2), one of which was the mango ACO gene, with a full length of 969 bp, and the specific sequence is shown in Appendix 2. The MiACO protein sequence was subjected to biochemical property analysis, and the results showed that the MiACO molecular formula was C2828H4746N912O1185S176, the length of the encoded protein was 322 amino acids, the protein molecular weight was 36656.05, the predicted isoelectric point was 5.39, the hydrophilicity index was -0.477 (<0, belonging to non-hydrophilic protein), the instability index was 30.22 (<40, belonging to stable protein), and the nucleotide sequence of the encoding gene was shown in SEQ ID NO.2.

[0080] Table 2 Screening of interacting proteins

[0081]

[0082] B. Yeast two-hybrid assay to verify the interaction between MiMYB44 and candidate interacting proteins

[0083] The positive control plasmid combination (pGADT7-T+pGBKT7-53, strong interaction control, expressing a pair of proteins that can interact in yeast), the negative control plasmid combination (pGADT7+pGBKT7, two plasmids empty, unable to express interacting proteins in yeast), pGADT7+pGBKT7-MiMYB44, pGADT7-MiACO+pGBKT7-MiMYB44 and other combinations were transferred into yeast competent cells, plated on 2-deficient culture medium, and picked up single colonies after 3 days. After identification, single yeast colonies containing recombinant plasmids were obtained and diluted 10 -1 , 10 -2 , 10 -3 Times, pipette 30 μL of each and drop it on the culture medium of Trp / -Leu and Trp / -Leu / -His / -Ade, and grow at 30°C for 3 days.

[0084] like Fig.14 As shown, it was observed that the positive control plasmid combination can grow normally on the culture medium with 2 or 4 deficiencies, while the negative control plasmid combination, pGADT7+pGBKT7-MiMYB44, etc. can only grow normally on the culture medium with 2 deficiencies, but cannot grow on the culture medium with 4 deficiencies; however, the pGADT7-MiACO+pGBKT7-MiMYB44 recombinant plasmid combination has the same growth trend as the positive control plasmid combination and can grow normally on the defective culture medium, which shows that MiMYB44 and MiACO are a pair of interacting proteins from the perspective of yeast two-hybrid.

[0085] The present invention demonstrates through qRT-PCR, colony count, physiological and biochemical indicators, and mango-related disease-resistant genes that MiMYB44 increases with the infection of Xcm and DC3000, and yeast two-hybrid experiments also confirm that there is an interactive relationship between MiMYB44 and MiACO, and MiMYB44 can enhance the resistance of mango to the infection of Xcm and DC3000 by promoting the synthesis of ethylene.

[0086] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principle of the present invention should be included in the protection scope of the present invention. Sequence Listing <120> A mango MYB transcription factor, interacting protein and its application in enhancing disease resistance <160> 10 <170> SIPOSequenceListing 1.0 <210> 1 <211> 744 <212> DNA <213> Artificial Sequence <400> 1 atgtatcaag accccaacgc ctttcactcg tcttcttcaa ctcactggaa ccgactcgag 60 gacaagctct ttgaacatgc ccttgttttg ttccctgatg aagttcccga tcgatggcag 120 aggatagccg atcaaattcc cgggaagtct ccccgcgatg tcaaggaaca ctatgaggtg 180 ctcgttcatg acgtcactga gattgactcg ggccgagttg agttgccttg ttatgctgat 240 gagtcagacg attgggactc cgccggtcag atctcgtttg ggtcaaagcc tgttaagcag 300 ggcgagcctg agaggaagaa gggtactccc tggacggaag aagaacacaa gttatttctg 360 attggattga gtaaatttgg caagggtgat tggaggagta tttcaaggaa tgtagtggtg 420 tcaagaacac caacccaggt ggctagtcat gcccagaagt atttccttcg ccaaactaac 480 atgaagaaag agcaaaaaag ggcgggcatc cacgacatta ccacagtgga tagcaagcca 540 attccactgg ctgtagacca gaactcaatg ggtctggctg tagaccagaa ctcaatgggt 600 ctgcctgttg accagaactg gaatcctccc cctgcagttt caggtcacca acagccacct 660 acatatcaac agtttcctcc accaaatcag tttctcagtc aaggagggtc aatggggtat 720 caaaactatg gctttcccat gtaa 744 <210> 2 <211> 969 <212> DNA <213> Artificial Sequence <400> 2 atggagagct tgagtttccc agtcattaac ttagagaaac taaatggtga ggagagagct 60 gccaccatgg agaagattaa agatgcttgt gagaactggg gcttctttga gttggtgaac 120 catgggatac ccattgagga acttgacact ctggagagga agacaaaaga gcactacaga 180 aaatgtatgg aacaaaggtt caaggaactt gtggctagta aagctttgga aggtgttcaa 240 actgagatca aagatatgga ctgggagagc accttctatg ttcgccatct ccctcagaca 300 aacttaaatg aactcccaga tctcgatgat gaatacagga aggtgatgaa ggaatttgca 360 tcaaaattgg agaaactagc agaggaactg ctggacttgt tatgtgagaa ccttggattg 420 gagaaaggtt acctgaagaa gaccttctat ggctcaaagg ggccaacttt tggcaccaag 480 gttagcaact acccaccatg tccacaaccc gatttgatca agggcctccg agcccacacc 540 gacgccggtg gcatcatctt actcttccag gatgataagg tgagcggcct ccagcttctg aaagatgggc agtggattga tgtgccccca atgcgccact ccattgtggt taaccttggt 660 gaccagcttg aggttatcac caatggcaaa tacaagagtg tggagcaccg agtagttgct 780. gcaacggccg catgtcaata gcttcattct acaaccctgg cagtgatgca gttatctatc cagcaccacc attgttgga aaagaagcag aggagaaga acaagtgtac 900. ccaaatttg tttttgaaga ctacatgaag ctgtatgccg gcttgaagtt ccaggcaaaa gaacccaggt ttgaagccat gaaagctgtt gaaaccactg ttaatctggg cccaattgcc actgcttaa 969 <210> 3 <211> 927 <212> DNA <213> Artificial Sequence <400> 3 atgaatcgag gagtagaagt tctgtctccg gcctcatatc tacagaattc aaactggttg tttcaagaaa gcaaaggac aagatggact cgacaagaga acaagctgtt tgagaatgct ttggctttgt atgataaaga tactcctgat cgatggatta aggtagcagc aatgattcca ggcaagacag tcggtgatgt catcaacaa tacaggaat tggaagga tgttagtgat 240 attgaagctg gtttaatttcc aattcctgtc tattgtaacg attctttcac ttggagtgg 300 gttaattgtg atcaagaatt tgatggattg agcaattgt atagtcctgg tgggaagaga 360 ggcaccacaa ctcggccttc tgatcagag agaagaaag ggattccatg gagagagaa 420 gaacacaggc aatttctaat gggtcttaaa aggtttggaa aagggactg gagaaatatc 480 tcccgcaattt ttgtgaccac tagacacca actcaggttg caagccatgc tcaaagtac 540 ttcaatcggc agcttactgg ggggaaggat agaggagat ctagcattca tgacattact 600 attgtcagtt tggaagtcac aggatattct tcgccagaaa atagtaacc ttcatctgca 660 aattcttcta cagctgtcat tcagctgcaa cagcaaccaa attgactag cataagaat 720 gagcgatttg attggaaacc accaatgaa ggagcagcca tggttttcaa cccaacaaat 780 agcaacgttt tcacacccc ttttgtgg atatcttcat atgcagctaa gttgcaggag 840 caaaatctgc tggtgaac tctcatgga tctcagtgtg acatgtacag cacattttat 900 ccgatgcaat caatgcaacg tcagtaa 927 <210> 4 <211> 711 <212> DNA <213> Artificial Sequence <400> 4 atgtgcacca gaggacattg gaggccggca gaagatgaaa agctcagaga gttggtggaa 60 cgttacggcc cgcataactg gaacgccata gctgaaaaac ttcaaggcag atcagggaaa 120 agttgtaggt taagatggtt caatcaactg gatcccagaa tcaacagaag ccctttcaca 180 gaagaagaag aggagcggct tttagcatct catcggatcc atggaaatcg atgggcagtg 240 atcgccagat tgttccccgg acgaactgat aatgcagtga agaatcattg gcatgtcatc 300 atggctcgaa gatgcagaga aagatcaaga ctttacgcca gaagagcggc tcaaaatctc 360 ctgaataatg agcagaaatc gccttcaaat ccagaaatct tctccgccta ttgccagaaa 420 aatctctaca gttttgctca gaatcataac catcttcttc atcagtgtta tccggtgcag 480 gttttcaaag aagtggaagc cgaaaataca ggccattgta tcaccatgaa tcaagataac 540 aagaaccagc gggtggagtt ttacgatttt cttcaagtga atacagattc aaccaagagt 600 gaggtgatag ataatgcgcg aagggacgat gaggaagtgg atcaagaagc gacggagcag 660 cagaaaacaa gggcacaatt tatcgacttc ttatcagtag gaggagatta a 711 <210> 5 <211> 1668 <212> DNA <213> Artificial Sequence <400> 5 atgagtcaca caacaagtga tagtgatgat agggtgcttt ccagaggtca gactgatttg 60 ccgttagtgg atgaaggtga caattctgtt caaggagtta tactgaagaa aggaccatgg 120 acatctgctg gagatgcaat tttaattgac tatgttaaga agcatggaga agggaactgg 180 aatgctgttc agaagaactc agggctgttc cgttgtggca aaagctgccg attgagatgg 240 gcaaatcacc tgaggccaag cttgaagaag ggcgcattta ctcaagaaga agagcagctg 300 attgttgagc tccatgccaa aatgggaaac aaatgggcac gcatggctgc acatttgccg 360 ggtcgcacag ataatgagat aaagaattac tggaatactc gaattaaacg acgtcaacgt 420 gctggcttgc ctctctatcc tccagaagtg tctttccaag cattggagga gagtcaaaac 480 caaaacattg gtggaatcaa taatggggat aaaggtcatc atgatatctt gcaggctaat 540 ggttatgaga tacctgatgt tgtacttgac agtttaaaag ccaatcaggg tgtcttacct 600 tatgtcgctg aacttcctga tatttcctgt agcaacatgc tgatgaaagg tctgggctct 660 ccattctgta gcttcatgcc accaacaatt catcgcgaga agcgtcttcg agaatctgtg 720 ggattattct ctggttatag tgaaagagca aagaacgagt tctgctcatt tgatcagttc 780 cagaatgata ctcctgatag atggctccac catttgggct gcctttcct ctggatcaga 840 tcctacgacc aaagcccttg agacttttgg gtaatacagg gtgcccagac cctttcaaat 900 ggcaatttct ctgcttctac gcccattca ggggctgtga agttggagct cccttcactc 960 caatatccag aaactgattt aagtagctgg ggtacatcat ctcccccaca acctttactt 1020 gaaacagttg actcttttat tcagtctcct ccaactggta ctgttgagtc agattgccct 1080 tcaccgcgca acagtggcct gcttgatgct ttactccatg aggcaaaaac tttgagcagt 1140 gcaaagaatc attcctctga caagagttca aattcatctt ctgttactcc tgatatagct 1200 gatagttgcc tgccgaatgt ttgtcaaaat gaatgggaag actactgtga ccccctttct 1260 ccactgggtc attctgcaac ctctctcttc aatgagtgca gtccactcag taccagtgga 1320 agttcattgg atgaacaagc tcccattgag ccatatgttg ggtgcaaggt gaaattagaa 1380 ccggttgacc aggcatggac tcctgataga ggaaaagaaa gcagtaacct gtatgatctt 1440 agtcaggctg atgctttact tagttcggac tggcttgagc aggggtctgc ttatgttaag 1500 aacccaactg tgattactga tgccatagca accttttttg gagatgattt gggcagtgaa 1560 tataagcaga tgacaacagg aacttcgaca gcaagtcaag gatggggctt tggttcttct 1620 gcatggaata acatgccggc agtgtgtcaa atgtctgaac tcccttga 1668 <210> 6 <211> 50 <212> DNA <213> Artificial Sequence <400> 6 atggccatgg aggccgaatt cccggaattc atgtatcaag accccaacgc 50 <210> 7 <211> 50 <212> DNA <213> Artificial Sequence <400> 7 ccgctgcagg tcgacggatc ccgcggatcc ttacatggga aagccatagt 50 <210> 8 <211> 26 <212> DNA <213> Artificial Sequence <400> 8 taagagtcac tttaaaattt gtatac 26 <210> 9 <211> twenty one <212> DNA <213> Artificial Sequence <400> 9 taatacgact cactataggg c 21 <210> 10 <211> 20 <212> DNA <213> Artificial Sequence <400> 10 agatggtgca cgatgcacag 20

Claims

1. A kind of mango MiMYB44 The use of a transcription factor, characterized in that: Mango MiMYB44 Application of transcription factors in enhancing plant disease resistance, wherein the disease resistance is resistance to mango bacterial black spot pathogen or Pseudomonas syringae, encoding the MiMYB44 The nucleotide sequence of the gene encoding the transcription factor is shown in SEQ ID NO.

1.

2. The use according to claim 1, characterized in that: The mango MiMYB44 Transcription factors positively regulate anthocyanin and ethylene content.

3. The use according to claim 1, characterized in that: The mango MiMYB44 Transcription factors positively regulate MDA content.

4. The use according to claim 1, characterized in that: The mango MiMYB44 Transcription factors positively regulate hydrogen peroxide levels and superoxide anion levels.

5. The use according to claim 1, characterized in that: The mango MiMYB44 The transcription factor positively regulates the disease resistance-related gene, wherein the disease resistance-related gene is AtICS1 , AtPR1 , AtPR2 , AtPR5 or AtACS Gene.

6. The use according to claim 1, characterized in that: Said MiMYB44 The transcription factor forms a protein complex with 1-aminocyclopropane-1-carboxylic acid oxidase ACO to activate the synthesis of endogenous ethylene, enhance the transcription of disease resistance-related genes, increase the synthesis and accumulation of disease resistance products, and improve the disease resistance of mangoes; the disease resistance-related genes are AtICS1 , AtPR1 , AtPR2 , AtPR5 or AtACS Gene.

7. A kind of mango MiMYB44 The use of a transcription factor, characterized in that: Mango MiMYB44 Application of a transcription factor in enhancing the defense response of mango to bacterial diseases, wherein the bacterial disease is mango bacterial black spot or a disease caused by Pseudomonas syringae, encoding the MiMYB44 The nucleotide sequence of the gene encoding the transcription factor is shown in SEQ ID NO.1.

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