CN113549134B - Leptospermum arborescens effect protein PBRA _6677 and application thereof - Google Patents

Abstract

The invention belongs to the fields of biotechnology, crop cultivation and disease control, and particularly discloses a plasmodiophora tumefaciens effect protein PBRA _6677, which can reduce the incidence rate of plasmodiophora tumefaciens by regulating and controlling rhizosphere microorganisms of tuber mustard and has application value in the aspect of controlling plasmodiophora.

Description

Leptospermum arborescens effect protein PBRA _6677 and application thereof

Technical Field

The invention relates to the field of biotechnology and crop cultivation and disease control, in particular to a plasmodiophora elata response protein and application thereof.

Background

Clubroot is the most destructive disease of cruciferous plants, and causes great economic loss worldwide. The pathogenic bacteria of the disease are Plasmodiophora brassicae (Plasmodiophora brassicae) which are obligately parasitic and develop in cells of the root of a host plant to form dormant spores. The plasmodiophora elata regulates the metabolic process of plants, stimulates the plants to secrete auxin, leads roots to form tumors, and leads to the reduction of the water and nutrient absorption capacity of the plants. The root nodule epidermis of the diseased plant cracks, other pathogenic fungi and bacteria in the soil enter the root, the root is rotten, and the plant dies. While a large number of dormant spores are released into the soil (Kageyama & Asano, 2009). Dormant spores of plasmodiophora elata can survive in soil for up to 20 years. Since the plasmodiophora elata belongs to endoparasitic bacteria and can be dormant in soil for a long time, the plasmodiophora elata is extremely difficult to prevent and cure, and once the plasmodiophora elata occurs, the plasmodiophora elata is difficult to eliminate. Along with the allocation and transportation of crucifer crop seedlings, the spreading range of clubroot in China is gradually enlarged, the clubroot is mainly distributed in northeast, china, southwest, middle and upstream of Yangtze river and other places (Lejinnu and the like, 2012), and the production of crucifer vegetables in China is seriously threatened.

Tumorous stem mustard (Brassica juncea var. Tubida. Tsen et Lee) is a Brassica brassicaceae Brassica mustard seed Brassica juncea subspecies Brassica juncea variety, and a mustard tuber processed by using the tumorous stem mustard as a raw material is one of three pickled vegetables in the world. "Fuling hot pickled mustard tuber" has become a distinctive agricultural product in China and creates great economic benefit (Gaomingquan, etc., 2002). However, the tumorous stem mustard is seriously damaged by clubroot in the cultivation process, which causes a great amount of yield and economic loss every year, and becomes a problem to be solved urgently in production. At present, the main control methods in production comprise crop rotation, removal and burning of diseased plants, soil pH adjustment by lime application, plant ash covering, chemical control and the like. In addition, the disease acts on the root of the stem nodule mustard and soil viscosity in Chongqing areas is high, more stones are generated, rainwater is sufficient in winter, the soil plowing difficulty is high, and pesticides are difficult to play a role. Meanwhile, the problems of pesticide residue and the like are increasingly prominent. The breeding of disease-resistant varieties provides an effective solution for the clubroot resistance of cruciferous plants, for example, rape, chinese cabbage and the like have bred the disease-resistant varieties, but the tumorous stem mustard belongs to wild tetraploid, so the breeding of the disease-resistant varieties is difficult, and the disease-resistant varieties are not bred at present. Therefore, the analysis of the pathogenesis of the tumorous stem mustard clubroot is particularly important for comprehensively preventing and treating the tumorous stem mustard clubroot from multiple aspects.

The life history of the plasmodiophora includes: dormant spore dormancy and germination stage; root hair and epidermal cell invasion stage; and (4) a cortex infection stage. The clubroot secretes protein effectors at every stage of life to promote its infestation of the host plant. The serine protease Pro1 is involved in dormant spore germination in the first stage (Feng et al, 2010). In the stage of root hair and epidermal cell infection, plasmodiophora rhizogenes may secrete protease and protease inhibitors, such as cysteine-rich protease and methyltransferase, inhibit the immune response of plants, prevent programmed death of host plant cells, and improve the infection success rate (Siemens et al, 2011). In the primary and secondary plasmogen formation stages, plasmodiophora hordei secretes a series of protein effectors that regulate phytohormone secretion, such as cytokinin biosynthesis, auxin homeostasis, salicylic and jasmonic acid metabolism (Malinowski et al, 2012), stimulate root cell growth, elongation and differentiation, and stimulate cells to form tumors. In the zoospore maturation stage, the plasmodiophora expressing effector has a chitin binding domain, which removes chitin fragments and inhibits the immune response elicited by the pathogen molecules of the plant. At the stage of secondary zoospores and secondary primordial formation, plasmodiophora may express effectors that control the plant defense response and interfere with plant meristem activity (Ludwig-muller et al, 2015). However, in the dormant spore soil dormant stage and the zoospore releasing infection stage, how the plasmodiophora falcata overcomes the influence of soil microorganisms and whether effector is secreted to improve the ecological competitive capacity with other microorganisms is to be disclosed.

Effectors are small molecules produced by phytopathogens during infestation of a plant that contribute to its infestation and colonization, regulating plant physiology or immunity to prevent plant resistance responses (Jones & Dangl, 2006). Most phytopathogen effectors belong to secreted proteins, are cysteine-rich, contain an N-terminal signal peptide with about 50-300 amino acid residues and high sequence specificity (Stergiopoulos et al,2009, bolton et al, 2010. In addition, secondary metabolites and small RNA molecules produced by phytopathogens also have effector functions (Wang et al, 2016). Certain endophytes, symbionts, and even saprophytes are also capable of producing effector homologous molecular species (Rovensich et al, 2014). The effector can help pathogenic bacteria break through the barrier of the plant and successfully infect the plant.

Snelders et al believe that plant pathogen effectors can be divided into three categories: plant target effectors, multi-target effectors (which may act on plants and microorganisms), and microbial target effectors (Snelders et al, 2018). The first class of effectors merely manipulate the physiological processes of the plant and regulate the process of the plant's disease-resistant response. The second class of protein effectors can regulate and control plants and rhizosphere microorganisms, have broad-spectrum activity, and regulate highly conserved physiological processes of plants and microorganisms. The third class of protein effectors specifically act on certain specific physiological processes of microorganisms, either to cause localized nutrient deficiencies or to block communication between plants and beneficial microorganisms. In addition, phytopathogens recruit cooperative microorganisms through such effectors to combat competitors or co-colonize plants.

A large number of microorganisms, particularly rhizosphere microorganisms, live around plants. Plants attract beneficial microorganisms through root secretions, forming a microbial barrier at the rhizosphere against pathogen infestation (Koprivova et al,2019, huangrappa et al,2008, berendsen et al, 2012. Before infestation of the plant, a barrier to plant microorganisms is first faced. Phytopathogens use protein effectors to inhibit certain antagonistic microorganisms, to modify the composition of the plant microbial community, and to break the microbial barrier, thereby successfully infecting plants (Snelders et al, 2018). Such effectors having a regulatory activity on microorganisms are referred to as microbial effectors. Microbial effectors play an important role in the parasitic and non-parasitic stages (saprophytic or dormant) of phytopathogen infestations. During the parasitic phase, the pathogenic bacteria use the microbial effectors to regulate the composition of plant rhizosphere microorganisms. During the saprophytic stage, pathogens may utilize effectors to inhibit other microorganisms, thereby maintaining their own survival advantage (Snelders et al, 2018). Therefore, the microbial effector has important significance in the infection and survival of soil-borne pathogens.

The soil-borne pathogenic bacteria live in soil, and live in a saprophytic or dormant state without the existence of a host, and the soil-borne pathogenic bacteria and microorganisms in the environment compete for the same ecological niche together. Microorganisms in the soil inhibit phytopathogens. The natural regression of wheat take-all is the increase of the number of pseudomonas in the soil, inhibiting the propagation of the specialized gaeumannomyces graminis wheat (Berendsen et al, 2012), and reducing the number of pathogenic bacteria in the soil. Phytopathogens also inhibit antagonistic microorganisms in order to occupy a dominant niche in the soil. The Zt6 effector of wheat leaf blight pathogenic bacteria has double functions, has pathogenicity on wheat and induces programmed death of wheat cells. The effector also has antibacterial activity, and can inhibit bacteria, yeast and filamentous fungi in soil. This suggests that this effector can help the pathogen infect wheat and can also participate in competition for other microbial niches (keys et al, 2017). The Verticillium dahliae secretes VdAve1 effector when infecting host plants, and inhibits antagonistic bacteria around the plants, such as sphingolipid, neosphingolipid, sphingomonas and sphingomonas. The Verticillium dahliae expresses VdAMP2 effector in saprophytic life, can inhibit the growth of Bacillus subtilis and Candida corrugatae in soil, improve the ecological adaptability of the Verticillium dahliae in soil and increase the relative biomass of the Verticillium dahliae in soil (Snelders et al, 2020). Therefore, the soil-borne pathogenic bacteria participate in ecological competition of soil microorganisms through effectors to obtain the dominant ecological niche. The clubroot is a typical soil-borne disease, can be dormant in soil for a long time, and can compete with other microorganisms in the soil, and the mechanism is to be revealed.

The plasmodiophora belongs to obligate parasites, lives in plant cells, and occupies the same ecological niche with endophytes of plants, so that nutrition and space competition exist, and certain influence is certainly caused on the endophytes of the plants. A research of Tian et al finds that the plasmodiophora infects tumorous stem mustard to change the composition of an endophytic fungus community and reduce the diversity of the endophytic fungus community, wherein the quantity of soil-borne pathogenic fungi is increased, such as fusarium (Tian et al, 2019). Lebreton et al found that plasmodiophora infects Chinese cabbage resulting in a change in rhizosphere and microbial community within the root. After infection with plasmodiophora, the number of bacilli with antibacterial activity in roots of rhizosphere, flavossobacter and Streptomyces, was significantly reduced (Lebreton et al, 2019). Wang et al showed that the clubroot infected tumorous stem mustard, regulated its physiological properties, and thereby altered the endophytic bacterial community structure. Rhodanobacter in healthy roots is the dominant endophyte, while the nodule dominant endophyte is Pseudomonas. The soluble sugars, soluble proteins, methanol, peroxidase and superoxide dismutase of the nodules were significantly higher than those of healthy root systems, indicating that plasmodiophora infection alters the physiological properties of the host, with soluble sugars, soluble proteins, methanol being closely related to the endogenous bacterial community of the nodule (Wang et al, 2020). Rhizosphere soil microorganisms can also affect the occurrence of clubroot. Daval et al found that rhizosphere microorganisms could regulate the interaction between Brassica napus and Plasmodium napus at the gene transcription level, and influence the expression of some pathogenic effectors of Plasmodium napus and rape disease resistance-related proteins, thereby alleviating the occurrence of clubroot (Daval et al, 2020).

The plasmodiophora elata can survive in soil for a long time, has strong stress resistance, and can be combined with other soil-borne pathogenic bacteria to infect hosts together. This suggests that the Plasmodium falciparum is able to alter the host rhizosphere microflora, recruit some microorganisms to co-fight competitors or co-colonize the host. Similar mechanisms may also exist for plasmodiophora given that soil-borne pathogens are capable of secreting effectors to regulate the composition of microorganisms. Therefore, it is necessary to deeply disclose a mechanism that the plasmodiophora elata co-infects the host by regulating the rhizosphere microbial community and combining with some microorganisms, and the method has important significance for subsequently developing plasmodiophora elata cooperative pathogenic bacteria as a novel target for controlling plasmodiophora elata and promoting the scientific control of plasmodiophora elata.

Reference documents

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Disclosure of Invention

According to the invention, 2 differential expression clubroot effector genes (PBRA _2565 and PBRA _ 6677) are screened and found in the early infection process of clubroot mustard by clubroot bacteria, and further research shows that the clubroot effector genes can reduce the incidence rate of clubroot by regulating and controlling rhizosphere microorganisms of tuber mustard, and have application value in the aspect of preventing and treating clubroot.

Therefore, the invention provides a plasmodiophora tumefaciens effector protein PBRA _6677, the amino acid sequence of which is shown in SEQ ID NO. 3.

Further provides the coding gene of the plasmodiophora tumefaciens effector protein PBRA _6677. More specifically, the nucleotide sequence is shown as SEQ ID NO. 4.

The invention provides a coding gene containing the coding gene, preferably an expression vector, more particularly a bacterial, fungal or plant expression vector, and further an escherichia coli and yeast expression vector.

Also provides a host cell containing the coding gene or the recombinant vector, more particularly a bacterial cell, a fungal cell or a plant cell, and further an Escherichia coli cell and a yeast cell.

The invention also provides application of the plasmodiophora effector protein PBRA _6677 in control of plasmodiophora brassicae crops.

In a particular embodiment, the Plasmodiophoromyces response protein PBRA _6677 is applied to the roots of said plants, preferably in combination with a fertilizer. Preferably, the crop is selected from brassica juncea, more particularly stem tumor mustard.

In another embodiment, the gene coding for the plasmodiophora tumefaciens effector protein PBRA _6677 is introduced into cruciferous crops for expression by transgenic technology, and more preferably, expression is targeted to the constitutive expression of the roots of the crops or induced when infected by pathogenic bacteria.

Drawings

FIG. 1 is the observation of root morphology of tumorous stem mustard infected by plasmodiophora.

Wherein, A: dormant spores of plasmodiophora elata; b: the plasmodiophora spores are adsorbed on the surface of the root system of the tumorous stem mustard; c: plasmodiophora primary zoospores; d: the plasmodiophora primary zoospores infect the tumorous stem mustard root hair; e: the plasmodiophora primary zoospores enter the interior of the tumorous stem mustard root hair (the part indicated by the asterisk); f: the root tumour produces secondary sporangia (sites indicated by asterisks) in the tumorous stem mustard root hair; g: root hair tips are broken and secondary sporangia are released (sites indicated by asterisks); h: secondary sporangia in division; i: small secondary sporangia resulting from division (sites indicated by asterisks); j: secondary spores released from secondary sporangia (sites indicated by asterisks); k: a fast swimming binuclear protoplasm mass (the part shown by an asterisk) formed by combining two secondary spores; l: secondary protoplasm mass infects tumorous stem mustard root hair and deforms the root hair (sites indicated by asterisks); m: the secondary protoplasm mass is propagated in mass in the cortex cells of the tumorous stem mustard; n: the dead tumorous stem mustard root cells are full of a large number of dormant spores of the plasmodiophora tumefaciens; o: swollen roots are produced after the tumorous stem mustard is infected by the plasmodiophora glabrata (the rightmost side is a control non-swollen root system, and the left side is a swollen root system after inoculation).

FIG. 2 is a diagram showing quantitative analysis of differentially expressed genes.

FIG. 3PBRA _2565and PBRA _6677 protein domain analysis.

FIG. 4 construction of PBRA_2565 gene fusion pGEX-4T-1 overexpression vector.

Wherein, A is a gel electrophoresis strip of a PBRA _2565 amplification product, and M is Marker D2000;1 is PBRA _2565 amplified band; b: the PCR result of the bacterial colony of the target vector pGEX4T-1 connected with the PBRA _2565 is detected, and M is Marker D2000;1-10 is 10 single colony PCR product amplification bands; n is a negative control, P is a positive control; c: the vector enzyme digestion detection is successfully constructed by PBRA _2565-pGEX4T-1, and M is Marker D2000;1 is a band after PBRA _2565-pGEX4T-1 double enzyme digestion, wherein a is an enzyme digested pGEX4T-1 vector, and b is an enzyme digested PBRA _2565 fragment; d: pGEX4T-1 vector map.

FIG. 5 is a construction of plasmodiophora tumefaciens PBRA _6677 gene fusion pGEX-4T-1 overexpression vector.

Wherein, A is a gel electrophoresis strip of the PBRA _6677 amplification product, and M is Marker D2000;1 is PBRA _6677 amplified band; b: detecting the PCR result of the bacterial colony of the PBRA _6677 connected with a target vector pGEX4T-1, wherein M is Marker D2000;1-9 are 9 single colony PCR product amplification bands; n is negative control, P is positive control; c: carrying out enzyme digestion detection on a vector successfully constructed by PBRA _6677-pGEX4T-1, wherein M is Marker D2000;1 is a band after PBRA _6677-pGEX4T-1 double enzyme digestion, wherein a is a pGEX4T-1 vector obtained by enzyme digestion, and b is a PBRA _6677 fragment obtained by enzyme digestion; d: pGEX4T-1 vector map.

FIG. 6PBRA _2565and PBRA _6677 proteins by polyacrylamide gel electrophoresis.

FIG. 7PBRA_2565 and PBRA _6677 crude protein bacteriostasis effect analysis.

Wherein, A, PBRA _2565 crude protein treatment; b: processing PBRA _6677 crude protein; CK: processing the empty carrier crude protein; t2: bacillus megaterium; t3: pseudomonas sp; t4: fictibacillus barbarecys; t5: pseudomonas aeruginosa; t8: fictibacillus enciensis; t12: massukua sp.; c1: acinetobacter pittii; c2: bacillus aryabhattai; c3: enterobacter hormechei; c7: enterobacter sp; c9: achromobacter animicus; c10: bacillus sp; c17: lelliotia leotgai; c19 Pseudomonas latetica.

Figure 8pbra _2565and PBRA _6677 interact with soil microorganisms from the root system of chlorambucil.

FIG. 9 the disease development of tumorous stem mustard after inoculation with Plasmodiophoromycete inoculum and Effector protein. Wherein, A, plasmodiophora tumefaciens is inoculated; b, inoculating plasmodiophora elata + PBRA _2565 crude protein; c, plasmodiophora hordei + PBRA _6677 crude protein).

Detailed Description

The present invention is further illustrated by the following specific embodiments, which are not to be construed as limiting the invention.

1.2 differential expression clubroot effector genes are found in the early infection process of clubroot by clubroot

The physiological process of infecting the root system of tumorous stem mustard with the plasmodiophora brassicae is observed by utilizing a water culture system. The specific process is as follows:

preparing 0.8% (m/v) agar culture medium with sterile water, sterilizing the seeds of the preserved szechuan pickle variety Yong' an lobule with 50% ethanol for 30 seconds, washing with sterile water for 5 times, uniformly spreading the seeds in the culture medium containing 0.8% agar, placing the sown culture dish in an illumination culture room for germination culture, wherein the culture temperature is 22 ℃, the illumination intensity is 6000 lux, and the light cycle is 16 hours of illumination/8 hours of darkness. The tuber mustard seeds can grow into seedlings containing 2 cotyledons after germinating and growing for 2 days in a culture dish, and the seedlings with the fully-extended cotyledons are used for the plasmodiophora brassicae inoculation experiment.

The physiological process that the clubroot infects the root system of the tuber mustard is observed by utilizing a water culture system. Firstly, extracting resting spores of plasmodiophora brassicae from tuber mustard and carrying out microscopic observation and identification (A in figure 1), then carrying out water culture inoculation on tuber mustard seedling root systems germinating for 3 days in a culture dish by using the extracted plasmodiophora brassicae, and observing the infection conditions of the plasmodiophora brassicae on the tuber mustard root systems at different time points. Observing that the plasmodiophora spores are adsorbed on the root surface of the tuber mustard (B in figure 1) 12 hours after inoculation; after 48 hours, zoospores released after germination, which are larger than the plasmodiophora elata resting spores, can be observed (C in figure 1), primary zoospores released are adsorbed on the surface of root hair, and the root hair of the tuber mustard is infected (D in figure 1); after 78 hours, the entry of the primary zoospores of Plasmodiophoromyces into the interior of the root hair cells was observed (E in FIG. 1); after 8 days, a plurality of subsequent infection processes can be observed, namely round spherical secondary sporangia are generated inside root hairs (F in figure 1), after the secondary sporangia are formed, the root hair cells of the tuber mustard are ruptured to release the sporangia to the external environment (G in figure 1), the released secondary sporangia continue to grow, more secondary spores are generated inside while the volume is increased, and the growing sporangia can be divided to generate a plurality of small secondary sporangia (H and I in figure 1); release of the secondary spores into the environment after maturation of the small secondary sporangia (J in fig. 1); the secondary spores released into the environment are pairwise qualitatively combined and fused into a binuclear protoplasm mass which is in a fusiform shape and can rapidly swim in water (K in figure 1); the binuclear raw biomass starts to infect the tuber mustard root hair, and in the process, a large amount of infected tuber mustard root hair is bent and deformed to wrap the binuclear raw biomass, so that the raw biomass enters the interior of the tuber mustard root hair (L in figure 1); after 14 days of inoculation, the thalli entering the interior of the root hair can reach the cortex part of the root system of the tuber mustard and multiply in the cortex (M in figure 1); if the root system of the host plant dies in the infection process, the plasmodiophora cells generate a large amount of dormant spores and enter a dormant stage (N in figure 1); after the tuber mustard is infected by the plasmodiophora brassicae for 28 days, the swelling of the root system of the tuber mustard can be observed, and a large number of swollen roots are generated, particularly the swollen roots are obvious in the plant root system close to the hypocotyl part (O in figure 1).

The observation results are shown in fig. 1, and the observation shows that 3 to 5 days after the inoculation of the plasmodiophora brassicae are the stage of mutual recognition of the plasmodiophora brassicae and the tumorous stem hair at the early stage of infection, the tumorous stem mustard root system sampling of 3 days after the inoculation of the plasmodiophora brassicae is subjected to transcriptome sequencing (the tumorous stem mustard root system which is treated by only adding equal amount of water for 3 days is taken as a control), and the expression change condition of the gene in the early stage of infection of the tumorous stem mustard by the plasmodiophora is detected through the transcriptome sequencing.

A total of 9210 genes were detected by transcriptome sequencing, with 9176 genes expressed in the control group (clubroot not infected with tumorigenic mustard), 8572 genes expressed in the treatment group (clubroot infected with tumorigenic mustard) and 8538 genes expressed in both the control and treatment groups. 247 differentially expressed genes were detected together, accounting for 2.68% of the total differentially expressed genes, with 42 genes up-regulated and 205 genes down-regulated (see fig. 2). Sequencing results of transcriptome show that compared with a control group, two effector genes in clubroot infected with tumorous stem mustard are significantly reduced in expression, namely PBRA _2565 and PBRA _6677. Expression level of PBRA2565 measured by transcriptome: FPKM value of treatment group was 4.75, control group FPKM value was 1.38, log2 (treatment/control) was-1.70; transcriptome-measured expression level of PBRA 6677: the FPKM value of the treated group was 4.32, that of the control group was 1.26, and that of log2 (treated group/control group) was-1.88.

2. Analysis of Leptoma nivea effector PBRA _2565 and PBRA _6677 genes

The CDS sequence of PBRA _2565 has the full length of 537bp, codes 178 amino acids, wherein the length of the signal peptide is 51, and codes 17 amino acids at the amino terminal; the CDS sequence of PBRA _6677 has a total length of 687bp, and encodes 228 amino acids, wherein the signal peptide has a length of 54, and 18 amino acids are encoded at the amino terminal.

The CDS sequence of PBRA _2565 is as follows:

ATGGCGTACTGGTTGGTCATCACGCTGGCGGTGCTCAGCGGCGCGGACGCGTTCCCGATCCCGTTTAGCAGTTGTGCAGGGAACCAGGCTGATGACGCGTCCCGCCTTGTGATCAAATCCATTGACATCTCGCCGTATCCCGTGAAGCCTGGTGGGTCGGCGCTGGCCAACATCGGCCTGTCCATCAAGAAGAAGGTGGACCATGGCAGCCACTACGACCTGAAGATCTACATGGACAAGTACGAGCTCGTACACGAAAGCGGCGACCTGTGCGCGCTCAGCGCCACCTTCACGTGCCCGAAGGACGCCGATGACAGCGCCGTTCTTCAGTATAAGTTCCAGTTTCCCAGAGTCCCGTTCGCCGGCAGCCTTCGCCTTCATCTAATGATTTGGAACCAGAACAACCAGGAGCTCTGCTGTGTCGACTTCTCGATCGACGTGAGGCTGGTCGACGGCAAGACCGTGATGGTGGAGAAGGACTACCTGATGGAAGAGGTGCAGAAGCTTATCGACGAGTACAGCGGCGCTGATATGTAG。

the amino acid sequence of PBRA _002565 is as follows:

MAYWLVITLAVLSGADAFPIPFSSCAGNQADDASRLVIKSIDISPYPVKPGGSALANIGLSIKKKVDHGSHYDLKIYMDKYELVHESGDLCALSATFTCPKDADDSAVLQYKFQFPRVPFAGSLRLHLMIWNQNNQELCCVDFSIDVRLVDGKTVMVEKDYLMEEVQKLIDEYSGADM。

the CDS sequence of PBRA _6677 is as follows:

ATGCTGGGCGCCACGACATTGCTGATCGCCTCCCTGATTGCTGCCTGCAACGGGCAGTCATGTCGAGGTCCAGGATTCATCATCACTGGATGTAACCCGGACTTGCCCATCACCGATCGCTGCGGTGTCGCCGCCGATGGAGCACCCTGTCCATTTGGAACCTGCTGCTCCGAATTCGGGTTTTGCGGGCGCAGCGCTGTTCACTGTGGTGGCGAGCAATACATTCCACAATGGGGTCCACCGCGAGACGACGGGCGGTGCGGAGAGGCATTCAGCTACGCTGGGTGCGATGACGATTTTATCTGCATCGCCACCTGGTGCGTCAGTTCCGACTCTGTCCCCGGTGCGTCACCCGTGAATCCGACGTCGGCAACGCCGACCGGAACGTCTGACACCACCAACCCACAACCTGTTCCTTCCGGCTCGGCTGAGGTCGACCCCAGTTCATCTGAGCATCCCAGATCAGGGAACAATGGTGTCACTAGGGAAGCGTCCAGCTCGGGAACAGGAAGCAGCACCAGCAGAGGAGGGCCTGCTCAACTAGCCGCGTTTGAAAGAGGTCAGGATATCACCGTCCCATATTCGACGGGTACCGACCAATCTTCAACTGTGCGTCGACACATCGCTCCATCCTGGACCTGGGCGCTTGTCACTACAGGCGCGTTCATCGTCATGGCCATGCAATAA。

the amino acid sequence of PBRA _006677 is as follows:

MLGATTLLIASLIAACNGQSCRGPGFIITGCNPDLPITDRCGVAADGAPCPFGTCCSEFGFCGRSAVHCGGEQYIPQWGPPRDDGRCGEAFSYAGCDDDFICIATWCVSSDSVPGASPVNPTSATPTGTSDTTNPQPVPSGSAEVDPSSSEHPRSGNNGVTREASSSGTGSSTSRGGPAQLAAFERGQDITVPYSTGTDQSSTVRRHIAPSWTWALVTTGAFIVMAMQ。

protein domains were predicted using on-line analytical software (https:// www. Ncbi. Nlm. Nih. Gov/Structure/cdd/wrpsb. Cgi) (see fig. 3), PBRA _2565 contains ML Super family, ML (MD-2-related lipid recognition) is a new domain found in MD-1, MD-2, GM2A, npc2 and various plant, animal and fungal proteins, ML protein is involved in LPS (lipopolysaccharide of gram-negative bacteria) signal transduction and lipid metabolism, PBRA _2565 effectors are not among the typical effector range known so far, and belong to other types of effectors; PBRA _6677 contains a chitin binding domain, and the effector protein AVR4 contains a chitin binding domain, which can prevent the phyllomycetes from degradation by tomato-produced chitinase (van Esse et al, 2007), which is presumed to belong to the AVR4 class of non-toxic effector proteins.

3. Prokaryotic expression and crude protein extraction of PBRA _2565 and PBRA _6677 effectors

A fusion expression vector of these two proteins was constructed by integrating the CDS sequences of PBRA _2565 and PBRA _6677 with the GST-tagged vector pGEX-4T-1 as the target vector (see FIGS. 4 and 5).

The successfully constructed vector is transferred into an escherichia coli BL21 strain, a positive clone is activated in an LB liquid culture medium containing ampicillin and then is subjected to liquid culture, IPTG induction expression with the final concentration of 1mmol/L is utilized, a small amount of thalli are taken for carrying out polypropylene gel electrophoresis detection on the protein expression condition, and the electrophoresis detection result shows that PBRA _2565 has an obvious induction band between 35-45kD and PBRA _6677 has a small induction band between 35-45kD (figure 6).

After protein expression is detected, strains which are induced to express and contain PBRA _2565, PBRA _6677 and empty vectors are inoculated again, after thalli are collected by centrifugation, the thalli are resuspended according to 20ml PBS/g, and the power of ultrasonication is as follows: 270W, ultrasound for 5s, interval for 5s, and 20min in the whole process, placing the sample in an ice bath in the whole ultrasound process, centrifuging for 15min at 4 ℃ at 12000g after ultrasound, and collecting supernatant for later use.

4. Analysis of the effects of PBRA _2565 and PBRA _6677 effectors on the regulation of soil microorganisms in the root system of Arabidopsis thaliana

Collecting soil samples of stem tumor mustard with different degrees of morbidity, uniformly mixing, respectively coating 100 mu L of soil suspension with different concentration gradients on an NB plate, and repeating each concentration gradient for 3 times. Culturing in 37 deg.C incubator for 24-48h, streaking the grown colony according to different forms (color, size, transparency, etc.), and selecting single colony for purification. Collecting single colonies subjected to multiple purification, performing 16S fragment amplification by adopting 27F and 1428R primers, inquiring and comparing nucleotide sequences by using NCBI database Blast after sequencing, and performing molecular identification on strains. After sequencing, the same strains were removed by Blast search and comparison, and 14 strains of genera, mainly bacillus, pseudomonas, acinetobacter, enterobacter, etc., were isolated in total (table 1).

TABLE 1 rhizosphere isolation of tumorous mustard culturable bacteria identification

Bacteria isolated from the rhizosphere of tumorous mustard were spread on NB plates, and the sonicated PBRA _2565, PBRA _6677, crude protein (OD) of empty vector, stored at-80 ℃ were removed ₆₀₀ = 0.3), pieces of filter paper were cut into a circle, sterilized and soaked in crude protein, then filter paper containing crude proteins of PBRA _2565 and PBRA _6677 (crude protein with empty carrier broken as a control) was inoculated on the bacteria-coated medium, and whether a zone of inhibition was formed was observed after 4 days.

The results show (fig. 7) that PBRA _2565 has an inhibitory effect on the strains numbered T8 and T12, both strains being (fibillus enciensis) and massukusp; the crude protein of PBRA _6677 had inhibitory effects on the strains numbered T2 and C19, which were Bacillus megaterium (Bacillus megaterium) and Pseudomonas bacteria (Pseudomonas bacteria), respectively.

After liquid culture activation of the bacterial strains isolated from the rhizosphere of chlorambucil, each strain was inoculated into liquid NB medium at a ratio of 1.

The result shows (figure 8), PBRA _2565 has obvious inhibition effect on the strain of T8 (Fictibacillus enciensis) and has extremely obvious growth promotion effect on C2 (Bacillus aryabhattai); PBRA _6677 has significant inhibitory effect on T2 (Bacillus megaterium), and has significant growth promoting effect on T4 (Bacillus barbaphys), T5 (Pseudomonas aeruginosa), C1 (Acinetobacter pittii), C2 (Bacillus aryabhattai) and C10 (Bacillus sp).

The experiment shows that the two effectors can inhibit and promote the growth of microorganisms at the root of the tuber mustard, and the infection and the morbidity of clubroot can be regulated and controlled by regulating (inhibiting or promoting the growth) the microorganisms at the root of the tuber mustard.

5. Correlation analysis of PBRA _2565 and PBRA _6677 effectors with Plasmodium septemlobum infection

Extracting dormant spores of plasmodiophora elata (Fr.) karst to obtain product with final concentration of OD ₆₀₀ =0.8 spore liquid for standby, quartz sand is used as a culture medium, and 1mL of plasmodiophora elata and 1mL of 1mL + OD of plasmodiophora elata are respectively inoculated in the quartz sand when seedlings grow to the size of two cotyledons ₆₀₀ Effector crude protein of = 0.3. After 30 days, the incidence and disease index (table 2 and figure 9) are observed and counted for 9 samples, the degree of the tumorous stem mustard inoculated by the plasmodiophora tumefaciens is the most serious, the incidence rate is up to 78 percent, and the disease index is 58.33; the incidence of tumorous stem mustard inoculated with plasmodiophora and PBRA _2566 effector crude protein is 33 percent, and the disease index is 22.22; the incidence of tumorous stem mustard inoculated with plasmodiophora tumefaciens and PBRA _6677 effector crude protein was 22% and the disease index was 19.44.

TABLE 2 statistical analysis of tumorous stem mustard disease

This suggests that these two effector proteins may reduce the disease index (i.e., extent of damage) of the incidence of clubroot, and that their mode of action may be through the regulation of tuber mustard rhizosphere microorganisms.

<110> Changjiang college of education

<120> plasmodiophora tumefaciens response protein PBRA _6677 and application thereof

<160>4

<210>1

<211>178

<212>PRT

<213> Plasmodiophora brassicae: CEP02598.1 hypothetical protein PBRA_002565

<400> 1

MAYWLVITLA VLSGADAFPI PFSSCAGNQA DDASRLVIKS IDISPYPVKP GGSALANIGL 60

SIKKKVDHGS HYDLKIYMDK YELVHESGDL CALSATFTCP KDADDSAVLQ YKFQFPRVPF 120

AGSLRLHLMI WNQNNQELCC VDFSIDVRLV DGKTVMVEKD YLMEEVQKLI DEYSGADM 178

<210>2

<211>537

<212>DNA

<213> Plasmodiophora brassicae: PBRA_2565 CDS

<400> 2

atggcgtact ggttggtcat cacgctggcg gtgctcagcg gcgcggacgc gttcccgatc 60

ccgtttagca gttgtgcagg gaaccaggct gatgacgcgt cccgccttgt gatcaaatcc 120

attgacatct cgccgtatcc cgtgaagcct ggtgggtcgg cgctggccaa catcggcctg 180

tccatcaaga agaaggtgga ccatggcagc cactacgacc tgaagatcta catggacaag 240

tacgagctcg tacacgaaag cggcgacctg tgcgcgctca gcgccacctt cacgtgcccg 300

aaggacgccg atgacagcgc cgttcttcag tataagttcc agtttcccag agtcccgttc 360

gccggcagcc ttcgccttca tctaatgatt tggaaccaga acaaccagga gctctgctgt 420

gtcgacttct cgatcgacgt gaggctggtc gacggcaaga ccgtgatggt ggagaaggac 480

tacctgatgg aagaggtgca gaagcttatc gacgagtaca gcggcgctga tatgtag 537

<210>3

<211>228

<212>PRT

<213> Plasmodiophora brassicae：CEO98563.1 hypothetical protein PBRA_006677

<400> 3

MLGATTLLIA SLIAACNGQS CRGPGFIITG CNPDLPITDR CGVAADGAPC PFGTCCSEFG 60

FCGRSAVHCG GEQYIPQWGP PRDDGRCGEA FSYAGCDDDF ICIATWCVSS DSVPGASPVN 120

PTSATPTGTS DTTNPQPVPS GSAEVDPSSS EHPRSGNNGV TREASSSGTG SSTSRGGPAQ 180

LAAFERGQDI TVPYSTGTDQ SSTVRRHIAP SWTWALVTTG AFIVMAMQ 228

<210>4

<211>687

<212>DNA

<213> Plasmodiophora brassicae：PBRA_6677 CDS

<400> 4

atgctgggcg ccacgacatt gctgatcgcc tccctgattg ctgcctgcaa cgggcagtca 60

tgtcgaggtc caggattcat catcactgga tgtaacccgg acttgcccat caccgatcgc 120

tgcggtgtcg ccgccgatgg agcaccctgt ccatttggaa cctgctgctc cgaattcggg 180

ttttgcgggc gcagcgctgt tcactgtggt ggcgagcaat acattccaca atggggtcca 240

ccgcgagacg acgggcggtg cggagaggca ttcagctacg ctgggtgcga tgacgatttt 300

atctgcatcg ccacctggtg cgtcagttcc gactctgtcc ccggtgcgtc acccgtgaat 360

ccgacgtcgg caacgccgac cggaacgtct gacaccacca acccacaacc tgttccttcc 420

ggctcggctg aggtcgaccc cagttcatct gagcatccca gatcagggaa caatggtgtc 480

actagggaag cgtccagctc gggaacagga agcagcacca gcagaggagg gcctgctcaa 540

ctagccgcgt ttgaaagagg tcaggatatc accgtcccat attcgacggg taccgaccaa 600

tcttcaactg tgcgtcgaca catcgctcca tcctggacct gggcgcttgt cactacaggc 660

gcgttcatcg tcatggccat gcaataa 687

Claims (6)

1. An application of plasmodiophora hordei effect protein PBRA _6677 in prevention and treatment of plasmodiophora brassicae crop, wherein the amino acid sequence of the plasmodiophora hordei effect protein PBRA _6677 is shown in SEQ ID NO. 3.

2. The use according to claim 1, wherein the Plasmodiophoromone PBRA _6677 is applied to the roots of said plants.

3. The use according to claim 1 or 2, wherein the Plasmodiophoromyces response protein PBRA _6677 is co-administered with a fertilizer.

4. The use of claim 1 or claim 2, wherein the crop is selected from brassica juncea.

5. The use of claim 4, wherein the crop is tumorigenic mustard.

6. The use according to claim 1, wherein the gene encoding the plasmodiophora tumefaciens effector protein PBRA _6677 is introduced into cruciferous crops for expression by transgenic technology.

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