CN117535317A - MAPK gene and application thereof in resisting poplar fungus infection - Google Patents

Abstract

The invention belongs to the technical field of plant biology, in particular relates to a MAPK gene and application thereof in resisting poplar fungal infection, and the invention discovers that PtMAPK3-1 gene is a poplar MAPK cascade gene family member and plays an important role in the stress resistance and disease resistance processes of poplar through bioinformatics analysis. According to the invention, through constructing an expression vector and carrying out agrobacterium tumefaciens transgenic over-expression MAPK3-1, the accumulation of ROS in poplar bodies after S.musiva infection is carried out on the over-expression PagMAPK3-1, nutrition is provided for further infection and propagation of pathogenic fungi in plant bodies, and the PagMAPK3-1 in K8 poplar is found to play a negative regulation role in poplar disease resistance reaction. The invention provides a molecular marker for screening the disease-resistant poplar varieties and a regulating and controlling means for improving the disease resistance of poplar.

Description

MAPK gene and application thereof in resisting poplar fungus infection

Technical Field

The invention belongs to the technical field of plant biology, and particularly relates to a MAPK gene and application thereof in resisting poplar fungus infection.

Background

With the increasing economy and population, the demand for wood product resources and bioenergy raw materials by humans is increasing, however, the productivity of forests is limited due to the rise in temperature, the limited availability of water resources and the increased probability of natural disasters occurring. The poplar has the advantages of high growth speed, strong adaptability, short rotation period and the like, and is widely planted in the global scope as a board and a paper pulp. However, long-term large-area poplar planting, poplar is also subject to a large number of insect pest infestations. Ulcers and leaf spots caused by Sphaerulinamusiva (sphaerella s. Musiva) can lead to premature defoliation, reduced photosynthetic area and stem breakage of poplar, not only reducing annual yield of wood, but also more seriously leading to death of poplar and failure of forestation. Poplar leaf spot is a disease caused by fungi and mainly infects poplar and related plants. A fungus of the genus sphaerella (sphaerulinaspp) is one of the pathogenic bacteria of the leaf spot, and can cause defoliation and rot of plants, leading to yield loss in light persons and death in heavy persons. Sphaerulatinosusiva can cause leaf spot and canker in poplar. It is native to eastern populus nigra (populus deltaides) and causes only leaf spot symptoms. On a hybrid poplar that is susceptible to infection, s.musiva can cause leaf necrosis, cause early leaf fall, and cause ulcers on stems and branches, thereby reducing the growth rate, making the tree susceptible to colonization by microorganisms. There is currently no way to prevent the spread of s.musiva in poplar planting areas, once s.musiva has spread into an area, it is difficult to remove the fungus. At present, chemical and biological control measures can greatly reduce the incidence of poplar, and in addition, the creation of disease-resistant transgenic poplar is another effective means for controlling poplar diseases. Chinese patent No. CN202210837131.X proposes over-expression of PtoCXE06 gene in poplar to enhance resistance of poplar to Populus rotten skin bacteria. Chinese patent CN202211588272.9 found the effector protein SmCSEP3 in s.musiva infection in poplar and proposed transient expression or overexpression of SmCSEP3 in poplar to enhance poplar resistance to s.musiva infection. Therefore, the genetic engineering technology and plant disease resistance are combined, the research of the disease resistance of poplar is developed, the relevant regulation and control mechanism of disease resistance is explored, and the method has important significance for improving the survival rate of artificial forest of poplar, increasing the wood yield and protecting landscape ecology.

Disclosure of Invention

At present, aiming at S.musiva infection, related infection, disease resistance genes and internal signal paths of poplar are explored, and researches for improving the antifungal capability of the poplar by adopting a genetic engineering method are relatively few and further penetration is needed. The invention aims at exploring related mechanism mechanisms and providing more means for improving the disease resistance of poplar.

Based on the above, the invention provides the following technical scheme:

in one aspect, the invention provides a negative regulation gene for poplar disease resistance, wherein the gene is PagMAPK3-1, and the nucleic acid sequence of the gene is shown as SEQ ID NO. 3.

In one aspect, the invention provides an expression vector, which contains PagMAPK3-1 genes, wherein the sequence of the PagMAPK3-1 genes is shown as SEQ ID NO. 3. The expression vector can be a prokaryotic expression vector or a eukaryotic expression vector.

In one aspect, the invention provides a genetically engineered bacterium, wherein the genetically engineered bacterium comprises the expression vector. Preferably, the engineering bacteria are one of escherichia coli and agrobacterium tumefaciens.

In one aspect, the invention provides the use of MAPK3-1 gene as a screening marker for poplar infection resistance to S.musiva bacteria. Preferably, the Yang Shuwei populus tomentosa or 84K populus tomentosa.

When PagMAPK3-1 gene expression of the tested variety is obviously higher than the expression level of wild poplar K8, the variety is a variety with poor S.musiva bacterial infection disease resistance; when PagMAPK3-1 gene expression of the tested variety is obviously lower than the expression level of wild poplar K8, the variety is a variety with better S.musiva bacterial infection resistance.

In one aspect, the invention provides a method for improving the infection resistance of poplar to S.musiva bacteria, wherein MAPK3-1 gene expression in poplar is reduced by transient transfection or stable transfection.

Advantageous effects

Through genomic analysis, ptMAPK3-1, ptMKK7, ptMKK9 and PtRaf23-1 were found to have significant changes in transcript levels under various biotic and abiotic stresses under salt, drought and M.brunnea fungal stresses, and in particular PtMAPK3-1 was able to respond to a variety of adverse effects as a new MAPK family member.

After S.musiva inoculation is carried out on the transgenic poplar of the over-expression PagMAPK3-1, the transgenic poplar shows more necrotic lesions, and physiological and biochemical indexes such as DAB dyeing, PAL activity, CAT activity, POD activity, MDA content and the like further indicate that the PagMAPK3-1 plays a negative regulation role in the disease resistance reaction of the poplar, so that a molecular marker is provided for screening the disease-resistant poplar varieties, and a regulation means is provided for improving the disease resistance of the poplar.

Drawings

Fig. 1: gel electrophoresis analysis after PCR amplification of PagMAPK3-1 gene.

Fig. 2: pagMAPK3-1 gene sequence and conserved domain prediction.

Fig. 3: and (5) carrying out PCR electrophoresis detection on the transgenic poplar genome.

Fig. 4: and detecting the expression level of PagMAPK3-1 over-expressed transgenic poplar genes.

Fig. 5: phenotypic analysis after PagMAPK3-1 transgenic poplar inoculation with S.musiva.

Fig. 6: physiological and biochemical index detection after two days of PagMAPK3-1 transgenic poplar inoculation with S.musiva.

Detailed Description

In the invention, plant material hybrid poplar 84K (Populus salba×Populus glandulosa) is given by a Wang Liujiang teacher of the national institute of forestry; other biological reagents are biological reagent companies on the market from which conventional reagents are derived except for special descriptions, and related methods of molecular biology operation are referred to in the guidelines for molecular cloning experiments (J. Sam Broker, D. W. Lassel, scientific Press) except for the special descriptions.

Example 1: identification of poplar MAPK cascade Gene family Member

Genomic data for the populus tomentosa genome assembly versions v1.0 and v3.0 were obtained from the Phycomsm database (http:// genome. Jgi-psf. Org/Poptr 1.Home. Html) and the Phytozome database (https:// phytzome. Jgi. Doe. Gov/pz/portal. Html). According to previous studies, protein sequences of 21 PtMAPKs and 11 PtMAPKKs in the poplar genome version v1.0 were obtained (Hamel L P, nicole M C, sritubtim S, et al, animal signature: comparative geno mics of plant MAPK and MAPKK gene families [ J ]. Trends in plant science,2006,11 (4): 192-198); ptMAPKs (i.e., the MAPKs of populus carpus) and PtMAPKKs gene IDs were obtained in the v3.0 version of the protein pool of populus carpus by BLAST tools in TBtools using these 32 protein sequences as query sequences. To determine potential members of the PtMA PKKK gene family, the genomic and protein sequences of populus tomentosa were downloaded from the plant genome database (https:// phytozome. Jgi. Doe. Gov/pz/portal. Html). BLAST searches of the protein database of Poplar were performed using the queried protein sequence of MAPKKK of Arabidopsis, jujube, jatropha, kiwi as query sequence. Subsequently, genes containing serine/threonine protein kinase domain (PF 00069) in populus tomentosa were searched using Hidden Markov Model (HMM). Comparing the genes obtained by BLAST search and HMM search, removing genes that cannot meet both conditions, and removing redundant transcripts. Finally, the gene numbers of 21 PtMAPKs and 11 PtMAP KKKs in the new version are obtained. In order to better understand the gene expression level of MAPK cascade gene response stress, the invention obtains transcriptome data related to poplar abiotic stress (drought and salt) and biological influence (Marssoninabrunea infection) from a GEO database of NCBI, and as a result, ptMPK3-1, ptMKK9, ptMKK7 and PtRaf23-1 are found to be significantly up-regulated after infection by pathogenic bacteria, and PtMAPK3-1 responds to various stresses. Based on the gene, the MAPK3-1 gene is an important candidate gene for stress resistance and disease resistance of poplar.

EXAMPLE 2 cloning and analysis of the PagMAPK3-1 Gene, a Poplar MAPK Cascade Gene family Member

The inventors studied the relationship of MAPK3-1 gene in the Chinese poplar variety 84K poplar, based on the results of the genomic analysis of the Populus deltoides in example 1, mainly in foreign poplar varieties.

PagMAPK3-1 (namely 84K poplar MAPK3-1 gene) was cloned by using the full-length cDNA of 84K poplar as a template, and related primers used in PCR experiments were as follows:

PagMAPK3-1-F：ATGGCGAATTATGCACAGGGAAATG(SEQ ID NO.1)

PagMAPK3-1-R：CTAGCATGCATATTCTGGATTAAGTGC(SEQ ID NO.2)

the PCR product was recovered by gel, and the recovered PCR product was ligated to cloning vector pEasy-BluntSimple to transform E.coli.

Analysis of results: the PCR products were isolated by 1% agarose gel electrophoresis gel purification, which showed a bright and single band between 2000bp and 1000bp (FIG. 1). The genome sequence annotation shows that the PagMAPK3-1 gene has a size of 1116bp, and the success of the PagMAPK3-1 gene amplification is primarily demonstrated. The bright gel blocks are cut out for gel recovery and then connected with cloning vectors, and the cloning vectors are sent to a sequencing company for sequencing. The sequencing results are compared through DNAMAN software, and the results show that the sequences are consistent, and the cloning success of the target gene is proved. Alignment of PagMAPK3-1 and PtMAPK3-1 sequences revealed that CDS sequences of MAPK3-1 in Populus deltoides and 84K Populus deltoides differ by 8 bases and that there is a difference of 3 amino acids after translation into proteins.

The sequence of the sequenced 84K poplar PagMAPK3-1 gene is shown as SEQ ID NO.3, the sequence is shown as (a) in FIG. 2, the PagMA PK3-1 gene size is 1116bp, and the structure conservation domain prediction analysis of the PagMAPK3-1 gene is shown as (b) in FIG. 2.

Example 3 identification of disease resistance function of overexpressed transgenic poplar

3.1 obtaining of overexpressing transgenic lines

(1) Vector construction

Constructing PagMAPK3-1 overexpression vector by adopting Gateway technology:

a. the gateway linker was added to the F, R end of the PagMAPK3-1 gene cloning primer, and the PagMAPK3-1 cloning vector constructed in example 2 was used as a template for cloning, and the sequence of the added linker primer was as follows:

then, carrying out PCR product gel recovery;

b. the PCR product was cloned into the intermediate vector pDONR207 by BP reaction, the reaction system was as follows:

reagent(s)	Dosage of
		PCR products	150ng
Carrier body	75ng
		BP enzyme	0.8μl

And (3) connecting at 25 ℃ for 6 hours, then carrying out PCR product gel recovery, transformation and sequencing.

c. After the construction and sequencing of the intermediate vector in the last step are correct, two genes are constructed on the overexpression vector pMDC32 by utilizing an LR reaction, and the reaction system is as follows:

the PCR products were recovered, transformed, sequenced, plasmid extracted and finally introduced into Agrobacterium competent cells GV3101 after 6h ligation at 25 ℃.

(2) Genetic transformation of poplar:

a. adding the agrobacterium obtained in the step (1) into LB culture medium containing corresponding Kan and Rif for amplification to ensure that the OD600 value reaches about 0.6;

b. placing the cut 84K poplar leaves with wounds into the bacterial liquid of the previous step in an ultra-clean workbench, and horizontally shaking the table for about 20min at a low speed;

c. the infected leaves are dried by sucking redundant bacterial liquid and then placed into a differentiation culture medium, and the infected leaves are subjected to dark culture for 2d at 24 ℃;

d. after 2d of dark culture, transferring the leaves into a screening culture medium containing hygromycin and timentin for screening, wherein resistant buds generally appear about 20 d;

e. cutting out the resistant bud seedling, transferring the cut resistant bud seedling into rooting culture medium containing the same resistance, propagating after rooting, and detecting the expression quantity.

Differentiation medium system 1L:

screening media system 1L: differentiation medium 1L, after sterilization, cooled to 50℃and hygromycin (0.0003 g/L) and timentin (0.2 g/L) were added under an ultra clean bench.

Rooting medium system 1L:

hygromycin (0.0003 g/L) and timentin (0.2 g/L) were added under an ultra clean bench after cooling to 50℃after sterilization.

(3) Genome PCR validation:

the poplar genome is extracted by a CTAB method, and then PCR experiments are carried out by taking the extracted genome as a template to detect PagMAPK3-1 genes. The positive control group was over-expression vector plasmid. After the PCR reaction is finished, gel electrophoresis detection is carried out, and the plants with PCR bands at the positions corresponding to the positive control can be positive plants (see figure 3).

(4) Analysis of the relative expression levels of positive lines:

RNA of positive plants is extracted and reverse transcribed respectively, and the obtained cDNA is used as a template for qRT-PCR experiments. The relative expression level of the target gene in each strain was detected by qRT-PCR experiments.

The results show that: in PagMAPK3-1 over-expressed transgenic lines, the expression level of PagMAPK3-1 genes of OE-1 and OE-3 was 20 times or more (see FIG. 4), so that these two lines were selected for subsequent analysis.

EXAMPLE 4PagMAPK3-1 transgenic poplar disease resistance study

Infection with musiva

S.musiva bacteria are separated from leaf-spot-disease populus tomentosa leaves, cultured by using a PDA culture medium, mycelium is scraped by a gun head or an inoculating needle, and dissolved by using a sterile 0.05% Tween 80 aqueous solution, the number of middle spores of spore suspension is regulated, healthy leaves of transplanted poplar seedlings are adopted, the healthy leaves are placed in a culture dish paved with wet gauze, the back surfaces of the leaves face upwards, and 25 mu l of prepared spore suspension is dripped at the positions, which avoid the veins, on the two sides of the leaves. The dishes were placed in a 25℃incubator for dark culture, and the onset of the disease was observed daily.

Results: observation of the condition of lesions after 4 days of inoculation of S.musiva spore suspensions on 84K poplar (wild type control WT), pagMAPK3-1 transgenic poplar leaves, respectively, as shown in FIG. 5, 84K poplar leaves appeared slightly spotted on the sixth day after inoculation with S.musiva, whereas the PagMAPK3-1 transgenic poplar leaves had more necrotic area than WT. After statistical analysis of the lesion size after the sixth day of inoculation of the germ, it was found that the lesion sizes of OE-1 and OE-3 were significantly higher than WT. The above results indicate that overexpression of PagMAPK3-1 reduces resistance of poplar to S.musiva.

4.2 measurement of physiological Biochemical index

Plants will rapidly become stressed by pathogenic bacteriaReactive Oxygen Species (ROS) accumulate, and the massive accumulation of reactive oxygen species can lead to death of plant cells. Thus, plants activate antioxidant enzymes such as Catalase (CAT), peroxidase (POD), and PAL (phenylalanine enzyme) in the body in order to clear excessive ROS. Malondialdehyde (MDA) is one of the common indicators of the extent to which plants are subjected to oxidative stress, and reflects the extent to which plant membrane lipids are peroxidized. DAB staining of leaves of WT, OE-1 and OE-3 plants the following day after S.musiva inoculation allows detection of ROS, H, in plants ₂ O ₂ Accumulation of conditions.

(1) CAT (catalase), POD (peroxidase) activity assay, PAL (phenylalanine enzyme), wherein POD, CAT detection method reference: maehly A C, chance B.the assay of catalases and peroxidases [ J ]. Methods of Biochemical Analysis,1954,1:357-424.PAL detection methods reference: beaudoin-Ega n LD, thorpe TA. Tyrosine and phenylalanine Ammonia Lyase activities during shoot initia tion in tobacco callus cultures.plant Physiol.1985;78:438-41.

(2) MDA content measurement: the malondialdehyde content was determined using the thiobarbituric acid method, and the specific experimental method was referred to the "plant physiology experiments course" published by 2017 science publishers, by Wang Sangen.

(3) DAB staining: the poplar leaves in the affected area are placed in a 50ml centrifuge tube, and DAB dye solution is added to submerge the leaves. Shaking the mixture for 4-7h under the condition of avoiding light at 25-28 ℃ and 80r/min, adding a decolorizing solution (the decolorizing solution is prepared by ethanol: acetic acid: glycerol=3:1:1) after removing the dye solution, and decolorizing the mixture for 15min by transferring the centrifuge tube into a constant temperature water tank (95 ℃), wherein the decolorizing solution can be replaced for 1-2 times.

Each of the above indicators was taken as a leaf of poplar in the disease area, 3 replicates per sample, and histogram and ANOVA difference significance analysis was presented with GraphPadPrism5 software (p <0.05; p < 0.01).

Results:

referring to FIG. 6 (b), the PAL, CAT and POD activity assays showed increased PAL and CAT activity for wild-type poplar (WT), while PAL and CAT activity for the overexpressed PagMAPK3-1 transgenic poplar decreased and were significantly lower than WT. POD activity of WT was increased the next day after inoculation, but POD activity of PagMAPK3-1 transgenic poplar was decreased and significantly lower than WT.

Referring to FIG. 6 (b), the MDA content of the transgenic lines after pathogen treatment was significantly higher than that of WT plants.

Referring to FIG. 6 (a), on the next day after S.musiva inoculation, yellow brown precipitations were generated in leaves of WT, OE-1 and OE-3 plants, indicating that after S.musiva infection of poplar, active oxygen accumulation of plants could be induced. Fewer precipitants in WT leaves than in two transgenic plants, indicating that overexpressing PagMAPK3-1 transgenic poplar caused more cell death and H after S.musiva invasion ₂ O ₂ The accumulation provides nutrition for further infection and propagation of pathogenic fungi in plants.

The foregoing is a further elaboration of the present invention in connection with the detailed description, and it is not intended that the invention be limited to the specific embodiments shown, but rather that a number of simple deductions or substitutions be made by one of ordinary skill in the art without departing from the spirit of the invention, should be considered as falling within the scope of the invention as defined in the appended claims.

Claims (8)

1. A negative regulation gene for poplar disease resistance is characterized in that the gene is PagMAPK3-1, and the nucleic acid sequence of the gene is shown as SEQ ID NO. 3.

2. The expression vector is characterized by comprising a PagMAPK3-1 gene, wherein the sequence of the PagMAPK3-1 gene is shown as SEQ ID NO. 3.

3. A genetically engineered bacterium, wherein the engineered bacterium comprises the expression vector of claim 2.

4. The engineered bacterium of claim 3, wherein the engineered bacterium is agrobacterium tumefaciens.

5. Use of a gene according to claim 1 as a screening marker for poplar infection against s.musiva bacteria.

6. Use according to claim 5, characterized in that said Yang Shuwei populus trichocarpa or 84K populus.

7. The use according to claim 5, wherein the strain is a strain with poor disease resistance to s.musiva bacterial infection when the PagMAPK3-1 gene expression of the test strain is significantly higher than the expression level of wild-type poplar K8; when PagMAPK3-1 gene expression of the tested variety is obviously lower than the expression level of wild poplar K8, the variety is a variety with better S.musiva bacterial infection resistance.

8. A method for improving the infection resistance of poplar to s.musiva bacteria, characterized in that MAPK3-1 gene expression in poplar is reduced by transient transfection or stable transfection.