CN117721145A - Application of GhMPK31 gene in regulation and control of upland cotton insect pest defense capability - Google Patents
Application of GhMPK31 gene in regulation and control of upland cotton insect pest defense capability Download PDFInfo
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Abstract
The invention provides application of GhMPK31 gene in regulating and controlling upland cotton insect pest defense capability, and belongs to the technical field of genetic engineering. The invention provides application of a GhMPK31 gene in regulating and controlling the insect pest defense capability of upland cotton, wherein the nucleotide sequence of the GhMPK31 gene is shown as SEQ ID NO. 1. The transgenic upland cotton is obtained by over-expressing the GhMPK31 gene in upland cotton, the over-expression of the gene in the leaf of the transgenic upland cotton leads to a cell death phenotype similar to anaphylactic reaction, the defending capability of the cotton to phytophagous insects is damaged, the correlation between the GhMPK31 gene and the defending capability of the upland cotton insect pests is shown, and valuable insight is provided for the interaction of MAPK family genes in plants and the phytophagous insects.
Description
Technical Field
The invention belongs to the technical field of genetic engineering, and particularly relates to application of a GhMPK31 gene in regulation and control of upland cotton insect pest defense capability.
Background
Cotton is an important source of human fiber, oil and feed, making a significant contribution to the world's development. During the long growth period of cotton, the cotton is affected by various phytophagous insects, the yield and the fiber quality of the cotton are seriously affected, and insect damage has become an important factor for limiting the development of the cotton industry. Traditional insect-resistant molecular breeding strategies are mainly to introduce exogenous bacillus thuringiensis (Bt) insecticidal protein genes into cotton genomes to enhance insect resistance of cotton. Although the method relieves the cotton production pressure caused by insect damage in a short time, the problems of evolution of target insect resistance, non-target secondary insect outbreaks and the like are gradually revealed along with long-term planting of Bt insect-resistant cotton, so that development of a new insect-resistant strategy for coping with insect attack is needed. At present, the research of gene expression in the field of genetic engineering has more reports on plant resistance or disease resistance, but the genes related to the insect pest defense capability of upland cotton have not been reported.
Disclosure of Invention
Therefore, the invention aims to provide the application of the GhMPK31 gene in regulating and controlling the insect pest defense capability of upland cotton, and the transgenic cotton over-expressing the GhMPK31 gene has reduced insect pest defense capability, so that the GhMPK31 gene has obvious correlation with the upland cotton insect pest defense, and theoretical basis and experimental materials are provided for subsequent upland cotton insect pest defense.
In order to achieve the above object, the present invention provides the following technical solutions:
the invention provides application of a GhMPK31 gene in regulating and controlling the insect pest defense capability of upland cotton, wherein the nucleotide sequence of the GhMPK31 gene is shown as SEQ ID NO. 1.
Preferably, overexpression of the GhMPK31 gene can improve the sensitivity of upland cotton pests.
The invention provides a recombinant expression vector containing the GhMPK31 gene.
Preferably, the receptor vector of the recombinant expression vector is pK2GW7.
Preferably, the primer group for amplifying the GhMPK31 gene comprises a forward primer and a reverse primer, and the nucleotide sequence is shown as SEQ ID NO.2-3 in sequence.
The invention provides a bioengineering bacterium containing the recombinant expression vector.
The invention provides a method for obtaining high-sensitivity upland cotton by pests, which is characterized in that the GhMPK31 gene is overexpressed in upland cotton.
Preferably, the recombinant vector containing the GhMPK31 gene is electrically transduced into bioengineering bacteria, and the transduced bioengineering bacteria are used for dying upland cotton.
The invention also provides application of the high-sensitivity upland cotton with high pest sensitivity obtained by the method in plant functional genomics research or crop breeding.
Compared with the prior art, the invention has the following beneficial effects:
aiming at MAPK gene families, the invention adopts a screening strategy based on the expression profile induced by cotton bollworm oral secretion, and the GhMPK31 gene is screened to be obviously induced. Then clone the gene and create the pest-sensing transgene material OM31 of the over-expression GhMPK31, which provides valuable resources for improving the pest-resistant breeding of upland cotton.
The invention proposes that the GhMPK31 gene can regulate and control the burst of Reactive Oxygen Species (ROS) in cotton body through interaction with GhRBOHB, so as to lead to programmed death of leaf cells, and simultaneously lead to reduction of cotton defensive metabolites, thereby leading to reduction of the defending ability of cotton pests. Therefore, the GhMPK31 gene plays a very important role in regulating and controlling the defenses of cotton to pests, provides theoretical and practical basis for breeding insect-resistant varieties, and has very important economic and social benefits for further researching molecular biological information related to the insect-resistant effect of cotton.
The over-expression GhMPK31 plant obtained by the invention provides precious materials for the research of cotton insect-resistant breeding, can be used as a mode plant with low insect-resistant capability, can be applied to the research of plant-insect-pest interaction relationship or insect-resistant drug screening, and the cultivated upland cotton leaf can also be used as a feed for experimental insects.
Drawings
FIG. 1 is a comprehensive analysis of the upland cotton MAPK gene family; (A) Phylogenetic analysis of the MAPK gene families of upland cotton, arabidopsis, rice and Raymond cotton; (B) Phylogenetic relationship, conserved protein motifs and genetic structure of GhMPKs; (C) Co-linearity analysis of GhMPK and MAPK genes of other three plants; (D) Colinear analysis and chromosome localization of the GhMPK gene family; (E) cis-acting elements of the GhMPKs promoter region; (F) prediction of GhMPKs phosphorylation site.
FIG. 2 is a graph showing expression patterns of cotton bollworm oral secretion induced GhMPK gene family.
FIG. 3 shows GhMPK31 overexpression and RNAi transgenic plant acquisition and phenotype identification; (a) GhMPK31 overexpression and construction of RNAi vectors; (B) genetic transformation of cotton; (C) Determination of expression level of GhMPK31 in T0 generation overexpression and RNAi transgenic plants; (D) Transgenic plants were compared to the WT plant phenotype, with red lines representing 1 cm; (E) OM31 with WT plant height, red line represents 10 cm.
FIG. 4 shows RNA-seq analysis revealing the effect of GhMPK31 on gene expression; (A) detecting the GhMPK31 expression level in T1 generation IM31 and OM31 plants; (B) The Venn plot shows the overlap between three sets of differentially expressed genes; (C) Identification of OM31 VS WT between and H by RNA-seq method 2 O 2 Related differentially expressed genes; (D) Three sets of differential gene volcanic charts, red for up-regulated differential expression genes and blue for down-regulated differential expression genes; (E) The KEGG enrichment pathway of OM31 VS WT up-or down-regulated differentially expressed genes; (F) GO enrichment of differentially expressed genes up-or down-regulated by OM31 vs WT.
FIG. 5 shows that overexpression of GhMPK31 results in H in plants 2 O 2 Is accumulated in the reactor; (a) OM31, IM31 and WT plant phenotypes of the TI generation; (B) DAB staining solution results of OM31, IM31 and WT plant leaves; (C) H in OM31, IM31 and WT blades 2 O 2 Concentration; (D) OM31, IM31 and WT plants H 2 O 2 And detecting the relative expression quantity of the related genes.
FIG. 6 shows the results of a study of the interaction of GhMPK31 with GhRBOHB; (a) subcellular localization map of GhMPK 31; (B) Y2H analysis of GhMAP31-GhRBOHB interactions, SD-T-L (-Trp/-Leu), SD-T-L-A-H (-Trp/-Leu/-His/-Ade) (C) BiFC analysis of GhMPK31-cYFP and GhRBOHB-nYFP in tobacco epidermal cells; (D) LCI analysis of GhMPK31-nLUC and GhRBOHB-cLUC in tobacco leaves, (E) three-dimensional protein structural model of GhMPK31 and GhRBOHB interactions.
FIG. 7 shows that overexpression of GhMPK31 reduces accumulation of defensive metabolites; (A) Volcanic plot of differential metabolites between OM31 and WT, red for up-regulated metabolites and green for down-regulated metabolites; (B) The number of differential metabolites for each class of compound in the OM31 mutant, orange for up-regulated metabolites and green for down-regulated metabolites; (C) The difference in levels of several defensive metabolites in OM31 and WT plants was compared, red for WT and green for OM31.
FIG. 8 shows that overexpression of GhMPK31 reduces the ability of cotton to defend against phytophagous insects; (A) Cotton bollworm selective feeding experiments, red lines represent 1 cm; (B) Leaf consumption statistics in cotton bollworm selective feeding experiments; (C) After the prodenia litura is non-selected to feed for 5 days, comparing the body types of larvae on the feeding WT leaves and the feeding OM31 leaves, wherein the red line represents 1 cm; (D) Average larval body weights on day 0 and day 5, mean ± standard error (n=8). Statistical analysis was performed using t-test, representing P <0.05, P <0.01, P <0.001. All experiments were repeated at least three times with similar results.
Detailed Description
The invention provides application of a GhMPK31 gene in regulating and controlling the insect pest defense capability of upland cotton, wherein the nucleotide sequence of the GhMPK31 gene is shown as SEQ ID NO. 1. According to the invention, the transgenic plant leaves are found through over-expression of GhMPK31 genes in upland cotton, so that a cell death phenotype similar to anaphylactic reaction (HR) appears relative to a normal plant, and the plant shows a dwarf structure. The invention discovers GhMPK31 and GhRBOHB (H) through Y2H, biFC and LCI experiments 2 O 2 Synthesis related genes) and overexpression of GhMPK31 enhances the expression of hydrogen peroxide related genes. The invention is based on the metabolome data of transgenic plants and normal plantsAnalysis showed that overexpression of GhMPK31 reduced accumulation of defense-related metabolites. According to the invention, through the experimental results of selective feeding of cotton bollworm larvae and non-selective feeding of prodenia litura larvae, the defending performance of the transgenic plant over-expressing GhMPK31 against pests is obviously lower than that of a normal plant.
The invention also provides a recombinant expression vector containing the GhMPK31 gene. The receptor carrier of the recombinant expression vector is pK2GW7. According to the invention, the GhMPK31 gene is inserted into the pK2GW7 vector through Gateway cloning technology, so that a recombinant expression vector containing the GhMPK31 gene is obtained. The cDNA sequence of upland cotton Jin668 strain is used as template to amplify the full-length CDS sequence (SEQ ID NO. 1) of GhMPK31, and the primer set used for amplification comprises a forward primer and a reverse primer, and the nucleotide sequence is shown as SEQ ID NO.2-3 in sequence. The amplification reaction system of the invention comprises: the total volume was 20. Mu.L, buffer solution 2. Mu. L, dNTP 0.3, 0.3. Mu. L, cDNA 1, forward primer 0.2. Mu.L, reverse primer 0.2. Mu.L, easy Taq 0.2. Mu.L, double distilled water 16.1. Mu.L. The amplification reaction program of the invention is as follows: pre-denaturation: 95 ℃ for 5min; denaturation: 30S at 95 ℃; annealing: extension at 59℃for 30S: cycling for 28 times at 72 ℃ for 1 min; and finally, extending: and at 72℃for 5min.
In the invention, the steps for obtaining the cDNA of the upland cotton Jin668 strain comprise the following steps: RNA from leaves was extracted with a plant RNA extraction kit (purchased from TIANGEN Co.) and then subjected to RNA reverse transcription.
The invention provides a bioengineering bacterium containing recombinant expression vector. The bioengineering bacterium is Agrobacterium, and more preferably an Agrobacterium (Agrobacterium) strain GV3101. The recombinant expression vector of the present invention is electrically transduced into Agrobacterium according to conventional methods.
The invention provides a method for obtaining high-sensitivity upland cotton for pests, which comprises the following steps: the GhMPK31 gene is overexpressed in upland cotton. The recombinant vector containing the GhMPK31 gene is electrically transduced into bioengineering bacteria, and the transduced bioengineering bacteria are used for dying upland cotton. The invention infects hypocotyl with bioengineering bacteria, then sequentially passes through a co-culture stage, a callus culture stage, a differentiation culture stage, a rooting culture stage, a nutrient solution culture stage and a transgenic plant in a greenhouse growth stage to obtain a transgenic plant over-expressing GhMPK31 genes.
In the invention, the upland cotton with high sensitivity to insect pests obtained by the method can be used as a model organism with low insect pest resistance, can be applied to research on interaction relation between plants and insect pests or screening of insect resistant medicines, and the cultivated upland cotton leaves can also be used as feed for experimental insects.
In the invention, the GhMPK31 gene can also be used as a marker for screening the insect-resistant upland cotton, and is used in the breeding of the insect-resistant upland cotton, if the GhMPK31 gene expression amount in the selected upland cotton is obviously higher than the GhMPK31 gene expression in a upland cotton Jin668 strain, the insect-resistant capability of the selected upland cotton is poorer. The invention measures the GhMPK31 gene expression in upland cotton by a qPCR method, and the related primers are QGhMPK31-F and QGhMPK31-R, and the nucleotide sequence is shown as sequence SEQ ID NO.4-5 in sequence; the amplification reaction system is as follows: 20. Mu.L of the total system, 2X ChamQ Blue Universal SYBR Qpcr Master Mix, 10. Mu. L, QGhMPK 31-F0.4. Mu. L, QGhMPK 31-R0.4. Mu. L, cDNA 1, 1. Mu.L of double distilled water, 8.2. Mu.L; the amplification procedure was: pre-denaturation: 95 ℃ for 2min; denaturation: 95 ℃ for 15S, annealing: and (5) cycling for 40 times at 60 ℃ for 30S.
The technical solutions provided by the present invention are described in detail below with reference to examples, but they should not be construed as limiting the scope of the present invention.
Example 1
1. Comprehensive analysis of upland cotton MAPK gene family
Genomic data from upland cotton (G.hirsutum) and Raymond cotton (G.raimondii) were from Cottongen (https:// www.cottongen.org /), and from Arabidopsis (A.thaliana) and rice (O.sativa) were from Ensembl Plants (http:// plant. Ensembl. Org/index. Html) and Rice Genome Annotation Project (http:// price. Uga. Edu/downloads_gad. Shtml), respectively.
MAPK protein coding sequences of arabidopsis, rice and Raymond cotton are used as query sequences, a BLASTP program is utilized to search upland cotton genomes (NBI) and (HAU), and a search based on a local Hidden Markov Model (HMM) is constructed according to the protein coding sequences to identify GhMPKs. The results of BLASTP and HMMER are then compared to remove redundant sequences, and finally the candidate gene is uploaded to SMART (http:// SMART. Emml. De /) and CDD (https:// www.ncbi.nlm.nih.gov/CDD /), confirming the presence of the conserved domain. The amino acid number, molecular Weight (MW) and isoelectric point (pI) were predicted by the ProtParam tool (https:// web. Expasy. Org/protParam /). Subcellular localization, transmembrane domain and signal peptide were predicted using the websites DeepLoc-2.0 (https:// services. Healthcare/services/DeepLoc-2.0 /), deepTMHMM (https:// DTU. Biological. Com/DeepTMHMM /) and SignalP-6 (https:// biological. Com/DTU/SignalP-6 /), respectively.
The MEGA 7.0 software is utilized to construct a phylogenetic tree based on the full-length protein sequences of Arabidopsis, rice, raymond cotton and upland cotton by adopting a Neighborhood Junction (NJ) method, and the phylogenetic tree is visualized by EvolView (https:// evolgenius. Info// evolvview-v 2). Conserved motifs of proteins were analyzed using the MEME program and visualized using Tbtools. The gene structure was analyzed by GSDS software (http:// GSDS. Gao-lab. Org /). Phosphorylation site analysis was done using NetPhos-3.1 (https:// services. Healthcare. Dtu. Dk/services/NetPhos-3.1 /). The cis-acting elements of the gene promoter region were analysed using the plantacare database (http:// bioinformation. Psb. Ugent. Be/webtools/plantacare/html /). The colinear analysis of upland cotton, arabidopsis, rice and Raymond cotton is carried out by using MCScanX software, and the visualization of the analysis is completed by using TBtools software. Mapping of the gene location on the chromosome and its whole genome gene replication events was performed using the Circos software.
As shown in FIG. 1, the candidate genes were verified by CDD and SMART software databases, and finally 55 GhMPKs family members were determined on the upland cotton genome. The GhMPK gene family is analyzed from the aspects of physicochemical property, family evolution relationship, gene structure, chromosome positioning, colinear analysis, promoter cis-acting element and phosphorus locus prediction and the like, so that the comprehensive understanding of the gene family is increased, and a foundation is laid for the functional analysis of the gene family in upland cotton.
2. Analysis of MAPK expression profiles in different tissues and under different stresses
Expression data of genes under different tissues and abiotic stress is derived from TM-1 transcriptome data. Transcriptome data of the gene at various times for cotton bollworm OS-induced expression was derived from NCBI Sequence ReadArchive (accession number: PRJNA 522889).
For different RNA sequencing reads, low quality reads were filtered using Trimmomatic (v.0.39) and then clean reads were mapped to the TM-1 reference genome using HISAT2 (v.2.2.1). Expression level (number of transcripts per million; TPM of genes calculated using StringTie (v.2.1.4) software. If TPM of one gene >0, it was considered to be expressed then, differential Expression Genes (DEGs) were identified using the DESeq2 package, false discovery rate (False Discovery Rate, FDR) <0.05, |log2 (fold change) |gtoreq 1. Heat map visualization was performed using Tbtools.
In transcriptome analysis of the GhMPK gene family response to cotton bollworm oral secretions, it was found that there was a fold change in 11 GhMPK gene differential expression of greater than 1.5 after cotton bollworm oral secretion induction compared to control at two time points of 30 minutes and 60 minutes (FIG. 2). Wherein, the expression level of GhMPK31 and GhMPK40 is higher than that of other genes at two time points. Binding to GhMPK31 is a direct homolog of SIPK and is involved in the regulation of plant defense against herbivorous insects. GhMPK31 was therefore selected for further study.
Example 2
1.GhMPK31 gene overexpression and RNAi vector construction
The upland cotton Jin668 strain (bred by cotton subject group guide in China university of agriculture (patent application number: 201510833618.0)) is used as a research material, RNA in leaves is extracted by using a plant RNA extraction kit (purchased by TIANGEN company), and then cDNA is obtained by RNA reverse transcription, wherein reagents for RNA reverse transcription comprise nuclease-free double distilled water, oligo (dT) 23VN, 4×gDNA viger Mix, random hexamers, 10×RT Mix and Hiscript II Enzyme Mix, and the reagents are purchased from Novirlaan company.
The cDNA template, such as the primer shown as SEQ ID NO.2-3, is used to amplify the full-length CDS sequence (SEQ ID NO. 1) of GhMPK31, and the full-length CDS sequence is inserted into the pK2GW7 vector by Gateway cloning technology to obtain the GhMPK31 gene overexpression vector (FIG. 3A). The cDNA was used as a template, and the primers shown in SEQ ID No.6-7 amplified an interference fragment of GhMPK31 (SEQ ID No. 8), and inserted into pHellgate 4 vector by Gateway cloning technique to obtain RNAi vector (FIG. 3A). The GhMPK31 gene overexpression vector and RNAi vector plasmid are electrically transduced into agrobacterium for genetic transformation of cotton.
2. Genetic transformation of cotton and identification of the Effect of GhMPK31 Gene expression on cotton
The constructed vector is subjected to a hypocotyl infection stage, a co-culture stage, a callus culture stage, a differentiation culture stage, a rooting culture stage, a nutrient solution culture stage and a transgenic plant growth stage in a greenhouse to obtain a transgenic plant. The agrobacterium-mediated genetic transformation process described above is shown in fig. 3B. T0 generation GhMPK31 gene over-expression plants (OM 31) and RNAi transgenic plants (IM 31) are obtained from the cotton genetic transformation process, and the transformed recipient plants J668 are used as controls (WT). OM31 with high expression and IM31 with low expression were selected from T0 generation plants by QRTPCR for subsequent study. Seed of the selected plant is planted, and the grown plant is T1 generation plant.
(1) The expression level of GhMPK31 in T0 generation GhMPK31 gene over-expression plants grown to the bud stage, RNAi transgenic plants and WT plants is measured by qPCR.
qPCR assay method: the RNA of the above plants was extracted using a plant RNA extraction kit (purchased from TIANGEN Co.) and reverse transcribed into cDNA by reverse transcription. qPCR amplification was performed using QGhMPK31-F (SEQ ID NO. 4) and QGhMPK31-R (SEQ ID NO. 5) as primers and reverse transcribed cDNA as a template; the amplification reaction system is as follows: 20. Mu.L of the total system, 2X ChamQ Blue Universal SYBR QpcrMasterMix (purchased by Novain Co.) 10. Mu. L, QGhMPK 31-F0.4. Mu. L, QGhMPK 31-R0.4. Mu. L, cDNA 1. Mu.L, 8.2. Mu.L of double distilled water; the amplification procedure was: pre-denaturation: 95 ℃ for 2min; denaturation: 95 ℃ for 15S, annealing: and (5) cycling for 40 times at 60 ℃ for 30S. Quantitative statistics of the amplified products were performed using GhUB7 expression as an internal reference.
(2) Phenotype and plant height comparisons were performed on T1 generation GhMPK31 gene overexpression plants (OM 31), RNAi transgenic plants (IM 31) and WT plants grown to seedling stage and bud stage.
Based on the expression level of GhMPK31, two independent RNAi lines (IM 31-19 and IM 31-28) and two independent overexpressing lines (OM 31-11 and OM 31-24) were identified, and the expression level of GhMPK31 was significantly higher than that of WT and RNAi lines for subsequent study (FIG. 3C).
Phenotypic analysis of OM31 lines showed necrotic spots on leaves of all lines and the plants presented a dwarf structure, whereas RNAi lines were not significantly different from wild type (fig. 3D and 3E). Necrotic lesions on the OM31 series leaves were also observed to appear during the seedling stage and persist throughout the cotton growth phase. Necrotic lesions do not appear on new leaves, but are usually present on old leaves. Thus, these results indicate that necrotic spots are not caused by pathogen infection, but rather are a phenomenon of Hypersensitivity (HR) -like cell death.
3. Transcriptome sequencing technology (RNA-seq) analysis of Differentially Expressed Genes (DEGs) between OM31, IM31 and WT
Seeds of OM31 line, IM31 line and WT plants were germinated simultaneously and cultured in a greenhouse at 28℃with 16h light/8 h dark and 60% relative humidity. Four weeks later, leaves were removed, RNA was extracted for transcriptome sequencing, and 2 biological replicates were performed for each sample. For RNA sequencing reads from different tissues, low quality reads were filtered using Trimmomatic (v.0.39) and then the filtered reads were mapped to the TM-1 reference genome using HISAT2 (v.2.2.1). Gene expression level (TPM) was calculated using the StringTie (v.2.1.4) software. If the TPM of a gene is >0, the gene is considered to have been expressed. Subsequently, DEGs were identified using the DESeq2 package. GO and KEGG enrichment analysis was performed on all DEGs using KOBAS 3.0 software (http:// KOBAS. Cbi. Pku. Edu. Cn/index. Php). The heatmap was visualized with Tbtools, and GO and KEGG were visualized with microbial messages (https:// www.bioinformatics.com.cn /).
The difference in gene expression between the T1 generation OM31, IM31 and WT cotton leaves was analyzed using RNA-seq technique, and the expression level of GhMPK31 is shown in FIG. 4A. In contrast to WT, 6038 DEGs were identified in the OM31 line, of which 2844 genes down-regulate expression and 3239 genes up-regulate expression. Comparing OM31 and IM31 difference basisA total of 3175 DEGs were found. In contrast, only 974 DEGs were identified between IM31 and WT plants, indicating that interference with GhMPK31 had no significant effect on the expression of other genes (fig. 4B and 4D). GO enrichment analysis and KEGG pathway enrichment analysis were performed on three groups (OM 31 vs WT, OM31 vs IM31 and IM31 vs WT) according to up-and down-regulated DEGs, respectively (fig. 4E and 4F). The first 15 key pathways that up-regulate deg enrichment, whether in the OM31 vs WT group or the OM31 vs IM31 group, contain similar pathways (carbon fixation, carbon metabolism, nitrogen metabolism, and peroxisomes in photosynthetic organisms). Furthermore, the same GO items were found in the first 15 upregulated DEG-enriched GO items of these two groups, including chloroplasts, peroxisomes, NADPH oxidase and H 2 O 2 Generating activity. In addition, some and H were found 2 O 2 The relevant key genes (RBOHA, RBOHD, CAT1 and CAT 2) significantly up-regulated expression in OM31 plants (fig. 4C). These results indicate that GhMPK31 is overexpressed in plants with plant carbon fixation, nitrogen metabolism and H 2 O 2 The generation and decomposition are associated with each other.
4. Overexpression of GhMPK31 results in cotton leaf H 2 O 2 Level increase
And simultaneously culturing the seeds of the T1 generation of OM31, IM31 and WT under the same environmental condition to obtain the plants of the T2 generation.
Six weeks later, DAB staining was performed on leaves of the same parts of three plants: detection of H with 3, 3-diaminobenzidine tetrahydrochloride (DAB) using endogenous peroxidase-dependent in situ histochemical staining 2 O 2 . After the isolated leaves are completely immersed in DAB staining solution, the isolated leaves are immersed for 15min under vacuum condition, so that the staining solution can conveniently enter the cell gap. After 8h of immersion in the dark, the solution was exposed to light for 1h. The DAB staining solution remaining on the leaves was rinsed with distilled water. Subsequently, the leaves were immersed in the decolorization solution (75% ethanol+5% glycerol) a plurality of times under dark conditions at 37℃until the chlorophyll was completely removed, and photographed.
By H 2 O 2 Quantification kit (#C 500069, sangon Biotech, shanghai, china) H was performed on T2-generation OM31, IM31 and WT leaves grown to six weeks 2 O 2 And (5) content testing.
Identification of Participation H by qPCR according to the conventional method 2 O 2 Synthesized and decomposed NADPH oxidase and catalase genes. Expression of GhUB7 was used as an internal control. The related primers comprise: primers GhRBOHB-QRT-S and GhRBOHB-QRT-A; ghRBOHC-QRT-S and GhRBOHC-QRT-A; ghCAT-S and GhCAT-A; the nucleotide sequence is shown in SEQ ID NO. 9-14.
The results showed that IM31 and WT plants were higher than OM31 and OM31 plants had brown spots on leaves (fig. 5A). Significant reddish brown precipitate accumulation occurred in the leaves of OM31, whereas no such accumulation occurred in the leaves of IM31 and WT (fig. 5B), and reddish brown precipitate formed in the transparent leaves demonstrated H 2 O 2 Is present. H 2 O 2 Content test shows that H in OM31 strain 2 O 2 Levels were significantly higher than IM31 and WT plants (fig. 5C). In addition, several of the involvement in H was identified by RT-qPCR 2 O 2 Synthesized and decomposed NADPH oxidase and catalase genes. The results showed that the expression levels of GhCAT1, ghRBOHB and GhRBOHC were significantly increased in OM31 line compared to WT and IM31 line (fig. 5D). These results indicate that overexpression of GhMPK31 results in H in cotton leaf 2 O 2 Elevated levels, resulting in HR-like cell death and dwarfing of OM31 plants.
5. Molecular interactions between GhMPK31 and GhRBOHB
The gene of interest used in this experiment was from cotton. To further explore the mechanism by which GhMPK31 regulates ROS, subcellular localization of GhMPK31 was first studied. The complete CDS of GhMPK31 was fused to the N-terminus of GFP and transiently expressed in tobacco epidermis, and fluorescent signals were observed by copolymerization Jiao Guangpu microscopy to appear in both cell membrane and nucleus (fig. 6A). Yeast two-hybrid (Y2H) experiments were then performed to screen for potential interacting proteins and found direct interactions of the GhMPK31 protein with NADPH oxidase (GhRBOHB) according to conventional methods (fig. 6B). Interaction between GhMPK31 and GhRBOHB was further confirmed by conducting luciferase complementation imaging (BiFC) and bimolecular fluorescence complementation (LCI) experiments in a conventional manner (fig. 6C and 6D). The three-dimensional structure of these two protein interaction models was constructed using a Y2H-AOS protein interaction prediction analysis, with a protein interaction confidence score of 9.6, demonstrating the high probability of such interactions (fig. 6E). These results reveal that GhMPK31 interacts with GhRBOHB in cotton.
6. Effect of GhMPK31 Gene overexpression on Cotton defensive metabolites
Metabolome analysis was performed on T1 generation OM31 and WT plants to determine the changes in defense-related metabolites between them and to investigate the effect of GhMPK31 on cotton defense against herbivores.
The identified metabolites were annotated using the KEGG database (https:// www.genome.jp/KEGG/path. Html), the HMDB database (https:// HMDB. Ca/metatemplates) and the LIPIDMaps database (http:// www.lipidmaps.org /). And a multivariate statistical analysis part for converting the data by using metabonomics data processing software metaX, and performing Principal Component Analysis (PCA) and partial least squares discriminant analysis (PLS-DA) to obtain the VIP value of each metabolite. Volcanic images are plotted using R-packet ggplot2, and the three parameters VIP, log2 (FoldChange) and-log 10 (p-value) of the metabolite can be integrated to screen the metabolite of interest. Cluster heatmaps, plotted with R package Pheatmap, were normalized with z-score for metabolite data. Correlation analysis (pearson correlation coefficient) between differential metabolites was performed using the R language.
The results showed that 698 total metabolites were detected, of which 185 exhibited significant differences (P < 0.05). 76.7% of the metabolites in OM31 line were significantly reduced compared to WT plants (fig. 7A). These differential metabolites are categorized into 21 different classes of compounds, most of which are categorized as amino acids and derivatives thereof, carbohydrates and derivatives thereof, organic acids and derivatives thereof, organic heterocyclic compounds, and the like. Known endogenous defensive metabolites of major interest include flavonoids, alkaloids and derivatives thereof, phenols and derivatives thereof, which are significantly reduced in OM31 plants (fig. 7B and 7C). It was shown that overexpression of GhMPK31 reduces accumulation of defensive metabolites in cotton, resulting in increased sensitivity to phytophagous insects
Example 3 experiment of Selective feeding of Heliothis armigera larvae and non-Selective feeding of Spodoptera litura larvae
The T1 generation transgenic plants and the WT plants were cultivated under the same field conditions. 3 leaf types were selected from OM31 strain: young leaves without necrotic spots, leaves with necrotic spots and old leaves with necrotic spots. Similarly, leaves of the corresponding parts of the WT plants were selected as controls. Cotton leaves of the T1-generation OM31 and WT plants are evenly distributed, and food can be ingested by the three-instar cotton bollworm larvae at will.
The feeding experiments were selected in 29X 44cm plastic boxes. Three-instar larvae were first starved for 6 hours, and then the removed leaves were symmetrically and evenly aligned. 4 starved larvae were placed in the middle of each plastic box, allowed to eat freely for 24 hours, and photographed at 0 and 24 hours (fig. 8A). The consumption of cotton leaves was calculated using ImageJ software. In the non-selective feeding experiments, 9cm round glass petri dishes were used to hold WT plants and OM31 leaves, respectively. 8 larvae with weight of 0.018-0.022g were selected, 1 per tray, and placed on leaves individually. The larval body weights were recorded on day 0 and day 5, respectively.
The results showed that all three types of leaves of OM31 strain were significantly increased in consumption compared to WT plants as observed over 24 hours (fig. 8B). In non-selective experiments, WT plants and OM31 plant leaves were fed with prodenia litura larvae of second age for 5 consecutive days. The results observed that the weight of the larvae fed OM31 leaves was significantly greater than that fed WT leaves (fig. 8C and 8D). Together, these results demonstrate that overexpression of GhMPK31 results in a reduction of various metabolites, including defenses, which impair cotton's defenses against phytophagous insects.
Together, these results demonstrate that GhMPK31 can regulate the burst of reactive oxygen species in cotton through interaction with GhRBOHB, resulting in apoptosis of leaf cells. At the same time, the defensive metabolites of the cotton are reduced, and the defensive ability of the cotton pests is reduced. The discovery enriches the research of MAPK in the insect-resistant function of upland cotton, and provides valuable reference for the insect-resistant breeding of upland cotton.
The foregoing is merely a preferred embodiment of the present invention and it should be noted that modifications and adaptations to those skilled in the art may be made without departing from the principles of the present invention, which are intended to be comprehended within the scope of the present invention.
Claims (9)
- The application of the GhMPK31 gene in regulating and controlling the insect pest defense capability of upland cotton is characterized in that the nucleotide sequence of the GhMPK31 gene is shown as SEQ ID NO. 1.
- 2. The use according to claim 1, wherein overexpression of the GhMPK31 gene increases sensitivity to upland cotton pests.
- 3. A recombinant expression vector comprising the GhMPK31 gene of claim 1.
- 4. The recombinant expression vector of claim 3, wherein the receptor vector of the recombinant expression vector is pK2GW7.
- 5. The recombinant expression vector according to claim 3, wherein the primer set for amplifying the GhMPK31 gene comprises a forward primer and a reverse primer, and the nucleotide sequences are shown in SEQ ID NO.2-3 in sequence.
- 6. A bioengineering bacterium comprising the recombinant expression vector according to any one of claims 3 to 5.
- 7. A method for obtaining high-sensitivity upland cotton by pests, characterized in that the GhMPK31 gene described in claim 1 or 2 is overexpressed in upland cotton.
- 8. The method of claim 7, wherein the recombinant vector containing the GhMPK31 gene is electrically transduced into a bioengineering bacterium, and the transduced bioengineering bacterium is used for dying upland cotton.
- 9. Use of the pest-highly sensitive upland cotton obtained by the method according to claim 7 or 8 in plant functional genomics research or crop breeding.
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