Application of GhSKS13 protein in cotton disease resistance

Abstract

The present invention discloses the application of GhSKS13 protein of upland cotton in cotton disease resistance. The present invention relates to the field of botany, and in particular to the application of GhSKS13 protein of upland cotton in cotton disease resistance. The protein GhSKS13 of the present invention is a protein with an amino acid sequence of SEQ ID No. 2, which is a protein that positively regulates cotton to resist Verticillium dahliae infection, can be used to regulate plant disease resistance, cultivate plants with altered disease resistance, and also has broad application prospects in the field of plant breeding.

Description

Technical Field

[0001] The present invention relates to the field of botany, and in particular to application of upland cotton GhSKS13 protein in cotton disease resistance. Background Art

[0002] Cotton is one of the most important economic crops in the world, providing us with natural fiber raw materials, oil and protein feed. At present, the most important cultivated variety is upland cotton (Gossypium hirsutum). However, cotton is often subjected to various biological and abiotic stresses during production. Cotton Verticillium wilt, mainly caused by Verticillium dahliae, is one of the main factors restricting cotton production, seriously damaging the yield and quality of cotton fiber. Exploring cotton resistance-related genes against V. dahiliae, analyzing its molecular mechanism and breeding disease-resistant varieties through genetic engineering technology is an effective strategy to control cotton Verticillium wilt.

[0003] The protein encoded by the SKS (skewed5 similar) gene belongs to the multi-copper-oxidase-like protein family and was first discovered in Arabidopsis thaliana. There are 19 SKS genes, including SKU5 genes and 18 SKU5-like genes. SKS proteins have a copper-redoxin domain similar to that in laccase and L-ascorbate oxidase, and are highly similar in sequence, but lack several conserved copper-binding motifs unique to copper oxidases, so they may not have the function of copper oxidases. Currently, this type of protein has also been found in plants such as rice, primrose, corn, and tobacco. Studies have found that SKS proteins play an important role in regulating plant growth and development: Jacobs et al. found that Arabidopsis SKS6 is involved in regulating the distribution of cotyledon veins, and the SKS6T-DNA insertion mutant significantly changed the shape of cotyledon veins; Zhou et al. found that Arabidopsis SKS1, SKS2, SKS3 and SKU5 proteins regulate root development by controlling cell polar expansion and cell wall synthesis; another study found that Arabidopsis SKS13 regulates the growth of pollen tubes in female tissues by regulating the biosynthesis of jasmonic acid and modifying cell walls. In SKS13 mutants, the JA content was reduced, the pollen tubes in the pistil grew slowly, and the number of silique seeds was reduced; Duan et al. found that Arabidopsis SKS11 and SKS12 also play an important role in the integrity, growth and guidance of pollen tubes. In recent years, studies have found that the SKS gene family is also involved in the process of plant resistance to biological stress: Chen et al. found that overexpression of the Arabidopsis SKS13 gene can improve the resistance of transgenic Arabidopsis to aphids (Myzus persicae), and the expression levels of disease-related genes in transgenic lines were significantly increased compared with the wild type. So far, research on the SKS gene family is still relatively scarce, and only the functional characteristics of a few SKS family members have been elucidated, while the functions of most SKS family members are still unclear. Summary of the invention

[0004] The technical problem to be solved by the present invention is how to improve the disease resistance of plants.

[0005] In order to solve the problems existing in the prior art, the present invention provides the use of a protein or a substance for regulating the expression of a gene or a substance for regulating the activity or content of the protein in regulating plant disease resistance.

[0006] The use of the protein or gene expression regulating substance provided by the present invention or the substance regulating the activity or content of the protein in any of the following:

[0007] 1) Application of proteins or substances regulating gene expression or substances regulating the activity or content of the proteins in regulating plant disease resistance;

[0008] 2) Use of proteins or substances that regulate the expression of genes or substances that regulate the activity or content of the proteins in the preparation of products that regulate plant disease resistance;

[0009] 3) Use of proteins or substances regulating gene expression or substances regulating the activity or content of the proteins in cultivating plants with altered disease resistance;

[0010] 4) Use of proteins or substances regulating gene expression or substances regulating the activity or content of the proteins in the preparation of products for cultivating plants with altered disease resistance;

[0011] 5) Application of proteins or substances regulating gene expression or substances regulating the activity or content of the proteins in plant breeding;

[0012] The protein is any of the following:

[0013] a1) a protein having an amino acid sequence of SEQ ID No. 2;

[0014] a2) a protein having the same function as the amino acid sequence shown in SEQ ID No. 2 after one or more amino acid residues are replaced and / or deleted and / or added;

[0015] a3) a protein having an amino acid sequence with more than 80% identity with any one of a1) to (a2) and having the same function;

[0016] a4) A fusion protein obtained by connecting a tag to the end of the protein defined in any one of a1) to (a3).

[0017] The name of the protein described in a1) above is SKS13.

[0018] In order to facilitate purification or detection of the protein in a1), a tag protein may be connected to the amino terminus or carboxyl terminus of the protein consisting of the amino acid sequence shown in SEQ ID No. 2 in the sequence listing.

[0019] The above proteins can be artificially synthesized, or their encoding genes can be synthesized first and then expressed biologically.

[0020] The tag protein includes but is not limited to: GST (glutathione sulfhydryltransferase) tag protein, His6 tag protein (His-tag), MBP (maltose binding protein) tag protein, Flag tag protein, SUMO tag protein, HA tag protein, Myc tag protein, eGFP (enhanced green fluorescent protein), eCFP (enhanced cyan fluorescent protein), eYFP (enhanced yellow-green fluorescent protein), mCherry (monomeric red fluorescent protein) or AviTag tag protein.

[0021] A person skilled in the art can easily mutate the nucleotide sequence encoding protein SKS13 of the present invention by using known methods, such as directed evolution or point mutation. Those artificially modified nucleotides having 75% or more identity with the nucleotide sequence of protein SKS13 isolated from the present invention are derived from the nucleotide sequence of the present invention and are equivalent to the sequence of the present invention as long as they encode protein SKS13 and have the function of protein SKS13.

[0022] The aforementioned 75% or more identity may be 80%, 85%, 90% or 95% or more identity.

[0023] Herein, identity refers to the identity of an amino acid sequence or a nucleotide sequence. The identity of an amino acid sequence can be determined using a homology search site on the Internet, such as the BLAST webpage on the NCBI homepage website. For example, in Advanced BLAST2.1, by using blastp as a program, setting the Expect value to 10, setting all Filters to OFF, using BLOSUM62 as a Matrix, setting Gap existence cost, Per residue gap cost and Lambda ratio to 11, 1 and 0.85 (default values) respectively and searching for the identity of a pair of amino acid sequences for calculation, the value (%) of identity can then be obtained.

[0024] Herein, the 80% or greater identity may be at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity.

[0025] Herein, the 90% or greater identity may be at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity.

[0026] In the above application, the protein is derived from upland cotton (Gossypium hirsutum).

[0027] Herein, the substance that regulates the activity and / or content of the protein may be a substance that regulates the expression of a gene encoding the protein SKS13.

[0028] In the above, the substance that regulates gene expression may be a substance that performs at least one of the following six types of regulation: 1) regulation at the transcription level of the gene; 2) regulation after transcription of the gene (that is, regulation of the splicing or processing of the primary transcript of the gene); 3) regulation of RNA transport of the gene (that is, regulation of the transport of the mRNA of the gene from the nucleus to the cytoplasm); 4) regulation of the translation of the gene; 5) regulation of the degradation of the mRNA of the gene; 6) post-translational regulation of the gene (that is, regulation of the activity of the protein translated from the gene).

[0029] In the present invention, the regulation may be up-regulation, enhancement or increase. The regulation may also be down-regulation, attenuation or reduction.

[0030] In the above application, the substance regulating the expression of the gene or the substance regulating the activity or content of the protein may be a biological material related to the protein mentioned above, and the biological material may be any of the following:

[0031] c1) a nucleic acid molecule encoding the protein described above;

[0032] c2) an expression cassette containing the nucleic acid molecule described in c1);

[0033] c3) a recombinant vector containing the nucleic acid molecule described in c1), or a recombinant vector containing the expression cassette described in c2);

[0034] c4) a recombinant microorganism containing the nucleic acid molecule described in c1), or a recombinant microorganism containing the expression cassette described in c2), or a recombinant microorganism containing the recombinant vector described in c3);

[0035] c5) a transgenic plant cell line containing the nucleic acid molecule described in c1), or a transgenic plant cell line containing the expression cassette described in c2);

[0036] c6) transgenic plant tissue containing the nucleic acid molecule described in c1), or transgenic plant tissue containing the expression cassette described in c2);

[0037] c7) a transgenic plant organ containing the nucleic acid molecule described in c1), or a transgenic plant organ containing the expression cassette described in c2);

[0038] e1) a nucleic acid molecule that inhibits, reduces or silences the expression of the protein encoding gene mentioned above;

[0039] e2) an expression cassette containing the nucleic acid molecule described in e1);

[0040] e3) a recombinant vector containing the nucleic acid molecule described in e1), or a recombinant vector containing the expression cassette described in e2);

[0041] e4) a recombinant microorganism containing the nucleic acid molecule described in e1), or a recombinant microorganism containing the expression cassette described in e2), or a recombinant microorganism containing the recombinant vector described in e3);

[0042] e5) a transgenic plant cell line containing the nucleic acid molecule described in e1), or a transgenic plant cell line containing the expression cassette described in e2);

[0043] e6) transgenic plant tissue containing the nucleic acid molecule described in e1), or transgenic plant tissue containing the expression cassette described in e2);

[0044] e7) A transgenic plant organ containing the nucleic acid molecule described in e1), or a transgenic plant organ containing the expression cassette described in e2).

[0045] In the above application, the nucleic acid molecule described in c1) can be any of the following DNA molecules,

[0046] d1) the nucleotide sequence is a DNA molecule shown in SEQ ID NO.3;

[0047] d2) The coding region sequence is the DNA molecule shown in SEQ ID NO.1 in the sequence listing;

[0048] d3) a DNA molecule having 90% or more identity with the nucleotide sequence defined in d1) or d2), derived from cotton and encoding the protein described above;

[0049] d4) A DNA molecule that hybridizes with the nucleotide sequence defined in d1) or d2) under stringent conditions and encodes the protein described above.

[0050] In the above application, the nucleic acid molecule described in e1) may be a DNA molecule whose nucleotide sequence is shown at positions 381-612 of SEQ ID No.1.

[0051] The nucleic acid molecule described herein can be DNA, such as cDNA, genomic DNA or recombinant DNA; the nucleic acid molecule can also be RNA, such as gRNA, mRNA, siRNA, shRNA, sgRNA, miRNA or antisense RNA.

[0052] The vectors described herein are well known to those skilled in the art, including but not limited to: plasmid, phage (such as lambda phage or M13 filamentous phage, etc.), cosmid (i.e., cosmid), Ti plasmid or viral vector. Specifically, it can be vector pYL156 vector.

[0053] The existing plant expression vector can be used to construct a recombinant expression vector containing the SKS13 gene. The plant expression vector includes, but is not limited to, binary Agrobacterium vectors and vectors that can be used for plant microprojectile bombardment. The plant expression vector may also contain the 3' non-translated region of the foreign gene, i.e., a polyadenylic acid signal and any other DNA fragments involved in mRNA processing or gene expression. The polyadenylic acid signal can guide the addition of polyadenylic acid to the 3' end of the mRNA precursor, such as, but not limited to, Agrobacterium crown gall induction (Ti) plasmid genes (such as rouge synthase Nos gene), plant genes (such as soybean storage protein gene) 3' end transcription non-translated regions have similar functions.

[0054] When the SKS13 gene is used to construct a recombinant plant expression vector, any enhanced promoter or constitutive promoter can be added before its transcription initiation nucleotide, including but not limited to the cauliflower mosaic virus (CAMV) 35S promoter and the ubiquitin promoter of corn. They can be used alone or in combination with other plant promoters. In addition, when the gene of the present invention is used to construct a plant expression vector, enhancers can also be used, including translation enhancers or transcription enhancers. These enhancer regions can be ATG start codons or adjacent region start codons, etc., but must be the same as the reading frame of the coding sequence to ensure the correct translation of the entire sequence. The sources of the translation control signal and the start codon are extensive and can be natural or synthetic. The translation initiation region can come from the transcription initiation region or the structural gene.

[0055] In order to facilitate the identification and screening of transgenic plant cells or plants, the plant expression vector used can be processed, such as adding genes that can be expressed in plants and encode enzymes or luminescent compounds that can produce color changes (GUS gene, luciferase gene, etc.), antibiotic markers with resistance (gentamicin marker, kanamycin marker, etc.) or chemical agent resistance marker genes (such as herbicide resistance genes), etc. Considering the safety of transgenic plants, no selective marker genes can be added, and transformed plants can be directly screened by adversity.

[0056] By using any vector that can guide the expression of foreign genes in plants, the SKS13 gene or gene fragment provided by the present invention is introduced into plant cells or recipient plants to obtain transgenic cell lines and transgenic plants with altered disease resistance. The expression vector carrying the SKS13 gene can be transformed into plant cells or tissues by conventional biological methods such as Ti plasmids, Ri plasmids, plant virus vectors, direct DNA transformation, microinjection, electroporation, Agrobacterium-mediated transformation, and the transformed plant tissues can be cultivated into plants.

[0057] Optionally, the expression cassette described in e2) is an expression cassette having a DNA molecule shown at positions 381-612 of SEQ ID No.1, for example, the expression cassette sequence is shown in SEQ ID No.4.

[0058] e2) The recombinant vector described may be a TRV:GhSKS13 vector.

[0059] The structure of the recombinant plasmid TRV:GhSKS13 is described as follows: a recombinant vector obtained by replacing the fragment between the BamHI and KpnI recognition sites of the pYL156 vector sequence with the DNA molecule shown in SEQ ID NO.4 while keeping the other nucleotides of the pYL156 vector unchanged.

[0060] The present invention also provides a method for improving plant disease resistance, which comprises step M, wherein step M is to enhance, increase or up-regulate the activity and / or content of the protein mentioned above in the target plant, or / and enhance, increase or up-regulate the expression level of the gene encoding the protein mentioned above to improve the plant disease resistance.

[0061] The present invention also provides a method for reducing plant disease resistance, which comprises step P, wherein step P is to inhibit, reduce or silence the activity and / or content of the protein mentioned above in the target plant, or / and, inhibit, reduce or silence the expression level of the gene encoding the protein mentioned above, so as to reduce the plant disease resistance.

[0062] In the above method, reducing the expression level and / or activity of the gene encoding the protein SKS13 in the target plant can be achieved by using gene mutation, gene knockout, gene editing or gene knockdown technology to reduce or inactivate the activity of the gene encoding the protein SKS13 in the genome of the target plant.

[0063] The present invention provides a method for cultivating plants with reduced disease resistance, comprising inhibiting or reducing or silencing the expression of the coding gene of the above-mentioned protein and / or the content and / or activity of the above-mentioned protein in the target plant, or / and inhibiting or reducing or silencing the activity and / or content of the coding gene of the above-mentioned protein, to obtain plants with reduced disease resistance.

[0064] In one embodiment of the present invention, the breeding method for cultivating plants with reduced disease resistance comprises the following steps:

[0065] (1) constructing a recombinant expression vector for inhibiting, reducing or silencing the gene encoding the protein described above;

[0066] (2) Transforming the recombinant expression vector constructed in step (1) into a recipient plant (such as a crop or cotton) to obtain a plant having lower disease resistance than the recipient plant.

[0067] In the present invention, the purpose of plant breeding may include cultivating plants with increased disease resistance.

[0068] In the present invention, the disease resistance may be resistance to cotton Verticillium wilt disease.

[0069] The cotton Verticillium wilt disease is caused by Verticillium dahliae.

[0070] The Verticillium dahliae may be Verticillium dahliae V991.

[0071] The proteins and / or biological materials mentioned above also fall within the scope of protection claimed by the present invention.

[0072] In the present invention, the plant may be any of the following:

[0073] N1) Dicotyledonous plants:

[0074] N2) Malvaceae;

[0075] N3) Malvaceae;

[0076] N4) cotton plants;

[0077] N5) Cotton.

[0078] In the above, the cotton may be the upland cotton variety TM-1.

[0079] Cotton is an important economic crop, and the study of cotton SKS family has important theoretical and practical value. This study identified members of the upland cotton SKS family at the whole genome level using bioinformatics methods, systematically analyzed their evolutionary relationships, gene structure, chromosome location, colinearity, and physical and chemical properties, and preliminarily verified the biological function of the Verticillium wilt resistance-related gene GhSKS13 using virus-induced gene silencing technology, laying the foundation for further in-depth research on the functional identification of upland cotton SKS genes and providing candidate genes for upland cotton disease resistance breeding. BRIEF DESCRIPTION OF THE DRAWINGS

[0080] Figure 1 The distribution and colinearity of the SKS gene family in upland cotton on chromosomes.

[0081] Figure 2 The conserved motif (A) and gene structure (B) of SKS family proteins in upland cotton.

[0082] Figure 3Expression pattern analysis of GhSKS13, where A: relative expression level of GhSKS13 in different tissues of upland cotton; B: expression of GhSKS13 in upland cotton roots after V. dahliae treatment.

[0083] Figure 4 The resistance analysis of GhSKS13 silenced plants to V. dahliae, where A: relative expression of GhSKS13 gene in silenced plants 14 days after Agrobacterium infection; BF: disease phenotype, disease index statistics, stem section, relative fungal biomass, and expression of genes related to the course of disease in TRV:00 plants and TRV:GhSKS13 plants 21 days after V. dahliae treatment. **: P<0.01; ***: P<0.001.

[0084] Figure 5 ROS detection in the leaves of TRV:00 and TRV:GhSKS13 plants 6 hours after V.dahliae treatment. DETAILED DESCRIPTION

[0085] The present invention is further described in detail below in conjunction with specific embodiments, and the examples provided are only for illustrating the present invention, rather than for limiting the scope of the present invention. The examples provided below can be used as a guide for further improvements by those of ordinary skill in the art, and do not constitute a limitation of the present invention in any way.

[0086] The experimental methods in the following examples, unless otherwise specified, are all conventional methods, and are performed according to the techniques or conditions described in the literature in the field or according to the product instructions. The materials, reagents, etc. used in the following examples, unless otherwise specified, can all be obtained from commercial channels.

[0087] The quantitative experiments in the following examples were performed in triplicate unless otherwise specified.

[0088] The upland cotton variety TM-1 in the following examples was provided by the Cotton Research Institute of the Chinese Academy of Agricultural Sciences and has been recorded in: Lu Huangjun, Ma Jiazhang. Research on the hybridization of upland cotton standard line TM-1 and diploid cotton [J]. China Cotton, 1990(03):6-7. The public can obtain the biological material from the applicant. The biological material is only used for repeating the experiments of the present invention and cannot be used for other purposes.

[0089] The strong deciduous Verticillium wilt pathogenic strain Verticillium dahliae V991 in the following examples was provided by Researcher Jian Guiliang of the Institute of Plant Protection, Chinese Academy of Agricultural Sciences, and has been recorded in: Zhang Xin, Jian Guiliang, Lin Ling, et al. Molecular detection method for deciduous cotton Verticillium wilt in soil [J]. Journal of Jiangsu Agricultural Sciences, 2011, 27(05): 990-995. The public can obtain the biological material from the applicant. The biological material is only used for repeating the experiments of the present invention and cannot be used for other purposes.

[0090] The virus-induced gene silencing vectors pYL156 and pYL192 in the following examples were provided by Professor Liu Yule of Tsinghua University and have been described in: Liu Y, Schiff M, Marathe R, Dinesh-Kumar SP. Tobacco Rar1, EDS1 and NPR1 / NIM1 like genes are required for N-mediated resistance to tobaccomosaic virus. Plant J. 2002 May; 30(4): 415-29. doi: 10.1046 / j.1365-313x.2002.01297.x. PMID: 12028572. The public can obtain the biological material from the applicant, and the biological material is only used for repeating the experiments of the present invention and cannot be used for other purposes.

[0091] Experimental material processing steps:

[0092] After delinting with concentrated sulfuric acid, the seeds of Upland Cotton TM-1 were soaked in water overnight, transferred to a culture dish, kept moist, and cultured in a 28°C incubator in the dark for 48h-72h. When the embryo germinated to about 2cm, they were planted in a mixture of nutrient soil and vermiculite (mass ratio of 2:1) or Hoagland's nutrient solution, and then transferred to a light incubator. Culture conditions: light 16h / dark 8h, relative humidity 60%, temperature 25°C.

[0093] Take out the Verticillium dahliae V991 stored at -80℃, spread it on a PDA plate, and culture it in a biochemical incubator at 28℃ in the dark for 5d-7d. Pick the germinated hyphae and put them into Czapek medium. Culture it at 28℃ in the dark for about 5d. Filter the bacterial solution with 4-6 layers of gauze, count it with a hemocytometer, and dilute the spore solution to a concentration of 1×10 with distilled water. 6 Pieces / mL.

[0094] Select the two-leaf and one-heart stage hydroponic TM-1 cotton seedlings with the same growth, and add the diluted spore solution of Verticillium dahliae (concentration diluted to 1×10 6 / mL) for 50 min and then put back into the nutrient solution. Using water treatment as control, root samples were taken at 0h, 1h, 3h, 6h, and 12h after treatment, quickly frozen in liquid nitrogen, and then placed at -80℃ for later use.

[0095] Example 1: Bioinformatics analysis and mining of SKS13 protein in upland cotton

[0096] 1. Identification and phylogenetic analysis of SKS gene family members in upland cotton

[0097] To identify the members of the cotton SKS gene family, the genome sequences of four cotton varieties, G. raimondii (JGI), G. arboreum (CRI), G. hirsutum (CRI), and G. barbadense (HAU), and the corresponding GFF3 format annotation files were downloaded from the CottonFGD (http: / / www.cottonfgd.org / ) website and converted into protein sequence files using the TBtools (https: / / github.com / CJ-Chen / TBtools) software. The protein sequences of 19 SKS gene families in Arabidopsis were obtained from the TAIR (http: / / www.arabidopsis.org) website. The protein sequences of the four cotton varieties were searched locally using the BLAST+ toolkit of NCBI (https: / / ftp.ncbi.nlm.nih.gov / blast / executables / blast+ / LATEST / ), and the E value cutoff was set to 1.0×e -5 , and the predicted cotton SKS proteins were obtained. Then these proteins were compared with the Arabidopsis proteome by blastp, and only the results with the most similar sequences of 19 Arabidopsis SKS proteins were retained. In order to further verify these proteins, domain verification was performed in the Pfam database, and only proteins containing three cupredoxin domains (Cu-oxidase, Cu-oxidase_2, Cu-oxidase_3) were retained.

[0098] The SKS gene sequences of Arabidopsis and four cotton species were aligned using the "Align by MUSCLE" software MEGA 11, and then the maximum likelihood method (ML) was selected to construct an evolutionary tree. The "JTT+G" model was selected, and 1000 bootstrap tests were performed. Other parameters used the default parameters. In addition, another neighbor-joining (NJ) tree was constructed in MEGA 11 using the "JTT+G" model and 1000 bootstrap tests as a control to verify the reliability of the ML tree.

[0099] By querying the annotation file in GFF3 format, the location of GhSKS gene family members on chromosomes, gene structure and gene distribution density on chromosomes were obtained. The colinearity between SKS genes was analyzed using the software MCScanX. The 48 GhSKS genes were submitted to the MEME website (http: / / meme-suite.org / ) to analyze the conservative motif. Finally, the above results were visualized using TBtools software. The online tool ProtParam (https: / / web.expasy.org / protparam / ) was used to predict the physicochemical properties of SKS proteins in upland cotton.

[0100] The SKS family genes of upland cotton are distributed on 19 chromosomes, and there are no genes on 7 chromosomes. These SKS genes are mostly concentrated near the ends of the chromosomes, suggesting that these genes have a high genetic exchange rate and belong to an active gene family ( Figure 1 ). In addition, SKS genes are distributed on chromosome A08, but not on chromosome D08, indicating that the genetic evolution of upland cotton from diploid cotton is uneven. MCScan software was used to analyze the collinearity of the SKS gene family in upland cotton, and TBtools software was used to visualize the results. The results showed that except for Gh_A08G092100, other genes had clear collinearity ( Figure 1 The collinearity relationship can be divided into three categories (Table 3): the first category is the whole genome duplication (WGD) between the corresponding genes of chromosomes A and D caused by the chromosome allopolyploidization event of upland cotton, with a total of 20 sets of duplications; the second category is the tandem duplication on the chromosome, that is, the duplication between consecutive genes on the chromosome, with a total of 3 sets; the third category is other duplications, including a total of 25 pairs of duplications. The K of these homologous gene pairs a / K s The values ​​were all less than 0.5, indicating that there was strong purifying selection in the SKS gene family of upland cotton during its evolution.

[0101] The GFF3 annotation file was used to analyze the structure of the SKS gene in upland cotton. The results showed that the SKS genes in Groups 1, 2, 4, and 5 usually have 6-8 introns, but most of the SKS genes in Group 3 contain 2 introns. The two genes with the most introns contain 4 introns, and one gene has no introns (i.e., Gh_A13G237600.1), which is significantly less than the number of introns in genes in other groups ( Figure 2The MEME online tool was used to analyze the conserved motifs in 48 GhSKS genes. The results showed that the GhSKS gene of upland cotton contained 8 motifs, and the motif composition of each member was basically the same ( Figure 2 Middle B). The highly similar motifs in the SKS gene family of upland cotton indicate that the functions of the SKS genes in upland cotton are relatively conservative, and there may be functional redundancy among the members. In addition, Gh_D05G294500.1 has a repeated motif, and the gene structure also suggests that the gene may be formed by the splicing of two consecutive genes.

[0102] Gh_A13G237600.1 belongs to Group 3, with a gene length of 1918bp, of which the CDS fragment length is 1653bp, and it has no introns. Evolutionary analysis showed that this gene was clustered in the same branch as Arabidopsis AtSKS13, and the gene was named GhSKS13 of upland cotton.

[0103] 2. RNA extraction, reverse transcription and qPCR

[0104] Take about 50-100 mg of different tissues of upland cotton (root, stem, and leaf tissues of hydroponic upland cotton in the two-leaf and one-heart period), grind them thoroughly with liquid nitrogen, and extract RNA using the Polysaccharide Polyphenol Plant Total RNA Extraction Kit (Qingke, TSP412). The RNA concentration was determined using NanoDrop 2000 (Thermo Scientific), and then reverse transcription was performed using a reverse transcription kit (Qingke, TSK302S). qPCR reactions were performed using qPCR Mix (Qingke, TSE401). Three technical replicates were set for each sample, and the amplification instrument used was a CFX96 fluorescent quantitative PCR instrument (Bio-Rad). The experimental data was imported into Excel and analyzed using 2 -ΔΔCt Methods Calculate relative gene expression

[15] The ubiquitin protein gene GhUBQ7 of upland cotton was used as an internal reference, and the amplification primers are shown in Table 1.

[0105] Table 1 List of primers used in this study

[0106]

[0107]

[0108] The results showed that the expression level of GhSKS13 was highest in the roots of upland cotton, followed by the stems, and lowest in the leaves, which was about 1 / 4 of the expression level in the roots ( Figure 3 (A).

[0109] To explore the expression pattern of GhSKS13 after infection with V.dahliae, cotton root samples were taken at 0, 1, 3, 6, and 12 h after inoculation with V.dahliae. The qPCR results showed that the expression of GhSKS13 increased after V.dahliae treatment, and was significantly higher than that of the control group after 3 h. The expression reached the highest value at 6 h, which was about 2.5 times that of the control group ( Figure 3 The above results suggest that GhSKS13 may be involved in regulating cotton's resistance to V. dahliae invasion.

[0110] Example 2: Study on the disease resistance of GhSKS13 protein in upland cotton

[0111] 1. Construction of VIGS vector for GhSKS13 target gene

[0112] The function of GhSKS13 was verified by VIGS technology. The coding sequence (CDS) of GhSKS13 gene in upland cotton TM-1 is SEQ ID No.1, and the encoded amino acid sequence is SEQ ID No.2 of GhSKS13 protein. In the genomic DNA of upland cotton TM-1, the genomic sequence encoding GhSKS13 protein is shown in SEQ ID No.3 of the sequence table. The online tool SGN VIGSTool (https: / / vigs.solgenomics.net / ) was used to select specific GhSKS13 gene fragments, and amplification primers were designed according to the GhSKS13 gene. The primers are as follows: V-GhSKS13-F: 5'-CCTCCATGGGGATCCATCCACAGCTTTACATAGGG-3'; V-GhSKS13-R: 5'-CGTGAGCTCGGTACCTTGTTTAACCCCAACTTTAC-3'.

[0113] The fragment was amplified by PCR technology, and the obtained PCR product was sequenced. The PCR product was positions 381-612 of sequence 1 in the sequence list.

[0114] The above PCR fragment was connected to the TRV:00(pYL156) vector by homologous cloning method to obtain the recombinant vector TRV:GhSKS13.

[0115] The structure of the recombinant plasmid TRV:GhSKS13 is described as follows: a recombinant vector obtained by replacing the fragment between the BamHI and KpnI recognition sites of the pYL156 vector sequence with the DNA molecule shown in SEQ ID NO.4 while keeping the other nucleotides of the pYL156 vector unchanged.

[0116] The identified recombinant plasmid TRV:GhSKS13 was transformed into Agrobacterium strain GV3101 by heat shock transformation method to obtain infection buffer of bacterial solution containing TRV:GhSKS13 vector plasmid and Agrobacterium GV3101 containing TRV:GhSKS13 vector plasmid, which were named Agrobacterium tumefaciens GV3101 / TRV:GhSKS13.

[0117] TRV:00 was transformed into Agrobacterium strain GV3101 by heat shock transformation method to obtain infection buffer of bacterial solution containing TRV:00 vector plasmid and Agrobacterium GV3101 containing TRV:00 vector plasmid, which were named Agrobacterium tumefaciens GV3101 / TRV:00.

[0118] TRV:PDS (positive control) was transformed into Agrobacterium strain GV3101 by heat shock transformation method to obtain infection buffer of bacterial solution containing TRV:PDS (positive control) vector plasmid and Agrobacterium GV3101 containing TRV:PDS (positive control) vector plasmid, which were named Agrobacterium tumefaciens GV3101 / TRV:PDS (positive control).

[0119] pYL192 was transformed into Agrobacterium strain GV3101 by heat shock transformation method to obtain infection buffer of bacterial solution containing pYL192 vector plasmid and Agrobacterium GV3101 containing pYL192 vector plasmid, which were named Agrobacterium tumefaciens GV3101 / pYL192.

[0120] 2. Obtaining GhSKS13 gene silenced plants

[0121] When the soil-cultured cotton cotyledons were fully expanded, Agrobacterium GV3101 / TRV:GhSKS13, GV3101 / TRV:00, GV3101 / TRV:PDS (positive control), and GV3101 / pYL192 were cultured overnight, and the recombinant Agrobacterium cells were collected by centrifugation. The bacterial solution OD was adjusted with MMA buffer (10 mM MES [pH 5.6], 10 mM MgCl2, 100 μM acetosyringone). 600to 1.2, and placed at room temperature for 3 hours. Then, the bacterial solutions of Agrobacterium TRV:GhSKS13, GV3101 / TRV:00, and GV3101 / TRV:PDS (positive control) were mixed with the bacterial solution containing the pYL192 vector in a 1:1 ratio, and injected into the lower epidermis of cotton cotyledons after mixing. The injected cotton seedlings were placed in the dark for 12 hours and then transferred to a light incubator for further cultivation to obtain cotton plants TRV:PDS, TRV:00, and TRV:GhSKS13. When the above-mentioned Agrobacterium was injected for 14 days, the cotton plants containing TRV:PDS (positive control) showed obvious albinism, indicating that the VIGS system was working properly. At this time, the second true leaves of TRV:00 and TRV:GhSKS13 were taken to detect the silencing efficiency of the GhSKS13 gene. The results showed that compared with the control group, the expression level of GhSKS13 in the TRV:GhSKS13 silenced plants was downregulated by about 71% ( Figure 4 (A).

[0122] 3. Study on disease resistance of transgenic cotton plants with GhSKS13 gene silenced

[0123] The TRV:00 control plants (referred to as TRV:00) and the TRV:GhSKS13 silenced plants (referred to as TRV:GhSKS13) were treated with V. dahliae spore solution by the root dipping method. After the treatment, the cotton seedlings were returned to the plug tray for further cultivation.

[0124] The DAB staining method was used to detect the burst and accumulation of ROS after cotton inoculation: 6 hours after inoculation, the second true leaves of the GhSKS13 silent plant and the negative control plant were placed in an appropriate amount of DAB staining solution (1 mg / mL) at a pressure of 0.1 Pa, and the vacuum pump was used to evacuate for 15 minutes. Then, the leaves were stained at room temperature and away from light for 12 hours. The DAB staining solution was rinsed with distilled water, and the leaves were placed in 95% ethanol, boiled to remove chlorophyll, and replaced with 95% ethanol. This was repeated three times. Finally, the leaves were placed in 70% glycerol and photographed with a stereo microscope. After 21 days of inoculation with V. dahliae, the disease phenotype was observed, the disease index was calculated, the color of the vascular bundle of the stem segment was observed, the relative fungal biomass and the expression of the disease course-related genes GhPR1 (Gh_D12G2819), GhPR2 (Gh_D06G2277), GhPR3 (Gh_D01G1683), and GhPR5 (Gh_A05G1689) were detected. The amplification primers are shown in Table 1.

[0125] The statistical standards for Verticillium wilt disease grades are:

[0126] Level 1: The plant is healthy, and the cotton leaves have no obvious yellowing or wilting, or the yellowing area of ​​the leaves is less than 10%;

[0127] Level 2: The yellowing degree of cotton plant leaves is greater than 10% and less than 40%;

[0128] Level 3: The yellowing degree of cotton plant leaves is greater than 40% and less than 70%;

[0129] Level 4: The cotton plant leaves turn yellow by more than 70% and even fall off, causing the plant to die.

[0130] Incidence rate (%) = number of diseased plants / total number of plants surveyed × 100%

[0131] Disease index (DI) = ∑ (number of infected plants × disease grade) / (total number of plants ×

[0132] 4) ×100%

[0133] 21 days after inoculation with V. dahliae, the TRV:GhSKS13 silenced plants showed severe leaf yellowing and wilting symptoms, while the TRV:00 control plants showed a milder degree of disease ( Figure 4 In addition, the disease index of TRV:GhSKS13 silenced plants was higher than that of the control group ( Figure 4 C), stem segment color observation revealed that the vascular bundles in the stem segments of TRV:GhSKS13 silenced plants were darker in color ( Figure 4 D), the relative fungal biomass results are consistent with the stem segment color observation results ( Figure 4 Middle E).

[0134] The expression of genes related to the course of disease was detected and it was found that after inoculation with V. dahlliae, the expression levels of genes related to the course of disease, GhPR1, GhPR2, GhPR3, and GhPR5 in TRV:GhSKS13 plants were significantly lower than those in the control group ( Figure 4 The above results indicate that silencing GhSKS13 reduces cotton resistance to V. dahliae, indicating that GhSKS13 is a gene that positively regulates cotton resistance to V. dahliae.

[0135] After 6 hours of V. dahliae treatment, the ROS burst was detected in the true leaves of the TRV:00 control and TRV:GhSKS13 silenced plants. The DAB staining results showed that there were more brown precipitates in the leaves of the control group, and less brown precipitates in the TRV:GhSKS13 silenced plants ( Figure 5 ), indicating that H2O2 changed significantly between the two materials, suggesting that silencing GhSKS13 inhibited the ROS burst induced by V. dahliae.

[0136] The present invention has been described in detail above. For those skilled in the art, without departing from the purpose and scope of the present invention, and without the need to carry out unnecessary experimental conditions, the present invention can be implemented in a wide range under equivalent parameters, concentrations and conditions. Although the present invention provides specific embodiments, it should be understood that the present invention can be further improved. In a word, according to the principles of the present invention, the application is intended to include any changes, uses or improvements to the present invention, including departure from the disclosed scope in the application, and changes made with conventional techniques known in the art.

Claims

1. Application of protein-related biomaterials in any of the following: 1) Application of protein-related biomaterials in reducing plant disease resistance; 2) Application of protein-related biomaterials in the preparation of products that reduce plant disease resistance; 3) Application of protein-related biomaterials in breeding plants with reduced disease resistance; 4) Use of protein-related biomaterials in the preparation of products for breeding plants with reduced disease resistance; The protein is any of the following: a1) a protein having an amino acid sequence of SEQ ID No. 2; a2) A fusion protein obtained by connecting a tag to the end of the protein defined in a1); The biological material is any of the following: e1) a nucleic acid molecule that silences the gene encoding the protein; e2) an expression cassette containing the nucleic acid molecule described in e1); e3) a recombinant vector containing the nucleic acid molecule described in e1), or a recombinant vector containing the expression cassette described in e2); e4) a recombinant microorganism containing the nucleic acid molecule described in e1), or a recombinant microorganism containing the expression cassette described in e2), or a recombinant microorganism containing the recombinant vector described in e3); The plant is cotton; The disease resistance is resistance to cotton Verticillium wilt disease; The cotton wilt disease is caused by Verticillium dahliae ( Verticillium dahliae )cause.

2. The use according to claim 1, characterized in that: The protein encoding gene is a DNA molecule as shown in any of the following, d1) The nucleotide sequence is the DNA molecule shown in SEQ ID NO.3; d2) The coding region sequence is the DNA molecule shown in SEQ ID NO.1 in the sequence listing.

3. A method for reducing plant disease resistance, characterized in that: The method comprises step P, wherein the step P is to silence the gene encoding the protein in claim 1 to reduce the disease resistance of the plant; The plant is cotton; The disease resistance is resistance to cotton Verticillium wilt disease; The cotton wilt disease is caused by Verticillium dahliae ( Verticillium dahliae )cause.

4. A method for breeding plants with reduced disease resistance, characterized in that: A method for silencing a gene encoding the protein of claim 1 in a receptor plant, wherein the plant with reduced disease resistance has lower disease resistance than the receptor plant; The plant is cotton; The disease resistance is resistance to cotton Verticillium wilt disease; The cotton wilt disease is caused by Verticillium dahliae ( Verticillium dahliae )cause.

5. The method according to claim 4, characterized in that: The steps include: (1) constructing a recombinant expression vector for silencing the gene encoding the protein described in claim 1; (2) Transforming the recombinant expression vector constructed in step (1) into a recipient plant to obtain a plant having lower disease resistance than the recipient plant.

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