Gossypium barbadense regulatory protein GbAGP6 as well as coding gene and application thereof

Abstract

The invention discloses a gossypium barbadense regulatory protein GbAGP6 as well as a coding gene and application thereof. The invention belongs to the field of botany, and particularly relates to a gossypium barbadense regulatory protein GbAGP6 as well as a coding gene and application thereof. The substance for regulating and controlling the expression of the coding gene of the protein GbAGP6 or the substance for regulating and controlling the activity or content of the protein can be applied to any one of the following aspects: 1) regulating and controlling the disease resistance and plant height of plants; 2) preparing a product for regulating the disease resistance and plant height of plants; 3) cultivating plants with changed disease resistance and plant height; the expression of GbAGP6 in susceptible sea island cotton is obviously higher than that of GbAGP6 in disease-resistant sea island cotton, after the expression of GbAGP6 in the susceptible sea island cotton is reduced by using a virus-induced gene silencing technology, the morbidity of the sea island cotton is obviously reduced, the plant height is also obviously increased, and the disease-resistant variety level is achieved.

Description

Technical Field

[0001] This invention belongs to the field of botany, specifically relating to the regulatory protein GbAGP6 of cotton sea island and its encoding gene and applications. Background Technology

[0002] Cotton is an important global economic crop, among which Sea Island cotton ( Gossypium barbadense L. (Bryophyllum oxysporum) is a primary natural fiber source for high-end textiles due to its exceptional fiber quality. However, during its cultivation, a fungus called Fusarium oxysporum (Bryophyllum oxysporum) presents a challenge. Fusarium oxysporum f. sp. vasinfectum Fusarium wilt, a devastating soil-borne vascular disease caused by [unspecified pathogen], severely restricts the growth, development, yield, and quality of island cotton. Currently, the control of this disease mainly relies on chemical pesticides, which are not only costly and environmentally polluting, but also have limited effectiveness. Therefore, exploring the inherent disease-resistant genes in cotton and cultivating new disease-resistant varieties through molecular breeding technology has become the most economical, environmentally friendly, and effective strategy for controlling Fusarium wilt.

[0003] Arabinogalactan proteins (AGPs) are a class of highly glycosylated cell wall-associated proteins widely distributed in plant cell membranes and cell walls. Currently, there are no reports of AGP family members playing a functional role in cotton wilt resistance. Therefore, identifying AGP genes regulating wilt resistance in island cotton is not only of significant theoretical importance but also provides valuable genetic resources for molecular breeding of cotton for disease resistance. Summary of the Invention

[0004] The technical problem to be solved by this invention is how to regulate the disease resistance and plant height of plants.

[0005] To address the problems existing in the prior art, the present invention provides a protein.

[0006] The protein provided by this invention is any of the following proteins: a1) A protein with the amino acid sequence SEQ ID No:1; a2) A protein having the same function as the amino acid sequence shown in SEQ ID No:1, but with one or more amino acid residues substituted and / or deleted and / or added. Proteins that share more than 75% identity with and have the same function of any of the amino acid sequences defined in a3), a1), or a2); The fusion protein is obtained by attaching a tag to the end of any of the proteins defined in a4), a1)-a3).

[0007] In the aforementioned proteins, the protein tag refers to a polypeptide or protein fused with the target protein using in vitro DNA recombination technology for expression, to facilitate the expression, detection, tracing, and / or purification of the target protein. The protein tag may be a Flag tag, His tag, MBP tag, HA tag, myc tag, GST tag, and / or SUMO tag, etc.

[0008] In the above-mentioned proteins, identity refers to the identity of the amino acid sequences. The identity of amino acid sequences can be determined using homology search sites on the Internet, such as the BLAST page on the NCBI homepage. For example, in Advanced BLAST 2.1, using blastp as the program, setting the Expect value to 10, setting all filters to OFF, using BLOSUM62 as the matrix, setting the Gap existence cost, Per residue gap cost, and Lambda ratio to 11, 1, and 0.85 (default values) respectively, and performing an identity search on a pair of amino acid sequences to calculate the identity value (%), then the identity value can be obtained.

[0009] In this article, the 75% or more identity can be 80%, 85%, 90% or 95% or more identity.

[0010] In this document, the 80% or more of identity can 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.

[0011] In this document, the above 90% identity can be at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity.

[0012] Of the proteins mentioned above, SEQ ID No. 1 consists of 166 amino acid residues. It is named GbAGP6 protein, and its encoding gene is... GbAGP6 Gene.

[0013] The protein is derived from sea island cotton ( Gossypium barbadense L.).

[0014] The present invention also provides biomaterials related to the above-mentioned proteins, said biomaterials may be any of the following: B1) Nucleic acid molecules that encode the proteins described above; B2) An expression cassette containing the nucleic acid molecule described in B1); B3) A recombinant vector containing the nucleic acid molecule described in B1), or a recombinant vector containing the expression cassette described in B2); B4) Recombinant microorganisms containing the nucleic acid molecules described in B1), or recombinant microorganisms containing the expression cassette described in B2), or recombinant microorganisms containing the recombinant vector described in B3); B5) A transgenic plant cell line containing the nucleic acid molecule described in B1), or a transgenic plant cell line containing the expression cassette described in B2); B6) Transgenic plant tissue containing the nucleic acid molecules described in B1), or transgenic plant tissue containing the expression cassette described in B2); B7) Transgenic plant organs containing the nucleic acid molecules described in B1), or transgenic plant organs containing the expression cassette described in B2); C1) Nucleic acid molecules that inhibit, reduce, or silence the expression of the genes encoding the proteins described above; C2) expresses the gene encoding the nucleic acid molecule described in C1); C3) contains an expression cassette containing the gene encoding described in C2); C4) A recombinant vector containing the encoding gene described in C2), or a recombinant vector containing the expression cassette described in C3); C5) A recombinant microorganism containing the encoding gene described in C2), or a recombinant microorganism containing the expression cassette described in C3), or a recombinant microorganism containing the recombinant vector described in C4); C6) A transgenic plant cell line containing the encoding gene described in C2), or a transgenic plant cell line containing the expression cassette described in C3), or a transgenic plant cell line containing the recombinant vector described in C4); C7) Transgenic plant tissue containing the encoding gene described in C2), or transgenic plant tissue containing the expression cassette described in C3), or transgenic plant tissue containing the recombinant vector described in C4); C8) A transgenic plant organ containing the encoding gene described in C2), or a transgenic plant organ containing the expression cassette described in C3), or a transgenic plant organ containing the recombinant vector described in C4).

[0015] In the above-mentioned biological materials, the nucleic acid molecule described in B1) may be a cDNA molecule or a DNA molecule whose coding sequence is SEQ ID No:2.

[0016] The DNA molecule shown in SEQ ID No:2 encodes the protein GbAGP6 of SEQ ID No:1.

[0017] The nucleotide sequence shown in SEQ ID No:2 is the nucleotide sequence of the gene encoding protein GbAGP6 (CDS).

[0018] The present inventionGbAGP6 Genes can be any nucleotide sequence that encodes the protein GbAGP6. Considering codon degeneracy and the codon preferences of different species, those skilled in the art can use codons suitable for expression in a specific species as needed.

[0019] B1) The nucleic acid molecule may also include a nucleic acid molecule obtained by codon preference modification based on the nucleotide sequence shown in SEQ ID No:2.

[0020] B1) The nucleic acid molecule may also include nucleic acid molecules that have a nucleotide sequence identity of more than 95% with the nucleotide sequence shown in SEQ ID No:2 and originate from the same species.

[0021] The nucleic acid molecules mentioned in this article can be DNA, such as cDNA, genomic DNA, or recombinant DNA; the nucleic acid molecules can also be RNA, such as gRNA, mRNA, siRNA, shRNA, sgRNA, miRNA, or antisense RNA.

[0022] The vectors described herein are well-known to those skilled in the art and include, but are not limited to: plasmids, bacteriophages (such as λ phage or M13 filamentous phage), granules (i.e., Cosmids), Ti plasmids, or viral vectors. Specifically, it may be the vector pCLCrVA.

[0023] Existing plant expression vectors can be used to construct structures containing... GbAGP6 Recombinant gene expression vectors. These plant expression vectors include, but are not limited to, binary Agrobacterium vectors and vectors suitable for plant microbombardment. The plant expression vectors may also contain the 3' untranslated region of the exogenous gene, i.e., containing a polyadenylate signal and any other DNA fragment involved in mRNA processing or gene expression. The polyadenylate signal can guide the addition of polyadenylate to the 3' end of the mRNA precursor; similar functions exist for the untranslated regions transcribed at the 3' end of genes including, but not limited to, Agrobacterium crown gall-inducing (Ti) plasmid genes (such as the Nos gene for lipase synthesis) and plant genes (such as the soybean storage protein gene).

[0024] use GbAGP6When constructing recombinant plant expression vectors, any type of enhancing promoter or constitutive promoter can be added before the transcription initiation nucleotide, including but not limited to the cauliflower mosaic virus (CAMV) 35S promoter and the maize ubiquitin promoter. These can be used alone or in combination with other plant promoters. Furthermore, when constructing plant expression vectors using the genes of this invention, enhancers, including translational enhancers or transcriptional enhancers, can also be used. These enhancer regions can be ATG start codons or adjacent region start codons, but they must be identical to the reading frame of the coding sequence to ensure correct translation of the entire sequence. The sources of the translation control signals and start codons are wide-ranging; they can be natural or synthetic. The translation initiation region can originate from the transcription initiation region or structural genes.

[0025] To facilitate the identification and screening of transgenic plant cells or plants, the plant expression vectors used can be processed, such as by adding genes that can be expressed in plants, encoding enzymes or luminescent compounds that produce color changes (GUS genes, luciferase genes, etc.), antibiotic resistance markers (gentamicin markers, kanamycin markers, etc.), or chemical reagent resistance marker genes (such as herbicide resistance genes). From a safety perspective, transgenic plants can be screened directly under stress without adding any selective marker genes.

[0026] The present invention also provides the use of a protein, a substance regulating the expression of a gene encoding said protein, or a substance regulating the activity or content of said protein in any of the following: 1) Application in regulating plant disease resistance and plant height; 2) Application in the preparation of products that regulate plant disease resistance and plant height; 3) Application in cultivating disease-resistant plants and altering plant height; 4) Application in the preparation of products that cultivate disease-resistant plants and alter plant height; 5) Application in plant breeding.

[0027] In this article, the substance that regulates the activity and / or content of the protein may be a substance that regulates gene expression, wherein the gene encodes the protein GbAGP6.

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

[0029] In this invention, the regulation can be increased, enhanced, or improved; the regulation can also be decreased, weakened, or reduced.

[0030] In this article, the enhancement, increase, or upregulation of the expression level of the coding gene of the aforementioned protein in the recipient plant, and / or the enhancement, increase, or upregulation of the activity and / or content of the coding gene of the aforementioned protein, is achieved by introducing the coding gene of the aforementioned protein into the recipient plant.

[0031] In this article, regulating the expression of the gene encoding the protein can be achieved by inhibiting, reducing, or downregulating the expression of the gene. Inhibition, reduction, or downregulation of the gene expression can be achieved through gene knockout or gene silencing.

[0032] Gene knockout refers to the phenomenon of inactivating a specific target gene through gene editing technology. Gene knockout inactivates a specific target gene by altering its DNA sequence, including but not limited to zinc-finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), and the CRISPR / Cas system. CRISPR (clustered regulatory interspaced short palindromic repeat) is a site in the genome containing multiple short repeat sequences, and the Cas9 protein can cleave target sequences recognized by crRNA–tracrRNA under RNA-mediated cleavage.

[0033] Gene silencing refers to the phenomenon of preventing or reducing gene expression without damaging the original DNA. Gene silencing presupposes no change in the DNA sequence, resulting in the absence or reduction of gene expression. Gene silencing can occur at two levels: transcriptional silencing due to DNA methylation, heterochromatinization, and position effects; and post-transcriptional gene silencing, which inactivates the gene at the post-transcriptional level through specific inhibition of target RNA. This includes antisense RNA, co-suppression, gene quelling, RNA interference (RNAi), and microRNA (miRNA)-mediated translational repression.

[0034] In the above applications, the substance that regulates the expression of the gene encoding the protein or the substance that regulates the activity or content of the protein can be a biological material related to the protein described above.

[0035] This invention also provides a method for altering plant disease resistance and plant height, the method comprising the following steps M or P: Step M is to inhibit, reduce, or silence the activity and / or content of the proteins mentioned above in the target plant, or / and to inhibit, reduce, or downregulate the expression level of the gene encoding the proteins mentioned above, in order to improve the plant's disease resistance and plant height.

[0036] Step P is to enhance, increase, or upregulate the activity and / or content of the proteins mentioned above in the target plant, or / and to enhance, increase, or upregulate the expression level of the encoding genes of the proteins mentioned above, in order to reduce the plant's disease resistance and plant height.

[0037] In the above method, reducing the expression level and / or activity of the gene encoding the protein GbAGP6 in the target plant can be achieved by using gene mutation, gene knockout, gene editing, or gene knockdown techniques to reduce or inactivate the gene encoding the protein GbAGP6 in the genome of the target plant.

[0038] In one specific embodiment, the inhibition, reduction, or silencing of the expression of the gene encoding the protein described above in the plant includes introducing the nucleic acid molecule, expression cassette, or recombinant vector described above into the target plant to obtain a plant with increased disease resistance and plant height.

[0039] The nucleic acid molecule that is inhibited, reduced, or silenced may be the DNA molecule shown in SEQ ID No:3.

[0040] As a specific embodiment, the recombinant vector is the recombinant vector pCLCrVA- GbAGP6 The recombinant vector pCLCrVA- GbAGP6 It is the sequence of the pCLCrVA vector (starting vector).Spe I and Pac The fragments between the I recognition sites are replaced with the DNA molecule shown in SEQ ID No:3, while keeping the other nucleotides of the pCLCrVA vector (starting vector) unchanged to obtain the recombinant vector.

[0041] In this article, the purpose of breeding includes cultivating plants with increased disease resistance and height; the purpose of breeding also includes cultivating plants with reduced disease resistance and height.

[0042] This invention provides a method for cultivating plants with increased disease resistance and height, comprising inhibiting, reducing, or silencing the expression level of the gene encoding the protein described above in the target plant, and / or the activity and / or content of the protein, thereby increasing the plant's disease resistance and height, wherein the disease resistance and height of the plant with increased disease resistance and height are higher than those of the target plant.

[0043] In the above applications or methods, the plant is any one of the following: N1) Dicotyledons; N2) Malvales (Malva orders) N3) Malvaceae family plants; N4) Plants of the genus *Gossypium*; N5) Cotton.

[0044] The cotton mentioned above may be Sea Island cotton (…). Gossypium barbadense L.).

[0045] This invention identified a gene from sea island cotton. GbAGP6 This gene GbAGP6 The exon contains a SNP site (Gbar_D03_1878030) that is highly associated with the incidence of Fusarium wilt, and this variation affects the amino acid sequence of the protein encoded by the gene. GbAGP6 The gene encodes an arabinogalactosin 6, a novel protein that regulates resistance to Fusarium wilt in sea island cotton. GbAGP6 The expression level was higher in susceptible Sea Island cotton varieties than in resistant varieties. Virus-induced gene silencing (VIGS) technology was used to reduce the expression level in Sea Island cotton. GbAGP6 After expression, the incidence rate decreased significantly and the plant height increased significantly, both reaching the level of disease-resistant varieties. This invention has important theoretical research significance and breeding application value for the study of wilt resistance and plant height of island cotton. Attached Figure Description

[0046] Figure 1 for GbAGP6The gene structure and the location of the SNP (Gbar_D03_1878030) significantly associated with the incidence of Fusarium wilt are shown. The light gray rectangle represents an exon, and a black line inside indicates a nonsynonymous SNP (Gbar_D03_1878030), located at the first base of the codon encoding the 130th amino acid. The reference type (Ref) is T, corresponding to the codon TCG, and the translated amino acid is serine (S). The variant type (Alt) is C, corresponding to the codon CCG, and the translated amino acid is proline (Pro).

[0047] Figure 2 To use qRT-PCR to detect GbAGP6 Differences in expression between disease-resistant and disease-susceptible Sea Island cotton varieties and VIGS expression in disease-susceptible varieties. GbAGP6 A bar chart expressing the levels. R_WT represents the wild-type control of disease-resistant island cotton varieties, and S_WT, S_pCLCrVA, and S_pCLCrVA- GbAGP6 These represent the wild-type control, VIGS empty control, and VIGS silent control of susceptible Sea Island cotton varieties, respectively. GbAGP6 Positive single plant.

[0048] Figure 3 To utilize VIGS silence GbAGP6 Incidence of Fusarium wilt in hindsea cotton (unit: %). R_WT represents the wild-type control of resistant hindsea cotton varieties, S_WT, S_pCLCrVA and S_pCLCrVA- GbAGP6 These represent the wild-type control, VIGS empty control, and VIGS silent control of susceptible Sea Island cotton varieties, respectively. GbAGP6 Positive single plant.

[0049] Figure 4 To utilize VIGS silence GbAGP6 Plant height of the later-developed Sea Island cotton (unit: cm). R_WT represents the wild-type control of the disease-resistant Sea Island cotton variety, S_WT, S_pCLCrVA and S_pCLCrVA- GbAGP6 These represent the wild-type control, VIGS empty control, and VIGS silent control of susceptible Sea Island cotton varieties, respectively. GbAGP6 Positive single plant. Detailed Implementation

[0050] The present invention will now be described in further detail with reference to specific embodiments. The given embodiments are merely illustrative of the invention and not intended to limit its scope. The embodiments provided below can serve as a guide for further improvements by those skilled in the art and do not constitute a limitation on the invention in any way.

[0051] Unless otherwise specified, the experimental methods used in the following examples are conventional methods, performed according to the techniques or conditions described in the literature in this field or according to the product instructions. Unless otherwise specified, the materials and reagents used in the following examples are commercially available.

[0052] Unless otherwise specified, the quantitative experiments in the following examples are all repeated three times, and the results are averaged.

[0053] The sea island cotton varieties described in the following examples are described in: Zhao N, et al. Genomic and GWASanalyses demonstrate phylogenomic relationships of Gossypium barbadense inChina and selection for fiber length, lint percentage and Fusarium wilt resistance. Plant Biotechnol J. 2022;20(4):691-710. The biological material is available to the public from the applicant and is intended solely for the purpose of repeating experiments of this invention and may not be used for any other purpose.

[0054] The pCLCrVA plasmid used in the following examples is described in: Zhao N, et al. Genomic and GWASanalyses demonstrate phylogenomic relationships of Gossypium barbadense inChina and selection for fiber length, lint percentage and Fusarium wilt resistance. Plant Biotechnol J. 2022;20(4):691-710. The biological material is available to the public from the applicant and is intended solely for the purpose of repeating experiments of this invention and may not be used for any other purpose.

[0055] The following examples use statistical software to process the data. The experimental results are expressed as mean values. T-tests are used, with * indicating statistical significance (P < 0.05), ** indicating highly statistical significance (P < 0.01), and *** indicating extremely significant statistical significance (P < 0.001). n.s. This indicates that the difference is not statistically significant.

[0056] Example 1: Obtaining the gene regulating resistance to Fusarium wilt in sea island cotton GbAGP6 The genes are derived from Sea Island cotton. GbAGP6 The gene coding sequence is SEQ ID No:2. GbAGP6 The genome sequence is the same as the coding sequence, and the protein it encodes is named GbAGP6 protein or protein GbAGP6. The amino acid sequence of GbAGP6 protein is shown in SEQ ID No:1.

[0057] The SNP (Gbar_D03_1878030) that is significantly associated with the incidence of Fusarium wilt in sea island cotton is located in the gene. GbAGP6 Within the exon (specifically at position 388 of SEQ ID No:2), it can cause changes in the amino acid sequence of the protein encoded by this gene. Figure 1 ).

[0058] Among the susceptible Sea Island cotton varieties... GbAGP6 The genotype at position 388 of the gene's coding sequence (SEQ ID No:2) is T, and the amino acid sequence of the encoded GbAGP6 protein (SEQ ID No:1) contains a serine residue (S, abbreviated as S) at position 130; among disease-resistant island cotton varieties, GbAGP6 The genotype at position 388 of the gene's coding sequence (SEQ ID No:2) is C, and the amino acid sequence of the encoded GbAGP6 protein is the sequence in SEQ ID No:1 where serine is mutated to proline (Pro, abbreviated as P). Therefore, it is inferred that... GbAGP6 It is a key gene that regulates resistance to Fusarium wilt in island cotton.

[0059] Example 2 GbAGP6 Gene expression differential analysis Disease-resistant and susceptible sea island cotton varieties were planted in fields in Korla, Xinjiang. After four weeks, leaves of both varieties were collected, RNA was extracted, and cDNA was synthesized using reverse transcriptase. Specific primers (forward primer 5'-TCTTATTGTTGTTGCACTTGTT-3'; reverse primer 5'-TATTGACAATGCCAATGATG-3') were designed using real-time PCR for detection. GbAGP6 Differences in expression between disease-resistant and disease-susceptible island cotton varieties.

[0060] turn out: GbAGP6 The expression level in disease-resistant sea island cotton varieties was significantly lower than that in disease-susceptible sea island cotton varieties. Figure 2 Therefore, it can be inferred that GbAGP6 Negative regulation of wilt resistance in island cotton.

[0061] Example 3, VIGS Silence GbAGP6Impact on disease resistance of sea island cotton 1. Silence GbAGP6 Construction of VIGS recombinant vector and acquisition of recombinant strains According to genes GbAGP6 The coding sequence was used to design primers for VIGS vector construction. A 5' extension was added to the 5' end of the forward primer F. Spe The I restriction site (ACTAGT) is specifically defined in the forward primer F as: 5'-TCTTATTGTTGTTGCACTTGTT-3'; the reverse primer R has an additional restriction enzyme at its 5' end. Pac The I restriction site (TTAATTAA) has a specific reverse primer nucleotide sequence of 5'-TATTGACAATGCCAATGATG-3'.

[0062] Recombinant plasmid pCLCrVA- GbAGP6 The structure is described as follows: To deliver the pCLCrVA vector sequence Spe I and Pac The fragments between the I recognition sites were replaced with the DNA molecule shown in SEQ ID No:3, while keeping the other nucleotides of the pCLCrVA vector unchanged to obtain the recombinant vector.

[0063] Recombinant vector pCLCrVA- GbAGP6 After being constructed, it was heat-shocked and transferred into Agrobacterium GV3101 strain to obtain a strain containing pCLCrVA- GbAGP6 The plasmid Agrobacterium GV3101 was named Agrobacterium tumefaciens GV3101 / pCLCrVA- GbAGP6 .

[0064] The empty vector pCLCrVA was heat-shocked into Agrobacterium GV3101 strain to obtain GV3101 / pCLCrVA as an empty vector control.

[0065] The vector pCLCrVB was heat-shocked into Agrobacterium GV3101 strain to obtain GV3101 / pCLCrVB. Before transformation, it was reacted with GV3101 / pCLCrVB- GbAGP6 Equal volumes of Agrobacterium tumefaciens culture of GV3101 / pCLCrVA were mixed.

[0066] 2. GbAGP6 Obtaining Silent Island Cotton Plants and Identifying Disease Resistance and Plant Height Disease-resistant and susceptible Sea Island cotton varieties were grown in soil as transgenic recipients. Two weeks later, the cotyledons were fully expanded, and genetic transformation was carried out.

[0067] Take the strain GV3101 / pCLCrVA- obtained in step 1 GbAGP6GV3101 / pCLCrVA and GV3101 / pCLCrVB were cultured at 28 ℃ to the logarithmic growth phase; the cells were collected by centrifugation at 8000 rpm for 5 min, and then resuspended in VIGS infection solution (10 mM MMES, 200 µM AS, 10 mM MgCl2), and the bacterial concentration was adjusted to OD200. 600 =Approximately 1.0; GV3101 / pCLCrVA- GbAGP6 GV3101 / pCLCrVA and GV3101 / pCLCrVB bacterial solutions were mixed at a volume ratio of 1:1 and allowed to stand at room temperature for 3 hours before being used to transform cotton leaves. The mixed bacterial solution of GV3101 / pCLCrVA and GV3101 / pCLCrVB was used as an empty control.

[0068] Using a 1 mL sterile syringe, draw up the bacterial suspension. First, make a tiny, non-penetrating wound on the underside of the cotyledon with a needle, then inject the inoculated solution using a needleless syringe. Inoculated sea island cotton plants were placed in a 28℃ greenhouse and cultured under a 16 h / 8 h light / dark cycle.

[0069] Two weeks later, wild-type controls of disease-resistant Sea Island cotton varieties (L_WT), wild-type controls of disease-susceptible Sea Island cotton varieties (S_WT), VIGS empty control (S_pCLCrVA), and VIGS silent controls were collected. GbAGP6 Single plant (S_pCLCrVA- GbAGP6 RNA was extracted from leaf samples, reverse transcribed into cDNA, and then the gene was detected using Real-time PCR. GbAGP6 The silencing efficiency (forward primer 5'-TCTTATTGTTGTTGCACTTGTT-3'; reverse primer 5'-TATTGACAATGCCAATGATG-3').

[0070] The results show that: 1) After VIGS treatment, the susceptible Sea Island cotton varieties showed a decrease in... GbAGP6 The expression of [something] was significantly suppressed ( Figure 2 ).

[0071] 2) The incidence of Fusarium wilt in the wild-type (S_WT) variety of susceptible sea-island cotton was significantly higher than that in the wild-type (R_WT) variety of resistant sea-island cotton; silencing infection was observed in susceptible sea-island cotton plants. GbAGP6 (S_pCLCrVA- GbAGP6 After that, the incidence of wilt disease decreased significantly, reaching the level of wild-type (R_WT) resistant island cotton varieties. Figure 3 ); 3) Furthermore, the plant height of the wild-type (S_WT) of the susceptible sea island cotton variety was significantly lower than that of the wild-type (R_WT) of the resistant sea island cotton variety, and the disease was silenced in the susceptible sea island cotton plants. GbAGP6 (S_pCLCrVA- GbAGP6 After that, its plant height also increased significantly, similar to that of the wild-type (R_WT) disease-resistant island cotton variety. Figure 4 ).

[0072] This confirms that GbAGP6 Negative regulation of disease resistance and plant height in island cotton.

[0073] The present invention has been described in detail above. Those skilled in the art will recognize that the invention can be practiced in a wide range of ways with equivalent parameters, concentrations, and conditions without departing from its spirit and scope, and without requiring unnecessary experiments. While specific embodiments have been provided, it should be understood that further modifications can be made to the invention. In summary, according to the principles of the invention, this application is intended to include any changes, uses, or improvements to the invention, including changes made using conventional techniques known in the art that depart from the scope disclosed herein.

Claims

1. A protein, wherein the protein is any of the following: A1) A protein with the amino acid sequence shown in SEQ ID No:1; A2) A protein that has more than 80% identity with and has the same function as the protein shown in A1) obtained by substituting and / or deleting and / or adding amino acid residues of the protein in A1). A3) A fusion protein obtained by attaching a protein tag to the N-terminus and / or C-terminus of A1) or A2).

2. The protein according to claim 1, characterized in that: The protein is derived from ( Gossypium barbadense L.).

3. A biomaterial relating to the protein of claim 1 or 2, wherein the biomaterial is any one of the following: B1) A nucleic acid molecule encoding the protein described in claim 1 or 2; B2) An expression cassette containing the nucleic acid molecule described in B1); B3) A recombinant vector containing the nucleic acid molecule described in B1), or a recombinant vector containing the expression cassette described in B2); B4) Recombinant microorganisms containing the nucleic acid molecules described in B1), or recombinant microorganisms containing the expression cassette described in B2), or recombinant microorganisms containing the recombinant vector described in B3); B5) A transgenic plant cell line containing the nucleic acid molecule described in B1), or a transgenic plant cell line containing the expression cassette described in B2); B6) Transgenic plant tissue containing the nucleic acid molecules described in B1), or transgenic plant tissue containing the expression cassette described in B2); B7) Transgenic plant organs containing the nucleic acid molecules described in B1), or transgenic plant organs containing the expression cassette described in B2); C1) A nucleic acid molecule that inhibits, reduces, or silences the expression of the gene encoding the protein described in claim 1 or 2; C2) expresses the gene encoding the nucleic acid molecule described in C1); C3) contains an expression cassette encoding the gene described in C2); C4) A recombinant vector containing the encoding gene described in C2), or a recombinant vector containing the expression cassette described in C3); C5) A recombinant microorganism containing the encoding gene described in C2), or a recombinant microorganism containing the expression cassette described in C3), or a recombinant microorganism containing the recombinant vector described in C4); C6) A transgenic plant cell line containing the encoding gene described in C2), or a transgenic plant cell line containing the expression cassette described in C3), or a transgenic plant cell line containing the recombinant vector described in C4); C7) Transgenic plant tissue containing the encoding gene described in C2), or transgenic plant tissue containing the expression cassette described in C3), or transgenic plant tissue containing the recombinant vector described in C4); C8) A transgenic plant organ containing the encoding gene described in C2), or a transgenic plant organ containing the expression cassette described in C3), or a transgenic plant organ containing the recombinant vector described in C4).

4. The biomaterial according to claim 3, characterized in that: B1) The nucleic acid molecule is a cDNA molecule or a DNA molecule whose coding sequence is SEQ ID No:

2.

5. The use of the protein or gene expression regulator or substance regulating the activity or content of said protein as described in claim 1 or 2 in any of the following: U1) Application in regulating plant resistance to wilt and plant height; U2) Application in the preparation of products that regulate plant resistance to wilt disease and plant height; U3) Application in cultivating plants with resistance to Fusarium wilt and increased plant height; U4) Application in the preparation of products for cultivating plants resistant to Fusarium wilt and with increased plant height; U5) Applications in plant breeding.

6. The application according to claim 5, characterized in that: The substance that regulates gene expression or the substance that regulates the activity or content of the protein is the biological material described in claim 3 or 4.

7. A method for regulating plant resistance to Fusarium wilt and plant height, characterized in that: This includes regulating the activity and / or content of the protein described in claim 1 or 2 in the recipient plant, and / or the expression level of the gene encoding the protein described in claim 1 or 2, to regulate plant wilt resistance and plant height.

8. The method according to claim 7, characterized in that: The regulation of the activity and / or content of the protein described in claim 1 or 2 in the recipient plant, or / and the expression level of the gene encoding the protein described in claim 1 or 2, includes introducing into the recipient plant an inhibitory, reduced, or silenced gene encoding the protein, to obtain a target plant with higher resistance to wilt disease and higher plant height than the recipient plant; the gene encoding the protein described in claim 1 or 2.

9. Methods for cultivating plants resistant to Fusarium wilt and altering plant height, including: 1) Inhibit or reduce or silence the expression level of the gene encoding the protein described in claim 1 in the receptor plant, and / or inhibit or reduce or silence the activity and / or content of the gene encoding the protein described in claim 1, to obtain plants with increased resistance to wilt disease and increased plant height. 2) Increase, enhance and / or upregulate the expression level of the gene encoding the protein described in claim 1 in the recipient plant, or / and increase, enhance and / or upregulate the activity and / or content of the gene encoding the protein described in claim 1, to obtain plants with wilt resistance and reduced plant height.

10. The application as described in claim 5 or 6, or the method as described in any one of claims 7-9, characterized in that, The plant is any one of the following: N1) Dicotyledons; N2) Malvales (Malva orders) N3) Malvaceae family plants; N4) Plants of the genus *Gossypium*; N5) Cotton.

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