Gossypium barbadense growth period regulation protein GbCET2 as well as coding gene and application thereof

Abstract

The invention discloses a gossypium barbadense growth period regulatory protein GbCET2 as well as a coding gene and application thereof. The invention belongs to the field of botany, and particularly relates to a gossypium barbadense growth period regulation protein GbCET2 as well as a coding gene and application thereof. The protein GbCET2 or a substance for regulating and controlling the expression of the coding gene of the protein or a substance for regulating and controlling the activity or content of the protein can be applied to any one of the following applications: 1) regulating and controlling the growth period of a plant; 2) preparing a product for regulating and controlling the growth period of plants; (3) cultivating plants with changed growth periods; the expression of the GbCET2 in the late-maturing sea island cotton variety with the long growth period is higher than that of the early-maturing sea island cotton variety with the short growth period, and after the expression of the GbCET2 in the sea island cotton with the long growth period and the expression of the GbCET2 in the sea island cotton with the short growth period are reduced through the virus-induced gene silencing technology, the growth period of the sea island cotton is remarkably shortened.

Description

Technical Field

[0001] This invention belongs to the field of botany, specifically relating to the growth period regulatory protein GbCET2 of sea island cotton and its encoding gene and applications. Background Art

[0002] Sea Island cotton is renowned worldwide for its superior fiber quality. Early maturity was the first trait to be artificially selected during sea island cotton breeding. The growth period, referring to the time from seedling emergence to boll opening, is a key factor determining cotton early maturity. Identifying genes regulating the growth period in sea island cotton and verifying their role in maturity regulation is of great significance for improving cotton variety maturity and creating early-maturing cotton germplasm. Currently, there are very few reports on proteins regulating the growth period in sea island cotton. Summary of the Invention

[0003] The technical problem to be solved by this invention is how to regulate the growth period of plants.

[0004] To address the problems existing in the prior art, the present invention provides a protein.

[0005] The use of the protein, the substance regulating the expression of the gene encoding the protein, or the substance regulating the activity or content of the protein provided by this invention in any of the following: 1) Application in regulating plant growth period; 2) Application in the preparation of products that regulate plant growth period; 3) Application in cultivating plants with altered growth periods; 4) Application in the preparation of products from plants with altered growth periods; 5) Application in plant breeding.

[0006] The protein is any of the following proteins: a1) A protein with the amino acid sequence SEQ ID No:3; a2) A protein having the same function as the amino acid sequence shown in SEQ ID No:3, but with one or more amino acid residues substituted and / or deleted and / or added. Proteins that have more than 80% identity and the same function of any of the amino acid sequences defined in (a3), (a1), and (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 the aforementioned proteins, the 80% or more identity can be at least 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 95%, 96%, 98%, 99%, or 100% identity.

[0010] Of the proteins mentioned above, SEQ ID No. 3 consists of 174 amino acid residues. It is named the GbCET2 protein, and its encoding gene is... GbCET2 Gene.

[0011] In the above applications, the protein is derived from sea island cotton (… Gossypium barbadense L.).

[0012] 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 GbCET2.

[0013] 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).

[0014] In this invention, the regulation can be increased, enhanced, or improved; the regulation can also be decreased, weakened, or reduced.

[0015] 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.

[0016] 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.

[0017] 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, where the Cas9 protein, mediated by RNA, can cleave target sequences recognized by crRNA–tracrRNA.

[0018] 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.

[0019] 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, and the biological material can be any of the following: c1) The nucleic acid molecule that encodes the protein described above; c2) An expression cassette containing the nucleic acid molecule described in c1); c3) A recombinant vector containing the nucleic acid molecule described in c1), or a recombinant vector containing the expression cassette described in c2); c4) Recombinant microorganisms containing the nucleic acid molecules described in c1), or recombinant microorganisms containing the expression cassette described in c2), or recombinant microorganisms containing the recombinant vector described in c3); 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); c6) Transgenic plant tissue containing the nucleic acid molecules described in c1), or transgenic plant tissue containing the expression cassette described in c2); 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); e1) Nucleic acid molecules that inhibit, reduce, or silence the expression of the protein-coding genes mentioned above; 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) Recombinant microorganisms containing the nucleic acid molecules described in e1), or recombinant microorganisms containing the expression cassette described in e2), or recombinant microorganisms containing the recombinant vector described in e3); 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); e6) Transgenic plant tissue containing the nucleic acid molecules described in e1), or transgenic plant tissue containing the expression cassette described in e2); 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).

[0020] In the above applications, c1) the nucleic acid molecule can be any of the following DNA molecules: d1) The nucleotide sequence is the DNA molecule shown in SEQ ID No:2; d2) The coding sequence is the DNA molecule shown in SEQ ID No:1; d3) has 90% or more identity with the nucleotide sequence defined by d1) or d2) and encodes a DNA molecule that encodes the protein described above; d4) A DNA molecule that hybridizes under strict conditions with a nucleotide sequence defined by d1) or d2) and encodes the protein described above.

[0021] In the above applications, the nucleic acid molecule described in e1) can be a DNA molecule with the nucleotide sequence shown in SEQ ID No:2.

[0022] 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.

[0023] The vectors described herein are 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., Cos plasmids), Ti plasmids, or viral vectors.

[0024] Existing plant expression vectors can be used to construct structures containing... GbCET2 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).

[0025] use GbCET2When 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.

[0026] In one specific embodiment, the vector may be the recombinant vector p35S:: GbCET2 - GFP .

[0027] The recombinant vector p35S:: GbCET2 - GFP It is the plasmid p35S:: GFP Restriction endonucleases Kpn I and Xba I. Replace the small fragments between the recognition sequences with the DNA molecule shown in Sequence 1 of the sequence listing (remove the stop codon TGA) while maintaining p35S:: GFP The recombinant expression vector is obtained by keeping the other sequences of the vector unchanged.

[0028] Recombinant plasmid p35S:: GbCET2 - GFP The recombinant plasmid p35S:: expresses the GbCET2 protein shown in sequence 3 of the sequence listing. GbCET2 - GFP An expression cassette containing the GbCET2-GFP fusion protein, in which the expression cassette is activated. GbCET2 The promoter for gene transcription is the 35S promoter.

[0029] The recombinant microorganism may specifically be recombinant Agrobacterium GV3101 / GbCET2-GFP.

[0030] The recombinant Agrobacterium GV3101 / GbCET2-GFP is derived from the recombinant vector p35S:: GbCET2 - GFP Recombinant bacteria obtained by introducing Agrobacterium tumefaciens GV3101.

[0031] 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.

[0032] The present invention also provides a method for altering the growth period of a plant, the method comprising the following steps M or P: Step M involves inhibiting, reducing, or silencing the activity and / or content of the aforementioned protein in the target plant, or / and inhibiting, reducing, or downregulating the expression level of the gene encoding the aforementioned protein, to shorten the plant's growth period; the shortened growth period of the plant is shorter than that of the target plant.

[0033] Step P is to enhance, increase, or upregulate the activity and / or content of the protein described above in the target plant, or / and enhance, increase, or upregulate the expression level of the gene encoding the protein described above, so as to prolong the growth period of the plant; the growth period of the plant with the extended growth period is longer than that of the target plant.

[0034] In the above method, reducing the expression level and / or activity of the gene encoding the protein GbCET2 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 GbCET2 in the genome of the target plant.

[0035] In one specific embodiment, inhibiting, reducing, or silencing 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 a shortened growth period.

[0036] The nucleic acid molecule that is inhibited, reduced, or silenced may be the DNA molecule shown in SEQ ID No:4.

[0037] As a specific embodiment, the recombinant vector is the recombinant vector pCLCrVA- GbCET2 The recombinant vector pCLCrVA- GbCET2 It is the sequence of the pCLCrVA vector (starting vector). Play I and Pac The fragments between the I recognition sites are replaced with the DNA molecule shown in SEQ ID No:4, while keeping the other nucleotides of the pCLCrVA vector (starting vector) unchanged to obtain the recombinant vector.

[0038] In this article, the purpose of breeding includes cultivating plants with shortened growth periods; the purpose of breeding also includes cultivating plants with extended growth periods.

[0039] The present invention also provides a method for shortening the growth period of plants, comprising inhibiting or 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 shortening the growth period of the plant.

[0040] 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.

[0041] The cotton mentioned above may be Sea Island cotton (…). Gossypium barbadense L.) Long-maturing late-maturing variety 9763И and short-maturing early-maturing variety Xinhai 44.

[0042] This invention identified a gene from sea island cotton. GbCET2 In genes GbCET2 The fourth exon contains a SNP site (Gbar_D07_15803388) that is highly associated with reproductive period, and this variation affects the amino acid sequence of the protein encoded by the gene. GbCET2 The expression level was higher in long-maturing late-maturing Sea Island cotton varieties than in short-maturing early-maturing Sea Island cotton varieties. Virus-induced gene silencing (VIGS) technology was used to reduce the expression level in both long-maturing and short-maturing Sea Island cotton varieties. GbCET2 After expression of this gene, the growth period was significantly shortened; at the same time, the flowering time was also significantly advanced. This gene encodes a phosphatidylethanolamine-binding protein, which is located on the cell membrane and nucleus. It is a novel protein that regulates the growth period of sea island cotton and has great theoretical and applied value. Attached Figure Description

[0043] Figure 1 for GbCET2The gene structure and the location of the SNP (Gbar_D07_15803388) significantly associated with reproductive period. Light gray rectangles represent exons, and black horizontal lines represent introns. A black line (black rectangle) within the fourth exon indicates a nonsynonymous SNP (Gbar_D07_15803388), located at the first base of the codon encoding the 113th amino acid. The reference type (Ref) is C, the corresponding codon is CCA, and the translated amino acid is proline (Pro, abbreviated as P); the variant type (Alt) is T, the corresponding codon is TCA, and the translated amino acid is serine (S, abbreviated as S).

[0044] Figure 2 To use qRT-PCR to detect GbCET2 Differences in VIGS expression between the long-maturing late-maturing variety 9763И and the short-maturing early-maturing variety Xinhai 44, and the post-VIGS expression in these two varieties. GbCET2 A bar chart representing the levels. EM_WT, EM_pCLCrVA, and EM_pCLCrVA- GbCET2 These represent the wild-type control, VIGS empty control, and VIGS silent control of the short-growing, early-maturing variety Xinhai 44, respectively. GbCET2 Positive single plants, LM_WT, LM_pCLCrVA and LM_pCLCrVA- GbCET2 These represent the wild-type control, VIGS empty control, and VIGS silent control of the long-maturing late-maturing variety 9763I, respectively. GbCET2 Positive single plants.

[0045] Figure 3 To utilize VIGS silence GbCET2 The growth period of sea island cotton after [the initial growth period]. EM_WT, EM_pCLCrVA and EM_pCLCrVA- GbCET2 These represent the wild-type control, VIGS empty control, and VIGS silent control of the short-growing, early-maturing variety Xinhai 44, respectively. GbCET2 Positive single plants, LM_WT, LM_pCLCrVA and LM_pCLCrVA- GbCET2 These represent the wild-type control, VIGS empty control, and VIGS silent control of the long-maturing late-maturing variety 9763I, respectively. GbCET2 Positive single plants.

[0046] Figure 4 To utilize VIGS silence GbCET2 Flowering time of post-sea island cotton. EM_WT, EM_pCLCrVA and EM_pCLCrVA- GbCET2 These represent the wild-type control, VIGS empty control, and VIGS silent control of the short-growing, early-maturing variety Xinhai 44, respectively. GbCET2Positive single plants, LM_WT, LM_pCLCrVA and LM_pCLCrVA- GbCET2 These represent the wild-type control, VIGS empty control, and VIGS silent control of the long-maturing late-maturing variety 9763I, respectively. GbCET2 Positive single plants.

[0047] Figure 5 Subcellular localization of the GbCET2 protein. Figure a shows the cellular localization of the recombinant plant p35S::GbCET2-GFP carrying the GbCET2-GFP fusion vector; Figure b shows the cellular localization of the recombinant plant p35S::GFP carrying the empty GFP vector, serving as a control. RFP signals indicate the cellular localization of the membrane marker protein CBLn. Detailed Implementation

[0048] 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.

[0049] Unless otherwise specified, the experimental methods in the following examples are conventional methods and were performed according to the techniques or conditions described in the literature in the field or according to the product instructions. The materials and reagents used in the following examples, unless otherwise specified, were all commercially available.

[0050] Unless otherwise specified, the quantitative experiments in the following examples are all repeated three times, and the results are averaged.

[0051] The p35S::GFP plasmid used in the following examples is described in: Zhang W, et al. Natural variations at TIG1 encoding a TCP transcription factor contribute to plant architecture domestication in Rice. Mol. Plant. 2019;12(8):1075-1089. This biological material is available to the public from the applicant and is intended solely for the replication of experiments of this invention and may not be used for any other purpose.

[0052] The recombinant Agrobacterium GV3101 / CBLn-RFP in the following examples is described in: Sun W, et al. Evolution and subfunctionalization of CIPK6Homologous genes in regulating cotton drought resistance. Nat Commun. 2024;15(1):5733. This biological material is available to the public from the applicant and is intended solely for the purpose of replicating experiments of this invention; it may not be used for any other purpose.

[0053] 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.

[0054] The long-growing, late-maturing Sea Island cotton variety 9763И and the short-growing, early-maturing Sea Island cotton variety XH44 (XH44) in the following examples are described in: Zhao N, et al. Genomic and GWAS analyses demonstrate phylogenomic relationships of Gossypium barbadense in China 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 of Benzoic tobacco ( Nicotiana benthamianaThe biological material is described in: Zhang W, et al. Natural variations at TIG1 encoding a TCP transcription factor contribute to plant architecture domestication in Rice. Mol Plant. 2019;12(8):1075-1089. The biological material is available to the public from the applicant and is intended solely for the purpose of repeating experiments of the present invention and may not be used for any other purpose.

[0056] The following examples use IBM SPSS_Statistics_25 statistical software to process the data. The experimental results are expressed as mean values. T-tests are used, and P < 0.05 (*) indicates a significant difference, P < 0.01 (**) indicates a highly significant difference, and P < 0.001 (***) indicates a highly significant difference.

[0057] Example 1: Obtaining genes regulating the growth period of sea island cotton GbCET2 The gene originates from the early-maturing island cotton variety Xinhai 44. GbCET2 The gene coding sequence (CDS) is SEQ ID No:1, the genome sequence is SEQ ID No:2, and the protein it encodes is named GbCET2 protein or protein GbCET2. The amino acid sequence of the encoded protein is shown in SEQ ID No:3.

[0058] The SNP (Gbar_D07_15803388) that is significantly associated with the growth period of sea island cotton is located in the gene GbCET2 Within the fourth exon (specifically located at position 337 of SEQ ID No:1), an amino acid sequence alteration can occur in the protein encoded by this gene. Figure 1 ).when GbCET2 When position 337 of the gene's coding sequence (SEQ ID No:1) is C, position 113 of the encoded GbCET2 protein's amino acid sequence (SEQ ID No:3) is proline (Pro, abbreviated as P); studies have found that when GbCET2 When the coding sequence of the gene (SEQ ID No:1) is T at position 337, the amino acid sequence of the encoded GbCET2 protein (SEQ ID No:3) is serine (S, abbreviated as S) at position 113.

[0059] Among the long-maturing late-maturing Sea Island cotton variety 9763I, GbCET2The genotype at position 337 of the gene's coding sequence (SEQ ID No:1) is C, and the amino acid sequence of the encoded GbCET2 protein (SEQ ID No:3) contains proline (Pro, abbreviated as P) at position 113; in the short-growing, early-maturing island cotton variety Xin Hai 44, GbCET2 The genotype at position 337 of the gene's coding sequence (SEQ ID No:1) is T, and the amino acid sequence of the encoded GbCET2 protein (SEQ ID No:3) contains a serine residue (S). Therefore, it can be inferred that... GbCET2 It is a major gene that regulates the growth period of sea island cotton.

[0060] Example 2 GbCET2 Gene expression differential analysis In fields in Korla, Xinjiang, two cotton varieties were planted: the long-growing, late-maturing island cotton cultivar 9763И and the short-growing, early-maturing island cotton cultivar XinHai 44. Four weeks later, leaves of both varieties were collected, RNA was extracted, and reverse transcriptase was used to synthesize cDNA. Using this cDNA as a template, specific primers were designed (forward primer 5'-ATATCCCCGGCACAACAGATGC-3'; reverse primer 5'-CACTGTTTGCCTGCCTTTTTGC-3'), and detection was performed by real-time PCR. GbCET2 Differences in expression between late-maturing and early-maturing island cotton materials.

[0061] turn out: GbCET2 The expression level in the long-maturing late-maturing island cotton variety 9763I was significantly higher than that in the short-maturing early-maturing island cotton variety Xinhai 44. Figure 2 Therefore, it can be inferred that GbCET2 It is regulating the growth period of island cotton.

[0062] Example 3, VIGS Silence GbCET2 Impact on the growth period of sea island cotton 1. Silence GbCET2 Construction of VIGS recombinant vector and acquisition of recombinant strains According to genes GbCET2 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. Play The I restriction enzyme site (ACTAGT) is specifically defined in the forward primer F as: 5'-GTTGGGGAGAGTGATTGGGG-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'-GCGTCTTCTAGCAGCTGTTTC-3'.

[0063] Recombinant plasmid pCLCrVA- GbCET2 The structure is described as follows: To deliver the pCLCrVA vector sequence Play I and Pac The fragments between the I recognition sites were replaced with the DNA molecule shown in SEQ ID No:4, while keeping the other nucleotides of the pCLCrVA vector unchanged to obtain the recombinant vector.

[0064] Recombinant vector pCLCrVA- GbCET2 After successful construction, the bacteria were heat-shocked and transferred into Agrobacterium EHA105 strain (Sangon Biotech Co., Ltd., catalog number B528432) to obtain a strain containing pCLCrVA- GbCET2 The plasmid of Agrobacterium EHA105 was named Agrobacterium tumefaciens EHA105 / pCLCrVA- GbCET2 .

[0065] The empty vector pCLCrVA was heat-shocked into Agrobacterium EHA105 strain to obtain EHA105 / pCLCrVA as an empty vector control.

[0066] The vector pCLCrVB was heat-shocked into Agrobacterium EHA105 strain to obtain EHA105 / pCLCrVB. Before transformation, it was reacted with EHA105 / pCLCrVB- GbCET2 Equal volumes of Agrobacterium tumefaciens culture of EHA105 / pCLCrVA were mixed.

[0067] 2. GbCET2 Obtaining Silent Island Cotton Plants and Identifying Their Growth Period The long-maturing, late-maturing sea island cotton variety 9763I and the short-maturing, early-maturing variety Xinhai 44 were used as transgenic recipients through soil cultivation. Two weeks later, when the cotyledons were fully expanded, genetic transformation was carried out.

[0068] Take the strain EHA105 / pCLCrVA- obtained in step 1 GbCET2 EHA105 / pCLCrVA and EHA105 / 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 OD. 600 =Approximately 1.0; EHA105 / pCLCrVA- GbCET2EHA105 / pCLCrVA and EHA105 / 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 EHA105 / pCLCrVA and EHA105 / pCLCrVB served as an empty control.

[0069] 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.

[0070] Two weeks later, wild-type control (EM_WT), VIGS empty control (EM_pCLCrVA), and VIGS silent control of the short-growing, early-maturing variety Xinhai 44 were collected. GbCET2 Single plant (EM_pCLCrVA- GbCET2 ) as well as the wild-type control (LM_WT), VIGS empty control (LM_pCLCrVA), and VIGS silent control of the long-maturing late-maturing variety 9763И. GbCET2 Single plant (LM_pCLCrVA- GbCET2 RNA was extracted from leaf samples, reverse transcribed into cDNA, and then the gene was detected using Real-time PCR. GbCET2 The silencing efficiency (forward primer 5'-ATATCCCCGGCACAACAGATGC-3'; reverse primer 5'-CACTGTTTGCCTGCCTTTTTGC-3').

[0071] The results show that after VIGS treatment, EM_pCLCrVA- GbCET2 and LM_pCLCrVA- GbCET2 In the plant GbCET2 Transcription of all samples was significantly suppressed. Figure 2 ); GbCET2 The growth period of silent sea island cotton plants was significantly shorter than that of control plants. Among them, the growth period of long-maturing late-maturing sea island cotton plants (LM_pCLCrVA-) was significantly shorter. GbCET2 The growth period of the early-maturing sea island cotton plants (EM_pCLCrVA-) was shortened by 5 days compared to the control plants (LM_WT and LM_pCLCrVA). GbCET2 The growth period of the EM_WT and EM_pCLCrVA plants was shortened by 2 days compared to the control plants. Figure 3 This confirms that GbCET2 Positive regulation of the growth period of island cotton.

[0072] also, GbCET2The flowering time of the silent late-maturing and early-maturing sea island cotton plants was significantly earlier by 5 days and 2 days, respectively, compared to the control plants. Figure 4 Therefore, it can be inferred that GbCET2 The growth period of island cotton can be regulated by influencing its flowering time.

[0073] Example 4: Subcellular localization of GbCET2 protein according to GbCET2 Based on the CDS sequence, primers were designed, and a 5' end of the forward primer was added. Kpn The I restriction site (GGTACC) has the following nucleotide sequence for the forward primer: 5'-ATGGCAAAACTGTCAGATCCTCTTGT-3'; the reverse primer has the following addition at the 5' end. Xba The I restriction site (TCTAGA) and the reverse primer nucleotide sequence are: 5'-GCGTCTTCTAGCAGCTGTTTCC-3'. Using leaf cDNA from *Cotton Island* as a template, the target sequence was amplified to obtain... GbCET2 The coding sequence of the gene (nucleotide sequence is positions 1 to 522 of SEQ ID No:1 (i.e., excluding the final stop codon TGA).

[0074] Purified GbCET2 The coding sequence of the gene and p35S:: GFP The plasmid DNA was double-digested with enzymes, and the digestion products were then ligated to construct p35S:: GbCET2-GFP The recombinant plasmid was transformed into Agrobacterium GV3101 to obtain recombinant Agrobacterium GV3101 / GbCET2-GFP.

[0075] p35S:: GbCET2-GFP The recombinant plasmid structure is described as follows: The plasmid p35S:: GFP Restriction endonucleases Kpn I and Xba The small fragments between the I-recognition sequences are replaced with the DNA molecules shown in positions 1 to 522 of SEQ ID No:1 in the sequence listing. Recombinant plasmid p35S:: GbCET2-GFP It can express the GbCET2 protein shown in SEQ ID No:3. Recombinant plasmid p35S:: GbCET2-GFP An expression cassette containing the GbCET2-GFP fusion protein, in which the expression cassette is activated. GbCET2 The promoter for gene transcription is the 35S promoter.

[0076] Using the same transformation method, p35S:: GFPThe plasmid was directly transformed into Agrobacterium GV3101 to obtain recombinant Agrobacterium GV3101 / GFP, with recombinant Agrobacterium GV3101 / GFP serving as an empty vector control. Recombinant Agrobacterium GV3101 / CBLn-RFP was used as a membrane marker.

[0077] Positive bacterial suspensions of recombinant Agrobacterium GV3101 / GbCET2-GFP, GV3101 / GFP, and GV3101 / CBLn-RFP were added to YEP liquid medium containing 50 μg / mL Kan and 50 μg / mL Rif, respectively. The strains were activated in a shaker (28℃, 160 rpm). 1 mL of the activated bacterial suspension was added to 20 mL of YEP liquid medium containing 50 μg / mL Kan and 50 μg / mL Rif, and cultured at 28℃, 160 rpm until OD was reached. 600 = 1.0, centrifuge at 5000 rpm for 5 min to collect the bacterial culture, add suspension (containing 10 mM MES, 10 mM MgCl2, pH = 5.2, 100 μM AS) to resuspend the bacterial cells to OD. 600 = 1.0, let stand at room temperature for 2-5 hours.

[0078] Select tobacco plants in good growing condition (Tobacco Benedict) Nicotiana benthamiana ), using a syringe to contain GbCET2-GFP The recombinant Agrobacterium GV3101 / GbCET2-GFP suspension was gently injected onto the underside of tobacco leaves to contain... GFP The empty vector Agrobacterium GV3101 / GFP suspension was used as a control, while the suspension containing... CBLn-RFP Recombinant Agrobacterium GV3101 / CBLn-RFP suspension was used as a membrane marker. Simultaneously, it was injected into the lower epidermis of *Nicotiana benthamiana* leaves. After 24 h of darkness treatment, the cells were cultured under light for 48 h. The subcellular localization of the GbCET2 protein was observed using a Zeiss LSM 900 inverted confocal fluorescence microscope with excitation light at 488 nm and emission light at 510 nm.

[0079] The results are as follows Figure 5 As shown in Figures a and b, the GFP signal of the GbCET2-GFP fusion protein is localized in the cytoplasmic membrane and the nucleus, indicating that GbCET2 plays a role in both the cytoplasmic membrane and the nucleus.

[0080] 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 further improvements can be made to the present invention. In short, according to the principles of the present invention, this application is intended to include any changes, uses or improvements to the present invention, including changes that depart from the disclosed scope in this application and are made using conventional techniques known in the art.

Claims

1. Use of a protein or a substance that regulates the expression of a gene encoding the protein or a substance that regulates the activity or content of the protein in any of the following; 1) Application in regulating plant growth period; 2) Application in the preparation of products for regulating plant growth period; 3) Application in the cultivation of plants with altered growth periods; 4) Use in the preparation of products for cultivating plants with altered growth periods; 5) Application in plant breeding; The protein is any of the following: a1) a protein having an amino acid sequence of SEQ ID No: 3; a2) a protein having the same function as the amino acid sequence shown in SEQ ID No: 3 after one or more amino acid residues are substituted and / or deleted and / or added; a3) a protein with an amino acid sequence identity of more than 80% with that specified in a1) or a2) and having the same function; a4) A fusion protein obtained by connecting a tag to the end of any one of the proteins defined in a1) to a3).

2. The use according to claim 1, characterized in that: The protein is derived from cotton.

3. The use according to claim 1 or 2, characterized in that: The substance that regulates gene expression or the substance that regulates the activity or content of the protein is a biological material related to the protein in the application according to claim 1 or 2, and the biological material is any one of the following: c1) a nucleic acid molecule encoding the protein; c2) an expression cassette containing the nucleic acid molecule described in c1); c3) a recombinant vector containing the nucleic acid molecule described in c1), or a recombinant vector containing the expression cassette described in c2); 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); 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); c6) transgenic plant tissue containing the nucleic acid molecule described in c1) or transgenic plant tissue containing the expression cassette described in c2); 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); e1) a nucleic acid molecule that inhibits, reduces or silences the expression of 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); 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); e6) transgenic plant tissue containing the nucleic acid molecule described in e1), or transgenic plant tissue containing the expression cassette described in e2); 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).

4. The use according to claim 3, characterized in that: c1) The nucleic acid molecule is a DNA molecule as shown below: d1) The nucleotide sequence is a DNA molecule shown in SEQ ID No: 2; d2) the coding sequence is the DNA molecule shown in SEQ ID No: 1; d3) a DNA molecule that has 90% or more identity with the nucleotide sequence defined in d1) or d2) and encodes the protein described in claim 1; d4) A DNA molecule which hybridizes with the nucleotide sequence defined in d1) or d2) under stringent conditions and encodes the protein described in claim 1.

5. A method for changing the growth period of a plant, characterized in that: The method comprises step M or P, wherein step M is to inhibit, reduce or silence the activity and / or content of the protein of claim 1 or 2 in the target plant, or / and, inhibit, reduce or down-regulate the expression of the gene encoding the protein of claim 1 or 2, so as to shorten the growth period of the plant; The method comprises step P, wherein step P is to enhance, increase or up-regulate the activity and / or content of the protein described in claim 1 or 2 in the target plant, or / and enhance, increase or up-regulate the expression level of the gene encoding the protein described in claim 1 or 2 to extend the growth period of the plant.

6. A method for cultivating plants with a shortened growth period, characterized in that: The method comprises inhibiting, reducing or silencing the expression level of the gene encoding the protein of claim 1 or 2 in the target plant, and / or the activity and / or content of the protein to obtain a plant with a shortened growth period, wherein the growth period of the plant with a shortened growth period is shorter than that of the target plant.

7. The method according to claim 6, characterized in that The inhibition, reduction or silencing of the expression of the gene encoding the protein of claim 1 or 2 in the plant comprises introducing the nucleic acid molecule e1) of claim 4, the expression cassette e2) or the recombinant vector e3) into the target plant to obtain a plant with a shortened growth period.

8. A method for cultivating plants with a prolonged growth period, characterized in that: The method comprises enhancing, increasing or up-regulating the expression level of the gene encoding the protein of claim 1 or 2 in the target plant, and / or the activity and / or content of the protein to obtain a plant with a low growth period, and the plant with an extended growth period has a growth period longer than that of the target plant.

9. The protein according to claim 1 or 2 and / or the biomaterial according to claim 3 or 4.

10. The method according to any one of claims 5 to 8, characterized in that: The plant is any of the following: N1) dicotyledonous plants; N2) Malvaceae; N3) Malvaceae; N4) cotton plants; N5) Cotton.

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