GhSAMDC10 gene and application thereof in regulating and controlling flowering time of upland cotton

Abstract

The invention discloses a GhSAMDC10 gene and application thereof in regulating and controlling the flowering time of upland cotton, and belongs to the technical field of plant genetic engineering. The invention provides the application of the GhSAMDC10 gene in any one of the following applications: the application in regulating and controlling the flowering time of a plant; application in preparation of products for regulating and controlling the flowering time of plants; application in regulation and control of plant floral organ development; application in preparation of products for regulating and controlling development of floral organs of plants; the nucleotide sequence of the GhSAMDC10 gene is as shown in SEQ ID NO. 1 (sequence identifier number 1). Experiments prove that the silent GhSAMDC10 gene leads to significant delay of the flowering period and change of the morphology of the floral organ, which indicates that the GhSAMDC10 gene is capable of regulating the flowering time of the plant and the development of the floral organ, and proves that the GhSAMDC10 gene plays an important role in the flowering development of the plant.

Description

Technical Field

[0001] The present invention relates to the technical field of plant genetic engineering, in particular to a GhSAMDC10 gene and an application thereof in regulating the flowering time of upland cotton. Background Art

[0002] Cotton (Gossypium spp.) is a major cash crop that provides fiber, oil, and plant protein. The cotton-growing areas of Xinjiang and Gansu have a short frost-free period and low effective accumulated temperature, making them suitable only for growing early-maturing cotton varieties. Early maturity is one of the most important traits in cotton breeding in this region, and flowering period is the main component of early maturity. It is closely related to traits such as harvest time, machine-picked cotton quality, and yield. The identification of key genes for flowering period and the analysis of their molecular regulatory mechanisms are the basis and key to the molecular improvement of early-maturing cotton, and have become a hot area of ​​scientific research in Northwest cotton. However, there are currently few studies on the molecular regulatory mechanisms of cotton flowering, and its regulatory mechanism remains unclear, which has restricted the development of molecular breeding for early-maturing cotton.

[0003] S-adenosylmethionine decarboxylase (SAMDC) catalyzes the conversion of S-adenosylmethionine (SAM) to the donor decarboxylated S-adenosylmethionine. This enzyme is the key rate-limiting enzyme in the synthesis of spermine (Spm) and spermidine (Spd), and is also a precursor for ethylene synthesis. Studies have shown that heterologous expression of OsSAMDC in rice can enhance SAMDC activity and lead to the accumulation of Spd and Spm in leaves. Overexpression of the OsSAMDC1 gene is positively correlated with salt tolerance in rice seedlings. Overexpression of this gene increases PA content and salt tolerance in rice. In transgenic Eremochloa ophiuroides (Munro) Hack, overexpression of CdSAMDC1 increases the synthesis of Spd and Spm, leading to increased polyamine oxidase activity, which in turn generates hydrogen peroxide, nitrate reductase activity, and nitric oxide, which in turn increases antioxidant activity. Overexpression of the TrSAMDC1 gene in Trifolium repens L. enhanced salt and drought tolerance in Arabidopsis thaliana. Overexpression of the ZmSAMDC gene significantly improved cold tolerance and yield in maize. Overexpression of the yeast SAMDC gene in tomato significantly increased Spd and Spm levels, enhanced antioxidant enzyme activity, and enhanced high temperature tolerance. Overexpression of SmSAMDC in Salvia miltiorrhiza Bunge improved drought tolerance in transgenic tobacco. These studies demonstrate that SAMDC genes play an important role in regulating plant responses to various abiotic stresses and microbial diseases.

[0004] Research on cotton SAMDC genes has also been reported. Heterologous overexpression of the GhSAMDC gene in Arabidopsis thaliana revealed that Spm synthesis, mediated by SA and leucine-related signals, enhanced resistance to Verticillium wilt. Overexpression of the Saccharomyces cerevisiae SAMDC gene in cotton revealed significantly increased Spm content and drought resistance in the overexpressed strains. Overexpression of the GhSAMDC gene in Arabidopsis thaliana also promoted the transition from vegetative to reproductive growth in cotton, enhancing resistance to stress and Verticillium wilt. However, there are currently no reports on whether GhSAMDC10 regulates flowering time or floral organ development in upland cotton. Summary of the Invention

[0005] The purpose of the present invention is to provide the GhSAMDC10 gene and its application in regulating the flowering time of upland cotton to solve the problems existing in the above-mentioned prior art. Silencing the GhSAMDC10 gene in the present invention leads to a significant delay in the flowering period and changes in the morphology of floral organs, indicating that the GhSAMDC10 gene positively regulates the flowering time and floral organ development of plants, proving the important role of the GhSAMDC10 gene in plant flowering development.

[0006] To achieve the above object, the present invention provides the following solutions:

[0007] The present invention provides the use of the GhSAMDC10 gene in any of the following:

[0008] Application in regulating flowering time of plants;

[0009] Application in the preparation of products for regulating plant flowering time;

[0010] Application in regulating the development of floral organs in plants;

[0011] Application in the preparation of products for regulating the development of plant floral organs;

[0012] The nucleotide sequence of the GhSAMDC10 gene is shown in SEQ ID NO.1.

[0013] The present invention also provides the use of the protein encoded by the GhSAMDC10 gene in any of the following:

[0014] Application in regulating flowering time of plants;

[0015] Application in the preparation of products for regulating plant flowering time;

[0016] Application in regulating the development of floral organs in plants;

[0017] Application in the preparation of products for regulating the development of plant floral organs;

[0018] The nucleotide sequence of the GhSAMDC10 gene is shown in SEQ ID NO.1;

[0019] The amino acid sequence of the protein is shown in SEQ ID NO.2.

[0020] The present invention also provides the use of a recombinant vector containing the GhSAMDC10 gene in any of the following:

[0021] Application in regulating flowering time of plants;

[0022] Application in the preparation of products for regulating plant flowering time;

[0023] Application in regulating the development of floral organs in plants;

[0024] Application in the preparation of products for regulating the development of plant floral organs;

[0025] The nucleotide sequence of the GhSAMDC10 gene is shown in SEQ ID NO.1.

[0026] The present invention also provides the use of a host bacterium containing the recombinant vector in any of the following:

[0027] Application in regulating flowering time of plants;

[0028] Application in the preparation of products for regulating plant flowering time;

[0029] Application in regulating the development of floral organs in plants;

[0030] Application in the preparation of products for regulating the development of plant floral organs.

[0031] Optionally, the regulation is positive regulation.

[0032] Optionally, the GhSAMDC10 gene regulates the flowering time or floral organ development of plants by regulating the synthesis of polyamines.

[0033] Optionally, the plant comprises Gossypium hirsutum.

[0034] The present invention also provides a method for delaying plant flowering, comprising the steps of silencing the GhSAMDC10 gene in the plant to delay flowering time;

[0035] The nucleotide sequence of the GhSAMDC10 gene is shown in SEQ ID NO.1.

[0036] The present invention also provides a method for cultivating a plant with delayed flowering time, comprising the steps of silencing the GhSAMDC10 gene in the plant to obtain a plant with delayed flowering time;

[0037] The nucleotide sequence of the GhSAMDC10 gene is shown in SEQ ID NO.1.

[0038] The present invention also provides a method for cultivating a plant with incomplete floral organ development, comprising the steps of silencing the GhSAMDC10 gene in the plant to obtain the plant with incomplete floral organ development;

[0039] The nucleotide sequence of the GhSAMDC10 gene is shown in SEQ ID NO.1.

[0040] The present invention discloses the following technical effects:

[0041] Experimental verification shows that silencing the GhSAMDC10 gene significantly delays flowering and alters floral organ morphology, demonstrating that the GhSAMDC10 gene positively regulates plant flowering time and floral organ development, confirming its important role in plant flowering and development. Delaying flowering can be used to mitigate adverse environmental stress and ensure stable yields; synchronize crop maturity and optimize mechanized harvesting; prolong vegetative growth and enhance biomass accumulation; and regulate flowering time to meet market demand.

[0042] Furthermore, the present invention further verified that gibberellin levels in young leaves at the stem apex of GhSAMDC10-silenced plants were significantly reduced, significantly delaying flowering. Exogenous application of spermine increased Spm and GA3 levels in GhSAMDC10-silenced plants, leading to earlier flowering. This study reveals that the GhSAMDC10 gene regulates endogenous hormone balance and thus flowering time through the polyamine metabolic pathway. This invention provides important genetic resources and theoretical support for cotton breeding, and has significant scientific significance and application value. BRIEF DESCRIPTION OF THE DRAWINGS

[0043] Figure 1 This is the positive identification of the TRV:GhSAMDC10 recombinant vector; from left to right are the results of target gene amplification, cloning vector bacterial solution PCR, expression vector bacterial solution PCR and expression vector double enzyme digestion;

[0044] Figure 2 For silencing efficiency detection; from left to right: Agrobacterium liquid PCR and target gene silencing efficiency;

[0045] Figure 3 Functional verification of GhSAMDC10; A: Phenotypes of plants expressing the empty vector TRV:00 and TRV:GhSAMDC10 silenced; B: Statistical analysis of bud appearance time; C: Statistical analysis of flowering time; D: Statistical analysis of the height of the first fruiting branch node; E: Statistical analysis of plant height;

[0046] Figure 4Cytological observation of flower buds; A: Detection of GhSAMDC10 silencing efficiency; B: Comparison of leaf morphology of plants silenced by empty vector TRV:00 and TRV:GhSAMDC10; C: Cytological observation of flower bud sections of plants silenced by empty vector TRV:00 and TRV:GhSAMDC10; red arrows indicate flower buds of TRV:00 plants, and blue arrows indicate flower buds of TRV:GhSAMDC10 plants;

[0047] Figure 5 Collinearity analysis; A: Collinearity analysis of SAMDC among three cotton species; B: Interspecific and intraspecific selection pressures of the SAMDC family;

[0048] Figure 6 This is the intraspecific SAMDC collinearity analysis of upland cotton;

[0049] Figure 7 For phylogenetic analysis;

[0050] Figure 8 Subcellular localization of GhSAMDC10 protein; scale bar is 20 μm;

[0051] Figure 9 Analysis of key enzyme activity and content in polyamine synthesis; A: Analysis of key enzyme activity in polyamine synthesis; B: Analysis of substrate and product content in polyamine synthesis;

[0052] Figure 10 Effects of exogenous spraying of Put on flowering and endogenous hormone changes in upland cotton; A: Flowering morphology of upland cotton after exogenous spraying of different concentrations of Put; B: Distribution of budding and flowering phenotypes of upland cotton after exogenous spraying of different concentrations of Put; C: Trends in changes of endogenous hormones (ABA, IAA, 6-BA, and GA3);

[0053] Figure 11 Exogenous spraying of Spm and Spd can promote early flowering of upland cotton and changes in endogenous hormones; A: Flowering morphology of upland cotton after exogenous spraying of different concentrations of Spm and Spd; B: Distribution of bud and flowering phenotypes of upland cotton after exogenous spraying of different concentrations of Spm and Spd; C: Trends in changes in endogenous hormones (ABA, IAA, 6-BA, and GA3);

[0054] Figure 12 Exogenous spraying of Spm significantly increased the endogenous Spm content in silenced plants and advanced the budding and flowering periods; A: Flowering morphology of empty vector and target gene silenced plants after exogenous spraying of Spm and Spd; B: Budding and flowering times of empty vector and target gene silenced plants after exogenous spraying of Spm and Spd; C: Endogenous Spm and Spd contents of empty vector and target gene silenced plants after exogenous spraying of Spm and Spd;

[0055] Figure 13Exogenous spraying of Spm significantly increased the GA3 content in silenced plants; A: Changes in endogenous hormone content in empty vector and target gene silenced plants after exogenous spraying of Spm and Spd; B: Changes in the ratio of endogenous hormone content in empty vector and target gene silenced plants after exogenous spraying of Spm and Spd. DETAILED DESCRIPTION

[0056] Previous studies have shown that the GhSAMDC10 gene may play a role in cotton growth and development. This study, combining bioinformatics analysis with experimental verification, has explored the gene's expression characteristics, functions, and evolutionary relationships. The nucleotide sequence of the GhSAMDC10 gene is shown in SEQ ID NO. 1.

[0057] SEQ ID NO.1:

[0058]

[0059] Among them, capital letters represent open reading frame sequences.

[0060] SEQ ID NO.2 (amino acid sequence encoded by the open reading frame of GhSAMDC10):

[0061] MMRMDMESPLPPSPIGFEGFEKRLEITFFDPPAFNDPNGYGLRALSRAQIDSILEPACCTIVSQISNSNFDSYVLSESSLFIYPNKIILKTCGTTKLLLSIPPILQLSNSISLTVSRVNYSRGSFIFPDHQPSPHRNFSEEVAMLNGYFTDFITEAYIIGNPKLQNRSRHIYSAVPKSSPPFVEDQRAEITLEMCMT GLNREKAGVFYKKSGDENHSAREMTKLSGIADIIPSHVICDFEFDPCGYSMNGIDGLAYSTVHVTPEDGFSYASYEFMGLDLETIKLEPLVKRVLTCFGPKEFSVAITCNGGVPVWFMEVADVEGYTSQYTVKQELPGGGCVVYRTYSSVGERCMVRIPTKLTMQQRCWEAAAEEEEEQEVAGGGAVVCQCISSA.

[0062] Example 1 Identification of GhSAMDC10 gene function

[0063] 1. Experimental Materials and Methods

[0064] 1.1 Plant materials

[0065] The upland cotton variety “Zhongmian 113” and Nicotiana benthamiana L. were both preserved and provided by the laboratory of the Evolutionary and Developmental Biology Team of Gansu Agricultural University.

[0066] 1.2 Test vectors and strains

[0067] The PCloneEZ-TA-AMP cloning vector and Agrobacterium tumefaciens competent vector GV3101 were purchased from Sino-US Taihe Biotechnology (Beijing) Co., Ltd.; the yeast two-hybrid competent vector AH109 was purchased from Beijing Cool Lab; the 207pCAMBIA1305-GFP subcellular localization expression vector was donated by the State Key Laboratory; the pGBKT7, PGADT7-T, and pGBKT7-53 vectors were donated by the research group of Henan Agricultural University; the virus-induced gene silencing (VIGS) system: TRV1 / TRV2 was preserved and provided by the laboratory of the Evolution and Developmental Biology Team of Gansu Agricultural University.

[0068] 1.3 Reagents and consumables

[0069] 2-Morpholineethanesulfonic acid (MES), acetosyringone (ACE), magnesium chloride (MgCl2), and sucrose were purchased from Beijing Solarbio Technology Co., Ltd. Primers designed for synthesis and Sanger sequencing in this study were synthesized at Sangon Biotech (Shanghai) Co., Ltd., and all chemicals used were domestically produced analytical grade.

[0070] 2. Experimental primer design

[0071] The cloning primers, qRT-PCR primers, and VIGS silencing primers (the fragment size of the VIGS silencing primer product is 300-500 bp) of the GhSAMDC10 gene were designed using NCBI Primer-BLAST (https: / / www.ncbi.nlm.nih.gov / tools / primer-blast / ). The primer sequences are shown in Table 1.

[0072] Table 1 List of primers used in the present invention

[0073]

[0074] Note: The bold uppercase letters represent the EcoR I and Kpn I restriction enzyme sites, respectively; the italicized part of the sequence is the vector terminal homology arm sequence, the bold lowercase letters represent the BamH I and Kpn I restriction enzyme sites, respectively; the underlined part represents the specific fragment primer for GhSAMDC10 gene cloning.

[0075] 3. Experimental methods

[0076] 3.1 Agrobacterium-mediated virus-induced gene silencing (VIGS)

[0077] 3.1.1 Construction of GhSAMDC10 expression vector

[0078] The specific sequence in the open reading frame of GhSAMDC10 was cloned and the cloning vector was constructed using PCloneEZ-TA-AMP vector. The primers are shown in Table 1. The plasmid with correct sequencing was named C-GhSAMDC10.

[0079] Using a sequenced positive plasmid as a template, PCR amplification was performed using primers containing restriction enzyme sites (EcoRI and XhoI) and protective bases. The target GhSAMDC10 fragment was inserted into the TRV2 vector by double digestion of the PCR product and the TRV2 vector, creating a TRV:GhSAMDC10 recombinant vector. After positive results were detected, the sequenced recombinant plasmid was transformed into Agrobacterium tumefaciens GV3101. The bacterial suspension was then stored at -80°C using glycerol.

[0080] 3.1.2 Agrobacterium-mediated VIGS

[0081] Conventional methods were used to grow Zhongmian 113. On the seventh day of growth, when the cotyledons had just flattened and the true leaves had not yet emerged, injection was prepared. The above-mentioned preserved bacterial solution was added to LB liquid medium containing antibiotics at a ratio of 1:100 to activate for 12-16 hours. Then, the bacterial solution was expanded at a ratio of 1:10 for 6-8 hours. Centrifuged at 6000 rpm / min for 10 minutes, the cells were retained and the supernatant was discarded. The cells were then resuspended in resuspension buffer (MMA) to an OD of 0. 600 =1.8, the strain containing pTRV1 was mixed with the strain containing pTRV2 or TRV:GhSAMDC10 in a ratio of 1:1, and incubated in the dark at 28°C for 3 h. The mixed Agrobacterium solution was then infiltrated into the back of the cotyledons of the cotton seedlings using the leaf syringe infiltration method to fill the abscess with the solution to more than 95%, producing GhSAMDC10-silenced cotton plants (TRV:GhSAMDC10) and negative (TRV:00) control plants, which were cultured in the dark for 24 h.

[0082] 3.1.3 Silencing efficiency detection

[0083] The first true leaves of TRV:00 and TRV:GhSAMDC10 were collected, quick-frozen in liquid nitrogen, and then stored at -80°C. The expression levels of the target genes were detected by qRT-PCR analysis compared with the control plants to evaluate the silencing efficiency. After two weeks, the plants were transplanted into pots for culture.

[0084] 3.1.4 Flower bud paraffin sections

[0085] Flower bud sections were made, and the differentiation process of cotton apical meristem was observed under an optical microscope. The differentiation period and morphological characteristics of each period after treatment were recorded and photographed.

[0086] 3.1.5 Collinearity Analysis

[0087] The whole-genome sequences and gff3 annotation files for Gossypium hirsutum, Gossypium arboreum, and Gossypium raimondii were downloaded from cottonFGD. CDS sequences were extracted using TBtools. The gff3 files were converted to BED files using Bedtools. Redundant IDs were removed from the BED files using seqkit to obtain a whole-genome non-repeated CDS file and BED file for each species. The JCVI-compara subcommand in the JCVI toolset was used to generate source files. Finally, collinearity plots were drawn using the jcvi.graphics.karyotype module.

[0088] 3.1.6 Phylogenetic tree construction

[0089] To construct a phylogenetic tree, the optimal evolutionary model was selected using the AIC (Akaike Information Criterion) and BIC (Bayesian Information Criterion) methods. Multiple sequence alignments were performed to select candidate evolutionary models. Each model was fitted using the maximum likelihood method (ML), and its log-likelihood value (L) and number of parameters (k) were calculated. The AIC and BIC values ​​for each model were then calculated using the AIC and BIC formulas. Finally, the selected optimal model was used in combination with the maximum likelihood method to construct an evolutionary tree, and the support rate of the tree nodes was evaluated using Bootstrap analysis. After construction, the tree structure can be displayed using visualization tools and biological analysis can be performed.

[0090] 3.2 GhSAMDC10 gene cloning and subcellular localization

[0091] 3.2.1 Construction of pCAMBIA1305-GhSAMDC10-GFP vector

[0092] The CDS region of the GhSAMDC10 gene was amplified using cDNA from leaves of Zhongmian 113 (see Table 1 for primer sequences). The sequenced plasmid was then subcloned into the pCAMBIA1305-GFP vector (see Table 1 for primer sequences). The recombinant vector that successfully sequenced was named pCAMBIA1305-GhSAMDC10-GFP (abbreviated as GhSAMDC10-GFP). The recombinant GhSAMDC10-GFP plasmid was transformed into Agrobacterium tumefaciens GV3101. After positive recombinant plasmid identification, it was mixed with glycerol at a 1:1 ratio and stored frozen at -80°C until further use.

[0093] 3.2.2 Agrobacterium infection of tobacco leaves

[0094] Agrobacterium carrying the GhSAMDC10-GFP recombinant plasmid was used to infect tobacco leaves, and Agrobacterium carrying the pCAMBIA1305-GFP empty vector was used as a control. Use a 2mL centrifuge tube to respectively aspirate 100μL of the empty vector and GhSAMDC10-GFP Agrobacterium culture solution that was frozen at -80℃ and thawed on ice into 1mL LB liquid culture medium, and activate at 28℃, 220r / min for 16h. Take 2.5mL of the activated bacterial solution and add it to 25mL LB liquid culture medium for expansion and culture for 6-8h. Centrifuge at 5000r / min at room temperature for 10min, discard the supernatant, add 1% MS liquid culture medium (add 10mM MES+150μM AS+10mM MgCl2) to resuspend the precipitated bacteria until OD 600 =0.4, and the bacterial solution was placed in the dark for 2 h before infection.

[0095] 3.2.3 Transient expression in tobacco leaves.

[0096] When the tobacco plants are four weeks old, select the second to fourth leaves from the bottom and infect the back of each leaf with the bacterial solution containing the recombinant plasmid or the empty vector using a 1 mL sterile syringe. Mark the injection area on the leaf. Incubate the infected tobacco plants in a dark incubator at 25°C for 36 hours.

[0097] 3.2.4 Observation of fluorescent proteins.

[0098] After culturing for 36 h, the marked infected leaves were cut away from the veins and fixed on a glass slide, and a coverslip was placed on the tobacco leaves. The complete epidermal cell field was observed using a laser confocal scanning microscope, and the distribution of green fluorescence signals was observed under the EGFP channel (excitation wavelength 488 nm).

[0099] 4. Results and Analysis

[0100] 4.1 Functional verification of candidate target genes of transcription factor GhAP1-D3

[0101] To verify the function of GhSAMDC10 in plant growth and development, this example used VIGS technology to perform functional loss analysis on the gene. Figure 1 ).

[0102] Agrobacterium-mediated infection of young cotton cotyledons was used. qRT-PCR was used to detect the expression levels of silenced plants. Compared with the empty vector (TRV:00) plants, the expression levels were all reduced to below 0.4, and the silencing efficiency reached 98%, indicating that the VIGS system effectively inhibited the expression of the target gene and successfully obtained gene-silenced plants ( Figure 2Phenotypic observation results showed that compared with TRV:00 plants, TRV:GhSAMDC10 silenced plants showed significant phenotypic changes ( Figure 3 ), including slower growth rate, delayed flowering, and increased first fruiting branch node height and plant height. The delayed flowering was particularly pronounced. Compared with TRV:00 plants, the bud appearance time and flowering time of silenced plants were delayed by 7.83 days and 6.5 days, respectively, and the first fruiting branch node height and plant height were significantly increased. In addition, GhSAMDC10 silencing also led to significant changes in floral organ morphology, manifested as yellowing of buds and incomplete stamen development. These results indicate that GhSAMDC10 silencing leads to significant phenotypic defects in upland cotton, especially in flowering time and floral organ development, showing obvious functional loss characteristics.

[0103] 4.2 Cytological Observation of GhSAMDC10 in Flower Bud Development

[0104] Comparison of phenotypic traits revealed that the growth of TRV:GhSAMDC10 plants slowed down, the leaves at the same location became smaller, and the development of flower buds was significantly affected, with some flower buds failing to fully differentiate or exhibiting deformities ( Figure 4 Specifically, flower bud development in GhSAMDC10-silenced plants was relatively slow, and the formation of stamens and pistils was significantly inhibited, with some flower buds showing incomplete differentiation or degeneration. At the cellular level, flower bud cells in silenced plants appeared looser and had thinner cell walls than those in the control group, with some cells exhibiting deformities or apoptosis. These results suggest that GhSAMDC10 plays a crucial role in flower bud development and may influence flowering in upland cotton by regulating cell proliferation and the degree of flower bud differentiation.

[0105] 4.3 Collinearity analysis of the evolutionary relationship of SAMDC among different cotton species

[0106] Through multi-genome comparison, it was found that the SAMDC gene has highly conserved homologous copies in tetraploid cotton (upland cotton) and its diploid ancestral species (Asian cotton and Raymond cotton). The results of interspecific synteny analysis showed that the SAMDC gene is highly conserved in the A t Subgenomes and D t Complete homologous gene pairs were retained in the subgenomes, indicating that no obvious gene loss events occurred during cotton polyploidization. t and D t The SAMDC genes in the subgenome showed an asymmetric differentiation pattern during evolution. t The SAMDC genes in the subgenome may dominate in terms of expression level and function ( Figure 5 Middle A).

[0107] Intraspecific synteny revealed that 22 pairs of segmental duplication (WGD) homologous gene pairs were identified in the upland cotton genome. These findings suggest that segmental duplications have dominated gene duplication during evolution ( Figure 6 To explore the functional differentiation and evolutionary selection pressure of the SAMDC family, the non-synonymous substitution rate (Ka), synonymous substitution rate (Ks) and their ratio (Ka / Ks) were calculated. The results showed that there was no significant difference in the Ka / Ks ratio between diploid and tetraploid cotton, and the ratio of all species was less than 1, indicating that these gene sequences are stable during the evolution of this family ( Figure 5 (B) Analysis of Ka / Ks ratios within upland cotton species showed that these ratios were consistently less than 1, with a peak distribution around 0.2. This suggests that the family has a similar and conservative evolutionary pattern, has experienced strong negative selection pressure, and has maintained relative stability in terms of function. These results suggest that SAMDC genes are highly conserved across cotton species, but have also undergone functional divergence during long-term evolution, particularly after polyploidization events, potentially evolving novel regulatory mechanisms in different subgenomes. These findings provide an important theoretical foundation for further understanding the role of SAMDC genes in cotton growth, development, and adaptive evolution.

[0108] 4.4 Phylogenetic analysis of the relationship between GhSAMDC10 in different species

[0109] The results showed that the phylogenetic tree was mainly divided into three branches according to the structure, and the bootstrap values ​​in each branch basically reached 70% or above, indicating that these genes may be highly conserved in function ( Figure 7 ). In addition, the phylogenetic tree also revealed the functional differentiation characteristics of SAMDC genes in the process of plant evolution. For example, the FaSAMDC gene clustered with GhSAMDC10 has been shown to be involved in polyamine metabolism to regulate the early ripening period of strawberry. In addition, wheat TaSAMDC is involved in the S-adenosylmethionine metabolic pathway and plays an important role in drought resistance and water conservation. This result suggests that GhSAMDC10 may have similar functions, especially in the reproductive growth and stress response of cotton. In summary, the phylogenetic analysis based on AIC and BIC model optimization not only clarified the position of GhSAMDC10 in the plant evolutionary tree, but also revealed its functional conservation and differentiation characteristics. These results provide an important basis for in-depth analysis of the biological function of GhSAMDC10 and its role in the adaptive evolution of cotton.

[0110] 4.5GhSAMDC10 fusion protein fluorescence detection

[0111] Laser confocal microscopy showed that the GFP signal of the empty vector was evenly distributed throughout the cell, including the cytoplasm, nucleus and cell membrane, while the fluorescence signal of the GhSAMDC10-GFP fusion protein was mainly concentrated in the nucleus and was also observed in the cell membrane ( Figure 8 These results indicate that the GhSAMDC10 protein is primarily localized in the cell nucleus, a characteristic that suggests the presence of interacting proteins involved in gene transcription regulation. These findings provide important experimental evidence for further analysis of the molecular function of GhSAMDC10 and its mechanism of action in cotton growth and development.

[0112] Example 2 Analysis of the molecular mechanism of GhSAMDC10 regulating early flowering of upland cotton

[0113] Putrescine (Put), spermine (Spm), and spermidine (Spd) used in this example were purchased from solarbio, GA3 standards, IAA standards, 6-BA standards, and ABA standards were purchased from Sigma, the overexpression vectors were M11-1 and pC2300s-HA-Flag, and the knockout vector was M9-2.

[0114] 1. Determination of key enzyme activities for spermine synthesis

[0115] The activities of key enzymes for spermine synthesis in the TRV:00 and TRV:GhSAMDC10 plants in Example 1 were determined.

[0116] The activity assay for spermine synthase (SPMS) is based on the production of S-adenosylmethionine (SAM), using homocysteine ​​and ATP as substrates and SAM detected by a chromogenic reagent. The activity assays for spermidine synthase (SPDS) and S-adenosylmethionine decarboxylase (SAMDC) reflect activity by detecting the release of ammonia during the enzymatic reaction, using SAM as a substrate and a chromogenic reagent. Enzyme solutions of varying concentrations are prepared by dissolving the enzyme in buffer. After mixing with the substrate solution, the reaction is terminated by adding the stop solution. A chromogenic reagent is then added for color development. The absorbance is measured using a microplate reader or spectrophotometer, and the enzyme activity (U / L) is calculated based on a standard curve.

[0117] 2. Exogenous polyamine spraying treatment

[0118] Normal plants, TRV:00 and TRV:GhSAMDC10 plants in Example 1 were treated.

[0119] Upland cotton seedlings grew to the first true leaf and were treated with five concentration gradients (0.5mM, 1mM, 1.5mM, 2mM, 2.5mM) of Put, Spm, and Spd, and the control group (MOCK) was treated with water. Each seedling was sprayed with 1mL, and each mL was sprayed for 2 minutes to prevent water droplets from forming on the leaves. 15 plants were treated for each treatment. After two days of growth in the incubator, they were transplanted into pots. When the plants grew to the three-leaf stage, 0.5g of the stem tip and young leaves were taken and stored at -80℃ for subsequent experiments.

[0120] 3. Determination of endogenous hormone content

[0121] 3.1 Standard curve determination

[0122] Accurately weigh IAA, GA3, 6-BA, and ABA standards and dissolve them in 80% methanol to prepare a 10 μg / mL stock solution. Pipette 250 μL of each hormone stock solution to prepare a mixed standard. Dilute the mixed standard to eight different concentrations of 1, 10, 50, 100, 200, 400, 600, and 800 μg / L. Detect each standard solution sequentially to obtain the corresponding peak area. Plot standard curves for the different hormones using peak area as the ordinate and concentration as the abscissa.

[0123] 3.2 Sample determination

[0124] 0.5 g of cotton tissue was quickly ground into powder using liquid nitrogen using an extract (n-propanol: distilled water: hydrochloric acid = 2:1:0.002), then placed in a 10 mL centrifuge tube and filled to 5 mL. The mixture was then shaken in a refrigerated shaker at 100 rpm and 4 °C for 30 min. Dichloromethane was added and shaken again for 30 min. The extract was centrifuged at 13000 r / min for 5 min. The supernatant was aspirated and placed at -80 °C for 14 h. The extract was dried to powder in a freeze concentrator and then diluted to 1 mL with methanol for reconstitution. The extract was filtered using a 0.22 μm organic microporous filter membrane and then tested on an instrument.

[0125] Chromatographic conditions: Agilent 1290-6460 chromatograph-mass spectrometer (LC-MS; Agilent, USA), chromatographic column: Eclipse plus C18 (2.1 × 50 mm, 1.8 μm); column temperature: 35°C, mobile phase: 0.1% formic acid aqueous solution and methanol; flow rate: 0.3 mL / min; injection volume: 2 μL.

[0126] 4. Genetic modification and gene editing

[0127] Creation of overexpression plants: Overexpression vectors were constructed and genetically transformed into Jin668 (wild type, WT) and Zhong75 materials using tissue culture and shoot apical meristem, respectively.

[0128] Gene-edited plant creation: Select sgRNA sequences with exon-specific editing sites, low off-target rates, and high specificity (Table 4). Construct a CRISPR / Cas9 gene-editing vector. Use Agrobacterium containing positive clones to infect cotton seedling stem tips to generate transgenic plants.

[0129] 5. Identification of transgenic lines

[0130] 5.1 Identification of overexpression-positive plants

[0131] OE strains were screened by PCR using specific primers (Table 3).

[0132] Table 3 Overexpression positive detection primer design

[0133]

[0134] 5.2 Identification of gene-edited positive plants

[0135] Using primers specific for NPTII and Cas9 in the M9-2 vector and designed target detection primers (Table 4), PCR analysis was performed on T0-generation ghsamdc10 mutant lines. Fragments containing the editing site were amplified from genomic DNA of both transgenic lines and WT lines, ligated into the PCloneEZ-TA-AMP cloning vector, and Sanger sequencing was performed to analyze the site of base change. Mutant material with target gene editing was identified, and combined with sequencing screening, natural mutant lines without Cas9 insertions were obtained.

[0136] Table 4. Design of primers for positive gene editing detection

[0137]

[0138] 6 Results and Analysis

[0139] 6.1 Analysis of Key Enzyme Activities and Contents of GhSAMDC10 in the Polyamine Pathway

[0140] In this example, the activities of key enzymes and the contents of their products and substrates in TRV:00 and TRV:GhSAMDC10 plants were determined, and the activities of S-adenosylmethionine decarboxylase (SAMDC), spermidine synthase (SPDS), and spermine synthase (SPMS) as well as the changes in the contents of S-adenosylmethionine (SAM), spermidine (Spd), and spermine (Spm) were analyzed. Figure 9). SAMDC, as a key enzyme in polyamine synthesis, determines the conversion rate of Spd to Spm. The results showed that in GhSAMDC10-silenced plants, the activity of SAMDC was significantly reduced, indicating that the silencing of GhSAMDC10 inhibited the initiation of the polyamine synthesis pathway. SPDS and SPMS are responsible for the synthesis of spermidine and spermine in the polyamine synthesis pathway. In GhSAMDC10-silenced plants, the activity of SPDS and SPMS decreased significantly, further confirming the important role of GhSAMDC10 in the polyamine synthesis pathway. SAM is a precursor substance for polyamine synthesis, and its content directly affects the synthesis rate of polyamines. The results showed that the SAM content in GhSAMDC10-silenced plants was significantly reduced, reflecting that the activity of SAMDC was weakened, thereby affecting the accumulation of SAM. Furthermore, we also analyzed the content of the products Spd and Spm in GhSAMDC10-silenced plants. The results showed that the Spd and Spm contents in TRV:GhSAMDC10 plants were significantly reduced compared with those in TRV:00 plants, indicating the regulatory effect of GhSAMDC10 on polyamine synthesis.

[0141] Based on the above results, GhSAMDC10 regulates the synthesis of SAM by regulating the enzyme activities of SAMDC, SPDS, and SPMS, thereby affecting the synthesis of polyamines and plant growth and development. Plants with GhSAMDC10 silenced showed blocked polyamine synthesis, affecting the growth and development process of plants.

[0142] 6.2 Effects of exogenous polyamine application on plant flowering

[0143] The experimental results showed that polyamine treatment significantly affected the budding and flowering time of plants, and showed a clear concentration dependence. At a concentration of 0.5mM (mmol / L) of Put, the budding and flowering time of plants were delayed by 6.3d and 7d, respectively. As the concentration increased, the delay effect gradually weakened. At 2.5mM, the flowering time was not significantly different from the control ( Figure 10 In contrast, Spd and Spm treatments at 1.5 mM concentration showed significant flowering-promoting effects. Spm treatment advanced the budding and flowering times of plants by 7.0 and 9.8 days, respectively; Spd treatment advanced the budding and flowering times by 8.6 and 10.3 days, respectively. However, when the concentration was increased to 2.5 mM, the budding and flowering times of treated plants were significantly delayed compared with the control group ( Figure 11 This result indicates that there is a concentration threshold effect of Spd and Spm in regulating plant flowering.

[0144] The results showed that 1.5mM Put treatment significantly reduced the GA3 content in plants ( Figure 10C), while the GA3 content was significantly increased after the same concentration of Spm treatment; Spd treatment significantly increased the level of auxin (IAA) ( Figure 11 These findings suggest that exogenous polyamines may affect the flowering process of plants by regulating endogenous hormone metabolic pathways.

[0145] In summary, this study confirms that exogenous polyamine treatment at an appropriate concentration (1.5 mM) can significantly promote plant flowering, with Spd being particularly effective. Furthermore, the changes in endogenous hormone levels induced by polyamine treatment provide important clues to the molecular mechanisms underlying their role in plant flowering regulation. These findings not only deepen our understanding of the physiological functions of polyamines but also provide theoretical support and practical guidance for regulating flowering timing in agricultural production.

[0146] 6.3GhSAMDC10 gene silencing and spermine application restored early flowering

[0147] result( Figure 12 ) found that after exogenous spraying of 1.50 mM concentration of Spm and Spd, the endogenous Spd and Spm contents in the young leaves of the stem tips of TRV:00 plants at the three-leaf stage were significantly increased, and the budding and flowering periods were significantly advanced. For TRV:GhSAMDC10 plants, after exogenous spraying of Spm, the endogenous Spm content increased significantly, the endogenous Spd content did not change significantly, and the budding and flowering periods were significantly advanced; after exogenous spraying of Spd, the endogenous Spd content increased significantly, and the endogenous Spm content did not differ significantly, the budding period was significantly advanced, and the flowering period did not differ significantly. Exogenous spraying of Spd can enable the higher Spd content in TRV:00 plants to be quickly converted into Spm, thereby promoting early flowering of cotton; while silencing GhSAMDC10 reduced the synthesis efficiency of Spd and Spm, and exogenous spraying of Spd made it difficult to quickly synthesize Spm, so it failed to significantly affect the flowering period of upland cotton. The above research results show that increased endogenous Spm content can promote early flowering of upland cotton, while increased endogenous Spd content fails to promote early flowering.

[0148] Effects of exogenous spermine on GA3 content after 6.4GhSAMDC10 gene silencing

[0149] Compared with TRV:00 plants, GhSAMDC10-silenced plants showed significantly decreased endogenous GA3, IAA, and 6-BA contents, while significantly increased ABA content. Hormone ratio analysis showed that the GA3 / IAA, (GA3+IAA) / ABA, and (GA3+IAA+6-BA) / ABA ratios in silenced plants decreased by 38.5%, 38.2%, and 40%, respectively, while the 6-BA / IAA ratio remained unchanged. Following application of Spm, GA3 and ABA contents in TRV:00 plants increased significantly, while IAA content decreased and 6-BA content remained unchanged. However, GhSAMDC10-silenced plants showed significantly increased GA3 and IAA contents, decreased ABA content, and no significant change in 6-BA content compared to MOCK plants. Hormone ratio analysis showed that Spm treatment significantly increased the GA3 / ABA, (GA3+IAA) / ABA, and (GA3+IAA+6-BA) / ABA ratios (by 44.8%, 55%, and 54%, respectively), but had no significant effect on the 6-BA / IAA ratio. Spd application significantly increased the IAA content of GhSAMDC10-silenced plants, but had no significant effect on the GA3 and 6-BA contents. The hormone ratio effect was weakened compared to Spm application ( Figure 13 ).

[0150] In summary, silencing the GhSAMDC10 gene significantly reduced GA3 and IAA content by interfering with the PAs metabolic pathway, while increasing ABA content, resulting in a significant decrease in the GA3 / ABA ratio. Application of Spm and Spd partially restored the endogenous hormone balance in silenced plants, with Spm's regulatory effect on the hormone ratio being more pronounced than that of Spd. GhSAMDC10 primarily regulates flowering in upland cotton by upregulating GA3 content and increasing the GA3 / ABA ratio through mediating Spm synthesis. These findings reveal the molecular mechanism by which the GhSAMDC10 gene regulates endogenous hormone balance through the polyamine metabolic pathway, providing important evidence for a deeper understanding of the role of the polyamine-hormone interaction network in plant growth and development.

[0151] 6.5 Creation of GhSAMDC10 gene mutant and overexpression lines in upland cotton

[0152] To obtain stably transformed lines, a CRISPR / Cas9 gene editing and overexpression vector for the GhSAMDC10 gene was constructed. GhSAMDC10 has an 1176-bp open reading frame (ORF) encoding a protein consisting of 391 amino acids (SEQ ID NO. 2). First, a CRISPR / Cas9 gene editing system was used to create GhSAMDC10 mutants. Using Zhongmian 75 as a template, a pair of sgRNAs was designed. Initially, 25 T0-generation lines were obtained. Based on detection using primers specific for NPTII and Cas9, 16 of these lines were mutants. To determine if there were base changes at the target sites, PCR was performed to confirm positive results. Sanger sequencing was then performed on all 16 lines, revealing that seven of these lines had base mutations and deletions at both target sites. PCR results for both the Cas9 and NPTII genes revealed that one mutant line lacked both genes. Therefore, this line (lacking both Cas9 and NPTII genes) was selected for further studies.

[0153] Secondly, GhSAMDC10 was overexpressed in the late-maturing cotton line Jin668 (wild type, WT) using tissue culture. Initially, 18 T0-generation overexpression lines were obtained. PCR analysis revealed 17 positive plants, one of which showed no positive bands using either primer pair and served as a negative control. Furthermore, GhSAMDC10 was overexpressed in the cotton variety Zhongbo 74 using shoot tip culture, resulting in 10 T0-generation lines. PCR analysis revealed positive bands in five of these lines, with the remaining plants, which did not, serving as negative controls.

[0154] The embodiments described above are merely descriptions of preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Without departing from the spirit of the present invention, various modifications and improvements made to the technical solutions of the present invention by persons skilled in the art should fall within the scope of protection defined by the claims of the present invention.

Claims

1. Use of the GhSAMDC10 gene in any of the following: Application in regulating flowering time of plants; Application in the preparation of products for regulating plant flowering time; Application in regulating the development of floral organs in plants; Application in the preparation of products for regulating the development of plant floral organs; The nucleotide sequence of the GhSAMDC10 gene is shown in SEQ ID NO.

1.

2. Use of the protein encoded by the GhSAMDC10 gene in any of the following: Application in regulating flowering time of plants; Application in the preparation of products for regulating plant flowering time; Application in regulating the development of floral organs in plants; Application in the preparation of products for regulating the development of plant floral organs; The nucleotide sequence of the GhSAMDC10 gene is shown in SEQ ID NO.1; The amino acid sequence of the protein is shown in SEQ ID NO.

2.

3. Use of a recombinant vector containing the GhSAMDC10 gene in any of the following: Application in regulating flowering time of plants; Application in the preparation of products for regulating plant flowering time; Application in regulating the development of floral organs in plants; Application in the preparation of products for regulating the development of plant floral organs; The nucleotide sequence of the GhSAMDC10 gene is shown in SEQ ID NO.

1.

4. Use of a host bacterium containing the recombinant vector according to claim 3 in any of the following: Application in regulating flowering time of plants; Application in the preparation of products for regulating plant flowering time; Application in regulating the development of floral organs in plants; Application in the preparation of products for regulating the development of plant floral organs.

5. The use according to any one of claims 1 to 4, characterized in that The regulation is positive regulation.

6. The use according to any one of claims 1 to 4, characterized in that The GhSAMDC10 gene regulates the flowering time or floral organ development of plants by regulating the synthesis of polyamines.

7. The use according to any one of claims 1 to 4, characterized in that The plants include Gossypium hirsutum.

8. A method for delaying flowering of a plant, characterized in that: including the steps of silencing the GhSAMDC10 gene in plants to delay flowering time; The nucleotide sequence of the GhSAMDC10 gene is shown in SEQ ID NO.

1.

9. A method for cultivating a plant with delayed flowering time, characterized in that: The method comprises the steps of silencing the GhSAMDC10 gene in a plant to obtain a plant with delayed flowering time; The nucleotide sequence of the GhSAMDC10 gene is shown in SEQ ID NO.

1.

10. A method for cultivating a plant with incomplete floral organ development, characterized in that: The method comprises the steps of silencing the GhSAMDC10 gene in plants to obtain plants with incomplete floral organ development; The nucleotide sequence of the GhSAMDC10 gene is shown in SEQ ID NO.1.

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