Application of cotton GhD05G2577 gene in breeding for increasing plant germination rate, biomass, seed size and thousand seed weight

Abstract

The invention provides a breeding application of a cotton GhD05G2577 gene in increasing the plant germination rate, the biomass, the seed size and the thousand seed weight. Through a transgenic technology, an arabidopsis thaliana homozygous line plant over-expressing the GhD05G2577 gene is obtained. Compared with a wild strain, the early germination rate of a transgenic plant in a 1 / 2 MS plate is obviously increased; in the seedling stage, the leaf number is increased, the leaf length and the leaf width are increased, the plant height is increased, the chlorophyll content SPAD is increased, and the fresh weight, the dry matter content and the biomass accumulation are remarkably increased; the fruit shows that the legume length and width are increased, the seed length and width are increased, and the thousand seed weight is increased, and all reach a significant level. When the gene is applied to high-yield and income-increasing breeding of plants, the germination rate of the plants can be increased, biomass accumulation of the plants can be increased, the seed size and thousand seed weight can be increased, and an alternative scheme is effectively provided for high-yield molecular breeding of the plants.

Description

Application of the cotton Gh_D05G2577 gene in breeding to increase plant germination rate, biomass, seed size, and thousand-seed weight. Technical Field

[0001] This invention belongs to the field of molecular biology and plant breeding technology, specifically relating to the application of the cotton Gh_D05G2577 gene in breeding to increase plant germination rate, biomass, seed size and thousand-seed weight. Background Technology

[0002] Cotton is one of the world's most important fiber, economic, and energy crops. In my country, the center of cotton production has shifted westward to the inland cotton-growing northwest region, represented by Xinjiang. Xinjiang is arid with little rainfall and severe soil salinization, which seriously affects early germination rate, biomass accumulation, seed size, and thousand-seed weight in cotton. Therefore, identifying genes that can improve germination rate, biomass accumulation, seed size, and thousand-seed weight is of great significance.

[0003] Plant germination rate, biomass, seed size, and thousand-seed weight are all key traits of concern in agricultural production. Increased germination rate improves early plant survival rates; increased biomass accumulation enhances plant tolerance to salinity and drought, increasing the yield of forage crops; and seed size and thousand-seed weight increase grain yield. Larger seeds in oilseed crops such as rapeseed, soybeans, and cotton increase oil production, making these traits crucial for promoting agricultural production. Plants are often affected by harsh environments such as drought, salt stress, and pests and diseases. Therefore, selecting varieties with strong germination rates, biomass, seed size, and thousand-seed weight can effectively improve plant resistance and adaptability, thereby achieving high yields.

[0004] Multiple studies have shown that bZIPs (Basic Leucine Zipper) transcription factors play a major role in seed germination, flower development, and responses to biotic and abiotic stresses. For example, the bZIPs transcription factor ABI5 participates in ABA or stress signaling, regulating seed size and development, seed germination and early seedling growth, as well as responses to abiotic stresses. Therefore, identifying bZIPs-like transcription factors in cotton, discovering genes, biomass, seed size, and thousand-seed weight, and exploring their application methods can help improve crop yield and quality, enhance plant germination rate, and increase resistance to stress. This will provide genetic resources and technical support for breeding new plant varieties with large seed weight and strong stress resistance, and has significant meaning and application value for improving crop agronomic traits and molecular breeding of high-yield and highly stress-resistant crops. Summary of the Invention

[0005] In view of the problems existing in the prior art, the present invention discloses the application of the cotton Gh_D05G2577 gene in breeding to increase plant germination rate, biomass, seed size and thousand-seed weight.

[0006] The technical solution of the present invention is as follows: In a first aspect, the present invention provides a cotton gene Gh_D05G2577, the mRNA nucleotide sequence of which is shown in SEQ ID No.1, the CDS sequence of which is shown in SEQ ID No.2, and the protein sequence of which is shown in SEQ ID No.3.

[0007] Secondly, this invention provides the application of the Gh_D05G2577 gene and biomaterials containing the Gh_D05G2577 gene in regulating the early germination rate of plant seeds.

[0008] As a preferred method, overexpression of the Gh_D05G2577 gene in plants significantly increases the early germination rate by more than 100%.

[0009] Thirdly, this invention provides the application of the Gh_D05G2577 gene and biomaterials containing the Gh_D05G2577 gene in regulating biomass such as the number of leaves, leaf length and width, plant height, chlorophyll content (SPAD), fresh weight, and dry matter content in plant seedlings.

[0010] Overexpression of the Gh_D05G2577 gene in plants resulted in increased leaf number, leaf length and width, plant height, SPAD chlorophyll content, an average increase of over 40% in fresh weight, an increase of over 30% in dry matter content, and a significant increase in biomass accumulation in transgenic plants during the seedling stage. Silencing the Gh_D05G2577 gene in plants resulted in a decrease in SPAD chlorophyll content.

[0011] Fourthly, this invention provides the application of the Gh_D05G2577 gene and biomaterials containing the Gh_D05G2577 gene in regulating plant seed size and thousand-seed weight.

[0012] By overexpressing the Gh_D05G2577 gene in plants, the length of transgenic plant seeds can be increased by 13%, the width by 9%, and the thousand-seed weight by more than 13%, all of which are significant.

[0013] Fifthly, the present invention provides the application of the gene Gh_D05G2577 in plant breeding, including the following steps: (1) obtaining the Gh_D05G2577 gene and connecting it with the expression vector plasmid to construct a recombinant vector containing Gh_D05G2577; (2) transforming the recombinant vector containing Gh_D05G2577 into competent Agrobacterium tumefaciens cells by freeze-thaw method, and screening to obtain positive transformants; (3) transfecting wild-type plants with bacterial solutions containing positive transformants, harvesting the T0 generation after the crop matures, detecting the gene expression level of positive plants in the T0 generation transgenic plants, and selecting positive transgenic plants with Gh_D05G2577 that are more than 4000 times higher than the control plants for subsequent experiments; (4) performing sexual or asexual reproduction on the transgenic plants, screening and harvesting Gh_D05G2577 homozygous lines with stable traits, and obtaining transgenic plants with increased germination rate and / or increased biomass accumulation and / or increased seed size and thousand-seed weight.

[0014] In the applications described in the second, third, and fourth aspects above, the biological material includes recombinant vectors or recombinant bacteria; in the applications described in the second, third, fourth, and fifth aspects above, the plant includes Arabidopsis thaliana and cotton, but is not limited to Arabidopsis thaliana and cotton.

[0015] Beneficial Effects: This invention identified a cotton Gh_D05G2577 gene, which can increase plant germination rate, biomass, seed size, and thousand-seed weight. This invention also provides the application of the cotton Gh_D05G2577 gene in breeding to increase plant germination rate, biomass, seed size, and thousand-seed weight. The Gh_D05G2577 gene was cloned from the upland cotton standard line TM-1 using PCR technology. Transgenic plants were created using overexpression, and it was found that the germination rate, biomass, seed size, and thousand-seed weight of the transgenic plants were superior to those of the wild-type control plants. Specifically, increasing the expression of Gh_D05G2577 can increase the germination rate, chlorophyll content, biomass accumulation, seed size, and thousand-seed weight of Arabidopsis thaliana, while decreasing the expression of Gh_D05G2577 can decrease the chlorophyll content in cotton. Therefore, the Gh_D05G2577 gene shows promise in increasing plant germination rate, chlorophyll content, biomass accumulation, seed size, and thousand-seed weight, providing an effective alternative for high-yield molecular breeding of plants. It also offers significant reference value for improving crop germination rate, biomass accumulation, and seed yield. The gene, biological materials, and application methods provided by this invention help increase crop yield and improve quality, enhance plant germination rate, and increase resistance to adverse conditions. They provide genetic resources and technical support for breeding new plant varieties with large seed weight and strong stress resistance, and have significant meaning and application value for improving crop agronomic traits and high-yield, highly stress-resistant molecular breeding. Attached Figure Description

[0016] Figure 1. Phylogenetic tree and protein conserved domain analysis of the cotton Gh_D05G2577 gene; (A) Evolutionary analysis of 13 different plants including the Gh_D05G2577 gene. (B) Conserved functional domain analysis of proteins from these 13 species. Gh_D05G2577 (Gossypium hirsutum: XM_041094519.1); GrVIP1 (Gossypium raimondii: XM_018125592.1); EgVIP1 (Eucalyptus grandis: XM_010063826.3); MeVIP1 (Manihot esculenta: XM_021749102.2); QsVIP1 (Quercusrubra: citriodora: XM_030609460.2); CmVIP1(Castanea mollissima: XM_075795124.1); CcVIP1 (Citrus clementina: brasiliensis: XM_058137962.1).

[0017] Figure 2. Screening of Arabidopsis thaliana lines overexpressing the Gh_D05G2577 gene; (A) Screening of positive seedlings in the T0 generation. (B) PCR detection of T0 generation transgenic lines, wells 1-7 are transgenic lines, and well 8 is the wild-type control (WT). (C) qRT-PCR detection of the expression level of Gh_D05G2577 in the T0 generation transgenic lines.

[0018] Figure 3 Germination rate analysis of Arabidopsis thaliana seedlings overexpressing the Gh_D05G2577 gene: (A) Phenotypic diagrams of wild-type plants and transgenic lines after 11 days of growth on normal 1 / 2 MS medium. (B) Statistical analysis of daily germination rate of wild-type plants and transgenic lines during 11 days of growth on normal 1 / 2 MS medium.

[0019] Figure 4. Phenotypic diagrams of Arabidopsis thaliana lines overexpressing the Gh_D05G2577 gene and wild-type WT lines after 23 and 31 days of normal growth in nutrient soil.

[0020] Figure 5. Phenotypic breakdown of Arabidopsis thaliana lines overexpressing the Gh_D05G2577 gene and wild-type WT lines after 31 days of normal growth in nutrient soil. (A) Rosette leaves of Arabidopsis thaliana after 31 days of growth in nutrient soil. (B) Number of rosette leaves for each line. (C) Rosette diameter. (D) Number of rosette leaves. (E) Chlorophyll content. (F) Leaf area. * indicates significant difference, ** indicates highly significant difference.

[0021] Figure 6. Biomass accumulation analysis of Arabidopsis thaliana lines overexpressing the Gh_D05G2577 gene and wild-type WT lines after 31 days of normal growth in nutrient soil. (A) Growth of each line after 31 days. (B) Fresh weight. (C) Dry weight. (D) Biomass accumulation. * indicates significant difference, ** indicates highly significant difference.

[0022] Figure 7. Phenotypic statistical analysis of Arabidopsis thaliana lines overexpressing the Gh_D05G2577 gene and wild-type WT lines at normal growth and maturity in nutrient soil. (A) Stem length phenotype. (B) Pod length phenotype. (C) Number of fruiting branches phenotype. (D) Stem length. (E) Number of fruiting branches. (F) Number of pods. (G) Pod length. * indicates significant difference, ** indicates highly significant difference.

[0023] Figure 8. Phenotypic statistical analysis of grains from Arabidopsis thaliana lines overexpressing the Gh_D05G2577 gene and wild-type WT lines. (A) Grain phenotype. (B) Grain length. (C) Grain width. (D) 1000-grain weight. ** indicates highly significant differences.

[0024] Figure 9. Phenotypic analysis of cotton VIGS-Gh_D05G2577 lines and wild-type lines. (A) TRV::GhPDS albino lines, TRV::00 empty control lines, and silent lines of TRV::Gh_D05G2577-1 and TRV::Gh_D05G2577-2. (B) Expression level analysis of Gh_D05G2577 gene in empty control lines and silent lines of TRV::Gh_D05G2577-1 and TRV::Gh_D05G2577-2. (C) Chlorophyll content analysis of empty control lines and silent lines of Gh_D05G2577-2. Detailed Implementation

[0025] To better explain the present invention, the following detailed description of the invention will be provided in conjunction with embodiments. Unless otherwise specified, the methods used in the following embodiments are conventional methods. Unless otherwise specified, the materials and reagents used in the following embodiments are commercially available.

[0026] Example 1: Phylogenetic tree and protein conserved domain analysis of the cotton Gh_D05G2577 gene.

[0027] Using the upland cotton standard line TM-1 as the research material, total RNA was extracted. Gene-specific primers were designed to reverse transcribe the mRNA sequence (SEQ ID No. 1) of the Gh_D05G2577 gene into cDNA, which was then amplified by RT-PCR to obtain the CDS coding sequence (SEQ ID No. 2) of Gh_D05G2577. Using the Gh_D05G2577 protein sequence (SEQ ID No. 3) as a template, a BLAST search was performed in the NCBI public sequence database to select VIP1 functional domain proteins from 13 species with high similarity, including Asian cotton, Gossypium raemondii, Gossypium seashore, and cocoa. Phylogenetic analysis of Gh_D05G2577 and these VIP1 proteins showed that Gh_D05G2577 is closely related to GrVIP1 (Gossypium Raymondii) and GaVIP1 (Gossypium Asiae) (Figure 1A). Furthermore, multiple sequence alignment indicated that the VIP1 protein domains are highly conserved during evolution, and Gh_D05G2577 has the highest amino acid homology with GrVIP1 / GaVIP1, at 99.69% / 99.38% (Figure 1B).

[0028] Example 2: Acquisition and Screening of Arabidopsis Lines Overexpressing the Gh_D05G2577 Gene. Based on the CDS coding region sequence of the Gh_D05G2577 gene, specific primers for full-length cloning were designed (forward primer: agaggatccccggggggtaccATGGACAAAATACCTCCTCG; reverse primer: ctaggttaaccatgtggtaccCTAGGCTCCCTGGTTAAAATC). Using cDNA from upland cotton TM-1 as a template, the full-length sequence of Gh_D05G2577 was amplified and cloned by PCR. The fragment was then recovered by agarose gel electrophoresis and ligated into the pCMBIA2300 vector digested with KpnI. The ligation product was transformed into *E. coli* DH5α, and positive strains were detected. Plasmids were extracted from positive strains with consistent sequencing alignment results. 2 μL of the constructed *Gh_D05G2577* overexpression vector was added to 20 μL of *Agrobacterium tumefaciens* GV3101 competent cells and incubated on ice for 30 minutes. The mixture was then flash-frozen in liquid nitrogen for 5 minutes, followed immediately by a 37°C water bath for 5 minutes. 600 μL of LB solution was added to the mixture, and the mixture was incubated at 28°C and 220 rpm for 2 hours. Finally, the mixture was evenly spread onto LB agar plates containing kanamycin and rifampicin. *Agrobacterium* positive transformants of the *Gh_D05G2577* gene were detected using colony PCR and used in subsequent transgenic experiments.

[0029] Screening of Arabidopsis thaliana lines overexpressing the Gh_D05G2577 gene.

[0030] Wild-type Arabidopsis thaliana Col-0 was sown in nutrient soil and grown under conditions of 20℃ and 16 h / 8 h light. When it reached its full flowering stage, Arabidopsis thaliana was infected with Agrobacterium tumefaciens transformed with the Gh_D05G2577 gene by flower dipping. After the Arabidopsis thaliana matured, T0 generation seeds were harvested. T0 generation seeds were sown on 1 / 2 MS solid medium containing Kana (50 mg / mL) for positive seedling selection. Non-positive seedlings exhibit a yellowing phenotype, while positive seedlings show normal green growth (Figure 2A). Normally growing green positive seedlings are transplanted into nutrient soil. After the plants mature, DNA is extracted from the leaves of each individual seedling line for PCR detection. Agarose gel electrophoresis is used to identify lines containing the target gene band (Figure 2B). During this process, RNA is extracted from the leaves of each individual seedling line, and the expression level of Gh_D05G2577 is detected by qRT-PCR (Figure 2C). At maturity, individual plants are harvested and recorded as T1 generation seeds. T1 generation seeds are then sown on 1 / 2 MS solid medium containing Kana (50 mg / mL). The ratio of positive to non-positive seedlings is counted, and single-copy lines meeting a segregation ratio of 3:1 are retained. After maturation, T2 generation seeds are obtained. T2 generation seeds were sown on 1 / 2 MS solid medium containing Kana (50 mg / mL), and lines containing only positive seedlings were transplanted to nutrient soil for further cultivation. After maturation, T3 generation homozygous transgenic seeds were obtained. Finally, three transgenic lines, OE11, OE13, and OE15, with expression levels more than 4000 times higher than the control plants, were selected for the next stage of experiments.

[0031] Example 3: Effect of Gh_D05G2577 gene overexpression on Arabidopsis thaliana germination rate. Two transgenic lines of Arabidopsis thaliana, OE11 and OE15, with the highest Gh_D05G2577 expression levels in the T3 generation, along with WT seeds, were surface-sterilized and sown into 1 / 2 MS solid medium. To break dormancy, the seeds were first incubated at 4°C for 48 h, then transferred to a 16 h light / 8 h dark cycle and grown at a constant temperature of 22°C. Germination was recorded daily for 11 days. Radicle penetration through the seed coat was defined as the germination standard, and the germination rate was calculated. Each treatment was repeated in triplicate.

[0032] Germination rate analysis of Arabidopsis thaliana overexpressing the Gh_D05G2577 gene.

[0033] In 1 / 2 MS plates, the early germination rate of the transgenic lines was significantly higher than that of the wild type (Figure 3A), especially on day 3, the seed germination rates of OE11 and OE15 were 77.1% and 60.4%, respectively, while the germination rate of WT was 32.6%, representing increases of 136.9% and 85.4%, respectively. On day 11, the average germination rate of the transgenic lines was 98.7%, compared to 92.1% for the wild type, an increase of 7.1% (Figure 3B). These results indicate that the Gh_D05G2577 gene can regulate the early germination rate of Arabidopsis seeds.

[0034] Example 4: Analysis of the effect of overexpression of Gh_D05G2577 gene on the vegetative growth of Arabidopsis thaliana.

[0035] Transgenic lines OE11, OE13, and OE15 (Gh_D05G2577) were sown together with wild-type Arabidopsis thaliana (WT) in nutrient soil. The growing conditions were 20℃ and 16 h / 8 h light / dark cycles. The aboveground vegetative growth status of each line was observed, and agronomic traits such as rosette diameter, number of rosette leaves, chlorophyll content, leaf area, fresh weight, dry weight, and biomass accumulation were recorded. Each treatment was replicated in triplicate.

[0036] Compared with WT, the transgenic lines showed significantly better growth at 23 days of Arabidopsis thaliana growth, with larger and thicker leaves. By day 31, the difference in growth between the transgenic lines and WT was even more significant (Figure 4), and various agronomic traits were statistically analyzed. The average rosette diameter of OE11, OE13, and OE15 was 5.64 cm, while that of WT was 4.97 cm, representing a relative increase of 13.6% for the transgenic lines (Figures 5A and 5C). Compared with WT, the number of rosette leaves in OE11, OE13, and OE15 lines also increased significantly (Figures 5B and 5D), chlorophyll content increased by 10.6% (Figure 5E), and leaf area increased by 46% (Figure 5F). Biomass analysis of the whole plants of WT and OE11, OE13, and OE15 revealed that the transgenic lines had longer stems than WT (Figure 6A). Fresh weight measurements showed that the average fresh weight of the transgenic lines was 3.35 g, while the average fresh weight of the WT lines was 2.40 g, an increase of 40% (Figure 6B). Dry weight measurements showed that the average dry weight of the transgenic lines was 0.16 g, while the average dry weight of the WT lines was 0.29 g, an increase of 85.5% (Figure 6C). Biomass accumulation was calculated, with an average of 8.42% for the transgenic lines and 6.50% for the WT lines, an increase of 29.5% (Figure 6D). These results indicate that overexpression of the Gh_D05G2577 gene can increase rosette diameter, number of rosette leaves, chlorophyll content, leaf area, fresh weight, dry weight, and biomass accumulation during the vegetative growth stage of Arabidopsis thaliana.

[0037] Example 5: Analysis of the effect of overexpression of Gh_D05G2577 gene on reproductive growth of Arabidopsis thaliana.

[0038] During the late reproductive growth stage of Arabidopsis thaliana, growth indicators of the transgenic lines OE11, OE13, and OE15 (Gh_D05G2577) and wild-type Arabidopsis thaliana (WT) were observed, with three replicates for each treatment. Results showed that the average stem length of OE11, OE13, and OE15 was 28.16 cm, while the average stem length of the WT lines was 25.97 cm, an increase of 8.4% (Figs. 7A and 7D). Furthermore, the number of fruiting branches in the OE11, OE13, and OE15 lines was significantly higher than that in the WT lines, averaging 7-8 branches compared to 5-6 branches in the WT lines, an increase of over 20% (Figs. 7C and 7E). Additionally, the average number of pods in the transgenic lines was 25, while the average number of pods in the WT lines was 21, an increase of 37%. Pod length measurements in each line revealed that the average pod length of the transgenic lines was 16.54 mm, while that of the WT lines was 15.15 mm, an increase of 9.2% (Figures 7B, F, G). After plant maturity, mature seeds from each line were collected, and seed development was measured. Results showed that compared to WT, the seeds of the OE11, OE13, and OE15 lines were 13.3% longer and 9.53% wider (Figures 8A-C). The thousand-seed weight was measured, with average thousand-seed weights of 21.2 mg, 21.47 mg, and 21.00 mg for OE11, OE13, and OE15, respectively, compared to 18.70 mg for the WT lines, an increase of 13.5% (Figure 8D). These results indicate that overexpression of the Gh_D05G2577 gene can improve the transformation efficiency of Arabidopsis reproductive growth and increase yields such as seed length, width, and thousand-seed weight.

[0039] Example 5: Analysis of the effect of silencing the Gh_D05G2577 gene on cotton seedling growth.

[0040] To further analyze the role of the Gh_D05G2577 gene in cotton, the expression of Gh_D05G2577 in cotton was reduced using VIGS technology. A gene-specific fragment to be inserted into the pTRV2 vector was designed using the SNGVIGS tool from the Sol Genomics Network website (https: / / vigs.solgenomics.net / ). After transformation into Agrobacterium GV3101, the bacterial suspension was adjusted to OD600 = 0.9–1.0 in a resuspension buffer containing MgCl2 (10 mM), MES (10 mM), and acetylsuccinone (200 μM). The pTRV1 (pYL192) resuspension was mixed with equal volumes of TRV::Gh_D05G2577, TRV::00 (pYL156, negative control), or TRV::GhPDS (positive control) resuspensions, respectively. The mixture was then injected into cotton seedlings at the cotyledon stage, with three replicates for each treatment. When plants injected with TRV::GhPDS exhibited albinism (Fig. 9A), RNA was extracted from plants injected with TRV::Gh_D05G2577 and TRV::00, and subsequently analyzed by reverse transcription and qRT-PCR to determine the silencing efficiency of the Gh_D05G2577 gene. The results showed that compared with TRV::00 plants, the expression level of the Gh_D05G2577 gene decreased by more than 60% in TRV::Gh_D05G2577 plants (Fig. 9B). The SPAD values ​​of chlorophyll in TRV::00 and TRV::Gh_D05G2577 plants were measured. The SPAD value of TRV::00 was 32.41, while that of TRV::Gh_D05G2577 plants was 28.01, a decrease of 13.7% (Figure 9C). These results indicate that when the expression level of Gh_D05G2577 in cotton decreases, its chlorophyll content also decreases accordingly, demonstrating that Gh_D05G2577 can positively influence the vegetative growth of cotton seedlings.

[0041] The above results indicate that the Gh_D05G2577 gene and its related biological materials can increase the early germination rate, chlorophyll content, leaf length, number of leaves, leaf area, biomass accumulation, grain length, grain width and thousand-grain weight of plants.

[0042] This invention cloned and identified a cotton bZIP-like transcription factor gene, Gh_D05G2577. By analyzing the sequence structure and protein evolution patterns of the cotton Gh_D05G2577 gene, it was found that Gh_D05G2577 is highly conserved in plant evolution. Subsequently, by cloning the Gh_D05G2577 gene and using transgenic technology, homozygous Arabidopsis lines overexpressing the Gh_D05G2577 gene were obtained. The growth of the transgenic lines and the control wild-type lines was analyzed. The results showed that: in 1 / 2 MS plates, the transgenic plants showed a significant increase in early germination rate, with a germination rate of 68% on the third day, which was twice that of the wild-type control plants; in the seedling stage, there was an increase in the number of leaves, leaf length and width, plant height, chlorophyll content (SPAD), fresh weight increased by an average of more than 40%, dry matter content increased by more than 30%, and biomass accumulation increased significantly; in the fruit stage, there was an increase in pod length and width, seed length increased by 13% and width increased by 9%, and thousand-seed weight increased by more than 13%, all of which reached a significant level. Applying the above genes to high-yield and yield-increasing plant breeding can improve the germination rate of plants, increase the accumulation of biomass such as dry weight and fresh weight, and increase seed size and thousand-seed weight to improve yield. It effectively provides alternative solutions for high-yield molecular breeding of plants and has important reference significance for improving crop germination rate, biomass accumulation and seed yield.

Claims

1. The cotton gene Gh_D05G2577, the mRNA nucleotide sequence of which is shown in SEQ ID No.

1.

2. The application of the gene Gh_D05G2577 as described in claim 1 and the biomaterial containing the Gh_D05G2577 gene in regulating the early germination rate of plant seeds.

3. The application of the gene Gh_D05G2577 as described in claim 1 and the biomaterial containing the Gh_D05G2577 gene in regulating the number of leaves, leaf length and width, fresh weight, dry matter content and biomass accumulation in plant seedlings.

4. The application of the gene Gh_D05G2577 as described in claim 1 and the biomaterial containing the Gh_D05G2577 gene in regulating plant seed size and thousand-seed weight.

5. The application as described in any one of claims 2-5, characterized in that, The biomaterials include recombinant vectors or recombinant bacteria.

6. The application as described in any one of claims 2-5, characterized in that, The plants mentioned include Arabidopsis thaliana and cotton.

7. The application as described in any one of claims 2-5, characterized in that, The regulation is achieved by overexpressing the gene Gh_D05G2577 in plants.

8. The application of gene Gh_D05G2577 as described in claim 1 in plant breeding, characterized in that, The process includes the following steps: (1) obtaining the Gh_D05G2577 gene and ligating it with the expression vector plasmid to construct a recombinant vector containing Gh_D05G2577; (2) transforming the recombinant vector containing Gh_D05G2577 into competent Agrobacterium tumefaciens cells using the freeze-thaw method and screening for positive transformants; (3) transfecting wild-type plants with bacterial solutions containing positive transformants, harvesting the T0 generation after the crop matures, detecting the expression level of the gene in the T0 generation transgenic plants, and selecting positive transgenic plants with a Gh_D05G2577 gene expression level that is more than 4000 times higher than that of the control plants for subsequent experiments; (4) performing sexual or asexual reproduction on the transgenic plants, screening and harvesting Gh_D05G2577 homozygous lines with stable traits, and obtaining transgenic plants with increased germination rate and / or increased biomass accumulation and / or increased seed size and thousand-seed weight.

9. The application of gene Gh_D05G2577 as described in claim 8 in plant breeding, characterized in that, The plant in question is Arabidopsis thaliana.

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