Application of GhDOF5.4 gene in regulation and control of cotton development
By introducing the GhDOF5.4 gene into cotton and using Agrobacterium-mediated transformation technology, the uncertainty of cotton fiber development regulation was solved, and the fiber length, cell wall thickness and fiber twist number were significantly improved, improving cotton quality and yield.
Patent Information
- Application Number
- CN202510471166.X
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-04-15
- Publication Date
- 2025-07-18
AI Technical Summary
The existing technology has not yet fully understood the genes and mechanisms involved in regulation during cotton fiber development, which has made it difficult to effectively improve fiber quality and yield.
By constructing the GhDOF5.4 gene overexpression plasmid, the GhDOF5.4 gene was introduced into cotton using Agrobacterium-mediated transformation technology, which promoted the increase in fiber length, cell wall thickness and fiber twist number.
The length, cell wall thickness and fiber twist number of cotton fibers are significantly improved, and the quality and yield of cotton fibers are improved.
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Figure CN120330241A_ABST
Abstract
Description
Technical Field
[0001] The present invention belongs to the field of cotton genetic engineering, and particularly relates to the application of the GhDOF5.4 gene in regulating cotton development. Background Art
[0002] Cotton fibers are developed from the polar elongated single cells of the ovule epidermis, and fiber development undergoes distinct and overlapping stages: initiation, elongation, secondary wall thickening, and maturation. The initiation and elongation stages of cotton fibers play an important role in the number, length, and thickness of the fibers, ultimately determining the yield and length of cotton. The biosynthesis of the secondary cell wall (SCW) of cotton fibers affects the cell wall thickness, and the cell wall thickness directly affects the fiber quality of cotton. Each stage of cotton fiber development is crucial for the entire fiber phenotype, and its development is inseparable from gene regulation. Multiple specific genes are involved in specific periods of cotton fiber development, constituting a complex regulatory network. However, the known regulation is limited, and all the genes involved and their regulatory mechanisms have not been fully understood. Therefore, a new method capable of regulating cotton fiber development is needed. Summary of the Invention
[0003] The purpose of the present invention is to provide the application of the GhDOF5.4 gene in regulating cotton development and a method for cultivating transgenic cotton.
[0004] To achieve the above purpose, the present invention adopts the following technical solutions:
[0005] In the first aspect of the present invention, the application of the GhDOF5.4 gene and its related genes, GhDOF5.4 protein, recombinant vector containing the GhDOF5.4 gene, and cell containing the recombinant vector in regulating cotton development is disclosed. The sequence of the CDS of the GhDOF5.4 gene is shown in SEQ ID NO:1. The related gene has at least 75% homology with the GhDOF5.4 gene and has the function of the GhDOF5.4 gene. Those skilled in the art can use known methods to mutate the nucleotide sequence of the gene GhDOF5.4 of the present invention. Those nucleotides obtained after artificial modification and having 75% or higher identity with the nucleotide sequence of the gene GhDOF5.4 of the present invention, as long as they encode the protein GhDOF5.4 and have the function of the gene GhDOF5.4, are all derived from the nucleotide sequence of the present invention and are equivalent to the sequence of the present invention.
[0006] Preferably, the cotton development is the development of cotton fiber cells.
[0007] Furthermore, overexpression of the GhDOF5.4 gene can increase the fiber length of cotton; specifically, the fiber length of cotton overexpressing the GhDOF5.4 gene at different stages after flowering is longer than that of the wild type.
[0008] Overexpression of the GhDOF5.4 gene can also increase the cell wall thickness of mature cotton fiber cells. Overexpression of the GhDOF5.4 gene can also increase the twist number of mature cotton fibers.
[0009] Preferably, the cotton development is the development of cotton plant height.
[0010] Furthermore, overexpression of the GhDOF5.4 gene can increase the height of mature cotton plants.
[0011] In the second aspect of the present invention, a method for cultivating transgenic cotton is disclosed, in which the coding gene GhDOF5.4 shown in SEQ ID NO:1 is introduced into recipient cotton to obtain transgenic cotton whose growth and development are different from that of the recipient cotton.
[0012] Preferably, the gene GhDOF5.4 is introduced into the recipient plant by introducing a recombinant plasmid containing the GhDOF5.4 gene into the recipient cotton.
[0013] Preferably, the backbone plasmid of the recombinant plasmid is pCAMBIA2300.
[0014] More preferably, the cotton is Gossypium hirsutum, Gossypium barbadense or Gossypium arboreum. For example, Gossypium hirsutum Ji 668 (J668) can be specifically used.
[0015] Compared with the prior art, the present invention has the following beneficial effects:
[0016] The present invention provides the application of the GhDOF5.4 gene in regulating cotton development. First, the coding sequence of GhDOF5.4 is cloned into the pCAMBIA2300 vector, transformed into Agrobacterium tumefaciens LBA4404, and used to infect cotton to obtain transgenic plants. By constructing an overexpression plasmid of GhDOF5.4 and performing Agrobacterium-mediated cotton transformation, fibers at 5 days, 10 days, 15 days, 20 days, and mature fibers were collected, and it was found that the fiber length of cotton overexpressing GhDOF5.4 was longer than that of the wild type at different stages after flowering. By detecting the relative expression level of GhDOF5.4 in 15DPA fibers of cotton overexpressing GhDOF5.4 by qPCR technology, it was found that it was significantly higher than that of wild-type cotton. By detecting the fiber length, plant height, fiber twist number, cell wall thickness of mature fiber cells, and cotton boll size of wild-type cotton and cotton overexpressing GhDOF5.4, it was found that overexpression of GhDOF5.4 could significantly increase the fiber length, cell wall thickness of mature fiber cells, and fiber twist number, indicating that the GhDOF5.4 gene regulates the fiber length and cell wall thickness of fiber cells. Transferring the GhDOF5.4 gene into cotton can promote the elongation of cotton fiber cells and increase the cell wall thickness of cotton fiber cells, which is of great significance for the research and improvement of cotton fiber quality. Description of the Drawings
[0017] Figure 1 It is a molecular identification result diagram of cotton plants overexpressing GhDOF5.4 transgenic plants; among them, A: PCR identification diagram of cotton lines overexpressing GhDOF5.4, B: relative expression level results of cotton plants overexpressing GhDOF5.4, C: protein enrichment diagram of cotton lines overexpressing GhDOF5.4.
[0018] Figure 2 It is a phenotypic diagram of fiber length at different development stages of GhDOF5.4 in cotton fibers; among them, A: phenotypes of cotton fibers at different development stages of GhDOF5.4 overexpression and wild type from 0 to 20 DPA, B: statistical results of fiber lengths of GhDOF5.4 overexpression and wild-type cotton fibers at different times. C: phenotypes of mature cotton fibers of GhDOF5.4 overexpression and wild type, D: statistical results of fiber lengths at the mature stage of each group of cotton fibers.
[0019] Figure 3 It is a phenotypic diagram of fiber twist number and plant height of GhDOF5.4 protein and wild-type cotton; among them, A: phenotypes of mature fiber twisting of wild-type cotton and cotton overexpressing GhDOF5.4, B: statistical results of twist numbers of 1 cm fiber cells of wild-type cotton and cotton overexpressing GhDOF5.4, C: plant heights of wild-type cotton and cotton overexpressing GhDOF5.4, D: statistical results of plant heights of wild-type cotton and mature cotton plants overexpressing GhDOF5.4.
[0020] Figure 4Phenotype diagrams of cell wall thickness of cotton fibers overexpressing GhDOF5.4 and at 15 DPA cotton bolls; A: Cross-sections of mature fibers of wild-type cotton and cotton overexpressing GhDOF5.4, B: Statistics of cell wall thickness of wild-type cotton and cotton overexpressing GhDOF5.4 based on A, C: Weights of 15 DPA cotton bolls of wild-type cotton and cotton overexpressing GhDOF5.4, D: Statistics of weights of 15 DPA cotton bolls of wild-type cotton and cotton overexpressing GhDOF5.4.
[0021] Figure 5 Phenotype diagrams of fiber initiation of cotton plants overexpressing GhDOF5.4 at 0 DPA; A: Number of fiber initiations of wild-type cotton and cotton overexpressing GhDOF5.4 at 0 DPA, B: Statistics of number of fiber initiations of wild-type cotton and cotton overexpressing GhDOF5.4 at 0 DPA. Detailed implementation manners
[0022] To make the objectives, technical solutions and advantages of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly described below in conjunction with the accompanying drawings in the embodiments of the present invention. Obviously, the described embodiments are some, but not all, of the embodiments of the present invention. All other embodiments obtained by those of ordinary skill in the art based on the embodiments of the present invention without creative efforts shall fall within the protection scope of the present invention.
[0023] In the following embodiments, the test methods are conventional methods unless otherwise specified; the materials, reagents, etc. used can be obtained from commercial channels unless otherwise specified; for quantitative experiments, three repeated experiments are set, and the results are averaged; cotton fiber tissues 15 days after flowering are abbreviated as 15 DPA in the text. Alternating light and dark culture means alternating light culture and dark culture. The specific culture cycle can specifically be: 16-hour light culture and 8-hour dark culture.
[0024] In the following embodiments, the cotton variety is Ji 668 (J668), and the public can obtain this biological material from ordinary channels.
[0025] In the following embodiments, Agrobacterium tumefaciens GV3101 is recorded in the following literature: Xiao Weimin, Zhao Mingchen, Zou Min, Su Chenggang, Du Xiaobing. Effects of Arabidopsis thaliana injury and inoculation with Agrobacterium tumefaciens GV3101 on transcription. Journal of Agricultural Biotechnology, 2013, 21(5): 537-545.; This bacterium can be a commercially available product.
[0026] In the following examples, the plasmid pCAMBIA2300 is described in the following literature: [1] Gong Yuanyong, Feng Yongkun, Ni Wanchao, et al. Construction and verification of plant expression vector pCAMBIA2300-35S-GUS-CaMVterm [J]. China Biotechnology Journal, 2013, 33(03): 86-91. DOI: 10.13523 / j.cb.20130314.; This plasmid is a commercially available product.
[0027] Example 1. Obtaining and phenotypic analysis of GhDOF5.4 transgenic cotton plants
[0028] I. Preparation of GhDOF5.4 overexpression recombinant Agrobacterium
[0029] 1. Design primers according to the CDS sequence of GhDOF5.4 (the sequence is shown in SEQ ID NO: 1), and the primer sequences are shown in SEQ ID NO: 2 and SEQ ID NO: 3 in Table 1. Take the cDNA of 10DPA fiber, a tissue with relatively high expression of GhDOF5.4 in cotton, as the PCR template, and use the PCR amplification system of 2×Phanta Flash Master Mix (Dye Plus) (Vazyme, china) to amplify the CDS sequence of GhDOF5.4.
[0030] 2. Use homologous recombination to ligate the GhDOF5.4 target gene fragment obtained in step 1 into the vector pCAMBIA2300 to obtain the recombinant plasmid pCAMBIA2300-GhDOF5.4.
[0031] 3. Introduce the recombinant plasmid PC2300S-GhDOF5.4 obtained in step 2 into Agrobacterium tumefaciens LBA4404 to obtain GhDOF5.4 overexpression recombinant Agrobacterium.
[0032] Table 1. Sequence list
[0033]
[0034] II. Obtaining transgenic plants
[0035] Respectively infect the hypocotyl segments of cotton J668 (J668) seedlings with the GhDOF5.4 overexpression recombinant Agrobacterium obtained in step I to obtain embryogenic callus, differentiate to obtain sterile seedlings, transplant them into sterilized substrate soil and then identify positive seedlings to obtain positive plants; Propagate by selfing to obtain T3 generation homozygous transgenic plants, namely GhDOF5.4 overexpression cotton lines.
[0036] III. Identification of transgenic plants
[0037] 1. Identification of GhDOF5.4 overexpression cotton
[0038] Using the GhDOF5.4 - overexpressing cotton (GhDOF5.4 - OE) as the experimental group and the wild - type cotton Ji668 (WT) as the control group, the DNA of cotton leaves in each group was extracted respectively, and PCR identification was carried out using primers (the sequences are shown as SEQ ID NO:5 and SEQ ID NO:6 in Table 1).
[0039] The results are shown in Figure 1 A. The transcriptional level of GhDOF5.4 in the GhDOF5.4 - overexpressing plants was overexpressed compared with that of the wild - type Ji668, indicating that the GhDOF5.4 - overexpressing vector had been transferred into cotton.
[0040] Using the wild - type cotton Ji668 (WT) as the control group and the GhDOF5.4 - overexpressing cotton (GhDOF5.4 - OE) as the experimental group, the gene expression levels of GhDOF5.4 - overexpressing cotton and wild - type cotton Ji668 were analyzed respectively. The fiber tissues at 15 days post - anthesis (15DPA) were taken, ground with liquid nitrogen, the RNA in each group was extracted and its concentration was detected, and cDNA of 15DPA was obtained by reverse transcription using a reverse transcription kit. The relative expression level of GhDOF5.4 was detected by qRT - PCR. In addition, using the wild - type cotton Ji668 (WT) as the control group and the GhDOF5.4 - overexpressing cotton (GhDOF5.4 - OE) as the experimental group, Western Blot experimental analysis was carried out on GhDOF5.4 - overexpressing cotton and wild - type cotton Ji668 respectively.
[0041] The results showed that the relative expression level of the GhDOF5.4 gene in wild - type cotton Ji668 was 0.0006, and the average relative expression level of GhDOF5.4 - overexpressing cotton was 1.13. The relative expression level of GhDOF5.4 - overexpressing cotton at 15 days post - anthesis was significantly higher than that of wild - type cotton, as Figure 1 shown in B, further indicating that the GhDOF5.4 - overexpressing vector had been transferred into cotton. In addition, the Western Blot results showed that the housekeeping gene Actin protein was normally detected in both wild - type cotton Ji668 and GhDOF5.4 - overexpressing cotton. The wild - type cotton Ji668 could not express the GhDOF5.4 protein carrying the flag tag, while the GhDOF5.4 - overexpressing vector normally expressed the GhDOF5.4 protein carrying the flag tag, as Figure 1 shown in C, and this result further indicated that the GhDOF5.4 - overexpressing vector had been transferred into cotton and was functioning normally.
[0042] IV. Phenotypic analysis of GhDOF5.4 transgenic cotton
[0043] Randomly select 10 mature fibers each from the GhDOF5.4 overexpressing cotton lines and the wild-type cotton line Ji 668 obtained in Step 3. Randomly pick 15 cotton bolls from each plant. After pulling the fibers of each group several times to make the fibers parallel, remove the attached fibers at the same time. Place the fiber bundles on a black velvet board, measure the length with a ruler, record the phenotypes of the mature fibers of each group of cotton, count the fiber length, identify the twist phenotypes of the mature fibers, count the number of twists; then perform cross-section slicing of the mature fibers, count the cell wall thickness of each group, and count the cotton cell wall thickness; determine the phenotypes of the mature plant heights of each group of cotton plants and count the mature plant heights.
[0044] Respectively select different stages of cotton fiber development of wild-type plants and GhDOF5.4 overexpressing plants and count the lengths. The results show that at different stages of fiber development, the wild-type fibers are significantly shorter than those of the GhDOF5.4 overexpressing plants, as Figure 2 shown in A and 2B.
[0045] In addition, the fiber length statistics results show that the average length of the mature fibers of wild-type plants is 25.75 mm, and the fiber length of GhDOF5.4 overexpressing plants is 28.45 mm. The fiber length of the overexpressing plants increased by 10% compared with that of the wild-type fibers, as Figure 2 shown in C and 2D. Overexpression of GhDOF5.4-OE increased the fiber length, demonstrating that the GhDOF5.4 gene regulates fiber cell development.
[0046] Figure 3 The results in A and 3B show that the number of twists of GhDOF5.4 overexpressing cotton is more than that of wild-type cotton. The average number of twists per 1 cm of mature fibers of wild-type cotton is about 90, and the number of twists per cm of mature fibers of GhDOF5.4 overexpressing cotton is about 240. The number of twists of the fibers of the overexpressing plants increased by 160% compared with that of the wild-type fibers, indicating that overexpression of GhDOF5.4-OE increased the number of twists of the fibers, demonstrating that the GhDOF5.4 gene regulates the degree of fiber twist.
[0047] Figure 3 The results in C and 3D show that the plant height of GhDOF5.4 overexpressing cotton is higher than that of wild-type cotton. The plant height of wild-type cotton is 94 cm, and the average plant height of GhDOF5.4 overexpressing cotton is 116 cm. The plant height of the GhDOF5.4 overexpressing plants increased by 23% compared with that of the wild-type mature plant height, indicating that overexpression of GhDOF5.4 increased the plant height, demonstrating that the GhDOF5.4 gene regulates the elongation of cotton plant height.
[0048] Figure 4The results of A and 4B showed that the cell wall of mature fibers in GhDOF5.4-overexpressing cotton was thicker than that in wild-type cotton. The cell wall thickness of mature fibers in wild-type cotton was about 2.79 μm, while the average cell wall thickness of mature fibers in GhDOF5.4-overexpressing cotton was about 4.34 μm. The cell wall thickness of mature fibers in GhDOF5.4-overexpressing plants increased by 55.5% compared with that of wild-type mature fibers, indicating that overexpression of GhDOF5.4 increased the cell wall thickness of cotton mature fibers and proving that the GhDOF5.4 gene promoted the development of fiber cell walls.
[0049] Figure 4 The results of C and 4D showed that the boll weight of GhDOF5.4-overexpressing cotton was lower than that of wild-type cotton. The average weight of 15DPA bolls in wild-type cotton was 14.7 g, while the average weight of 15DPA bolls in GhDOF5.4-overexpressing cotton was 7.3 g. The average weight of 15DPA bolls in GhDOF5.4-overexpressing plants decreased by 101.3% compared with that of wild-type 15DPA bolls, indicating that overexpression of GhDOF5.4 reduced the average weight of 15DPA bolls in cotton and proving that the GhDOF5.4 gene regulated the ovule development of bolls.
[0050] Figure 5 The results of A and 5B showed that there was no significant difference in the protrusions of 0DPA fiber cells between GhDOF5.4-overexpressing cotton and wild-type cotton. The average starting numbers of WT in every 0.01 mm 2 were 172, 193, and 184 respectively; the average starting numbers of GhDOF5.4-OE were 205, 188, and 197 respectively, and there was no statistical significance in the analysis. It indicated that overexpression of GhDOF5.4 could not increase or decrease the number of fiber cell protrusions during the fiber initiation period of cotton, proving that the GhDOF5.4 gene did not participate in regulating the development of fiber cell numbers.
[0051] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those of ordinary skill in the art should understand that they can still modify the technical solutions described in the foregoing embodiments, or perform equivalent replacements for some of the technical features; and these modifications or replacements do not make the essence of the corresponding technical solutions deviate from the spirit and scope of the technical solutions of the embodiments of the present invention.
Claims
1. GhDOF5.4 Genes and their related genes, GhDOF5.4 protein, GhDOF5.4 the application of a recombinant vector containing the gene, and a cell containing the recombinant vector in regulating cotton development, characterized in that The GhDOF5.4 CDS sequence of the gene is shown in SEQ ID NO:
1.
2. The application according to claim 1, wherein The cotton development is the development of cotton fiber cells.
3. The application according to claim 2, characterized in that Overexpression GhDOF5.4 The gene can increase the fiber length of cotton.
4. The application according to claim 2, wherein Overexpression GhDOF5.4 The gene can increase the cell wall thickness of mature cotton fiber cells.
5. The application according to claim 2, characterized in that Overexpression GhDOF5.4 The gene can increase the twist number of mature cotton fibers.
6. The application according to claim 1, characterized in that, The cotton development is the development of cotton plant height.
7. The application according to claim 6, wherein Overexpression GhDOF5.4 The gene can increase the height of mature cotton plants.
8. A method for cultivating transgenic cotton, characterized in that, Introduce the coding gene shown in SEQ ID NO:1 into recipient cotton GhDOF5.4 to obtain transgenic cotton with growth and development different from that of the recipient cotton.
9. The method according to claim 8, characterized in that By introducing a recombinant plasmid containing GhDOF5.4 gene into recipient cotton, the introduction of gene GhDOF5.4 into the recipient plant is achieved.
10. The method according to claim 9, wherein The backbone plasmid of the recombinant plasmid is pCAMBIA2300.