Gh rsl1-2 gene, method for cultivating transgenic plants and application in regulating cotton development

Abstract

The application discloses a GhRSL1-2 gene, a method for cultivating a transgenic plant and application in regulation of cotton development, and belongs to the field of cotton genetic engineering. It is found that the GhRSL1-2 is located in the nucleus. By overexpressing and knocking out the GhRSL1-2 gene and transferring into cotton, it is found that the relative expression amount of the cotton overexpressing the GhRSL1-2 is significantly higher than that of the wild type cotton at 20 days after flowering. By detecting the fiber length, cell wall thickness, fiber twist number and seed quality of the wild type cotton, the cotton overexpressing the GhRSL1-2 and the cotton strain knocking out the GhRSL1-2 gene, it is found that the overexpression of the GhRSL1-2 can significantly increase the fiber length, the cell wall thickness, the fiber twist number and the seed weight, and it is indicated that the GhRSL1-2 gene regulates the fiber length and the cell wall thickness of the fiber cell. The GhRSL1-2 gene transferred into the cotton can promote the elongation of the cotton fiber cell and increase the cell wall thickness of the cotton fiber cell, and it has great significance for the research and improvement of the cotton fiber quality.

Description

Technical Field

[0001] This invention belongs to the field of cotton genetic engineering, specifically involving GhRSL1-2 Genes, methods for breeding transgenic plants, and their application in regulating cotton development. Background Technology

[0002] Cotton fibers are seed trichomes, extremely long single cells originating from the epidermal cells of the ovule. Their development involves distinct but overlapping stages: initiation, elongation, secondary cell wall synthesis, and maturation. The initiation and elongation stages of cotton fibers significantly influence the number, length, and fineness of the fibers, ultimately determining cotton yield and length. The biosynthesis of the secondary cell wall (SCW) in cotton fibers affects cell wall thickness, which directly impacts cotton fiber quality. Each stage of cotton fiber development is crucial for the overall fiber phenotype, and its development is inseparable from gene regulation. Multiple specific genes participate in specific periods of cotton fiber development, forming a complex regulatory network. However, known regulations are limited, and all involved genes and their regulatory mechanisms are not yet fully understood. Therefore, a new way to regulate cotton fiber development is needed. Summary of the Invention

[0003] In order to overcome the shortcomings of the prior art, the present invention aims to provide... GhRSL1-2 Genes, methods for breeding transgenic plants and their application in regulating cotton development.

[0004] To achieve the above objectives, the present invention employs the following technical solution: The first aspect of the present invention is disclosed. GhRSL1-2 The gene, which regulates cotton development, has the sequence shown in SEQ ID NO.1.

[0005] The second aspect of the present invention is disclosed. GhRSL1-2 The application of genes and related genes in regulating cotton development GhRSL1-2 The gene sequence is shown in SEQ ID NO.1, and the related gene has the same characteristics as... GhRSL1-2 Genes that share at least 75% homology and whose coding genes have GhRSL1-2 The function of genes.

[0006] Preferably, the cotton develops into fibroblast development.

[0007] Preferably, overexpression GhRSL1-2 Genes that can increase cotton fiber length, knockout GhRSL1-2 Genes can suppress the length of cotton fibers.

[0008] Preferably, overexpression GhRSL1-2Genes that increase the cell wall thickness of mature cotton fibers, knocked out GhRSL1-2 Genes can reduce the cell wall thickness of mature cotton fibers.

[0009] Preferably, overexpression GhRSL1-2 Genes can increase the weight of mature cotton seeds.

[0010] Preferably, overexpression GhRSL1-2 Genes that can increase the twist number of cotton fibers, knocked out GhRSL1-2 Genes can reduce the twist number of cotton fibers.

[0011] A third aspect of the present invention discloses a method for cultivating transgenic plants by introducing a gene encoding the gene shown in SEQ ID NO. 1 into a recipient plant. GhRSL1-2 Transgenic plants with growth and development different from the recipient plant were obtained.

[0012] Preferably, by introducing into the recipient plant a substance containing GhRSL1-2 Recombinant plasmids of genes enable the introduction of genes into recipient plants. GhRSL1-2 .

[0013] Preferably, the recombinant plasmid is pBI121- GhRSL1-2 pCAMBIA2300- GhRSL1-2 or pHellsgate4- GhRSL1-2 .

[0014] Compared with the prior art, the present invention has the following beneficial effects: This invention provides GhRSL1-2 The application of genes in regulating cotton development firstly involves... GhRSL1-2 The coding sequence was cloned into the pCAMBIA2300-GFP vector, and its localization in tobacco cells was detected as the nucleus; through investigation... GhRSL1-2 The evolutionary relationship between proteins and those in the model plant Arabidopsis thaliana indicates that... GhRSL1-2 The protein is most closely related to the AtRHD6 protein. Next, overexpression and knockout were performed separately. GhRSL1-2 The gene was transferred into cotton and screened to obtain GhRSL1-2 Genetically modified cotton was then used to detect overexpression using qRT-PCR technology. GhRSL1-2 The relative expression levels of cotton and wild-type cotton 20 days after fiber flowering (secondary cell wall development stage) revealed overexpression. GhRSL1-2 The relative expression level of cotton fibers 20 days after flowering was significantly higher than that of wild-type cotton, and GhRSL1-2 It is located in the cell nucleus. Finally, by detecting wild-type cotton, GhRSL1-2 Overexpression of cotton and GhRSL1-2Fiber length, cell wall thickness, fiber twist number, and seed weight of gene knockout cotton lines were measured, and overexpression was found. GhRSL1-2 It significantly increases fiber length, cell wall thickness, fiber twist number, and seed weight, while GhRSL1-2 Gene knockout significantly inhibited fiber length, cell wall thickness, fiber twist number, and seed quality, indicating that... GhRSL1-2 Genes regulate fiber length and cell wall thickness in fibroblasts. Therefore, GhRSL1-2 The transfer of genes into cotton can promote the elongation of cotton fiber cells and increase the thickness of cotton fiber cell walls, which is of great significance for the research and improvement of cotton fiber quality. Attached Figure Description

[0015] Figure 1 for GhRSL1-2 Subcellular location diagram in tobacco leaves; Figure 2 for GhRSL1-2 Evolutionary relationship diagram between proteins and proteins in the model plant Arabidopsis thaliana; Figure 3 for GhRSL1-2 Image showing the molecular identification results of overexpressing transgenic cotton plants; Figure 4 for GhRSL1-2 Relative expression levels of overexpression in 20DPA fibers; Figure 5 for GhRSL1-2 -Graph showing the molecular identification results of CRISPR-Cas9 transgenic cotton plants; Figure 6 This is a diagram showing the phenotypic identification results of genetically modified cotton; where A: wild-type cotton, GhRSL1-2 overexpression and GhRSL1-2 The mature fiber phenotype of gene knockout cotton, B: wild-type cotton. GhRSL1-2 overexpression and GhRSL1-2 Fiber length statistics of gene knockout cotton, C: wild-type cotton, GhRSL1-2 overexpression and GhRSL1-2 Gene knockout cotton mature fiber twisting phenotype, D: wild-type cotton, GhRSL1-2 overexpression and GhRSL1-2 Twist count of 1 cm fiber cells in gene knockout cotton, E: wild-type cotton, GhRSL1-2 overexpression and GhRSL1-2 Cross sections of mature cotton fibers from gene knockout cotton were stained with calcium fluoride white. F: Wild-type cotton based on E. GhRSL1-2 overexpression and GhRSL1-2 Statistics on cell wall thickness in gene knockout cotton, G: wild-type cotton, GhRSL1-2 overexpression and GhRSL1-2Mature fiber sections of gene knockout cotton stained with Pontamine FastScarlet 4B, H: wild-type cotton based on G. GhRSL1-2 overexpression and GhRSL1-2 Statistics on cell wall thickness in gene knockout cotton, I: GhRSL1-2 Phenotype of mature cotton seeds with high expression, J: GhRSL1-2 Phenotype of mature seeds from gene knockout cotton plants, K: wild-type cotton, GhRSL1-2 overexpression and GhRSL1-2 Statistical analysis of the weight of 100 mature cotton kernels from gene knockout cotton. Detailed Implementation

[0016] The present invention will be further described in detail below with reference to specific implementation methods. The embodiments given are only for illustrating the present invention and are not intended to limit the scope of the present invention.

[0017] In the following examples, unless otherwise specified, the experimental methods are conventional; the materials and reagents used are commercially available unless otherwise specified; quantitative experiments are all performed in triplicate, and the average value is taken; the material selected is cotton fiber tissue 20 days after flowering, referred to in the text as 20DPA, etc. Alternating light and dark culture means alternating between light and dark culture, and the specific culture cycle can be: 14 hours of light culture / 10 hours of dark culture.

[0018] In the following examples, the cotton variety is Xuzhou Cotton 142, and the public can obtain this biomaterial through ordinary means.

[0019] In the following examples, Agrobacterium tumefaciens GV3101 is described in the following literature: Xiao Weimin, Zhao Mingchen, Zou Min, Su Chenggang, Du Xiaobing. Effects of injury and inoculation with Agrobacterium tumefaciens GV3101 on transcription in Arabidopsis thaliana. Journal of Agricultural Biotechnology, 2013, 21(5):537-545. In the following examples, plasmid pCAMBIA2300 is described in the following literature: Gong Yuanyong, Feng Yongkun, Ni Wanchao, Shu Hongmei, Guo Shuqiao, Liu Laihua.

[0020] For the construction and verification steps of the plant expression vector pCAMBIA2300-35S-GUS-CaMVterm in the following examples, please refer to "Chinese Journal of Biotechnology", 2013, 33(3):86-91.

[0021] Those skilled in the art can use known methods to analyze the genes of the present invention. GhRSL1-2 The nucleotide sequence is mutated, and those artificially modified sequences have the same characteristics as the gene of this invention. GhRSL1-2 Nucleotide sequences with 75% or higher nucleotide identity are required, as long as the encoding gene is...GhRSL1-2 And possesses genes GhRSL1-2 The functions of all of them are derived from the nucleotide sequences of this invention and are equivalent to the sequences of this invention.

[0022] Example 1 GhRSL1-2 Gene analysis one, GhRSL1-2 Position in tobacco 1. Select two single restriction enzyme sites on the pCAMBIA2300-GFP vector, based on the target gene. GhRSL1-2 (The sequence is shown as SEQ ID NO.1 in Table 1) Design primers ， Furthermore, the primers need to include restriction enzyme sites and homologous arms (primer sequences are shown in SEQ ID NO.2 and SEQ ID NO.3 in Table 1). The restriction enzyme sites are KpnI and XbaI. The target gene fragment containing the restriction enzyme sites is amplified in a PCR instrument according to the forward and backward induction.

[0023] Table 1 Sequence List

[0024] 2. The pCAMBIA2300-GFP vector was double-digested with KpnI and XbaI enzymes, the same as those added to the gene primers. The target gene fragment containing the enzyme sites was then ligated into the pCAMBIA2300-GFP vector and then transformed into E. coli (DH5α) competent cells.

[0025] 3. Select single clones. After PCR reaction, select positive strains for sequencing. Once sequencing is successful, extract the plasmid from the corresponding strain. Simultaneously, select strains without the target gene. GhRSL1-2 The plasmid was used as a control.

[0026] 4. Each group of plasmids was introduced into the GV3101 Agrobacterium tumefaciens strain and transiently co-expressed in tobacco leaves. The cell nuclei were stained with 4',6-diamidinyl-2-phenylindole (DAPI), and the green fluorescent protein (GFP) fluorescence signal was detected using a confocal laser scanning microscope.

[0027] The results are as follows Figure 1 As shown, the cells are in good condition, as indicated by fluorescence. GhRSL1-2 Its location in tobacco is the cell nucleus.

[0028] II. Evolutionary Relationship between GhRSL1-2 Protein and Proteins in the Model Organism Arabidopsis thaliana The amino acid sequence of the GhRSL1-2 protein (SEQ ID NO.4) was compared with the amino acid sequences of proteins from the model organism Arabidopsis thaliana obtained from the database, and a phylogenetic tree was constructed. The cotton RHD was named according to its phylogenetic relationship with AtRHD. The results are as follows: Figure 2 As shown, this indicates that the GhRSL1-2 protein is most closely related to AtRHD6.

[0029] Example 2 GhRSL1-2 Obtaining and phenotypic analysis of transgenic cotton plants I. Recombinant plasmid PC2300S- GhRSL1-2 Construction of overexpression 1. According to GhRSL1-2 Primers were designed based on the CDS sequence (as shown in SEQ ID NO.1), and the primer sequences are shown in Table 1 as SEQ ID NO.2 and SEQ ID NO.3. (The text abruptly ends here, likely due to an incomplete sentence or missing information.) GhRSL1-2 Using cDNA from tissues with relatively high expression levels as PCR templates, the PCR amplification was performed using the KOD–Plus-Neo PCR amplification system. GhRSL1-2 CDS sequence.

[0030] 2. Use homologous recombination to transform the product obtained in step 1. GhRSL1-2 The target gene fragment was ligated into the vector pCAMBIA2300 to obtain the recombinant plasmid pCAMBIA2300- GhRSL1-2 .

[0031] 3. The recombinant plasmid PC2300S- obtained in step 2... GhRSL1-2 Introducing Agrobacterium tumefaciens LB4404, yielding GhRSL1-2 Overexpression of recombinant Agrobacterium.

[0032] II. Recombinant plasmid CRISPR-Cas9- GhRSL1-2 Construction of gene knockout vector 1. Find GhRSL1-2 The specific nucleotide sequences of approximately 20 bp were selected as the two target sites for knockout, as shown in SEQ ID NO. 9 and SEQ ID NO. 10 in Table 1. Target sites were designed for the cotton gene knockout sgRNA (sequence shown in SEQ ID NO. 7 in Table 1), employing a single gene dual-target approach.

[0033] 2. Primers were designed based on the target, and sgRNA and tRNA fragments were amplified on the pGTR4 vector. The tRNA sequence is shown in SEQ ID NO. 8 in Table 1. The first PCR amplification used this vector as a template to obtain two fragments: tRNA + target 1 (primers shown in SEQ ID NO. 11 and SEQ ID NO. 12) and target 1 + sgRNA1 + tRNA + target 2 (SEQ ID NO. 13 and SEQ ID NO. 14). The second amplification used overlap extension PCR, using the two fragments obtained in the first amplification as templates, with primer sequences shown in SEQ ID NO. 15 and SEQ ID NO. 16, to obtain tRNA + target 1 + sgRNA1 + tRNA + target 2 containing the homologous arm of the expression vector. The expression vector was PRGEB32, which contains the U6 promoter and gRNA2, and has two BsaI restriction sites. The PRGEB32 vector was double-digested with BsaI.

[0034] 3. The tRNA+target1+sgRNA1+tRNA+target2 obtained by homologous recombination and the enzyme-digested vector PRGEB32 were ligated and transformed into Trans1-T1 competent cells. The cells were plated on kanamycin-resistant medium and cultured. Positive colonies were picked for PCR detection, and plasmids were extracted from the identified positive colonies.

[0035] 4. The recombinant plasmid PRGEB32- GhRSL1-2 -gRNA was introduced into Agrobacterium tumefaciens GV3101 to obtain GhRSL1-2 Knock out recombinant Agrobacterium.

[0036] GhRSL1-2 In the knockout experiment, GhRSL1-2 The silencing mechanism is as follows: Cas9 protein is naturally present in plants. When Agrobacterium tumefaciens transfects a plant, the recombinant plasmid PRGEB32- GhRSL1-2 -gRNAs, through the localization of sgRNAs and the transport of tRNAs, bind to substances in the plant body. GhRSL1-2 Cas9 binds to a self-recognizing PAM site (Cas9 / gRNA can target any DNA site containing 5'-N20-NGG-3' (N = A,T,G,C) or 5'-CCN-N20-3', where NGG is the PAM required for Cas9 recognition, and it precisely cleaves the target DNA double strand approximately 3 bp before the PAM). When Cas9 binds to a portion of its sequence, the Cas9 protein knocks out this target site bound to the sgRNA. After the target is cleaved by the CRISPR system, the organism initiates its own repair mechanism, resulting in a mutation and thus achieving gene silencing.

[0037] III. Obtaining Transgenic Plants Take the results obtained in step one respectively GhRSL1-2 Overexpression of recombinant Agrobacterium and the product obtained in step two GhRSL1-2 Knockout of the hypocotyl segment of recombinant Agrobacterium tumefaciens infected with Xuzhou cotton 142 seedlings yielded embryogenic callus and sterile seedlings. Positive seedlings were identified after grafting, resulting in positive plants. Further propagation yielded T3 generation homozygous transgenic plants. GhRSL1-2 Overexpression of cotton lines and GhRSL1-2 Knock out cotton lines.

[0038] IV. Identification of Transgenic Plants 1. GhRSL1-2 Identification of cotton overexpression by GhRSL1-2 Overexpression of cotton ( GhRSL1-2-OE The experimental group was a wild-type cotton variety, Xuzhou Cotton 142 (WT), which was used as the control group. DNA was extracted from the leaves of each group and identified by PCR using gene primers (sequences shown in SEQ ID NO.5 and SEQ ID NO.6 in Table 1).

[0039] See results Figure 3 The extracted DNA bands showed GhRSL1-2 Overexpression of plant GhRSL1-2 Compared to Xuzhou cotton 142, which is overexpressed, this indicates... GhRSL1-2 The overexpression vector has been transferred into cotton.

[0040] Using wild-type cotton Xuzhou cotton 142 (WT) as the control group, GhRSL1-2 Overexpression of cotton ( GhRSL1-2-OE () as the experimental group. (The following is a list of subjects / groups) GhRSL1-2 Gene content analysis was performed on overexpressing cotton and wild-type cotton, Xuzhou cotton 142. Fibers were collected 20 days after flowering, ground with liquid nitrogen, and RNA was extracted from each group and its concentration was determined. cDNA from the 20-day-old fibers was obtained by reverse transcription using a reverse transcription kit and detected by qRT-PCR. GhRSL1-2 The relative expression level.

[0041] See results Figure 4 Wild-type cotton, Xuzhou cotton 142 GhRSL1-2 The relative expression level of the gene was 0.3. GhRSL1-2 The average relative expression level of overexpression in cotton was 21. GhRSL1-2 The relative expression level of overexpression in cotton was significantly higher than that in wild-type cotton 20 days after fiber flowering, further indicating that... GhRSL1-2 The overexpression vector has been transferred into cotton.

[0042] 2. GhRSL1-2 Identification of cotton knockout Using wild-type cotton Xuzhou cotton 142 (WT) as the control group, GhRSL1-2 The cotton was knocked out as the experimental group. The cotton obtained in step two was selected. GhRSL1-2 Knockout recombinant Agrobacterium monoclonal samples were generated by designing primers near the target site on the genome sequence, followed by PCR. The product was then purified, linked to a T-vector, and sequenced. The sequence was compared with the genome sequence to identify the knockout transgene.

[0043] See the identification results Figure 5 , GhRSL1-2 The knockout recombinant sgRNA1 and sgRNA2 each had one base inserted compared to the wild-type sequence, proving that they had been knocked out.

[0044] five, GhRSL1-2 Phenotypic analysis of genetically modified cotton Randomly select the result obtained in step three GhRSL1-2 Overexpression of cotton lines, GhRSL1-2 Ten mature fiber plants from the gene knockout cotton line and ten wild-type cotton line Xuzhou Cotton 142 were collected. Fifteen cotton bolls were randomly selected from each plant. The fibers were pulled several times to make them parallel, and any attached fibers were removed. The fiber bundles were placed on a black felt board, and their lengths were measured with a ruler. The phenotype of mature cotton fibers in each group was recorded, fiber lengths were counted, the twisting phenotype of mature fibers was identified, and the number of twists was counted. Then, cross-sections were stained with calcium fluoride white, and the cell wall thickness of each group was counted. Pontamine Fast Scarlet 4B staining was performed on mature fiber sections, and the cotton cell wall thickness was counted. The phenotype of mature seeds from each cotton plant was determined, and the mature grain weight was counted.

[0045] Figure 6 Results from A and B showed that the average fiber length of the wild-type plants was 27 mm. GhRSL1-2 The fiber length of the overexpressing plants was 29 mm. GhRSL1-2 The fiber length of the gene knockout line was 23 mm, and the fiber length of the overexpressing plant was increased by 7% compared with the wild type. GhRSL1-2 The gene knockout lines showed a 15% reduction in fiber length compared to the wild-type lines, indicating overexpression. GhRSL1-2 -OE increases fiber length, while knocking out CRISPR-Cas9- GhRSL1-2 The gene significantly suppresses fiber length, indicating GhRSL1-2 Genes regulate fibroblast development.

[0046] Figure 6 Results from C and D show that wild-type cotton has an average twist count of approximately 57 per cm of mature fiber. GhRSL1-2 Overexpression showed that the number of twists per cm of mature cotton fiber was approximately 66. GhRSL1-2In gene knockout cotton, the number of twists per cm of mature fiber is approximately 18, while in overexpression plants, the number of fiber twists increases by 16% compared to wild-type fibers. GhRSL1-2 Gene knockout lines showed a 68% reduction in fiber twist compared to wild-type lines, indicating overexpression. GhRSL1-2 -OE increases the twist number of the fiber, while knocking out CRISPR-Cas9- GhRSL1-2 The gene significantly reduced the fiber twist number, indicating GhRSL1-2 Genes regulate the degree of fiber distortion.

[0047] Figure 6 Cross sections of cotton from varieties E and F were stained with calcium fluoride, revealing that the cell wall thickness of mature wild-type cotton fibers was approximately 5.5 μm. GhRSL1-2 The average cell wall thickness of mature cotton fibers overexpressing this gene was approximately 7.1 μm. GhRSL1-2 The cell wall thickness of mature cotton fibers with gene knockout was approximately 4.3 μm, while the cell wall thickness of mature fibers from overexpressing plants increased by 29% compared to that of wild-type mature fibers. GhRSL1-2 The gene knockout line showed a 22% reduction in cell wall thickness compared to wild-type fibers. Figures G and H show mature fiber sections stained with Pontamine Fast Scarlet 4B, and the statistically significant cell wall thickness is consistent with that in Figures E and F, ensuring data reliability and indicating that overexpression... GhRSL1-2 -OE increases the cell wall thickness of mature cotton fibers, while knocking out CRISPR-Cas9- GhRSL1-2 The gene significantly reduced the cell wall thickness of mature fibers, indicating that... GhRSL1-2 Genes regulate cell wall development in fibers.

[0048] Figure 6 Statistics on the mature seed weights of plants in categories I, J, and K show that the average mature seed weight of wild-type plants is approximately 7.5 g. GhRSL1-2 The average weight of mature seeds from overexpressed plants was approximately 9.3 g. GhRSL1-2 The average mature seed weight of the gene knockout lines was approximately 7.2 g, while the mature seed weight of the overexpressing plants increased by 24% compared to the wild-type and knockout types; indicating that overexpression... GhRSL1-2 -OE increases the weight of mature seeds, indicating GhRSL1-2 Genes regulate seed development.

[0049] The above content is only for illustrating the technical concept of the present invention and should not be construed as limiting the scope of protection of the present invention. Any modifications made to the technical solution based on the technical concept proposed in this invention shall fall within the scope of protection of the claims of this invention.

Claims

1. GhRSL1-2 The application of genes in regulating cotton development is characterized by, GhRSL1-2 The gene sequence is shown in SEQ ID NO.1, and it is overexpressed. GhRSL1-2 Genes that increase the cell wall thickness of mature cotton fibers, knocked out GhRSL1-2 Genes can reduce the cell wall thickness of mature cotton fibers.

2. GhRSL1-2 The application of genes in regulating cotton development is characterized by, GhRSL1-2 The gene sequence is shown in SEQ ID NO.1, and it is overexpressed. GhRSL1-2 Genes can increase the weight of mature cotton seeds.

3. GhRSL1-2 The application of genes in regulating cotton development is characterized by, GhRSL1-2 The gene sequence is shown in SEQ ID NO.1, and it is overexpressed. GhRSL1-2 Genes that can increase the twist number of cotton fibers, knocked out GhRSL1-2 Genes can reduce the twist number of cotton fibers.

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