CN117004614A - Gene GhTPR_A12 that regulates cotton fiber elongation and its application - Google Patents

Abstract

The invention belongs to the field of cotton genetic engineering and discloses a gene GhTPR_A12 that regulates the elongation and development of cotton fibers and its application. The nucleotide sequence of the gene is shown in SEQ ID No. 1. The present invention knocks out the gene GhTPR_A12 from cotton and performs silencing to obtain transgenic cotton with shortened mature fibers. The invention provides new ideas for the fiber development mechanism of cotton, and is of great significance in cultivating excellent cotton fiber varieties or researching.

Description

Gene GhTPR_A12 that regulates cotton fiber elongation and its application

Technical field

The invention belongs to the field of cotton genetic engineering, and specifically relates to the GhTPR_A12 gene that regulates cotton fiber elongation and its application in cotton fiber cell elongation; this gene can positively regulate upland cotton fiber elongation growth, and is an important factor in improving cotton fiber quality. Genes with important value can be used in molecular breeding research to improve cotton fiber quality through hybridization, transgenic and other technical methods.

Background technique

The TPR (Tetratricopeptide repeat) protein family consists of a class of proteins containing TPR conserved motifs. It is widely distributed in animals, plants and microorganisms. It is considered to be involved in the regulation of protein interactions and plays an important role in the formation of some protein complexes. role ^[1] . Each TPR conserved motif consists of two antiparallel α-helical subunits of 34 amino acids. TPR proteins have been reported frequently in animals and are mainly involved in animal cell cycle regulation, transcription inhibition, stress response, protein transport, RNA splicing, protein folding and other processes ^[2] . Relatively speaking, there are few reports on plant TPR proteins, which are mainly related to cell meiosis, growth and development, and signal transduction. They are crucial for the regulation of plant hormone pathways such as auxin, gibberellin, and ethylene ^[3,4] . For example, the TPR family protein SSR1 (short and swollen root 1), located in mitochondria, regulates Arabidopsis root development. In the ssr1 knockout mutant, primary root growth is significantly affected due to severe impairment of cell proliferation and cell elongation. inhibition. Auxin measurement found that the auxin level in the roots of the ssr1 knockout mutant was significantly reduced. Further studies found that SSR1 mainly affects the accumulation and degradation of the polar auxin export carrier PIN2 in the vacuole to regulate root growth and development ^[5] . The TPR family protein Spindly (SPY) is a negative regulator of the gibberellin pathway. Its regulatory function requires the formation of a complex with two other transcription factors through its TPR motif ^[6] . In Arabidopsis thaliana, the SPY protein regulates biological processes such as flowering, biological rhythms, and hypocotyl elongation by interacting with the GIGANTEA protein ^[1] . GmTPR is a member of the soybean TPR protein family. This protein can regulate the ethylene signaling pathway by interacting with GmETR1-1, an important ethylene receptor protein in the soybean ethylene signaling pathway ^[7] . The tomato TPR family protein SlTPR1 is located in the plasma membrane and nuclear membrane and can interact with ethylene receptors NR and LeETR1 to regulate the ethylene metabolism pathway ^[1] . The TPR family protein ATRTP1 (AtTPR130) is the only homolog of the SlTPR1 protein in Arabidopsis thaliana and can also preferentially interact with ethylene receptors to regulate the ethylene signaling pathway [108]. These results indicate that TPR protein plays an important role in regulating hormone signaling pathways such as auxin, gibberellin, and ethylene. In cotton, there are few reports on TPR family proteins. Only Fang et al ^{. [8]} reported that after the mutation of a TPR protein family gene: Ghir_A12G008870 on the cotton A12 chromosome induced by EMS mutagenesis technology, the mature fiber length of the mutant strain was significantly shorter. Research on the response regulation of cotton TPR protein family members to auxin, gibberellin and ethylene signaling pathways and fiber development has not yet been reported.

references:

[1] Zhou X, Zheng Y, Cai Z, et al. Identification and FunctionalAnalysis of Tomato TPR Gene Family[J]. International Journal of MolecularSciences, 2021, 22(2).

[2] Yan X, Tian D, Peng R, et al. Tetratricopeptide repeat domain 36protects renal tubular cells from cisplatin-induced apoptosis potentially viamaintaining mitochondrial homeostasis[J]. Tissue Cell, 2022, 76: 101749

[3] Schapire A L, Valpuesta V, Botella M A TPR Proteins in PlantHormone Signaling[J]. Plant Signaling&Behavior, 2006, 1(5): 229-230.

[4] Sparks J A, Kwon T, Renna L, et al. HLB1 Is a TetratricopeptideRepeat Domain-Containing Protein That Operates at the Intersection of theExocytic and Endocytic Pathways at the TGN/EE in Arabidopsis[J]. The PlantCell, 2016, 28 (3): 746-769.

[5] Zhang M, Wang C, Lin Q, et al. A tetratricopeptide repeat domain-containing protein SSR1 located in mitochondria is involved in root development and auxin polar transport in Arabidopsis[J]. The Plant Journal, 2015, 83(4) : 582-599.

[6] Greenboim-Wainberg Y, Maymon I, Borochov R, et al. Cross talk between gibberellin and cytokinin: the Arabidopsis GA response inhibitorSPINDLY plays a positive role in cytokinin signaling[J]. The Plant Cell, 2005, 17(1): 92-102.

[7] Niu Y Y, Chen M, Zhao-Shi X U, et al. Characterization ofEthylene Receptors and Their Interactions with GmTPR-A NovelTetratricopeptide Repeat Protein (TPR) in Soybean (Glycine max L.)[J].Journal of Integrative Agriculture, 2013, 000(004): 571-581.

[8] Fang DD, Naoumkina M, Thyssen GN, et al. An EMS-inducedmutation in a tetratricopeptide repeat-like superfamily protein gene ( Ghir_ A12G008870 ) on chromosome A12 is responsible for the liy short fiberphenotype in cotton[J]. Theoretical and Applied Genetics, 2020, 133(1): 271-282.

Contents of the invention

The purpose of the present invention is to provide a gene GhTPR_A12 that can regulate cotton fiber elongation development, and to preliminarily prove that gene GhTPR_A12 positively regulates cotton fiber elongation.

The technical solution of the present invention is as follows:

GhTPR_A12 , a gene that regulates plant development, has a nucleotide sequence as shown in SEQ ID No. 1.

Further, the protein sequence encoded by the GhTPR_A12 gene is shown in SEQ ID No. 2.

Further, the present invention also provides the expression cassette, recombinant expression vector and transgenic cell line of the GhTPR_A12 gene; the recombinant expression vector is a plant expression vector, preferably the gene fragment is connected to the vector pBI121 or pCAMBIA3301 to obtain recombinant Plasmids pBI121- GhTPR_A12 , pCAMBIA3301-GhTPR_A12 and pCAMBIA3301- GhTPR_A12 pro-GUS.

Furthermore, the present invention also provides the application of the GhTPR_A12 gene or recombinant expression vector in regulating plant development.

Further, the application is an application in regulating the development of plant fiber cells, preferably an application in regulating the elongation of plant fiber cells.

Further, the application is to introduce the GhTPR_A12 gene into the target plant and perform overexpression to obtain a transgenic plant with elongated fiber cells.

Further, the plant is one of a dicotyledonous plant, a monocotyledonous plant, a gramineous plant, and a cruciferous plant; preferably, the plant is cotton; more preferably, the plant is Asian cotton, Gossypium hirsutum , or one of the Raymond type cottons.

The beneficial effects of the present invention: 1. Provides a full-length gene sequence and the encoded protein sequence of a new cotton gene GhTPR_A12 ; the yeast system detects that the protein does not have transcription activation activity; 2. Analyzes the subcellular expression of the protein GhTPR_A12 Localization confirmed that the protein is located in the nucleus and cell membrane; 3. Using cotton genetic transformation combined with CRISPR/Cas9 technology to knock out the gene GhTPR_A12 in cotton, it was found that the fiber length of the gene-edited plants was significantly shorter. This shows that GhTPR_A12 positively regulates the elongation and growth of cotton fiber cells and has potential application value in improving cotton fiber quality. The present invention can be used for molecular breeding research on cotton fiber quality improvement through hybridization, transgene and other methods, provides new ideas for the fiber development mechanism of cotton, and is of great significance in cultivating excellent cotton fiber varieties or in research.

Description of the drawings

Figure 1: Analysis of protein domain of gene GhTPR_A12 .

Figure 2: Subcellular localization of gene GhTPR_A12 in tobacco cells.

Figure 3: Analysis of transcriptional activation activity of gene GhTPR_A12 .

Figure 4: GUS staining of GhTPR_A12 pro:GUS transgenic Arabidopsis thaliana, where a: root and rosette leaves; b: stem; c: inflorescence; d: silique.

Figure 5: GhTPR_A12 pro:GUS Arabidopsis leaf staining.

Figure 6: Comparison of cotton fiber length between wild-type Jin668 and T ₁ generation gene knockout plants, where, a: WT, b: GhTPR_A12 - DK1, c: GhTPR_A12 -DK2, d: GhTPR_A12 -DK3, e: GhTPR_A12 -DK4, f: GhTPR_A12 -DK5, g: GhTPR_A12 -DK6.

Detailed ways

The present invention is described in detail below through the drawings and examples, but does not limit the scope of the present invention.

1. Genomic DNA sequence and cDNA isolation and cloning of cotton gene GhTPR_A12

RNA extraction and reverse transcription from cotton fiber tissue 5 days after flowering. Prepare a number of mortars, clean and dry them, add an appropriate amount of 95% alcohol to sterilize them by burning, and after cooling, add liquid nitrogen to pre-cool them. Take out the plant tissue from the −80 ℃ ultra-low temperature refrigerator (the tissue is cotton fiber tissue 5 days after cotton flowering, put it in the refrigerator in advance), put it into a mortar and add liquid nitrogen to grind it quickly. After grinding it thoroughly into powder, use liquid Put the powder into a pre-cooled 2 mL centrifuge tube with a nitrogen-precooled spoon, and perform RNA extraction immediately or store it in an ultra-low temperature refrigerator. RNA was extracted using the RNAprep Pure polysaccharide polyphenol plant total RNA extraction kit from Tiangen Biochemical Technology (Beijing) Co., Ltd. (Cat. No.: DP441). Please refer to the kit instruction manual for the operation process. Reverse transcription used the AT341 reverse transcription kit from Beijing Quanshijin Biotechnology Co., Ltd. For the operation, please refer to the kit instruction manual. Using gene-specific primers ( GhTPR_A12 -F 5'-ATGGTTGCCTTTTACAAGTGC-3' and GhTPR_A12 -R 5'-CTATAATCCTCTATCCAACATCA-3') and high-fidelity DNA polymerase PFU, the PCR reaction program is: pre-denaturation at 98°C for 3 minutes; 95°C Denaturation for 20 s, renaturation at 55°C for 20 s, and extension at 72°C*s (2 kb/min, *extension time is determined according to the length of the target gene) for 35 cycles; extension at 72°C for 5 min, and storage at 10°C, amplification was obtained A DNA fragment of approximately 1.5kb ( GhTPR_A12 ). Insert this fragment into the T vector. The target DNA fragment was sequenced using the universal primers M13F and M13R on the plasmid to obtain the sequence, as shown in SEQ ID No. 1. The results showed that the full length of GhTPR_A12 gene sequence is 1569bp, with only one exon region and no introns. ATG is the start codon and TAG is the stop codon. The protein sequence encoded by the cotton gene GhTPR_A12 is shown in SEQ ID No. 2, and the protein domain of the gene is shown in Figure 1.

2. Subcellular localization analysis of GhTPR_A12 protein

①Construct the pBI121- GhTPR_A12 :EGFP2 fusion expression vector, transform the vector into Agrobacterium GV3101 competent cells, and activate positive single colonies on a shaker at 28°C overnight.

②Put the activated bacterial liquid into the liquid LB medium containing kanamycin and tetracycline, and culture it on a shaking table at 30°C. When the OD600 is around 1, centrifuge at 5000 rpm/min for 10 minutes. Discard supernatant.

③ Mix the Agrobacterium solution with the prepared infiltration buffer (10 μM magnesium chloride, 0.5% glucose, 200 μM AS, 10 μM MES), and then resuspend at room temperature in the dark for about 2 hours.

④Select young and tender tobacco leaves and use a syringe to inject the bacterial liquid into the young tobacco leaves through the stomata. Cultivate in the dark in a culture room for 2 days.

⑤Tear off the epidermal cells of tobacco leaves, observe and take pictures under a confocal laser microscope. The results showed that the GhTPR_A12-GFP fusion protein was localized in the nucleus and cell membrane (Figure 2).

3. GhTPR_A12 transcriptional activation activity detection

The pGBKT7 empty vector, pGBKT7-p53 (a transcription factor with confirmed transcriptional activation activity) and the pGBKT7-GhTPR_A12 vector were transformed into the yeast strain Y2H through the LiAC/PEG method. After the positive colonies were confirmed by PCR detection, the pGBKT7 empty vector, pGBKT7- AH109 colonies of p53 and pGBKT7-GhTPR_A12 vectors were spotted on the medium containing X-α-gal SD/-Trp/-Leu, SD/-Trp/-Leu/-His and SD/-Trp/-Leu/-Ade, After 2-3 days of growth, observe whether new colonies grow. The results are shown in Figure 3. Y2H containing pGBKT7-GhTPR_A12 vector can only grow on SD/-Trp/-Leu medium, but cannot grow on SD/-Trp/-Leu/-His and SD/-Trp/-Leu. /-Ade selective medium growth, indicating that the protein does not have transcriptional activation activity.

4. Obtaining and phenotypic identification of Arabidopsis thaliana GUS vector

Use primers to amplify from cotton genomic DNA to obtain four sets of gene sequences, approximately 500bp, 1000bp, 1500bp, and 2000bp upstream of the GhTPR_A12 gene. The primer sequences used are shown in the table below:

name sequence TPR-GUS-R acaggacgtaacatGGTCGGTACCTTTCAGAGAACGAAGATTGTAA 3TPR-GUS-F500 gagctcggtacccggggatccTTACTTTCATATATAAGCTCAGT 3TPR-GUS-F1000 gagctcggtacccggggatccTCTTATCTAGAACAAGATTGTG 3TPR-GUS-F1500 gagctcggtacccggggatccAATCGTAATGAGGTACATGTTGAT 3TPR-GUS-F2000 gagctcggtacccggggatccAATCCTTATCCTCTAATAATATAT

The promoter (whose nucleotide sequence is shown in SEQ ID No. 3) is connected to the upstream of the GUS encoding sequence through homologous recombination to form the recombinant vector pCAMBIA3301-GhTPR_A12pro-GUS. The recombinant vector was transferred into Agrobacterium competent GV3101 to form recombinant Agrobacterium. Wild-type Arabidopsis was transfected through Agrobacterium-mediated flower dipping method. After screening to obtain positive seedlings, they were transplanted into nutrient soil to obtain T ₃ generations. After the seeds. The T ₃ generation seeds were planted in nutrient soil for histochemical staining. The results are shown in Figure 4 and Figure 5. The GUS gene driven by the GhTPR_A12 promoter is expressed in the epidermal trichomes of leaves, flowers, roots and stems. The fiber development of cotton is similar to that of Arabidopsis thaliana. It is inferred that this gene may affect the development of epidermal trichomes. Development of cotton fiber cells.

5. Creation and phenotypic analysis of gene GhTPR_A12 CRISPR/Cas9 transgenic cotton

The open reading frame (ORF) of gene GhTPR_A12 was predicted on the gene editing website (http://crispr.hzau.edu.cn/), and an editing site (Site1: CGACAGGTAAACAAACCAAACGG) was selected for gene editing. , construct the candidate target sequence sgRNA+NGG into the CRISPR/Cas9 (p7n-Cas9) binary expression vector. After sequencing to verify that the sequence is correct, Agrobacterium GV3101 is transformed, and clones that are positive in PCR detection (the size is consistent with the target gene ), after shaking and propagation, the bacteria were preserved in glycerol and stored in an ultra-low temperature refrigerator at −80°C. Then Agrobacterium-mediated infection of cotton hypocotyls was performed, and the transformed explants were screened and cultured to select resistant calli. Then, the embryogenic callus is induced to differentiate on the differentiation medium, and then transferred to the embryo induction medium to induce the differentiation of somatic embryos. The seedlings are regenerated and transplanted to the field to grow, mature, flower and set fruit. After the cotton matures, the upper half of the mature cotton bolls are collected for fiber quality testing (Cotton Fiber Quality Inspection Supervision and Testing Center (Cotton Research Institute of the Ministry of Agriculture)). Phenotypic analysis shows that the transgenic cotton plant height, plant type, leaf size and shape, seed setting rate, boll size, and seed size are not significantly different from the wild type, but its fiber length is significantly shorter than the wild type (Figure 6 and the table below GhTPR_A12 -Cotton fiber length of DK-T ₁ , T ₂ , and T ₃ generation gene-edited plants).

strain T ₁ length L/mm T ₂ length L/mm T ₃ length L/mm WT-Jin668 27.00 27.00 27.88 GhTPR-DK1 25.67 25.78 25.16 GhTPR-DK2 25.90 25.90 24.81 GhTPR-DK3 26.79 25.46 24.62 GhTPR-DK4 25.40 26.73 26.31 GhTPR-DK5 25.74 25.93 25.64 GhTPR-DK6 26.14 26.08 25.43

These results indicate that GhTPR_A12 plays an important regulatory role in the elongation growth of cotton fiber cells and the formation of related quality traits, and has potential application value in improving cotton fiber quality.

In short, the name of the gene provided by the present invention is GhTPR_A12 . The full-length sequence of the gene is 1569 bp, encoding a protein sequence of 522 amino acids. The gene has a typical Tetratricopeptide repeat (TPR) domain (Figure 1).

We analyzed the subcellular localization of the protein, constructed the pBI- GhTPR_A12 : GFP fusion expression vector, transformed Agrobacterium, and then injected the Agrobacterium containing the expression vector into tobacco leaves. After 2 days, the lower epidermis of the tobacco leaves was taken and placed under laser confocal The fluorescence was observed under a microscope, and the results showed that the GhTPR_A12 -GFP fusion protein was located in the nucleus and cell membrane (Figure 2). At the same time, the pGBKT7- GhTPR_A12 vector was constructed, transformed into yeast strain Y2H, and the yeast system was used to detect the transcriptional activation activity of the protein. The results are shown in Figure 3. Y2H containing pGBKT7- GhTPR_A12 vector can only grow on SD/-Trp/-Leu medium, but cannot grow on SD/-Trp/-Leu/-His and SD/-Trp/-Leu. /-Ade selective medium growth, indicating that the protein does not have transcriptional activation activity. Secondly, the present invention determined the possible expression region of the GhTPR_A12 gene. The results proved that the GUS gene driven by the GhTPR_A12 promoter was expressed in the epidermal hairs of leaves, flowers, roots and stems. The results are shown in Figures 4 and 5. Further, we constructed a gene editing (CRISPR/Cas9) vector, transformed cotton, and obtained GhTPR_A12 transgenic gene-edited cotton. Phenotypic analysis of transgenics in T ₁ , T ₂ and T ₃ generations showed that the fiber length of transgenic cotton was significantly shorter than that of wild type (Figure 5 and Table 1), indicating that GhTPR_A12 plays an important role in regulating the elongation growth of cotton fiber cells and the formation of related quality traits. It has potential application value in improving the quality of cotton fiber.

Claims (10)

1. Gene regulating plant developmentGhTPR_A12The nucleotide sequence is shown as SEQ ID No. 1.

2. The gene according to claim 1GhTPR_A12The coded protein sequence is shown as SEQ ID No. 2.

3. Comprising the composition of claim 1GhTPR_A12Expression cassettes for genes, recombinant expression vectors, and transgenic cell lines.

4. The recombinant expression vector of claim 3, wherein said gene fragment is ligated into vector pBI121 or pCAMBIA3301 to obtain recombinant plasmid pBI121-GhTPR_A12 、pCAMBIA3301-GhTPR_A12And pCAMBIA3301-GhTPR_A12pro-GUS.

5. The method of claim 1GhTPR_A12Use of a gene, or of a recombinant expression vector according to claim 4, for regulating plant development.

6. The use according to claim 5, wherein the use is in modulating plant fibre cell development.

7. The use according to claim 6, wherein the use is in regulating plant fibre cell elongation.

8. The application of claim 7, wherein the application is to beGhTPR_A12The gene is introduced into target plant and over-expressed to obtain transgenic plant with elongated fiber cell.

9. The use according to claim 8, wherein the plant is one of dicotyledonous plants, monocotyledonous plants, plants of the family poaceae, cruciferous plants.

10. The use of claim 9, wherein the plant is cotton and the cotton is one of asian cotton, upland cotton, or raymond cotton.