Application of GhHD-ZIP7 protein and coding gene thereof in regulation and control of fiber length

Abstract

The invention discloses application of GhHD-ZIP7 protein and a coding gene thereof in regulation and control of fiber length, and belongs to the technical field of plant genetic engineering. An overexpression vector is constructed for the GhHD-ZIP7 gene to transform arabidopsis thaliana and cotton, and it is found that the length of a main root of the transgenic arabidopsis thaliana is remarkably increased compared with that of a control (Columbia ecotype), the length of root hair in a mature region is remarkably shortened compared with that of the control, and the fiber length of an overexpression cotton material is remarkably reduced compared with that of a control (Zhong cotton No. 113). A knockout vector is constructed for the GhHD-ZIP7 gene to transform cotton, and it is found that the length of mature fibers of transgenic cotton with the GhHD-ZIP7 gene knocked out is remarkably increased compared with that of a control group. The research results show that the GhHD-ZIP7 gene regulates and controls the elongation of cotton fiber cells.

Description

Technical Field

[0001] This invention relates to the field of plant genetic engineering technology, and in particular to the application of the GhHD-ZIP7 protein and its encoding gene in regulating fiber length. Background Technology

[0002] Cotton is the world's most important natural fiber crop. Improving fiber quality has become the main goal of cotton breeding at present. The main indicators for measuring high-quality cotton include larger fiber length (≥30 mm), higher specific strength (≥30 cN·tex-1), and moderate micronaire value (3.5 - 4.5).

[0003] The period from 1 to 20 days after cotton flowering is a critical time for determining fiber length, and the regulatory role of homologous domain-leucine motif (HD-ZIP) transcription factors in fiber development during this period has not yet been revealed. Summary of the Invention

[0004] The purpose of this invention is to provide the application of GhHD-ZIP7 protein and its encoding gene in regulating fiber length, so as to solve the problems existing in the prior art.

[0005] To achieve the above objectives, the present invention provides the following solution: One of the technical solutions of the present invention is the application of the GhHD-ZIP7 gene in increasing the length of cotton fibers, wherein the nucleotide sequence of the GhHD-ZIP7 gene is shown in SEQ ID NO.1.

[0006] The second technical solution of the present invention is a method for increasing cotton fiber length by silencing or knocking out the GhHD-ZIP7 gene to increase cotton fiber length; the nucleotide sequence of the GhHD-ZIP7 gene is shown in SEQ ID NO.1.

[0007] The third technical solution of the present invention is the application of the GhHD-ZIP7 gene in the cultivation of cotton varieties with increased fiber length, wherein the nucleotide sequence of the GhHD-ZIP7 gene is shown in SEQ ID NO.1.

[0008] The fourth technical solution of the present invention is a method for cultivating cotton varieties with increased fiber length, comprising the following steps: silencing or knocking out the GhHD-ZIP7 gene to increase the fiber length of cotton; the nucleotide sequence of the GhHD-ZIP7 gene is shown in SEQ ID NO.1.

[0009] Based on the above technical solution, the present invention has the following technical effects: This invention, through the transformation of Arabidopsis thaliana and cotton using an overexpression vector of the GhHD-ZIP7 gene, revealed that the transgenic Arabidopsis had a significantly longer taproot length and a significantly shorter root hair length in the mature zone compared to the control (Columbia ecotype). Furthermore, the fiber length of the overexpressed cotton material was significantly reduced compared to the control (Zhongmian 113). Additionally, by constructing a silencing vector for the GhHD-ZIP7 gene and transforming it into cotton, the transgenic cotton with the silenced GhHD-ZIP7 gene showed a significantly longer mature fiber length compared to the control group. These findings indicate that the GhHD-ZIP7 gene regulates cotton fiber cell elongation. Attached Figure Description

[0010] Figure 1 This is a clone of the GhHD-ZIP7 gene.

[0011] Figure 2 To perform in vitro ligation of the plasmid pCAMBIA1300 digested with KpnI-SalI with the target gene GhHD-ZIP7.

[0012] Figure 3 To screen positive plants that overexpress Arabidopsis thaliana.

[0013] Figure 4 To identify the overexpression of Arabidopsis thaliana. Specifically: a. PCR detection of GhHD-ZIP7 overexpression positive lines in Arabidopsis thaliana; b. GhHD-ZIP7 gene expression level in the overexpression positive lines in Arabidopsis thaliana; c. Western blot detection of GhHD-ZIP7 overexpression positive lines in Arabidopsis thaliana.

[0014] Figure 5 The study included statistics on the taproot length of Arabidopsis thaliana. Specifically: a. Observation of taproot length in GhHD-ZIP7 Arabidopsis thaliana overexpression lines; b. Statistical analysis of taproot length.

[0015] Figure 6 This study included the statistical analysis of root hair length in Arabidopsis thaliana. Specifically: a. Observation of root hair length in GhHD-ZIP7 Arabidopsis thaliana overexpression lines; b. Statistical analysis of root hair length.

[0016] Figure 7 Identification of GhHD-ZIP7 positive plants. A. DNA identification results; b. GhHD-ZIP7 relative expression level detection results.

[0017] Figure 8 The fiber length of GhHD-ZIP7 overexpression lines was measured in vitro after 20 PDA culture. Specifically, a. fiber length was observed in WT and GhHD-ZIP7 transgenic lines; b. the mature fiber length of wild-type and transgenic lines was statistically analyzed.

[0018] Figure 9The study analyzed the mature fiber length of GhHD-ZIP7 overexpression lines. Specifically, a. the mature fiber length of WT and GhHD-ZIP7 overexpression lines was observed, and b. the mature fiber length of wild-type and overexpression lines was statistically analyzed.

[0019] Figure 10 Analysis of mature fiber length in GhHD-ZIP7 knockout lines. Specifically, a. mature fiber length was observed in WT and GhHD-ZIP7 knockout lines; b. mature fiber length was statistically analyzed in wild-type and knockout lines. Detailed Implementation

[0020] Unless otherwise specified, the technical solutions described in this invention are all conventional solutions in the field, and the reagents or raw materials used are all purchased from commercial channels or are publicly available unless otherwise specified.

[0021] This invention provides the application of the GhHD-ZIP7 gene in increasing cotton fiber length, and the nucleotide sequence of the GhHD-ZIP7 gene is shown in SEQ ID NO.1.

[0022] In some specific implementations, silencing or knocking out the GhHD-ZIP7 gene increases the fiber length of cotton.

[0023] This invention also provides a method for increasing cotton fiber length by silencing or knocking out the GhHD-ZIP7 gene to increase cotton fiber length; the nucleotide sequence of the GhHD-ZIP7 gene is shown in SEQ ID NO.1.

[0024] This invention also provides the application of the GhHD-ZIP7 gene in cultivating cotton lines with increased fiber length, the nucleotide sequence of which is shown in SEQ ID NO.1.

[0025] In some specific implementations, silencing or knocking out the GhHD-ZIP7 gene increases the fiber length of cotton.

[0026] This invention also provides a method for cultivating cotton varieties with increased fiber length, comprising the following steps: silencing or knocking out the GhHD-ZIP7 gene to increase the fiber length of cotton; the nucleotide sequence of the GhHD-ZIP7 gene is shown in SEQ ID NO.1.

[0027] Example 1 1. Cloning of the cotton GhHD-ZIP7 gene 1.1 Required tools, enzymes, reagent kits, and chemicals (1) Enzymes and kits: KOD-Plus-Neo high-fidelity PCR amplification enzyme, Target Clone-Plus blunt-end A-addition enzyme, T4 DNA Ligase, and SYBR® Green Realtime PCR qRT-PCR fluorescent quantitative enzyme were purchased from TOYOBO Biotechnology Co., Ltd.; Gel Extraction Kit was purchased from Omega Biotechnology Co., Ltd.; DNA Purification Kit PCR product purification kit and PrimeScript™ RTase RNA reverse transcription kit were purchased from TaKaRa Biotechnology Co., Ltd.; DNA Marker III and GelStain EB substitutes were purchased from TransGen Biotechnology Co., Ltd.; RNAprep pure plant total RNA extraction kit was purchased from Tiangen Biotechnology Co., Ltd.; RNAprep pure yeast total RNA extraction kit was purchased from Promega Biotechnology Co., Ltd.; primers used in the experiment were synthesized by GENEBITDA Biotechnology Co., Ltd. in Beijing; Escherichia coli competent cells (DH5α) were purchased from Shanghai Sangon Biotech Co., Ltd.; restriction endonucleases BamHI and SacI were purchased from NEB.

[0028] (2) Other drugs: Agarose was purchased from TransGen Biotech Co., Ltd., peptone, yeast extract, chloroform, isoamyl alcohol, ethanol, isopropanol, sodium chloride and other drugs were domestically produced analytical grade, and ampicillin and other drugs were purchased from Boge Biotechnology Co., Ltd. (Dalian).

[0029] (3) Solution preparation: All reagents mentioned in this article but not listed were prepared according to the method in the third edition of Molecular Cloning Laboratory Manual. Biochemical reagents were of analytical grade or higher.

[0030] (4) LB liquid medium: 10 g / L tryptone, 5 g / L yeast extract, 10 g / L sodium chloride (NaCl); LB solid medium: 10 g / L tryptone, 5 g / L yeast extract, 10 g / L sodium chloride (NaCl), 15 g / L agar powder, bring the volume to 1 L; LB selective medium: before plate the LB medium, add the corresponding concentration of antibiotics when the medium has been autoclaved and cooled to 55 °C, shake well and then plate.

[0031] (5) Main instruments: PCR amplification instrument (BIO-RAD), high-speed centrifuge (Hettich MIKRO 200R), electrophoresis equipment (BIO-RAD), gel imaging system (BIO-RAD), and real-time PCR instrument (ABI7500).

[0032] 1.2 Cloning of GhHD-ZIP7 The reference sequence for GhHD-ZIP7 was derived from the TM-1 genome (Nanjing Agricultural University), with the gene number Gh_A11G107200. Primers were designed using Oligo 6 software, and a complete open reading frame (ORF) of 720 bp encoding 239 amino acid residues was amplified from upland cotton material Zhongmian Institute 36 using PCR (Polymerase Chain Reaction) technology.

[0033] The gene ORF sequence is (SEQ ID NO.1): >GhHD-ZIP7 .

[0034] The amino acid sequence is (SEQ ID NO.2): >GhHD-ZIP7 MLDGEEYREEMGEPFSSVAQVTPTKKKKNKNKRRFSDEQIKSLELMFESETRLEPRKKLQVAKELGLQPRQVAIWFQNKRARWKSKQLERDYTILQANYDLLASKYESLKREKQALLTQ LQKLNDLIKKPKEEEQCCGQVNGMRCSEGASDKGETTVKSDSEGQLSLSMGRSEHALGALSDDDSAIRTDYFGLEEEPNLMSMVDPADGSLSSPEDWRSLDSDGLFDQSPCGYQWWDFWS .

[0035] 1.3 The specific process of gene cloning (1) The experimental material, No. 113 of the Cotton Research Institute, Chinese Academy of Agricultural Sciences, was planted in the experimental field and managed as usual. Fiber tissue was taken at 5, 10 and 15 days after flowering. The collected materials were quickly frozen in liquid nitrogen and stored at -70℃ for later use. Total RNA was extracted from the plants using a kit from TIANGEN.

[0036] (2) 500 ng RNA was reverse transcribed into cDNA, and the reverse transcription product cDNA solution was diluted 4 times as a PCR reaction template.

[0037] Using total RNA extracted from cotton fibers at different stages as a template, the TaKaRa reverse transcription kit was used to reverse transcribe it into cDNA. The reaction system is shown in Table 1.

[0038] Table 1 Reaction System

[0039] Note: The reaction process is first incubated at 37°C for 15 min in the PCR instrument, then at 85°C for 5 s, and finally stored at -20°C for later use.

[0040] (3) Perform PCR reaction to amplify the target gene Primers for GhGLU18A were designed using Oligo6 software. The mixed cDNA obtained from reverse transcription was used as a template for PCR amplification of the cotton GhGLU18A gene. The reaction system is shown in Table 2.

[0041] Table 2 Target Gene Amplification System

[0042] The PCR reaction program was as follows: pre-denaturation at 94℃ for 2 min; denaturation at 98℃ for 10 s, annealing at 52℃ for 15 s, extension at 68℃ for 70 s, for 35 cycles; final extension at 68℃ for 10 min.

[0043] Primer sequences: GhHD-ZIP7 - F (SEQ ID NO.3): 5′-ATGTTAGATGGGGAAGAATATAGAGA-3′; GhHD-ZIP7 - R (SEQ ID NO. 4): 5′-TCAAGACCAAAAATCCCACC-3′.

[0044] (4) After the reaction is complete, store at 4℃ and detect by 1% agarose gel electrophoresis. If the band size meets the expected design, it is considered a valid result. Figure 1 ).

[0045] (5) The target fragment was cut and recovered using a gel recovery kit.

[0046] (6) The product recovered from the above gel was linked to the PMD18-T vector and transformed into Escherichia coli.

[0047] (7) Overnight culture at 37°C: Pick single clones from the resistant LB medium and culture them in 600 μL LB medium containing Amp at 37°C for 4 h.

[0048] (8) PCR verification of bacterial culture: send bacterial culture containing the target gene fragment size band for sequencing. The sequencing results are consistent with the gene sequence.

[0049] Construction and transfection of pCAMBIA1300-35S:: GhHD-ZIP7 Arabidopsis expression vector 2.1 Plasmid extraction and enzyme digestion Plasmid extraction was performed using an OMEGA plasmid extraction kit. The plasmid concentration was measured to be 180 ng / μl. Agarose gel electrophoresis showed no protein contamination, meeting the experimental requirements. KpnI-SalI was selected as the restriction enzyme for double digestion.

[0050] 2.2 Construction of vectors overexpressing Arabidopsis thaliana Target gene ORF sequence amplification: Based on the map of the final vector pCAMBIA1300, KpnI-SalI was designed as the insertion site and primers were synthesized. The primer sequences are as follows: In-GhHD-ZIP7 -F (SEQ ID NO.5): 5′- ggacgagctcggtaccATGTTAGATGGGGAAGAATATAGAGA -3′ (containing enzyme cleavage site KpnI); In-GhHD-ZIP7 - R (SEQ ID NO.6): 5′- ctcctttactcatgtcgacTCAAGACCAAAAATCCCACC -3′ (containing the SalI restriction site).

[0051] The target fragment was obtained by amplification using a high-fidelity enzyme.

[0052] Table 3 Target Gene Amplification System

[0053] (1) PCR reaction procedure: Pre-denaturation at 98℃ for 5 min; cycle 98℃ denaturation for 10 s, annealing at 60℃ for 30 s, extension at 68℃ for 1 min, for a total of 30 cycles; extension at 68℃ for 5 min.

[0054] (2) Construction of integrated expression carriers, such as Figure 2 As shown.

[0055] 2.3 The connection system is shown in Table 4.

[0056] Table 4. Ligation system of target gene and pET32a recombinant plasmid

[0057] a. Whisk until well mixed, centrifuge briefly, then add a drop of mineral oil; b. Connect at 16℃ for 2 hours; c. After connection, store in a 4°C refrigerator overnight.

[0058] 2.4 Conversion of Linkage Products a. Sterilize in a clean bench for 30 min, take out 100 μL of competent cells from a -70℃ ultra-low temperature freezer, place on ice, and pre-cool for 10 min; b. Take out an Eppendorf tube, mark it, place it on ice, and add 80 μL of competent cells (operate on ice); c. Then add 10 μL of the ligation product, mix well with a pipette, and incubate on ice for 30 min. d. After the ice bath, heat shock it in a constant temperature water bath at 42℃ for 90 seconds, then quickly put it into ice and ice bath for 2 minutes; e. Pipette 500 μL of Amp-free LB liquid culture medium into an Ep tube, mix well, and place in a shaker at 160 rpm and 37°C for 1 h. f. Remove the Ep tube after shaking, centrifuge at 2000-3000 rpm for 5 min, discard 300 μL of supernatant, gently aspirate and mix the remaining bacterial culture, add it to an LB solid culture dish containing Amp, spread it evenly with a glass spreader, and let it dry. g. Incubate at 37℃ for 16–20 h; h. Randomly select single clones for PCR verification.

[0059] 2.5 Transformation and identification of Col-0 Arabidopsis thaliana When Arabidopsis thaliana is in full bloom, infection can be achieved through the inflorescence inoculation method. Sufficient water should be applied to the Arabidopsis thaliana one day in advance. After infection, the Arabidopsis thaliana should be placed in darkness for 24 hours, then removed and placed in a greenhouse to allow for normal growth.

[0060] After maturity, the seeds were harvested and dried at 37°C for one week, then sterilized with 75% alcohol and vernalized at 4°C for 3 days. The seeds were then spotted in 1 / 2 MS medium containing hygromycin and placed horizontally in an incubator (16 hours light / 8 hours darkness, 22°C). After two weeks of growth, seedlings with true leaves and normal root development were considered positive plants and transferred to a greenhouse for further growth.

[0061] DNA, RNA, and protein were extracted from positive Arabidopsis thaliana plants to further determine the expression level of GhHD-ZIP7. DNA extraction was performed using the CTAB method. Results are as follows: Figure 4 As shown, this indicates that Arabidopsis thaliana plants overexpressing GhHD-ZIP7 were successfully obtained. Propagation continued until homozygous lines were obtained.

[0062] 3. Phenotypic identification of Arabidopsis thaliana overexpressing GhHD-ZIP7 Five days after seed germination, the taproot length of wild-type Arabidopsis thaliana and three overexpression lines was observed. The average taproot length of GhHD-ZIP7 Arabidopsis thaliana (1.38 cm) was significantly increased compared to the control (1.23 cm). Figure 5 ).

[0063] Meanwhile, root hair length was observed under a research-grade stereomicroscope, and statistical analysis revealed that the root hair length of Arabidopsis thaliana overexpressing GhHD-ZIP7 was significantly lower than that of the control. Figure 6 ).

[0064] 4. Validation of the function of GhHD-ZIP7 overexpression in cotton transformation 4.1 Transformation and Identification of GhHD-ZIP7 Overexpression in Cotton Will GhHD-ZIP7The CDS was constructed into the WMV067 vector, and after correct Sanger sequencing, it was transformed into Agrobacterium GV3101. Genetic transformation was performed using cotton seed apical meristem stem cells as the target. The explants were placed in Agrobacterium infection solution and sonicated, then shaken at room temperature for 50 min. The explants were then dried on sterile filter paper, transferred to clean filter paper containing culture medium, and incubated in the dark at 23°C for 2-3 days. Next, the explants were transferred to recovery medium and incubated at 35°C for 3 days. Finally, the culture dishes were incubated at 25°C for approximately 15 days until positive seedlings appeared. The obtained overexpression positive lines were identified: GhHD-ZIP7 Design specific primers based on CDS sequence, extract total DNA, such as Figure 7 As shown in Figure A, the target sequence was amplified by PCR. Then, RNA was extracted from the leaves for expression level determination. Figure 7 (B). After one generation of self-pollination, T1 generation seeds were obtained and used for subsequent phenotypic observations.

[0065] 4.2 Phenotypic Observation of GhHD-ZIP7 Overexpression in Cotton The T1 generation overexpression-positive lines were planted at the experimental farm of the Cotton Research Institute, Chinese Academy of Agricultural Sciences, in Anyang, Henan Province. Ovules from the day of flowering were selected for in vitro culture experiments. Culture media, auxin, gibberellin, and glucose were all purchased from Coollab Biotechnology Co., Ltd.

[0066] The specific steps are as follows: 1. Collect ovules on the day of flowering, remove petals, and retain ovary and pedicel.

[0067] 2. Disinfect with 75% alcohol, disinfect with anhydrous ethanol for 30 seconds, and finally rinse thoroughly with sterile water.

[0068] 3. Use a scalpel to cut open the shell, being careful not to damage the ovule. Carefully remove the ovule with forceps. Place it in BT liquid medium and incubate in the dark at 30°C. The working concentrations of IAA and GA are both 5 μM. The entire process is performed in a clean bench.

[0069] When the ovules were cultured to 20 DPA, this invention found that the surface of the ovules of the overexpression line generally turned yellowish-brown, and their growth was restricted. At this time, this invention measured the fiber length and found that the fiber length of the overexpression line was significantly shorter than that of the control. Figure 8 The average length of 20DPA fibers in OE-8 and OE-13 strains was 18.32 mm, while the average length of 20DPA fibers in Zhongmian 113 strains was 21.14 mm.

[0070] Simultaneously, the present invention also measured the mature fiber length of the overexpression line and the control material, and the data further showed that the fiber length of the overexpression line was significantly reduced compared with the control. Figure 9The average fiber length of the overexpression lines planted in the field was 25.17 mm, while the average fiber length of Zhongmian 113 was 27.61 mm.

[0071] Finally, this invention also constructed a GhHD-ZIP7 gene knockout vector and performed genetic transformation using cotton seed apical meristem stem cells as the target. The results showed that the mature fiber length of the GhHD-ZIP7 gene knockout line was significantly longer than that of the control group. Figure 10 The average fiber length of the greenhouse-grown knockout lines was 31.13 mm, while the average fiber length of Zhongmian 113 was 29.49 mm.

[0072] The above findings indicate that GhHD-ZIP7 is a key gene regulating cotton fiber length.

[0073] Obviously, the above embodiments of the present invention are merely examples for clearly illustrating the present invention, and are not intended to limit the implementation of the present invention. For those skilled in the art, other variations or modifications can be made based on the above description. It is neither necessary nor possible to exhaustively describe all embodiments here. Any modifications, equivalent substitutions, and improvements made within the spirit and principles of the present invention should be included within the scope of protection of the claims of the present invention.

Claims

1. Application of GhHD-ZIP7 gene in improving cotton fiber length, characterized in that, The nucleotide sequence of the GhHD-ZIP7 gene is shown as SEQ ID NO.

1.

2. Use according to claim 1, characterized in that, Silencing or knocking out the GhHD-ZIP7 gene can improve the fiber length of cotton.

3. A method of increasing the length of cotton fibers, characterized by, Silencing or knocking out the GhHD-ZIP7 gene can improve the fiber length of cotton; the nucleotide sequence of the GhHD-ZIP7 gene is shown as SEQ ID NO.

1.

4. Use of the GhHD-ZIP7 gene in breeding cotton lines with increased fiber length, characterized in that, The nucleotide sequence of the GhHD-ZIP7 gene is shown as SEQ ID NO.

1.

5. Use according to claim 4, characterized in that, Silencing or knocking out the GhHD-ZIP7 gene can improve the fiber length of cotton.

6. A method of breeding a cotton line having increased fiber length, comprising, The method comprises the following steps: Silencing or knocking out the GhHD-ZIP7 gene can improve the fiber length of cotton; the nucleotide sequence of the GhHD-ZIP7 gene is shown as SEQ ID NO. 1.

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