Use of cotton hd-zip transcription factor ghhb16 in improving lint percentage of cotton

Abstract

The application provides application of cotton HD-ZIP transcription factor GhHB16 in improving lint percentage of cotton, and belongs to the technical field of plant genetic engineering. The application provides application of HD-ZIP transcription factor GhHB16 or a gene encoding the HD-ZIP transcription factor GhHB16 in negatively regulating cotton lint percentage and / or cultivating cotton with an advantage of lint percentage. GhHB16 The lint percentage is significantly improved. Therefore, the HD-ZIP transcription factor GhHB16 or the gene encoding the HD-ZIP transcription factor GhHB16 can improve the lint percentage of cotton, provide important genetic resources for cultivating excellent cotton varieties with high lint percentage and high yield, and have wide application value.

Description

Technical Field

[0001] This invention relates to the field of plant genetic engineering technology, specifically to the application of a cotton HD-ZIP transcription factor GhHB16 in improving cotton lint percentage. Background Technology

[0002] Cotton, belonging to the genus Gossypium of the Malvaceae family, is an important natural fiber crop and one of the most important oil crops. With the development of the global textile industry and population growth, the demand for cotton continues to increase, and cotton production and economic benefits directly determine the sustainable development of the cotton industry.

[0003] Cotton yield is mainly affected by traits such as boll number per plant, boll weight, and lint percentage. Among these, lint percentage (the percentage of lint cotton to seed cotton) is a crucial indicator of cotton's economic yield. A high lint percentage means that more lint cotton can be obtained with the same seed cotton yield, directly increasing lint cotton production and economic benefits. Therefore, improving the yield and lint percentage of cotton varieties is a key task in enhancing cotton production efficiency and economic value. In recent years, improving crop yield traits through molecular biology methods has become a research hotspot; however, reports on key genes regulating cotton yield and lint percentage remain relatively scarce. Summary of the Invention

[0004] In view of this, the present invention provides the application of HD-ZIP transcription factor GhHB16 or the gene encoding said HD-ZIP transcription factor GhHB16 in negatively regulating cotton lint percentage and / or cultivating cotton with lint percentage dominance.

[0005] To achieve the above objectives, the present invention provides the following technical solution: This invention provides the application of HD-ZIP transcription factor GhHB16 or the gene encoding said HD-ZIP transcription factor GhHB16 in regulating cotton lint percentage and / or cultivating cotton with lint percentage advantage, wherein said HD-ZIP transcription factor GhHB16 or said gene increases cotton lint percentage through negative regulation. The HD-ZIP transcription factor GhHB16 includes GhHB16A and GhHB16D; The amino acid sequence of GhHB16A is shown in SEQ ID NO: 1; The amino acid sequence of GhHB16D is shown in SEQ ID NO: 2.

[0006] This invention provides the application of sgRNA or a derivative containing said sgRNA in increasing cotton lint percentage and / or cultivating cotton with lint percentage advantage; The nucleotide sequence of the DNA molecule targeted by the sgRNA is shown in SEQ ID NO: 3; The derivatives containing the sgRNA include recombinant vectors and / or recombinant bacteria; The application includes transferring a derivative containing the sgRNA into a recipient plant.

[0007] This invention provides a method for increasing cotton lint percentage, comprising: inhibiting the biological function of HD-ZIP transcription factor GhHB16 in plants or inhibiting the expression of the gene encoding said HD-ZIP transcription factor GhHB16 to increase cotton lint percentage; The HD-ZIP transcription factor GhHB16 includes GhHB16A and GhHB16D; The amino acid sequence of GhHB16A is shown in SEQ ID NO: 1; The amino acid sequence of GhHB16D is shown in SEQ ID NO: 2.

[0008] Preferably, a reagent containing sgRNA or a derivative containing said sgRNA is transferred into a recipient plant to inhibit the biological function of the HD-ZIP transcription factor GhHB16 or the expression of said gene in the plant. The derivatives containing the sgRNA include recombinant vectors and / or recombinant bacteria; The nucleotide sequence of the DNA molecule targeted by the sgRNA is shown in SEQ ID NO: 3.

[0009] This invention provides a method for breeding and / or assisting in the breeding of cotton with superior lint content, comprising: detecting the gene encoding the HD-ZIP transcription factor GhHB16 in the genome of a plant, and selecting plants with mutations in the gene as breeding materials for breeding; The HD-ZIP transcription factor GhHB16 includes GhHB16A and GhHB16D; The preferred nucleotide sequence of the mutated gene encoding GhHB16A is shown in SEQ ID NO: 6, and the preferred nucleotide sequence of the mutated gene encoding GhHB16D is shown in SEQ ID NO: 7.

[0010] This invention provides a type of cotton in which the gene encoding the HD-ZIP transcription factor GhHB16 is knocked out; The HD-ZIP transcription factor GhHB16 includes GhHB16A and GhHB16D; The amino acid sequence of GhHB16A is shown in SEQ ID NO: 1; The amino acid sequence of GhHB16D is shown in SEQ ID NO: 2.

[0011] Compared with the prior art, the present invention has the following advantages: This invention provides the application of HD-ZIP transcription factor GhHB16 or the gene encoding said HD-ZIP transcription factor GhHB16 in regulating cotton lint percentage and / or cultivating cotton with lint percentage dominance. The HD-ZIP transcription factor GhHB16 or the gene increases cotton lint percentage through negative regulation. The HD-ZIP transcription factor GhHB16 includes GhHB16A and GhHB16D. The amino acid sequence of GhHB16A is shown in SEQ ID NO: 1; the amino acid sequence of GhHB16D is shown in SEQ ID NO: 2. Examples have verified that knocking out the gene encoding said HD-ZIP transcription factor GhHB16 in cotton (…) GhHB16 The percentage of lye cells increased significantly after HD-ZIP. Therefore, the transcription factors GhHB16 and... GhHB16 Genes can be used to increase cotton lint percentage, and this invention provides important genetic resources and an ideal approach for breeding high-yield cotton varieties. Attached Figure Description

[0012] Figure 1 The spectrum of the knockout vector pRGEB32-GhU6.7-GhHB16; Figure 2 for GhHB16 The gel image shows the positive detection results of Cas9 knockout lines. Lane M represents the marker electrophoresis results, from top to bottom: 100bp, 250bp, 500bp, 750bp, 1000bp, 2000bp, 3000bp and 5000bp. Lane CR1 represents the electrophoresis results of GhHB16 knockout line GhHB16-CR. Lane P represents the electrophoresis results of positive plasmids. Lane N represents the electrophoresis results of wild-type recipient material. Figure 3 for GhHB16 Gel images showing the NPTII positive detection results of the knockout strain; lane M represents the marker electrophoresis results, from top to bottom: 100bp, 250bp, 500bp, 750bp, 1000bp, 2000bp, 3000bp and 5000bp, lane CR1 represents the GhHB16-CR electrophoresis results, lane P represents the positive plasmid electrophoresis results, and lane N represents the wild-type receptor material electrophoresis results. Figure 4 for GhHB16Gel image showing the positive results of sgRNA knockout line; lane M represents the marker electrophoresis result, from top to bottom: 100bp, 250bp, 500bp, 750bp, 1000bp, 2000bp, 3000bp and 5000bp, lane CR1 represents the GhHB16-CR electrophoresis result, lane P represents the positive plasmid electrophoresis result, and lane N represents the wild-type receptor material electrophoresis result. Figure 5 for GhHB16 Image of the detection results of the knockout strain Hi-TOM; Figure 6 for GhHB16 A bar chart showing the lint percentage statistics of knockout lines; where T1, T2, and T3 represent different generations of transgenic cotton. This indicates that P < 0.05; This indicates that P < 0.01; Figure 7 for GhHB16 Scanning electron microscope image of fibrous protrusions in knockout strains; Figure 8 for GhHB16 A statistical chart showing the number of fibrous protrusions in knockout strains; among which... This means P < 0.001. Detailed Implementation

[0013] This invention provides the application of HD-ZIP transcription factor GhHB16 or the gene encoding said HD-ZIP transcription factor GhHB16 in regulating cotton lint percentage and / or cultivating cotton with lint percentage dominance. The HD-ZIP transcription factor GhHB16 or said gene increases cotton lint percentage through negative regulation. The HD-ZIP transcription factor GhHB16 comprises GhHB16A and GhHB16D. The amino acid sequence of GhHB16A is shown in SEQ ID. NO: 1 (MKRVGSSHSLGAMMSICPISDDNQIYSREFQSILDGLDEEEGVEESGYVAEKKRRLNVDQVKALEKDFEVENKLDPGRKLKLAQQLGLRPRQVAVWFQNRRARWKTKQLEKDYGLLKNRYETLKLNYDNLQHDNQVLLKQIEEVKAKLNGKDNVS VKEEVNVTKTANRTLEQSEAPVEVKYESLKNNLFLDLKDGSSDSDSSAVLNEDNNNGSNYVGVSSSGVLQSQHVWMSPTTAPSHNFNSSSSSSMKCFQPQQFVKMEEKNFFSADEACKFFSDEEAPSLHWYCPEHWN); the amino acid sequence of GhHB16D is as SEQ ID NO: 2 shown (MKRVGSSHSLGAMMSICPISDDNQIYSREFQSILDGLDEEEGVEESGYVAEKKRRLNVDQVKALEKDFEVENKLDPGRKLKLAQQLGLRPRQVAVWFQNRRARWKTKQLEKDYGLLKNRYETLKLNYDSLQHDNQVLLKQIEEVKAK LNGKNNVSVKEEVNVTKTANRTLEQSEAPVEVKYESLKNNSKGSNGAILFLDLKDGSSDSDSSAVLNEDNNNGSNYVGVSSSGVLQSQHVWMSPTTASSLNFNSSSSMKCLQPQQFVKMEEQNFFSADEACKFFSDEEAPSLHWYCPEHWN).

[0014] In the present invention, the nucleotide sequence of the gene encoding said GhHB16A is preferably as shown in SEQ ID NO: 4 (ATGAAAAGAGTTGGCAGCTCACATTCCTTGGGTGCTATGATGTCCATCTGCCCAATCTCAGATGACAACCAGATTTACAGTAGAGAGTTTCAGTCGATTTTAGATGGATTAGATGAAGAAGAGGGCGTGGAAGAATCAGGGTATGTTGCAGAAAAGAAGAGGCGATTGAATGTAGATCAAGTGAAGGCGTTGGAGAAGGATTTCGAGGTGGAAAACAAACTTGATCCTGGAAGAAAATTGAAACTAGCTCAACAACTTGGCTTGCGACCTCGACAAGTTGCTGTCTGGTTCCAAAACCGCCGTGCTAGGTGGAAGACCAAACAACTGGAGAAGGATTATGGGCTTCTCAAAAACAGATATGAAACTCTTAAGCTCAATTATGATAACCTCCAGCATGATAATCAAGTTCTTCTTAAACAGATAGAGGAGGTGAAGGCAAAGCTGAATGGAAAGGACAATGTTTCAGTGAAGGAGGAGGTGAATGTGACTAAAACTGCTAATAGAACACTAGAACAAAGTGAAGCACCAGTAGAAGTGAAGTATGAGAGCTTGAAGAACAACCTTTTCCTAGACTTAAAAGACGGTTCATCAGATAGTGACTCTAGCGCTGTGTTGAATGAAGACAACAACAACGGCTCGAACTACGTGGGTGTATCTTCATCTGGGGTTCTACAAAGCCAACATGTTTGGATGTCACCGACAACAGCCCCTTCACACAATTTCAACTCCTCTTCATCTTCTTCTATGAAGTGCTTCCAACCCCAGCAATTTGTGAAGATGGAAGAAAAAAATTTCTTCAGTGCAGATGAAGCTTGCAAGTTCTTCTCAGATGAAGAAGCTCCAAGTCTTCATTGGTATTGTCCTGAACACTGGAACTAA);Preferably, the nucleotide sequence of the gene encoding said GhHB16D is as shown in SEQ ID NO: 5 (ATGAAAAGAGTTGGCAGCTCACATTCCTTGGGTGCTATGATGTCCATCTGCCCAATCTCAGATGACAACCAGATTTACAGTAGAGAGTTTCAGTCGATTTTAGATGGATTAGATGAAGAAGAGGGCGTGGAAGAATCAGGGTATGTTGCAGAAAAGAAGAGGCGATTGAATGTAGATCAAGTGAAAGCGTTGGAGAAGGATTTCGAGGTGGAAAACAAACTTGATCCTGGGAGGAAATTGAAACTAGCTCAACAACTTGGCTTGCGACCTCGACAAGTTGCTGTCTGGTTCCAAAACCGCCGTGCTAGGTGGAAGACTAAACAACTGGAGAAGGATTATGGGCTTCTCAAAAACAGATATGAAACTCTTAAGCTAAATTATGATAGCCTCCAGCATGACAATCAAGTTCTTCTTAAACAGATAGAGGAGGTGAAGGCAAAGCTGAATGGAAAGAACAATGTTTCAGTGAAGGAGGAGGTGAATGTGACTAAAACTGCTAATAGAACACTAGAACAAAGTGAAGCACCAGTAGAAGTGAAGTATGAGAGCTTGAAGAACAACAGCAAGGGATCAAATGGGGCCATCCTTTTCCTAGACTTAAAAGACGGTTCATCAGATAGTGACTCAAGCGCTGTCTTGAATGAAGACAACAACAACGGCTCGAATTACGTGGGTGTATCTTCATCTGGGGTTCTACAAAGCCAACATGTTTGGATGTCACCGACAACAGCCTCTTCACTCAATTTCAACTCTTCATCTTCTTCTATGAAGTGCCTCCAACCCCAGCAATTTGTGAAGATGGAAGAACAAAATTTCTTTAGTGCAGATGAAGCTTGCAAGTTCTTCTCAGATGAAGAAGCTCCAAGTCTTCATTGGTATTGCCCTGAACACTGGAACTAA).;

[0015] In this invention, the HD-ZIP transcription factor GhHB16 or the gene increases the number of ovule protrusions and cotton lint percentage through negative regulation. This invention does not specifically limit the variety of cotton; the cotton can be upland cotton and / or sea island cotton, for example, JIN668, Lumianyan 28, Tongza 411, Ezamian 29, and Xinluzao 42. In the embodiments of this invention, comparisons were made... GhHB16 The changes in phenotype between gene knockout lines and wild-type lines showed that, compared with the wild-type, GhHB16 The significantly increased lint percentage in the gene knockout lines indicates that the HD-ZIP transcription factor GhHB16 or the gene itself increases cotton lint percentage through negative regulation.

[0016] This invention provides the application of sgRNA or a derivative containing said sgRNA in increasing cotton lint percentage and / or cultivating cotton with lint percentage advantage; The nucleotide sequence of the DNA molecule targeted by the sgRNA is shown in SEQ ID NO: 3 (CGACAAGTTGCTGTCTGGTT). The derivatives containing the sgRNA include recombinant vectors and / or recombinant bacteria; The application includes transferring a derivative containing the sgRNA into a recipient plant.

[0017] In this invention, the recombinant vector is preferably a backbone vector containing the sgRNA. The backbone vector preferably includes a CRISPR / Cas9 vector, more preferably a pRGEB32-GhU6.7 vector (disclosed in the prior art Wang P, Zhang J, Sun L, et al. High efficient multisites genome editing in allotetraploidcotton (Gossypium hirsutum) using CRISPR / Cas9 system[J]. Plant biotechnologyjournal, 2018, 16(1): 137-150.), and the multiple cloning site of the backbone vector is preferably... Bsa I. This invention references the cotton genome sequence and the CRISPR-P website (http: / / crispr.hzau.edu.cn / cgi-bin / CRISPR2 / CRISPR) to search for targets, designs primers for sgRNA for PCR amplification, and utilizes... BsaThe empty vector pRGEB32-GhU6.7 was digested with enzymes, purified, and then ligated with an Exnase enzyme to construct the recombinant vector (pRGEB32-GhU6.7-GhHB16). After obtaining the recombinant vector, the process preferably includes heat shock transformation of *E. coli* TOP10, identification of positive clones, and propagation. The recombinant bacteria of this invention are preferably host bacteria containing the recombinant vector. The host bacteria preferably include *Agrobacterium*, more preferably *Agrobacterium* EHA105. The recombinant vector is preferably transformed into the host bacteria by electroporation to obtain the recombinant bacteria. The recombinant bacteria can transfer the recombinant vector into plants, and... GhHB16 Genes undergo targeted mutations.

[0018] In this invention, transferring the sgRNA or its derivative into plants can [achieve the desired effect]. GhHB16 Genes undergo targeted mutations to obtain GhHB16 Gene knockout lines increase cotton lint percentage.

[0019] This invention provides a method for increasing cotton lint percentage, comprising: inhibiting the biological function of HD-ZIP transcription factor GhHB16 in plants or inhibiting the expression of the gene encoding said HD-ZIP transcription factor GhHB16 to increase cotton lint percentage; said HD-ZIP transcription factor GhHB16 includes GhHB16A and GhHB16D; the amino acid sequence of said GhHB16A is shown in SEQ ID NO: 1; the amino acid sequence of said GhHB16D is shown in SEQ ID NO: 2.

[0020] In this invention, the cotton is preferably the same as in the above applications, and will not be repeated here. In this invention, the method preferably involves transferring a reagent containing the sgRNA or the derivative thereof into a recipient plant to inhibit the biological function of the HD-ZIP transcription factor GhHB16 or the expression of the gene in the plant; the nucleotide sequence of the DNA molecule targeted by the sgRNA is shown in SEQ ID NO: 3; the derivative containing the sgRNA is preferably the same as the derivative containing the sgRNA described in the above applications.

[0021] The method for transferring the transplanted plant preferably includes Agrobacterium-mediated genetic transformation. The recipient plant preferably includes a cotton hypocotyl. After the recipient plant is transferred, positive transformants are preferably screened using antibiotics, and then the positive transformants are used to obtain regenerated plants through tissue culture. The antibiotic preferably includes kanamycin. The working concentration of kanamycin is preferably 100 mg / L. Following antibiotic screening, gene detection and high-throughput sequencing are also performed.

[0022] In this invention, the gene detection reagent includes at least one of the following: a Cas9 primer pair, an NPTII primer pair, and an sgRNA primer pair. The Cas9 primer pair is designed for the Cas9 protein and includes Cas9-F (SEQ ID NO: 11) and Cas9-R (SEQ ID NO: 12), with a target gene fragment length of 999 bp. The NPTII primer pair is designed for the NPTII resistance gene and includes NPTII-F (SEQ ID NO: 13) and NPTII-R (SEQ ID NO: 14), with a target gene fragment length of 750 bp. The sgRNA primer pair preferably includes sgRNA-F (SEQ ID NO: 15) and sgRNA-R (SEQ ID NO: 16), with a target gene fragment length of 290 bp. When the gene detection reagent is used to perform PCR amplification on the target plant, if the corresponding band is amplified, it indicates that the reagent containing the sgRNA has been successfully transferred into the recipient plant. The preferred PCR amplification system is 20 μL: 16 μL ddH2O; 2 μL 10× EasyTaq Buffer; 0.4 μL dNTP; 0.2 μL Forward / Reverse Primer; 0.2 μL EasyTaq enzyme; 1 μL DNA. The preferred PCR amplification program is: 95℃ pre-denaturation for 5 min; 95℃ for 30 sec, 58℃ for 30 sec, 72℃ for 50 sec, 28 cycles; extension at 72℃ for 5 min. The high-throughput sequencing method preferably includes Hi-tom high-throughput sequencing technology. This embodiment of the invention utilizes Hi-tom high-throughput sequencing technology to analyze the editing sites of mutants. Detailed results of the mutant gene editing site analysis can be found in [link to relevant documentation]. Figure 5 .

[0023] In this invention, after the transfer into the recipient plant, the gene encoding the HD-ZIP transcription factor GhHB16 in the recipient plant is knocked out. The nucleotide sequence of the knocked-out gene encoding GhHB16A is preferably as shown in SEQ ID NO: 6 (ATGAAAAGAGTTGGCAGCTCACATTCCTTGGGTGCTATGATGTCCATCTGCCCAATCTCAGATGACAACCAGATTTACAGTAGAGAGTTTCAGTCGATTTTAGATGGATTAGATGAAGAAGAGGGCGTGGAAGAATCAGGGTATGTTGCAGAAAAGAAGAGGCGATTGAATGTAGATCAAGTGAAGGCGTTGGAGAAGGATTTCGAGGTGGAAAACAAACTTGATCCTGGAAGAAAATTGAAACTAGCTCAACAACTTGGCTTGCGACCTCGATGCTGTCTGGTTCCAAAACCGCCGTGCTAGGTGGAAGACCAAACAACTGGAGAAGGATTATGGGCTTCTCAAAAACAGATATGAAACTCTTAAGCTCAATTATGATAACCTCCAGCATGATAATCAAGTTCTTCTTAAACAGATAGAGGAGGTGAAGGCAAAGCTGAATGGAAAGGACAATGTTTCAGTGAAGGAGGAGGTGAATGTGACTAAAACTGCTAATAGAACACTAGAACAAAGTGAAGCACCAGTAGAAGTGAAGTATGAGAGCTTGAAGAACAACCTTTTCCTAGACTTAAAAGACGGTTCATCAGATAGTGACTCTAGCGCTGTGTTGAATGAAGACAACAACAACGGCTCGAACTACGTGGGTGTATCTTCATCTGGGGTTCTACAAAGCCAACATGTTTGGATGTCACCGACAACAGCCCCTTCACACAATTTCAACTCCTCTTCATCTTCTTCTATGAAGTGCTTCCAACCCCAGCAATTTGTGAAGATGGAAGAAAAAAATTTCTTCAGTGCAGATGAAGCTTGCAAGTTCTTCTCAGATGAAGAAGCTCCAAGTCTTCATTGGTATTGTCCTGAACACTGGAACTAA),The nucleotide sequence of the mutated GhHB16D-encoding gene is preferably as shown in SEQ ID NO: 7 (ATGAAAAGAGTTGGCAGCTCACATTCCTTGGGTGCTATGATGTCCATCTGCCCAATCTCAGATGACAACCAGATTTACAGTAGAGAGTTTCAGTCGATTTTAGATGGATTAGATGAAGAAGAGGGCGTGGAAGAATCAGGGTATGTTGCAGAAAAGAAGAGGCGATTGAATGTAGATCAAGTGAAAGCGTTGGAGAAGGATTTCGAGGTGGAAAACAAACTTGATCCTGGGAGGAAATTGAAACTAGCTCAACAACTTGGCTTGCGACCTCGATCAAGTTGCTGTCTGGTTCCAAAACCGCCGTGCTAGGTGGAAGACTAAACAACTGGAGAAGGATTATGGGCTTCTCAAAAACAGATATGAAACTCTTAAGCTAAATTATGATAGCCTCCAGCATGACAATCAAGTTCTTCTTAAACAGATAGAGGAGGTGAAGGCAAAGCTGAATGGAAAGAACAATGTTTCAGTGAAGGAGGAGGTGAATGTGACTAAAACTGCTAATAGAACACTAGAACAAAGTGAAGCACCAGTAGAAGTGAAGTATGAGAGCTTGAAGAACAACAGCAAGGGATCAAATGGGGCCATCCTTTTCCTAGACTTAAAAGACGGTTCATCAGATAGTGACTCAAGCGCTGTCTTGAATGAAGACAACAACAACGGCTCGAATTACGTGGGTGTATCTTCATCTGGGGTTCTACAAAGCCAACATGTTTGGATGTCACCGACAACAGCCTCTTCACTCAATTTCAACTCTTCATCTTCTTCTATGAAGTGCCTCCAACCCCAGCAATTTGTGAAGATGGAAGAACAAAATTTCTTTAGTGCAGATGAAGCTTGCAAGTTCTTCTCAGATGAAGAAGCTCCAAGTCTTCATTGGTATTGCCCTGAACACTGGAACTAA).

[0024] This invention provides a method for breeding and / or assisting in the breeding of cotton with superior lint content, comprising: detecting the gene encoding the cotton HD-ZIP transcription factor GhHB16 in the genome of a plant, and selecting gene-knockout plants as breeding materials for breeding; the HD-ZIP transcription factor GhHB16 includes GhHB16A and GhHB16D; the amino acid sequence of GhHB16A is shown in SEQ ID NO: 1; the amino acid sequence of GhHB16D is shown in SEQ ID NO: 2. The nucleotide sequence of the knockout gene encoding GhHB16A is preferably shown in SEQ ID NO: 6, and the nucleotide sequence of the knockout gene encoding GhHB16D is preferably shown in SEQ ID NO: 7.

[0025] The present invention provides a cotton in which the gene encoding the HD-ZIP transcription factor GhHB16 is knocked out; the HD-ZIP transcription factor GhHB16 includes GhHB16A and GhHB16D; the amino acid sequence of GhHB16A is shown in SEQ ID NO: 1; the amino acid sequence of GhHB16D is shown in SEQ ID NO: 2.

[0026] In this embodiment of the invention, comparisons were made. GhHB16 The changes in phenotype between gene knockout lines and wild-type lines showed that, compared with the wild-type, GhHB16 The number of ovule protrusions and the amount of lining were significantly increased in gene knockout lines.

[0027] To further illustrate the present invention, the solutions provided by the present invention will be described in detail below with reference to the accompanying drawings and embodiments, but these should not be construed as limiting the scope of protection of the present invention.

[0028] The culture formula used in the examples: MGL medium: tryptone 5 g / L, NaCl 5 g / L, MgSO4·7H2O 0.1 g / L, KH2PO4 0.25 g / L, mannitol 5 g / L, glycine 1 g / L, and distilled water to make up to 1 L; 2,4-D induction medium: MS medium was used as the basal medium, with the addition of 0.1 mg / L 2,4-D, 0.1 mg / L cytokinin (KT), 30 g / L glucose, and 2.5 g / L Phytagel, and then diluted to 1 L with distilled water. The pH was adjusted to 5.9. Embryo differentiation medium: MS medium was used as the basal medium, supplemented with 1.9 g / L KNO3, 0.1 mg / L KT, 30 g / L glucose, 1.0 g / L Gln, 0.5 g / L Asn, and 2.5 g / L Phytagel, and diluted to 1 L with distilled water. The pH was adjusted to 5.9. Rooting medium: Use 1 / 2 MS as the basal medium, add 15 g / L glucose and 2.5 g / L Phytagel, and make up to 1 L with distilled water. Adjust the pH to 5.9; The MS medium formulation is as follows: macroelements (KNO3 1.9 g / L, KH2PO4 0.17 g / L, MgSO4·7H2O 0.37 g / L, CaCl2·2H2O 0.44 g / L), microelements (KI 0.83 mg / L, H3BO3 6.2 mg / L, MnSO4·4H2O 22.3 mg / L, ZnSO4·4H2O 22.3 mg / L, ZnSO4·4H2O 22.3 mg / L). 4﹒ 7H2O 8.6 mg / L, Na2MoO4·2H2O 0.25 mg / L, CuSO4·5H2O 0.025 mg / L, CoCl2 0.025 mg / L, iron salts (Na2·EDTA 37.3 mg / L, FeSO4·7H2O 27.8 mg / L), organic components (inositol 100 mg / L, Gly 2 mg / L, VB1 0.1 mg / L, VB6 0.5 mg / L, VB5 0.5 mg / L).

[0029] Example 1 Construction of the knockout vector pRGEB32-GhU6.7-GhHB16 A single sgRNA sequence was recombined into the pRGEB32-GhU6.7 vector, which is disclosed in existing technology (Wang P, Zhang J, Sun L, et al. High efficient multisites genome editing inallotetraploid cotton (Gossypium hirsutum) using CRISPR / Cas9 system[J]. Plantbiotechnology journal, 2018, 16(1): 137-150). The vector contains a kanamycin resistance gene, and the specific construction method is as follows.

[0030] Referring to the CRISPR-P website http: / / crispr.hzau.edu.cn / cgi-bin / CRISPR2 / CRISPR, we searched for targets and selected sgRNA sequences located in exon regions with high scores. The nucleotide sequence of the DNA molecule targeted by the sgRNA is shown in SEQ ID NO: 3. Based on the sequence shown in SEQ ID NO: 3, we designed the primers used for vector construction as follows: pRGEB32-GhU6.7-F:AAGCATCAGATGGGCAAACAAAGCACCAGTGGTCTAG (SEQ ID NO: 8); CR-HB16-R:TTCTAGCTCTAAAACCGACAAGTTGCTGTCTGGTTTGCACCAGCCGGGAAT (SEQ ID NO: 9).

[0031] Using pGTR bacterial culture (disclosed in existing technology Wang P, Zhang J, Sun L, et al. High efficient multisites genome editing in allotetraploid cotton (Gossypium hirsutum) using CRISPR / Cas9 system[J]. Plant biotechnology journal, 2018, 16(1): 137-150.) as a template, PCR products containing tRNA-sgRNA were amplified using primers pRGEB32-GhU6.7-F and CR-HB2-R. Bsa The backbone vector pRGEB32-GhU6.7 was digested with enzymes at 37℃ for 6 h. Following digestion, gel electrophoresis was performed to recover the large fragment of the pRGEB32-GhU6.7 backbone vector. The PCR product and the linearized expression vector were then infused using exnase to obtain the ligation product containing the knockout vector pRGEB32-GhU6.7-GhHB16. The image of the knockout vector pRGEB32-GhU6.7-GhHB16 is shown below. Figure 1 The vector backbone pRGEB32-GhU6.7 contains two independent resistance gene systems: one for screening bacterial colonies containing the plasmid and the other for screening successfully transformed plants. Both systems use the same antibiotic, kanamycin.

[0032] The ligation reaction product was transformed into E. coli competent cells TOP10 (Sangon Biotech). After 10-12 hours, single clones were picked for PCR positive detection. Positive single clones were amplified and plasmids were extracted to obtain the knockout vector pRGEB32-GhU6.7-GhHB16.

[0033] The primers used for positive detection are U6-7S (TGTGCCACTCCAAAGACATCAG, SEQ ID NO: 10) and CR-HB16-R (SEQ ID NO: 9).

[0034] The PCR reaction system consisted of 20 μL: 16 μL ddH2O; 2 μL 10× EasyTaq Buffer; 0.4 μL dNTP; 0.2 μL each of forward and reverse primers; 0.2 μL EasyTaq enzyme; and 1 μL bacterial culture.

[0035] The PCR reaction conditions were as follows: 95 °C pre-denaturation for 5 min; 95 °C for 30 sec, 58 °C for 30 sec, 72 °C for 20 sec, 28 cycles; 72 °C extension for 5 min.

[0036] Example 2 Genetic transformation and screening and identification of knockout lines of the knockout vector pRGEB32-GhU6.7-GhHB16 A. Genetic transformation of the knockout vector pRGEB32-GhU6.7-GhHB16 Genetic transformation was performed using Agrobacterium-mediated transformation. The knockout vector pRGEB32-GhU6.7-GhHB16 from Example 1 was transformed into Agrobacterium EHA105 (Sangon Biotech) to obtain recombinant Agrobacterium EHA105, which was then cryopreserved for later use. The tested plant material was the upland cotton variety (JIN668). Plump and uniform JIN668 seeds were selected, the seed coat was removed, and the seeds were sterilized with 0.1% mercuric chloride solution for 10-12 minutes with continuous shaking. The seeds were then rinsed three times with sterile water and placed on the surface of MS medium. After incubation in the dark at 30°C for 1 day, the seedlings were propped up and incubated in the dark for another 5 days.

[0037] The glycerol tubes of recombinant Agrobacterium EHA105 stored in the ultra-low temperature freezer were thawed on ice. 10 μl of the thawed bacterial culture was added to 2 mL of LB broth containing 100 mg / L kanamycin and cultured at 28°C with shaking for 1 day. 20 μl of the activated bacterial culture was added to 20 mL of fresh LB broth containing 100 mg / L kanamycin and cultured at 28°C with shaking overnight. 1 mL of the turbid bacterial culture was transferred to a 2 mL sterile centrifuge tube and centrifuged at 12000 rpm for 30 s to collect the bacterial cells. The bacterial cells were resuspended in 20 mL of medium containing 50 mg / L acetylsyringone (AS) and cultured at 28°C with shaking for 30 min for use in infecting hypocotyls.

[0038] The specific steps of Agrobacterium-mediated transformation of cotton hypocotyls are as follows: (1) In a clean bench, take 30 sterile seedlings, cut the hypocotyl into 0.5-0.8cm segments on sterile filter paper and put them into a 50 mL sterile conical flask. Add the activated recombinant Agrobacterium EHA105 bacterial solution and infect for 5 min, shaking several times during the process. (2) Discard the bacterial solution, place the hypocotyl on sterile filter paper to absorb the surface bacterial solution, place it on a clean bench and blow it for 10 min, then inoculate it onto 2,4-D induction medium without antibiotics, and co-culture at 19℃ in the dark for 48 h. (3) After co-culture, hypocotyl segments were inoculated into 2,4D induction medium containing kanamycin (100 mg / L) and cephalosporin (100 mg / L), and incubated at 28℃ under weak light (cold light source 135 μmol m) -2 s -1 Cultured under [a specific culture medium], and continuously subcultured until embryogenic callus appears; (4) Embryogenic callus tissues were successively inoculated into embryo differentiation medium until somatic embryos matured, and mature cotyledon embryos were inoculated into rooting medium to germinate until complete plants were obtained.

[0039] B. Identification of transgenic plants (1) Positive detection of transgenic T0 generation plants Genomic DNA was extracted from young leaves of intact plants. DNA extraction was performed using a plant genomic DNA extraction kit from Tiangen Biotech (Beijing) Technology Co., Ltd. Specific operating procedures are detailed in the kit's instruction manual. Cas9 primer pairs, NPTII primer pairs, and sgRNA primer pairs were designed based on the knockout vector pRGEB32-GhU6.7-GhHB16 sequence. The Cas9 primer pairs were designed to target the Cas9 protein, the NPTII primer pairs to target the NPTII resistance gene, and the sgRNA primer pairs to target the sequences at both ends of the sgRNA on the pRGEB32-GhU6.7-GhHB16 vector.

[0040] The forward primer for PCR amplification of Cas9 positive detection of transgenic materials is Cas9-F (GCTTGTGCGTTTCGATTTGA, SEQ ID NO: 11), and the reverse primer is Cas9-R (CCGCTCGTGCTTCTTATCCT, SEQ ID NO: 12). The forward primer for PCR amplification of transgenic material NPTII positive detection is NPTII-F (GCTTGGGTGGAGAGGCTATTC, SEQ ID NO: 13), and the reverse primer is NPTII-R (GAAGAACTCGTCAAGAAGGCG, SEQ ID NO: 14). The forward primer for PCR amplification of transgenic material sgRNA positive detection is sgRNA-F (TATAAGCGAAAGAAGCATCAGA, SEQ ID NO: 15), and the reverse primer is sgRNA-R (GACCCGAATTTGTGGACC, SEQ ID NO: 16).

[0041] The PCR reaction mixture consisted of 20 μl: 16 μL ddH2O; 2 μL 10× EasyTaq Buffer; 0.4 μL dNTPs; 0.2 μL each of forward and reverse primers; 0.2 μL EasyTaq enzyme; and 1 μL DNA. The PCR reaction conditions were: 95℃ pre-denaturation for 5 min; 28 cycles of 95℃ for 30 sec, 58℃ for 30 sec, and 72℃ for 50 sec; and a final extension at 72℃ for 5 min.

[0042] PCR was performed using three primer pairs to detect the presence of corresponding T-DNA insertion. The positive control showed amplified bands, while the wild-type did not. Positive test results for transgenic plants are shown below. Figure 2 , Figure 3 and Figure 4 . Figure 2 for GhHB16 gel image of Cas9 positive test results in knockout strains; Figure 3 for GhHB16 gel image of NPTII positive test results of knockout strain; Figure 4 for GhHB16 Gel image of sgRNA positive detection results from the knockout line. The results showed that all three primer pairs amplified the corresponding bands, with GhHB16-CR being the most effective. GhHB16 Knockout strains.

[0043] Example 3 GhHB16 Knockout line editing site rate detection The method for extracting DNA from cotton leaves was the same as in Example 2. Because CRISPR-Cas9 technology can result in multiple edit types on a single plant and Sanger sequencing cannot identify multiple mutation types, high-throughput Hi-TOM sequencing technology was used. The specific steps are as follows: (1) Design primers according to the conventional PCR primer design principles (16-20 nt); the target site should be within 10-100 bp of the left or right primer. For independent single-plant materials with gene knockout, design site-specific primers with common adapter sequences (5′-ggagtgagtacggtgtgc-3′, SEQ ID NO: 17; 5′-gagttggatgctggatgg-3′, SEQ ID NO: 18) to amplify the genome sequence of the target gene in the first round of PCR.

[0044] The first round of PCR reaction system (20 μL) contains: 50 ng genomic DNA, 0.2 µL 10 µmol / L forward and reverse primers, and 10 µL 2×Taq Master Mix, with ddH2O added to bring the total to 20 μL.

[0045] The first round of PCR reaction program was as follows: 95℃ pre-denaturation for 5 min; 95℃ for 30 sec, 58℃ for 30 sec, 72℃ for 20 sec, 29 cycles; extension at 72℃ for 5 min.

[0046] (2) The second round of PCR uses the first round of PCR products as templates and adds primers containing barcode and index for PCR amplification.

[0047] The second round PCR reaction system (20 μL) includes: 1 μL of the first round PCR product, 0.4 nmol / L of forward and reverse barcode primers, 200 nmol / L of forward and reverse index primers, and 10 µL of 2×Taq Master Mix, with ddH2O added to bring the total to 20 μL.

[0048] The second round of PCR reaction program was as follows: 95℃ pre-denaturation for 5 min; 95℃ for 30 sec, 58℃ for 30 sec, 72℃ for 30 sec, 30 cycles; 72℃ extension for 5 min.

[0049] (3) The second-round PCR products were mixed in equal volumes from each well and purified using a purification kit (OMEGA, D2500-2). Each batch of 96 individual samples was sequenced using a next-generation sequencing platform, yielding 1 G of data. The data were analyzed using the Hi-TOM analysis website (http: / / www.hi-tom.net / hi-tom / ). The sequenced data were compared with the reference genome sequence to evaluate the final editing efficiency.

[0050] Using a barcode-based approach, for each individual mutant of the knockout material, a barcode is attached to the 5' end of both primers targeting the target site, via a linker. This results in a unique barcode pair for each mutant. These barcode-containing primers are then used to amplify the target site sequences of individual mutants, constructing a mixed DNA library for high-throughput sequencing. Sequencing results are sorted according to the barcode-labeled primers to obtain the sequencing results for each individual mutant. After removing repetitive and low-quality sequences, the sequencing results are compared with a reference gene sequence to complete the process of detecting mutations at the target gene site in individual mutants.

[0051] The barcode primers used during detection are as follows: F-1 GCTTGCGTTGGAGTGAGTACGGTGTGC (SEQ ID NO: 19); F-2 GCTTGTAGTGGAGTGAGTACGGTGTGC (SEQ ID NO: 20); F-3 GCTTACGCTGGAGTGAGTACGGTGTGC(SEQ ID NO:21); F-4 GCTTCTCGTGGAGTGAGTACGGTGTGC(SEQ ID NO:22); F-5 GCTTGCTCTGGAGTGAGTACGGTGTGC(SEQ ID NO:23); F-6 GCTTAGTCTGGAGTGAGTACGGTGTGC(SEQ ID NO:24); F-7 GCTTCGACTGGAGTGAGTACGGTGTGC(SEQ ID NO:25); F-8 GCTTGATGTGGAGTGAGTACGGTGTGC(SEQ ID NO:26); F-9 GCTTATACTGGAGTGAGTACGGTGTGC(SEQ ID NO:27); F-10 GCTTCACATGGAGTGAGTACGGTGTGC(SEQ ID NO:28); F-11 GCTTGTGCTGGAGTGAGTACGGTGTGC(SEQ ID NO:29); F-12 GCTTACTATGGAGTGAGTACGGTGTGC(SEQ ID NO:30); R-A CCATCCAGCATCCAACTCAACGCACAG(SEQ ID NO:31); R-B CCATCCAGCATCCAACTCACTACACAG(SEQ ID NO:32); R-C CCATCCAGCATCCAACTCAGCGTACAG(SEQ ID NO:33); R-D CCATCCAGCATCCAACTCACGAGACAG(SEQ ID NO:34); R-E CCATCCAGCATCCAACTCAGAGCACAG(SEQ ID NO:35); R-F CCATCCAGCATCCAACTCAGACTACAG(SEQ ID NO:36); R-G CCATCCAGCATCCAACTCAGTCGACAG(SEQ ID NO:37); RH CCATCCAGCATCCAACTCACATCACAG (SEQ ID NO: 38); The index primers used for detection were 2P-F-1 (AATGATACGGCGACCACCGAGATCTACACAGGAACCTACACTCTTTCCCTACACGACGCTCTT, SEQ ID NO: 39) and 2P-R-1 (CAAGCAGAAGACGGCATACGAGATCTTAGCCAGTGACTGGAGTTCAGACGTGTGCTCTT, SEQ ID NO: 40).

[0052] The PCR reaction conditions were as follows: 95℃ pre-denaturation for 5 min; 30 cycles of 95℃ for 30 sec, 58℃ for 30 sec, and 72℃ for 30 sec; extension at 72℃ for 5 min.

[0053] Take equal amounts of the PCR product, purify it, and then send it for sequencing. (Cotton) GhHB16 The genome is divided into subgroup A and subgroup D, and sgRNA targets subgroups A and D. Figure 5 To edit the site detection results image, sequencing results show that, GhHB16 The gene of the knockout line GhHB16-CR-1 was mutated. Subgroup A had a 5-base deletion, and the nucleotide sequence after mutation is shown in SEQ ID NO: 6. Subgroup D had a 1-base insertion, and the nucleotide sequence after mutation is shown in SEQ ID NO: 7.

[0054] Example 4 Using gene editing technology GhHB16 Gene functional verification In Example 3 GhHB16 Materials from the knockout line GhHB16-CR and the wild-type material (WT) were planted in the transgenic experimental field, with 30 plants of each line. At the same time point, cotton bolls from the same part of the transgenic and control lines were harvested for fiber quality determination, with 3 biological replicates for each line.

[0055] 1. GhHB16 Knockout strains and fiber quality identification Mature cotton bolls from the middle of the plant at the same time were picked and machine-ginned. The mature fiber seed cotton and the ginned fiber were weighed and the lint percentage was calculated (the formula for calculating the lint percentage is 100% × fiber weight / seed cotton weight). The weight of each fiber sample was about 10 g. The sorted samples were tested for five indicators: average fiber length of the upper half, uniformity index, micronaire value, breaking strength and elongation. The specific measurement methods are disclosed in the existing technology (Li et al., (2016). GbEXPAtr, a species-specific expansin, enhances cotton fibre elongation through cell wall restructuring. Plant biotechnology journal, 14(4), 951-963.). The instrument used was a high volume cotton fiber tester (High Volume Instrument, HFT9000, Premier, India). Each transgenic line was replicated at least three times. The obtained data were analyzed using Prism software for multiple comparisons. The results are shown in Table 1. The results showed that compared with the wild type, the knockout GhHB16 Genes do not significantly alter cotton fiber quality, but they can increase cotton lint percentage. Figure 6 ).

[0056] Table 1 Measurement results of five indicators of cotton

[0057] Note: ns indicates insignificant; T1 and T2 represent different generations of genetically modified cotton.

[0058] 2. GhHB16 Scanning electron microscopy analysis of knockout strains At 8:00 AM on the day of flowering, five flowers from each line were randomly selected, and the ovules in the center of each ovary were carefully removed and placed in 2.5% glutaraldehyde, then stored at 4°C until sample preparation. The specific steps are as follows: (1) Dehydration: Dehydrate by stepwise application of 30%, 50%, 70%, 85%, 95% and two 100% ethanol solutions. Each dehydration step takes 10-15 minutes. If the sample volume is large, shake it appropriately to ensure that it is dehydrated.

[0059] (2) Intermediate solution replacement: First soak in isoamyl acetate / ethanol (1:1, v / v) for 10 min, then replace with isoamyl acetate and soak for 10 min, and shake appropriately.

[0060] (3) Critical point drying: After substitution, the sample is transferred into the sample basket (with filter paper), placed in the sample chamber of the pre-cooled (-10 ℃) critical point dryer, and liquid CO2 is injected after the chamber is closed to submerge the sample. The temperature is adjusted to 38 ℃ to allow it to vaporize. After the liquid has completely vaporized, the gas is slowly released. After the gas is released, the cover is opened and the sample is taken.

[0061] (4) Paste the sample: Use conductive adhesive to paste the sample onto the stage.

[0062] (5) The samples were observed after being coated by ion sputtering. The instrument used for sample observation was a JSM-6390 / LV scanning electron microscope (Jeol, Japan).

[0063] The number of ovule protrusions was counted using ImageJ software. The results showed that, compared to the wild type, the knockout... GhHB16 Genes lead to an increase in the number of fiber initiation points ( Figure 7 and Figure 8 The number of ovule protrusions is related to the number of fibers per unit area, which proves that knocking out... GhHB16 Genes can increase the lining content by increasing the number of ovule protrusions.

[0064] Although the above embodiments have provided a detailed description of the present invention, they are only some embodiments of the present invention, and not all embodiments. Other embodiments can be obtained based on these embodiments without creative effort, and these embodiments all fall within the protection scope of the present invention.

Claims

1. The application of an HD-ZIP transcription factor GhHB16 or a gene encoding the HD-ZIP transcription factor GhHB16 in regulating cotton lint percentage and / or cultivating cotton with a high lint percentage, wherein the HD-ZIP transcription factor GhHB16 or the gene increases cotton lint percentage through negative regulation. The HD-ZIP transcription factor GhHB16 includes GhHB16A and GhHB16D; The amino acid sequence of GhHB16A is shown in SEQ ID NO: 1; The amino acid sequence of GhHB16D is shown in SEQ ID NO:

2.

2. The application of an sgRNA or a derivative containing said sgRNA in increasing cotton lint percentage and / or cultivating cotton with superior lint percentage; The nucleotide sequence of the DNA molecule targeted by the sgRNA is shown in SEQ ID NO: 3; The derivatives containing the sgRNA include recombinant vectors and / or recombinant bacteria; The application includes transferring a derivative containing the sgRNA into a recipient plant.

3. A method for increasing cotton lint percentage, characterized in that, include: Inhibiting the biological function of HD-ZIP transcription factor GhHB16 in plants or inhibiting the expression of the gene encoding HD-ZIP transcription factor GhHB16 increases cotton lint percentage. The HD-ZIP transcription factor GhHB16 includes GhHB16A and GhHB16D; The amino acid sequence of GhHB16A is shown in SEQ ID NO: 1; The amino acid sequence of GhHB16D is shown in SEQ ID NO:

2.

4. The method according to claim 3, characterized in that, The reagent containing sgRNA or a derivative containing said sgRNA is transferred into the recipient plant to inhibit the biological function of said HD-ZIP transcription factor GhHB16 or the expression of said gene in the plant. The derivatives containing the sgRNA include recombinant vectors and / or recombinant bacteria; The nucleotide sequence of the DNA molecule targeted by the sgRNA is shown in SEQ ID NO:

3.

5. A method for selecting and / or assisting in the selection of cotton with a superior lint content, characterized in that, include: The gene encoding the HD-ZIP transcription factor GhHB16 in the genome of plants was detected, and plants with the gene knocked out were selected as breeding materials for breeding. The HD-ZIP transcription factor GhHB16 includes GhHB16A and GhHB16D; The amino acid sequence of GhHB16A is shown in SEQ ID NO: 1; The amino acid sequence of GhHB16D is shown in SEQ ID NO:

2.

6. A type of cotton, characterized in that, The gene encoding the HD-ZIP transcription factor GhHB16 in the cotton was knocked out; The HD-ZIP transcription factor GhHB16 includes GhHB16A and GhHB16D; The amino acid sequence of GhHB16A is shown in SEQ ID NO: 1; The amino acid sequence of GhHB16D is shown in SEQ ID NO: 2.

Images