Application of the cotton GoYP gene in regulating plant petal size and / or petal color

Abstract

This invention provides cotton GoYP The application of genes in regulating plant petal size and / or petal color belongs to the field of genetic engineering technology. In this invention, GoYP Genes include GhYP Genes and / or GaYP Genes; among which, GhYP Genes can regulate the biosynthesis of flavonoids (anthocyanins and / or flavonols) in plant petals and promote petal elongation by promoting the biosynthesis of brassinosteroids (BRs). GhYP This gene encodes the R2R3-MYB regulatory factor, which has a dual function of promoting the biosynthesis of flavonoids and BRs, providing a new gene resource and a more convenient method for the synergistic improvement of the color and size of ornamental plant petals. Simultaneously, through petal-specific expression promoters... proGaFLS Driver genes GaYP Overexpression of the gene in tobacco petals resulted in redder and larger petals, further demonstrating the gene's efficacy. GoYP Its application value.

Description

Technical Field

[0001] This invention belongs to the field of genetic engineering technology, and particularly relates to cotton. GoYP Application of genes in regulating plant petal size and / or regulating plant petal color. Background Technology

[0002] As one of the important floral organs, petals vary significantly in size and color. For ornamental plants, flower size and color have always been core objectives for horticulturists in breeding, as they are key traits for meeting the needs of different consumers and markets.

[0003] To date, no gene has been discovered that simultaneously regulates petal size and petal color, resulting in a relative scarcity of genetic resources for molecular breeding of flowers. Summary of the Invention

[0004] The purpose of this invention is to provide GoYP The application of genes in regulating plant petal size and / or regulating plant petal color. GoYP Genes can simultaneously regulate both petal size and petal color.

[0005] This invention provides cotton GoYP The application of genes in regulating plant petal size and / or regulating plant petal color, the GoYP Genes include GhYP Genes and / or GaYP Genes; the stated GhYP The amino acid sequence of the protein encoded by the gene is shown in SEQ ID NO.1 or SEQ ID NO.2; GaYP The amino acid sequence of the protein encoded by the gene is shown in SEQ ID NO.3.

[0006] Preferably, the regulation of plant petal color includes regulating the content of anthocyanins and / or flavonols in plant petals.

[0007] Preferably, the regulation of anthocyanin and / or flavonol content in plant petals includes negative regulation. GhYP Genes that reduce the content of anthocyanins and / or flavonols in plant petals; and / or, the regulation of anthocyanin and / or flavonol content in plant petals includes positive regulation. GaYP Genes increase the anthocyanin content in plant petals.

[0008] Preferably, the regulation of plant petal size includes negative regulation. GhYP Genes reduce plant petal size and / or positively regulate GaYP Genes that increase plant petal size; the regulation of petal color includes positive regulation. GaYP Genes make the petals more vibrant in color.

[0009] Preferably, the reduction in plant petal size is achieved by reducing the size of plant petal epidermal cells; the increase in plant petal size is achieved by increasing the size of plant petal epidermal cells.

[0010] The present invention also provides GhYP The application of the gene in regulating the content of brassinosteroids in plant petals, characterized in that... GhYP The amino acid sequence of the protein encoded by the gene is shown in SEQ ID NO.1 or SEQ ID NO.2.

[0011] Preferably, the regulation of brassinosteroid content in plant petals includes negative regulation. GhYP Genes reduce the content of brassinosteroids in plant petals.

[0012] This invention also provides a method for improving the ornamental value of plants, comprising the following steps: overexpressing in plants GaYP Genes; the enhancement of plant ornamental value includes making the plant's petals more vibrant in color and / or larger in size; the GaYP The amino acid sequence of the protein encoded by the gene is shown in SEQ ID NO.3.

[0013] This invention also provides negative regulation GhFLS The application of genes in increasing the content of anthocyanins in plant petals, the aforementioned GhFLS The amino acid sequence of the gene-encoded protein is shown in SEQ ID NO.4 or SEQ ID NO.5.

[0014] The present invention also provides a genetically modified cotton or a genetically modified tobacco, wherein the genome of the genetically modified cotton has been knocked out. GhYP Gene or GhFLS Gene; the genome of the genetically modified tobacco plant was inserted with GaYP Genes; the stated GhYP The amino acid sequence of the protein encoded by the gene is shown in SEQ ID NO.1 or SEQ ID NO.2; GaYP The amino acid sequence of the protein encoded by the gene is shown in SEQ ID NO.3; GhFLS The amino acid sequence of the gene-encoded protein is shown in SEQ ID NO.4 or SEQ ID NO.5.

[0015] This invention provides cotton GoYP The application of genes in regulating plant petal size and / or regulating plant petal color, the GoYP Genes include GhYP Genes and / or GaYP Genes; the stated GhYPThe amino acid sequence of the protein encoded by the gene is shown in SEQ ID NO.1 or SEQ ID NO.2; GaYP The amino acid sequence of the protein encoded by the gene is shown in SEQ ID NO.3. In this invention, GhYP Genes can regulate the biosynthesis of anthocyanins and / or flavonols in plant petals, and promote petal elongation by promoting the biosynthesis of brassinosteroids (BRs). GhYP The gene encodes an R2R3-MYB regulatory factor that promotes both flavonoid and BR biosynthesis, providing a new genetic resource and a more convenient method for the synergistic improvement of petal color and size in ornamental plants. Simultaneously, through petal-specific expression promoters... proGaFLS drive GaYP The gene was overexpressed in tobacco petals, resulting in redder and larger petals, further proving... GoYP The application value of genes. Attached Figure Description

[0016] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0017] Figure 1 for GhYP KO and GhFLS Obtaining KO gene-edited cotton; where A is the gene encoding the R2R3-MYB transcription factor. GhYP And the gene encoding flavonol synthase GhFLS Gene structure; gRNA sequence is indicated by a red box; exons and introns are indicated by black boxes and black solid lines, respectively; B, GhYP KO (2# and 11#) and GhFLS Alignment of mutant sequences in KO (10# and 13#) plants; green shaded bases are used to distinguish At and Dt subgenomes; PAM and gRNA target sequences are indicated by yellow and blue shades, respectively; inserted and deleted bases are indicated by underline and dashed lines, respectively. Figure 2 for GhYP KO (2# and 11#) and GhFLS KO (10# and 13#) cotton flowers, 0 DPA, 11:00, scale = 1cm; Figure 3 The graph shows the results of flavonol content determination in petals (0 DPA, 11:00); statistical analysis was performed using Tukey's test, with identical and different lowercase letters indicating no significant difference and significant difference, respectively.P <0.05); Figure 4 for GhYP KO (2# and 11#) and GhFLS KO (10# and 13#) cotton bolls, 0 DPA, 18:00, scale = 1cm; Figure 5 for GhYP KO (2# and 11#) and GhFLS KO (10# and 13#) cotton petals (0 DPA, 18:00) anthocyanin extract; Figure 6 for GhYP KO (2# and 11#) and GhFLS The results of anthocyanin content determination in the petals (0 DPA, 18:00) of KO (10# and 13#) cotton were shown in the figure. Statistical analysis was performed using Tukey's test. Identical and different lowercase letters indicate no significant difference and significant difference, respectively. P <0.05); Figure 7 for GhYP KO and GhFLS A single petal of KO cotton (0 DPA, 11:00). Figure 8 Analysis of the area of ​​a single petal; Figure 9 A schematic diagram showing the sampling location for petal epidermal cells; Figure 10 Microscopic observation of petal epidermal cells at different locations; scale bar = 50 μm; Figure 11 Analysis of petal epidermal cell length at different locations; statistical analysis was performed using Tukey's test, with identical and different lowercase letters indicating no significant difference and significant difference, respectively. P <0.05); Figure 12 Analysis of petal epidermal cell width at different locations; statistical analysis was performed using Tukey's test, with identical and different lowercase letters indicating no significant difference and significant difference, respectively. P <0.05); Figure 13 Analysis of epidermal cell area at different locations of petals; statistical analysis was performed using Tukey's test, with identical and different lowercase letters indicating no significant difference and significant difference, respectively. P <0.05); Figure 14 for GhYP KO compared to WT and GhFLS Wayne analysis of downregulated genes expressed in KO petals; Figure 15 forGhYP KEGG enrichment analysis of downregulated genes expressed in KO petals; Figure 16 The content of BRs in cotton petals was determined; statistical analysis was performed using the Tukey test, with the same and different lowercase letters indicating no significant difference and significant difference, respectively. P <0.05); Figure 17 For qRT-PCR detection of transgenic tobacco petals GaYP The amount of expression; Figure 18 for GaYP Genetically modified tobacco flower; scale bar = 1 cm; Figure 19 for GaYP Transgenic tobacco petals; scale bar = 1 cm; Figure 20 for GaYP Microscopic observation of the upper part of the transgenic tobacco flower (upper half, scale bar = 1 cm) and its epidermal cells (lower half, scale bar = 50 μm); the area indicated by the white box is the microscopic observation area; Figure 21 Tobacco petal area statistics were performed; Tukey's test was used for statistical analysis, with the same and different lowercase letters indicating no significant difference and significant difference, respectively. P <0.05); Figure 22 The area of ​​epidermal cells in tobacco petals was statistically analyzed; Tukey's test was used for statistical analysis, with the same and different lowercase letters indicating no significant difference and significant difference, respectively. P <0.05). Detailed Implementation

[0018] This invention provides cotton GoYP The application of genes in regulating plant petal size and / or regulating plant petal color, characterized in that... GoYP Genes include GhYP Genes and / or GaYP Genes; the stated GhYP The amino acid sequence of the protein encoded by the gene is shown in SEQ ID NO.1 or SEQ ID NO.2; GaYP The amino acid sequence of the protein encoded by the gene is shown in SEQ ID NO.3.

[0019] The present invention GoYP The gene is an R2R3-MYB transcription factor encoding the Sg6 subgroup, which can simultaneously promote the biosynthesis of petal flavonoids and BRs, making the petals of plants such as cotton and tobacco more colorful and larger, providing a new approach for the synergistic improvement of the color and size of petals in ornamental plants (roses, chrysanthemums, etc.).

[0020] In this invention, the GhYP The amino acid sequence of the protein GhYP_At encoded by the gene is shown in SEQ ID NO.1. It is derived from upland cotton variety Yuzao 1 (YZ1), specifically: MGGVAWTEEEDQLLKKCIERYGEGKWHRVPLLAGLNRCRKSCRLRWINYLRPNIKRGTFAEDEVQLIIKLHKLLGNKWSLIAGRLPGRTANDVKNYWNCHLSKKLNNAQETEEEYQNGM KMETLKPQPRCSAMTGSIKPRTQDHFAPKNQPTIQESTMPTPFNFAEVVKQQGHEIVKEEEDIEEEAAVGVFFGDLATEDHQFGQLDEVNVLSSTNEGCSKWDWDDLMLDMDLWTDSL.

[0021] As one implementation method, when the GhYP When the amino acid sequence of the gene-encoded protein GhYP is as shown in SEQ ID NO. 1, the... GhYP The nucleotide sequence of the gene is shown in SEQ ID NO.6, specifically: atgggaggcgttgcttggaccgaagaagaagatcagttgctcaagaaatgtatagagcgttatggagaagggaagtggcatcgggtacctcttttggctggtctaaacaggtgtcgtaaaagctgtagactgagatggataaactacctacgcccaaacatcaaaagaggaacctttgcagaagatgaagttcagctcatcattaagcttcacaagctcctcggaaacaaatggtcattgatagctgggagactcccaggaagaacagcaaatgatgtaaagaattattggaactgccacctgagcaaaaagctcaacaatgctcaagaaaccgaagaggaatatcaaaatggcatgaaaatggaaaccctaaaaccccagccacgatgcagtgcaatgacgggctcaattaaaccaagaacccaagatcattttgctcctaagaaccagccaacaatacaagaaagcaccatgcccacacctttcaacttcgctgaagttgttaaacaacaaggccacgaaattgtgaaagaagaagaagacatcgaagaggaggctgcagttggtgtgttttttggtgatttggcaacggaagatcatcagtttgggcagcttgatgaagttaacgtattgagtagtactaatgaaggatgcagcaaatgggattgggatgatttgatgttagacatggatttatggactgattcattgtga。

[0022] In the present invention, the GhYP amino acid sequence of the protein GhYP_Dt encoded by the gene is as shown in SEQ ID NO.2, derived from Gossypium hirsutum YZ1, specifically: MGGVAWTEEEDQLLKKCIERYGEGKWHRVPLLAGLNRCRKSCRLRWLNYLRPNIKRGTFAEDEVQLIIKLHKLLGNKWSLIAGRLPGRTANDVKNYWNCHLSKKLNNAQETEEEYQNG MKMETLKPQPRCIAVTGSIKPRTQDHFAPKNQPTIQESTMPTPFNFVEVDKQQGQELVKEEEDIREEAAGGVFFGDLATEDHQFGQLDEVNVLSSTEGCSKWDWDDLMLDMDLWTDSL.

[0023] As one implementation method, when the GhYP When the amino acid sequence of the gene-encoded protein GhYP is as shown in SEQ ID NO.2, the... GhYP The nucleotide sequence of the gene is shown in SEQ ID NO.7, specifically: atgggaggcgttgcttggaccgaagaagaagatcagttgctcaagaaatgtatagagcgttatggagaagggaagtggcatcgggtacctcttttggctggtctaaacaggtgtcgtaaaagctgtagactgagatggctaaactacctacgcccaaacatcaaaagaggaacctttgcagaagatgaagttcagctcatcattaagcttcacaagctcctcggaaacaaatggtcattgatagctgggagactcccaggaagaacagcaaatgatgtaaagaattattggaactgccacctgagtaaaaagctcaacaatgctcaagaaaccgaagaggaatatcaaaatggcatgaaaatggaaaccctaaaaccccagccacgatgcattgcagtgacgggctcaattaaaccaagaacccaagatcattttgctcctaagaaccagccaacaatacaagaaagcaccatgcccacacctttcaacttcgttgaagttgataaacaacaaggccaggagcttgtgaaagaagaagaagacatcagagaggaggctgcaggtggtgtgttttttggtgatttggcaacggaagatcatcagtttgggcagcttgatgaagttaatgtattgagtagtactgaaggatgcagcaaatgggattgggatgatttgatgttagacatggatttatggactgattcattgtga。

[0024] In the present invention, the GaYP amino acid sequence of the protein GaYP encoded by the gene is as shown in SEQ ID NO.3, and it is derived from Gossypium arboreum Changshu Long variety, specifically: MGGVAWTEEEDQLLKKCIERYGEGKWHRVPLLAGLNRCRKSCRLRWLNYLRPNIKRGTFAEDEVQLIIKLHKLLGNKWSLIAGRLPGRTANDVKNYWNCHLSKKLNNAQETEEEYQNGM KMETLKPQPRCSAMTGSIKPRTQDHFAPKNQPTIQESTMPTPFNFAEVDKQQGHEIVKEEEDIKEEAAVGVLFGDLATEDHQFGQLDEVNVLSSTNEGCSKWDWDDLMLDMDLWTDSL.

[0025] As one implementation method, when the GaYP When the amino acid sequence of the gene-encoded protein GaYP is as shown in SEQ ID NO.3, the... GaYP The nucleotide sequence of the gene is shown in SEQ ID NO.8, specifically: atgggaggcgttgcttggaccgaagaagaagatcagttgctcaagaaatgtatagagcgttatggagaagggaagtggcatcgggtacctcttttggctggtctaaacaggtgtcgtaaaagctgtagactgagatggctaaactacctacgcccaaacatcaaaagaggaacctttgcagaagatgaagttcagctcatcattaagcttcacaagctcctcggaaacaaatggtcattgatagctgggagactcccaggaagaacagcaaatgatgtaaagaattattggaactgccacctgagcaaaaagctcaacaatgctcaagaaaccgaagaggaatatcaaaatggcatgaaaatggaaaccctaaaaccccagccacgatgcagtgcaatgacgggctcaattaaaccaagaacccaagatcattttgctcctaagaaccagccaacaatacaagaaagcaccatgcccacacctttcaacttcgctgaagttgataaacaacaaggccacgaaattgtgaaagaagaagaagacatcaaagaggaggctgcagttggtgtgctttttggtgatttggcaacggaagatcatcagtttgggcagcttgatgaagttaacgtattgagtagtactaatgaaggatgcagcaaatgggattgggatgatttgatgttagacatggatttatggactgattcattgtga。

[0026] In the present invention, the GhFLS amino acid sequence of the protein GhFLS_At encoded by the gene is as shown in SEQ ID NO.4 and is derived from upland cotton YZ1, specifically: MEVERVQEIANFSKETIPEEFIRTTYEQPALTTVQGTVLEVPVIDLSDPDEEKVVRTIVDASSNWGMFQVVNHGIPHETVRKLKEVGKAFFELPPQEKEAYAKSPGSQSIEGYGTKLQREVEGKKAWVDHLFHKIWPPSEINYQFWPKNPPSYRKNNEEYAKHMHGV VDKLFRCLSLGLGLEGNEMKEAMGGDNMVYLLKINYYPQCPQADLALGVPAHTDMSAITILVPNDVQGLQACKDDHWYDVEYIPNALIVHIGDQIEIVSNGKYKSVLHRAKVNKEKTRISWPVFVEPPSDLEVGPHPKLVNEANPPKFKSKKYSQYCFCKLNKTPQ.

[0027] As one implementation method, when the GhFLS When the amino acid sequence of the gene-encoded protein GhFLS is as shown in SEQ ID NO. 4, the... GhFLS The nucleotide sequence of the gene is shown in SEQ ID NO.9, specifically:

[0028] In this invention, the GhFLS The amino acid sequence of the gene-encoded protein GhFLS_Dt is shown in SEQ ID NO.5 and is derived from upland cotton YZ1, specifically: MGVVRVQEIANFSKETIPEEFIRTTYEQPALTTVQGTVLEVPVIDLSDPDEEKVVRAIVDASSNWGMFQVVNHGIPRETVGKLKEVGKAFFELPPHEKEAYAKSPGFQSIEGYGTKLQREVEGKKAWVDHLFHKIWPPSEINYQFWPKNPPSYRKNNEEYAKHMHGV VNKLFRCLSLGLGLEGNEMKEAMGGDSMVYLLKINYYPQCPRPDLALGVPAHTDMSAITILVPNDVQGLQACKDDHWYDVEYIPNALIVHIGDQVEIVSNGKYKSVLHRAKVNKEKTRISWPVFVEPPSDLEVGPHPKLVNEANPPKFESKKYSQYCFCKLNKTPK.

[0029] As one implementation method, when the GhFLS When the amino acid sequence of the gene-encoded protein GhFLS is as shown in SEQ ID NO. 5, the... GhFLS The nucleotide sequence of the gene is shown in SEQ ID NO.10, specifically:

[0030] As one implementation method, the regulation of plant petal color includes regulating the content of anthocyanins and / or flavonols in plant petals.

[0031] As one implementation, the regulation of anthocyanin and / or flavonol content in plant petals includes negative regulation. GhYP Genes that reduce the content of anthocyanins and / or flavonols in plant petals; and / or, the regulation of anthocyanin and / or flavonol content in plant petals includes positive regulation. GaYP Genes increase the anthocyanin content in plant petals.

[0032] In this invention, when the regulation of anthocyanin and / or flavonol content in plant petals is negatively regulated... GhYP When a gene reduces the content of anthocyanins and / or flavonols in the petals of a plant, the plant includes cotton.

[0033] In one embodiment, the flavonols include one or more of quercetin, myricetin, kaempferol, 6-hydroxykaempferol, and zeaxanthin.

[0034] In this invention, knockout is performed on upland cotton. GhYP Genes caused the petals to turn white, and the content of petal pigments and flavonols was significantly reduced.

[0035] In this invention, when the regulation of plant petal pigment content is positively regulated... GaYP When genes increase the anthocyanin content in plant petals, the plant includes tobacco.

[0036] As one implementation method, the regulation of plant petal size includes negative regulation. GhYP Genes reduce plant petal size and / or positively regulate GaYP Genes that increase plant petal size; the regulation of petal color includes positive regulation. GaYP Genes make the petals more vibrant in color.

[0037] As one implementation, more vibrant petal color includes more red petal color.

[0038] In one implementation, the petal size includes the petal area.

[0039] In this invention, when the regulation of plant petal size is negative... GhYP When genes reduce the size of plant petals, the plant in question includes cotton.

[0040] In this invention, when the regulation of plant petal size is positive... GaYP When genes increase the size of plant petals, the plant includes tobacco.

[0041] In one implementation, the reduction of plant petal size is achieved by reducing the size of plant petal epidermal cells; the increase of plant petal size is achieved by increasing the size of plant petal epidermal cells.

[0042] In this invention, knockout is performed on upland cotton. GhYP Genes showed that the epidermal cells of the petals were significantly smaller, and the petal elongation was severely hindered.

[0043] The present invention also provides GhYP The application of genes in regulating brassinosteroid content in plant petals, the aforementioned GhYP The amino acid sequence of the protein encoded by the gene is shown in SEQ ID NO.1 or SEQ ID NO.2.

[0044] In this invention, knockout is performed on upland cotton. GhYP Genes showed a significant decrease in the content of brassinosteroids (BRs) in the petals.

[0045] In one embodiment, the brassinosteroid includes one or more of brassinosteroidone, 6-deoxybrassinosteroidone, 28-demethylbrassinosteroidone, 28-homostassinosteroidone, and typhasterol.

[0046] As one implementation method, the regulation of brassinosteroid content in plant petals includes negative regulation. GhYP Genes reduce the content of brassinosteroids in plant petals.

[0047] In this invention, when the brassinosteroid content in the petals of the plant is negatively regulated... GhYP When the gene reduces the brassinosteroid content in plant petals, the plant includes cotton.

[0048] As one implementation method, the negative regulation includes knockout.

[0049] As one implementation method, the positive regulation includes overexpression.

[0050] As one implementation method, overexpression GhYP The promoter used in the gene is proGaFLS; The proGaFLS The nucleotide sequence is shown in SEQ ID NO.11, and it is derived from the Asian cotton variety Changshulong, specifically:

[0051] In this invention, a petal-specific promoter is expressed. proGaFLS Driver genes GaYP Overexpression in tobacco petals can produce more vibrant colors and larger flowers.

[0052] In one embodiment, the plant includes cotton or tobacco; the cotton includes upland cotton, and further includes upland cotton Yuzao 1 (YZ1); the tobacco variety includes Honghua Dajinyuan.

[0053] This invention also provides a method for improving the ornamental value of plants, comprising the following steps: overexpressing in plants GaYP Genes; the enhancement of plant ornamental value includes making the plant's petals more vibrant in color and / or larger in size; the GaYP The amino acid sequence of the protein encoded by the gene is shown in SEQ ID NO.3.

[0054] This invention also provides negative regulation GhFLS The application of genes in increasing the content of anthocyanins in plant petals, characterized in that... GhFLS The amino acid sequence of the gene-encoded protein is shown in SEQ ID NO.4 or SEQ ID NO.5.

[0055] The present invention also provides a genetically modified cotton or a genetically modified tobacco, wherein the genome of the genetically modified cotton has been knocked out. GhYP Gene or GhFLS Gene; the genome of the genetically modified tobacco plant was inserted with GaYP Genes; the stated GhYP The amino acid sequence of the protein encoded by the gene is shown in SEQ ID NO.1 or SEQ ID NO.2; GaYP The amino acid sequence of the protein encoded by the gene is shown in SEQ ID NO.3; GhFLS The amino acid sequence of the gene-encoded protein is shown in SEQ ID NO.4 or SEQ ID NO.5.

[0056] To further illustrate the present invention, the cotton provided by the present invention will be described below with reference to the accompanying drawings and embodiments. GoYP The application of genes in regulating plant petal size and / or petal color is described in detail, but should not be construed as limiting the scope of protection of this invention.

[0057] Example 1 1. Plant materials The recipient varieties are upland cotton variety Yuzao 1 (YZ1) and tobacco variety Honghua Dajinyuan.

[0058] 2. Strains and plasmids The gene knockout vector used in this experiment is pRGEB32-GhU6.7 (Provided by Huazhong Agricultural University), the overexpression vector is... pLGN (Preserved by this laboratory); the large intestine competent cells used E.coli DH5α was purchased from Nanjing Novizan Biotechnology Co., Ltd., and Agrobacterium tumefaciens strain LBA4404 was preserved in our laboratory.

[0059] 3. Main reagents High-fidelity amplification enzyme 2×Phanta Flash Master Mix was purchased from Nanjing Novizan Biotechnology Co., Ltd.; FastKing cDNA first-strand synthesis kit and FastUniversal rapid real-time PCR premix reagent were purchased from Tiangen Biotech (Beijing) Co., Ltd.; restriction endonucleases Bsa I. Hind III. BamH I. EcoR I. Purchased from Thermo Fisher Scientific.

[0060] 4. Carrier Construction by pRGEB32-GhU6.7 Gene knockout vectors are constructed based on the vector backbone, in which... GhU6.7 This is the promoter. Bsa I. Digest the above backbone vector with enzymes to extract the gene. GhYP_At / Dt (The nucleotide sequences are shown in SEQ ID NO.6 and SEQ ID NO.7) GhFLS_At / Dt Each of the CDS regions (nucleotide sequences shown in SEQ ID NO. 9 and SEQ ID NO. 10) can select one nucleotide to target its respective At / Dt subgenome sequence. gRNA Sequence (see) Figure 1 ), constructing knockout vectors pRGEB32-GhU6.7-GhYP and pRGEB32-GhU6.7-GhFLS The two vectors were respectively transformed into Agrobacterium tumefaciens strain LBA4404, with upland cotton YZ1 as the recipient, and the cotton was genetically transformed using our laboratory's mature Agrobacterium tumefaciens-infected cotton hypocotyl transformation method.

[0061] by pLGN Carrier skeleton ( pCambia Based on the modified version, an overexpression carrier is constructed. Hind III. BamH I. Enzyme digestion pLGN Promoter sequence amplified from the DNA sequence of Asian cotton Changshu dragon seed. proGaFLS , construct carrier pLGN - proGaFLS ; BamH I. EcoR I. Enzyme digestion pLGN -proGaFLS amplification of cDNA sequence from Asian cotton Changshu dragon variety GaYP (The nucleotide sequence is shown in SEQ ID NO.8), construct an overexpression vector. pLGN - proGaFLS - GaYP The above vectors were transformed into Agrobacterium tumefaciens strain LBA4404, with Tobacco safflower as the recipient, and genetic transformation of tobacco was achieved through our laboratory's mature Agrobacterium tumefaciens transformation method involving infection of tobacco leaf discs.

[0062] 5. Genetic transformation in cotton and tobacco Genetic transformation of cotton and tobacco was performed using Agrobacterium tumefaciens-mediated transformation. The culture medium formulations used are shown in Tables 1-3. The specific methods are as follows: 1) Cotton For the dehulling treatment of plump cotton seeds of wild-type upland cotton YZ1, a small amount (about 20-40 seeds) of dehulled seeds were placed in a sterilized 100mL Erlenmeyer flask. The seeds were first pre-washed with 75% alcohol for 1 minute, then the alcohol was gently poured off. Next, 10% H2O2 was added for sterilization for about 12 minutes (while continuously shaking the Erlenmeyer flask for sterilization). The H2O2 was gently poured off, and the seeds were rinsed with sterile water about 10 times. The sterilized seeds were placed on germination medium. When the radicle grew to about 1cm (about 2 days), the radicle was gently inserted into the germination medium and cultured in the dark at about 30℃ until the hypocotyl reached about 7cm (about 7 days). About 20 hours before infecting the hypocotyl, a single colony of Agrobacterium carrying the genetic transformation vector was inoculated into liquid YEB medium containing 50mg / L Km and 125mg / L Sm, and cultured on a shaker (200rpm) at 28℃ for about 20 hours. The OD value of the bacterial solution was then measured. 600 ), OD 600 Transformation is suitable at a pH of 0.4–0.6. Collect the activated Agrobacterium suspension, centrifuge at 8000 rpm for 1 min, discard the supernatant, and resuspend the cells in an equal volume of MGL liquid medium (containing 100 μmol / L AS). Collect the resuspended cells in a sterile 100 mL Erlenmeyer flask and incubate on a shaker (28℃, 100 rpm) for approximately 40 min. Cut the hypocotyl into 0.8–1 cm segments using a sterile scalpel, then place them in the resuspension and incubate on a shaker (28℃, 100 rpm) for 40 min. Discard the liquid, remove the hypocotyl segments, and gently place them on the surface of a solid co-culture medium. Incubate in the dark for approximately 48 h. After dark incubation, transfer the hypocotyl segments to a solid sieve medium (30℃, 16 h light / 8 h dark, the same below). After 30 days, transfer to solid IBA medium. Subculture every 30 days until green embryos form on the callus. Transfer the green embryos to a solid differentiation medium for further culture. Once the embryo has grown to about 3cm, insert it into the rooting medium until the seedling emerges. All of the above operations must be performed under strict aseptic conditions.

[0063] 2) Tobacco Wild-type red-flowered Tobacco Dajinyuan seeds were placed in sterilized 50mL Erlenmeyer flasks, sterilized using the same procedure as for cotton seeds. After sterilization, the seeds were placed on germination medium and cultured for one week (30℃, 16h light / 8h dark, the same applies below). Individual seedlings were then carefully transplanted to new germination medium and cultured for approximately one month (to a height of about 7cm). Leaves from the sterile Tobacco Dajinyuan seeds were taken, cut into 4-6mm leaf discs, placed in sterile bottles, and Agrobacterium resuspension containing the genetic transformation vector was added (Agrobacterium culture and resuspension steps were the same as for cotton). Infection was carried out for 10 minutes, the liquid was discarded, and the leaf discs were gently placed on the surface of a solid co-culture medium and cultured in the dark for approximately 48 hours. After dark culture, the leaf discs were transferred to a solid first selection medium. After 30 days, the regenerated buds around the wound on the leaf disc were cut off with a sterile scalpel and transferred to a second selection medium for 30 days. Finally, the Tobacco seedlings were inserted into a rooting medium to root and mature.

[0064] Antibiotic, hormone and other reagent formulations: Cefotaxime sodium (Cef, 200 mg / mL, 10 mL): Dissolve 2 g of cefotaxime sodium powder in 10 mL of sterile distilled water and filter through a 0.22 μm filter head for sterilization. Store at -20℃ for later use.

[0065] Kanamycin (Km, 50mg / mL, 10mL): Dissolve 0.5g of kanamycin powder in 10mL of sterile distilled water, filter through a 0.22μm filter head for sterilization, and store at -20℃ for later use.

[0066] Streptomycin (Sm, 125 mg / mL, 10 mL): Dissolve 1.25 g of streptomycin powder in 10 mL of sterile distilled water, filter sterilize using a 0.22 μm filter head, and store at -20 °C for later use.

[0067] Indolebutyric acid (IBA, 1 mg / mL, 100 mL): Dissolve 0.1 g of indolebutyric acid powder in 100 mL of distilled water and store at 4 °C for later use.

[0068] Kinetin (KT, 0.5 mg / mL, 100 mL): Dissolve 0.05 g of kinetin powder in 100 mL of distilled water and store at 4 °C for later use.

[0069] 2,4-Dichlorophenoxyacetic acid (2,4-D, 0.1 mg / mL, 100 mL): Dissolve 0.01 g of 2,4-dichlorophenoxyacetic acid powder in 100 mL of distilled water and store at 4 °C for later use.

[0070] 6-Benzylaminopurine (6-BA, 2.0 mg / mL, 100 mL): Dissolve 0.2 g of 6-benzylaminopurine powder in 100 mL of distilled water and store at 4 °C for later use.

[0071] Naphthaleneacetic acid (NAA, 0.1 mg / mL, 100 mL): Dissolve 0.01 g of naphthaleneacetic acid powder in 100 mL of distilled water and store at 4 °C for later use.

[0072] Acetyleugenol (As, 200mM, 25mL): Dissolve 1g of acetyleugenol powder in 25mL of dimethyl sulfoxide, filter sterilize using a 0.22μm filter head, and store at -20℃ for later use.

[0073] Mother liquor formula: Table 1. Mother cultures of cotton and tobacco tissue cultures

[0074] Subculture medium formulation: Table 2 Cotton Culture Medium

[0075] Table 3 Tobacco Culture Medium

[0076] 6. Extraction and content determination of anthocyanins, flavonols, and brassinosteroids (BRs) 1) Anthocyanins extract: (1) Take about 0.15g of cotton petals, and make 3 replicates for each sample. Grind them into fine powder in liquid nitrogen.

[0077] (2) Add 1.5 mL of 80% methanol (v / v) solution (containing 1% hydrochloric acid, w / v), shake for 30 sec, and extract overnight at 4°C.

[0078] (3) Repeat step (2) twice until the precipitate is colorless, and combine the supernatants from the three precipitations. The anthocyanin extract was then used to detect the absorbance using an ELISA reader.

[0079] Measurement: (1) Take a transparent microplate, and set up 3 replicates for each sample. Add 100 μL of supernatant to each well.

[0080] (2) The absorbance values ​​at 530 nm and 657 nm were read using a Varioskan LUX microplate reader, and the anthocyanin content was calculated. Q = [A530−(0.25×A657)]× M −1 M represents the fresh weight of the petals.

[0081] 2) Flavonols extract: (1) Take about 0.1g of cotton petals, and make 3 replicates for each sample. Grind them into fine powder in liquid nitrogen.

[0082] (2) Add 1 mL of 0.2% formic acid-methanol (v / v) solution, shake for 30 sec, and sonicate for 20 min. Centrifuge at 12000 rpm for 10 min and collect the supernatant.

[0083] (3) Repeat step (2) twice until the precipitate is colorless, and combine the supernatants from the three precipitations. Filter the supernatant through a 0.22 μm filter membrane to obtain the flavonol extract for LC-MS / MS detection.

[0084] Measurement: (1) Prepare the mobile phase: Phase B is 100% methanol and Phase A is 0.1% formic acid water.

[0085] (2) The chromatographic separation column was an Eclipse XDB-C18 column (150×4.6mm, 5μm) with a column temperature of 30℃.

[0086] (3) The elution program is as follows: 0 min, 10% B; 15 min, 40% B; 30 min, 55% B; 45-54 min, 100% B; 55 min, 10% B, with a flow rate of 0.16 mL / min.

[0087] (4) MS / MS detection uses positive ion mode, and other detection parameters are shown in Table 4.

[0088] Table 4 MS / MS detector parameters for flavonol determination

[0089] 3) Brassinosteroids (BRs) extract: (1) Take about 0.1g of cotton petals, and make 3 replicates for each sample. Grind them into fine powder in liquid nitrogen.

[0090] (2) Weigh the ground fresh sample under liquid nitrogen, weigh 20 mg of the ground powder, add 10 μL of internal standard mixed working solution with a concentration of 10 ng / mL, extract with 1 mL of acetonitrile vortex for 15 min, centrifuge at 4°C and 12000 r / min for 10 min, and take the supernatant into a brown sample bottle.

[0091] (3) Add 200 μL of 4-DMAPBA, react at 75°C for 1 h, and then dry with nitrogen.

[0092] (4) Redissolve in 100 μL acetonitrile solution, filter through a 0.22 μm filter membrane, place in the inner liner of the sample vial, and use for LC-MS / MS analysis.

[0093] Measurement: LC-MS / MS analysis was performed by Wuhan Maiwei Metabolism Co., Ltd.

[0094] 7. Results Gene knockout in upland cotton using gene editing techniques GhYP (Asian cotton gene) GaYP Homologous genes in upland cotton (nucleotide sequences shown in SEQ ID NO. 6 and SEQ ID NO. 7) and GhFLS (The nucleotide sequences are shown in SEQ ID NO. 9 and SEQ ID NO. 10) Figure 1 Gene-edited cotton was obtained. GhYP KO-2, GhYP KO-11 and GhFLS KO-10 GhFLS KO-13.

[0095] Observing the color of cotton petals on the day of flowering (0 DPA, 11:00), it was found that the petals of wild-type upland cotton (WT) were pale yellow. GhYP KO and GhFLS KO cotton petals are predominantly white. Flavonol content analysis results show that, compared to WT petals, GhYP The contents of quercetin, 6-hydroxykaempferol, and gossypol in KO petals were all significantly reduced, while GhFLS The contents of all five flavonols in KO petals were significantly reduced. Analysis of petals at another time point (0 DPA, 18:00) showed that, compared to WT petals, GhYP The red area at the top of the KO petals is smaller, and the content of anthocyanins is significantly reduced. GhFLS The accumulation of pigments in KO petals showed a similar pattern to... GhYP KO petals show the opposite trend ( Figures 2 to 6 ).

[0096] Besides discovering significant differences in petal color, compared to WT petals, GhYP The area of ​​the KO petals was significantly reduced ( Figure 7 and Figure 8 ),and GhFLS The size of the KO petals did not change significantly, therefore the difference in pigmentation is not... GhYP The reason why the petals of the KO flower become smaller is... GhYP It directly regulates the normal development of petals. Analysis of epidermal cells from different parts of the petal showed that ( Figure 9 ), WT and GhFLS The size of the epidermal cells in the KO petals showed no difference, while GhYP The epidermal cells at the base, middle, and top of the KO petals were significantly smaller. Figures 10 to 13 ),This is GhYP The direct cause of the reduction in the size of the KO petals.

[0097] For WT, GhYP KOGhFLS Transcriptome analysis was performed on KO petals. GhYP KO compared to WT and GhFLS KO downregulated 1956 genes. Figure 14 KEGG enrichment analysis revealed that the above genes were significantly enriched in pathways such as fatty acid elongation; cutin, suberin and wax biosynthesis; brassinosteroid (BRs) biosynthesis; and unsaturated fatty acid biosynthesis. Figure 15 BRs, as plant hormones that promote cell elongation, have received significant attention.

[0098] Next, six brassinosteroids were detected in cotton petals, including two bioactive brassinosteroids (BRs): brassinosteroidone and brassinolide, and four intermediates: 6-deoxybrassinosteroidone, 28-demethylbrassinosteroidone, 28-homoprassinosteroidone, and typhasterol. All five brassinosteroids were detected except for brassinolide. Compared with WT and... GhFLS Compared to KO petals, GhYP The contents of brassinosteroids (the bioactive BR), 6-deoxybrassinosteroids, 28-homoprassinosteroids, and 28-demethylbrassinosteroids in KO petals were all significantly reduced. Figure 16 ),This is GhYP The molecular reasons for the reduction in KO petals.

[0099] To promote GaYP Its application in the petals of ornamental plants is firstly to... GaYP (The nucleotide sequence is shown in SEQ ID NO. 8) is overexpressed in the petals of the model crop tobacco. Figure 17 The study found that genetically modified tobacco petals accumulated more pigment and were redder in color. Figures 18 to 20 Furthermore, the epidermal cells and petal area of ​​the petals significantly increased, ultimately increasing the area of ​​tobacco petals by 23% to 35%. Figures 18 to 22 ).

[0100] 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. People can obtain other embodiments based on these embodiments without creative effort, and these embodiments all fall within the protection scope of the present invention.

Claims

1. Cotton GoYP The application of genes in regulating plant petal size and / or regulating plant petal color is characterized by, The GoYP Genes include GhYP Genes and / or GaYP Genes; the stated GhYP The amino acid sequence of the protein encoded by the gene is shown in SEQ ID NO.1 or SEQ ID NO.2; GaYP The amino acid sequence of the protein encoded by the gene is shown in SEQ ID NO.

3.

2. The application according to claim 1, characterized in that, The regulation of plant petal color includes regulating the content of anthocyanins and / or flavonols in plant petals.

3. The application according to claim 2, characterized in that, The regulation of anthocyanin and / or flavonol content in plant petals includes negative regulation. GhYP Genes that reduce the content of anthocyanins and / or flavonols in plant petals; and / or, the regulation of anthocyanin and / or flavonol content in plant petals includes positive regulation. GaYP Genes increase the anthocyanin content in plant petals.

4. The application according to claim 1, characterized in that, The regulation of plant petal size includes negative regulation. GhYP Genes reduce plant petal size and / or positively regulate GaYP Genes that increase plant petal size; the regulation of petal color includes positive regulation. GaYP Genes make the petals more vibrant in color.

5. The application according to claim 4, characterized in that, The reduction in plant petal size is achieved by reducing the size of plant petal epidermal cells; the increase in plant petal size is achieved by increasing the size of plant petal epidermal cells.

6. GhYP The application of genes in regulating the content of brassinosteroids in plant petals is characterized by, The GhYP The amino acid sequence of the protein encoded by the gene is shown in SEQ ID NO.1 or SEQ ID NO.

2.

7. The application according to claim 6, characterized in that, The regulation of brassinosteroid content in plant petals includes negative regulation. GhYP Genes reduce the content of brassinosteroids in plant petals.

8. A method for improving the ornamental value of plants, characterized in that, Includes the following steps: Overexpression in plants GaYP Genes; the enhancement of plant ornamental value includes making the plant's petals more vibrant in color and / or larger in size; the GaYP The amino acid sequence of the protein encoded by the gene is shown in SEQ ID NO.

3.

9. Negative regulation GhFLS The application of genes in increasing the content of anthocyanins in plant petals is characterized by, The GhFLS The amino acid sequence of the gene-encoded protein is shown in SEQ ID NO.4 or SEQ ID NO.

5.

10. A genetically modified cotton or genetically modified tobacco, characterized in that, The genome of the genetically modified cotton was knocked out GhYP Gene or GhFLS Genes; the genome of the genetically modified tobacco plant was inserted with... GaYP Genes; the stated GhYP The amino acid sequence of the protein encoded by the gene is shown in SEQ ID NO.1 or SEQ ID NO.2; GaYP The amino acid sequence of the protein encoded by the gene is shown in SEQ ID NO.3; GhFLS The amino acid sequence of the protein encoded by the gene is shown in SEQ ID NO.4 or SEQ ID NO.5.

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