Application of GhFL gene in regulating flowering time and plant type in cotton

Abstract

The present invention provides GhFL The application of genes in regulating the flowering period and plant type of cotton belongs to the field of genetic engineering technology. GhFL The GhFL protein is located in the cell nucleus and plays an important role in regulating the flowering and plant type of plants. After silencing the gene in cotton, the growth of the top of the main stem of the plant is inhibited, the growth point is replaced by the flower bud, flowering is advanced, and GhFL Gene silencing in plants GhAP1 and GhCAL The expression level of GhFL Genes may affect flowering time and plant type by affecting the expression of flowering-related genes. GhFL Genes and their encoded proteins can regulate the flowering time and plant type of plants, providing new molecular targets for improving early maturity and plant type of cotton.

Description

Technical Field

[0001] The present invention relates to the field of genetic engineering technology, and in particular to GhFL The application of genes in regulating flowering time and plant type in cotton. Background Art

[0002] Cotton is an important cash crop. It not only provides important natural fiber raw materials for the textile industry, but also has important uses in national defense, medicine, automobile industry, etc. It has an irreplaceable position in the development of the national economy. Plant type is one of the important traits of cotton. Cotton varieties with different plant types have obvious differences in yield, quality, stress resistance, etc. The ideal plant type can improve cotton yield and quality. The cotton plant type structure mainly includes plant height, total number of fruit branches, branching pattern, fruit branch length, number of fruit branch nodes, internode length of fruit branches, fruit branch angle, leaf size, and arrangement of leaves and fruit branches, which are important traits affecting cotton yield. Improving cotton plant type through genetic means is one of the effective means to enhance the international competitiveness of domestic cotton. Plant type breeding can adjust the characteristics of cotton plant structure and fruit distribution, making it more suitable for mechanized picking and improving harvesting efficiency and quality.

[0003] In cotton, plant morphology and flowering time are critical for mechanized management and harvesting. Compact plant shape and highly concentrated boll distribution are considered desirable traits that facilitate high-density planting and are amenable to mechanized production. FL Genes play an important role in the flower development regulatory network, controlling the initiation of floral meristems, the maintenance of floral meristem characteristics, and the formation of floral organs. They are key regulatory factors in the transition from growth development to reproductive development. Studies have shown that both the gibberellin (GA) pathway and the photoperiod pathway regulate FL The expression of genes controls flowering. FL Genes integrate external environmental and internal signals to regulate flowering in plants 。FL Mutations in the gene can cause the floral organs to transform into leaf-like structures.

[0004] although FL As a characteristic gene of floral meristem, its expression range is not limited to floral tissue or tissues directly related to flowering. FL The gene was expressed not only in leaf primordia but also in leaves throughout the vegetative growth stage. FL The gene causes flowering to occur earlier and encourages lateral buds to transform into flower buds. In cucumbers, CsFL The gene is expressed in the shoot apical meristem, floral meristem, and floral organ primordium. Studies have shown that CsFL maintains the growth of the shoot apical meristem by interacting with CsWUS and activates CsAP3 and CUM1to promote flower development. However, in cotton FL The function of the gene is still unclear, but this invention reveals GhFL Its function in regulating cotton plant development is GhFL Genes provide the basis for improving cotton plant type. Summary of the invention

[0005] The object of the present invention is to provide GhFL The application of genes in regulating cotton flowering period and plant type provides new gene targets for improving cotton maturity and plant type.

[0006] The inventors screened a mutant with shorter plant height and inhibited apical growth from an Asian cotton mutant library and found that the mutant GaFL Gene mutation causes changes in traits. In order to further study the function of the FL gene in upland cotton, the inventors used Asian cotton GaFL Based on the gene, homologous genes were searched in the genome of upland cotton, and the gene with the highest homology was named GhFL The gene ID is Gh_A07G051000. The cDNA of this gene contains an open reading frame of 1221 bp, encoding a protein of 406 amino acids. The expression level of this gene in different developmental stages and different tissues of cotton was analyzed and it was found that GhFL The expression level of the gene in leaves gradually increased from the one-leaf stage to the five-leaf stage. It was expressed in different tissues, but the highest expression level was in the apical meristem. Further studies showed that the GhFL protein was localized in the nucleus and may function as a transcription factor.

[0007] For research GhFL The inventors used virus-induced gene silencing (VIGS) technology to silence the gene in the upland cotton variety TM-1. GhFL Gene silencing, found that in cotton plants injected with VIGS, GhFL The gene expression level is reduced. GhFL After gene silencing, the growth of the top of the main stem of the plant was inhibited, and the growth point was replaced by flower buds, resulting in a decrease in plant height. GhFL The gene silenced lines flowered earlier, and this result indicated that GhFL Genes can change the growth habits of cotton and thus regulate the plant shape.

[0008] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions:

[0009] The present invention provides GhFL Gene or contains GhFL The use of genetically derived biological material in at least one of the following:

[0010] (1) Application in regulating cotton flowering period and plant type;

[0011] (2) Application in cotton breeding;

[0012] (3) Application in breeding new cotton germplasm with improved plant traits;

[0013] Said GhFL The nucleotide sequence of the gene is shown in SEQ ID NO.1.

[0014] Preferably, the regulation is inhibition or downregulation GhFL The expression level of the gene is changed, cotton flowers bloom earlier, the growth of the top of the main stem is inhibited, the growth point is replaced by flower buds, and the plant height becomes shorter.

[0015] Preferably, the comprising GhFL Genetic biological materials include recombinant vectors, recombinant microorganisms or expression cassettes.

[0016] Preferably, by inhibiting the GhFL Genes, regulate cotton plant type, and cultivate cotton germplasm with improved plant type.

[0017] The present invention also provides a method for reducing cotton plant height, changing cotton plant type and flowering period, comprising the following steps:

[0018] S1. Design primers and amplify GhFL Specific sequences of genes;

[0019] S2. GhFL The coding sequence of the gene was connected to the pTRV2 plasmid vector to obtain the pTRV2-GhFL recombinant plasmid;

[0020] S3. Introduce the pTRV2-GhFL recombinant plasmid into GV3101 Agrobacterium, and culture the Agrobacterium until the bacterial solution OD600 = 1.2~1.8;

[0021] S4. Collect the GV3101 Agrobacterium bacterial suspension and resuspend it with an equal volume of resuspension solution;

[0022] S5. The resuspension containing GV3101 Agrobacterium was kept in dark and then mixed with the resuspension containing the auxiliary vector pTRV1 in a volume ratio of 1:1 to obtain a mixed solution;

[0023] S6. Select cotton seedlings with flattened cotyledons and no true leaves for injection. Scratch the back of the cotton cotyledons with the needle of a syringe and inject the mixed solution from the wound until the entire cotyledon is filled. After the injection, culture in the dark for 22 to 26 hours and then culture normally.

[0024] Preferably, amplification GhFLThe primers for the gene coding sequence are shown in SEQ ID NO.9 and SEQ ID NO.10. GhFL The specific sequence of the gene is shown in SEQ ID NO.11.

[0025] Preferably, the culture conditions of the recombinant Agrobacterium are: 26-30°C, 200-240 rpm.

[0026] Preferably, the resuspension contains 8-12 mM MgCl2, 8-12 mM MES, 140-160 μM acetosyringone, and pH=5-6.

[0027] Preferably, the light-proof standing time is 2.5 to 3.5 h. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] Figure 1 for GhFL The expression levels of genes in terminal buds and different tissues of upland cotton TM-1 at different stages.

[0029] Figure 2 for GhFL Subcellular localization results of the protein.

[0030] Figure 3 for GhFL Cotton plant phenotype, flowering time and plant height statistics after gene silencing.

[0031] Figure 4 for GhFL Flowering-related genes in cotton plants after gene silencing GhSOC1 , GhAP1 and GhCAL Expression changes. DETAILED DESCRIPTION

[0032] The technical solutions provided by the present invention are described in detail below in conjunction with the embodiments, but they should not be construed as limiting the protection scope of the present invention.

[0033] Example 1. GhFL Gene cloning

[0034] GhFL The gene ID number is Gh_A07G051000, obtained from CottonFGD (http: / / www.cottonfgd.org / ) GhFL The CDS sequence and the encoded amino acid sequence of the gene, whose CDS sequence is 1221 bp and encodes 406 amino acids, are shown in SEQ ID NO.1 and SEQ ID NO.2 respectively.

[0035] SEQ ID NO.1:

[0036]

[0037] SEQ ID NO.2:

[0038] MDPETFASGSFFKWDPRGMMAPTPARLMEAVAAVPQPQTVAVAAAAAYMGRPRELGGIEELFQAYGIRYYTAAKIAELGFTVNTLLGMKEEELDEMMNSVSQIFRWELLVGERYGIKAAVRAERRRLEEEDSRRRHLVLGDTTTTTTAANALDALSQEGLSEEPVQQEKEAAGSGGWGTWEMVIGGGRRKQQRRRKGQKKVVE VDNGDEFEGGDDDDENGDGGGGGYERQREHPFIVTEPGEVARGKKNGLDYLFHLYEQCRDFLIQVQNIAKDRGEKCPTKVTNQVFRYAKKAGASYINKPKMR HYVHCYALHCLDEEASNALRRAFKERGENVGAWRQACYKPLVSIAARQGWDIDAIFNAHPRLAIWYVPTKLRQLCHAERNGAAVVAAASSSVSGGHDHMGF.

[0039] Example 2 GhFL Analysis of gene expression in upland cotton TM-1

[0040] 1. Design amplification GhFL Gene-specific primers and internal reference gene primers:

[0041] GhHistonePF: TCAAGACTGATTTGCGTTTCCA (SEQ ID NO.3);

[0042] GhHistonePR: GCGCAAAGGTTGTGTCTTC (SEQ ID NO.4);

[0043] GhFLPF2: GCAGTGTCGGGATTTCTTGA (SEQ ID NO.5);

[0044] GhFLPR2: AGGCAATGTAGGGCGTAGCA (SEQ ID NO. 6).

[0045] 2. Extraction of total RNA from cotton tissue.

[0046] Leaves of cotton TM-1 from the 1-leaf stage to the 5-leaf stage, as well as roots, stems, stem tips, petals, anthers, styles, and pollen tissues were collected and placed in liquid nitrogen and stored in a -80°C refrigerator for total RNA extraction. RNA extraction was performed according to the instructions of the kit FastPure Plant Total RNA Isolation Kit (NoviZan, Nanjing, China).

[0047] 3. The extracted total RNA was synthesized by reverse transcription cDNA according to the instructions of the kit StarScript II RT Mixwith gDNA Remover (Beijing Kangrun Chengye Biotechnology Co., Ltd.).

[0048] 4. Using cDNA as a template, gene-specific primers and 2× RealStar Fast SYBR qPCR Mix (Genstar, China), and then using Applied Biosystems 7500 for fluorescence quantitative PCR: the PCR program was set according to the two-step method: pre-denaturation: 95°C, 2min; 95°C, 5s; 60°C, 30s; a total of 40 cycles, the PCR reaction system is shown in Table 1; the melting curve was automatically set by the instrument, and the data was processed using Microsoft Excel 2019 software to calculate the expression level of the gene, and the graph was plotted using GraphPad Prism 8 software. The results are shown in Table 1. Figure 1 The results show that GhFL The expression level of the gene in leaves gradually increased from the one-leaf stage to the five-leaf stage. It was expressed in different tissues, but the expression level was highest in the apical meristem, indicating that the gene may be involved in the developmental regulation of cotton flowering and apical growth points.

[0049] Table 1 PCR reaction system:

[0050]

[0051] Example 3. Subcellular localization analysis of GhFL protein

[0052] Will GhFL The full-length CDS fragment of the gene was inserted into the pRI101-An-GFP vector to obtain the pRI101-An-GhFL-GFP fusion protein vector (sequencing verified). The preparation steps of the pRI101-An-GhFL-GFP fusion protein vector are as follows:

[0053] The genomic DNA of upland cotton TM-1 was extracted, and PCR primers were designed to amplify the full length of the CDS of the gene. The primers used for amplification were:

[0054] GhFLPF1:ATACATATGCCCGTCGACATGGACCCTGAGACT (SEQ ID NO.7);

[0055] GhFLPR1: AACCGCCTCCACCGGATCCGAACCCCATATGAT (SEQ ID NO. 8).

[0056] The PCR amplification reaction system and reaction procedure were the same as in Example 2. GhFL The CDS sequence fragment of the gene was connected to the pRI101-An-GFP vector by homologous recombination, and the restriction sites SalI and BamHI were added at the start codon and stop codon, respectively. ® The Ultra One Step Cloning Kit (Nanjing Novozyme Biotechnology Co., Ltd.) was used in strict accordance with the instructions. Positive clones were selected for sequencing verification. GhFL The full-length sequence of the gene was completely consistent with the reference sequence, and the pRI101-An-GhFL-GFP fusion protein vector was obtained.

[0057] The vector was transferred into Agrobacterium GV3101 by freezing method, and then Agrobacterium was injected into young tobacco leaves. After 2 days of dark treatment, GFP fluorescence was observed under laser confocal microscope. The results are as follows: Figure 2 The results showed that GhFL protein was localized in the cell nucleus and may play a regulatory role as a transcription factor.

[0058] Example 4 Silence GhFL Observation of cotton plant phenotype and changes in flowering gene expression

[0059] Design primers for amplification GhFL The specific sequence of the gene (SEQ ID NO.11) was cloned into the pTRV2 plasmid vector. After sequencing confirmed that the sequence was correct, the pTRV2-GhFL vector was obtained and amplified. GhFL The primers for the gene-specific sequences are:

[0060] GhFLPF4:GTGAGTAAGGTTACCGAATTCATGGAAGCAGTTGCCGCC (SEQ ID NO.9);

[0061] GhFLPR4:CGTGAGCTCGGTACCGGATCCAAGCCGTCTCCTTTCAGCTC (SEQ ID NO. 10);

[0062] VIGS specific sequence:

[0063] ATGGAAGCAGTTGCCGCCGTACCTCAGCCGCAGACGGTGGCTGTGGCTGCTGCTGCTGCTTACATGGGGAGGCCGAGGGAACTTGGGGGTATTGAAGAGTTATTTCAAGCTTATGGAATCAGGTACTATACTGCGGCGAAGATAGCTGAGTT AGGGTTCACTGTAAACACCCTTTTAGGCATGAAAGAAGAAGAGTTAGACGAGATGATGAATAGTGTATCACAGATATTCAGGTGGGAACTCCTCGTCGGTGAAAGGTACGGTATTAAAGCCGCCGTTAGAGCTGAAAGGAGACGGCTT (SEQ ID NO.11).

[0064] The pTRV2-GhFL vector was introduced into GV3101 Agrobacterium and cultured in a constant temperature shaker at 28 °C and 220 rpm for 12 h until the OD 600 =1.5, room temperature, centrifuge at 6000 rpm for 10 min, collect the bacteria, resuspend them with an equal volume of resuspension solution (10mMMgCl2, 10mM MES, 150 μM acetosyringone, pH=5.6), keep them away from light for 3 h, mix them with GV3101 Agrobacterium resuspension solution containing auxiliary vector pTRV1 at a volume ratio of 1:1, select cotton seedlings with uniform growth, flat cotyledons and no true leaves for injection, gently scratch the back of the cotton cotyledons with a syringe needle, inject the bacterial solution from the wound until the entire cotyledon is filled, and culture them in the dark for 24 h after the injection, and then culture them normally.

[0065] Two weeks after VIGS bacterial injection, newly grown true leaves were selected, RNA was extracted, cDNA was synthesized, and quantitative PCR was used to detect the silencing efficiency of the GhSP2 gene. Plants with high silencing efficiency were selected for phenotypic observation. The results are shown in Figure 3 shown. Figure 3 show, GhFL After gene silencing, the growth of the top of the main stem of the plant is inhibited, the growth point is replaced by flower buds, flowering is early, and the plant height becomes shorter. GhFL The silencing of the gene led to changes in the plant shape of cotton plants and early flowering, which may play a role in the establishment of cotton plant shape.

[0066] For further analysis GhFL The mechanism of gene regulation of cotton plant shape and flowering, and the detection of flowering-related genes in silenced plants GhSOC , GhAP1 andGhCAL The expression level of GhFL The cDNA of the gene silenced plant was used as a template, and the primer sequences were:

[0067] GhAP1PF: AGGAGCAAAACGCCATGCTA (SEQ ID NO. 12);

[0068] GhAP1PR:ATTCAAACAAGGATGCGGC (SEQ ID NO. 13);

[0069] GhSOCPF: CGACGGAACATGCACAACAT (SEQ ID NO. 14);

[0070] GhSOCPR: GGCTTCGTAACTCCCTCCA (SEQ ID NO. 15);

[0071] GhCALPF: TATAGGCGGCATACAAAGGA (SEQ ID NO. 16);

[0072] GhCALPR: TTCCATGATTTTGTTCCACA (SEQ ID NO. 17);

[0073] The reaction procedure and reaction system are the same as in Example 2.

[0074] The amplification results are as follows Figure 4 Compared with the control group, GhFL Gene silencing in plants GhAP1 and GhCAL The expression level of GhFL Genes may affect flowering time and plant type by influencing the expression of flowering-related genes.

[0075] As can be seen from the above embodiments, the present invention provides GhFL Application of genes in regulating cotton flowering period and plant type, down-regulation constructed by the present invention GhFL Cotton lines expressing the gene can provide germplasm materials for breeding dwarf and compact cotton.

[0076] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. Silence G JZ Gene or silence G JZ The use of genetically derived biological material in at least one of the following: (1) Application in regulating cotton plant type; the regulation is: silencing G JZ Gene, the growth of the top of the cotton main stem is inhibited, and the height of the cotton plant becomes shorter; (2) Application in breeding cotton germplasm with improved plant traits; G JZ Gene, reduce cotton plant height, cultivate cotton germplasm with improved plant type; Said G JZ The nucleotide sequence of the gene is shown in SEQ ID NO.

1.

2. A method for reducing cotton plant height, characterized in that: The following steps are involved: S1. Design primers and amplify G JZ Specific sequences of genes; S2. G JZ The specific sequence of the gene was connected with the pTRV2 plasmid vector to obtain the pTRV2-GhFL recombinant plasmid; S3. Introduce the pTRV2-GhFL recombinant plasmid into GV3101 Agrobacterium and culture the recombinant Agrobacterium until the bacterial liquid OD 600 =1.2~1.8; S4. Collect the recombinant Agrobacterium bacterial solution and resuspend it with an equal volume of resuspension solution; S5. After the resuspension containing the recombinant Agrobacterium was kept in the dark, it was mixed with the resuspension containing the auxiliary vector pTRV1 in a volume ratio of 1:1 to obtain a mixed solution; S6. Select cotton seedlings with flattened cotyledons and no true leaves for injection. Use the needle of a syringe to scratch the back of the cotton cotyledons. Inject the mixed solution from the wound until the entire cotyledon is filled. After the injection, culture in the dark for 22-26 hours and then culture normally. Said G JZ The nucleotide sequence of the gene is shown in SEQ ID NO.1; Said G JZ The specific sequence of the gene is shown in SEQ ID NO.

11.

3. The method according to claim 2, characterized in that Amplification G JZ The primers of the gene coding sequence are shown in SEQ ID NO.9 and SEQ ID NO.

10.

4. The method according to claim 3, characterized in that The culture conditions of recombinant Agrobacterium are: 26~30 ℃, 200~240 rpm.

5. The method according to claim 4, characterized in that The resuspension solution contains 8-12 mM MgCl2, 8-12 mM MES, 140-160 μM acetosyringone, and pH=5-6.

6. The method according to claim 2, characterized in that The light-proof standing time is 2.5 to 3.5 h.

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