Application of GhCOMT gene in regulation and control of plant height

Abstract

The invention provides application of a GhCOMT gene in regulating and controlling the plant height of a plant, and belongs to the technical field of molecular breeding. The GhCOMT gene comprises GhCOMTAt and GhCOMTDt, the nucleotide sequence of the GhCOMTAt is as shown in SEQ ID NO. 1, and the nucleotide sequence of the GhCOMTAt is as shown in SEQ ID NO. 2. The nucleotide sequence of the GhCOMTDt is as shown in SEQ ID NO. 2. A CRISPR / Cas9 system is used for constructing a recombinant vector, an agrobacterium infection method is used for transforming a receptor plant, a plant with the GhCOMTAt gene and the GhCOMTDt gene knocked out is obtained, then through investigation of plant height changes of cotton in different growth periods, it is found that after the GhCOMTAt and the GhCOMTDt are knocked out, the plant height of the cotton can be remarkably reduced, and great significance is achieved for dwarf cotton variety cultivation and production practice.

Description

Technical Field

[0001] The invention belongs to the field of molecular breeding, and in particular relates to the application of GhCOMT gene in regulating plant strain growth. Background Art

[0002] As one of the world's important cash crops, cotton (Gossypium spp.) has always been in the leading position in the world in terms of planting area and output. Cotton is not only the basic raw material of the textile industry, but also an important strategic material and economic pillar of many countries. With the growth of the global population and the increase in demand for cotton, the cotton industry faces huge challenges and opportunities. In the cotton production process, the planting link occupies a vital position. In order to enhance the competitiveness of the cotton industry and achieve efficient and sustainable production methods, the application of mechanized planting and harvesting technology has become a key breakthrough in the development of the industry. Mechanized planting can not only greatly improve production efficiency and reduce labor costs, but also ensure the quality of cotton to a certain extent, thereby enhancing the overall competitiveness of the industry. Especially in the global market, the promotion of mechanization can help the cotton industry better adapt to changes in market demand and enhance its competitiveness in the international market. In addition, the promotion of mechanized planting and harvesting technology has a far-reaching impact on ensuring the sustainable development of the national cotton industry. It not only helps to improve production efficiency and ensure a stable supply of cotton, but also provides impetus for the development of related industrial chains. By improving technical level and management efficiency, the cotton industry can better cope with challenges such as climate change and land resource shortage, thereby achieving long-term economic benefits and social value.

[0003] Mechanized harvesting places specific requirements on the plant height of cotton, which is generally required to be controlled between 85 and 100 cm to ensure the smooth progress of mechanized operations and improve harvesting efficiency. Appropriate plant height not only helps the accuracy and efficiency of mechanical operations, but also promotes the optimization of cotton plant type and improves the utilization rate of light energy, thereby improving the growth quality and final yield of cotton. In my country's main cotton producing areas, cotton planting generally adopts a dwarfing and dense planting model. This model helps to improve the yield and quality of cotton, while facilitating mechanized harvesting, thereby significantly improving work efficiency and reducing harvesting costs. Mechanized harvesting can reduce manpower requirements, reduce losses and costs during harvesting, and is an important means to promote the modernization of the cotton industry. However, in the actual production process of cotton, in order to achieve the ideal plant height, plant growth regulators such as mesotrione are usually used to limit the excessive growth of plant height. Although this practice can effectively control the plant height of cotton, the frequent use of mesotrione also brings many challenges. First, over-reliance on such chemical regulators increases production and labor costs; second, the use of these chemicals will cause certain harm to the environment, especially if they are used improperly or excessively, which may cause the cotton plants to be too short or even cause the bolls to fall off, resulting in yield losses. Therefore, it is particularly important to explore and tap the key genes that control cotton plant height and apply them to the cultivation and production practices of new varieties. Through gene regulation technology, the use of chemical regulators can be reduced, while ensuring the optimization of cotton plant type, thereby improving the production efficiency of cotton and promoting the cotton industry to develop in a more environmentally friendly and sustainable direction. This can not only effectively reduce production costs, but also reduce negative impacts on the environment, providing guarantees for the long-term stability and efficiency of cotton production. Summary of the invention

[0004] The purpose of the present invention is to provide a method for regulating plant height, and to obtain dwarf plants through gene regulation technology.

[0005] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions:

[0006] The present invention provides an application of a GhCOMT gene in regulating plant strains, wherein the GhCOMT gene includes GhCOMT_At and GhCOMT_Dt;

[0007] The nucleotide sequence of GhCOMT_At is shown in SEQ ID NO.1;

[0008] The nucleotide sequence of GhCOMT_Dt is shown in SEQ ID NO.2.

[0009] The amino acid sequence encoded by the GhCOMT_At gene is shown in SEQ ID NO.3;

[0010] The amino acid sequence encoded by the GhCOMT_Dt gene is shown in SEQ ID NO.4.

[0011] Preferably, dwarf plants are obtained by knocking out the GhCOMT gene.

[0012] The invention also provides application of the GhCOMT gene in dwarf cotton breeding.

[0013] The present invention utilizes the CRISPR / Cas9 system to construct a recombinant vector and utilizes the Agrobacterium infection method to transform a recipient plant, thereby obtaining a plant in which the GhCOMT_At gene and the GhCOMT_Dt gene are knocked out. Then, by investigating the plant height changes of cotton in different growth stages and detecting the lignin content, it is found that knocking out GhCOMT_At and GhCOMT_Dt can significantly reduce the plant height of cotton, which is of great significance for the breeding of dwarf cotton varieties and production practice. BRIEF DESCRIPTION OF THE DRAWINGS

[0014] Figure 1 This is the gene structure diagram of cotton caffeic acid-O-methyltransferase gene GhCOMT;

[0015] Figure 2 is the alignment of the nucleotide sequences of cotton caffeic acid-O-methyltransferase gene GhCOMT At and GhCOMT Dt;

[0016] Figure 3 is the alignment of the amino acid sequences encoded by the caffeic acid-O-methyltransferase genes GhCOMT_At and GhCOMT_Dt;

[0017] Figure 4 This is the gene editing status of mutant cotton KO1 and KO2;

[0018] Figure 5 It is the signal distribution location of caffeic acid-O-methyltransferase genes GhCOMT_At and GhCOMT_Dt in cells;

[0019] Figure 6 is the expression of caffeic acid-O-methyltransferase genes GhCOMT_At and GhCOMT_Dt in different tissues in the qPCR real-time fluorescence quantitative experiment;

[0020] Figure 7 It is the expression of caffeic acid-O-methyltransferase genes GhCOMT_At and GhCOMT_Dt in transcriptome data at different stages and in different tissue locations.

[0021] Figure 8 This is the comparison of plant height between the wild type WT and the knockout mutants KO1 and KO2 in the greenhouse;

[0022] Fig. 9 It is the statistical situation of plant height of WT, KO1 and KO2 at different periods after sowing in the field; DETAILED DESCRIPTION

[0023] In the present invention, the plant material is the allotetraploid upland cotton recipient material W0;

[0024] Escherichia coli DH5α and Agrobacterium competent GV3101 were maintained in our laboratory;

[0025] Plasmid vectors pRGEB32 and pBinGFP4 were maintained by our laboratory;

[0026] Experimental reagents Total RNA extraction kit (SteadyPure Plant RNA Extraction Kit, Catalog No.: AG21019) and reverse transcription kit (Evo M-MLV Reverse Transcription Premix Kit, Catalog No.: AG11728) were purchased from Aikerui Biotech Co., Ltd.;

[0027] DNA polymerase (ApexHF HSDNApolymerase, catalog number: AG12204) was purchased from Aikerui Biotechnology Co., Ltd.

[0028] DNA ligation kit (OK Clon DNA ligation kit, catalog number: AG11808) was purchased from Acryl Biotech Co., Ltd.;

[0029] Fluorescence quantitative kit (ChamQ Universal SYBR qPCR Master Mix, catalog number: Q711-02) was purchased from Nanjing Novezan Biotechnology Co., Ltd.;

[0030] 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.

[0031] Example 1

[0032] The structure and amino acid sequence of cotton caffeic acid-O-methyltransferase gene GhCOMT Figure 1 As shown, the cotton caffeic acid-O-methyltransferase gene GhCOMT is located in the 87246359-87249073 bp interval of chromosome 08 of subgroup A and the 39175105-39177825 interval of chromosome 08 of allotetraploid upland cotton, and the CDS sequence is 1098 bp. The nucleotide sequence of GhCOMT_At is shown in SEQ ID NO.1:

[0033] ATGGGTTCAACCGGTGAAACCCAAATGACTCCCACCCAAGTCTCGGAT

[0034] GAGGAAGCCAACTTATTCGCCATGCAACTCACCAGTGCCTCAGTCCTT

[0035] CCGATGGTCCTCAAGTCGGCCATAGAACTTGACCTGCTGGAGATCATG

[0036] GCCAAAGCTGGCCCAGGTGCTTTCCTCTCCCCAAAAGAATTGGCTTCC

[0037] CAGCTCCCCACCAACAACCCCGATGCACCTGTCATGCTAGACCGCATC

[0038] TTGCGCCTCCTGGCTACCTACTCCATCCTCACTTGCTCCTTGCGCACTC

[0039] TTCCTGATGGCAAAGTGGAGAGACTCTATGGTCTTGGCCCTGTCTGCA

[0040] AATTCTTGACCAAGAATGAAGATGGCGTCACTCTTTCCGCCCTCAGTCT

[0041] CATGAATCAAGACAAGGTCCTTATGGAGAGCTGGTACTACTTGAAAGA

[0042] TGCCGTGCTGGAAGGTGGAATTCCCTTCAACAAAGCCTATGGTATGAC

[0043] CGCATTCGAGTATCATGGCACGGATCCTAGATTCAACAAGGTTTTCAAC

[0044] AGGGGAATGTCTGATCACTCTACCATTACCATGAAGAAGATTCTTGAGA

[0045] CCTATGATGGCTTTGAGGGACTCAAAACACTGGTCGATGTTGGCGGCG

[0046] GTACTGGAGCTACGCTTAACATGCTTGTCACCAAGCACCCTTCTATAAA

[0047] GGGCATCAACTTTGATTTGCCTCATGTCATTGAGGATGCTCCTGCTTATC

[0048] CTGGTGTGGAGCATGTTGGTGGGAGACATGTTTGAAAGTGTTCCAAAAG

[0049] GAGACGCCATCTTCATGAAGTGGATATGCCACGATTGGAGCGATGAGC

[0050] ACTGCTCCAAGTTTTTGAAGAAGTGCTACGAAGCTTTACCGGACAACG

[0051] GAAAAGTAATTGTCGCAGAATGTATTCTCCCAGATTACCCAGATCCTAG

[0052] CCTTGACACAAAGTTAGTTGTCCATATTGATTGCATCATGTTGGCTCAC

[0053] AATCCTGGTGGGAAAAGAGAGGACTGAGAAAGAATTCGAGGCCTTGGC

[0054] AAGGAGTGCTGGGTTTCAAGGTTTCCAAGTAAAATGCTGCGCTTTCGGCACATACATCATGGAGTTTCTCAAAAGGGTTTGA;

[0055]

[0056] The amino acid sequence encoded by GhCOMT_At is SEQ ID NO.3: MGSTGETQMTPTQVSDEEANLFAMQLTSASVLPMVLKSAIELDLLEIMAKAGPGAFLSPKELASQLPTNNPDAPVMLDRILRLLATYSILTCSLRTLPDGKVERLYGLGPVCKFLTKNEDGVTLSALSLMNQDKVLMESWYYLKDAVLEGGIPFNKAYGMTAFEYHGTDPRFNKVFNRGMSDHSTITMKKILETYDGFEGLKTLVDVGGGTGATLNMLVTKHPSIKGINFDLPHVIEDAPAYPGVEHVGGDMFESVPKGDAIFMKWICHDWSDEHCSKFLKKCYEALPDNGKVIVAECILPDYPDPSLDTKLVVHIDCIMLAHNPGGKERTEKEFEALARSAGFQGFQVKCCAFGTYIMEFLKRV;

[0057] The amino acid sequence encoded by GhCOMT_Dt is SEQ ID NO.4:

[0058] MGSTGETQMTPTQVSDEEANLFAMQLTSASVLPMVLKSAIELDLLEIMAK

[0059] AGPGAFLSPKELASQLPTSNPDAPVMLDRILRLLATYSILTCSLRTLPDGKV

[0060] ERLYGLGPVCKFLTKNEDGVTLSALSLMNQDKVLMESWYYLKDAVLEG

[0061] GIPFNKVYGMTAFEYHGTDPRFNKVFNRGMSDHSTITMKKILETYDGFEG

[0062] LKTLVDVGGGTGATLNMIVTKHPSIKGINFDLPHVIEDAPAYPGVEHVGG

[0063] DMFESVPKGDAIFMKWICHDWSDEHCSKFLKKCYEALPDSGKVIVAECIL

[0064] PDYPDPSLATKLVVHIDCIMLAHNPGGKERTEKEFEALARSAGFQGFQVKCCAFGTYIMEFVKRV.

[0065] like Figure 2 and Figure 3 As shown, the similarity between the nucleotide sequence and amino acid sequence of the two is 98%.

[0066] The GhCOMT amino acid sequence was submitted to the Expasy website (https: / / web.expasy.org / ) for amino acid sequence analysis and prediction. The results showed that the isoelectric point of GhCOMT_At was 5.27, and it was speculated to be a hydrophilic protein; the isoelectric point of GhCOMT_Dt was 5.35, and it was speculated to be a hydrophilic protein.

[0067] Example 2

[0068] Acquisition and propagation of mutant materials

[0069] The synthesis of lignin is closely related to plant growth and development. Changes in lignin content will affect plant height, so we selected the caffeic acid-O-methyltransferase gene GhCOMT in the lignin metabolic pathway for the next step of experiment and exploration. A vector for knocking out the GhCOMT gene was constructed to obtain homozygous plants with the GhCOMT gene knocked out. The specific process is briefly as follows:

[0070] The target site base sequence was selected as: 5'-ACACTGGTCGATGTTGGCGG-3' (SEQ ID NO.5). The target sequence of the GhCOMT_At gene was located at the 1976bp-1995bp position of the nucleotide sequence of the gene; the target sequence of the GhCOMT_Dt gene was located at the 1981bp-2000bp position of the nucleotide sequence of the gene.

[0071] The CRISPR / Cas9 system was used to construct a recombinant vector and transform the recipient plant W0 (WT). The specific steps are as follows:

[0072] Construct the pRGEB32_COMT vector and design primers according to the plasmid vector and target nucleotide sequence. The primer sequences are shown in Table 1:

[0073] Table 1

[0074]

[0075] Upland cotton W0 was used as the research material, and the RNA in the leaves of the cotton material was extracted using a plant RNA extraction kit (SteadyPure plant RNA extraction kit). The above RNA was reverse transcribed (Evo M-MLV reverse transcription premix kit) to obtain cDNA, and the above cDNA was used as a template for PCR amplification, and the product was purified and recovered.

[0076] The PCR amplification reaction is shown in Table 2:

[0077] Table 2

[0078] Reagents Volume (25μL system) KODOneTMPCRMasterMix 12.5μL Primer1 0.3μL Primer2 0.3μL Template cDNA 1.0μL ddH2O 10.9μL Total 25μL

[0079] The PCR instrument program settings are shown in Table 3:

[0080] Table 3

[0081]

[0082]

[0083] Note: The annealing temperature is determined by the primer Tm value, and in practice 58°C is generally used; the extension time is determined by the length of the amplified fragment.

[0084] The enzyme digestion reaction system is shown in Table 4:

[0085] Table 4

[0086] Reagents volume Plasmid vector 1-2 μg 10×CutSmartBuffer 5μL BnS 1μL water Up to 50 μL

[0087] The homologous recombination reaction conditions are as follows in Table 5:

[0088] Table 5

[0089] Reagents volume PCR purification product 3μL pRGEB32 digested vector 1μL 2.5XOKClonMasterMIX 4μL RNase-free water 2μL

[0090] The PCR product was cloned into the pRGEB32 vector, and the recombinant plasmid was transformed into E. coli DH5α to obtain a single clone colony and send it for testing, thereby obtaining a recombinant plasmid with correct sequencing. The recombinant plasmid with correct sequencing was transformed into Agrobacterium competent GV3101 and stored in a -80℃ refrigerator.

[0091] Use seedling culture medium to culture upland cotton W0 sterile etiolated seedlings, take the sterile etiolated seedling hypocotyls and cut them into stem segments of about 0.5 cm, with neat cuts; shake and infect the prepared stem segments in Agrobacterium solution for 8 minutes, take out the stem segments and dry them on sterilized filter paper, and evenly place them in the co-culture medium for dark culture for 48 hours. After co-culture, place the hypocotyl stem segments on the embryonic callus induction medium to induce callus tissue, and add 50 μg / mL kanamycin and timentin to the medium to inhibit the growth of Agrobacterium. Obtain callus tissue, and further cultivate and screen to obtain T0 generation plants.

[0092] The DNA of T0 generation plants was extracted, the gene was amplified by PCR, the PCR products were recovered and identified using a high-throughput Tracking Of Mutations (Hi-TOM), the effectively edited strains were screened, propagated and the above sequencing identification was repeated.

[0093] Example 3 Identification and screening of homozygous mutant materials

[0094] The editing type of the GhCOMT gene in the obtained homozygous plants was detected and identified, and finally homozygous plants with GhCOMT gene knockout were obtained in the T2 generation. The specific process is as follows:

[0095] 1. Use Hi-Tom high-throughput sequencing to detect the type of gene editing in homozygous strains with GhCOMT knockout. Figure 4 a is a schematic diagram of gene editing types.

[0096] 2. The first generation sequencing method was used to detect the gene editing type in the homozygous strain with GhCOMT knockout. The test results were as follows: Figure 4 As shown in b. In GhCOMT#KO1, one base C was missing from the DNA of GhCOMT_At, and two bases GG were missing from the DNA of GhCOMT_Dt; in GhCOMT#KO2, eight bases GGCGGTAC were missing from the DNA of GhCOMT_At, and one base C was missing from the DNA of GhCOMT_Dt; both resulted in frameshift knockout of genes in the A and D families. Therefore, two homozygous editing lines were obtained, both of which were effective editing lines of GhCOMT.

[0097] Subcellular localization

[0098] Construct 35S-GhCOMT-GFP vector, design primer sequences according to GhCOMT nucleotide sequence, the primer sequences are shown in Table 6:

[0099] Table 6

[0100]

[0101] Upland cotton W0 was used as the research material, and the RNA in the leaves of the cotton material was extracted using a plant RNA extraction kit (SteadyPure plant RNA extraction kit). The above RNA was reverse transcribed (Evo M-MLV reverse transcription premix kit) to obtain cDNA, and the above cDNA was used as a template for PCR amplification, and the product was purified and recovered.

[0102] The vector construction steps are the same as in Example 2.

[0103] Agrobacterium infection of tobacco: shake overnight, prepare 10 mL of turbid Agrobacterium culture for each carrier, centrifuge at 4000 rpm for 10 min, collect the bacteria at the bottom of a 50 mL centrifuge tube, drain the liquid, resuspend the bacteria with an appropriate amount of infiltration buffer (containing 10 mM MgCl2, 10 mM MES, 200 μM acetosyringone), and measure the OD 600 The value was adjusted to between 1.50-1.80, wrapped in tin foil for dark treatment, and left at 28℃ for 3-5h. The Agrobacterium resuspension was injected into Nicotiana benthamiana / nuclear localization tobacco leaves of appropriate size with a syringe, and cultured in the dark for 48h. Take the tobacco leaves injected with Agrobacterium, make a glass slide with the back of the leaves facing up, and observe the back of the leaves with a laser confocal microscope (Zeiss LSM980, Germany) and take pictures. Figure 5 As shown, the control GFP was localized in the cell membrane and nucleus, and the GhCOMT_At-GFP and GhCOMT_Dt-GFP signals were distributed on the cell membrane.

[0104] Real-time fluorescence quantitative analysis of different tissues

[0105] Taking upland cotton W0 as the research material, the first stem node, young leaves, stem meristem and roots about 25 days after sowing (four-leaf stage) and ovules and petals on the 0th day of flowering period were taken respectively. RNA in the cotton material was extracted using a plant RNA extraction kit, and the RNA of each tissue was reverse transcribed to obtain cDNA. Real-time fluorescence quantitative primers and internal reference gene primers were designed (as shown in Table 7):

[0106] Table 7

[0107]

[0108]

[0109] The fluorescence quantitative kit ChamQ Universal SYBR qPCR MasterMix (Vazyme, Nanjing) was used to detect the expression level of GhCOMT gene in different tissue parts of W0. The reaction system is shown in Table 8.

[0110] Table 8

[0111] Reagents volume 2×ChamQ Universal SYBR qPCR Master Mix 10μL PrimerF 0.4μL PrimerR 0.4μL Template cDNA 1μL RNase-free ddH2O 8.2μL

[0112] The PCR amplification program is shown in Table 9:

[0113] Table 9

[0114]

[0115] Three biological replicates were set. The relative expression data were expressed as 2 ^(-ΔC) The relative expression level is Figure 6 As shown, GhCOMT_At and GhCOMT_Dt genes were expressed at relatively high levels in the stems and roots of cotton, and this pair of genes may be involved in the regulation of plant height during the growth period of cotton. Figure 7 The relative expression data of GhCOMT_At and GhCOMT_Dt transcriptomes are from the cotton gene mutant library of the College of Agriculture and Biotechnology, Zhejiang University ( http: / / cotton.zju.edu.cn / ), the GhCOMT_At and GhCOMT_Dt genes in the data also have relatively high expression levels in the stems and roots of Upland Cotton TM-1 cotton, which again indicates that this pair of genes may be involved in the regulation of plant height during the growth period of cotton.

[0116] Example 4 Investigation of changes in plant height at different growth stages

[0117] At different developmental stages of the plants, the plant height changes of the wild type WT and the two homozygous mutant plants KO1 and KO2 screened in Example 3 were statistically analyzed, and the specific contents are as follows:

[0118] like Figure 8 As shown in the figure, the standard ruler is 10 cm. The plant height of the wild type WT grown in the greenhouse is significantly different from that of the mutants KO1 and KO2. The plant heights of WT and KO plants were measured 20 days, 30 days, 40 days, 50 days and 60 days after sowing in the field, with ten plants in each group. The statistical results are shown in Fig. 9 As shown, there are obvious differences between the mutant KO and the wild-type WT at each stage, and at each stage, the wild-type WT plants are significantly taller than the two mutant KO1 and KO2 plants. The GhCOMT gene knockout plants can significantly reduce the plant height of cotton plants and can be used to breed dwarf cotton varieties.

[0119] 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. The application of GhCOMT gene in regulating plant strains, characterized in that: The GhCOMT gene includes GhCOMT_At and GhCOMT_Dt; The nucleotide sequence of GhCOMT_At is shown in SEQ ID NO.1; The nucleotide sequence of GhCOMT_Dt is shown in SEQ ID NO.

2.

2. The use according to claim 1, characterized in that: The amino acid sequence encoded by the GhCOMT_At gene is shown in SEQ ID NO.3; The amino acid sequence encoded by the GhCOMT_Dt gene is shown in SEQ ID NO.

4.

3. The use according to claim 1, characterized in that: Dwarf plants were obtained by knocking out the GhCOMT gene.

4. Application of GhCOMT gene in dwarf cotton breeding.

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