Application of Unicaulis gene for regulating and controlling growth of tomato axillary buds in tomato breeding

Abstract

The invention relates to the technical field of plant molecular biology, in particular to application of a Unicaulis gene for regulating and controlling growth of tomato axillary buds in tomato breeding. The Uniicaures gene is used for positively regulating and controlling the elongation and growth of lateral branches of tomatoes, and the nucleotide sequence of the Uniicaures gene is as shown in SEQ ID NO. 1; the amino acid sequence of the encoding protein of the Unicaulis gene is as shown in SEQ ID NO. 2. The tomato lateral branch elongation growth gene Unicaulis is positively regulated and controlled by constructing a genetic linkage group, finely positioning and cloning, and by overexpressing the Unicaulis gene in a tomato lateral branch-free plant or increasing the content of the encoding protein of the Unicaulis gene, the accumulation amount of auxin at the axilla part of a transgenic plant is reduced, and the lateral branch of the tomato can normally sprout and obviously grow. The Unicaulis gene or the encoded protein thereof provided by the invention can significantly reduce the accumulation amount of auxin in the axillary part of the tomato plant, relieve the apical dominance of the tomato plant to lateral branches, accelerate the extension and growth of axillary buds of the axillary part of the tomato plant into lateral branches, and provide gene resources and new germplasm for cultivating tomato varieties with strong lateral branches.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of plant molecular biology, and particularly relates to a gene for regulating axillary bud growth of tomato and application of the gene in tomato breeding. Unicaulis BACKGROUND

[0002] Plant architecture is extremely important for its own growth, and plays an important role in photosynthesis optimization, nutrient and water utilization, and response to biological stress. Plant morphogenesis is a complex and delicate process, and lateral branches, as an important component of the aboveground part of the plant, play a key role in light capture, space utilization, and reproduction of the plant.

[0003] The growth and development of plant lateral branches is a complex process, which is comprehensively regulated by multiple factors, and hormones play a crucial role. As chemical messengers in the plant body, hormones can accurately regulate the initiation, growth, and branching pattern formation of lateral branches at each stage, and maintain the balance and coordination of plant morphogenesis. When the external environmental factors change, plant hormones can achieve a new balance through their own adjustment and then initiate the downstream signal conduction to regulate the germination or dormancy of plant lateral branches. Auxin is mostly synthesized at the top of the plant and transported downward to the leaf axil to inhibit the elongation growth of axillary buds. In previous studies, a series of key genes that can regulate lateral bud formation and later growth have been identified. For example, the key gene TEOSINTE BRANCHED1 TB1 for regulating maize tillering, MOC1 transcription factors, etc. The gene LAX1 in rice encodes a bHLH transcription factor, lax1 mutants exhibit fewer panicles and reduced tiller number. The Arabidopsis branching regulatory gene RAX1 belongs to the R2R3-MYB gene family, RAX1 affects the expression of STM by regulating CUP-SHAPED COTYLEDON2 ( CUC2 ), thereby inhibiting the formation of lateral meristems and reducing branching.

[0004] Tomato lateral branches are one of its important agronomic traits, with the characteristics of continuous growth and branching. During the entire growth period of tomato, the axillary buds have strong germination ability, each axillary bud can form a lateral branch, and multiple lateral branches can grow at the same time. However, too many small lateral branches can break the balance between vegetative growth and reproductive growth of the plant, causing intensified competition for nutrients, unbalanced light utilization, and poor ventilation conditions, which affects the excellent growth of the tomato plant itself. However, there are relatively few genes for regulating the growth of tomato axillary buds or lateral branches in the prior art. SUMMARY

[0005] ​​In view of this, this invention mainly uses the tomato variety LA0580 with few lateral branches and the variety LA0533 with normal lateral branches as two parents. By constructing a genetic linkage population, the gene for positively regulating the elongation of tomato lateral branches is precisely mapped and cloned. Unicaulis (This invention is referred to as " Uni The study identified the genes that regulate the elongation of tomato axillary buds, providing genetic resources and new germplasm for breeding tomato varieties with strong lateral branches.

[0006] Specifically, the present invention provides the following technical solutions: In a first aspect, the present invention provides a method for regulating the growth of tomato axillary buds. Uni The application of genes in tomato breeding, wherein, the Uni The gene positively regulates the elongation of tomato lateral branches. Uni The amino acid sequence of the gene-encoded protein is shown in SEQ ID NO.2.

[0007] Furthermore, the aforementioned Uni The nucleotide sequence of the gene is shown in SEQ ID NO.1.

[0008] Furthermore, the aforementioned Uni The gene significantly promotes the elongation of lateral branches in the leaf axils of tomatoes.

[0009] Furthermore, the application includes overexpressing the [specific ingredient] in tomato plants. Uni The gene significantly promotes the elongation of lateral branches. Preferably, the tomato plant is a tomato variety without lateral branches.

[0010] Secondly, this invention provides a method for regulating the growth of tomato axillary buds. Uni The application of biological materials with overexpression of the gene or its encoded protein in the positive regulation of lateral branch elongation in tomatoes, wherein... Uni The amino acid sequence of the gene-encoded protein is shown in SEQ ID NO.2.

[0011] Furthermore, the biomaterial includes any one or more of the following: 1) Contains Uni Expression cassettes of gene ORF sequences; 2) Contains Uni Recombinant expression vectors of gene ORF sequences; 3) A recombinant expression vector containing the expression cassette described in 1); 4) Contains Uni Engineered bacteria with ORF sequences of genes; 5) Engineered bacteria containing the expression cassette described in 1); 6) Engineered bacteria containing the recombinant expression vector described in 2) or 3).

[0012] Preferably, the backbone vector of the recombinant expression vector is the pSAK277 vector.

[0013] Thirdly, the present invention provides a method for constructing a recombinant expression vector, comprising the steps of: S1, using cDNA reverse transcribed from total RNA of LA0533 tomato tissue as a template, was amplified using amplification primers. Uni The ORF region of the gene is used to obtain the target gene fragment; wherein the amplification primers include an upstream primer FW with a nucleotide sequence as shown in SEQ ID NO.19 and a downstream primer RW with a nucleotide sequence as shown in SEQ ID NO.20; S2, the target gene fragment is ligated into a linearized pSAK277 backbone vector, and after transformation, the desired gene is obtained. Uni Gene overexpression vectors.

[0014] Fourthly, the present invention provides a method for creating tomato varieties with strong lateral branches, comprising: increasing the number of lateral branches in tomato varieties without lateral branches. Uni The content of the gene-encoded protein was used to obtain the tomato variety with strong lateral branches; wherein, the Uni The amino acid sequence of the gene-encoded protein is shown in SEQ ID NO.2.

[0015] Furthermore, the auxin content in the leaf axils of the tomato varieties with strong lateral branches was significantly lower than that of the parent varieties LA0580 and LA0533.

[0016] Fifthly, the present invention provides a method for creating a tomato variety with strong lateral branches, comprising: promoting the growth of tomato varieties without lateral branches... Uni Overexpression of the gene yields a tomato variety with strong lateral branches; wherein, the Uni The nucleotide sequence of the gene is shown in SEQ ID NO.1.

[0017] Furthermore, the auxin content in the leaf axils of the tomato varieties with strong lateral branches was significantly lower than that of the parent varieties LA0580 and LA0533.

[0018] Therefore, the present invention provides Uni The gene or its encoded protein can be used to regulate the elongation of tomato axillary buds, through overexpression in tomato varieties without lateral branches. Uni Genes or increase Uni The content of the gene-encoded protein reduces the accumulation of auxin in the leaf axils of the tomato plant, eliminates the apical dominance of the tomato plant on the lateral branches, accelerates the elongation of axillary buds in the leaf axils of the tomato plant into lateral branches, and the tomato lateral branches can emerge and grow normally and significantly, with a greater lateral branch growth than varieties with normal lateral branches; thus providing effective genetic resources for breeding tomato varieties with strong lateral branches. Attached Figure Description

[0019] Uni Figure 1 is a diagram of BSA mapping results of the gene; Figure 1 Uni Figure 2 is a diagram of genetic linkage and fine mapping of the gene; Figure 2 Figure 3 is a diagram of gene expression in different tissue parts of transgenic plants and transgenic background material LA0580; Uni Figure 3 Figure 4 is a diagram of gene expression in different tissue parts of transgenic plants and transgenic background material LA0580; Uni-OE#1 Figure 5 is a diagram of gene expression in different tissue parts of transgenic plants and transgenic background material LA0580; Uni Figure 4 Figure 6 is a diagram of auxin (IAA) content in leaf axils of OE#1 and parent LA0580 and LA0533; Uni Figure 7 is a diagram of gene overexpression lines of parent LA0580, parent LA0533 and Figure 5 Uni Figure 8 is a diagram of OE#1, Uni Figure 9 is a diagram of OE#12, Uni Figure 10 is a diagram of OE#15.

[0020] Uni SEQ ID NO. 1 is the nucleotide sequence of the gene of tomato; Uni SEQ ID NO. 2 is the amino acid sequence of the protein encoded by the gene of tomato. Uni DETAILED DESCRIPTION

[0021] In order to make the objectives, technical solutions and advantages of the present application clearer, the technical solutions of the present application will be described clearly and completely below in combination with embodiments of the present application. The present application can be embodied in many different forms of embodiments, and the protection scope of the present application is not limited to the embodiments mentioned herein. The experimental methods described in the embodiments of the present application are all conventional methods, and the materials, reagents, culture media and the like used in the following embodiments are all commercially available, unless otherwise specified.

[0022] In the present application, the less-branching tomato variety LA0580 and the normal-branching tomato variety LA0533 are both provided by the Tomato genetics resource center.

[0023] The main culture medium formulae used are as follows: 1) 1 / 2 MS medium: MS salt (with vitamins) 2.2 g / L, sucrose 15 g / L, agar 7.4 g / L;​​​​​​ 2) 0.2 MS medium: MS salt (without vitamins) 4.4 g / L, myo-inositol 0.1 g / L, thiamine hydrochloride (VB1) 0.4 mg / L, sucrose 20 g / L; 3) KCMS medium: MS salt (without vitamins) 4.4 g / L, myo-inositol 0.1 g / L, thiamine hydrochloride (VB1) 1.3 mg / L, 2,4-Dichlorophenoxyacetic acid 0.2 mg / L, KH2PO4 0.2 g / L, Kinetin KT 0.1 mg / L, sucrose 30 g / L, agar 7.4 g / L; 4) 2 Z medium: MS salt (with vitamins) 4.4 g / L, sucrose 30 g / L, agar 7.4 g / L, Zeatin (TRZ) 0.2 mg / L, Kan (kanamycin) 0.08 g / L, Timentin 0.3 g / L, Indoleacetic acid (IAA) 0.2 mg / L; 5) 0.2 Z medium: MS salt (with vitamins) 4.4 g / L, sucrose 30 g / L, agar 7.4 g / L, Zeatin (TRZ) 0.2 mg / L, Kan (kanamycin) 0.08 g / L, Timentin 0.3 g / L, pH=5.82; 6) R rooting medium: MS salt (with vitamins) 4.4 g / L, sucrose 30 g / L, agar 7.4 g / L, Kan (kanamycin) 0.08 g / L, Timentin 0.3 g / L, Indolebutyric acid (IBA) 2 mg / L.

[0024] The various MS salts used in the above medium are from Beijing Coolab Technology Co., Ltd.

[0025] The nucleotide sequences of the primers used in the embodiments of the application are shown in Table 1.

[0026] Table 1 Nucleotide sequences of the primers used in the application Uni Uni Uni Uni Uni Uni Uni Uni Uni Uni Uni Uni Uni Uni Uni Table Uni Uni Sequence No. Primer Name Sequence No. Primer Name Sequence No. Primer Name SEQ ID NO.3 Caps1-F SEQ ID NO.11 Caps10-F SEQ ID NO.19 FW SEQ ID NO.4 Caps1-R SEQ ID NO.12 Caps10-R SEQ ID NO.20 RW SEQ ID NO.5 Caps3-F SEQ ID NO.13 Caps11-F SEQ ID NO.21 pSAK277-F SEQ ID NO.6 Caps3-R SEQ ID NO.14 Caps11-R SEQ ID NO.22 pSAK277-R SEQ ID NO.7 Caps6-F SEQ ID NO.15 Caps12-F SEQ ID NO.23 Q_Uni-F The application uses LA0580, a less lateral branch tomato variety, and LA0533, a normal lateral branch variety, as parents, crosses the two parent varieties to obtain F1, and self-crosses F1 to obtain a F2 provisional genetic population. Through BSA analysis and molecular marker development on the two parent materials and the lateral branch non-elongation and lateral branch elongation two extreme pools in the F2 population, a regulatory gene for regulating tomato lateral branch elongation is finally located SEQ ID NO.8 is Caps6-R, the gene function annotation on NCBI is AUX / IAA family of auxin regulation genes SEQ ID NO.16 In LA0580 tomato material Caps12-R overexpressing the gene, created SEQ ID NO.24 OE#1 Q_Uni-R OE#12 SEQ ID NO.9 The lateral branch length of OE#15 plant has a very significant increase compared with LA0580 plant, and also has a significant increase compared with normal lateral branch plant LA0533. The present application has certain innovativeness in the aspect of regulating gene for enriching tomato leaf axillary bud growth, and provides effective gene resources for cultivating strong lateral branch tomato varieties.

[0027] Example 1 Positioning of tomato axillary bud growth regulating Caps8-F gene 1.1 Preliminary positioning of tomato lateral branch elongation growth regulating gene To obtain the gene regulating tomato axillary bud elongation growth, the present experiment uses the no-lateral branch tomato variety LA0580 (the material is obtained by radiation mutagenesis from LA0533) and the lateral branch tomato variety LA0533 as the parent material to obtain F1 by hybridization, and obtains F2 genetic population by selfing F1. Through the two parent materials, F2 genetic population, the no-lateral branch and lateral branch two extreme pools, and each pool contains 30 plants, respectively, the DNA is extracted for tomato genome resequencing, the sequencing fold of two parent materials is 30x, and the sequencing fold of each single plant in the pool is 1x. Based on the SNP and phenotype information in the genomes of two parent materials and F2 two pool materials, BSA analysis is carried out, and the gene regulating lateral branch elongation growth is preliminarily positioned in the 0.29 Mb interval of SL4.0 ch09: 65,483,943-65,777,357 of the 9th chromosome, as shown in SEQ ID NO.17 .

[0028] 1.2 Fine positioning of tomato Indel3-F gene According to the genomic sequence in the initial positioning interval SL4.0 ch09: 65,483,943-65,777,357, based on the SNP and indel information in the initial positioning interval, 7 Caps markers (Caps1, Caps3, Caps6, Caps8, Caps10-12) and 1 Indel marker (Indel3) were developed using Primer Premier 5.0 software, and the primer nucleotide sequences of the 8 molecular markers are shown as SEQ ID NO. 3-18. The DNA of each single plant in the parent and F2 population was used as template DNA, and the primers of the 8 markers were subjected to PCR amplification, respectively. The amplification products were subjected to polyacrylamide gel electrophoresis, and the difference band data were recorded. The QTL IciMapping 4.2 software was combined with the phenotype of the plant to construct a genetic linkage map, as shown in SEQ ID NO.25 , and the genotype and phenotype information of the F2 population were combined to finally fine map the Q_FW gene between Caps12 and Caps1 markers. Based on the tomato genome annotation information, it was found that there were 15 genes between the Caps12 and Caps1 markers, among which SEQ ID NO.10 LA0533 and LA0580 two tomato materials were significantly differentially expressed in leaf axils, and the remaining 14 genes were not differentially expressed, so Caps8-R was selected as the candidate gene of the tomato SEQ ID NO.18 gene. The nucleotide sequence of the coding region of the tomato Indel3-R gene is shown in the sequence table SEQ ID NO. 1, and the amino acid sequence is shown in the sequence table SEQ ID NO. 2.

[0029] Example 2 Overexpression vector of tomato SEQ ID NO.26 gene and construction method thereof 2.1 Cloning of tomato Q_RW gene The total RNA of the leaf tissue of LA0533 tomato plant was extracted, and the cDNA was reversely converted. The cDNA was used as a template, and the primers FW and RW with nucleotide sequences shown in SEQ ID NO. 19-20 were used to clone the Uni gene. The PCR amplification system is shown in Table 2, and the PCR amplification program is shown in Table 3. After amplification, the target band of about 710 bp was recovered.

[0030] Table 2 PCR amplification system Solyc09g083290 SlIAA14 Uni 25 Uni- 2 , Uni- 2 and Uni- 1 ddH2O 20 Table 3 PCR amplification program 2.2 Uni Overexpression vector of tomato The pSAK277 vector is single-enzyme cut by EcoR I, and the linearized vector is recovered by cutting gel. The recovered PCR amplified target fragment and linearized vector are subjected to homologous recombination. The homologous recombination system is a 10 μL system: 2 μL of 5X CE II Buffer, 1 μL of Exnase II, 3 μL of target fragment, and 2 μL of linearized vector, and the rest is supplemented with dd H2O. Add the components in order, then mix the reaction solution by gently pipetting, and then place it in a PCR instrument for reaction at 37°C for 30 min for homologous recombination.

[0031] The ligation product is transferred into E. coli, and the E. coli monoclonal with correct sequencing results is expanded. The recombinant plasmid is extracted using a DNA small amount extraction kit to obtain the overexpression vector pSAK277 of the Uni gene. Figure 1 .

[0032] The positive detection of the recombinant plasmid is detected by Taq-PCR, and the universal primer of the pSAK277 plant expression vector is used to detect the plasmid. The primer pair has a nucleotide sequence of SEQ ID NO. 21-22, the Taq-PCR detection system is shown in Table 4, and the Taq-PCR reaction program is shown in Table 5.

[0033] Table 4 Taq-PCR detection system Uni Figure 2 Uni 10 Solyc09g083290 1 Solyc09g083290 1 Uni 2 ddH2O 6 Table 5 Taq-PCR reaction program Example 3 Tomato Uni Application of the gene in tomato breeding The present example provides a method for creating a tomato variety with strong lateral branches, which is mainly based on Uni The overexpression of the gene is obtained by genetic transformation of a positive transgenic line, and the specific steps are as follows: (1) Tomato wild type LA0580 sterile seedling culture: select full and uniform tomato seeds, soak them in distilled water for 30 min, then sterilize them with 75% ethanol for 1 min, then surface sterilize them with 5% sodium hypochlorite solution for 15 min, and finally wash them with sterile ddH2O for 3-4 times, and sow them on 1 / 2 MS medium, and place them in a tissue culture room with a light cycle of 16 h light / 8 h dark for 7-8 d.

[0034] (2) Agrobacterium activation and culture: the recombinant vector pSAK277- UniThe Agrobacterium containing the target vector is inoculated into 20 mL of LB liquid medium containing 50 mg / L spectinomycin and 35 mg / L rifampicin, and cultured at 200 r / min, 28°C, and in the dark for 14-20 h until the OD600 of the bacterial solution is 1. 1.5 mL of the bacterial solution is centrifuged at 5000 rpm for 2 min in a sterilized 1.5 mL centrifuge tube, and the supernatant is removed. The bacterial solution is suspended in 0.2 MS liquid medium and diluted to an OD600 of 0.1-0.3 for standby use.

[0035] (3) The cotyledon explants of tomato LA0580 are infected with Agrobacterium containing the pSAK277- Uni The cotyledon explants of tomato LA0580 are infected with Agrobacterium containing the pSAK277- vector: sterile seedlings cultured for 7-8 d are selected, and the cotyledons are cut off from the petioles. The leaves are cut into 2-3 segments with a blade, and the cut cotyledons are placed flat on a sterilized KCMS medium with the back of the leaves facing up. The culture dish is sealed with a sealing film to prevent bacterial and fungal invasion, and is placed in a dark culture room for 1 d. The high-temperature sterilized 0.2 MS medium is poured into the culture dish, and the cotyledon segments cultured for 1 d are suspended in 0.2 MS liquid medium. Finally, 250 μL of the Agrobacterium suspension with an OD600 of 0.1-0.3 is added, the culture dish is covered, and the culture dish is gently rotated by hand. The infection time is 3 min. After the infection is completed, the bacterial solution is poured out, the bacterial solution on the cotyledons is absorbed with a sterile filter paper, and the infected cotyledons are placed back on the original KCMS medium and cultured in a dark culture room for 2 d.

[0036] (4) The explants are screened until callus grows, and the callus is subcultured until growth points grow: the infected cotyledons are transferred to the corresponding 2Z medium for regeneration, the front of the cotyledons faces up, the leaf surface fully contacts the medium, and the appropriate distance between the cotyledons is maintained. The density is moderate, and the culture is placed in a culture room for about 2 weeks, during which a large amount of callus and bud points are generated. To reduce the bud point malformation rate, the callus with bud points is transferred to new 0.2 Z medium for subculture.

[0037] (5) Obtaining and transplanting of transgenic plants: when the bud points grow on the 0.2 Z medium for about 2 weeks, the young shoots with complete growth points are selected and cut from the stem base, and are cultured for rooting on R medium. After about 1 week of culture, the transgenic plants start to root. After 2 weeks of rooting culture, the sealing film is removed for acclimation. After 2 d of acclimation in a culture room, the regenerated seedlings are transplanted into 12 cm diameter flowerpots containing nutrient soil, and are subjected to normal cultivation management.

[0038] (6) Transgenic positive plant identification: RNA was extracted from transgenic line plants grown in nutrient soil, reverse transcribed into cDNA, and the PCR primers shown in SEQ ID NO. 23-34 were used to identify the positive plants using Q_Uni-F and Q_Uni-R primers, System components The expression level was significantly higher than that of the non-transgenic plant LA0580, i.e. Volume (total 50 μL) Positive transgenic line plants 2xPhanta Flash Master Mix OE#1, FW (10 μmol / L) OE#12, RW (10 μmol / L) OE#15. Among them, the wild type tomato line LA0580 and the positive transgenic line LA0533 cDNA OE#1, Uni OE#12, Uni OE#15 were sown for 8 weeks, and the length of the lateral branches of the plants was measured, as shown in Table 6 and System components

[0039] Table 6 Length of lateral branches of wild type LA0580 and created line LA0533 (mm) Volume (total 20 μL) 2xTaq Plus Mix pSAK277-F (10 μmol / L) pSAK277-R (10 μmol / L) Plasmid pSAK277- Uni Uni Uni Uni Uni Uni Uni- Uni- Uni- Uni- Uni- Uni- Figure 5 Uni-OE# Plant No. Node 1 (mm) Node 2 (mm) Node 3 (mm) LA0580-1 <1 1.6 1.8 LA0580-2 <1 2.2 1.4 LA0580-3 <1 1.6 2.6 OE#1 71.7 54.3 30.9 OE#12 49.8 44.1 40.6 OE#15 36.6 6.4 25.4 As can be seen from Table 6 and Figure 5 Compared with the wild type LA0580 and LA0533, Uni the length of the lateral branches of the transgenic overexpression line was significantly longer than that of the wild type, especially the positive transgenic line Uni- OE#1 had a significantly longer lateral branch length.

[0040] Example 4 Length-related performance test of positive transgenic line Uni- OE#1 4.1 Expression level detection of the gene in the plant Uni The expression level of the gene in the positive transgenic line OE#1 created in Example 3 was detected, and the RNA was extracted from the roots, stems, leaves, flowers, fruits, etc. of the transgenic plants, and then reverse transcribed and subjected to fluorescent quantitative PCR detection. Specifically, the following steps are included: Uni (1) RNA extraction (using the "Plant RNA Extraction Kit" of Seville Biology), including the following steps: Uni (2) cDNA synthesis (using the "cDNA synthesis kit" of Seville Biology), including the following steps: (3) Fluorescent quantitative PCR detection (using the "cDNA fluorescent quantitative PCR kit" of Seville Biology), including the following steps: (4) Data analysis​1) Plant tissue sample lysis: Take 50 mg fresh plant tissue and quickly transfer into a 2.0 mL Nuclease-free grinding tube with 3-4 4 mm steel beads and 500 μL Buffer PRL1. Place the grinding tube in a grinder and grind (grinding program: frequency 50 HZ, temperature 4°C, 30 s each time, grind 3 times, interval 3 s) until homogenate. After sufficient grinding, incubate at 56°C for 15 min, and invert to mix every 5 min; 2) 12,000 rpm, room temperature centrifugation for 5 min, transfer the supernatant to a new 1.5 mL Nuclease-free centrifuge tube, add 1 / 5 supernatant volume of Buffer PRL2, and mix well by inverting; 3) 2,000 rpm, 4°C centrifugation for 5 min, transfer the supernatant to a new 2.0 mL Nuclease-free centrifuge tube, add 500 μL of Buffer PRL3 (add DTT Solution before use), and mix well by inverting; 4) Add 1 / 2 volume of anhydrous ethanol to the mixture of the previous step, and mix well by inverting; 5) Transfer the above mixture to the RNA Spin Column; 6) 12,000 rpm, room temperature centrifugation for 1 min, discard the filtrate. Put the RNA Spin Column back into the Collection Tube; 7) Add 500 μL Buffer RWA to the RNA Spin Column, 12,000 rpm, room temperature centrifugation for 1 min, discard the filtrate; 8) Add 600 μL Buffer RWB to the RNA Spin Column (please add Buffer RWB along the tube wall, which helps to flush the salt residues on the tube wall), 12,000 rpm, room temperature centrifugation for 1 min, discard the filtrate; 9) DNase digestion: a) Preparation of DNase reaction solution: Take 5 μL 10×DNase Buffer, 5 μL DNase, 40 μL Nuclease-free Water into a new 1.5 mL Nuclease-free centrifuge tube, and mix well; b) Add 50 μL DNase reaction solution to the center of the RNA Spin Column, and stand at room temperature for 15 min; c) Add 500 μL Buffer RWB to the RNA Spin Column, centrifuge at 12,000 rpm for 30 s at room temperature, discard the filtrate, and place the spin column back into the collection tube; 10) Repeat operation step 8); 11) Place the RNA Spin Column into the Collection Tube, centrifuge at 12,000 rpm for 2 min at room temperature, and remove the residual liquid; 12) Place the RNA Spin Column into a new 1.5 mL Nuclease-free centrifuge tube, and let it stand at room temperature for 3-5 min to completely evaporate the residual ethanol in the RNA Spin Column; 13) Add 50-100 μL Nuclease-free Water to the center of the membrane of the RNA Spin Column, let it stand at room temperature for 5 min, centrifuge at 12,000 rpm for 2 min at room temperature to elute the RNA. Re-add the first eluent to the RNA Spin Column, let it stand at room temperature for 5 min, centrifuge at 12,000 rpm for 2 min at room temperature to collect the RNA again.

[0041] 14) After detecting the quality of the RNA by agarose gel electrophoresis and determining the concentration of the RNA by absorbance method, store the RNA at -80°C for standby use.

[0042] (2) RNA reverse transcription The RNA provided in step (1) is subjected to RNA reverse transcription under the reverse transcription reaction system shown in Table 7 according to the reverse transcription reaction procedure shown in Table 8; after the reaction procedure is completed, the product is immediately used for qPCR reaction after the concentration is adjusted to 50 ng / μL, or is stored at 20°C.

[0043] Table 7 Configuration of reverse transcription reaction system System components Volume (20 μΐ total) 4 x All-in-one qRT SuperMix 5 Template RNA 2 RNase-free ddH2O 13 Table 8 Reverse transcription reaction procedure Temperature Time 50℃ 15 min 85℃ 5s (3) Tomato Uni Relative expression amount determination of gene The reaction system shown in Table 9 and the reaction procedure shown in Table 10 are used to express tomato Uni gene and the internal reference gene Slactin , and the relative expression amount of the tomato Uni gene is determined; wherein, the detection UniThe primer pair for gene expression level determination was the upstream primer Q_Uni-F shown in SEQ ID NO. 23 and the downstream primer Q_Uni-R shown in SEQ ID NO. 24; the upstream primer Q_FW shown in SEQ ID NO. 25 and the downstream primer Q_RW shown in SEQ ID NO. 26 were used to detect the expression level of the internal reference gene; the relative gene expression level was tested according to the conventional detection method; the detection results are as follows. Figure 3 As shown.

[0044] Table 9 Uni Relative gene expression level response system System components Volume (20 μΐ total) SYBR Mix 5 Q_Uni-F 0.2 Q_Uni-R 0.2 cDNA (50 ng / ul) 4 ddH2O 0.6 Table 10 Uni Relative gene expression response program from Figure 3 It can be seen that, compared with the wild-type plant LA0580, the transgenic line Uni-OE#1... Uni The relative expression level of the gene was significantly increased.

[0045] 4.2 Determination of IAA content in transgenic plants Determination of wild-type plant LA0580 and transgenic lines Uni- The axillary auxin (IAA) content of OE#1. Specific operating steps: 1) Take out the cryopreserved biological samples and grind them in liquid nitrogen using a grinder (30 Hz, 1 min) until they are powdered; 2) Weigh 50 mg of the ground sample in liquid nitrogen, add 10 μL of internal standard mixed solution with a concentration of 100 ng / mL and 1 mL of methanol / water / formic acid (15:4:1, v / v / v) extractant, and mix well; 3) Vortex for 10 min, centrifuge at 4°C and 12000 r / min for 5 min, and transfer the supernatant to a new centrifuge tube for concentration; 4) After concentration, redissolve in 100 μL of 80% methanol / water solution, filter through a 0.22 μm filter membrane, and place in a sample vial for LC-MS / MS analysis. The results are as follows: Figure 4 As shown. The data acquisition instrument system mainly includes ultra-high performance liquid chromatography (UPLC) (ExionLC™ AD, https: / / sciex.com.cn / ) and tandem mass spectrometry (MS / MS) (QTRAP® 6500+, https: / / sciex.com.cn / ).

[0046] Figure 4 This indicates that the transgenic strain ( Uni The auxin (IAA) content in the transgenic plant (-OE#1) was significantly lower than that in the wild-type plant (LA0580), with a decrease of 56.99%. The reduction in auxin accumulation in the lateral branches relieved the inhibition of auxin flow in the main stem on the axillary buds in the leaf axils, and promoted the elongation of lateral branches, which is consistent with the phenotype of the transgenic plant.

[0047] In summary, this invention constructs genetic linkage populations to finely locate and clone genes that positively regulate tomato lateral branch elongation. Uni By overexpressing in tomato plants without lateral branches Uni Genes or increase Uni The genetically encoded protein content was reduced, resulting in decreased auxin accumulation in the leaf axils of transgenic plants, and normal and significant growth of tomato lateral branches. This invention provides... Uni Uni The gene or its encoded protein can significantly reduce the accumulation of auxin in tomato plants, relieve the apical dominance of tomato plants on lateral branches, and accelerate the elongation of axillary buds in the leaf axils of tomato plants into lateral branches, providing genetic resources and new germplasm for breeding tomato varieties with strong lateral branches.

[0048] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and not to limit them; although the present invention has been described in detail with reference to preferred embodiments, those skilled in the art should understand that modifications can still be made to the specific implementation of the present invention or equivalent substitutions can be made to some technical features without departing from the spirit of the technical solutions of the present invention, and all such modifications and substitutions should be covered within the scope of the technical solutions claimed in the present invention.

Claims

1. A method for regulating the growth of tomato axillary buds Unicaulis The application of genes in tomato breeding is characterized by, The Unicaulis The gene positively regulates the elongation of tomato lateral branches. Unicaulis The amino acid sequence of the gene-encoded protein is shown in SEQ ID NO.

2.

2. The application according to claim 1, characterized in that, The Unicaulis The nucleotide sequence of the gene is shown in SEQ ID NO.

1.

3. The application according to claim 1 or 2, characterized in that, The Unicaulis The gene significantly promotes the elongation of lateral branches in the leaf axils of tomatoes.

4. The application according to claim 3, characterized in that, It includes overexpressing the aforementioned in tomato plants. Unicaulis Gene.

5. A method for regulating the growth of tomato axillary buds Unicaulis The application of biological materials with overexpression of genes or their encoded proteins in the positive regulation of lateral branch elongation in tomatoes, among which... The Unicaulis The amino acid sequence of the protein encoded by the gene is shown in SEQ ID NO.

2.

6. The application according to claim 5, characterized in that, The biomaterials include any one or more of the following: 1) Contains Unicaulis Expression cassettes of gene ORF sequences; 2) Contains Unicaulis Recombinant expression vectors of gene ORF sequences; 3) A recombinant expression vector containing the expression cassette described in 1); 4) Contains Unicaulis Engineered bacteria with ORF sequences of genes; 5) Engineered bacteria containing the expression cassette described in 1); 6) Engineered bacteria containing the recombinant expression vector described in 2) or 3).

7. A method for constructing a recombinant expression vector, comprising the following steps: S1, using cDNA reverse transcribed from total RNA of LA0533 tomato tissue as a template, was amplified using amplification primers. Unicaulis The ORF region of the gene is used to obtain the target gene fragment; among which, The amplification primers include an upstream primer FW with a nucleotide sequence as shown in SEQ ID NO.19 and a downstream primer RW with a nucleotide sequence as shown in SEQ ID NO.20; S2, the target gene fragment is ligated into a linearized pSAK277 backbone vector, and after transformation, the desired gene is obtained. Unicaulis Gene overexpression vectors.

8. A method for creating a tomato variety with strong lateral branches, comprising: Increase the number of tomato varieties without side branches Unicaulis The content of the protein encoded by the gene is used to obtain the tomato variety with strong lateral branches; wherein, the Unicaulis The amino acid sequence of the gene-encoded protein is shown in SEQ ID NO.

2.

9. A method for creating a tomato variety with strong lateral branches, comprising: Promote the growth of tomato varieties without side branches Unicaulis Overexpression of the gene yields a tomato variety with strong lateral branches; wherein, the Unicaulis The nucleotide sequence of the gene is shown in SEQ ID NO.

1.

10. The method of creation according to claim 8 or 9, characterized in that, The auxin content in the leaf axils of the tomato varieties with strong lateral branches was significantly lower than that of their parent varieties LA0580 and LA0533.

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