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Sl1ARF10 gene and Sl1miR160b regulating tomato lateral bud development and application thereof

A technology of slarf10-srdx and lateral buds, applied in the fields of application, genetic engineering, plant genetic improvement, etc., can solve problems such as undiscovered effects, and achieve the effect of increasing the incidence

Inactive Publication Date: 2019-03-12
YANGTZE NORMAL UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the role of miRNA160 or SlARF10 gene in the development of tomato lateral buds has not yet been discovered

Method used

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  • Sl1ARF10 gene and Sl1miR160b regulating tomato lateral bud development and application thereof
  • Sl1ARF10 gene and Sl1miR160b regulating tomato lateral bud development and application thereof
  • Sl1ARF10 gene and Sl1miR160b regulating tomato lateral bud development and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0038] Embodiment 1 Tomato SIARF10 promoter cloning and GUS staining analysis

[0039] (1) Sequence analysis and cloning of SIARF10 promoter

[0040] Using PlantCARE (http: / / bioinformatics.psb.ugent.be / webtools / plantcare / html / ) online software to analyze the sequence of about 2.5kb of the upstream promoter pSlARF10 of the tomato SlARF10 gene, the pSlARF10 promoter contains a variety of hormone-regulated response elements, Including auxin response element AUX, gibberellin response element GA3, ethylene response element ERE, etc., the results are as follows figure 1 shown.

[0041] According to the nucleotide sequence of pSIARF10 (SEQ ID NO.4), design specific primers pSIARF10-F and pSIARF10-R, and use CTAB method (RTG2405-01, Zhongke Tairui) to extract tomato genomic DNA as a template for conventional PCR amplification target fragment.

[0042] The primer sequences are as follows:

[0043] pSlARF10-F: 5'-CAATTTACATATACAACACATGCCTCA-3'

[0044] pSlARF10-R: 5'-GCTAATCCAATAGT...

Embodiment 2

[0067] Example 2 Construction of recombinant expression vector P35S-SlmiR160b-NOS and genetic transformation of tomato

[0068] (1) Cloning of tomato SlmiR160b

[0069] Through bioinformatics analysis, it was found that in the tomato genome, in addition to the published SlmiR160a Genbank (NR_107984.1) or miRBase (MI0008357), miR160 also has another site SlmiR160b on chromosome 12 (GenBank: HG975524.1) (SEQ ID NO. 1). Utilize The mfold Web Server online service software RNA Folding Form (version 2.3energies) to analyze the SlmiR160b hairpin structure such as Figure 4 shown.

[0070] According to the precursor nucleotide sequence of SlmiR160b (SEQ ID NO.1), specific primers SlmiR160b-F and SlmiR160b-R were designed, and the tomato genomic DNA was extracted by CTAB method (RTG2405-01, Zhongke Tairui) as a template for routine PCR amplification of target fragments. The primer sequence is:

[0071] SlmiR160b-F: 5'-CCAAGTCCAAGAAATGAAATTG-3'

[0072] SlmiR160b-R: 5'-GTTGCCAATTGG...

Embodiment 3

[0102] Example 3 Construction of recombinant expression vector P35S-SIARF10-RNAi-NOS and genetic transformation of tomato

[0103] (1) Cloning of tomato SIARF10-RNAi target fragment

[0104] Take Micro Tom tomato plants in flowering stage as material, use TRIzol TM Plus RNA Purification Kit (12183555, Invitrogen TM ), extract tomato total RNA according to the instructions, and use DNase I (18047019, Invitrogen TM ) to remove residual traces of DNA, and use a spectrophotometer to measure the concentration of RNA for later use.

[0105] About 2.0 μg of tomato total RNA was taken, and the first-strand cDNA was synthesized using PrimeScript II first-strand cDNA synthesis kit (6210A, Takara) according to the instructions.

[0106] According to the tomato SIARF10 gene sequence (SEQ ID NO.2), a specific segment was found by comparing homologous gene sequences, and specific primers SIARF10-RNAi-F and SIARF10-RNAi-R were designed. Using cDNA as a template, the specific fragment of...

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Abstract

The invention discloses an Sl1ARF10 gene and Sl1miR160b regulating tomato lateral bud development and application thereof. The nucleotide sequence of the precursor of Sl1miR160b is as shown in SEQ IDNO.1, the nucleotide sequence of the Sl1ARF10 gene is as shown in SEQ ID NO.2, and the coded amino acid sequence of the Sl1ARF10 gene is as shown in SEQ ID NO.3. Expression of the Sl1ART10 gene is inhibited by utilizing overexpression of Sl1miR160b or utilizing the chimeric repressor gene-silencing technology (CRES-T), and the obtained transgenic plant promotes the occurrence rate of the lateral bud without changing the occurrence mode of the lateral bud. The invention reveals that Sl1miR160b and Sl1ARF10 genes are the key target spots to regulate lateral bud development, meanwhile, the lateral bud occurrence number of the transgenic plant is in the positive relation with the expression abundance of Sl1miR160b in specific range, therefore the plant morphology with different lateral bud occurrence size and number can be screened out according to the transcription expression level of Sl1miR160b in the transgenic plant system. The regulatory effect of Sl1miR160b and Sl1ARF10 genes is commonly suitable for herbaceous plant, an effective technical means is provided for regulating lateral bud development of the herbaceous plant, and Sl1ARF10 gene and Sl1miR160b have the wide applicationvalue.

Description

technical field [0001] The invention belongs to the technical field of plant genetic engineering, and in particular relates to SlARF10 gene and SlmiR160b for regulating the development of tomato lateral buds and applications thereof. Background technique [0002] Side branches are an extremely important factor in plant morphology, representing the plasticity of plants to adapt to environmental changes. As the external environment changes, the appearance of plants will also change accordingly. In the landscaping of horticultural plants or the production of vegetables and field crops, side branches have an irreplaceable position. For example, in the field of landscape gardens, increasing the number of side buds can increase the width of the canopy and play a shading effect. In agricultural production, crop branching has an important impact on the yield of crops. In actual production, people use measures such as topping cotton, marigolds, grapes, eggplants, pruning fruit trees...

Claims

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Application Information

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IPC IPC(8): C12N15/113C12N15/29C07K14/415C12N15/82A01H5/00A01H6/20A01H6/82
CPCC07K14/415C12N15/113C12N15/8218C12N15/8226C12N15/8294C12N2310/141
Inventor 符勇耀杨利平李昌满徐文姬朱艺勇
Owner YANGTZE NORMAL UNIVERSITY
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