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Seedless tomato with characteristics of regular cracks and bright color, and breeding method thereof

A bright-colored, tomato technology, applied in the field of plant genetic engineering, can solve the problems of single gene function loss homologous gene function compensation failure, high coding sequence homology, gene function redundancy and other problems, so as to overcome the inhibition effect is not significant. Problems, simple inheritance of offspring, the effect of vigorous growth

Active Publication Date: 2019-02-01
YANGTZE NORMAL UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the role of these transcriptional regulators in controlling fruit parthenocarpy and overall fruit quality remains largely unknown.
[0004] At present, gene knockout, antisense technology, RNA interference technology, etc. are often used to identify and analyze the biological functions of these genes, but the coding sequence homology among the transcription factor members of the gene family is very high, and the function loss of a single gene is often Can fail due to functional compensation of homologous genes (gene function redundancy phenomenon)

Method used

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  • Seedless tomato with characteristics of regular cracks and bright color, and breeding method thereof
  • Seedless tomato with characteristics of regular cracks and bright color, and breeding method thereof
  • Seedless tomato with characteristics of regular cracks and bright color, and breeding method thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0031] Example 1 Tomato Gene SlARF10A Promoter cloning and GUS histochemical staining analysis

[0032] (1) SlARF10A Cloning of gene promoter

[0033] Using CTAB method (RTG2405-01, Zhongke Tairui) to extract tomato genomic DNA, according to SEQ ID NO.3 pSlARF10A Nucleotide sequence, design primers pSlARF10A -F and pSlARF10A -R uses the extracted genomic DNA as a template to pSlARF10A -F (forward primer) and pSlARF10A -R (reverse primer) is the primer for gene SlARF10A The amplification.

[0034] The primer sequence is as follows:

[0035] pSlARF10A -F: 5’- CAATTTACATATACAACACATGCCTCA-3’

[0036] pSlARF10A -R: 5’- GCTAATCCAATAGTTTTTCCCCTTC-3’

[0037] P CR amplification system: high-fidelity amplification enzyme P rime STAR HS (R010A, TaKaRa) 0.25 μL, 5× P rimeSTAR Buffer (Mg 2+ P lus) 5 μL, forward primer (10 μM) 0.5 μL, reverse primer (10 μM) 0.5 μL, template (DNA) 1 μL, dNT P (2.5mM) 2μL, sterile ddH 2 Make up O to 25μL.

[0038] Reaction procedure: pre-denaturation 95℃, 5m...

Embodiment 2

[0059] Example 2 Tomato SlARF10A-SRDX Fusion gene cloning and Agrobacterium-mediated genetic transformation

[0060] (1) SlARF10A-SRDX Cloning of Fusion Fragments

[0061] Take the Micro Tom tomato mixed sample (leaf, flower and fruit) as the material, TRIzol™ Plus RNA PurificationKit (12183555, Invitrogen™), follow the instructions to extract total tomato RNA, and use DNase I (18047019, Invitrogen™) to remove residual trace DNA , And use a spectrophotometer to determine the concentration of RNA for use.

[0062] Take about 2.0μg tomato total RNA, use P rimeScri p t II first-strand cDNA synthesis kit (6210A, Takara), and follow the instructions to synthesize the first strand of cDNA.

[0063] According to the tomato shown in SEQ ID NO.1 SlARF10A Gene sequence, design specific primers SlARF10A -F, SlARF10A- 3’SRDX- R (without termination password) and SRDX -R. Use cDNA as a template to SlARF10A -F (forward primer) and SlARF10A - 3’SRDX- R (reverse primer) is the primer for th...

Embodiment 3

[0102] Example 3 Tomato SlARF10B-SRDX Cloning of fusion gene and genetic transformation mediated by Agrobacterium

[0103] (1) SlARF10B-SRDX Cloning of Fusion Fragments

[0104] Take the Micro Tom tomato mixed sample (leaf, flower and fruit) as the material, TRIzol™ Plus RNA PurificationKit (12183555, Invitrogen™), follow the instructions to extract total tomato RNA, and use DNase I (18047019, Invitrogen™) to remove residual trace DNA , And use a spectrophotometer to determine the concentration of RNA for use.

[0105] Take about 2.0μg tomato total RNA, use P rimeScri p t II first-strand cDNA synthesis kit (6210A, Takara), and follow the instructions to synthesize the first strand of cDNA.

[0106] According to the tomato shown in SEQ ID NO.3 SlARF10B Gene sequence, design specific primers SlARF10B -F, SlARF10B- 3’SRDX- R (without termination password) and SRDX -R. Use cDNA as a template to SlARF10B -F (forward primer) and SlARF10B - 3’SRDX- R (reverse primer) is the primer ...

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Abstract

The invention discloses a seedless tomato with characteristics of regular cracks and bright color, and a breeding method thereof, wherein the auxin response factor gene S1ARF10A or SlARF10B of tomatois subjected to fusion inhibition expression, the nucleotide sequence of the S1ARF10A is represented by SEQ ID. NO.1, and the nucleotide sequence of the SlARF10B is represented by SEQ ID NO.3. According to the present invention, by using the chimeric repressor gene-silencing technology (CRES-T), the tomato auxin response factor gene S1ARF10A or SlARF10B is fused to the SRDX inhibition region, andthen is introduced into the tomato genome, such that the obtained transgenic tomato has characteristics of regular cracks, bright color, size consistent with the wild type, stable trait inheritance and independence of asexual reproduction, and contains less seeds (containing 1-5 seeds) or has a seedless form of more than 84% (average seedless form accounts for 37.8%); and the seedless tomato withcharacteristics of regular cracks and bright color is obtained by regulating the expression of the tomato gene SlARF10A or SlARF10B through the chimeric repressor gene-silencing technology (CRES-T), such that the new idea is provided for the molecular breeding in the future, and the theoretical basis and materials are provided for the shape establishment, the variety cultivation and the like in the research on tomato.

Description

Technical field [0001] The invention belongs to the technical field of plant genetic engineering, and specifically relates to a seedless tomato with regular cracks and bright color and a cultivation method thereof. Background technique [0002] Tomato fruits are rich in vitamins and minerals, which have a protective effect on the cardiovascular system; the lycopene in the fruits has unique antioxidant capacity and can resist aging; the malic acid or citric acid contained in tomatoes helps gastric juices Digestion of fat and protein, etc. Therefore, in the process of tomato growth and development, it is of great significance to study the regulation of tomato fruit. [0003] In recent years, the role of auxin in gene expression and regulation has attracted more and more attention. In Arabidopsis and other plant species, a large number of genes that may be regulated by auxin and may play a role in growth and development have been identified. Among these genes, members of the auxin ...

Claims

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Application Information

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IPC IPC(8): C12N15/82C12N1/21A01H5/00A01H6/82C12R1/01
CPCC07K14/415C12N15/8218
Inventor 符勇耀杨利平徐文姬杨韦高双
Owner YANGTZE NORMAL UNIVERSITY
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