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A seedless tomato with regular cracks and bright color and its cultivation method

A technology with bright colors and cracks, 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, etc., to achieve low cost and future generations inheritance Simple, Vigorous Effects

Active Publication Date: 2021-09-07
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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  • A seedless tomato with regular cracks and bright color and its cultivation method
  • A seedless tomato with regular cracks and bright color and its cultivation method
  • A seedless tomato with regular cracks and bright color and its cultivation method

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0031] Example 1 Tomato Gene SlARF10A Cloning of the promoter and analysis of GUS histochemical staining

[0032] (1) SlARF10A Cloning of gene promoters

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

[0034] The primer sequences are as follows:

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

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

[0037] P CR amplification system: high-fidelity amplification enzyme Pprime STAR HS (R010A, TaKaRa) 0.25 μL, 5× P primeSTAR 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 O to m...

Embodiment 2

[0059] Example 2 tomato SlARF10A-SRDX Fusion Gene Cloning and Genetic Transformation Mediated by Agrobacterium

[0060] (1) SlARF10A-SRDX Cloning of fusion fragments

[0061] Take Micro Tom tomato mixed samples (leaves, flowers and fruits) as materials, use TRIzol™ Plus RNAPurification Kit (12183555, Invitrogen™), extract tomato total RNA according to the instructions, and use DNaseI (18047019, Invitrogen™) to remove residual trace DNA, And use a spectrophotometer to measure the concentration of RNA for later use.

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

[0063] According to the tomato shown in SEQ ID NO.1 SlARF10A Gene sequence, design of specific primers SlARF10A -F, SlARF10A- 3'SRDX- R (without the termination password) and SRDX -R. Using cDNA as a template, with SlARF10A -F (forward primer) and SlARF10A - ...

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 Micro Tom tomato mixed samples (leaves, flowers and fruits) as materials, use TRIzol™ Plus RNAPurification Kit (12183555, Invitrogen™), extract tomato total RNA according to the instructions, and use DNaseI (18047019, Invitrogen™) to remove residual trace DNA, And use a spectrophotometer to measure the concentration of RNA for later use.

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

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

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Abstract

The invention discloses a seedless tomato with regular cracks and bright color and a cultivation method thereof. The tomato includes auxin response factor gene of tomato SlARF10A or SlARF10B Expression is suppressed by the fusion, the SlARF10A The nucleotide sequence is shown in SEQ ID NO.1, the SlARF10B The nucleotide sequence of is shown in SEQ ID NO.3. Using chimeric suppressor gene silencing technology (CRES‑T), the tomato auxin response factor gene SlARF10A or SlARF10B and SRDX After the fusion of the suppression region was introduced into the tomato genome, the obtained transgenic tomato had regular cracks, bright color, and more than 84% of the seedless (including 1-5 seeds) or seedless form (the average seedless form accounted for 37.8%), and the size was similar to that of the wild Consistent type, stable inheritance of traits, independent of asexual reproduction. Regulation of tomato genes by chimeric suppressor gene silencing technology (CRES‑T) SlARF10A or SlARF10B According to the expression, the seedless tomato with regular cracks and bright color was obtained, which provides new ideas for molecular breeding in the future, and provides theoretical basis and materials for the study of tomato fruit morphogenesis and variety cultivation.

Description

technical field [0001] The invention belongs to the technical field of plant genetic engineering, in particular to a seedless tomato with regular cracks and bright color and a cultivation method thereof. Background technique [0002] Tomato fruit is rich in vitamins and mineral elements, which has a protective effect on cardiovascular; the lycopene in the fruit has a unique antioxidant capacity, which can resist aging; the malic acid or citric acid contained in tomato is helpful for the gastric juice to Digestion of fat and protein. Therefore, it is of great significance to study the regulation of tomato fruit during tomato growth and development. [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 have been identified that are likely to be regulated by auxin and may play a role in growth and development. Among these genes, members of the auxin ...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/82C12N15/29C12N1/21A01H5/00A01H6/82C12R1/01
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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