Fragaria ananassa vacuole processing enzyme coding gene FaVPE3 and application thereof

A technology that encodes genes, strawberries, applied in the field of plant biology

Active Publication Date: 2019-11-29
SHANGHAI ACAD OF AGRI SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

There are no related reports on the involvement of genes encoding strawberry vacuolar processing enzymes in the regulation of fruit quality

Method used

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  • Fragaria ananassa vacuole processing enzyme coding gene FaVPE3 and application thereof
  • Fragaria ananassa vacuole processing enzyme coding gene FaVPE3 and application thereof
  • Fragaria ananassa vacuole processing enzyme coding gene FaVPE3 and application thereof

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Embodiment 1

[0047] Embodiment 1, the construction of FaVPE3 gene interference RNAi vector

[0048] The gene FaVPE3 of the present invention is derived from strawberry (Fragaria×ananassa Duchase), and the full-length FaVPE3 gene and the 5'265bp fragment of the specific sequence of FaVPE3 are cloned by using Gateway homologous recombination cloning technology.

[0049] According to the full-length gene information of the vacuolar processing enzyme gene in the forest strawberry in the GDR database (www.rosaceae.org), design the full-length primer and add the linker sequence required for the BP recombination reaction at the 5' end, such as SEQ ID No.3, SEQ ID Shown in No.4. Using octoploid 'Hongyan' strawberry cDNAs as a template, high-fidelity enzyme (PrimeSTAR MaxDNA Polymerase) was used for PCR amplification, and the full-length FaVPE3 fragment that met the expected target size was purified and then processed by BP recombinase (Gateway@BP Clonase TM II Enzyme Mix) was cloned into the pDO...

Embodiment 2

[0053] Embodiment 2, the acquisition of FaVPE3 gene interference expression strawberry

[0054] Using the big green fruit of the "beautiful" strawberry in the intelligent artificial greenhouse as the material, use a marker pen to draw a vertical line from the middle to divide it into two parts. Agrobacterium strains containing target gene interference expression vector pK7GWIWG2D(II)-FaVPE3-RNAi and empty vector pK7GWIWG2D(II) were inoculated in 5mL YEP medium (containing 50μg / mL Kan, 50μg / mL Rif and 50μg / mLGen) cultured overnight at 28°C at 250 rpm, and the next day, 500 μl of bacterial liquid was drawn from it and transferred to 50 mL of YEP medium (containing 10 mM MES, 20 μM AS, 50 μg / mL Kan, 50 μg / mL Rif and 50 μg / mL Gen). Cultivate overnight at ℃, shake the bacteria to the logarithmic phase (OD value 0.6-0.8), and collect the bacteria by centrifugation at 4000rpm for 10min. Infection buffer (containing 10mM MES, 200μMAS, 10mM MgCl 2 ) to resuspend the bacteria, adjus...

Embodiment 3

[0055] Example 3 Molecular Identification of Strawberry with Decreased Expression of RNA Interference FaVPE3 Gene

[0056] About one week to 10 days after Agrobacterium microinjection, the fruit matures. Comparing the strawberry fruit side of the experimental group FaVPE3 gene RNA interference with the strawberry fruit side of the control group injected with the empty vector, it was found that the two were basically the same in appearance, and there was no visible difference ( figure 2 ).

[0057] Design a new specific primer pair for FaVPE3 at the 3' end of the gene, the forward primer

[0058] 5'-GCTCAAGCTCAAAAAGAA-3' SEQ ID No.7 and reverse primer

[0059] '-CATCCCATACTGAGAAAGTG-3' SEQ ID No.8, the amplified product is 222bp.

[0060] The effect of FaVPE3 gene interference expression was identified at the transcriptional level by RT-PCR. The internal reference gene GAPDH-specific primer set was 5'-TCCATCACTGCCACCCAGAAGACTG-3'SEQ ID No.9 and 5'-AGCAGGCAGAACCTTTCCGACAG...

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Abstract

The invention discloses a fragaria ananassa vacuole processing enzyme coding gene FaVPE3 and an application thereof. A nucleotide sequence and a protein sequence of the gene are provided, and furtheran RNA interference expression vector of the FaVPE3 gene is also constructed. An interference expression vector is transferred into agrobacterium tumefaciens, large fragaria ananassa green fruits areinfected by a microinjection method, and a transgenic fragaria ananassa strain of which the FaVPE3 gene expression is adjusted downwards is established. The gene has critical adjusting and controllingeffects on the accumulation of metabolism products of cane sugar, levulose, glucose, citric acid, malic acid and the like of fragaria ananassa fruits. A base is established for sugar acid quality adjusting and controlling mechanism of the fragaria ananassa fruits, genetic resources are provided for adjusting and controlling the sugar acid accumulation of the fragaria ananassa fruits through genetic engineering means, and important technique means are provided for development of functional SNP molecular markers and cultivation of superior fragaria ananassa.

Description

technical field [0001] The invention relates to the field of plant biotechnology, in particular to a strawberry fruit sugar-acid regulating gene FaVPE3 and its application in fruit sugar-acid quality regulation. Background technique [0002] Strawberry (Fragaria×ananassa Duchase) belongs to the Rosaceae Fragaria plant, and the accumulation and regulation of sugar and acid during fruit development are crucial for fruit development and quality formation. The flavor of the fruit largely depends on the content and ratio of sugar and acid. Generally, the fruit with high sugar and high acid has the richest taste and good flavor. The taste of sour fruit is very sour and few people can accept it. As for the low-sugar and low-acid fruit, it is bland and tasteless. The sugar composition of strawberry fruit is mainly composed of sucrose, glucose and fructose, but it varies from variety to variety. Most varieties mainly accumulate sucrose, and there are also varieties whose fruits main...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/57C12N9/64C12N15/82A01H5/00A01H6/74
CPCC12N9/6472C12Y304/22034C12N15/8245C12N15/8243C12N15/8218
Inventor 段可吕文远刘海婷杨静高清华邹小花田书华
Owner SHANGHAI ACAD OF AGRI SCI
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