Strawberry vacuolar processing enzyme encoding gene favpe3 and its application

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

Active Publication Date: 2022-07-29
SHANGHAI ACAD OF AGRI SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

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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  • Strawberry vacuolar processing enzyme encoding gene favpe3 and its application
  • Strawberry vacuolar processing enzyme encoding gene favpe3 and its application
  • Strawberry vacuolar processing enzyme encoding gene favpe3 and its application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

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

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

[0049] According to the full-length gene information of vacuolar processing enzymes in forest strawberry in the GDR database (www.rosaceae.org), design full-length primers and add the linker sequence required for the BP recombination reaction at the 5' end, such as SEQ ID No.3, SEQ ID No.4 shown. The octaploid 'Hongyan' strawberry cDNAs were used as templates for PCR amplification with a high-fidelity enzyme (PrimeSTAR MaxDNA Polymerase). TM II Enzyme Mix) was cloned into pDONR221 vector and transformed into E. coli DH5α competent cells for overnight culture. After identification by colony PCR, plasmid PCR and plasmid enzyme digestion,...

Embodiment 2

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

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

Embodiment 3

[0055] Example 3. Molecular identification of strawberry with reduced expression of FaVPE3 gene by RNA interference

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

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

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

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

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

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Abstract

The invention discloses a strawberry vacuole processing enzyme encoding gene FaVPE3 and its application; provides the nucleic acid sequence and protein sequence of the gene; also constructs an RNA interference expression vector of the FaVPE3 gene; Microinjection method was used to infect strawberry large green fruit, and a transgenic strawberry line with down-regulated FaVPE3 gene expression was established. This gene plays a key regulatory role in the accumulation of sucrose, fructose, glucose, and metabolites such as citric acid and malic acid in strawberry fruit. The invention lays a foundation for the regulation mechanism of sugar and acid quality in strawberry fruit, provides genetic resources for regulating the accumulation of sugar and acid in strawberry fruit by means of genetic engineering, and provides important technical means for the development of functional SNP molecular markers and the cultivation of high-quality strawberries.

Description

technical field [0001] The invention relates to the field of plant biotechnology, in particular to a strawberry fruit sugar-acid regulation gene FaVPE3 and its application in fruit sugar-acid quality regulation. Background technique [0002] Strawberry (Fragaria×ananassa Duchase) belongs to the Rosaceae family Strawberry, and the accumulation and regulation of sugar and acid in fruit development is crucial for fruit development and quality formation. The flavor of fruit depends largely on the content and ratio of sugar and acid. Generally, fruits with high sugar and high acid have the richest taste and good flavor, while fruits with high sugar and low acid have pure sweet taste, and the flavor is too mild and thin. The taste of sour fruit is very sour and few people can accept it, and the fruit with low sugar and low acid is bland and tasteless. The sugar composition of strawberry fruit is mainly composed of sucrose, glucose and fructose, but it varies with the variety. Mos...

Claims

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Application Information

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Patent Type & Authority Patents(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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