Method for realizing gene transient expression in peach fruits

A transient expression and fruit technology, applied in genetic engineering, biochemical equipment and methods, using vectors to introduce foreign genetic material, etc., can solve the problems of lack of regeneration system and transgenic technology, etc., achieve reliable results, high expression level, and simple operation Effect

Active Publication Date: 2017-07-04
ZHEJIANG UNIV
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  • Abstract
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  • Claims
  • Application Information

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Problems solved by technology

However, due to the lack of regeneration system and transgenic technology, there is an urgent need to

Method used

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  • Method for realizing gene transient expression in peach fruits
  • Method for realizing gene transient expression in peach fruits
  • Method for realizing gene transient expression in peach fruits

Examples

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Embodiment 1

[0025] The present invention will be further described below in conjunction with specific embodiments and accompanying drawings, but the embodiments do not limit the protection scope of the present invention. Embodiment 1: Cloning of peach fruit PpTPS1 gene (SEQ: NO.1)

[0026] (1) Experimental method

[0027] Taking the amino acid sequence of AtTPS14, which has the function of linalool synthesis in Arabidopsis thaliana, as a reference sequence, the blastp algorithm was used to search for peach homologous sequences in the peach genome database Peach Genome V2.0. Select the sequence with the highest matching degree, design primer pair SEQ:NO.2 and SEQ:NO.3, use peach cDNA as template, carry out PCR amplification to obtain the sequence numbered as Prupe.4G030400 (SEQ:NO.1), named as PpTPS1. The PCR reaction system was 50 μl, including 0.5 μl Taq enzyme (Roche), 5 μl buffer (10×), 4 μl dNTP (2.5 mM), 2 μl each of upstream and downstream primers (10 μM, Invitrogen), 4 μl cDNA, 3...

Embodiment 2

[0030] Example 2: Transient expression of PpTPS1 in the fruit of 'Zhonghua Shoutao' promotes the synthesis of aroma substance linalool

[0031] (1) Experimental method

[0032] 1. Agrobacterium transformation culture containing the target gene PpTPS1

[0033] The target gene is constructed on the plant binary expression vector PGreen0029 62-SK, the primers used in PCR are aligned to SEQ: NO.4 and SEQ: NO.5, and the PCR product is connected to the vector by double enzyme digestion, and the enzyme digestion site is BamHI and SalI were transformed into Escherichia coli, cultured overnight, single clones were picked and sequenced for verification, and then the recombinant plasmid was electrotransformed into Agrobacterium GV3101::pSoup. After culturing in solid medium containing kanamycin (50mg / L) and gentamycin (50mg / L) at 28°C for 2 days, select monoclonal strains, add 5ml of LB containing kanamycin and gentamicin antibiotics after PCR test middle. After overnight culture, exp...

Embodiment 3

[0049] Embodiment 3: Cloning of peach fruit PpTPS2 gene (SEQ: NO.13)

[0050] (1) Experimental method

[0051] Taking the apple MdAFS-RG1 sequence with the function of α-Farnesene synthesis as a reference sequence, the blastp algorithm was used to search for homologous sequences in the peach genome database Peach Genome V2.0, and the sequence with the highest matching degree was selected. SEQ: NO.11 and SEQ: NO.12. The PpTPS2 sequence SEQ: NO.13 obtained by PCR amplification was verified by sequencing. The PCR system is the same as in Example 1.

[0052] (2) Experimental results

[0053] After sequencing verification, the PpTPS2 sequence SEQ: NO.13 matching the peach fruit genome data was obtained

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Abstract

The invention provides a method for realizing gene transient expression in peach fruits. According to the method, genes are excessively expressed in the pulp tissue through a vacuum infiltration infection method, and the method can be used for researching the gene functions. The method comprises the following steps: (1) selecting the ripeness of the peach fruits; (2) carrying out competence culture on the peach pulp tissue for pre-infection; (3) soaking the pulp in an agrobacterium infection solution containing a gene sequence; (4) applying vacuum to the pulp tissue; (5) releasing the vacuum for realizing transgenosis; and (6) carrying out further culture on the pulp tissue after infection. The established gene transient expression method is suitable for the peach fruits with thin peel and without cavities in the pulp tissue, the operation is simple, the expression level is high, the result is reliable, and a technical support is provided for developing gene function identification and selecting functional genes.

Description

technical field [0001] The invention belongs to the fields of plant molecular biotechnology and genetic engineering, and relates to a method for realizing transient expression of a target gene in peach fruit. Background technique [0002] With the completion of more and more plant genome sequencing and the development of transcriptome sequencing technology, the mining of functional genes is becoming an important part of plant biology research. Transgenic technology is the main means to carry out research on the function of target genes. But for most plants, especially perennial fruit trees, except for apples, citrus and grapes, most fruit trees have not yet established a stable transgenic technology. The lack of transgenic technology has become a limiting factor for the mining of fruit tree functional genes, and it is also a bottleneck factor affecting the development of fruit tree molecular biology. Compared with the stable expression characteristics of transgenic technol...

Claims

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Application Information

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IPC IPC(8): C12N15/82
CPCC12N15/8205
Inventor 张波柳洪入陈昆松
Owner ZHEJIANG UNIV
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