Peach fruit polygalacturonase inhibitory protein PpPGIP1 gene as well as cloning method and application thereof

A polygalactose and inhibitory protein technology is applied in the field of peach fruit polygalacturonase inhibitory protein PpPGIP1 gene and its cloning to achieve the effects of inhibiting VIN activity, reducing decomposition and reducing chilling damage

Active Publication Date: 2021-04-16
NINGBO UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, there is no successful application of transiently silencing PpPGIP1 in peach fruit. Therefore, it is particularly important to transiently silence the target gene PpPGIP1 in peach fruit by Agrobacterium transformation.

Method used

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  • Peach fruit polygalacturonase inhibitory protein PpPGIP1 gene as well as cloning method and application thereof
  • Peach fruit polygalacturonase inhibitory protein PpPGIP1 gene as well as cloning method and application thereof
  • Peach fruit polygalacturonase inhibitory protein PpPGIP1 gene as well as cloning method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

specific Embodiment 1

[0023] Cloning and Sequence Analysis of PpPGIP1 Gene of Peach Fruit Polygalacturonase Inhibitor Protein

[0024] 1. Total RNA was extracted from "Yulu" peach fruit and reversed into cDNA for PCR template; details are as follows: TIANGEN's RNA prep Pure Plant Kit (Tiangen, Beijing, China) was used to extract total RNA from polysaccharide and polyphenol plants Kit, extract total RNA from peach fruit, and then use HiScript® II Q Select RT Super Mix for qPCR (Vazyme, Nanjing, China) kit to reverse transcribe into cDNA as a template for PCR reaction;

[0025] 2. Use the online website NCBI-PRIMER (https: / / www.ncbi.nlm.nih.gov / tools / primer-blast / ) to design specific amplification primers for the CDS region of peach fruit PpPGIP1 gene (Gene ID: LOC18769194), Upstream primer sequence: 5´-CCCGCAATCACATTTCTTATCC-3´, downstream primer sequence: 5´-CACTCCCAAGCTGCAAATAA-3´;

[0026] 3. PCR amplification: The PpPGIP1 gene amplification product was obtained by PCR amplification. The reactio...

specific Embodiment 2

[0033] Confirmation of protein-protein interaction between PpPGIP1 and PpVIN2 using yeast two-hybrid system (Y2H)

[0034] 1. Construction and identification of bait recombinant vector pGBKT7-PpVIN2 and prey recombinant vector pGADT7-PpPGIP1

[0035] Use the online website NCBI-PRIMER (https: / / www.ncbi.nlm.nih.gov / tools / primer-blast / ) to design the specific amplification of peach fruit PpPGIP1 gene and PpVIN2 gene (Gene ID are LOC18769194, LOC18776102, respectively) Primers, with appropriate restriction sites and homologous sequences of the expression vector at both ends of the primers (Table 1). The standard of primers is specific to the forward and reverse directions of the target fragment according to the requirements of homologous recombination enzyme Clon Express ®II One Step Cloning Kit (Vazyme, Nanjing, China) and high-fidelity enzyme 2×Phanta Max Master Mix (Vazyme, Nanjing, China). The 5' end of the primer is introduced into the homologous sequence at both ends of th...

specific Embodiment 3

[0049] Silencing of PpPGIP1 in peach fruit by Agrobacterium transient transformation and virus-induced gene silencing (VIGS) significantly inhibits VIN activity

[0050] The tested variety "Yulu" peach (Prunus persica L. Batsch) was extracted from Fenghua Peach Research Institute, Ningbo City, Zhejiang Province. The green ripe peach fruits with uniform size and no damage from diseases, insect pests and mechanical damage were selected for Agrobacterium infection. dye.

[0051] 1. In order to improve the silencing efficiency and reduce the possibility of non-target genes being silenced, use the online software SGNVIGS (https: / / vigs.solgenomics.net / ) to predict the target gene for PpPGIP1 (Gene ID: LOC18769194) PpPGIP1 The specific silencing sequence (300bp) was subcloned into the pTRV2 vector to obtain the pTRV2-PpPGIP1 recombinant vector, and the control group was pTRV1+pTRV2.

[0052] Construction and identification of the recombinant vector pTRV2-PpPGIP1: the specificity of...

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Abstract

The invention discloses a peach fruit polygalacturonase inhibitory protein PpPGIP1 gene as well as a cloning method and an application thereof, and is characterized in that the peach fruit polygalacturonase inhibitory protein PpPGIP1 has a nucleotide sequence as shown in SEQIDNO: 1; the amino acid sequence of the protein encoded by the peach fruit polygalacturonase inhibitory protein PpPGIP1 is shown as SEQIDNO: 2, and the cloning method comprises the following steps: (1) extracting peach fruit total RNA, and carrying out reverse transcription to obtain cDNA as a template; (2) designing primers according to the gene sequence of the PpPGIP1; and (3) PCR amplification: obtaining a PpPGIP1 gene amplification product through PCR amplification. The method has the advantages that PpPGIP1 and PpVIN2 have a protein interaction relationship, the PpPGIP1 expression quantity is effectively inhibited in peach fruits, the activity of acid invertase PpVIN2 can be remarkably reduced, and sucrose decomposition is reduced.

Description

technical field [0001] The invention belongs to the field of plant molecular biology, and in particular relates to a peach fruit polygalacturonase inhibitory protein PpPGIP1 gene regulating peach fruit acid invertase PpVIN2, a cloning method and application thereof. Background technique [0002] Peach( Prunus persica ) belongs to the family Rosaceae and the genus Peach, and is prone to chilling damage at 2-8 °C. The main symptoms include fruit browning, failure to ripen normally, cells under the epidermis shrinking to a spongy shape, and loss of inherent flavor. Low temperature stress is often accompanied by changes in soluble sugar content and metabolism-related enzyme activities. In plants, sucrose is the strongest cell cryoprotectant, which protects plants by regulating osmotic pressure, maintaining the liquid crystal state of cell membrane bilayers and stabilizing proteins. Our previous study showed that under low temperature stress, the degradation of sucrose in peach...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/29C12N15/10C07K14/415C12N15/84A01H5/08A01H6/74
CPCC07K14/415C12N15/8218C12N15/8205
Inventor 邵兴锋韦莹莹毛艺慧陈义姜舒
Owner NINGBO UNIV
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